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SEMEN CHARACTERISTICS, PRESERVATION AND ITS USE

IN ARTIFICIAL INSEMINATION OF RED JUNGLE FOWL

(Gallus gallus murghi)

BUSHRA ALLAH RAKHA

03-arid-779

Department of Wildlife Management

Faculty of Forestry, Range Management and Wildlife

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi,

Pakistan

2017

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SEMEN CHARACTERISTICS, PRESERVATION AND ITS USE

IN ARTIFICIAL INSEMINATION OF RED JUNGLE FOWL

(Gallus gallus murghi)

by

BUSHRA ALLAH RAKHA

(03-arid-779)

A thesis submitted in partial fulfillment of

the requirement for the degree of

Doctor of Philosophy

in

Wildlife Management

Department of Wildlife Management

Faculty of Forestry, Range Management and Wildlife

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi,

Pakistan

2017

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I dedicate PhD dissertation to

my husband

Dr. Muhammad Sajjad Ansari

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CONTENTS

Page

List of Figures xii

List of Tables xxi

List of Abbreviations xxiv

Acknowledgement xxv

Abstract xxvii

1. GENERAL INTRODUCTON 1

1.1. RED JUNGLE FOWL 1

1.2. CONSERVATION STRATEGIES 2

1.3. SEMEN BANKING AND ITS SCOPE IN AVIAN

SPECIES

3

1.4. STORAGE OF SEMEN 4

1.5. EVALUATION OF EXTENDERS FOR LIQUID

(SHORT-TERM) STORAGE OF SEMEN

5

1.6. EVALUATION OF EXTENDERS FOR LONG-TERM

STORAGE OF SPERMATOZOA

6

1.7. CRYOPROTECTANTS FOR AVIAN SEMEN 9

1.8. POTENTIAL OF STORED SEMEN FOR ARTIFICIAL

INSEMINATION

10

2. SEMEN CHARACTERISTICS, COLLECTION

FREQUENCY AND TIMING IN INDIAN RED JUNGLE

FOWL (GALLUS GALLUS MURGHI)

13

2.1. INTRODUCTION 13

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2.2. REVIEW OF LITERATURE 14

2.2.1. Semen Characteristics 15

2.2.2. Semen Collection Frequency 17

2.2.3. Suitable Timing of Semen Collection 17

2.3. MATERIALS AND METHODS 18

2.3.1. Experimental Birds 18

2.3.2. Semen Collection and Quantitative Evaluation 20

2.3.3. Experimental Design 21

2.3.4. Extender Preparation 21

2.3.5. Semen Quality Assays 22

2.3.5.1. Motility 22

2.3.5.2. Plasma membrane integrity 22

2.3.5.3. Sperm viability 23

2.3.5.4. Acrosomal integrity 23

2.3.6. Sperm Abnormalities 24

2.3.7. Sperm Morphometrics 25

2.3.7. Statistical Analysis 25

2.4. RESULTS 29

2.4.1. Semen Characteristics and Morphology 29

2.4.2. Impact of Ejaculate Frequencies on the Semen

Characteristics of Indian Red Jungle Fowl

30

2.4.3. Influence of Semen Collection Timing (Morning vs

Evening)

38

2.5. DISCUSSION 38

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2.6. CHAPTER SUMMARY 43

3. IDENTIFICATION OF SUITABLE EXTENDERS FOR

LIQUID STORAGE (SHORT-TERM) AND

CRYOPRESERVATION (LONG-TERM) OF INDIAN RED

JUNGLE FOWL SEMEN

45


3.2. REVIEW OF LITERARURE 46

3.2.1. Liquid Storage of Semen 47

3.2.2. Cryopreservation/Long-Term Storage of Semen 47



3.3.2. Semen Collection and Quantitative Evaluation 49

3.3.3. Extender Preparation and Processing 50

3.3.4. Liquid Storage of Semen 50

3.3.5. Freezing and Thawing Procedure 51

3.3.6. SEMEN QUALITY ASSAYS 52


3.3.6.2. Sperm membrane integrity 52

3.3.6.3. Viability 53


3.3.7. Recovery Rate for Semen Quality Parameters 54

3.3.8. Absolute Livability Index 54

3.3.9. Fertility Measurements 55

3.4. STATISTICAL ANALYSIS 56

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3.5. RESULTS 56

3.5.1. Evaluation of Extenders for Liquid (Short-Term) Storage

of Semen

56

3.5.2. Evaluation of Extenders for Cryopreservation of Indian

Red Jungle Fowl Semen

58

3.5.3. Efficiency of Extenders for the Absolute Livability Index 64

3.5.4. Fertility Potential of Cryopreserved semen 64

3.6. DISCUSSION 72


4 EFFECT OF GLYCEROL CONCENTRATIONS ON

SPERMATOZOA OF THE INDIAN RED JUNGLE FOWL

78





4.3.2. Extender Preparation 82

4.3.3. Semen Collection and Evaluation 82

4.3.4. Semen Processing, Freezing and Evaluation 83





4.3.5.4. Sperm acrosome integrity 85

4.3.6. Recovery Rate of Semen Quality Parameters 85

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4.3.7. Absolute Livability Index 86

4.3.8. Artificial Insemination 86


4.4. RESULTS 88

4.4.1. Effect of Glycerol Concentrations on Motility, Plasma

Membrane Integrity, Viability and Acrosome Integrity at

Different Post-thaw Intervals (after 0, 2 and 4 Hours of

Incubation)

88

4.4.2. Effect of Glycerol Concentrations on Sperm Quality

Parameter Recovery Rates

89

4.4.3. Effect of Glycerol Concentrations on Absolute Livability

Index

89

4.4.4. Effect of Glycerol Concentrations on Fertility Attributes 90

4.5. DISCUSSION 90


5. EFFECT OF CRYOPROTECTANTS ON QUALITY AND

FERTILITY OF INDIAN RED JUNGLE FOWL SEMEN

102




5.3.1. Experimental Animals 110

5.3.2. Experimental Design 111

5.3.3. Semen Collection and Dilution with Freezing Extender 111

5.3.4. Cryopreservation of Semen 112

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5.3.5.1. Motility 112




5.3.6. Artificial insemination 115


5.4. RESULTS 116

5.4.1. Effect of Different Concentrations of DMA and Stages

of Cryopreservation on Quality of Indian Red Jungle

Fowl Spermatozoa

116

5.4.2. Effect of Different Concentrations of DMSO and Stages

of Cryopreservation on Quality of Indian Red Jungle

Fowl Spermatozoa

119

5.4.3. Effect of Different Concentrations of DMF and Stages of

Cryopreservation on Quality of Indian Red Jungle Fowl

Spermatozoa

132

5.4.4. Effect of Different Concentrations of PVP and Stages of


Spermatozoa

140

5.4.5. Effect of Different Concentrations of Egg Yolk and

Stages of Cryopreservation on Quality of Indian Red

Jungle Fowl Spermatozoa

147

5.5. DISCUSSION 154

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6. GENERAL DISCUSSION 166

SUMMARY 183

LITERATURE CITED 186

APPENDICES 232

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LIST OF FIGURES

Fig. No. Page

2.3.6.1 Sperm head abnormalities in Indian red jungle fowl fresh semen

ejaculate (lense = 100x oil immersion x eye piece = 10x).

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2.3.6.2 Sperm tail abnormalities in Indian red jungle fowl fresh semen


27

2.3.6.3 Sperm mid-piece abnormalities in Indian red jungle fowl fresh semen


28

3.6.1.1. Effect of extenders [Beltsville poultry semen extender (BPSE), Red

fowl extender (RFE), Lake extender (LE), EK extender (EKE),

Tselutin poultry extender (TPE) and Chicken semen extender (CSE)]

on the motility of Indian red jungle fowl spermatozoa stored at 5°C

(n=8). Bars with different letters differ significantly (P < 0.05) within

a given period of storage.

59




on the plasma membrane integrity of Indian red jungle fowl

spermatozoa stored at 5°C (n=8). Bars with different letters differ

significantly (P < 0.05) within a given period of storage.

60

3.6.1.3. Effect of extenders [Beltsville poultry semen extender (BPSE), Red 61

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on the viability of Indian red jungle fowl spermatozoa stored at 5°C

(n=8). Bars with different letters differ significantly (P < 0.05) within

a given period of storage.




on the acrosomal integrity of Indian red jungle fowl spermatozoa

stored at 5°C (n=8). Bars with different letters differ significantly (P <

0.05) within a given period of storage.

62

3.6.2.1.1

.

Effect of extenders [Beltsville poultry semen extender (BPSE), Red



on motility (%) of Indian red jungle fowl spermatozoa post-thaw at 0,

2 and 4 hours of incubation at 37 ºC (n=5). The bars with different

letters showed significant differences (P > 0.05) among all the

extenders at 0, 2 and 4 hours of Incubation.

65

3.6.2.1.2

.




on plasma membrane integrity (%) of Indian red jungle fowl

spermatozoa post-thaw at 0, 2 and 4 hours of incubation at 37ºC (n=5).

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The bars with different letters showed significant differences (P >

0.05) among all the extenders at 0, 2 and 4 hours of Incubation.

3.6.2.1.3

.




viability (%) of Indian red jungle fowl spermatozoa post-thaw at 0, 2

and 4 hours of incubation at 37 ºC (n=5). The bars with different

letters showed significant differences (P>0.05) among all the

extenders at 0, 2 and 4 hours of Incubation.

67

3.6.2.1.4

.

Effect of extenders [Beltsville poultry semen extender (BPSE),

Red fowl extender (RFE), Lake extender (LE), EK extender

(EKE), Tselutin poultry extender (TPE) and Chicken semen

extender (CSE)] on sperm acrosomal integrity (%) of Indian red

jungle fowl spermatozoa post-thaw at 0, 2 and 4 hours of

incubation at 37 ºC (n=5). The bars with different letters showed

significant differences (P > 0.05).

68

4.7.1.1. Effect of different levels of glycerol on spermatozoa motility (%) of

Indian red jungle fowl post-thaw at 0, 2 and 4 hours of incubation at

37 ºC (n=5). The bars with different letters showed significant

differences at a given stage at 0, 2 and 4 hours of Incubation.

91

4.7.1.2. Effect of different levels of glycerol on spermatozoa Plasma

membrane integrity (%) of India red jungle fowl post-thaw at 0, 2 and

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4 hours of incubation at 37 ºC (n=5). The bars with different letters

showed significant differences at a given stage at 0, 2 and 4 hours of

Incubation.

4.7.1.3. Effect of different levels of glycerol spermatozoa viability (%) of

Indian red jungle fowl post-thaw at 0, 2 and 4 hours of incubation at

37 ºC (n=5). The bars with different letters showed significant

differences at a given stage at 0, 2 and 4 hours of Incubation.

93

4.7.1.4. Effect of different levels of glycerol on spermatozoa acrosomal

integrity (%) of Indian red jungle fowl post-thaw at 0, 2 and 4 hours of

incubation at 37 ºC (n=5). The bars with different letters showed

significant differences at a given stage at 0, 2 and 4 hours of

Incubation.

94

5.4.1.1. Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of

dimethyleacetamide (DMA) on the spermatozoa motility (%) at

various stages of cryopreservation in Indian red jungle fowl. The

results are expressed as mean ± SEM, n=5. Different superscripts

indicate statistically significant differences (P < 0.05).

120


dimethyleacetamide (DMA) on the sperm plasma membrane integrity

(%) at various stages of cryopreservation in Indian red jungle fowl.

The results are expressed as mean ± SEM, n=5. Different superscripts


121

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dimethyleacetamide (DMA) on the sperm viability (%) at various

stages of cryopreservation in Indian red jungle fowl. The results are

expressed as mean ± SEM, n=5. Different superscripts indicate

statistically significant differences (P < 0.05).

122


dimethyleacetamide (DMA) on the sperm acrosomal integrity (%) at


results are expressed as mean ± SEM, n=5. Different superscripts


123


dimethylsulfoxide (DMSO) on the sperm motility (%) at various


expressed as mean ± SEM, n=5. Different superscripts indicate


127


dimethylsulfoxide (DMSO) on the sperm plasma membrane integrity


The results are expressed as Mean ± SEM, n=5. Different superscripts


128


dimethylsulfoxide (DMSO) on the sperm viability (%) at various


129

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expressed as Mean ± SEM, n=5. Different superscripts indicate



dimethylsulfoxide (DMSO) on the sperm acrosomal integrity (%) at


results are expressed as Mean ± SEM, n=5. Different superscripts


130


dimethylformamide (DMF) on the sperm motility (%) at various




134


dimethylformamide (DMF) on the sperm plasma membrane integrity




135


dimethylformamide (DMF) on the sperm viability (%) at various




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dimethylformamide (DMF) on the sperm acrosomal integrity (%) at




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polyvinylpyrrolidone (PVP) on the sperm motility (%) at various




141


polyvinylpyrrolidone (PVP) on the sperm plasma membrane integrity




142


polyvinylpyrrolidone (PVP) on the sperm viability (%) at various




143


polyvinylpyrrolidone (PVP) on the sperm acrosomal integrity (%) at

144

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5.4.5.1. Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of egg

yolk on the sperm motility (%) at various stages of cryopreservation in

Indian red jungle fowl. The results are expressed as Mean ± SEM,

n=5. Different superscripts indicate statistically significant differences

(P < 0.05).

148


yolk on the sperm plasma membrane integrity (%) at various stages of

cryopreservation in Indian red jungle fowl. The results are expressed

as Mean ± SEM, n=5. Different superscripts indicate statistically

significant differences (P < 0.05).

149


yolk on the sperm viability (%) at various stages of cryopreservation

in Indian red jungle fowl. The results are expressed as Mean ± SEM,

n=5. Different superscripts indicate statistically significant differences

(P < 0.05).

150


yolk on the sperm acrosomal integrity (%) at various stages of

cryopreservation in Indian red jungle fowl. The results are expressed

as Mean ± SEM, n=5. Different superscripts indicate statistically

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significant differences (P < 0.05).

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LIST OF TABLES

Table No.

Page

2.1. Semen characteristics (mean ±SEM) of Indian red jungle fowl

(n=40)

31

2.2. Sperm abnormalities (mean ± SEM) in semen of Indian red jungle

fowl (n=40)

32

2.3. Correlation between different semen quality parameters of Indian

red jungle fowl spermatozoa (n=40)

33

2.4. Morphometrics (mean ± SEM) of normal spermatozoa collected

from eight birds of Indian red jungle fowl (n=50 per

bird/measurement)

34

2.5. Effect of collection frequency (mean ± SEM) on characteristics of

Indian red jungle fowl spermatozoa (n=40)

35

2.6. Effect of semen collection time (morning vs. evening) on

spermatozoal quality parameters (mean ± SEM) of Indian red

jungle fowl spermatozoa (n=40)

36

3.1. Composition of extenders used for cryopreservation of Indian red

jungle fowl semen

69

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3.2. Absolute livability index (mean ± SEM) and recovery rate (%) of

semen quality parameters of Indian red jungle fowl after freeze-

thawing (n=5)

70

3.3. Fertility attributes of Indian red jungle fowl spermatozoa after

artificial insemination in hens (n=25) with freshly diluted and

frozen semen

71

4.1. Recovery rates (%; mean ± SEM) of sperm parameter after

cryopreservation with different levels of glycerol (n=5)

95

4.2. Multivariate regression for sperm parameters of Indian red jungle

fowl frozen with different levels of glycerol during incubation at

37°C using hours as independent variable (n=5)

96

4.3. Fertility outcomes of Indian red jungle fowl after artificial

insemination with different levels of glycerol in extender (n=50)

97

5.1. Comparison of dimethyleacetamide (DMA; 6%) with Glycerol

(20%; control) for fertility and hatchability parameters of Indian red


124

5.2. Comparison of dimethylsulfoxide (DMSO; 8%) with Glycerol



131

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5.3. Effect of DMF (dimethylformamide; 8%) vs Control (Glycerol

20%) on the fertility of spermatozoa and hatchability in Indian red

jungle fowl (n = 50)

138

5.4. Comparison of polyvinylpyrrolidone (PVP; 8%) with Glycerol


jungle fowl spermatozoa

145

5.5. Comparison between the effects of Egg yolk (15%) and Glycerol

(20%; control) on fertility and hatchability parameters of Indian red


152

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LIST OF ABBREVIATIONS

RJF Red Jungle Fowl

DMA Dimethyleacetamide

DMSO Dimethylsulfoxide

DMF Dimethylformamide

IRJF Indian red jungle fowl

PVP Polyvinylpyrrolidone

AI Artificial Insemination

BPSE Beltsville Poultry Semen Extender

LE Lake Extender

TSE Turkey Semen Extender

RFE Red Fowl Extender

CSE Chicken Semen Extender

EKE EK extender

TPE Tselutin Poultry Extender

ATP Adinosine Tri-phosphate

HOST Hypo-osmotic Swelling Test

LA Absolute index of Livability

LSD Least Significant Difference

DF Degree of Freedom

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ACKNOWLEDGEMENTS

“In the name of ALLAH, The Merciful and The Compassionate” who

bestowed upon me the will and the ability to carry this task, who blessed me with

capabilities to complete this study and be always with me at every stage of my life.

Countless salutations are upon the Holy Prophet “Muhammad (Peace be upon

him) who is beacon of enlightened benefactor the mankind ever had and who

enabled me to recognize my Creator and declared it to be an obligatory duty of

every Muslim to acquire knowledge.

I feel highly privileged in taking opportunities to express my profound

gratitude, sincere thanks and sense of obligations to my respectable and caring

supervisor, Prof. Dr. Iftikhar Hussain, Department of Wildlife Management for

his keen interest, skilful guidance, valuable suggestions, kind behaviour and timely

help during the entire study program.

I take this opportunity to express my deep gratitude and most sincere thanks

to my honorable Dean Prof. Dr. Maqsood Anwar, Department of Wildlife

Management for his dexterous help, impetuous guidance ,valuable and expert

suggestions and sympathetic attitude throughout the progress of this study.

I also owe debt of gratitude to members of my supervisory committee, Dr.

Tariq Mahmood, Assistant Professor, Department of Wildlife Management and

Dr. Shamim Akhter, Chairperson, Department of Zoology, PMAS-Arid

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Agriculture University, Rawalpindi, for their timely help, suggestions, special

attention and sympathetic attitude while completing this work. My deep and

heartfelt thanks to Prof. Dr. Elisabeth Blesbois and Dr. Francois Seigneurin,

INRA, UMR85 Physiologie de la Reproduction et des Comportements, F-37380

Nouzilly, France for providing me necessary initial guidance, help, cooperation and

encouragement for this task.

Words are lacking to express my thanks and feelings for my husband Dr.

Muhammad Sajjad Ansari, Associate Professor, University of Sargodha,

Faisalabad, who was always with me throughout the study period and helped in the

study design, experiment work and completion of this work.

Special thanks to my students Mr. Amir Naseer, Dr. Rabea Ejaz, Dr.

Saima Qadeer, Ms. Asma ul Husna, Mr. Imtiaz Ahmed Khan, Ms. Sumbull

Gill Ms. Zartasha Zafar Butt and Ms. Samia Bashir for their assistance in

laboratory work and field trials of artificial insemination. Thanks are due to Mr.

Muhammad Haider Shah, Mr. Muhammad Amir and Mr. Muhammad

Shabbir for helping me in keeping and management of birds for experimental

purpose.

I am indebted to my affectionate parents and parents in law and brothers

and sisters, brothers in law and sisters in law and other family members for their

prayers and moral support.

BUSHRA ALLAH RAKHA

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ABSTRACT

The Indian red jungle fowl (IRJF; Gallus gallus murghi) native to South-

Asia is facing threats in its natural habitat and needs immediate conservation

employing ex-situ and in-situ approaches. For ex-situ, in vitro conservation of

IRJF, semen banking is one of the potential techniques that require an extender

with appropriate cryoprotectants having adequate retrieval capacity for functional

spermatozoa. Therefore, study was designed to evaluate semen production capacity

(semen characteristics, timing, frequency of collection and seasonal changes),

identification of efficient extender, cryo-damage estimation, permeable (glycerol,

DMA, DMSO and DMF) for short and long term storage of semen and the use of

non-permeable cryoprotectants (PVP and egg yolk) and fertility outcomes for ex

situ in vitro conservation of IRJF germplasm. Semen was collected from eight male

birds of IRJF (housed at main campus PMAS-AAUR) in a graduated plastic tube

through abdominal massage and transferred to the laboratory for assessment of

semen volume, initial motility and concentration. The ejaculates having at least

60% motility were processed for further experimentation. The qualifying ejaculates

were studied for motility, volume, concentration, plasma membrane integrity,

viability and acrosomal integrity of spermatozoa. Semen production in IRJF was

quite low compared to domestic fowl but could be collected safely on daily basis

preferentially in the evening. A number of diluents (Beltsville poultry, turkey,

Lake, EK, Tselutin poultry and chicken semen extender) were tested against their

efficacy to preserve IJRF spermatozoa in liquid (5ºC) and frozen state (LN2; -

196ºC). The turkey semen extender was found superior compared to all

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experimental extenders for storage in liquid and frozen state showing remarkably

higher fertility rate. Cryopreservation with 11% glycerol caused maximum loss to

motility (50%) followed by plasma membrane integrity (45%), viability (25%) and

minimal to acrosomal integrity (20%). Glycerol optimization (11, 15 and 20%)

study demonstrated the superiority (P < 0.05) of 20% glycerol in freezing for

cryopreservability and fertility after artificial insemination. Various levels of DMA,

DMSO, DMF and PVP (4, 6, 8, and 10%) were evaluated; 6% DMA, 8% DMSO,

8% DMF and 6% PVP maintained higher (P < 0.05) semen quality and fertility

after artificial insemination compared to control treatment where spermatozoa were

exposed to 20% glycerol. Among four levels of egg yolk (10, 15, 20 and 25%)

evaluated; 15% egg yolk was superior (P < 0.05) for semen quality and fertility

compared to the control. It is concluded that IJRF semen with maximum efficiency

can be collected once in a day either in the morning or evening. The germplasm of

IJRF can be conserved in liquid (for two days) and in frozen state (for indefinite

period) using cryopreservation protocol based on DMA or DMF or DMSO or PVP

and egg yolk can be used efficiently in artificial breeding program for conservation.

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Chapter 1

GENERAL INTRODUCTION

1.1. RED JUNGLE FOWL

The red jungle fowl (Gallus gallus), an important member of Phasiandae

family, is one of the ancestor of domestic chicken (Delacour, 1951). Male cock‟s

average body length is 60 cm with body weight ranging between 671 and 1451 g,

while females‟ body length and weight range is 41-45 cm and 484-1051 g,

respectively. The species utilizes a variety of habitats, but are thought to prefer

extensive, undisturbed mixed forests for foraging as well as breeding purposes (del

Hoyo et al., 2001; Ali and Ripley, 1989). In nature, red jungle fowl lives in small

flocks during non-breeding season. However, during breeding season (spring and

summer), one cock makes harem of three to five hens. Hens lay five to seven eggs

in a clutch. The females have the ability to retain the spermatozoa of dominant

male or reject of recessive male, so that their chicks grow up to be the leaders

(Peterson and Bribson, 1999).

The red jungle fowl is diversified into five sub-species; Cochin-Chinese red

jungle fowl (G. g. gallus) found in China, Burmese red jungle fowl (G. g.

spadiceus) found in Thailand, Tonkinese red jungle fowl (G. g. jabouillei) found in

North Vietnam, Javan red jungle fowl (G. g. bankiva or G. g. ferrugineus) found in

Indonesia and Indian red jungle fowl (G. g. murghi) found in lower ranges of the

Himalayas and Southern Kashmir (Johnsgard, 1999).

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The Indian red jungle fowl (IRJF) is a native sub-species of southern Asia

(Genome Sequence Center, 2006) and in Pakistan has a limited distribution in Deva

Vatala National Park, Azad Jammu and Kashmir (Subhani et al., 2010). The global

status of this species is least concern (IUCN, 2008), while in Pakistan, it has a

threatened status attributed to habitat destruction, poaching, egg collection,

predation and genetic hybridization (Subhani et al., 2010). However, Birdlife

International (2016) has declared that the IRJF is facing extreme decline

throughout its range and is reported to be found only in small fragments. The avian

populations facing threats in their natural habitat are unable to breed due to

selection pressure and threatening events in their natural habitat (Gautier, 2002).

The captive breeding/propagation of the red jungle fowl is a viable activity that

might be helpful in the conservation of this bird (Malik et al., 2013).

1.2. CONSERVATION STRATEGIES

Conservation can be done through ex situ or in situ methods. Although

highest priority should be given to in situ conservation that enables the populations

for maintaining in their native or adaptive environment, however, the success of in

situ conservation among avian species is very low due to habitat destruction

(Williams and Hoffman, 2009). Various captive breeding programs for endangered

avian species were initiated in Pakistan, Nepal, South-east Asia but none of these

have been successful, for instance the cases of cheer pheasant (Catreus wallichii;

Grahame, 1980) in Pakistan and Nepal, and demoiselle crane (Grus virgo) in

Pakistan (Ali et al., 2011; Acharya, 2007). The major reason of failure of captive

breeding program is failure of birds to copulate naturally even living together for a

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number of years (Blanco et al., 2009). There are many ex-situ in vitro conservation

strategies which are helpful in maintaining the viable populations through

cryopreservation of animal genetic resources (gametes, embryos, other tissue

samples or DNA) and artificial insemination (Andrabi and Maxwell, 2007).

1.3. SEMEN BANKING AND ITS SCOPE IN AVIAN SPECIES

Semen banking is one of the techniques of artificial insemination that can

be used to improve genetic structure by avoiding inbreeding depression for limited

and at risk populations (Zhang, 2000; Jalme et al., 2003). However, successful

application of such reproductive biotechnologies require prior knowledge of sperm

physiology, response to low temperature, semen extenders and the effect of adding

cryo-protectants (Malecki et al., 1997; Klimowicz et al., 2005).

Semen characteristics differ among different groups of birds and even

within the same species (Riaz et al., 2004; Zhang, 2006; Blanco et al., 2009). A

number of studies have reported variations in semen characteristics of poultry

(Lake, 1962), emu (Malecki et al., 1997), cheer pheasant (Jalme et al., 2003),

turkeys (Zahraddeen et al., 2005), white backed vulture (Umapathy et al., 2005),

tragopan (Zhang, 2006), ducks (Ghonim et al., 2009), norfa cocks (Gebriel et al.,

2009) and rhea (Goes et al., 2010). Semen characteristics are also affected by the

frequency and timing of collection (Riaz et al., 2004). To get the impact of semen

collection on semen quality, it is necessary to investigate the maximum output of

the birds, so that it will not affect the birds in the long run (Ghonim et al., 2009). In

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poultry birds, collection of semen three times a week had better quality as

compared to twice daily or five times a week collection (McDaniel and Sexton,

1977), however in another study the broiler breeder showed better semen quality at

twice daily frequency of collection (Riaz et al., 2004).

1.4. STORAGE OF SEMEN

The importance of avian semen preservation for indefinite preservation of

genetic material especially for at risk populations has been recognized (Long,

2006). To date, most of the work has been done on domestic chicken and duck

spermatozoa, while relatively little work is performed on other non-domestic avian

species (Han et al., 2005). The preservation of spermatozoa in such species could

play an important role in breeding and genetic resources conservation as an aid to

economizing or to impart greater flexibility in special breeding programs. To

maximize the use of semen collected through artificial means, there is a need to

store the semen either in liquid form (short-term storage) or frozen form (long-term

storage) for artificial insemination rather than to exchange animals to overcome the

problem of inbreeding in small and isolated populations (Łukaszewicz et al., 2004;

Siudzinska and Łukaszewicz, 2008; Mukesh et al., 2013). The possibility of

dilution and storage of avian semen would help to make decisions about the

propagation of small populations by inseminating females where present and

utilization of semen from superior males (Siudzinska and Łukaszewicz, 2008;

Malik et al., 2013).

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1.5. EVALUATION OF EXTENDERS FOR LIQUID (SHORT-TERM)

STORAGE OF SEMEN

The liquid storage of semen has many benefits over cryopreservation;

however there is emergent need to develop the strategy for long term storage with

minimum loss to spermatozoa fertilizing ability. The liquid storage is the best

option only for short term storage of semen. It has been reported that semen stored

more than 6 hours in avian species have much reduced fertility (Blesbois et al.,

2007). This might be due to the production of reactive oxygen species and

metabolites from the dead spermatozoa which deteriorate the quality of semen

stored in liquid form (Donoghue, 1996). The most common procedure for short-

term storage (hours to days at refrigerator temperature) of fowl semen requires

suspending sperm in an extender to retain their viability in vitro. An appropriate

semen extender has to provide an energy source for spermatozoa and to maintain

pH and osmolarity levels identical to those of seminal plasma, the natural medium

for sperm.

The possibility of dilution and storage of sperm would enhance the

longevity by reducing metabolic activity through controlled cooling (Rakha et al.,

2013). The storage of semen, at low temperature, often results in the fertilizing

potential of spermatozoa to decline with the passage of time in a species specific

manner. However, the use of liquid semen is still the method of choice for many

avian species because the avian sperm have small quantity of cytoplasm as well as

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nuclear contents that badly affect sperm acrosomal integrity during freeze thawing

process (Blesbois et al., 2008).

Several extenders have been developed for the storage of semen in avian

species. Glutamic acid is the major component of extenders (Lake and McIndoe,

1959). Many buffered salts and zwitterionic molecules such as BES (N,N-bis (2-

hydroxyethyl)-2-aminoethanesulfonic acid), TES (N-[Tris (hydroxymethyl)

methyl]-2-aminoethanesulfonic acid) are constituent of extenders (Bootwala and

Miles, 1992; Siudzinska and Łukaszewicz, 2008). The extenders developed for

avian species include Turkey semen extender (Blanco et al., 2000), Beltsville

poultry semen extender (BPSE) (Sexton and Fewlass, 1978), EK extender

(Łukaszewicz, 2002), Lake diluent (Lake, 1960), Tselutin extender (Tselutin et al.,

1995) and chicken semen extender (Blanco et al., 2000). The interaction and ability

of each extender to protect the integrity of sperm are highly specialized and

species-specific (Blesbois et al., 2005).

1.6. EVALUATION OF EXTENDERS FOR LONG-TERM STORAGE

OF SPERMATOZOA

Germplasm storage for an indefinite period after cryopreservation is the

best option for ex situ conservation for any species facing high risks in their native

habitat (Blanco et al., 2000, Lukaszewicz et al., 2004; Blesbois et al., 2007; 2008;

Blanco et al., 2011; 2012; Rakha et al., 2013). The anatomy and physiology of bird

oocytes and embryos cause serious hindrance in their use for cryopreservation

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(Blanco et al., 2000, Blesbois et al., 2007; Lukaszewicz et al., 2004), thus,

cryopreservation of sperm, that is not invasive, has been proved the most

appropriate technique in ex situ in vitro conservation of Indian red jungle fowl

germplasm (Blesbois et al., 2007).

The sperm cryopreservation in combination with artificial insemination are

acknowledged for great worth in the ex situ conservation of threatened species

(Blanco et al., 2000, Lukaszewicz et al., 2004; Ansari et al., 2010; Rakha et al.,

2013). The success of these techniques is determined by the quality of frozen-

thawed semen multiplying with outcomes of the fertility attributes after artificial

insemination (Blesbois et al., 2008; Ansari et al., 2012; Rakha et al., 2013).

The cryopreservation of avian spermatozoa is not as successful as many

mammalian species due to specific reproductive physiology and high variability

from species to species and breed/strains that resulted in different

behavior/response of spermatozoa during freezing (Blanco et al., 2000, 2009; 2012;

Lukaszewicz et al., 2004; Blesbois et al., 2006; 2007; 2008; Long, 2006).

Therefore, the cryopreservation protocol for some avian species is highly specific

to achieve optimum quality of frozen-thawed semen (Blanco et al., 2000;

Lukaszewicz et al., 2004; Han et al., 2005; Blesbois et al., 2006; 2008). For

example, the ability of sperm and its response in an extender during dilution,

cooling, equilibration and freeze-thawing phases is highly variable from species to

species (Han et al., 2005; Blanco et al., 2009; 2012). One essential requirement of

the cryopreservation process is dilution of semen in an extender which can

maintain structural as well as functional integrity of the frozen-thawed semen (Han

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et al., 2005; Siudzinska and Lukaszewicz, 2008). The characteristics of an ideal

extender include provision of energy source to sperm cell metabolism, maintenance

of pH and osmolarity to sustain viability and cell functions.

Each extender has its own constituents and composition with precise limits

of physical properties that alter in a specific sequence with a minor change in

temperature and depends on solute to solute or solute to solvent interaction (Holt,

2000a,b; Rakha et al., 2013). However, the ability of an extender to maintain the

integrity of sperm at cellular level is highly variable with respect to species/breeds

and strains (Blanco et al., 2000; Holt, 2000a,b; Blesbois et al., 2008; Rakha et al.,

2013). The variation in bio-physical characteristics of sperm cell of a certain

species is responsible for interaction with a certain extender in highly specialized

manner with reduction in temperature (Blanco et al., 2000; Holt, 2000a,b; Han et

al., 2005; Rakha et al., 2013). These interactions may cause alterations in the

structural and functional status of the sperm ranging from beneficial to harmful

outcomes (Holt, 2000a).

Nevertheless, owing to the species related differences in fresh semen

quality and spermatozoa susceptibility to freezing, there exist no single

comprehensive cryopreservation method which could be effective and applicable

for all birds species (Łukaszewicz et al., 2004; Blanco et al., 2000; Holt, 2000b;

Polge, 1980). Therefore, it is necessary to identify a suitable extender for the

cryopreservation of IRJF that suits its physiology and maintain cellular integrity

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during changes in temperatures encountered in the process of cryopreservation and

thawing.

1.7. CRYOPROTECTANTS FOR AVIAN SEMEN

It is believed that avian sperm has low surface:volume ratio and elongated

tail that makes it more vulnerable to osmotic, chemical, thermal and physical

stresses during cryopreservation process compared to mammalian sperm (Long,

2006; Donoghue and Wishart, 2000). Freeze-thawing process not only causes

irreversible damage to mitochondria, mid-piece and perforatorium of spermatozoa

(Xia et al., 1988), but also induces physical and chemical changes that alter

physiological processes. Sperm may lose ATP due to energy metabolism

(Soderquist et al., 1991) and glyco-proteins or glycol-lipids necessary for transport

and maturation (Pelaez and Long, 2005; Long, 2006).

In the freezing of biological cells, addition of cryo-protectant is necessary

to minimize the loss and harmful effect of water crystallization (Łukaszewicz,

2001). A number of cryo-protectants have been used for domestic avian species

(Mitchell et al., 1977; Sexton, 1979; Fujihara and Buckland, 1987) and wild avian

species (Gee, 1983; 1985; Brock et al., 1983; Hargrove, 1986; Parks et al., 1986;

Gee and Sexton, 1990) sperm such as dimethylacetamide (DMA; molecular

weight: 87), dimethylsulfoxide (DMSO; molecular weight: 78; Blanco et al.,

2000), dimethyformamide (DMF; molecular weight: 73; Tselutin et al., 1999),

glycerol (molecular weight: 92; Long and Kolkarni, 2004), polyvinylpyrrollidone

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(PVP; molecular weight: 10,000; Holt, 2000a) and egg yolk (Makhafola et al.,

2009). Tselutin et al. (1999) studied the comparative effect of these cryo-

protectants on the fowl spermatozoa and found that glycerol and PVP has less

deleterious effects on spermatozoa survival than DMSO and DMA. Another study

on chicken spermatozoa showed higher motility in the presence of egg yolk as a

cryoprotectant compared to other extenders with no egg yolk (Wilcox, 1960).

1.8. POTENTIAL OF STORED SEMEN FOR ARTIFICIAL

INSEMINATION

The stored semen can artificially be inseminated through everted cloaca of

the hen, and sperm are placed inside the vagina near the sperm storage tubules.

Approximately 50 µL semen sample can be inseminated without having large

portions of the inseminate efflux from the hen‟s reproductive tract (Amann et al.,

1997). A single dose of semen sample should contain 200-300 million fresh or

frozen/thawed sperm (Philips et al., 1996). Sperm can be inseminated every other

day or even multiple times a day, but maximum fertility can only be achieved using

fresh semen (Blesbois et al., 2007).

Fertility with the frozen thawed semen is always compromised when having

glycerol as a cryoprotectant. However, fertility can be restored when diluted with

glycerol and removed just before artificial insemination (Shaffner, 1964). Purdey et

al. (2009) reported that glycerol must be removed slowly through step wise dilution

method from the sperm to avoid osmotically-induced damage and reduce its

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contraceptive effect. However, other cryoprotectants like DMA, DMF, DMSO,

PVP and egg yolk need not to be removed at the time of artificial insemination and

yield better results compared to glycerol (Blanco et al., 2000).

Although in vitro semen quality assays can predict much about sperm

physiology, yet is unable to unveil spermatozoan fertilizing ability (Graham and

Moce, 2005). The only way to test the success of frozen-thawed semen is to

perform fertility trial. Fertility measurements include the number of eggs laid,

fertile eggs laid, percent fertility and hatchability of the fertile eggs (Wilson et al.,

1997). Fertility trials can be conducted at any time and hens should be inseminated

once in 8 days to assess the longevity of the cryopreserved sperm in the hen‟s

reproductive tract and eggs collected daily (Blackburn et al., 2009).

Significant progress has been made in developing semen diluents and

preservation procedures for the avian semen in the 1970‟s and 1980‟s (Blesbois et

al., 2007). However, sperm survival and motility rate remained low after

cryopreservation. According to Dumpala et al. (2006) chicken semen is highly

concentrated with low volume. Therefore, extension of neat semen with a proper

diluent is required prior to artificial insemination (AI) and storage.

The critical steps in successful cryopreservation of fowl semen are:

selection of suitable semen extender, proper cryo-protectant, as well as freezing-

thawing method (Suidzinska and Lukaszewicz, 2008; Bellagamba et al., 1993;

Dumpala et al., 2006). Successful semen collection from the subspecies of

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Malaysian red jungle fowl (G. gallus) has been little documented (Malik et al.,

2013) and is lacking in red jungle fowl (Gallus gallus murghi). Present study was

conducted with the following objectives;

1. To study the characteristics of red jungle fowl semen

2. Identification of appropriate semen collection frequencies and timings in

red jungle fowl

3. Identification of suitable extenders for liquid storage and cryopreservation

of red jungle fowl semen

4. Evaluation of permeable and non-permeable cryo-protectants for red jungle

fowl semen

5. Fertility rate of liquid and cryopreserved red jungle fowl semen

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Chapter 2

SEMEN CHARACTERISTICS, COLLECTION FREQUENCY

AND TIMING IN INDIAN RED JUNGLE FOWL (Gallus gallus

murghi)

2.1. INTRODUCTION

The IRJF (Gallus gallus murghi) is the native gallus sub-species of

Southern Asia having limited distribution in Deva Vatala National Park, Azad

Jammu and Kashmir, Pakistan (Johnsgard, 1999; Subhani et al. 2010; Eriksson et

al., 2008). The global status of this species is least concern (IUCN 2008), while in

Pakistan it is threatened by many factors including habitat destruction, poaching,

egg collection, predation and genetic hybridization (Subhani et al., 2010).

The captive breeding/propagation of the IRJF is a viable activity that might

be helpful in the conservation of this unique bird. One key factor to develop

conservation programs is the management of the reproduction capacities of the

individuals that requires, for the males, a good knowledge of the gamete production

capacities (semen characteristics, impact of collection frequency and timing of

semen collection) and fertilization ability including fertilization after artificial

insemination. These studies may lead to the development of semen

cryopreservation programs to manage genetic diversity with the use of artificial

insemination (AI). Successful application of the AI/semen banking requires high

quality semen from the breeding males (Malecki et al., 1997; Klimowicz et al.,

2005). It is known that semen characteristics and frequency of collection differs

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among breeds and species (Riaz et al., 2004). In poultry, collection of semen three

times a week had better semen quality compared to twice daily or five times a week

collection (McDaniel and Sexton, 1977). However, the Hubbard broiler breeder

showed better semen quality at twice daily collections (Riaz et al., 2004). Although

semen can be collected on daily basis, it may affect semen output of the cocks in

terms of quality and quantity (Ghonim et al., 2009). According to available

information, no study is available on the impact of semen collection frequency and

timing of collection on the semen characteristics and need to be built for the IRJF.

Therefore, in order to prepare a conservation program, this study was

conducted to report for the first time, the successful semen collection and semen

characteristics, impact of collection frequency and the timing of semen collection

in the daily cycle of the IRJF.

2.2. REVIEW OF LITERATURE

IRJF lives in small mixed flocks during non-breeding season. However

during breeding season (spring and summer), one male maintains a territory with

three to five hens. Hens produce four to seven eggs per clutch (Delacour, 1951)

while in captivity, the clutch size increases up to 10-15 eggs if egg are removed

daily from the pen (personnel observation). Reproduction phenomenon in IRJF is a

complex with multiple environmental and physiological factors contributing to

successful fertilization.

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Successful semen collection from this subspecies of jungle fowl carries a

little documentation (Malik et al., 2013) and is lacking in IRJF. There is a need to

develop suitable techniques/strategies for the captive breeding/propagation of IRJF.

Efforts to save the red jungle fowl in the USA have remained unsuccessful

(Peterson and Brisbin, 1999) similar to captive breeding programs for endangered

avian species viz; cheer pheasant (Catreus wallichii) and demoiselle crane

(Anthropoides virgo; Acharya, 2007). The major reason of failure in captive

breeding programs is reduction in capacity of the birds to copulate naturally even

after living together for a number of years (Blanco et al., 2009). This necessitates

the conservation of red jungle fowl through the use of assisted reproductive

technologies like artificial insemination (AI) and semen banking. AI and/or semen

banking have been applied in the order Falconiformes, Gruiformes, Anseriformes

(Gee and Temple, 1978), sand hill crane (Grus Canadensis; Gee et al., 1985), emu

(Dromaius novaehollandia; Malecki et al., 1998), penguins (Sphenisciformes;

O‟Brien et al., 1999), ostrich (Struthio camelus; Rozen-boim et al., 2003), cheer

Pheasant (Catreus wallichii; Jalme et al., 2003), large parrots (Psittaciformes;

Stelzer et al., 2005) and cabot‟s tragopan (Tragopan caboti; Zhang, 2006).

Successful application of AI and/or semen banking requires high quality semen

from breeding males (Malecki et al., 1997; Klimowicz et al., 2005).

2.2.1. Semen Characteristics

Semen characteristics of many bird species such as domestic chickens

(Lake, 1966; Saeid and Al-Soudi, 1975; Tuncer et al., 2006; Malik et al., 2013),

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turkeys (Burrows and Quinn, 1937) and pheasants (Mantovani et al., 1993; Jalme

et al., 2003) have previously been studied. They include quantitative and

qualitative traits. The quantitative traits lead to define the daily, weekly or seasonal

number of sperm produced by the animals and the mean capacity of the testis

production and length of maturation of sperm in the epididymis and deferent ducts.

These production parameters differ between species (De Reviers, 1972; De

Reviers, 1980; Brillard and De Reviers, 1981; Etchu and Egbunike, 2002;

Adeyemo et al., 2007).

The gamete‟s capacities are evaluated by in vitro semen evaluation of so

called quality traits that may (at least partly) predict fertility (Saacke et al., 1980;

Blesbois et al., 2008). Some quality parameters for avian species such as semen

volume and membrane integrity were found to be the best variables for predicting

the fertilization potential in black Castellana roosters (Santiago-Moreno et al.,

2009). Most of the parameters are derived from criteria applied to mammalian

species such as bulls, rams, boars and stallions (Foote, 1978). However, some of

these are specific to avian species such as the number of sperm measured on the

vitelline membrane or the number of holes made by spermatozoa on these

membranes (Rabbani et al., 2006; Stewart et al., 2004). The quality tests used to

estimate the fertility of the semen after natural mating or artificial insemination

need to destroy the eggs produced and are not encouraged for rare breeds or

subspecies such as the IRJF. The only way to test the efficacy of the technique is to

maintain the highest quality of semen after post-thawing. Thus complementary

quantitative and noninvasive qualitative traits are joined in order to ensure the

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highest possible number of sperm of the highest possible quality for insemination

and conservation programs. The standard non invasive sperm parameters include

motility, sperm concentration, live or dead counts, acrosomal status, plasma

membrane integrity and morphology of spermatozoa (Malik et al., 2013).

2.2.2. Semen Collection Frequency

It is known that frequency of collection differs among breeds and species

(Riaz et al., 2004). In poultry, collection of semen three times a week had better

quality as compared to twice daily or five times a week collection (McDaniel and

Sexton, 1977). However, in another study the Hubbard broiler breeder showed

better semen quality at twice daily frequency of collection (Riaz et al., 2004).

Although, semen can be collected on daily basis, it may affect semen output of

male birds in terms of quality and quantity in the long run (Ghonim et al., 2009).

2.2.3. Suitable Timing of Semen Collection

Semen collection in avian species can be done at any time of the day

without affecting semen quality (Blesbois et al., 2008). However, semen volume

can vary at the morning time compared to evening time. Because the birds do not

drink water at night and may produce lower volume of semen in the morning. The

evening time has the advantage over morning time, as the birds can drink and eat

all the day and may produce higher volume of semen (Peterson et al., 2003). Riaz

et al. (2004) have reported that semen collection timing had no effect on the semen

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volume or concentration. The birds generally copulate in the evening time, and the

cocks may produce good quality ejaculate in the evening (Pizzari and Birkhead,

2001). In nature, the birds live in flocks and copulate more intensively in evening

time compared to morning. When the birds were dissected to obtain semen sample

directly from epidydmus, it was observed empty at early morning due to intense

mating at night (Peterson et al., 2003). This may be the indirect effect, either the

birds are producing low quality semen at morning time compared to evening time

or may be due the water retention at night when the birds do not eat or drink. The

scientists are unable to answer this question, and the response of birds with respect

to time is highly variable among all groups of birds (Hoolihan and Burnham,

1985). However, this needs to be studied for IRJF to know production capacity and

daily cycle of sperm production.

2.3. MATERIALS AND METHODS

2.3.1 Experimental Birds

IRJF chicks were obtained from the local poachers living in the vicinity of

Deva Vata National Park, Azad Jammu and Kashmir, Pakistan. It was done by

following snow ball sampling method (Setianto et al., 2017) which describes to

contact local persons involved in egg collection from wild and domestication of the

birds, with subsequent verification of these persons from the communities residing

inside and at the perimeter of the park. Futher, the genetic purity of the IRJF was

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confirmed by phenotypical characters of the species following Peterson and Brisbin

(1999), Brisbin and Peterson (2007) and Setianto et al. (2017).

These studies described that appearance of eclipse plumage during molting

period is a reliable characteristic of a pure wild population of IRJF. During eclipse

plumage molt, the birds loose their long, fine and often brightly colored feathers at

the neck and back (hackles), which are replaced by dull and black feathers. The

birds continue to molt, until they become indistinguishable from the female

(Johnsgard, 1986).

In present study, 2-3 days old chicks of IRJF were obtained from the local

paochers in 2012 and maintained in rearing pens under control conditions by

maintaining day and night temperature at 30 °C.

The chicks were offered commercially available poultry cock feed

(10g/day) and were exposed to 16 light hours a day. The birds were trained for

semen collection on reching six month age. The molting was started in June 2012

and continued till the end of October 2012. During the molting, the male birds lost

the hackles and were difficult to distinguish them from the females. Bsed on this

phenotypic confirmation of the genetic purity of the IRJF (Peterson and Brisbin,

1999; Brisbin and Peterson, 2007; Setianto et al., 2017), I used these birds for

further experimentation. At the time of semen collection, the experimental birds

had mean body weight of 1.7 kg (1.4-1.7 kg) and aged 1.5 years. Fresh water was

available to the birds all the day long throughout the experimental period.

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2.3.2. Semen Collection and Quantitative Evaluation

The birds were subjected to semen collection training by abdominal

massage as described by Burrows and Quinn (1935). The abdominal message was

carried out to get the birds prepared at the age of 6 months for semen collection as

well as to collect neat and clean ejaculate by avoiding fecal contaminations. This

training was continued until the cocks were mature (1.5 years of age) and able to

produce desired quantity of semen required for further processing.

The successful ejaculate (having at least 60% motility and free from fecal

contamination) was available after 4 weeks of training. Semen was collected from

individual birds in a graduated plastic tube. Semen volume was measured in

microlitres using micropipette. Initial sperm motility of each ejaculate was

determined as described by Zemjanis (1970) by mixing 10 µl semen samples in 500

µl of phosphate buffer saline (pH 7.2, 300 mosm).

The percentage of motile spermatozoa was determined by putting a drop of

semen sample on a pre-warmed glass slide (37oC) under phase contrast microscope

(x400, Olympus BX20, Japan). Sperm concentration was measured by taking 1µl

of semen and 200 µl of formal citrate solution (1 mL of 37% formaldehyde in 99

mL of 2.9% (w/v) sodium citrate) with Neubauer haemocytometer (Marienfeld,

Germany) under phase contrast microscope (x400, Olympus BX20, Japan). Total

sperm per ejaculate was obtained by multiplying the total volume with the

concentration. Daily (total sperm output multiplied by one) and weekly sperm

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output (total sperm output multiplied by seven) was calculated by unitary method

(De Reviers and Williams, 1981).

2.3.3. Experimental Design

Eight male birds of IRJF were used in the study. The study was conducted

during December 01, 2013 to June 22, 2014; five replicates for each male for each

experiment i.e. semen characteristics, collection frequency and timing of collection,

were studied. For the experiments on semen characteristics and morphology, semen

was collected once daily. For the experiment on effect of ejaculation frequencies,

the male cocks were subjected to four semen collection frequencies of twice a day,

once a day, after alternate days and after three days. Collection was done at 7:00

am and 7:00 pm for experiment on the effect of ejaculation frequencies and for the

experiment on the effect of collection time (morning vs evening). The ejaculate

volume, concentration, motility, membrane integrity and acrosome integrity of the

sperm cells and the morphological abnormalities were recorded for each sample.

2.3.4. Extender Preparation

The Beltsville Poultry Semen Extender (BPSE) was used as a diluent

(Sexton 1982). The extender composed of 60 mL distilled water, potassium citrate

(0.0384g), sodium glutamate (0.5202g), magnesium chloride (0.0204g), fructose

(0.3g), di-potassium hydrogen phosphate (0.7620g), potassium di-hydrogen

phosphate (0.039g), TES (0.3170g) and sodium acetate (0.2580g). The pH of this

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diluent was 7.3 having osmotic pressure 330 mOsm/kg. All samples were diluted

1:5 with BPSE and were processed for further investigations. All chemicals used in

this study were from Sigma-Aldrich, Co., 3050 Spruce Street, St Louis, USA.

2.3.5. Semen Quality Assays

2.3.5.1. Motility

Sperm motility was assessed by placing a drop of sample, previously

diluted to 1:5 (v:v) in the BPSE on a pre-warmed (37oC) glass slide under a phase

contrast microscope (400x) (Zemjanis 1970).

Sperm motility was determined by following 5-point scale (5: 80 to 100%,

vigrious rectilinear progressive movement; 4: 60 to 80%, rectilinear but not very

vigorous movement; 3: 40 to 60%, progressive but not rectilinear movement; 2: 20

to 40%, only circular movements; 1: below 20%, tail movment but not

progressive). Individual sperm motility was given as percentage of motile sperm

(Bielanski, 1972).

2.3.5.2.Plasma membrane integrity

Plasma membrane integrity was assessed by using hypo-osmotic swelling

test (HOS) as described by (Santiago-Moreno et al. 2009). The HOS solution was

prepared by adding 1 g of sodium citrate to 100 mL of distilled water. Previously

diluted 25 µl semen was mixed with 500 µl of HOS solution (100 mOsm/kg) and

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incubated at 37oC for 30 minutes. A drop of incubated solution was placed on a pre

warmed (37oC) slide and fixed in buffered 2% glutaraldehyde. The spermatozoa

showing swollen heads, swollen and coiled tails were classified as normal

spermatozoa having intact plasma membrane. A total of 200 spermatozoa were

counted at four separate fields under a phase-contrast microscope (lense = 100x oil

immersion x eye piece = 10x).

2.3.5.3. Sperm viability

Viability (% live / total sperm) was examined by adding eosin-nigrosin to

the lake glutamate solution. Lake‟s glutamate solution (Bakst and Cecil 1997) was

prepared by adding sodium glutamate (0.01735g), potassium citrate (0.00128g),

sodium acetate (0.0085g) and magnesium chloride (0.000676) in 100 mL distilled

water. Water soluble nigrosin (5g) and water soluble Eosin-bluish (1g) were added

into Lake‟s glutamate solution. Twelve drops of stain were mixed with 1 drop of

semen. A smear was made on a glass slide, fixed and air dried. A total of 200

spermatozoa were assessed per slide under a phase-contrast microscope (lense =

100x oil immersion x eye piece = 10x). The mixture provides a clear background in

the smear to enhance the contrast of white, unstained “live” sperm or the pinkish

stained “dead” sperm.

2.3.5.4. Acrosomal integrity

Acrosomal integrity was assessed by giemsa stain (Jianzhong and Zhang

2006). The stain was prepared by adding giemsa (3g) and phosphate buffer saline

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at pH 7.0 (2 mL) into 35 mL water. Smear was prepared by taking a drop of semen

sample on a clean glass slide, dried and fixed in neutral formal-saline (5%

formaldehyde) for 30 min.

The fixed slides were kept in giemsa stain for 1.5 hours. Sperm with normal

acrosome appeared to be evenly stained; abnormal spermatozoa were unevenly

stained while spermatozoa having ruptured acrosome remained unstained. A total

of 200 spermatozoa were observed at least in four separate fields under a phase-

contrast microscope (x400; Olympus BX20, Japan) at magnification of lense =

100x oil immersion x eye piece = 10x.

2.3.6. Sperm Abnormalities

Sperm abnormalities were assessed after fixing the semen sample in formal

citrate solution (prepared by adding 1 ml of 37% commercial formaldehyde in 99

ml of 2.9% (w/v) sodium citrate). The images were captured by Nikon Eclipse

E600 (Tokyo, Japan) microscope attached to a Nikon interfaced camera and

computer.

Sperm were studied for head abnormalities (macro-heads, acephalic, round

head, bent, deformed and detached heads), mid piece abnormalities (defective and

shorter) and tail abnormalities (tail coiled below the head, tail loose, coiled,

deformed, multiflagellate and disjoined) as described by Alkan et al.( 2002).

24

4

4

2.3.7. Sperm Morphometrics

The morphometrics of the various parts of the sperm (head length, head

width, mid-piece length, tail length and total length of sperm) were determined by

measuring a minimum of 50 cells from each bird (Santiago-moreno et al., 2016).

A drop of semen sample was placed on a glass slide and mixed with a drop

of lake glutamate solution to make a thin smear and air dried. Ten slides were

prepared for each individual bird, and five unstained and morphologically normal

spermatozoa were studied for morphometric measurements. All measurements

(micrometer) were taken by using a caliberated ocular micrometer at (lense = 100x

oil immersion x eye piece = 10x).

2.3.8. Statistical Analysis

Prior to analysis, all percentage data were normalized with an arcsine

transformation. Results are reported as non-transformed means (±SEM). The

differences between males or between ejaculation frequencies or between morning

and evening were analysed by Analysis of Variance using (MSTAT-C, Version

1.42 Michigan State University, East Lansing, MI, USA).Post hoc comparison

between the means was done though Fisher‟s protected LSD test. Pearson

correlation estimates for semen quality trait were also performed with Megastat

Version 7.25 Mc-Graw-Hill New Media, New York, for excel. Data on size of

sperm head, mid-piece and tail were analyzed for individual birds and presented as

means (±SEM).

25

4

4

Figure: 2.3.6.1: Sperm head abnormalities in Indian red jungle fowl fresh

semen ejaculate (lense = 100x oil immersion x eye piece = 10x).

A. Acephalic: spermatozoa having no head

B. Macrocephalic: Spermatozoa having large head mean that mid-piece is not

differentiated

C. Sperm showing head bent

D. Spermatozoa showing round head

E. Sperm showing deformed head

F. Sperm showing detached head

26

4

4

Figure: 2.3.6.2: Sperm tail abnormalities in IRJF fresh semen ejaculate (lense

= 100x oil immersion x eye piece = 10x).

A. Spermatozoa have coiled tail

B. Tail coiled below the head

C. Sperm have deformed tail

D. Sperm have tail loosely attached to head

E. Multiflagellate sperm

F. Detached tails

G. Loose tail

27

4

4

(a) (b)

Figure: 2.3.6.3: Sperm mid-piece abnormalities in IRJF fresh semen ejaculate

(lense = 100x oil immersion x eye piece = 10x).

A. Sperm having shorter mid-piece

B. Spermatozoa have defective mid-piece

C. Sperm have bent mid-piece

D. Normal spermatozoa

(b) Normal spermatozoa stained with Eosin-Red (lense = 100x oil immersion x

eye piece = 10x).

28

4

4

2.4. RESULTS

2.4.1. Semen Characteristics and Morphology

Semen collection from eight mature cocks of IRJF was routinely done at the

frequency of two ejaculates per week. The data on semen volume (mean 19.3 µl),

concentration (800 x 106 sperm/mL); total sperm per ejaculate (mean 0.015

billion), motility (mean 63.5%), viability (mean 92.4 %), intact acrosomes (mean

75.5%) and plasma membrane integrity (mean 89.2%) are shown in Table 2.1.

Sperm motility was positively correlated with acrosomal integrity (r=0.34;

P < 0.05) and plasma membrane integrity (r=0.41; P < 0.05). Semen volume was

negatively correlated with sperm motility, concentration, acrosomal integrity,

sperm viability and plasma membrane integrity (Table 2.3).

The data on the sperm morphological abnormalities are shown in Table 2.2.

The total amount of abnormal sperm found in the semen ejaculate of IRJF was

quite low i.e. 8.1% out of which the maximum abnormalities were mid-piece

abnormalities (56.17%) followed by tail abnormalities (22.2%) and head

abnormalities (21.8%).

The IRJF spermatozoa were observed typically filiform, consisting of head

and tail with vague neck (Figures 2.3.6.1 to 2.3.6.3). The spermatozoa were

generally straight/gently curled head and constrict anteriorly, consist of clearly

29

4

4

defined nucleus crowned by a small pointed acrosome, which appears on white

illumination.

The nuclear material was homogeneous in appearance and appeared as

white condensed nuclear content in elongated form. The tail was elongated and

varied in length and tapered at end. However, the mid-piece was continuous with

the base of head and start of tail. The nuclear contents in the head and proximal end

of mid-piece have similar diameter, but thinner than head. The mid-piece was

difficult to study under light microscope, but appeared clearer under phase contract

microscope.

The total average sperm length was recorded as 81.3 ± 0.5 µm as shown in

Table 2.3. The average lengths of different parts of IRJF sperm are 14.4 ± 0.4 µm

for head, 4.9 ± 0.1 µm for mid-piece and 62.0 ± 0.6 µm for tail. Based on

observations from the above measurements, the total head length to tail length ratio

was 1:4. There were no statistically significant differences (P > 0.05) observed

between any of the morphometric measurement for the sperm part among

individual birds.

2.4.2. Impact of Ejaculate Frequencies on the Semen Characteristics of

Indian Red Jungle Fowl

The data on the impact of ejaculation frequencies on the quality of IRJF

spermatozoa are given in Table 2.4. Total spermatozoa per ejaculate (million) were

30

32

Table 2.1. Semen characteristics (mean ±SEM) of Indian red jungle fowl (n=40)

Bird

ID

Semen quality parameters

Volume

(µl)

Concentration

(million/ml)

Total Sperm

Per Ejaculate

(million)

Motility

(%)

Acrosomal

Integrity

(%)

Plasma

membrane

integrity

(%)

Sperm

viability

(%)

1 11.6 ± 2.2 730 ± 0.3 8.0 64.0 ± 7.5 75.9 ± 3.7 87.7 ± 2.6 91.6 ± 2.5

2 17.3 ± 4.3 280 ± 0.2 4.0 60.0 ± 8.9 73.6 ± 5.5 87.8 ± 3.0 92.4 ± 1.8

3 23.2 ± 9.6 680 ± 0.2 15.0 44.0 ± 9.8 76.1 ± 6.8 90.2 ± 3.1 89.6 ± 2.2

4 14.4 ± 3.4 1620 ± 0.8 23.0 64.0 ± 7.5 80.1 ± 2.9 92.4 ± 2.5 92.0 ± 1.8

5 11.3 ± 2.2 1160 ± 0.6 13.0 72.0 ± 15 79.3 ± 11 92.0 ± 3.2 94.7 ± 1.8

6 30.2 ± 10.4 360 ± 0.2 11.0 52.0 ± 12 65.3 ± 6.7 86.9 ± 5.6 89.6 ± 1.6

7 14.5 ± 2.4 1110 ± 0.2 16.0 76.0 ± 9.8 77.9 ± 5.2 93.1 ± 3.6 95.9 ± 1.6

8 32.0 ± 6.6 470 ± 0.3 15.0 76.0 ± 4.0 75.8 ± 3.4 83.5 ± 8.7 93.7 ± 2.0

Overall 19.3 ± 2.9 800 ± 0.2 15.0 63.5 ± 4.0 75.5 ± 1.6 89.2 ± 1.2 92.4 ± 0.8

(Table 1; Appendices)

31

32

Table 2.2.: Sperm abnormalities (mean ± SEM) in semen of Indian red jungle fowl

(n=40)

Abnormalities Type Percentage

Head Macrocephalic 1.1 ± 0.1

Acephalic 0.9 ± 0.1

Round 1.6 ± 0.1

Bent 3.0 ± 0.2

Deformed 1.2 ± 0.1

Detached 2.8 ± 0.2

Overall 1.77 ± 0.37

Mid piece Defective 5.0 ± 0.5

Shorter 4.1 ± 0.5

Overall 4.55 ± 0.5

Tail Loose 1.3 ± 0.1

Coiled 3.3 ± 0.4

Muliflagellate 1.0 ± 0.1

Deformed 1.2 ± 0.2

Disjoined 2.2 ± 0.2

Overall 1.8 ± 0.43

Total Abnormalities (%) 8.1 ± 0.7

33

Table 2.3: Correlation between different semen quality parameters of Indian red jungle fowl spermatozoa (N=40)

Volume Motility Concentration Acrosomal Integrity Plasma membrane integrity

Motility -0.110

Concentration -0.239 0.084

Acrosomal Integrity -0.077 *0.342 0.249

Plasma membrane integrity -0.270 0.185 0.266 0.027

Viability -0.314 **0.412 0.019 -0.077 0.167

*Significant at P<0.05 ** Significant at P<0.01

34

Table 2.4: Morphometrics (mean ± SEM) of normal spermatozoa collected from eight birds of Indian red jungle fowl (n=50

per bird per measurement)

Measurements

Bird ID Head (µm) Mid-Piece (µm) Tail (µm) Total length (µm)

Length (µm) Width (µm) Perimeter (µm) Area (µm)

1 13.3 ± 0.3 1.2 ± 0.0 28.9 ± 0.5 15.4 ± 0.6 5.0 ± 0.2 62.9 ± 1.6 81.2 ± 1.8

2 14.2 ± 0.4 1.2 ± 0.0 30.7 ± 0.8 16.4 ± 0.2 4.7 ± 0.2 62.3 ± 1.2 81.1 ± 1.8

3 15.3 ± 1.2 1.2 ± 0.0 32.8 ± 0.7 17.7 ± 0.9 4.9 ± 0.2 61.6 ± 1.3 81.7 ± 1.4

4 14.4 ± 0.5 1.1 ± 0.0 31.2 ± 1.0 16.7 ± 0.8 5.4 ± 0.1 63.8 ± 1.6 83.6 ± 1.9

5 15.5 ± 0.4 1.0 ± 0.1 33.3 ± 0.8 17.9 ± 0.8 4.4 ± 0.2 58.9 ± 1.5 78.8 ± 1.7

6 13.3 ± 0.3 1.0 ± 0.1 28.9 ± 0.6 15.5 ± 0.2 5.0 ± 0.2 62.9 ± 1.6 81.2 ± 1.7

7 15.6 ± 0.2 1.1 ± 0.0 33.4 ± 0.4 16.9 ± 0.7 4.5 ± 0.2 60.4 ± 1.3 80.5 ± 1.3

8 13.3 ± 0.3 1.2 ± 0.0 28.9 ± 0.5 15.3 ± 0.6 5.2 ± 0.2 63.5 ± 1.6 82.0 ± 1.8

Total 14.4 ± 0.4 1.1 ± 0.0 31.0 ± 0.7 16.5 ± 0.4 4.9 ± 0.1 62.0 ± 0.6 81.3 ± 0.5

34

35

Table 2.5: Effect of collection frequency (mean ± SEM) on characteristics of Indian

red jungle fowl spermatozoa (n=40)

Sperm parameters Collection Frequency

12 hour 24 hours 48 hours 72 hours

Total Sperm per Ejaculate

(million)

102 ± 0.01c 303 ± 0.03

b 340 ± 0.07

b 555 ± 0.03

a

Daily Sperm output (million) 204 ± 0.03b 303 ± 0.03

a 170 ± 0.04

b 185 ± 0.01

b

Weekly Sperm output (million) 1263 ± 0.20b 2122 ± 0.21

a 1191 ± 0.27

b 1292 ± 0.09

b

Concentration (million/ml) 1390 ± 0.1c 2230 ± 0.3

b 3050 ± 0.3

a 4130 ± 0.8

a

Motility (%) 65.0 ± 2.8b 85.0 ± 2.1

a 84.3 ± 2.5

a 81.0 ± 6.3

a

Acrosomal integrity (%) 78.4 ± 1.4a 73.9 ± 2.8

a 77.5 ± 2.6

a 77.5 ± 5.1

a

Plasma membrane integrity (%) 85.5 ± 1.8a 90.9 ± 0.8

a 84.7 ± 1.3

a 91.5 ± 3.8

a

Sperm viability 89.8 ± 0.7a 84.7 ± 1.5

b 85.3 ± 1.7

b 85.0 ± 3.0

b

The values having different superscripts differ significantly (P<0.05) across the row

for a given parameter. (Table 2-9; Appendices)

36

Table 2.6: Effect of semen collection time (morning vs. evening) on spermatozoal

quality parameters (mean ± SEM) of Indian red jungle fowl spermatozoa (N=40)

Timing of semen collection

Morning Evening

Volume (µl) 74.8 ± 5.6a 74.0 ± 5.7

a

Motility (%) 71.7 ± 3.2a 65.6 ± 3.8

a

Concentration

(million/ml)

1730 ± 0.20a 1050 ± 0.2

b

Acrosomal integrity

(%)

69.4 ± 1.9a 87.7 ± 1.1

b

Plasma membrane

integrity (%)

78.6 ± 3.2a 92.7 ± 0.6

b

Sperm viability (%) 89.8 ± 1.2a 89.8 ± 0.8

a

The values (mean ± SEM) with different superscripts across the row differ

significantly (P<0.05). (Table 10: Appendices)

37

significantly higher (one way ANOVA, F3,21 = 26.67, P < 0.001) when semen was

collected after 72 hours compared to the 24 and 48 hours collections.

Daily sperm output (one way ANOVA, F3,21 = 2.90, P = 0.06) and weekly

sperm output (one way ANOVA, F3,21 = 3.49, P < 0.05) was higher (P < 0.05) at 24

hours of collection compared to 12, 48 and 72 hours of collection. There was an

overall increase (one way ANOVA, F3,201 = 15.43, P < 0.001) in sperm

concentration as the interval between the collection of semen was increased from

12 hours (1.39 ± 0.1) to 24 hours (2.23 ± 0.3), 48 hours (3.05 ± 0.3) and 72 hours

(4.13 ± 0.8).

Sperm motility (%) was significantly improved (one way ANOVA, F3,181 =

13.30, P < 0.001) when time interval between the collection was increased from 12

h to 24 h (85.0 ± 2.1), while it remain unchanged when semen was collected at 24 h

(85.0 ± 2.1), 48 h (84.3 ± 2.5) and 72 h (81.0 ± 6.3) of intervals.

The sperm acrosomal integrity (one way ANOVA, F3,200= 0.77, P > 0.5)

and plasma membrane integrity (one way ANOVA, F3,200 = 2.49, P > 0.05) of IRJF

spermatozoa remained similar when semen was collected at 12h, 24 h, 48 h and 72

h intervals.

The percentage of live sperm was recorded significantly higher (one way

ANOVA, F3,200 = 4.50, P < 0.01) at 12 hours (89.8 ± 0.7) of collection compared to

38

24 hours (84.7 ± 1.5), 48 hours (85.3 ± 1.7) and 72 hours (85.0 ± 3.0) of collection

intervals.

2.4.3.Influence of Semen Collection Timing (Morning vs Evening)

The data on the effect of semen collection time (7 am and 7 pm) on

spermatozoa quality parameters of IRJF is given in the Table 2.5. Semen volume

(t-test (pooled variance), t98 = -0.08, P > 0.5), motility (t-test (pooled variance), t98 =

-1.18, P > 0.5) and viability (t-test (pooled variance), t98 = 0.42, P > 0.5) did not

differ significantly at either time. However the sperm concentration was higher

when semen was collected in morning time. While sperm viability and plasma

membrane integrity was significantly higher in the semen collected at evening

time.

2.5. DISCUSSION

Assessment of semen characteristics, ejaculate frequency and timing of

semen collection are important indicators of the reproductive potential required for

the genetic exploitation of the breeding individuals and also for conservation of

threatened species (Marzoni et al., 2000; Bah et al., 2001; Tuncer et al., 2006;

Peters et al., 2008; Madeddu et al., 2009; Ajayi et al., 2011; Malik et al., 2013).

Semen quality tests (sperm motility, plasma membrane integrity, viability and

acrosomal integrity) are used in routine semen evaluation for artificial insemination

39

(Graham et al., 1990; Froman et al., 1999; Parker et al., 2000; Snook, 2005;

Partyka et al., 2012).

The present study for the first time reported the successful semen collection

and characteristics of semen production in the IRJF. Semen collected by the

massage methods showed sperm concentration, volumes and total number of sperm

per ejaculate generally lower of what is usually observed in the domestic chicken,

Malaysian red jungle fowl and broiler breeder (Malik et al., 2013; McDanieal,

1995). The proportion of motile sperm was relatively lower when compared to

commercial parents. However, sperm viability, acrosome integrity and plasma

membrane integrity was equivalent to those reported in studies on different breeds

of chicken (Graham et al., 1990; Froman et al., 1999; Parker et al., 2000; Snook,

2005; Partyka et al., 2012).

It is interesting that sperm motility, plasma membrane integrity, viability

and acrosomal integrity was not significantly different (P > 0.05) in eight

experimental birds. It is suggested that these finding may be due to similar age,

uniform food and management conditions provided in captivity. The data showed

positive correlation between motility, acrosome integrity and plasma membrane

integrity. Similar trends have been reported in Rhode Island red, white breeder

cocks (Nwagu et al., 1996), seven strains of chicken (Peters et al., 2008) and

Spanish breeds of chicken (Prieto et al. 2011). IRJF sperm closely resemble those

of other galliform (avian) species including chicken (Grig and Hodge, 1948), goose

(Ferdinand, 1992) and turkey (Wakely and Kosin, 1951). Knowledge of sperm

40

morphometrics may elucidate adaptation of avian species and function of

spermatozoa. Variation found in the size and shape and nuclear contents of the

sperm cells in an ejaculate may be related to the degree of maturity; which

ultimately predict the fertilizing capacity of spermatozoa (Maroto-Morales et al.,

2012). The shape of spermatozoa varies even among Galliformes, Passeriformes

and Falconiformes. The results of the present study showed that IRJF sperm have

vermiform (long and narrow) head as that of other avian species (chicken, quail

and partridge).

The length of spermatozoa in various avian species may range between 30-

300 µm and is not associated with species size and weight. The length of IRJF

sperm is about 81 µm, which is longer than emu (47 µm; Korn et al., 2000), duck

(55.6 µm; Zawadzka and Lukaszewicz, 2012), ostrich (56.7 µm; Soley and

Roberts, 1994) and equal to closely related chicken species (82 µm; Grigg and

Hodge, 1949). The sizes of different parts of the sperm (head, mid-piece and tail)

are variable between the species (Santiago-moreno et al., 2016).

IRJF has larger and narrow head while the falconiformes have shorter and

wider head and have greater percentage of abnormal spermaotozoa (55-60%). This

high percentage of abnormal spermatozoan in ejaculate of falconiformes birds is an

indicative that these are monogamous and competition between the sperm is very

low (Blanco et al., 2001). As IRJF is a polygamous bird, the sexual strategy

(polygamy) poses greater influence on the morphological and functional

characteristics of spermatozoa (Santiago-moreno et al., 2016). As previously

41

reported for polygynous avian species (Galliformes), due to high sperm

competition within the sperm storage tubules (SSTs), the cocks may produce high

quality ejaculate with very low abnormailities. These findings confirm our study

that IRJF has very low percentage of abnormal spermatozoa. Sperm competition

within SSTs is highly selective force that promotes the spermatozoa in terms of

larger and faster swimming flagellum. It is now evident from various studies that

spermatozoa having large heads have greater ability to beat/swim at large pace

compared to other species having short headed spermatozoa (Santiago-moreno et

al., 2015). It is well acknowledged that sperm ejaculates of various avian species

are positively correlated to the environmental conditions or the habitats. The avian

species found in temperate and tropical climates have comparatively large and

narrow head compared to those found in arid or semi-arid sub-tropical zones

(Santiago-moreno et al., 2016). The present study confirm the findings of this

study, as IRJF is a resident of temperate and tropical climatic conditions and

exhibits similar degree of morphology.

It is well recognized in domestic birds that quality of semen differs due to

ejaculation frequency among breeds and species (Santayana 1985; Fan et al., 1988;

Riaz et al., 2004; Peters et al., 2008; Ghonim et al., 2009; Zahraddeen et al., 2005).

Semen quality was higher with daily semen collection in Taiwan chicken breed

(Fan et al., 1988) and Domyati ducks (Ghonim et al., 2009). In the present study it

was observed that the optimum semen output can be achieved with daily semen

collection frequency in IRJF. Daily and weekly sperm output decreased but overall

sperm concentration increased with the increase in collection interval. The

42

progressive decline in the spermatozoa concentration was recorded with the

increase in frequency of collection which is in agreement to the previous studies on

poultry birds (Santayana, 1985) and Turkey (Zaharddeen et al., 2005).

The acrosomal and plasma membrane integrity of IRJF spermatozoa

remained similar (P > 0.05) in all collection frequencies according to previous

studies in other poultry birds (Bilgilli and Renden, 1984; Bilgilli et al., 1985;

Donoghue et al., 1995; 1996). It is the same for the proportion of morphologically

normal sperm. However, changes in motility (decrease) and viability (increase) was

noted with the highest collection frequency (12h) that indicates distortions in the

maturation process (non-complete motility acquisition, less selection of the “good”

sperm) in the epididymis and deferent ducts and suggests that sperm do not stay

enough time in these compartments with this “too high” frequency of each 12h.

In the present study, semen volume, sperm motility and viability remained

the same if the semen was collected in morning or evening. The sperm

concentration was recorded higher when semen was collected in morning, while

sperm viability and plasma membrane integrity were significantly higher with

semen collected in evening. These results do not confirm previous studies, which

showed that timing of semen collection had no effect on semen volume and

concentration (Riaz et al., 2004). It has been reported that artificial insemination in

domestic fowl at evening time could result in higher fertilization rate compared to

morning. It was based on the assumption that male copulate naturally more

intensively at the evening time as compared to morning time and sperm

43

competition is also increased at evening which could result in the better ejaculate

having more volume and concentration (Pizzari and Birkhead, 2001; Preston et al.,

2003).

The other explanation for the higher concentration but the lower semen

quality in early morning collections compared to evening collections could be

related to environmental factors in our study. Indeed drinking water was available

to the animals through out the day but birds did not drink or eat during the night.

This could directly affect the volume of semen that would be lower in the early

morning because of water retention due to lack of drinking water during the night

and thus increaseing the sperm concentration. Whatever the case, it is clear from

present results that good quality semen can be collected in the evening at regular

daily collection and these can be used for artificial insemination for captive

conservation of this bird. From a general point of view, if the different semen

characteristics were very homogeneous in each experiment, they varied between

each experiment (i.e. of a factor 5 for sperm concentration). This leads to suggest

that the animals were not exactly in the same physiological states over the six

month of experiment.

2.6. CHAPTER SUMMARY

Semen characteristics, impact of collection frequencies on IRJF ejaculate

and timing of collection were evaluated. The results of this chapter describe that

the IRJF ejaculates have mean sperm concentration of 800 million/ mL, total sperm

per ejaculate 0.015 billion, motility 63.5%, live/total sperm 92.4%, intact acrosome

44

75.5% and plasma membrane integrity 89.2%. Percentage of abnormal sperm

(head, mid-piece and tail) was 8.1% and it consists of mainly the mid-piece

abnormalities. The motile sperm percentage was positively correlated with intact

acrosomes (r=0.34) and plasma membrane integrity (r=0.41). Total sperm per

ejaculate (0.015 billion) were found maximum at 72 hours of collection followed

by 24 and 48 hours of collection. Daily and weekly sperm production (billion) was

found maximum at 24 hours of collection compared to 12, 48 and 72 hours of

collection. Sperm motility was higher at 24, 48 and 72 hours of collection

compared to 12 hours of collection, but the number of live sperm were higher at 12

hours of collection compared to 24, 48 and 72 hours. Sperm concentration was

better in the morning time, while the values for sperm viability and plasma

membrane integrity were higher in the semen collected at evening time. Hence, it is

concluded that IRJF semen can be collected effectively with abdominal massage

method, have reasonable production in terms of quality and quantity. Optimum

semen output can be achieved with daily semen collection frequency practiced

preferentially at the evening time.

45

Chapter 3

IDENTIFICATION OF SUITABLE EXTENDERS FOR LIQUID

STORAGE (SHORT-TERM) AND CRYOPRESERVATION

(LONG-TERM) OF INDIAN RED JUNGLE FOWL SEMEN

3.1. INTRODUCTION

The IRJF is found in restricted/fragmented areas in its distribution range

and chance of collapse of genetic diversity always exists (Peterson and Brisbin,

1999; Gautier, 2002; Mukesh et al., 2013; Kumar et al., 2015). Due to its decline in

natural habitat, researchers have frequently emphasized on adopting suitable ex situ

conservation strategies for its conservation (Peterson and Brisbin, 1999; Gautier,

2002; Mukesh et al., 2013).

Germplasm storage either at liquid sate or for an indefinite period after

cryopreservation is a best option for ex situ conservation for any species facing

high risks in its native habitat (Blanco et al., 2000, Lukaszewicz et al., 2004;

Blesbois et al., 2007; 2008; Blanco et al., 2011; 2012; Rakha et al., 2013).

The anatomy and physiology of bird oocytes and embryos cause serious

hindrance in their use for cryopreservation (Blanco et al., 2000, Blesbois et al.,

2007; Lukaszewicz et al., 2004), thus, cryopreservation of sperm, that is not

invasive, is the most applicable technique in ex situ in vitro conservation of Indian

red jungle fowl germplasm (Blesbois et al., 2007). For this purpose semen needs to

45

46

be collected from males and stored either in liquid (short-term) or frozen (long-

term) state (Lake, 1960; Wambeke, 1967; Lake and Ravie, 1981; Lukaszewicz,

2002; Lukaszewicz et al., 2004; Rakha et al., 2013).

A wide range of extenders viz; Beltsville poultry semen (BPSE; Gieson and

Sexton, 1983), red fowl (RFE; developed by modifying extender suggested by

Lake, 1978), Lake extender (LE; Lake, 1960), EK extender (EKE; Lukaszewicz,

2002), Tselutin poultry extender (TPE; Tselutin et al., 1995) and Chicken semen

extender (CSE; Lake, 1966) are available.

The present study was designed to identify the suitable extender for liquid

(short-term) or frozen (long-term) preservation of IRJF spermatozoa. The aim was

to obtain success in fertilizing by using fresh spermatozoa as well as cryopreserved

spermatozoa in best evolved extender from a range of extenders.

3.2. REVIEW OF LITERARURE

The importance of preservation of avian semen has been recognized for

indefinite preservation of genetic material, especially for at risk populations (Long,

2006). To date, most of the work has been done on domestic chicken and duck

spermatozoa, while relatively little work has been reported on other non-domestic

avian species (Han et al., 2005). The preservation of spermatozoa could play an

important role in breeding and genetic resources conservation as an aid to

economizing or greater flexibility in special breeding programs.

47

3.2.1. Liquid Storage of Semen

The possibility of dilution and storage of sperm would enhance the

longevity by reducing metabolic activity through controlled cooling/freezing

procedures (Rakha et al., 2013). The storage of semen, even at low temperature,

often results in the fertilizing potential of spermatozoa to decline with the passage

of time in a species specific manner. However, the use of liquid semen is still the

method of choice for many avian species (Blesbois et al., 2008). The most common

procedure for liquid storage requires suspending sperm in extender to retain their

structural and functional integrity. An appropriate semen extender has to provide

an energy source for spermatozoa and maintain pH and osmolarity levels identical

to those of seminal plasma, the natural medium for sperm (Siudzinska and

Lukaszewicz, 2008). The interaction and ability of each extender to protect the

integrity of sperm are highly specialized and species-specific (Blesbois et al.,

2005).

3.2.2. Cryopreservation/Long-Term Storage of Semen

The sperm cryopreservation in combination with artificial insemination are

acknowledged for great worth in the ex situ conservation of threatened species

(Blanco et al., 2000, Lukaszewicz et al., 2004; Ansari et al., 2010; Rakha et al.,

2013). The success of these techniques is determined by the quality of frozen-

thawed semen multiplying with outcomes of the fertility attributes after artificial

insemination (Blesbois et al., 2008; Ansari et al., 2012; Rakha et al., 2013). The

48

cryopreservation of avian spermatozoa is not as successful as many mammalian

species due to specific reproductive physiology and high variability from species to

species and breed/strains that resulted in different behavior/response of

spermatozoa during freezing (Blanco et al., 2000, 2009, 2012; Lukaszewicz et al.,

2004; Blesbois et al., 2006, 2007; 2008; Long, 2006). The cryopreservation

protocol for certain avian species is highly specific to achieve optimum quality of

frozen-thawed semen (Blanco et al., 2000; Lukaszewicz et al., 2004; Han et al.,

2005; Blesbois et al., 2006, 2008). For example, the ability of sperm and its

response in an extender against low temperature during dilution, cooling,

equilibration and freeze-thawing phases is highly variable from species to species

(Han et al., 2005; Blanco et al., 2009, 2012).

Each extender has its own constituents and composition with precise limits

of physical properties that alter in a specific sequence manner with a minor change

in temperature and depends on solute to solute or solute to solvent interaction

(Holt, 2000a,b; Rakha et al., 2013). However, the ability of an extender to maintain

the integrity of sperm at cellular level is highly variable with respect to

species/breeds and strains (Blanco et al., 2000; Holt, 2000a,b; Blesbois et al., 2008;

Rakha et al., 2013). The variation in bio-physical characteristics of sperm cell of a

certain species is responsible for interaction with a certain extender in highly

specialized manners which are undergone transitional changes with reduction in

temperature (Blanco et al., 2000; Holt, 2000a,b; Han et al., 2005; Rakha et al.,

2013). These interactions may cause alterations in the structural and functional

status of the sperm from beneficial to harmful outcomes (Holt, 2000). Therefore, it

is necessary to identify a suitable extender for the cryopreservation of IRJF sperm

49

that suits its physiology and maintain cellular integrity during changes in

temperatures encountered in the process of storage either in liquid or cryopreserved

state.


3.3.1. Experimental Birds

Eight mature male birds of IRJF were used in this study (as previously

described in chapter 1 at page 18). The experiment was done between 18-06-2014

to 11-08-2014. The animals were housed individually in pens of 192.20 cm x

102.34 cm at Avian Research Center, Pir Mehr Ali Shah Arid Agriculture

University Rawalpindi, Pakistan. The birds were offered commercially available

poultry cock breeder feed (Islamabad Poultry Feed®) 100g/day and were exposed

to natural light hours. Fresh water was available ad libitum throughout the

experimental period.

3.3.2. Semen Collection and Quantitative Evaluation

The semen was collected through abdominal massage as described by

Burrows and Quinn (1935) in a graduated plastic tube. For semen volume

measurement, the graduated plastic tube was measured before and after semen

collection. The final volume (µL) was obtained by subtracting the initial weight of

tube from the filled tube. Initial motility was determined as described by Zemjanis

50

(1970) by mixing 10 µL semen sample in 500 µL of phosphate buffer saline (pH

7.2; 300 mOsm/kg). The percentage of motile spermatozoa was determined by

putting a drop of semen sample on a pre-warmed glass slide (37oC) under phase

contrast microscope (x400, Olympus BX20, Japan). Sperm concentration was

measured by taking 1µL of semen and 200 µL of formal citrate solution (1mL of

37% formaldehyde in 99 mL of 2.9% (w/v) sodium citrate) with Naubauer

haemocytometer (Marienfeld, Germany) under phase contrast microscope (x400,

Olympus BX20, Japan).

3.3.3. Extender Preparation and Processing

Semen samples having motility >65% were pooled, evaluated for motility,

plasma membrane integrity, viability and acrosome integrity, and divided into six

aliquots for dilution 1:5 in each extender BPSE (Gieson and Sexton, 1983), RFE

(developed by modifying extender suggested by Lake, 1978), LE (Lake, 1960),

EKE (Lukaszewicz, 2002), TPE (Tselutin et al., 1995) and CSE (Lake,1966). The

extenders composition is given in the Table 3.1.

3.3.4. Liquid Storage of Semen

The extended semen was cooled from 37°C to 5°C in a gradual 2 hours

process by keeping the tubes in ice water and then stored in preset 5 °C temperature

of the refrigerator (Dawlance Refrigerator 9188 WBM, Pakistan) for 48 hours.

51

Motility (%), plasma membrane integrity (%), viability (%) and acrosomal integrity

(%) of IRJF spermatozoa were assessed for 0, 3, 6, 24 and 48 hours of storage.

3.3.5. Freezing and Thawing Procedure

The diluted semen (final concentration at 1.72 x 109/mL) was cooled from

37°C to 4°C in @-0.275°C min-1

. After cooling 20% glycerol was added to each

extender at 4°C; the volume of glycerol was measured by modifying (by cutting 3

mm of 1 ml eppendorf tube at the distal end; futher the volume of glycerol was

confirmed and calibertaed by weighing through digital balance) pipetting apparatus

caliberated with volumetric measurements.

The sample was equilibrated for 10 minutes at 4°C, filled in 0.5mL French

straws in cold cabinet unit, kept over liquid nitrogen vapours (5cm above the level

of LN2) for 10 minutes to freeze from 4 to -80°C at the rate of -8.4 °C min-1

and

plunged into liquid nitrogen for storage (-196 °C). After 24 hours, the straws were

thawed for 30 sec in a water bath at 37 °C and held at 37 °C for 4 hours to assess

motility, plasma membrane integrity, viability, and acrosomal integrity at 0, 2 and

4 hours.

For further fertility measurements, glycerol was removed at thawing by

following stepwise dilution protocol as suggested by Purdy et al. (2009). In brief,

phosphate buffer saline was added to frozen thawed semen sample at 5ºC after

every minute; 10 x 10 µl (10 dilutions of 10 µl per dilution), 10 x 20 µl, 10x 50 µl,

52

and 5 x 100 µl (Tajima et al., 1990), and centrifuged at 300 x g for 25 min at 5ºC.

After centrifugation, the supernatant was discarded; pellet was re-suspended and

measured to determine volume and sperm concentration with a digital photometer

ACCUREAD® (IMV Technologies, France).


3.3.6.1. Motility

Sperm motility was assessed by placing a drop of semen sample, on a pre-

warmed (37oC) glass slide under a light microscope (400x) (Zemjanis, 1970).

Percentage of motile sperm was subjectively evaluated as described at page 22 in

chapter 2.

3.3.6.2. Sperm membrane integrity

Sperm membrane integrity was assessed by using hypo-osmotic swelling

test (HOS) as previously described by Santiago-Moreno et al. (2009). The HOS

solution was prepared by adding 1 g of sodium citrate to 100 mL of distilled water.

Previously diluted semen (25 µL) was mixed with 500 µL of HOS solution (100

mOsm/kg) and incubated at 25oC for 30 minutes.

A drop of incubated solution was placed on a pre warmed (37oC) slide and

fixed in buffered 2% glutaraldehyde. The spermatozoa showing swollen heads,

swollen and coiled tails were classified as normal spermatozoa having intact

53

plasma membrane. A total of 200 spermatozoa were counted at four separate fields

under a phase-contrast microscope (lense = 100x oil immersion x eye piece = 10x).

3.3.6.3. Viability

Viability was examined by adding eosin-nigrosin to the Lake glutamate

solution. Lake‟s glutamate solution (Bakst and Cecil, 1997) was prepared by

adding sodium glutamate (0.01735g), potassium citrate (0.00128g), sodium acetate

(0.0085g) and magnesium chloride (0.000676g) in 100 mL distilled water. Water

soluble nigrosin (5g) and water soluble Eosin-bluish (1g) were added into Lake‟s

glutamate solution. Twelve drops of stain were mixed with one drop of semen. A

smear was made on a glass slide, fixed and air dried. A total of 200 spermatozoa

were assessed per slide under a phase-contrast microscope (lense = 100x oil

immersion x eye piece = 10x). The mixture provides a clear background in the

smear to enhance the contrast of white, unstained “live” sperm or the pinkish



Acrosomal integrity was assessed through giemsa stain (Jianzhong and

Zhang, 2006). The stain was prepared by adding giemsa (3g) and phosphate buffer

saline at pH 7.0 (2 mL) into 35 mL water. Smear was prepared by taking a drop of

semen sample on a clean glass slide, dried and fixed in neutral formal-saline (5%

formaldehyde) for 30 min. Fixed slides were kept in giemsa stain for 1.5 hrs.

54

Sperm with normal acrosome appeared to be evenly stained; abnormal spermatozoa

were unevenly stained while spermatozoa having ruptured acrosome remained

unstained. A total of 200 spermatozoa were observed atleast in four separate fields

under a phase-contrast microscope (x400, Olympus BX20, Japan) at magnification

lense = 100x oil immersion x eye piece = 10x.

3.3.7. Recovery Rate for Semen Quality Parameters

The recovery rate of sperm motility, plasma membrane integrity, viability

and acrosomal integrity were calculated through following formula (Dubos et al.,

2008);

Recovery rate =Value of a given parameter in frozen semen

Value of a given paramter in fresh semen x100

3.3.8. Absolute Livability Index

The absolute livability index of sperm was calculated as a measure of time

dependant decrease in sperm motility after thawing at 37°C. The samples were

incubated at 37 ºC after thawing and examined for sperm motility after every two

hours for 4 hours. The absolute index of livability of spermatozoa was computed

by the following equation (Melovenof, 1962).

La=Σ(TR)

Where La = absolute index of livability, T = time interval (h) between the

two consecutive observations, R = average of motility percentage for two

consecutive observations starting from the time of incubation.

55

3.3.9. Fertility Measurements

The fertility attributes of IRJF spermatozoa in best evolved extender i.e.

Red fowl extender (RFE) was measured by inseminating 25 hens with freshly

diluted (at 4°C) and frozen-thawed semen. The study of artificial insemination was

carried out at a private facility; M/S Abdullah Pheasantry, 8 km from Thokar Niaz

Beg, Multan Road, Lahore, Pakistan. The female birds of IRGF were also

confirmed for genetic purtity by reliable phenotypical characters i.e the absence of

comb, wattle and having dark, slender legs following Delacour (1951), Brisbin et

al. (2002), Brisbin and Peterson (2007) Setianto et al. ( 2017). Intravaginal

artificial inseminations were performed at depth of 4cm with AI gun (IMV

Technologies, France) having fitted glass pipette with a dose consisted of 270-300

million sperm within 10 minutes after the removal of glycerol (Purdey et al., 2009).

The egg formation completes in the oviduct within 24-26 hours post insemination.

Therefore, the eggs were collected day after insemination for five days. The eggs

were set

in an automatic incubator at temperature 99-100°F and relative humidity 86-90%.

Eggs were rotated on daily basis with an automatic rotator and the rotator was

stopped three days before the expected hatching. Eggs fertility was evaluated by

candling after 7 days of incubation. The chicks were hatched out at 24 days of

incubation. The fertility attributes were calculated with the following formulae

(Blanco et al., 2012):

Fertility (%) = Total Number of fertile eggs

Total Number of eggs laidx100

56

Hatch (%) = Toatal Number of chicks hatched


Hatchability of fertile eggs = Total Number of chicks hatched

Total Number ofFetile eggs x100

3.4. STATISTICAL ANALYSIS

The effect of extenders on motility plasma membrane integrity, viability

and acrosome integrity were analyzed in Completely Randomized Design by

Analysis of Variance using MSTAT-C® (Version 1.42 Michigan State University,

East Lansing, MI, USA) and are presented as means (± SE). When F-ratio was

found significant (P<0.05), post hoc comparison between the means was done

though Fisher‟s protected LSD test. The data on fertility attributes of IRJF

spermatozoa were analyzed by chi-square test using Megastat® (Version 7.25

McGraw-Hill New Media, New York) for excel.

3.5. RESULTS

3.5.1. Evaluation of Extenders for Liquid (Short-Term) Storage of Semen

The data on sperm motility, plasma membrane integrity and acrosomal

integrity are presented in the Figures 3.6.1.1 to 3.6.1.4. The sperm motility was

recorded significantly higher (two factor ANOVA, Extender; F5,210 = 24.59, P <

0.001) in red fowl, Lake and EK semen extender compared to Beltsville poultry,

57

Tselutin poultry and chicken semen extender. The sperm motility (Hours; F4,210 =

334.94, P < 0.001) was significantly decreased during the post-thaw hours of

storage from 0 and 3 hours of storage at 5°C. At 6 hours of storage, red fowl and

Lake semen extender maintained higher (two factor ANOVA, F4,210 = 334.94, P <

0.001) sperm motility compared to all other experimental extenders. However, red

fowl extender maintained the highest (two factor ANOVA, F5,210 = 24.59, P <

0.001) motility compared to Lake, EK, Beltsville poultry, Tselutin poultry and

Chicken semen extender at 24 and 48 hours of storage at 5 °C. The interaction

between extender and hours of storage was also significant (Extender x Hours;

F20,210 = 0.95, P < 0.01) that suggests, experimental extenders have different ability

to maintain sperm motility during different hours of storage.

The sperm plasma membrane integrity was recorded higher (two factor

ANOVA, Extender; F5,210 = 80.91, P < 0.001) in red fowl extender compared to

Lake, EK, Beltsville poultry, Tselutin poultry and Chicken semen extender. The

PMI was significantly (Hours; F4,210 = 1069.85, P < 0.001) reduced from 0 to 48

hours of storage at 5 °C. The interaction between extenders and hours of storage

was significant (Extender x Hours; F20,210 = 2.89, P < 0.001) that suggests that

different experimental extenders have different abililty to protect the plasma

membrane of spermatozoa during different hours of storage.

The sperm viability was recorded higher (two factor ANOVA, Extender;

F5,210 = 59.29, P < 0.001) in red fowl extender compared to Lake, EK, Beltsville

poultry, Tselutin poultry and Chicken semen extender. However, the storage hours

have significantly (Post-thaw Hours; F4,210 = 901.71, P < 0.001) reduced the

58

percentage of viable sperm, as the duration of storage increases from 0 to 48 hours

at 5 °C. Is is clear from the ANOVA results that the interation between extender

and storage hours was not significant (Extender x Hours; F20,210 = 1.33, P > 0.05)

and suggest that all extenders have similar ability to maintain the percentage of

viable sperm during storage hours.

The sperm acrosomal integrity was recorded significantly higher (two factor

ANOVA, Treatment; F5,210 = 49.59, P < 0.001) in red fowl extender compared to

Lake, EK and Beltsville poultry compared to Tselutin poultry and Chicken semen

extender. However, at 0 hours of storage, acrosome integrity of spermatozoa was

recorded higher in red fowl, Lake, EK and Beltsville Poultry compared to the

Tselutin Poultry and Chicken semen extenders (P = 0.000; df = 239; F = 49.5991)

at 5°C. It is relevant to mention that sperm acrosome integrity was recorded higher

(Hours; F4,210 = 1617.49, P < 0.001) in red fowl semen extender compared to Lake,

EK and Beltsville poultry, Tselutin poultry and Chicken semen extenders at 3, 6,

24 and 48 hours of storage at 5 °C. The interaction between experimental extenders

and hours of storage have also significant effect (Extender x Hours; F20,210 = 0.95,

P > 0.001) that suggest that the extenders have different ability to protect the

acrosome integrity of spermatozoa during different hours of storage.

3.5.2. Evaluation of Extenders for Cryopreservation of Indian Red Jungle

Fowl Semen

The data on the effect of extenders on post-thaw motility, plasma

membrane, viability and acrosome integrity of IRJF spermatozoa assessed at 0, 2

56

Figure 3.6.1.1: Effect of extenders [Beltsville poultry semen extender (BPSE), Red fowl extender (RFE), Lake extender (LE), EK

extender (EKE), Tselutin poultry extender (TPE) and Chicken semen extender (CSE)] on the motility of Indian red jungle fowl

spermatozoa stored at 5°C (n=8). Bars with different letters differ significantly (P < 0.05) within a given period of storage. (Table 11;

Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

0 3 6 24 48

Hours

Mot

ility

(%)

BPSE RFE LE EKE TPE CSE

bcd

aab

abc

cd

deff

cde cde

deff

ghh

ef fg

gh

h

iij

h

i

ij

i

jk

l

jk

kl

ll

l

59

57


extender (EKE), Tselutin poultry extender (TPE) and Chicken semen extender (CSE)] on the plasma membrane integrity of Indian red

jungle fowl spermatozoa stored at 5°C (n=8). Bars with different letters differ significantly (P < 0.05) within a given period of storage.

(Table 12; Appendices)

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

0 3 6 24 48

Hours

PLas

ma

Mem

bran

e Int

egrit

y (%

)BPSE RFE LE EKE TPE CSE

ab a

b b

c

ef de

b

ffg

gh gh fg

cd

hiij i

jk

l

k

l l lm

no

lm

nno no o

60

58


extender (EKE), Tselutin poultry extender (TPE) and Chicken semen extender (CSE)] on the viability of Indian red jungle fowl

spermatozoa stored at 5°C (n=8). Bars with different letters differ significantly (P < 0.05) within a given period of storage. (Table 13;

Appendices)

0.0

50.0

100.0

150.0

200.0

250.0

300.0

0 3 6 24 48

Hours

Via

bilit

y (%

)BPSE RFE LE EKE TPE CSE

ba

c c c

c

e

d

fg

ghfg

gh ghi

ef

jkljkl

hij

jklkl

gh

n mn

lmmn

op

ijk

pp

no

op

61

59


extender (EKE), Tselutin poultry extender (TPE) and Chicken semen extender (CSE)] on the acrosomal integrity of Indian red jungle

fowl spermatozoa stored at 5°C (n=8). Bars with different letters differ significantly (P < 0.05) within a given period of storage. (Table

14; Appendices)

0.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

0 3 6 24 48

Hours

Sper

m a

cros

ome i

nteg

rity

(%)


ab aabc abc

bcd cde defabc

ef fg fg gh

hi

ef

hihij ij j

lm

k

l

m m m

n

m

nono

o

p

62

63

and 4 hours of incubation at 37°C following cryopreservation are presented in

Figures 3.6.2.1-4. The higher sperm motility (two factor ANOVA, Treatment; F5,72

= 13.76, P < 0.001), plasma membrane integrity (two factor ANOVA, Treatment;

F5,72 = 41.88, P < 0.001), viability (two factor ANOVA, Treatment; F5,72 = 38.75, P

< 0.001) and acrosome integrity (two factor ANOVA, Treatment; F5,72 = 38.45, P <

0.001) were recorded higher in RFE compared to Lake, EK, Beltsville poultry,

Tselutin poultry and chicken semen extender. The BPSE and CSE gave the lowest

levels of motility. The BPSE was at lowest level for plasma membrane integrity

while EK extender was the less efficient for sperm viability. However, all of the

above parameters viz; sperm motility (Post-thaw Hours; F2,72 = 23.83, P < 0.001),

PMI (Post-thaw Hours; F2,72 = 58.34, P < 0.00), viability (Post-thaw Hours; F2,72 =

14.00, P < 0.001) and acrosome integrity (Post-thaw Hours; F2,72 = 47.18, P <

0.001) were significantly redcued at 0, 2 and 4 hours of post-thaw incubation at

37°C.

The interaction between the extenders and post-thaw hours was found non-

signifcant for sperm motility (Extender x Hours; F10,72 = 0.67, P > 0.05), PMI

(Extender x Hours; F10,72 = 1.05, P > 0.05), viability (Extender x Hours; F10,72 =

0.21, P > 0.05) and acrosome integrity (Extender x Hours; F10,72 = 0.46, P > 0.05)

that suggest the extenders and post-thaw incubation hours work independtly for all

of the above mentioned parameters.

The data on the effect of extenders on recovery rate of motility, plasma

membrane integrity, viability and acrosome integrity of frozen-thawed IRJF

64

permatozoa are given in Table 3.2. The higher recovery rate of motility (one way

ANOVA, F5,24 = 3.70, P < 0.05), plasma membrane integrity (one way ANOVA,

F5,24 = 3.56, P < 0.05), viability (one way ANOVA, F5,24 = 2.38, P = 0.06) and

acrosome integrity (one way ANOVA, F5,24 = 3.72, P < 0.05) were recorded in

RFE compared to Beltsville poultry, turkey, Lake, EK, Tselutin poultry and

chicken semen extender but the levels of significance depended on the parameter

measured. The sperm recovery rate was significantly higher (one way ANOVA,

F5,114 = 3.30, P < 0.05) for plasma membrane integrity and acrosomal integrity with

RFE compared to BPSE and CSE, however for viability significant difference was

recorded only with TPE and CSE.

3.5.3. Efficiency of Extenders for the Absolute Livability Index

The data on the effect of extenders on absolute livability index of Indian red

jungle spermatozoa are given in Table 3.2. The higher (one way ANOVA, F5,24 =

5.90, P < 0.001) absolute livability index was recorded in RFE compared to Lake,

EK, Beltsville poultry, Tselutin poultry and chicken semen extenders. Following all

these results, the diluent RFE was used for further fertility test.

3.5.4. Fertility Potential of Cryopreserved Semen

The results of fertility obtained after artificial insemination of either frozen-

thawed or unfrozen semen diluted in red fowl extender are given in Table 3.3. The

65

Figure 3.6.2.1.1: Effect of extenders [Beltsville poultry semen extender (BPSE), Red fowl extender (RFE), Lake extender (LE), EK

extender (EKE), Tselutin poultry extender (TPE) and Chicken semen extender (CSE)] on motility (%) of Indian red jungle fowl

spermatozoa post-thaw at 0, 2 and 4 hours of incubation at 37 ºC (n=5). The bars with different letters showed significant differences

(P > 0.05) among all the extenders at 0, 2 and 4 hours of Incubation. (Table 15; Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 Hours 2 Hours 4 Hours

Post-thaw hours

Sper

m M

oilit

y (%

)


efgh

a

bcdebcbcd

efghefghi

b

cde

cdef

cdefg

fghi

i

cdef

defgh

highi

i

65

66


extender (EKE), Tselutin poultry extender (TPE) and Chicken semen extender (CSE)] on plasma membrane integrity (%) of Indian

red jungle fowl spermatozoa post-thaw at 0, 2 and 4 hours of incubation at 37ºC (n=5). The bars with different letters showed

significant differences (P > 0.05) among all the extenders at 0, 2 and 4 hours of Incubation. (Table 16; Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0


Post-Thaw hours

Sper

m P

lasm

a M

embr

ane

Inte

grity

(%)


fg

a

cdbc

bc

cd

hi

b

efgdef

defdef

i

cde

gh ghgh

fg

66

67


extender (EKE), Tselutin poultry extender (TPE) and Chicken semen extender (CSE)] viability (%) of Indian red jungle fowl

spermatozoa post-thaw at 0, 2 and 4 hours of incubation at 37 ºC (n=5). The bars with different letters showed significant differences

(P>0.05) among all the extenders at 0, 2 and 4 hours of Incubation. (Table 17; Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0


Post-Thaw Hours

Sper

m V

iabi

lity

(%)


bc

a

de

efg

de

cdde

ab

ef

fg g

de

de

ab

ef

fg g

de

67

68


extender (EKE), Tselutin poultry extender (TPE) and Chicken semen extender (CSE)] on sperm acrosomal integrity (%) of Indian red

jungle fowl spermatozoa post-thaw at 0, 2 and 4 hours of incubation at 37 ºC (n=5). The bars with different letters showed significant

differences (P > 0.05). (Table 18; Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0


Post-Thaw Hours

Sper

m A

cros

omal

Inte

grity

(%)


b

a

defcd

bc

defgcd

a

efghdefg de

efgh fgh

bc

i

ghi ghihi

68

69

Table 3.1: Composition of extenders used for cryopreservation of Indian red jungle

fowl semen

Items Extenders*


Fructose 0.3000 1.15 0.2 0.8 0.8

Potassium citrate 0.0384 0.128 0.14

Sodium glutamate 0.5202 2.1 1.35 1.4 1.92

Magnesium Chloride 0.0204

di-Potassium Hydrogen phosphate 0.7620

TES 0.3170

potassium di-hydrogen phosphate 0.0390

sodium acetate 0.2580 0.51

PVP 0.6 0.1 0.3 0.38

Glycine 0.2

Potassium acetate 0.5 0.5

Magnesium acetate 0.08

Glucose 0.8 0.35

Inositol 0.7

Protamin-sulphate 0.02 0.32 0.5

Anhydrous sodium hydrogen

phosphate

0.98

Anhydrous sodium di-hydrogen

phosphate

0.21

pH 7.3 7.0 7.2 7.5 7.05 6.85

Osmotic Pressure (mOsmol/kg) 330 380 310 390 320 310

*Values are given in grams per 100 mL of double distilled water.

RFE, Red fowl extender; BPSE, Beltsville poultry semen extender; LE, Lake

extender, EKE, EK extender, TPE, Tselutin poultry extender; CSE, Chicken semen

extender.

70

Table 3.2: Absolute livability index (mean ± SEM) and recovery rate (%) of semen

quality parameters of Indian red jungle fowl after freeze-thawing (n=5)

Extender

Recovery rate (%) Absolute livability

index Motility Plasma membrane

Integrity Viability

Acrosome

Integrity

RFSE 63.9 ± 3.7a 74.1 ± 4.8

a 87.0 ± 2.7

a 93.8 ± 1.0

a 164.0 ± 2.4

a

BPSE 26.1 ± 6.4b 54.5 ± 2.4

c 76.1 ± 5.4

ab 86.8 ± 5.5

ab 71.0 ± 11.2

bc

LE 46.2 ± 10.0ab

75.2 ± 6.5a 86.4 ± 3.3

a 80.9 ± 2.0

bc 110.0 ± 17

b

EKE 56.6 ± 8.4a 71.9 ± 5.3

ab 75.8 ± 3.0

ab 82.2 ± 3.6

bc 107.0 ± 5.4

bc

TPE 40.8 ± 12.1ab

66.6 ± 3.3abc

71.7 ± 6.2b 81.5 ± 4.2

bc 104.0 ± 26

bc

CSE 26.1 ± 4.6b 60.8 ± 2.2

bc 71.3 ± 5.3

b 72.8 ± 3.5

c 68.0 ± 10.1

c

The values (mean ± SEM) with different superscripts in a column differ

significantly (P < 0.05). (Table 19-23; Appendices)

71

Table 3.3: Fertility attributes of Indian red jungle fowl spermatozoa after artificial

insemination in hens (n=25) with freshly diluted and frozen semen

Items State

Day after insemination

Mean ± SEM

2 3 4 5 6

Number of laid eggs Liquid* 25 25 23 24 22 23.8 ± 0.7a

Frozen* 22 23 24 20 22 22.2 ± 0.7a

Number of fertilized

eggs

Liquid 20 22 21 20 20 20.6 ± 0.4a

Frozen 13 11 12 14 13 12.6 ± 0.5b

Fertility (%) Liquid 80.0 88.0 91.3 83.3 90.9 86.7 ± 2.2a

Frozen 59.1 47.8 50.0 70.0 59.1 57.2 ± 3.9b

Number of hatched

chicks

Liquid 17 20 18 20 16 18.2 ± 0.8a

Frozen 11 9 10 10 10 10.0 ± 0.3b

Hatch (%) Liquid 68.0 80.0 78.3 8.3. 72.7 76.5 ± 2.7a

Frozen 50.0 39.1 41.7 50.0 45.5 45.3 ± 2.2b

Hatchability of

fertilized eggs (%)

Liquid 85.0 90.9 85.7 100.0 80.0 88.3 ± 3.4a

Frozen 84.6 81.8 83.3 71.4 76.9 79.6 ± 3.4a

“*Liquid: diluted unfrozen sperm”. *Frozen: frozen-thawed sperm. The values

(mean ± SEM) with different superscripts for a given parameter in a column differ

significantly (P < 0.05). (Table 24; Appendices)

72

number of fertilized eggs (mean/day: unfrozen-diluted, 20.6 ± 0.4; Frozen, 12.6 ±

0.5), percent fertility (86.7 ± 2.2; 57.2 ± 3.9), number of hatched chicks (18.2 ± 0.8;

10.0 ± 0.3), percent hatch (76.5 ± 2.7; 45.3 ± 2.2) and hatchability of fertilized eggs

(88.3 ± 3.4; 79.6 ± 3.4) were recorded higher after AI with freshly diluted semen in

red fowl extender compared to frozen-thawed semen. More than 60% of the initial

fertility was kept after sperm cryopreservation.

3.6. DISCUSSION

The components of the extenders i.e. solutes and solvents affect the

physical properties of the extender (Farrant et al., 1969; Freshney, 2000). Each

solute dissolved in water at different proportions showed highly specific physical

properties that undergo exchanges in terms of quality and quantity (Wolfe and

Bryant, 2001). The interaction between solute to solute or solute to solvent alters

with the change in kinetic energies of the solutions that affect its colligative

properties (Williams, 1983). The transformation in the behavior of the solution in

terms of colligative properties varies in a very specific manner during

cryopreservation in which systemic changes occur in temperatures (Wolfe and

Bryant, 2001). These all factors affect the ability of extender for protecting the

quality of semen during freeze-thawing process.

The distinctive physiology and bio-chemical characteristics of the avian

spermatozoa cause inherent hindrance in the success of cryopreservation larger

than mammalian spermatozoa (Long, 2006; Blesbois et al., 2008). Furthermore,

73

high variability in biochemical properties from species to species and breed/strain

add in complexity (Blanco et al., 2000). It is known that morphological and bio-

physical characteristics of the sperm change its behavior during low temperature

that encounters during cryopreservation. Therefore the behavior of the sperm of

certain species/breed in a particular extender at low temperatures is different that

affects its preservability (Gee, 1995; Fujihara and Xi, 2000; Lukaszewicz, 2002;

Siudzinska and Lukaszewicz, 2008).

In the present study, significantly (P<0.05) higher values for sperm

motility, viability, plasma membrane and acrosome integrity were recorded and

successful AI was achieved with sperm cryopreserved in the best of the diluent

tested i.e. RFE. Post-thaw motility, viability and acrosome integrity of sperm

assessed at 0, 2 and 4 hours of incubation at 37°C and their recovery rates were

recorded higher in red fowl extender. The same observation was recorded by using

absolute livability index that give some rough indirect clue about the longevity

and/or surviving ability of the sperm at ambient temperature. The results of this

study are in line with the previous similar study on liquid stored IRJF spermatozoa

that showed higher motility, plasma membrane integrity, viability and acrosome

integrity in red fowl extender at 0, 3, 6, 24 and 48 hours of storage at 5°C

compared to Lake, EK, Beltsville poultry, Tselutin poultry and chicken semen

extenders.

The composition of extender RFE is slightly different (PVP 6g was used in

RFE instead 3g) to diluent used by Blanco et al. (2000, 2011, 2012) in different

74

bird species, domestic or wild. However, other diluents such as the BPSE or Lake‟s

diluents have been successful for chicken semen (Lake 1960; Lake 1966; Sexton,

1981; Lake 1978; Lake and Ravie, 1982). The BPSE has however been reported to

have detrimental effects on motility of duck semen (Han et al., 2005) and higher

percentages of live duck spermatozoa were recorded in EK extender compared to

Lake and Tselutin poultry extender (Han et al., 2005). These different results

illustrate well the point that ability of sperm to respond to huge media changes,

temperature and osmotic variations during dilution, cooling, equilibration and

freeze-thawing phases is highly variable from species to species (Blanco et al.,

2009, 2012).

The usability of an extender found superior on the basis of laboratory tests

for routine practices is always questionable until supported with satisfactory

outcomes for fertility (Ansari et al., 2012). The red fowl extender showed fertility

rates of 86.7 ± 2.2% vs. 57.8 ± 3.9% in IRJF with unfrozen diluted and frozen-

thawed semen, respectively. Although there was a decrease in fertility after

cryopreservation, about 61% fertility rate of IRJF showed its positive signs of

potential application. The observed fertility rates with frozen semen are

comparatively higher than in domestic chicken, knowing that, here, it was

measured after a single insemination of less than 300 sperm at the opposite of the

practice in domestic chicken and other species where successive inseminations are

needed to get good fertility results (Wishart and Staines, 1999; Tselutin et al.,

1999; Blanco et al., 2012; Seigneurin et al., 2013; Getachew, 2016). This attests of

the success of the methodology that we have chosen with the IRJF sperm.

75

The red fowl extender has osmolarity 380 mOsmol/kg that probably offers

better protection during cryopreservation as higher osmolarity (350-450

mOsmol/kg) are considered suitable for fertility of chicken sperm (Harris et al.,

1963; Graham and Brown, 1971; Bakst, 1980). The availability of the energy is

highly associated with ability of sperm to reach the site of fertilization (Saeki,

1960; Tsukunaga, 1971) and fertilization process itself (Lake, 1960; Sexton, 1976).

It is suggested that higher osmolarity and availability of energy source i.e. fructose

both had positive contributions in fertility attributes of IRJF spermatozoa.

The distinctive physiology of the avian semen (high sperm concentration,

elongated size, very less cytoplasm and seminal plasma) showed increased

oxidative stress intensiveness and lower potential of antioxidant defense system

(Partyka et al., 2012). In addition, presence of dead and/or injured sperm

(comparatively higher in avian semen than mammalian semen) in semen sample

during freeze-thawing increases levels of reactive oxygen species (Nagy et al.,

2003; Partyka et al., 2012).

It is believed that decrease in motility, plasma membrane and acrosome

integrity is associated with decrease in antioxidant potential of frozen-thawed

semen (Partyka et al., 2012; Ansari et al., 2011, 2012). Glutathione is naturally

occurring antioxidant in all type of animal cells including sperm (Ansari et al.,

2012). Furthermore, decreased level of glutathione in frozen-thawed semen is

believed to be associated with semen quality and fertilizing ability of the

spermatozoa (Ansari et al., 2012). Glutamate and glycine both are involved in the

76

biosynthesis of glutathione at the cellular level (Wu et al., 2009). This is suggested

that presence of higher level of glutamate and glycine may support the IRJF

spermatozoa against the oxidative stress-mediated damages especially lipid

peroxidation of the plasma membrane during freeze-thawing process (Partyka et

al., 2012).

The osmotic changes and ice crystal formation are considered the major

causes of cryo-induced damages during cryopreservation (Qadeer et al., 2013,

2015). The colligative properties (freezing point depression and osmotic pressure)

of the polymers in water and the changes during continuously decreasing

temperatures well suit the physiology of animals cell and enhance its

cryopreservability by causing efflux of some water molecules and squeezing the

cytoplasm (Wolfe and Bryant, 2001). These physical processes may contribute in

reduction of the intra and extracellular ice-crystals formation. It is also believed

that PVP, present in RFE diluents in present study, reduced the surface energy of

the solution below the cell that may have resulted in the formation of stable

interface at the plasma membrane surface, hindered the leakage from the cytoplasm

and hid the membrane defects (Williams, 1983). It is also proposed that PVP has a

protecting capacity by interacting directly to fill the gap were lipids have been lost

during freeze-thawing process in the lipid-bilayer of cell (Nash, 1966). Obviously,

PVP has the ability to support biological cells during low temperatures through

different mechanism by colligative properties, coating of the cell membrane and

depressing the freezing point of the water (Farrant et al., 1969; Nash, 1966;

Freshney, 2000). It is suggested that high level of PVP in red fowl extender

77

protected the functionality of the IRJF by espousing multiple mechanisms as

discussed.


The extenders (Beltsville poultry, RFE, Lake, EK, Tselutin poultry and

Chicken semen extender) were evaluated for long-term and short-term storage of

IRJF (Gallus gallus murghi) spermatozoa in this chapter. Percentages of motility,

plasma membrane integrity, viability and acrosome integrity were higher (P < 0.05)

in Red fowl extender at 0, 2 and 4 hours of incubation post-thaw and liquid semen

at 0, 3, 6, 24 and 48 hours of storage. After cryopreservation and post-thawing at

37°C, the highest (P < 0.05) recovery rates and absolute livability index was also

recorded in red fowl extender that was thus used for further artificial insemination

of cooled-diluted (Liquid) and cryopreserved sperm. The no. of fertilized eggs

(Liquid, 20.6 ± 0.4; Cryopreserved, 12.6 ± 0.5), percent fertility (86.7 ± 2.2; 57.2 ±

3.9), no. of hatched chicks (18.2 ± 0.8; 10.0 ± 0.3), percent hatch (76.5 ± 2.7; 45.3

± 2.2) and hatchability of fertilized eggs (88.3 ± 3.4; 79.6 ± 3.4) were higher with

sperm freshly cooled-diluted or cryopreserved, respectively in red fowl extender. In

conclusion, the present study shows the first success of fertility obtained with IRJF

cryopreserved sperm. This was obtained with the use of RFE as diluent. It is to

believe that higher levels of fructose, glutamate, PVP and presence of glycine in

red fowl extender contributed to make it superior extender for liquid state at 5 ºC

and cryopreservation of IRJF spermatozoa compared to Lake, EK, Beltsville

poultry, Tselutin poultry and chicken semen extender.

78

Chapter 4

EFFECT OF GLYCEROL CONCENTRATIONS ON

SPERMATOZOA OF THE INDIAN RED JUNGLE FOWL

4.1. INTRODUCTION

Cryopreservation of sperm in avian species is under the process of

experimental phase and is yet not sufficiently advanced for practical application in

the conservation of non-domestic species and only few examples are available

(Jalme et al., 2003; Sontakke et al., 2004). There are various problems associated

with the technique of cryopreservation i.e., choice of diluent, choice of

cryoprotectant to reduce cell damage, stages of cryopreservation, thawing

procedure and artificial insemination (Lake et al., 1980). Cryoprotectant

identification is necessary for freeze-thawing success and thus to develop the

technique of freezing avian semen.

Glycerol was the cryoprotectant that was the first time identified for fowl

semen. However, the improvement in the post thaw semen quality could only be

obtained after the addition of 10-15% glycerol (Smith and polge, 1950). Various

studies have shown that glycerol containing diluent could maintain post-thaw

semen quality but not fertility. Hence, the contraceptive effect of glycerol was first

time identified when artificial insemination was done in the turkey (Clark and

Shaffner, 1960; Lake et al., 1980). The glycerol is capable of penetrating across the

sperm cell membrane during equilibration and freezing and cause efflux of the

78

79

water molecules hindering the intra-cellular ice-formation (Fuller and Paynter,

2004). The usefulness of any permeable cryoprotectant including glycerol in

protecting the sperm integrity depends on exposure time, concentration, adding

temperature and interaction with solute constituents (Sexton, 1975; Gao et al.,

1995; Saragusty et al., 2005; Johnston et al., 2006; Baust et al., 2007). In addition,

biochemical and physical properties of the sperm plasma membrane vary with

species/breed/strain and affect the penetrability of the permeable cryoprotectant

(Gao et al., 1992; Gao et al., 1995; Holt et al., 1996; Holt et al., 2005). The

optimum level of glycerol which can provide maximum protection to sperm of a

species/breed/strain during equilibration and freezing-thawing is highly specific

and must be investigated (Tselutin et al., 1999; Holt, 2000a,b).

The IRJF is a wild endemic sub-species of genus Gallus and considered

ancestor of the domesticated chicken (Genome Sequence Center, 2006). Recently,

in an attempt to preserve IRJF spermatozoa to establish semen bank and artificial

insemination for extended period of time (Rakha et al., 2015a,b; 2016a,b), semen

was cryopreserved with 11% glycerol (added at 4°C, equilibrated for 10 minutes

before freezing) following protocol developed for domestic poultry (Rakha et al.,

2017). The decline was observed in motility, plasma membrane integrity, viability

and acrosome integrity during different stages of cryopreservation (Rakha et al.,

2017). Consequently, optimized cryopreservation protocol with appropriate level of

glycerol is required for red jungle fowl sperm to reduce cry-induce damages. It was

hypothesized that use of higher concentration of glycerol may result in enhanced

survivability of IRJF sperm during freeze-thawing.

80

The present study was designed to evaluate the cryoprotective effect of

different glycerol concentrations (11%, 15% and 20%) on post-thaw quality

(motility, plasma membrane integrity, viability and acrosome integrity) at 0, 2 and

4 hours of incubation (at 37°C), recovery rates, absolute livability index and

fertility attributes (No. of fertile eggs, fertility (%), No. of hatched chicks, percent

hatch and hatchability of fertilized eggs) of IRJF semen.


The current success in the field of cryopreservation is principally based on

the discovery of glycerol as a cryoprotectant for the spermatozoa (Polge et al.,

1949). It provides protection to the sperm bio-membrane system during cooling,

equilibration and freeze-thawing stages (Holt, 2000). The mechanism involved in

the protection offered by glycerol is associated with lowering the freezing point of

the diluent and hindering the formation of intracellular ice-crystallization (Gao et

al., 1995; Holt, 2000, Medeiros et al., 2002; Andrabi, 2009). Ice-crystallization

during cryopreservation of sperm is considered one of the major reasons which

cause disruption of the membrane and resulted in reduced motility, plasma

membrane integrity, viability and acrosome integrity of semen (Baust et al., 2007;

Qadeer et al., 2013, 2015). It is worth mentioning that glycerol is least toxic to fowl

sperm viability and integrity when compared to DMSO and DMA (Tselutin et al.,

1999). The physiology of the avian sperm is very unique have smaller surface area-

to-volume ratio, cylindrical head, minute cytoplasm, elongated flagellum provide

less chance to any permeable cryoprotectant including glycerol to accumulate in

81

the sperm cell (Blanco et al., 2000; Partyka et al., 2010; 2012a,b; Long, 2013). It is

known that during the process of cryopreservation, majority of the injuries occur to

sperm tail, thus lengthened flagellum of avian sperm make it more prone to cryo-

induced damages (Holt, 2000). This might be a reason of comparatively higher

magnitude of structural and functional losses that are observed during

cryopreservation of avian semen cryopreservation.

The studies on the use of glycerol as cryoprotectant in emu revealed that

glycerol become toxic even at 6 and 9% compared to DMA and DMSO, because

glycerol penetrates more readily in sperm membrane (Malecki et al., 1997).

Glycerol addition (11%) in a diluent was harmful for chicken spermatozoa

compared to DMA (Moce et al., 2010). In another study on turkey semen, it was

revelaed that addition of glycerol at 11% in a diluent exhibited higher post-thaw

semen quality compared to 6% DMA (Long et al., 2014). However, no study is

available on the use of glycerol as a cryoprotectant for IRJF spermatozoa. The

present study was designed with the objective to evaluate different concentration of

glycerol 11%, 15% and 20% on post-thaw semen quality and fertility.


4.3.1. Experimental Birds

Eight mature male birds of IRJF of 1.5 year for age were used in this study.

The experiment was conducted between 16-09-2014 to 17-12-2014. The animals

82

were housed at Avian Research Center, Pir Mehr Ali Shah Arid Agriculture

University Rawalpindi. The birds were offered commercially available poultry

cock breeder feed (100g/day). Fresh water was available throughout the

experimental period.

4.3.2. Extender Preparation

The diluent was prepared by adding fructose (1.15g), sodium glutamate

(2.1g), PVP (0.6g), glycine (0.2g) and potassium acetate (0.5g) to 100mL of double

distilled water (pH, 7.0; osmotic pressure 380 mOsm/kg. All the chemicals were

purchased from Sigma-Aldrich, Co., 3050 Spruce street, St Louis, USA.

4.3.3. Semen Collection and Evaluation

Semen was collected through abdominal massage as described by Burrows

and Quinn (1935) from individual birds in a graduated plastic tube. Semen volume

was measured in microlitres using micropipette. Initial sperm motility of each

ejaculate was determined as described by Zemjanis (1970) by mixing 10 µl semen

samples in 500 µl of phosphate buffer saline (pH 7.2, 300 mosm). The motility was

determined by putting a drop of semen sample on a pre-warmed glass slide (37oC)

under phase contrast microscope (x400, Olympus BX20, Japan). Sperm

concentration was measured by taking 1µl of semen and 200 µl of formal citrate

solution (1mL of 37% formaldehyde in 99 mL of 2.9% (w/v) sodium citrate) with

83

Neubauer haemocytometer (Marienfeld, Germany) under phase contrast

microscope (x400, Olympus BX20, Japan).

4.3.4. Semen Processing, Freezing and Evaluation

Semen ejaculates from eight mature cocks having >70% motility were

pooled. The pooled sample was divided into three portions, diluted (1:5; at 37 °C)

and cooled to 4 °C in two hours (-0.275 min-1

). Glycerol was added at 11, 15 and

20% to each of the portion at 4 °C, and equilibrated for 10 minutes. After this, the

cooled semen was filled in 0.5 mL French straws (IMV, L‟Aigle, France), kept

over liquid nitrogen vapors (5cm above the level of LN2) for 10 minutes and

plunged into liquid nitrogen for storage. After 24 hours, the straws were thawed

individually at 37 °C for 30 seconds in water bath (Memmert GmbH +Co.Kg,

Germany) and incubated at 37°C, until all the spermatozoa got dead. The

thawed/incubated semen samples were further evaluated for motility, plasma

membrane integrity, viability and acrosome integrity. The study was

repeated/replicated for five times. All the chemicals used in this study were from

Sigma®

-Aldrich or otherwise mentioned.


4.3.5.1. Motility

Sperm motility was assessed under phase contrast microscope at 400x by

examining a cover-slipped drop of semen sample on pre-warmed (37oC) glass slide

84

(Zemjanis, 1970). Percentage of motile sperm was subjectively evaluated on a scale

ranging from 1 to 5 (see page 22: chapter 2).

4.3.5.2. Plasma membrane integrity

Sperm plasma membrane integrity was assessed with hypo-osmotic

swelling test (HOS) as described by Santiago-Moreno et al. (2009). The HOS

solution was prepared by adding 1 g of sodium citrate to 100 mL of distilled water.

Semen (25 µl) was mixed with 500 µl of a HOS solution (100 mOsm/kg) and

incubated at 37oC for 30 minutes. A drop of incubated solution was placed on a pre

warmed (37oC) slide, fixed in buffered 2% glutaraldehyde and cover-slipped to

examine under phase contrast microscope at lense = 100x oil immersion x eye

piece = 10x. The spermatozoa showing swollen heads, swollen and coiled tails

were classified as normal spermatozoa having intact plasma membrane. A total of

200 spermatozoa were counted at four separate fields.


Sperm viability was assessed by adding eosin-nigrosin to the lake glutamate

solution. Lake‟s glutamate solution (Bakst and Cecil, 1997) was prepared by


(0.0085g) and magnesium chloride (0.000676) in 100 mL distilled water. Water

soluble nigrosin (5g) and water soluble Eosin-bluish (1g) were added into Lake‟s

glutamate solution. Twelve drops of stain were mixed with 1 drop of semen. A


85






4.3.5.4. Sperm acrosome integrity

Sperm acrosome integrity was evaluated with giemsa stain (Jianzhong and

Zhang, 2006). The stain was prepared by adding giemsa (3g) and phosphate buffer

saline at pH 7.0 (2 mL) into 35 mL water. Smear was prepared by placing 5µl of

semen sample on a clean glass slide, air-dried and fixed in neutral formal-saline

(5% formaldehyde) for 30 minutes. Fixed slides were kept in giemsa stain for 1.5

hours. Sperm with normal acrosome appeared to be evenly stained; abnormal

spermatozoa were unevenly stained while spermatozoa having ruptured acrosome

were remained unstained. A total of 200 spermatozoa were observed under phase

contrast microscope at lense = 100x oil immersion x eye piece = 10x in at least in

four separate fields.

4.3.6. Recovery Rate of Semen Quality Parameters

The recovery rate of sperm motility, plasma membrane integrity, viability

and acrosome integrity were calculated through following formula (Dubos et al.,

2008).

86

Recovery rate =Value of a given parameter in frozen semen

Value of a given paramter in fresh semen x100

4.3.7. Absolute Livability Index

The absolute livability index of sperm was calculated as a measure of time

dependant decrease in sperm motility after post-thaw incubation at 37°C. The

absolute index of livability of spermatozoa was computed by the following

equation (Melovenof, 1962).

La=Σ(TR)

Where La = absolute index of livability, T = time interval (h) between the

two consecutive observations, R = average of motility percentage for two

consecutive observations starting from the time of incubation.

4.3.8. Artificial Insemination

Before insemination with frozen semen, glycerol was removed at thawing

by following stepwise dilution protocol as suggested by Purdy et al. (2009). The

fertility attributes of IRJF spermatozoa were measured by inseminating 50 hens

with semen frozen with 11, 15 and 20% glycerol. Intravaginal artificial

inseminations were performed at depth of 4cm with AI gun (IMV Technologies,

France) having fitted glass pipette with a dose consisted of 270-300 million sperm

87

within 10 minutes. The eggs were collected day after insemination for five days

incubated for 24 days. Eggs fertility was examined by candling after 7 days of

incubation. The eggs were hatched out after 19-21 days. The fertility attributes

were calculated with the following formulae:

Fertility (%) = Total Numbe r of fertile eggs





Total Number of fetile eggs x100


The data (mean ± SEM) on the effect of glycerol in a diluent on sperm

motility, plasma membrane integrity, viability and acrosome integrity was analyzed

by Analysis of Variance in completely randomized factorial design using MSTAT-

C® (version 1.42, Michigan State University, East Lansing, MI, USA). When the F-

value was significant, the post-hoc comparison between the means was done

though Fisher‟s protected LSD test. Multiple regression analysis was preformed for

sperm quality parameters post-thaw during thermo-resistance test at 37°C using

storage hours as independent variable with statistical package Past® (version 3.12,

Copyright: Øyvind Hammer, Natural History Museum, University of Oslo,

88

Norway). The data on fertility rate was analyzed by ANOVA by MegaStat®

(version 7.25, Mc-Graw-Hill New Media, New York, for Excel, 2007).

4.4. RESULTS

4.4.1. Effect of Glycerol Concentrations on Motility, Plasma Membrane

Integrity, Viability and Acrosome Integrity at Different Post-thaw

Intervals (after 0, 2 and 4 Hours of Incubation)

The data on the effect concentrations of glycerol on motility, plasma

membrane integrity, viability and acrosome integrity of IRJF spermatozoa at 0, 2

and 4 hours post-thaw is given in Figure 4.7.1.1 to 4.7.1.4.

Percentages of motility (0 hours; one way ANOVA, F2,12 = 20.18, P <

0.001; 2 hours; one way ANOVA, F2,12 = 17.33, P < 0.001; 4 hours; one way

ANOVA, F2,12 = 25.44, P < 0.001), plasma membrane integrity (0 hours; one way

ANOVA, F2,12 = 11.81, P < 0.01; 2 hours; one way ANOVA, F2,12 = 22.04, P <

0.001; 4 hours; one way ANOVA, F2,12 = 34.30, P < 0.001), viability (0 hours; one

way ANOVA, F2,12 = 23.21, P < 0.001; 2 hours; one way ANOVA, F2,12 = 41.34, P

< 0.001; 4 hours; one way ANOVA, F2,12 = 60.68, P < 0.001) and acrosome

integrity (0 hours; one way ANOVA, F2,12 = 55.45, P < 0.001; 2 hours; one way

ANOVA, F2,12 = 57.63, P < 0.001; 4 hours; one way ANOVA, F2,12 = 30.53, P <

0.001) were recorded higher (P<0.05) with 20% glycerol at 0, 2 and 4 hours of

incubation post-thaw compared to 11% and 15% glycerol.

89

Multivariate regression analysis (Table 4.2) showed that incubation hours

negatively affected the aforementioned semen quality parameters; the calculated

value of R2 showed that the effect of hours on semen quality parameters was least

in a diluent having 20% glycerol (0.72) followed by 15% (0.75) and 11% glycerol

(0.81).

4.4.2. Effect of Glycerol Concentrations on Sperm Quality Parameter

Recovery Rates

The data on the effect of glycerol on recovery rates (%) for motility, plasma

membrane integrity, viability and acrosome integrity after cryopreservation is

given in Table 4.1. The highest (one way ANOVA, F2,12 = 38.73, P < 0.001)

recovery rates were obtained in a diluent with 20% glycerol compared to 15% and

11% glycerol for motility (one way ANOVA, F2,12 = 16.02, P < 0.001), plasma

membrane integrity (one way ANOVA, F2,12 = 26.35, P < 0.001), viability (one

way ANOVA, F2,12 = 4.59, P < 0.05) and acrosome integrity (one way ANOVA,

F2,12 = 19.54, P < 0.001).

4.4.3. Effect of Glycerol Concentrations on Absolute Livability Index

The data on the effect of glycerol concentrations on absolute livability

index of IRJF spermatozoa at 37°C is given in Table 4.1. Absolute livability index

was recorded higher (one way ANOVA, F2,12 = 38.73, P < 0.001) with 20%

90

glycerol (164.0 ± 2.4) compared to 15% (139.0 ± 7.3) and 11% glycerol (98.0 ±

5.1).

4.4.4. Effect of Glycerol Concentrations on Fertility Attributes

The data on the effect of glycerol on fertility attributes of IRJF sperm is

given in Table 4.3. The results based on eggs collected for five days after

insemination with cryopreserved semen showed significantly higher fertility

(P<0.05) with 20% glycerol followed by 15% and 11% glycerol in terms of number

of fertile eggs (one way ANOVA, F2,12 = 66.04, P < 0.001), fertility (one way

ANOVA, F2,12 = 47.50, P < 0.001), number of hatched chicks (one way ANOVA,

F2,12 = 7.47, P < 0.01), percent hatch (one way ANOVA, F2,12 = 82.24, P < 0.001)

and hatchability of fertilized eggs (one way ANOVA, F2,12 = 8.44, P < 0.01).

4.5. DISCUSSION

The fertilizing ability of the avian sperm is generally assessed indirectly

based on motility, plasma membrane integrity, viability and acrosome integrity in

the laboratory experiments (Blesbois et al., 2008). Nevertheless, fertility assessed

at 5-6 day of incubation through candling gives very precise and direct information

(Tselutin et al., 1998). In a recent study, during cryopreservation of IRJF sperm,

following routine poultry protocol using 11% glycerol, a massive loss was

observed in motility, plasma membrane integrity, viability and acrosome integrity.

Therefore, present study was designed to evaluate different concentration of

91

Figure 4.7.1.1: Effect of different levels of glycerol on spermatozoa motility (%)

of Indian red jungle fowl post-thaw at 0, 2 and 4 hours of incubation at 37 ºC

(n=5). The bars with different letters showed significant differences at a given stage

at 0, 2 and 4 hours of Incubation. (Table 25-27; Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0 hours 2 hours 4 hours

Moti

lity

(%

)

Post-Thaw Hours

11% 15% 20%

c

b

a

c

b

a

c

b

a

92

Figure 4.7.1.2: Effect of different levels of glycerol on spermatozoa Plasma

membrane integrity (%) of India red jungle fowl post-thaw at 0, 2 and 4 hours of

incubation at 37 ºC (n=5). The bars with different letters showed significant

differences at a given stage at 0, 2 and 4 hours of Incubation. (Table 28-30;

Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0


Pla

sma

Mem

bra

ne

Inte

gri

ty (

%)

Post-Thaw Hours

11% 15% 20%

c

b

a

c

b

a

c

b

a

93

Figure 4.7.1.3: Effect of different levels of glycerol spermatozoa viability (%) of

Indian red jungle fowl post-thaw at 0, 2 and 4 hours of incubation at 37 ºC (n=5).

The bars with different letters showed significant differences at a given stage at 0, 2

and 4 hours of Incubation. (Table 31-33; Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0


Sp

erm

Via

bil

ity

(%

)

Post-Thaw Hours

11% 15% 20%

c

b

a

cb

a

c

b

a

94

Figure 4.7.1.4: Effect of different levels of glycerol on spermatozoa acrosomal

integrity (%) of Indian red jungle fowl post-thaw at 0, 2 and 4 hours of incubation

at 37 ºC (n=5). The bars with different letters showed significant differences at a

given stage at 0, 2 and 4 hours of Incubation. (Table 34-36; Appendices)

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0


Sp

erm

Acr

oso

ma

l In

teg

rity

(%

)

Post-Thaw Hours

11% 15% 20%

c

b

a

b b

a

b

b

a

95

Table 4.1: Recovery rates (%; mean ± SEM) of sperm parameter after

cryopreservation with different levels of glycerol (n=5)

Glycerol (%) in diluent

11 15 20

Sperm motility (%) 40.8 ± 5.2b 52.8 ± 5.9

b 84.2 ± 5.6

a

Sperm plasma membrane integrity (%) 41.3 ± 4.0c 64.6 ± 5.8

b 80.8 ± 6.1

a

Sperm viability (%) 57.7 ± 4.7b 64.6 ± 5.8

b 80.8 ± 6.1

a

Sperm acrosome integrity (%) 55.0 ± 4.3c 63.8 ± 3.9

b 86.0 ± 2.3

a

Absolute livability index 98.0 ± 5.1c 139.0 ± 7.3

b 164.0 ± 2.4

a

The values (mean ± SEM) having different superscripts differ significantly (P <

0.05) along the row. (Table 37-41; Appendices)

96

Table 4.2: Multivariate regression for sperm parameters of Indian red jungle fowl

frozen with different levels of glycerol during incubation at 37°C using hours as

independent variable (n=5)

Glycerol Sperm

parameter

Slope Error Intercept Error P-

value

Statistics

11%

Motility -5.5 0.6 34.7 1.7 0.00 R2=0.81

MSE=18.1

F=74.24

P=0.00

df1 = 4

df2 = 10

Plasma

membrane

integrity

-6.3 0.7 49.8 1.9 0.00

Viability -3.7 0.6 56.8 1.4 0.00

Acrosome

integrity

-4.0 0.8 65.0 2.0 0.00

15%

Motility -4.8 0.9 43.5 2.3 0.00 R2=0.75

MSE=28.6

F=23.0

P=0.00

df1 = 4

df2 = 10

Plasma

membrane

integrity

-7.0 0.8 59.8 2.0 0.00

Viability -4.0 0.7 63.4 1.9 0.00

Acrosome

integrity

-5.0 1.0 72.0 2.5 0.00

20%

Motility -6.5 0.7 54.0 1.7 0.00 R2=0.72

MSE=24.9

F=38.3

P=0.00

df1 = 4

df2 = 10

Plasma

membrane

integrity

-5.2 0.8 65.0 2.0 0.00

Viability -1.8 1.1 80.8 2.8 0.13

Acrosome

integrity

-3.8 0.5 91.7 1.3 0.00

97

Table 4.3: Fertility outcomes of Indian red jungle fowl after artificial insemination

with different levels of glycerol in extender (n=50)

Items Glycerol

(%)

Day after insemination Mean ± SEM

2 3 4 5 6

Number of laid eggs 11 45 50 50 45 40 46.0 ± 1.9a

15 48 49 50 45 45 47.4 ± 1.0 a

20 45 50 50 50 45 48.0 ± 1.2 a

Number of fertile eggs 11 10 12 15 8 9 10.8 ± 1.2c

15 13 15 20 15 15 15.6 ± 1.2b

20 30 35 29 30 28 28.4 ± 0.9a

Fertility (%) 11 22.2 30.0 40.0 17.8 22.5 26.5 ± 3.9c

15 27.1 30.6 40.0 33.3 33.3 32.9 ± 2.1b

20 66.7 70.0 58.0 60.0 62.2 63.4 ± 2.2a

Number of hatched chicks 11 7 9 10 3 5 6.8 ± 1.3c

15 10 10 15 10 12 11.4 ± 1.0b

20 25 22 25 25 24 24.2 ± 0.6a

Hatch (%) 11 15.6 18.0 20.0 6.7 12.5 14.5 ± 2.3c

15 20.8 20.4 30.0 22.2 26.7 24.0 ± 1.9b

20 55.6 44.0 50.0 50.0 53.3 50.6 ± 2.0a

Hatchability of fertile

eggs (%)

11 70.0 75.0 66.7 37.5 55.6 60.9 ± 6.7b

15 76.9 66.7 75.0 66.7 80.0 73.0 ± 2.7b

20 83.3 88.0 86.2 83.3 85.3 85.3 ± 0.9a

The values (mean ± SEM) having superscript differ significantly (P < 0.05) along

the row for a given parameter. (Table 42-46; Appendices)

98

glycerol (11%, 15% and 20%) to get optimal post-thaw quality and fertility of IRJF

semen. For IRJF sperm; 20% glycerol exhibited better post-thaw motility, plasma

membrane integrity, viability and acrosome integrity at 0, 2 and 4 hours. Moreover,

recovery rates of aforementioned parameters and absolute livability index was

recorded higher with 20% glycerol. Results based on eggs collected for five days

after insemination with cryopreserved semen demonstrated higher No. of fertile

eggs, fertility (%), No. of hatched chicks, percent hatch and hatchability of

fertilized eggs (%) with 20% glycerol followed by 15% and 11% glycerol.

In a previous study, higher motility and viability and their recovery rates

were reported with 7% glycerol for Egyptian local strains (El-Salam and Dokki-4).

Although fertility and hatchability rates were recorded 38% and 88%, respectively

in El-Salam strain with 7% glycerol but in Dokki-4, zero fertility and hatchability

rate was observed (Roushdy et al., 2014). It suggests the major source of variation

in fertility results in former study may be due to difference in genetics of the strains

(Roushdy et al., 2014; Sexton, 1979). Rooster sperm from four different breeds

when cryopreserved with same level of glycerol resulted in variable post-thaw

semen quality (Siudzińska and Łukaszewicz, 2008). In a study on Muscovy

spermatozoa, higher mobility was observed when semen was cryopreserved in

HIA-1 and AU extender with 5% and 7% glycerol (Gerzilov, 2010).

Interestingly, an increasing trend in motility was reported with the increase

in glycerol concentration in a diluent which attained maximum value at 7%,

remained stable upto 13% and declined afterward in Leghorns rooster frozen-

99

thawed semen (Terada et al., 1988). Moreover, volume of frozen-thawed sperm

cell decreased with increasing concentration of glycerol upto 7%, and may observe

increase in cell volume with further increase in glycerol level. All these studies

suggest that the level of glycerol to achieve optimum post-thaw quality and fertility

greatly varies with species, breeds and strains.

In a systematic study, the toxicity extent of glycerol, DMA and DMSO

level from 4-11% remained similar for fowl spermatozoa, while glycerol was found

least toxic following DMA and DMSO for sperm viability and integrity (Tselutin et

al., 1998). In contrast, 10% of DMSO provided better protection to duck

spermatozoa during cryopreservation compared to 4, 6, 8 and 10% of glycerol (Han

et al., 2005). DMSO may have shown more toxic effects for fowl sperm compared

to glycerol due to lower molecular weight and higher penetrability (Tselutin et al.,

1998). However, in Taiwan native rooster, 20% DMSO maintained satisfactory

post-thaw quality based on mass motility, mitochondria polarization, plasma

membrane and acrosome integrity with slow, moderate and fast cooling rates. It is

to believe that protection provided to sperm during cryopreservation by permeable

cryoprotectants is a multi-factor and complex phenomenon (Donoghue and

Wishart, 2000; Long, 2006; Sieme et al., 2016).

The levels of glycerol which provide optimum semen quality differ in

different species and depend upon certain factors that include, concentration of

glycerol, adding temperature, equilibration time, biochemical composition of

plasma membrane and solute constituent (Hammerstedt and Graham, 1992;

100

Tselutin et al., 1999; Blanco et al., 2000; Holt, 2000). The composition of the

plasma membrane is highly variable from species to species and breeds/strains that

affect the penetrability of glycerol and efflux of the water molecules (Hammerstedt

and Graham, 1992). The glycerol level which produced better post-thaw quality in

a certain species failed to repeat it even in closely related species/breed or strains.

It is pertinent to mention that glycerol added in a cryodiluter at ambient

temperature even at lower concentration of 11% is highly deleterious to IRJF

sperm and resulted in zero motility after cryopreservation (unpublished data by the

author). This signifies the uniqueness that Indian red jungle fowl sperm membrane

that could not withstand osmotic shock when glycerol is added at ambient

temperature in diluent.

The addition of glycerol at lower temperature may possibly eliminate the

toxic effects of glycerol during cryopreservation (Blesbois et al., 2008). For

cryopreservation of IRJF spermatozoa, an approach with slow cooling rate and

higher concentration of glycerol added at low temperature with short equilibration

period is effective in recovering acceptable motility, viability, plasma membrane

and acrosome integrity.


The different glycerol concentrations (11%, 15% and 20%) were evaluated

for post-thaw quality, recovery rates, absolute livability index and fertility

101

attributes (number of fertile eggs, percent fertility, no. of hatched chicks, percent

hatch and hatchability of fertilized eggs) of IRJF semen were assessed in this

chapter. Percentages of motility, plasma membrane integrity, viability and

acrosome integrity were recorded higher (P < 0.05) at 0, 2 and 4 hours post-

thawing at 37°C with glycerol 20% compared to 15% and 11% glycerol. Likewise,

recovery rates (%) of aforementioned parameters and absolute livability index after

cryopreservation were observed highest (P < 0.05) with 20% glycerol. Multivariate

regression analysis showed least negative effect of hours of incubation on semen

quality in diluent with 20% glycerol followed by 15% and 11% glycerol. The

fertility outcomes (number of fertile eggs, fertility (%), no. of hatched chicks,

percent hatch and hatchability of fertilized eggs) were recorded higher (P < 0.05)

with 20% glycerol followed by 15% and 11% glycerol. It is worth mentioning to

highlight that data on fertility rate and hatchability of fertilized eggs also support

the results of post-thaw in vitro semen quality parameters. In conclusion, 20%

glycerol added to diluent at 4°C improved the cryopreservability and fertility

attributes (No. of fertile eggs, fertility, No. of hatched chicks, percent hatch and

hatchability) of IRJF semen.

102

Chapter 5

EFFECT OF CRYOPROTECTANTS ON QUALITY AND

FERTILITY OF INDIAN RED JUNGLE FOWL SEMEN

5.1. INTRODUCTION

Cryopreservation provides safety net against the stochastic events of nature;

continuously at work and playing role in the deletion of valuable genetic resource

(Holt et al., 2014). During the last few decades, increasing human population has

resulted in extreme exploitation and selection pressure in poultry birds that pushed

indigenous or local species/breeds in the background disposing of valuable genes

(Tisdell, 2003; Allendorf et al., 2008). According to Domestic Animal Diversity

Information System, about 50% of the poultry species have been enlisted in

endangered category (Hoffmann, 2005).

The cryo-banking has great potential of application in ex situ in vitro

conservation in Gallus sub-species (Abouelezz et al., 2015). Semen

cryopreservation is the most feasible method in birds as cryopreservation of oocyte

or embryo is not possible because of large size, high lipid content and polar

organization. The most feasible method for avian species is the semen banking

(Blesbois et al., 2008). Cryopreservation is one of the techniques of semen

banking. The process of cryopreservation is a very stressful process that readies the

sperm to undergo various thermal and osmotic changes and ultimately decreases

the sperm viability by lipid peroxidation, production of reactive oxygen species

102

103

(Ros) molecules (Moce et al., 2010), increase in cell volume before

cryopreservation, and significant decrease in cell volume post-thawing. However,

during post-thawing osmotic shrinkage of sperm cells occurred due to increase in

external solute concentration; thus making the sperm osmotically shrinked and

damged (Mazur et al., 1981).

Siginifcant decrease in cell volume and oxgygen consumption has been

recorded in chicken semen during freeze-thawing due to rapid cooling from 37 to

20 ºC in just 20 min and then from 20 ºC to 4 ºC within in 2 hours. This rapid

cooling reduces the proportion of solute within the cell compared to outside and

makes the sperm exposed to extracellular ice crystals (Terada et al., 1983). Thus,

addition of cryoprotectant in an extender restores the sperm viability by providing

osmotic balance between solute and solvent concentration during freezing

(Blesbois et al., 2007). The decrease in post-thaw volume thus favored in terms of

increased semen quality due to increase in the level of cryoprotectant up to a

certain level. The cryoprotectants dehydrate the sperm cell by increasing loss of

intracellular water and favor the sperm to avoid damge due to ice-crystallization

within the cell during freeze-thawing (Qadeer et al., 2015).

Recently, tremendous loss was observed to viability of IRJF during freezing

process; following cryopreservztion protocol routinely used for fowl semen i.e.

11% glycerol as cryoprotactnet added at 4°C. The higher variability has already

been reported in success of cryopreservation for even in closely related

species/breeds or strains (Blesbois et al., 2006; Blanco et al., 2011, 2012; Holt,

104

2000, 2001). This needs an optimized procedure and cryoprotectants for the

successful freezing of IRJF spermatozoa to achieve satisfactory fertility and

hatching of fertilized eggs. All the available cryoprotectants including glycerol

differ in their ability for preservation of sperm even in closely related avian species

due to penetrability, membrane permeability and toxicity to organelles at cellular

level (Sieme et al., 2016). It is noted that a cryoprotectant that showed highest

toxicity to sperm of a certain species may have been beneficial for sperm of other

allied species/breeds and strains (Moce et al., 2010; Malecki et al., 1997; Long et

al., 2014).

The intensity of cryo-induced changes to sperm are highly species-specific

(Blanco et al., 2011; Roushdy et al., 2014; Massip et al., 2004) and previous study

on IRJF showed different response of sperm to different phases of cryopreservation

(dilution, cooling, equilibration, freezing and thawing). Considering the importance

of semen cryo-banking for conserving valuable genetic resources of IRJF, the

present study was planned to elucidate the effect of different concentrations of

dimethyleacetamide (DMA), dimethylformamide (DMF), dimethylsulfoxide

(DMSO), polyvinylpyrrolidone (PVP) and egg yolk on quality and fertility of IRJF

spermatozoa.


Cryopreservation negatively affects semen quality; because various stages

of cryopreservation have different impact on semen quality. The first part of

105

damage occurs at the time of dilution probably due to osmotic disturbance and then

at the stage of cooling. Ice crystallization at the stage of equilibration makes the

sperm more vulnerable to damage (Long, 2006; Qadeer et al., 2015). At the stage

of post-thawing, re-crystallization due to temperature transition from -196 ºC to 37

ºC occurs (Qadeer et al., 2015). Thus making avian sperm more sensitive to cryo-

damages compared to domestic species (Blesbois et al., 2007).

Generally, it is believed that avian sperm has lower surface to volume ratio

and elongated tail that makes it more vulnerable to osmotic, chemical, thermal and

physical stresses during cryopreservation process compared to mammalian sperm

(Long, 2006; Donoghue and Wishart, 2000). Freeze-thawing process not only

causes irreversible damage to mitochondria, mid-piece and perforatorium of

spermatozoa (Xia et al., 1988), but also induces physical and chemical changes that

alter physiological processes. Sperm may lose ATP due to energy metabolism

(Soderquist et al., 1991) and glyco-proteins or glycol-lipids necessary for transport

and maturation (Pelaez and Long, 2005; Long, 2006).

There is great variation in the success of cryopreservation of avian semen

even in closely related species breeds/strains due to unique morphology,

physiology, biochemistry of sperm of each species, cryoprotectant used and

freezing protocols (Blesbois et al., 2006; Blanco et al., 2011, 2012; Holt, 2000,

2001). Each procedure has its own particular variables; diluents, dilution rates,

cooling rate, nature of cryoprotectant, rate and timing of cryoprotectant used,

freezing conditions, choice of packaging during freezing and thawing procedures

106

(Tselutin et al., 1999). It is suggested that development of optimized

cryopreservation protocols is obligatory that confirms to the IRJF sperm

physiology, morphology and biochemistry that contends the freeze-thaw

cryodamages and results in remarkable post-thaw quality and fertility outcomes.

A variety of cryoprotectants; glycerol, dimthylsulfoxide (DMSO),

dimethylacetamide (DMA) and dimethylformamide (DMF), PVP and egg yolk

have been used in the past (Sexton, 1978; Lake et al., 1980; Lake and Ravie, 1982,

1984; Tselutin et al., 1999). Any given cryoprotectant can be acceptable due to its

colligative properties; less damage to sperm cells during cryopreservation and

fertility results are adequate after artificial insemination (Seigneurin and Blesbois,

1995; McGann, 1978). Glycerol and dimethyleacetamide (DMA) are considered

more adequate of all available cryoprotectants (Woelders et al., 2006; Blesbois et

al., 2007). Glycerol is least toxic and more effective cryoprotectant for low fertility

lines of poultry (Blesbois et al., 2007). However, for endangered species, it is not

effective, because of its contraceptive ability and need to be removed prior to

artificial insemination (Blanch et al., 2012; Blesbois, 2011; Long and Kulkarni,

2004). Glycerol is also known to interact with the metabolism of spermatozoa, alter

lipid-bilayer that possibly influence the acrosome of sperm to react and contribute

to low fertilizing ability (Hammerstedt and Graham, 1992; Blesbois and

Seigneurin, 2010).

The DMA is a permeable cryoprotectant, not contraceptive and is not

removed prior to artificial insemination; thus helpful in reducing damage due to


107

cryoprotectant removal (Blesbois et al., 2007). The DMA have capacity of

penetrability by increasing plasma membrane fluidity through the rearrangement of

lipids and proteins that cause reduction in formation of ice-crystallization at low

temperature (Holt, 2000; Blanco et al., 2011). DMA has been used as a

cryoprotectant in various avian species, namely, houbara bustard (Hartley et al.,

1999), fowl (Tselutin et al., 1999), poultry, eagle, and peregrine falcon (Balnco et

al., 2000), duck (Han et al., 2005), landese gender (Dubos et al., 2008), sandhill

crane and turkey (Blanco et al., 2012), Japanese quail (Kowalczyk, 2008), rooster

sperm (Purdy et al., 2009) and emu (Sood et al., 2012).

DMSO is one of the permeable cryoprotectants and has the ability to

precipitate solution below the freezing point rather than depressing freezing point

colligatively and promote vitirification of water to prevent intracellular ice

crystallization (McGann, 1978). The possible mechanism of DMSO in reducing ice

crystallization is due to thermodynamic stability of the solution that reduces

nucleation and subsequent ice formation as the temperature decreases during

cryopreservation process (Mandumpal et al., 2011).

The DMSO is a standard cryoprotectant for poultry sperm cryopreservation

and gives higher postthaw quality and fertility outcomes (Sontakke et al., 2004).

The DMSO usage in poultry semen alleged to have lower toxicity (Lake and Ravie,

1984), but it penetrates the cell membrane and may become toxic at higher

concentrations at higher temperatures (Mphaphathi et al., 2012). The DMF is a low

molecular weight cryoprotectant and shows colligative properties at higher

108

concentrations when exposed to severe temperature regimes (Santarius and

Stocking, 1969). The DMF addition in extender decreases the freezing point of the

mixture (semen and extender) during freeze-thaw process. Due to specific

interaction with sperm membranes; the cryoprotectant not only provides protection

from severe temperature regimes, but it is also responsible for protection to sperm

from post-thaw damage during thawing process (Mazur, 1970). It was observed

that methyl containing cryoprotectants did not pose any damage to sperm motility

when used at lower concentrations. However, at higher concentrations of

cryoprotectant, not only the post thaw sperm motility was affected but the damage

was evident during different phases (post-dilution, post cooling and post-thawing)

of cryopreservation (Yang et al., 2010). Further studies are suggested on IRJF to

improve the post thaw semen quality and fertility by adding different

concentrations of DMF as cryoprotectant.

The polyvinylpyrrolidone (PVP), a polymeric non-permeable

cryoprotectant; had capacity to successfully preserve a variety of biological cells

(Wolfe and Bryant, 2001; Noskov et al., 2002; Li et al., 2005; Thirumala et al.,

2009). The mechanism through which PVP protect the cell integrity is poorly

understood, nevertheless, several assumptions explain the cryoprotective capacity

of PVP i.e. colligative properties during freezing (Freshney, 2000), developing cell

membrane coating (Nash, 1966; Freshney, 2000) and freezing point depression

(Farrant et al., 1969; Freshney, 2000). In a study, PVP maintained acceptable post-

thaw quality of rooster and ring-necked pheasant and red-tailed hawks semen

109

(Herrera et al., 2005). The remarkable fertility of 39% in rooster and 57% in ring-

necked pheasant was recorded in semen frozen with PVP as cryoprotectant.

Egg-yolk is the essential component of extender for semen cryopreservation

the livestock species (Andrabi et al., 2008). Egg yolk is an impermeable

cryoprotectant that provides protection to sperm during freeze-thaw process. The

possible mechanism of protection of egg yolk may be due to the presence of low

density lipoproteins that adhere to the sperm plasma membrane and stabilize the

membrane externally. While the other possible mechanism of protection is that low

density lipoproteins form a protective coating over the sperm membrane or replace

the sperm plasma membrane phospholipids that are lost during the freeze-thaw

process (Foulkes et al., 1980).

A number of workers reported the use of egg yolk as cryoprotectant for

chicken spermatozoa (Munro, 1938; Ishikawa, 1930; Xumasi, 1957) and found that

egg yolk has maintained the sperm motility. Smith (1949) and Takeda (1954) found

that egg yolk addition to the freezing extender may enhance the survival of sperm

post-thawing in chicken. However, in another study, egg yolk addition in

phosphate buffer was found to decreases the sperm motility in fowl (Wilcox,

1960). Similarly, low fertility in turkey was attributed to the the addition of egg

yolk to the freezing extender (Bajpai and Brown (1963). Traditionally, the egg yolk

has been used as cryoprotectant for various livestock species due to its easy

availability and protection to sperm (Kumaresan et al., 2005). The improvement or

decline in post-thaw sperm motility has been attributed to the chemical

110

composition of the egg yolk and species for which they are being used (Bathgate et

al., 2006). The egg yolk as non-permeable cryoprotectant helps to protect the

sperm against cold shock, and maintain viability. The phospholipids cholesterol

and the low density lipoprotein content of chicken egg yolk specifically have been

identified as the protective components (Pace and Graham, 1974; Watson and

Martin, 1975). Successful semen preservation of IRJF requires the identification of

a suitable cryoprotectant and its levels to be used for the development of a freezing

regime. However, little research has been done on the use of cryoprotectants in the

genus gallus. The present study is designed to test different levels of DMA, DMF,

DMSO, PVP and egg yolk on fertility of IRJF for conservation and propagation.


5.3.1. Experimental Animals

The semen used in the present study was obtained from sexually mature

cocks of IRJF. The birds used in this were maintained at Avian Research Station

The experiment was conducted between 18-12-2014 to 26-04-2016. The birds were

housed in single sand floor pen (8 m2) with roof cover and kept under natural

photoperiod and temperature conditions. They were fed on the commercial feed

containing corn 61.0%, rice polish 4%, corn gluten 1%, canola meal 5%, rapeseed

meal 2%, soybean meal 13%, sunflower meal 4%, limestone 8%, DL. Methionine

0.10%, soda bicarb 0.10%, salt 0.30%, vitamin and min. premix 0.40%, MDCP 1%

and was available over the experimental period ad libitum.

111

5.3.2. Experimental Design

The study consisted of five experiments involving the cryopreservation of

daily collected semen samples from all cocks. Experiment involved the addition of

different concentrations of DMA, DMSO, DMF, PVP (at 4%, 6%, 8% and 10%),

egg yolk (at 10%, 15%, 20% and 25%) and glycerol (20%) as a control. The semen

quality parameters were studied at different stages of cryopreservation; post-

dilution, post-cooling, post-equilibration and post thawing. The best freezing

extender containing 6% DMA, 8% DMSO, 8% DMF, 6% PVP and 15% egg yolk

and control having 20% glycerol was used in artificial insemination. For this

purpose, 50 hens were inseminated with each experimental extender and fertility

was estimated through the fertilized and unfertilized eggs collected up to 5 days

following insemination.

5.3.3. Semen Collection and Dilution with Freezing Extender

Semen was collected twice a week as described by Burrows and Quinn

(1935) in a graduated plastic tube using massage technique. Semen was pooled on

each occasion. Each pool was immediately diluted 1:1 (v/v) using diluent

comprised of D-fructose (1.15g), sodium glutamate (2.1g), polyvinylpyrrolidone

(0.6g), glycine (0.2g) potassium acetate (0.5g) and distilled water (100mL) to final

pH (7.0) and osmolarity 380 mOsm/kg. Precaution of the temperature shock was

taken, and thus the tubes containing diluents were kept in water bath at 37°C. The

diluent was divided into experimental extenders containing 4%, 6%, 8%, and 10%

112

(DMA, DMF, DMSO, PVP) and egg yolk (10%, 15%, 20% and 25% and 20%) as

a cryoprotectant and 20% glycerol was used as control. All diluents and media

were prepared in the laboratory using analytical grade chemicals purchased from

Sigma-Aldrich, Co., 3050 Spruce street, St Louis, USA.

5.3.4. Cryopreservation of Semen

The semen was pooled and diluted to a concentration of 1200 x 106

sperm/mL in freezing extenders containing 1 (4% of DMA, DMF, DMSO and PVP

or 10% egg yolk); 2 (6% of DMA, DMF, DMSO and PVP or 15% egg yolk); 3

(8% of DMA, DMF, DMSO and PVP or 20% egg yolk); 4 (10% of DMA, DMF,

DMSO and PVP or 25% egg yolk); 5 (20% Glycerol). The diluted semen was

cooled to 4 °C in two hours (0.275 min-1

) and equilibrated for 10 min at 4°C. Cooled

semen was filled in 0.5 mL French straws (IMV, L‟Aigle, France) and kept over

liquid nitrogen vapors for 10 minutes and plunged into liquid nitrogen for storage.

The straws were thawed individually at 37°C for 30 seconds in water bath for

further evaluation of post-thaw semen quality.


5.3.5.1. Motility

The percentage of motile spermatozoa and the quality of sperm were

assessed subjectively [see page 22: chapter 2 (Santiago-moreno et al., 2011)]. A

113

semen sample was placed on a pre-warmed (37 ºC) glass slide and observed under

a phase contrast microscope (400x) at different stages of cryopreservation; post-

dilution, post-cooling, post-equilibration and post-thawing.

5.3.5.2. Plasma membrane integrity

Plasma membrane integrity of IRJF spermatozoa at different stages of

cryopreservation viz; pre-dilution, post-dilution, post-cooling, post equilibration

and post freeze-thawing was assessed by using hypo-osmotic swelling test (HOST)

as described by Santiago-Moreno et al. (2009). The HOS solution composed of

sodium citrate (1g) and distilled water (100 mL). Previously diluted 25 µl semen

was mixed with 500 µl of a HOS solution (100 mOsm/kg) and incubated at 37oC

for 30 minutes. The slides were fixed in buffered 2% glutaraldehyde. The plasma

membrane integrity of spermatozoa was scored on the basis of swollen heads,

swollen and coiled tails. The spermatozoa that had taken the HOS solution become

swollen and coiled and were classified as normal spermatozoa having intact plasma

membrane and those which have not taken solution and remain straight were

abnormal and dead. A total of 200 spermatozoa were counted at four separate fields

under a phase-contrast microscope (lense = 100x oil immersion x eye piece = 10x).


Viability (% Live / total sperm) of IRJF spermatozoa at different stages of

cryopreservation viz; pre-dilution, post-dilution, post-cooling, post equilibration

and post freeze-thawing was examined by adding eosin-nigrosin to the lake


114

glutamate solution. Lake‟s glutamate solution (Bakst and Cecil, 1997), prepared by


(0.0085g) and magnesium chloride (0.000676) in 100 mL distilled water. Water

soluble nigrosin (5g) and water soluble eosin-bluish (1g) were added into Lake‟s

glutamate solution. Twelve drops of stain were mixed with 1 drop of semen. A







The percentage of spermatozoa having intact acrosome was determined by

phase contrast microscopy, examining 200 giemsa blue-stained cells under the

magnification of lense = 100x oil immersion x eye piece = 10x as described by

Jianzhong and Zhang (2006) at different stages of cryopreservation viz; pre-

dilution, post-dilution, post-cooling, post equilibration and post freeze-thawing.

Giemsa staining involved spreading of 5µl of diluted semen on to a glass slide,

allowing them to dry and then fixed in neutral formal-saline (5% formaldehyde) for

30 min followed by air-drying. Fixed slides were kept in giemsa stain for 1.5 hours

and washed with distilled water and air-dried. The staining solution was prepared

by adding geimsa stain (3g), phosphate buffer saline (2mL) and distilled water (35

mL). Spermatozoa classified as not showing acrosome integrity were those with

115

hooked, swollen, thinned, unstained and missing acrosome. While all sperm with

normal acrosome were those with evenly stained showed acrosome integrity. A

total of 200 spermatozoa were observed at least in four separate fields.

5.3.6. Artificial Insemination

The fertilization capacity of the sperm cells was estimated from the results

of two consecutive intravaginal artificial inseminations involving 300 million

sperm per female at each insemination. A total of 50 hens of IRJF were

inseminated in each experiment (having 6% DMA, 8% MDSO, 8%DMF, 6% PVP

and 15% Egg yolk or 20% glycerol). All inseminations were performed between

12:00 and 14:00 h as described by Burrows and Quinn (1939). Eggs were collected

from day after insemination up to 5 days. Fertility was determined by candling of

eggs on the 7th

day of incubation. The eggs were hatched out after 19-21 days. The

fertility attributes were calculated with the following formulae:

Fertility (%) = Total Number of fertile eggs





Total Number ofFetile eggs x100


The data was presented as means (±SEM). The effect of different stages

cryopreservation and the cryoprotectant concentration on IRJF spermatozoa were

116

analyzed by Factorial design using Mstat C. When F-ratio was found significant (P

< 0.05), post hoc comparison between the means was done though Fisher‟s

protected LSD test.

5.4. RESULTS

5.4.1. Effect of Different Concentrations of DMA and Stages of


Spermatozoa

The data on the effect of DMA as cryoprotectant on motility, plasma

membrane integrity, viability and acrosomal integrity of IRJF spermatozoa are

presented in figures 5.4.1.1 to 5.4.1.4. The sperm motility (%) was significantly

higher (two factor ANOVA, Treatment; F4,60 = 38.45, P < 0.001) in an extender

having 6% DMA compared to 4% DMA in extender and control. The stage of

cryopreservation poses significant (Stage; F3,60 = 157.50, P < 0.001) damage to

sperm motility. Only 4% cryopreservation induced damage to sperm motility

occurred from post-dilution to post-cooling in an extender having 6% DMA

compared to extenders having 4% (10% damage), 8% (8.7% damage) and 10%

(10% damage) DMA respectively and control (6.2% damage). In the next phase,

from post-cooling to post-equilibration, 6% DMA caused less damage compared to

the concentrations of DMA at 4% (13.7% damage), 8% (21.2% damage), 10%

(20,7% damage) and control (17% damage). Lastly, the freeze-thaw process itself

induced damage to sperm motility that was again significantly less (P < 0.05) (22.5

117

%) with 6% DMA compared to the control (37.5%) and with 4%, 8% and 10%

DMA (23.7%, 27.5% and 27.5% damages, respectively). The interaction between

the treatment and stage was also found significant (Treatment x Stage; F12,60 = 2.76,

P < 0.01) that suggest different concentration of DMA affect sperm motility at all

stages of cryopreservation.

The sperm plasma membrane integrity significantly (two factor ANOVA,

Treatment; F4,60 = 36.68, P < 0.001) higher in an extender having 6% DMA

compared to other concentration of DMA (4%, 8%, 10%). However, the stage of

cryopreservation significantly (Stage; F3,60 = 96.43, P < 0.001) affect the recovery

of the intact sperm plasma membrane. Extremely low damage to sperm PMI was

noted from post-dilution to post-cooling stage and post-equilibration stage of

cryopreservation in an extender having 6% DMA compared to 4%, 8%, 10% and

control. The freeze-thawing process was more injurious (P < 0.05) to the intactness

of plasma membrane in an extender containing 20% glycerol (control) and 4%

DMA, followed by 8% and 10% DMA and much less damage was observed with

the extender having 6% DMA. The interaction between the treatment and stage of

cryopreservation was non-significant (Treatment x Stage; F12,60 = 1.05, P > 0.05)

that suggest that all of different concentrations of DMA have similar ability to

protect the membrane integrity of spermatozoa at all stages of cryopreservation.

The percentage of live sperm vary significantly (two factor ANOVA,

Treatment; F4,60 = 12.84, P < 0.001) in freezing extender containing 6% DMA

compared to other concentrations of DMA (4%, 8% and 10%) and control. The live

118

sperm recovery rate was significantly higher (Stage; F3,60 = 67.23, P < 0.001) in

extender containing 6% DMA compared to extenders containing 4%, 8%, 10%

DMA and control at post-cooling and post-equilibration stage. The freeze-thawing

process resulted in the higher (P < 0.05) recovery of percent live sperm in a diluent

having 6% DMA compared to 4%, 8% and 10% DMA and much less in a diluent

having 20% glycerol (Control). The interaction between different concentrations

of DMA and the stage of cryopreservation was found non-significant (Treatment x

Stage; F12,60 = 0.84, P > 0.05), that suggest that recovery of live spermatozoa was

independent and did not vary due to different concentrations of DMA and stages of

cryopreservation.

The sperm having intact and functional acrosome was significantly higher

(two factor ANOVA, Treatment; F4,60 = 6.83, P < 0.001) in extender having 6%

DMA compared to all other experimental extenders having 4%, 8% and 10% DMA

and control. The damage to sperm acrosome integrity at post-dilution stage of

cryopreservation was non significant in all the experimental extenders containing

DMA (4%, 6%, 8% and 10%) and control. Although a decline in the number of

spermatozoa having functional acrosome was recorded but not statistically

significant in all the experimental extenders at the stage of post-cooling except

higherin an extender having 6% DMA. However, at the equilibration stage, sperm

recovery was higher at 6% DMA but not significantly different (P > 0.05) from the

remaining extenders. The post-thawed sperm showed better (Stage; F3,60 = 37.04, P

< 0.001) acrosome intactness in freezing extender having 6% DMA compared to

4%, 8%, 10% and control 20%. The interaction between treatment and stage of

119

cryopreservation was found non-significant (Treatment x Stage; F12,60 = 0.17, P >

0.05) that suggest that damage to sperm acrosome intactness was independent from

different concentrations of DMA and stages of cryopreservation.The data on the

effect of DMA on fertility attributes of IRJF sperm is given in Table 5.1. Results

based on eggs collected for five days after insemination with cryopreserved semen

showed higher (P < 0.05) no. of fertile eggs (32.0 ± 1.4, 25.4 ± 1.1), percent

fertility (68.1 ± 3.0, 56.9 ± 4.1), no. of hatched chicks (28.2 ± 0.9, 20.0 ± 0.6),

percent hatch (60.1 ± 2.1, 44.7 ± 2.2) and percent hatchability of fertilized eggs

(88.4 ± 2.4, 81.5 ± 2.2) with 6% DMA compared to 20% glycerol.

5.4.2. Effect of Different Concentrations of DMSO and Stages of


Spermatozoa

The data on the effect of DMSO on motility, plasma membrane integrity,

viability and acrosomal integrity of IRJF spermatozoa at post-dilution, cooling,

equilibration and freeze-thawing are given in Figure 5.4.2.1-4. Sperm motility was

recorded highest (two factor ANOVA, Treatment; F4,60 = 3.93, P < 0.001) in

diluent with 8% DMSO at post-dilution (85.0 ± 2.9), cooling (80.0 ± 3.5),

equilibration (71.3 ± 4.3) and freeze-thawing (60.0 ± 1.3) compared to diluent with

4%, 6% and 10% DMSO and control. However, at all stages of cryopreservation,

the addition of DMSO have significantly (Stage; F3,60 = 140.66, P < 0.001) reduced

sperm motility between 25-42%. At cooling stage (20ºC), the sperm motility

107

Figure 5.4.1.1: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethyleacetamide (DMA) on the spermatozoa

motility (%) at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as mean ± SEM, n=5. Different

superscripts indicate statistically significant differences (P < 0.05). (Table 47; Appendices)

0

10

20

30

40

50

60

70

80

90

100

Post-dilution Post-cooling Post-equilibration Post-thawing

Stage of cryopreservation

Sper

m m

otil

ity

(%)

0% 4% DMA 6% DMA 8% DMA 10% DMA

cde

bca

ab ab

fg efg

ab

def ef

ij

gh

bcd

hihi

l

ij

gh

k jk

120

108

Figure 5.4.1.2: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethyleacetamide (DMA) on the sperm plasma

membrane integrity (%) at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as mean ± SEM,

n=5. Different superscripts indicate statistically significant differences (P < 0.05). (Table 48; Appendices)

0

10

20

30

40

50

60

70

80

90


Stage of cryopreseration

Pla

sma

mem

bran

e in

tigr

ity

(%)

0% 4% DMA 6% DMA 8% DMA 10% DMA

ef

bc

a

bcdab

gh

cdef

abcde

cdef

hi

def

bc

efg fg

kjk

efg

hi

ij

121

109

Figure 5.4.1.3: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethyleacetamide (DMA) on the sperm viabiltiy

(%) at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as mean ± SEM, n=5. Different


0

10

20

30

40

50

60

70

80

90



Sper

m V

iabi

lity

(%)

0% 4% DMA 6% DMA 8% DMA 10% DMA

cdef

abc a

cdef

ab

defgbcde

abc

defgdefg

g

efg

abcd

efg fg

i

h

fg

hih

122

110

Figure 5.4.1.4: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethyleacetamide (DMA) on the sperm acrosomal

integrity (%) at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as mean ± SEM, n=5. Different


0

10

20

30

40

50

60

70

80

90



Sper

m a

cros

ome

inte

grity

(%)

0% 4% DMA 6% DMA 8% DMA 10% DMA

abcdabc

a

abcd

ab

bcdef

bcde

a

abcde

bcd

fg

cdef

abcd

cdef def

h

gh

ef

gh gh

123

124

Table 5.1: Comparison of dimethyleacetamide (DMA; 6%) with Glycerol (20%;

control) for fertility and hatchability parameters of Indian red jungle fowl

spermatozoa (n=50)

Items Glycerol

Or

DMA

Day after insemination Mean ± SEM

1 2 3 4 5

Number of laid eggs Glycerol 50 45 47 48 45 45.0 ± 1.4a

DMA 45 47 48 40 45 47.0 ± 0.9a

Number of fertilized eggs Glycerol 30 32 34 36 28 25.4 ± 1.1a

DMA 27 22 24 28 26 32.0 ± 1.4b

Fertility (%) Glycerol 60.0 46.8 50.0 70.0 57.8 56.9 ± 4.1b

DMA 60.0 71.1 72.3 75.0 62.2 68.1 ± 3.0a

Number of hatched chicks Glycerol 22 18 20 20 20 20.0 ± 0.6b

DMA 28 30 28 30 25 28.2 ± 0.9a

Hatch (%) Glycerol 48.9 38.3 41.7 50.0 44.4 44.7 ± 2.2b

DMA 56.0 66.7 59.6 62.5 60.1 60.1 ± 2.1a

Hatchability of fertilized eggs

(%)

Glycerol 93.3 93.8 82.4 83.3 89.3 81.5 ± 2.2a

DMA 81.5 81.8 83.3 71.4 76.9 88.4 ± 2.4b

The values (mean ± SEM) having superscript differ significantly (P < 0.05) in a

row for a given parameter. (Table 51; Appendices)

125

reduced from 5 to 12 percent. However, at the 2 hours equilibration, the motility

was further reduced from 10 to 20 percent. The further reduction in sperm motility

occurred at the stage of post-thawing which was ranged 8 to 23 percent. The

interaction between different concentration of DMSO in an extender and stage of


that suggest all of the experimental extenders have similar ability to retain sperm

motility during different stages of cryopreservation.

Sperm plasma membrane integrity was recorded highest (two factor

ANOVA, Treatment; F4,60 = 12.93, P < 0.001) in diluent with 8% DMSO at post-

dilution (79.5 ± 3.8%), cooling (75.3 ± 2.4%), equilibration (72.8 ±3.3%) and

freeze-thawing (60.3 ± 2.8%) compared to diluent with 4%, 6% and 10% DMSO

and control.

At all the stages of cryopreservation, the addition of DMSO significantly

(Stage; F3,60 = 55.72, P < 0.001) reduced sperm plasma membrane integrity to 20-

21 percent. At cooling stage (20ºC), the sperm plasma membrane integrity was

reduced to 3-6 percent. However, at the end of 2 hours equilibration, the plasma

membrane integrity was further reduced to 9-17 percent. The lowest levels (P <

0.05) of plasma membrane integrity were observed after thawing. The interaction

between different concentration of DMSO and stages of cryopreservation was non-

significant (Treatment x Stage; F12,60 = 0.40, P > 0.05) that suggest all of the

experimental extenders have similar ability to protect PMI during different stages

of cryopreservation.

126

Sperm viability was recorded highest (two factor ANOVA, Treatment; F4,60

= 12.69, P < 0.001) in diluent with 8% DMSO at post-dilution (80.8 ± 4.6%),

cooling (75.5 ± 2.9%), equilibration (71.0 ± 7.6%) and freeze-thawing (58.8 ±

1.3%) compared to diluent with 4%, 6% and 10% DMSO and control. At all the

stages, the addition of DMSO significantly reduced (Stage; F3,60 = 55.63, P <

0.001) sperm viability to 20-27 percent. At cooling stage (20ºC), the sperm

viability reduced to 2-6 percent. However, at the end of 2 hours equilibration, the

sperm viability was further reduced to 8-15 percent. The lowest levels of sperm

viability were observed after post-thawing. The interaction between treatment and

stages was non-significant (Treatment x Stage; F12,60 = 0.22, P > 0.05) that suggest

all of the extenders having different concentrations of DMSO have similar potential

in terms of recovery of live and viable spermatozoa at different stages of

cryopreservation.

Sperm acrosomal integrity was recorded highest (two factor ANOVA,

Treatment; F4,60 = 6.42, P < 0.001) in diluent with 8% DMSO at post-dilution (76.3

± 2.4%), cooling (72.0 ± 6.0%), equilibration (62.5 ± 4.3%) and freeze-thawing

(55.0 ± 3.2%) compared to diluent with 4%, 6% and 10% DMSO and control. At

all the stages, the addition of DMSO significantly (Stage; F3,60 = 34.63, P < 0.001)

reduced sperm acrosomal integrity to 17-28 percent. At cooling stage (20ºC), the

sperm viability reduced to 3-8 percent. However, at the end of 2 hours equilibration

the sperm viability was further reduced to 10-12 percent.

114

Figure 5.4.2.1: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethylsulfoxide (DMSO) on the sperm motility (%)

at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as mean ± SEM, n=5. Different superscripts

indicate statistically significant differences (P < 0.05). (Table 52; Appendices)

0

10

20

30

40

50

60

70

80

90

100



Sper

m m

otil

ity

(%)

Control 4% DMSO 6% DMSO 8% DMSO 10% DMSO

ab a a ababc

cdef

bcde bcdabc

cdef

fgef f def

f

j ij

ghhiij

127

115

Figure 5.4.2.2: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethylsulfoxide (DMSO) on the sperm plasma

membrane integrity (%) at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as Mean ± SEM,


0

10

20

30

40

50

60

70

80

90



Plas

ma

mem

bran

e in

tigri

ty (%

)


bcdeabc abc

a

abc

defgbcdbcd

ab

bcde

fgh efgefg

abccdef

ij

hi hi

fgh

ij

128

116

Figure 5.4.2.3: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethylsulfoxide (DMSO) on the sperm viability

(%) at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as Mean ± SEM, n=5. Different


0

10

20

30

40

50

60

70

80

90



Sper

m V

iabi

lity

(%

)


bcdeabc

abca

bcde

def

bcdebcde

ab

cde

gh

fg efg

bcd

fg

i

hi

hi

fg

hi

129

117

Figure 5.4.2.4: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethylsulfoxide (DMSO) on the sperm acrosomal

integrity (%) at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as Mean ± SEM, n=5. Different


0

10

20

30

40

50

60

70

80

90



Sper

m a

cros

omal

inte

grit

y (%

)


abcdbcde

abcd

aabc

cdef cdefbcde

ab

cdef

fgh

cdefdef bcde

defg

i

ghi hi

efg

hi

130

131

Table 5.2: Comparison of dimethylsulfoxide (DMSO; 8%) with Glycerol (20%;


spermatozoa (n=50)

Items Glycerol Day after insemination Mean ± SEM

1 2 3 4 5


DMSO 98 99 93 98 93 96.2 ± 1.3a

Number of fertile eggs Glycerol 55 58 47 43 42 49.0 ± 3.2b

DMSO 75 80 71 74 52 70.4 ± 4.8a

Fertility (%) Glycerol 64.7 62.4 48.0 43.9 46.7 53.1 ± 4.3a

DMSO 76.5 80.8 67.3 75.5 55.9 73.0 ± 4.4b

Number of hatched

chicks

Glycerol 48 25 38 33 30 34.8 ± 3.9b

DMSO 68 73 65 68 45 63.8 ± 4.9a

Hatch (%) Glycerol 56.5 26.9 38.8 33.7 33.3 37.8 ± 5.0b

DMSO 69.4 73.7 69.9 69.4 48.4 66.2 ± 4.5a

Hatchability of fertile

eggs (%)

Glycerol 87.3 43.1 80.9 76.7 71.4 71.9 ± 7.6b

DMSO 90.7 91.3 91.5 91.9 86.5 90.4 ± 1.0a

The values (mean ± SEM) having superscript differ significantly (P < 0.05) across

the row for a given parameter. (Table 53; Appendices)

132

The lowest levels of sperm acrosomal integrity were observed post-thawing. The

interaction between the treatement and stage was non-significant (Treatment x

Stage; F12,60 = 0.44, P > 0.05) that suggest all of the experimental extenders have

similar potential to retain viable spermatozoa with intact acrosome at all stages of

cryopreservation.

The data on fertility attributes of IRJF spermatozoa cryopreserved with 8%

DMSO and 20% glycerol (control) are given in Table 5.2. The higher values for no.

of fertile eggs (70.4 ± 4.8, 49.0 ± 3.2%), percent fertility (73.0 ± 4.4, 53.1 ± 4.3),

no. of hatched chicks (63.8 ± 4.9, 34.8 ± 3.9), percent hatch (66.2 ± 4.5, 37.8 ± 5.0)

and percent hatchability (90.4 ± 1.0, 71.9 ± 7.6) were recorded after artificial

insemination with semen cryopreserved in 8% DMSO compared to control (20%

glycerol).

5.4.3. Effect of Different Concentrations of DMF and Stages of


Spermatozoa

The data on the effect of DMF on motility, plasma membrane integrity,

viability and acrosomal integrity of IRJF spermatozoa at post-dilution, cooling,

equilibration and freeze-thawing are given in Figure 5.4.3.1-4. Sperm motility was

recorded highest (two factor ANOVA, Treatment; F4,60 = 14.72, P < 0.001) in

diluent with 8% DMF at post-dilution (85.0 ± 2.9), cooling (80.0 ± 2.0),

equilibration (76.3 ± 1.3) and freeze-thawing (61.3 ± 1.3) compared to diluent with

133

4%, 6% and 10% DMF and control. The stages of cryopreservation pose significant

damage (Stage; F3,60 = 158.75, P < 0.001) to sperm motility. However, the

interaction between the addition of different concentrations of DMA in an extender

and stage of cryopreservation was also found significant (Treatment x Stage; F12,60

= 2.73, P < 0.01) that shows sperm motility was affected in a different manner in

all experimental extenders at all stages of cryopreservation.


ANOVA, Treatment; F4,60 = 27.17, P < 0.001) in diluent with 8% DMF compared

to all other extenders having 4%, 6% and 10% DMF and control. The PMI

significantly (Stage; F3,60 = 92.23, P < 0.001) differ at all stages of cryopreservation

viz; post-dilution (88.0 ± 3.0), cooling (79.3 ± 3.9), equilibrium (76.0 ± 1.5) and

post-thawing (67.0 ± 1.2). The interaction between experimental extenders and

stages of cryopreservation was found non-significant (Treatment x Stage; F12,60 =

1.56, P > 0.05) that suggest all of the extenders have similar ability to protect PMI

during all stages of cryopreservation. Sperm viability was recorded higher (two

factor ANOVA, Treatment; F4,60 = 11.08, P < 0.001) in diluent with 8% DMF

compared to other extenders with 4%, 6% and 10% DMF and control. However,

the stages of cryopreservation had significant (Stage; F3,60 = 36.71, P < 0.001)

affect on sperm viability. The maximum recovery of viable sperm was recorded at

the stage of post-dilution (76.3 ± 4.5), cooling (73.5 ± 2.2), equilibrium (71.0 ± 3.6)

and very less at post- thawing (60.5 ± 0.9) stage of cryopreservation.

120

Figure 5.4.3.1: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethylformamide (DMF) on the sperm motility (%)

at various stages of cryopreservation in Indian red jungle fowl. The results are expressed as Mean ± SEM, n=5. Different superscripts


0

10

20

30

40

50

60

70

80

90

100



Sper

m m

otili

ty (%

)

Control 4% DMF 6% DMF 8% DMF 10% DMF

aa a a ab

c

abab ab

ab

de

c c

b

c

g

f

efcd

f

134

121

Figure 5.4.3.2: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethylformamide (DMF) on the sperm plasma



0

10

20

30

40

50

60

70

80

90

100



Plas

ma

mem

bran

e in

tigri

ty (%

)


cde

abbc

aab

efg

cde

fgh

bc

ghihij

def

hi

bcd

hij

jkij

jk

efg

k

135

122

Figure 5.4.3.3: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethylformamide (DMF) on the sperm viability



0

10

20

30

40

50

60

70

80

90



Sper

m V

iabi

lity

(%)


abc abab

aab

bcd abcd abcab

deef

bcdbcd

ab

ef

g

ef

efg

cde

fg

136

123

Figure 5.4.3.4: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of dimethylformamide (DMF) on the sperm acrosomal



0

10

20

30

40

50

60

70

80

90



Sper

m a

cros

mal

inte

grity

(%)


aabc ab ab a

bcdcde

bcd abcd

efggh

fgdef

bcd

fgh i highi

efg

i

137

138

Table 5.3: Effect of DMF (dimethylformamide; 8%) vs Control (Glycerol 20%) on

the fertility of spermatozoa and hatchability in Indian red jungle fowl (n=50)

Items Cryoprotectant Day after insemination Mean ± SEM

1 2 3 4 5


DMF 98 99 93 98 93 96.2 ± 1.3a


DMF 67 75 70 72 57 68.2 ± 3.1a


DMF 68.4 75.8 75.3 73.5 61.3 70.8 ± 2.7a


DMF 59 70 65 66 52 62.4 ± 3.1a

Hatch (%) Glycerol 52.9 48.4 40.8 36.7 38.9 43.5 ± 3.1b

DMF 60.2 70.7 69.8 67.3 55.9 64.8 ± 2.9a

Hatchability of fertile eggs

(%)

Glycerol 83.3 83.3 85.1 83.3 83.8 83.8 ± 0.3b

DMF 88.0 93.3 92.9 91.7 91.4 91.4 ± 0.9a

The values (mean ± SEM) having different superscript differ significantly (P <

0.05) across the row for a given parameter. (Table 61; Appendices)

139

The interaction between different concentrations of DMF and stages of


that is suggestive of equal recovery of viable sperm in all experimental extenders at

all stages of cryopreservation.

Sperm acrosomal integrity was recorded higher (two factor ANOVA,

Treatment; F4,60 = 6.32, P < 0.001) in diluent with 8% DMF compared to diluents

having 4%, 6%, 10% DMF and control. The recovery of viable sperm with intact

acrosome was found higher (Stage; F3,60 = 69.88, P < 0.001) at post-dilution (73.0

± 4.3) compared to cooling (68.8 ± 2.4), equilibrium (65.8 ± 2.7) and post-thawing

(56.3 ± 1.6).

The interaction between treatment and stage of cryopreservation was found

non-significant (Treatment x Stage; F12,60 = 1.65, P > 0.05) that suggest all of the

experimental extenders have equal efficiency in terms of recovery of viable

spermatozoa during different stages of cryopreservation.

The data on fertility attributes of IRJF spermatozoa cryopreserved with 8%

DMF and 20% glycerol (control) are given in Table 5.3. The higher values for no.

of fertile eggs (68.2 ± 3.1, 48.0 ± 2.6), fertility (70.8 ± 2.7, 52.0 ± 3.7), no. of

hatched chicks (62.4 ± 3.1, 40.2 ± 2.1), percent hatch (64.8 ± 2.9, 43.5 ± 3.1) and

hatchability (91.4 ± 0.9, 83.8 ± 0.3) were recorded after artificial insemination with

semen cryopreserved in 8% DMSO compared to control.

140

5.4.4. Effect of Different Concentrations of PVP and Stages of

Cryopreservation on Quality of Indian Red Jungle Fowl Spermatozoa

The data on the effect of PVP on motility, plasma membrane integrity,

viability and acrosomal integrity of IRJF spermatozoa at different stages of

cryopreservation are given in Figure 5.4.4.1-4. Sperm motility was recorded

highest (two factor ANOVA, Treatment; F4,60 = 16.81, P < 0.001) in diluent with

6% PVP compared to diluent with 4%, 6% and 10% PVP and control. However,

maximum (Stage; F3,60 = 158.03, P < 0.001) sperm motility was recorded at post-

dilution stage of cryopreservation (87.5 ± 2.5) compared to cooling (85.0 ± 2.9),

equilibration (81.3 ± 2.4) and freeze-thawing (67.5 ± 1.4). The interaction between

treatment and stages of cryopreservation was significant (Treatment x Stage; F12,60

= 2.30, P < 0.05) that indicates the potential of all experimental extenders in terms

of their ability to retain higher percentage of motile sperm during different stages



ANOVA, Treatment; F4,60 = 16.98, P < 0.001) in diluent with 6% PVP compared to

diluent with 4%, 6% and 10% PVP and control. Maximum (Stage; F3,60 = 65.29, P

< 0.001) recovery of spermatozoa with intact plasma membrane was recorded at

post-dilution (80.5 ± 4.6%), cooling (77.5 ± 3.2%), equilibration (75.3 ± 1.0%) and

very less at freeze-thawing (66.5 ± 2.2%) stage of cryopreservation. The interaction

between treatment and stages of cryopreservation was found non-significant

126

Figure 5.4.4.1: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of polyvinylpyrrolidone (PVP) on the sperm motility



0

10

20

30

40

50

60

70

80

90

100

Post-dilution Post-cooling Post-Equilibration Post-thawing

Stage of Cryopreservation

Sper

m m

otili

ty (%

)

Control 4% PVP 6% PVP 8% PVP 10% PVP

ab a aab

bcd

ef

cde

ababc

cde

fef

abcd

efde

h

g

f

ghgh

141

127

Figure 5.4.4.2: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of polyvinylpyrrolidone (PVP) on the sperm plasma



0

10

20

30

40

50

60

70

80

90

100



Plas

ma

mem

bran

e in

tigri

ty (%

)


bcdefg

abcdab a

cdefgh

ghi

abcdeabc

bcdef

defghi

ij

efghi

abcd

fghihij

l

kl

efghi

jk k

142

128

Figure 5.4.4.3: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of polyvinylpyrrolidone (PVP) on the sperm viability



0

10

20

30

40

50

60

70

80

90

100



Sper

m V

iaib

ility

(%)


bcd abcaba ab

fgdef

bcd

efg efg

hi

efg cde

ghefg

j

ij ghij

j

143

129

Figure 5.4.4.4: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of polyvinylpyrrolidone (PVP) on the sperm acrosomal



0

10

20

30

40

50

60

70

80

90



Sper

m a

cros

ome

inte

grity

(%)


bcab

aa

ab

defg

bc

a

de def

efg

cd

ab

def def

i

fg

cd gh

h

144

145

Table 5.4: Comparison of polyvinylpyrrolidone (PVP; 8%) with Glycerol (20%;


spermatozoa


1 2 3 4 5


PVP 45 50 48 45 49 47.4 ± 1.0a


PVP 34 35 36 38 34 35.4 ± 0.7a


PVP 75.6 70.0 75.0 84.4 69.4 74.9 ± 2.7a


PVP 30 32 33 38 34 32.0 ± 0.9a

Hatch (%) Glycerol 54.0 57.8 52.1 44.9 44.4 50.6 ± 2.6b

PVP 66.7 64.0 68.8 77.8 61.2 67.7 ± 2.9a


(%)

Glycerol 87.1 86.7 92.6 78.6 90.9 87.2 ± 2.4a

PVP 88.2 91.4 91.7 92.1 88.2 90.3 ± 0.9a


0.05) across the row for a given parameter. (Table 66; Appendices)

146

(Treatment x Stage; F12,60 = 1.59, P > 0.05) that showed recovery of spermatozoa

with intact plasma membrane is independent of experimental extenders and stages


Sperm viability was recorded highest (two factor ANOVA, Treatment; F4,60

= 12.01, P < 0.001) in diluent with 6% PVP compared to diluent with 4%, 6% and

10% PVP and control. The stage of cryopreservation had significant (Stage; F3,60 =

113.16, P < 0.001) affect on the recovery of live spermatozoa. Maximum live

spermatozoa were recovered at post-dilution (81.5 ± 1.7%) compared to cooling

(77.5 ± 2.5%), equilibration (73.5 ± 2.5%) and freeze-thawing (63.8 ± 1.3%). The

interaction between addition of different concentrations of PVP in an extender and

stages of cryopreservation was non-significant (Treatment x Stage; F12,60 = .164, P

> 0.05) that propose all of the experimental extenders have similar ability in terms

of recovery of live sperm during different stages of cryopreservation.

Sperm acrosomal integrity was recorded highest (two factor ANOVA,

Treatment; F4,60 = 31.21, P < 0.001) in diluent with 6% PVP compared to diluent

with 4%, 6% and 10% PVP and control. The stages of cryopreservation differ

significantly (Stage; F3,60 = 79.06, P < 0.001) from each other. The recovery of

viable spermatozoa with intact acrosome was found maximum (P > 0.05) at post-

dilution (81.5 ± 1.2%) compared to cooling (79.5 ± 2.6%), equilibration (75.8 ±

2.2%) and freeze-thawing (66.3 ± 1.3%) stage of cryopreservation. The interaction

between treatment and stages of cryopreservation was incurred non-significant

(Treatment x Stage; F12,60 = 1.47, P > 0.05) that suggest all of the experimental

147

extenders have similar ability to protect integrity of acrosome during stages of

cryopreservation.

The data on fertility attributes of IRJF spermatozoa frozen with 6% PVP

and 20% glycerol (control) is given in Table 5.4. The higher values for no. of

fertile eggs (35.4 ± 24.6 ± 2.2), fertility (74.9 ± 2.7, 58.2 ± 3.0), no. of hatched

chicks (32.0 ± 0.9, 24.0 ± 1.3), percent hatch (67.7 ± 2.9, 50.6 ± 2.6) and

hatchability (90.3 ± 0.9, 87.2 ± 2.4) were recorded after artificial insemination with

semen cryopreserved in 6% PVP compared to control.

5.4.5. Effect of Different Concentrations of Egg Yolk and Stages of

Cryopreservation on Quality of Indian Red Jungle Fowl Spermatozoa

The data on the effect of different concentrations of egg yolk as

cryoprotectant in a freezing extender on sperm motility, plasma membrane

integrity, viability and acrosomal integrity at different stages of cryopreservation

viz; post-dilution, cooling, equilibration and post-thawing are given in Figures

5.4.5.1-4. Sperm motility was recorded higher (two factor ANOVA, Treatment;

F4,60 = 37.93, P < 0.001) in an extender containing 15% egg yolk compared to

extenders having 10%, 20% and 25% egg yolk and control. Maximum (Stage; F3,60

= 136.60, P < 0.001) sperm motility was recorded at post dilution (90.0 ± 0.0) stage

of cryopreservation compared to cooling (86.3 ± 2.4), equilibration (81.3 ± 1.3) and

post-thawing (67.5 ± 2.5).

132

Figure 5.4.5.1: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of egg yolk on the sperm motility (%) at various stages

of cryopreservation in Indian red jungle fowl. The results are expressed as Mean ± SEM, n=5. Different superscripts indicate

statistically significant differences (P < 0.05). (Table 67; Appendices)

0

10

20

30

40

50

60

70

80

90

100



Sper

m m

otili

ty (%

)

Control 10% Egg yolk 15% Egg yolk 20% Egg yolk 25% Egg yolk

cdebc

aab ab

fg efg

ab

def ef

ij

fg

bcd

hi hi

l

hijgh

k jk

148

133

Figure 5.4.5.2: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of egg yolk on the sperm plasma membrane integrity



0

10

20

30

40

50

60

70

80

90


Stage of cryopreseration

Pla

sma

mem

bran

e in

tigr

ity

(%)


efg

abca

bcdab

ghij

cdef

ab

def

hijkjkl

fgh

bcde

fghi

ijkl

m lm

fgh

ijklkl

149

134

Figure 5.4.5.3: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of egg yolk on the sperm viability (%) at various stages

of cryopreservation in Indian red jungle fowl. The results are expressed as Mean ± SEM, n=5. Different superscripts indicate

statistically significant differences (P < 0.05). (Table 69; Appendices)

0

10

20

30

40

50

60

70

80

90



Sper

m V

iabi

lity

(%

)


bc

ab a

bc cde

fgh

cdbc

def

fgh ghiefg

cd

fgh

k jk

fghjk

k

150

135

Figure 5.4.5.4: Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of egg yolk on the sperm acrosomal integrity (%) at

various stages of cryopreservation in Indian red jungle fowl. The results are expressed as Mean ± SEM, n=5. Different superscripts


0

10

20

30

40

50

60

70

80

90



Sper

m a

cros

ome

inte

grity

(%)


bcdabc

a

abc bcdcde

cdeab

bcdcde

efgcde

abc

cdefdef

hi

fgh

def

ghii

151

152

Table 5.5: Comparison between the effects of Egg yolk (15%) and Glycerol (20%;

control) on fertility and hatchability parameters of Indian red jungle fowl

spermatozoa (n=50)


1 2 3 4 5

Number of laid eggs Glycerol 50 45 48 45 45 46.6 ± 1.0

Egg-yolk 45 50 45 49 50 47.8 ± 1.2


Egg-yolk 30 30 25 28 20 26.6 ± 0.7a

Fertility (%) Glycerol 40.0 66.7 50.0 48.9 48.9 50.9 ± 4.3a

Egg-yolk 66.7 60.0 55.6 57.1 40.0 55.9 ± 4.4a


Egg-yolk 25 27 22 24 20 23.6 ± 1.0a

Hatch (%) Glycerol 30.0 44.4 37.5 33.3 40.0 37.1 ± 2.5b

Egg-yolk 55.6 54.0 48.9 49.0 40.0 49.5 ± 3.2a


(%)

Glycerol 75.0 83.3 81.8 68.2 90.0 79.7 ± 3.7b

Egg-yolk 50 45 48 45 45 46.6 ± 1.0


0.05) across the row for a given parameter (Table 71; Appendices)

153

The interaction between extenders having different egg yolk concentration and

stages of cryopreservation was recorded significant (Treatment x Stage; F12,60 =

3.42, P < 0.001) that suggests, experimental extenders have different ability to

protect sperm motility during different stages of cryopreservation.

Sperm plasma membrane integrity was recorded higher (two factor

ANOVA, Treatment; F4,60 = 32.48, P < 0.001) in an extender containing 15% egg

yolk compared to extenders having 10%, 20% and 25% egg yolk and control. The

recovery of spermatozoa with intact plasma membrane was recorded higher (Stage;

F3,60 = 56.99, P < 0.001) at post dilution (83.5 ± 1.5) compared to cooling (78.8 ±

3.1), equilibration (73.3 ± 2.0) and post-thawing (66.3 ± 2.4) stage of

cryopreservation. However, the interaction between these two treatments was

found significant (Treatment x Stage; F12,60 = 2.27, P < 0.05) that indicates, all of

the experimental extenders have different ability to protect PMI during different

stages of cryopreservation.

Sperm viability was recorded higher (two factor ANOVA, Treatment; F4,60

= 19.17, P < 0.001) in an extender containing 15% egg yolk compared to extenders

having 10%, 20% and 25% egg yolk and control. Viability of spermatozoa was

recorded higher (Stage; F3,60 = 77.69, P < 0.001) at post dilution (79.3 ± 1.4)

compared to cooling (71.3 ± 1.3), equilibration (67.5 ± 1.4) and post-thawing (58.8

± 1.3) stage of cryopreservation. The interaction between these two treatments was

recorded non-significant (Treatment x Stage; F12,60 = 1.11, P > 0.05) that suggest

154

all of the diluents having different concentrations of egg yolk have similar ability in

terms of recovery of live sperm during different stages of cryopreservation.

Sperm acrosomal integrity was recorded higher (two factor ANOVA,

Treatment; F4,60 = 10.03, P < 0.001) in an extender containing 15% egg yolk

compared to extenders having 10%, 20% and 25% egg yolk and control. Maximum

(Stage; F3,60 = 19.32, P < 0.001) number of viable spermatozoa with intact

acrosome were recovered at post dilution (79.0 ± 2.0) compared to cooling (76.5 ±

2.5), equilibration (71.3 ± 1.3) and post-thawing (60.8 ± 2.0) stage of

cryopreservation. The interaction of these above two treatments was recorded non-

significant (Treatment x Stage; F12,60 = 0.39, P > 0.05) that suggest all of the

extenders have similar ability to protect acrosome integrity of spermatozoa during

the different stages of cryopreservation. The data on fertility attributes of IRJF

spermatozoa frozen with 15% egg yolk and 20% glycerol (control) is given in

Table 5.5. The higher values for no. of fertile eggs (22.4 ± 1.2, 21.6 ± 0.7), fertility

(50.9 ± 4.3, 47.6 ± 3.5), no. of hatched chicks (18.0 ± 1.0, 17.2 ± 1.0), percent

hatch (37.8 ± 3.2, 37.1 ± 2.5) and hatchability (80.0 ± 2.2, 79.7 ± 3.7) were

recorded after artificial insemination with semen cryopreserved in 15% egg yolk

compared to control.

5.5. DISCUSSION

Gamete cryopreservation technique has been developed for a wide range of

species over the last sixty five years (Polge, 1951). The success of freeze-thawing

155

of spermatozoa depends on the type of cryoprotectant, packaging method, freezing

and thawing rates for avian species (Donoghue and Wishart, 2000; Blesbois et al.,

2008) that are responsible for the alteration of structure and function of sperm

(Bailey et al., 2003). Cryoprotectants help prevent enzyme denaturation, protein

destabilization, and ice crystal formation during freeze thaw process (Chao, 1991).

The most commonly used cryoprotectants for avian sperm are glycerol (Seigneurin

and Blesbois, 1995), DMA (Blesbois and Grasseau, 2001), DMSO (Hammerstedt

and Graham, 1992), DMF (Han et al., 2005), PVP (Tiersch et al., 1994) and egg

yolk (Bajpai and Brown, 1963). The present study was conducted to compare the

efficacy of glycerol and different levels of DMA, DMF, DMSO, PVP and egg yolk

for their cryoprotective ability at different stages of cryopreservation viz; dilution,

cooling, equilibration, freeze-thawing and fertility following artificial insemination

using optimized concentration of DMA (6%), DMF (8%), DMSO (8%), PVP (6%),

egg yolk (15%) and previously established glycerol (20%).

Both glycerol and DMA are penetrating cryoprotectants that provide intra

and extracellular protection, dehydrating the sperm by an osmotically driven flow

of water (Purdy, 2006). Penetrating cryoprotectants improve membrane fluidity and

thus increase ability to survive post-thawing (Holt, 2000). However, the permeable

cryoprotectants have toxic effect on sperm, that is directly related to cryoprotectant

concentration used and time to cell exposure (Swain and Smith, 2010; Iaffaldano et

al., 2014). In the present study, the optimum evolved concentration of DMA was

6%. The previous work on DMA usage at 6% concentration in Houbara bustard

(Hartley et al., 1999) and domestic bird (Blesbois et al., 2005) confirm that lower

156

concentration of penetrating cryoprotectant is more favorable to sperm during

cryopreservation process (Abouelezz et al., 2015; Tselutin et al., 1999; Han et al.,

2005). It is assumed that cryoprotectant in hand is able to increase osmolarity,

suitably dehydrates cells and thus avoids ice crystal formation during

cryopreservation process (Blanch et al.., 2012). Cryopreservation stages (post-

dilution, post-cooling, post-equilibration and post-thawing) do affect sperm

survival and are highly dependable on the diluent and cryoprotectant used. It has

been observed that fertility rates [66.8% (6% DMA) vs. 56.4% (control)] were

significantly higher in the diluent in hand compared to previous studies on chicken

(Blesbois et al., 2008) duck (Han et al., 2005) and turkey (Iaffaldano et al., 2016)

which used the same cryoprotectant, but having different diluents. Sexton (1977)

suggested that the diluent having glutamic acid as a major constituent would better

maintain sperm quality and fertility, which is in favor of the present results.

No study is available on the use of DMA as a cryoprotectant for freezing

IRJF spermatozoa. The published literature on other avian species suggest that

semen processing and in vitro sperm quality were always variable (Bakst and

Sxeton, 1979); Blesbois et al., 2008). Use of DMA (6%) as cryoprotectant in

relative permeability presumably explains that cryopreservation efficiency is

dependent on the diluent used and would induce less damage at the tested stages of

cryopreservation compared to previously (chapter 5) used glycerol (20%) protocol

for IRJF spermatozoa. In the present study the damage from post-dilution to post-

equilibration ranged 4-15%, but post-thawing exerted damage ranging from 22-

37% in the present study. The damage reported during cryopreservation was almost

157

40-60% in the previous studies on chicken (Santiago-moreno et al., 2012;

Iaffaldano et al., 2016), duck (Han et al., 2005) and turkey (Long et al., 2014). The

possible reason of damage at post-thawing is that re-crystallization of ice occurs

which is imposed by the disruption of proteins that causes more cell damage (Cao

et al., 2003; Mazur, 2004).

The present study has demonstrated the usefulness of DMSO in freezing

protocol for IRJF sperm in comparison with protocol based on glycerol. Results

showed 8% DMSO in extender resulted in highest motility, plasma membrane

integrity, viability and acrosome integrity at post-dilution, cooling, equilibration

and freeze-thawing compared to 4%, 6%, 10% DMSO and control (with glycerol).

It is suggested that DMSO being permeable to sperm membrane offers extracellular

and intracellular protection to spermatozoa by causing efflux of water molecules

and maintaining cell volume through osmosis (Purdey, 2006).

The results of the present study are in agreement to the study on chicken

semen in which, addition of 10% DMSO significantly decreased the post-thaw

semen quality compared to 8% DMSO. The addition of 8% DMSO in extender

may possibly have been able to increase the process of osmosis, dehydrate the

sperm cells and avoid ice crystallization during the process of cryopreservation.

The other reason might be that optimized concentration of DMSO (8%) enabled to

maintain physico-chemical structures of lipid bi-layer and provided greater

permeability as compared to lower concentrations and glycerol (control). DMSO

has been used as a cryoprotectant in many avian species such as Sandhill crane,

158

domestic fowl and poultry and revealed that 4% DMSO is the most effective

cryoprotectant for conserving post-thaw sperm motility (Sexton and Gee, 1978;

Penfold et al., 2000). Another study on chicken blastoderm cells revealed that

addition of 10% DMSO significantly improved the survival rate (Sawicka et al.,

2015). The study on turkey semen revealed that 8% and 10% DMSO depress the

post thaw quality and fertility (Graham, 1980).

Lierz et al. (2013) investigated the role of DMSO in turkey semen and

found that 10% DMSO has significantly resulted in higher post thaw motility and

viability compared to 4% DMSO. However, as for as the American kestrel is

concerned, there was no significant difference between 4%, 6%, 8% and 10%

DMSO in terms of sperm motility at all stages of cryopreservation and fertility

(Gee et al., 1993). A study on fowl spermatozoa showed toxic effect of DMSO

even at lower concentration and did not significantly differ at DMSO 4%, 6%, 8%

and 11% (Tselutin et al., 1999).

It is evident from literature that variable concentrations of cryoprotectant

affect the tolerance of sperm in each species breed/strain (Blanco et al., 2011,

2012). The results of Venda chicken are in agreement to the present study and

revealed that addition of 8% DMSO in an extender improves the post thaw semen

quality (Mphaphati, et al., 2012). However, DMSO has greater advantage in terms

of fertility obtained after artificial insemination. It is always safe when inseminated

compared to glycerol (contraceptive effect) and provide greater viability in semen

storage tubules (SSTs). Live and viable sperm diluted with DMSO were recovered

159

after 6 days following insemination compared to glycerol diluted semen having

viability of only 3 days following insemination (Sexton and Gee, 1978; Lake and

stewart, 1978; Lake and Ravie, 1984). These results are in agreement to the present

study and showed that DMSO (8%) yielded better fertility compared to glycerol.

The permeable cryoprotectants stabilize the membrane potential of sperm

through rearrangement of lipids and proteins, thus making the membrane more

fluid, providing greater dehydration at lower temperatures and increasing viability

of spermatozoa after freeze-thawing (Holt, 2000, 2001; Papahadjopoulos et al.,

1976). Permeable cryoprotectants at higher concentrations may affect sperm

possibly due to membrane destabilization, protein and enzyme denaturation (Swain

and Smith, 2010; Iaffaldano et al., 2014, 2015). The present study has

demonstrated the usefulness of DMF in freezing protocol for IRJF sperm and

comparison with protocol based on glycerol. Results showed 8% DMF in extender

resulted in highest motility, plasma membrane integrity, viability and acrosome

integrity at post-dilution, cooling, equilibration and freeze-thawing compared to

4%, 6%, 10% DMF and control (with glycerol) and fertility after artificial

insemination. The usage of DMF as cryoprotectant for chicken spermatozoa has

resulted in the improved semen quality and fertility (Tselutin et al., 1999; Woelders

et al., 2006; Hanzawa et al., 2006; Schramm, 2008; Sasaki et al., 2010) which are

in agreement to the present study. In another study on duck semen, DMF at 8% in

extender improved the post-thaw semen quality and fertility (Han et al., 2005)

compared to 4%, 6%, and 10% DMF. The results of the previous experiment on

gander are in contradiction to the present study and showed that addition of DMF

160

to the freezing extender reduced the post thaw semen quality and fertility

(Lukaszewicz, 2001). In another study on geese showed 20% decline in fertility

after artificial insemination using DMF as cryoprotectant in extender compared to

glycerol with a different extender. The decline in fertility was due to the diluent

used and diluent-cryoprotectant posed great effect on post-thaw sperm quality and

fertility in geese semen (Lukaszewicz and Fujihara, 1999; Lukaszewicz, 2001).

In another study on stallion sperm, DMF addition in a freezing diluent

yielded higher post-thaw semen quality and fertility compared to glycerol, which

are in agreement to the present study. The superior post-thaw results with DMF

might be due to reduction in osmotic stress that the sperm observed during different

phases of cryopreservation (Gibb et al., 2013). The DMF is a low molecular weight

compound compared to glycerol and passes more frequently across the sperm

plasma membrane and reduces the time of exposure of sperm to the hypertonic

environment (Ball, 2008). Osmotic stress during the different phases of

cryopreservation (post-dilution, cooling, equilibration and freeze-thawing) not only

damages the sperm plasma membrane but also results in the production of reactive

oxygen species (Carvalho et al., 2010; Bochenek et al., 2005). The use of DMF to

reduce DNA damage during cryopreservation process is associated with osmotic

stress and seems an appealing strategy to improve the post-thaw semen quality and

fertility of Indian red jungle spermatozoa.

The water soluble polymers are capable of preventing ice-crystal formation,

and the concentration at which maximum protection is attained for various

161

biological cells generally have lesser toxicity compared to similar concentration of

glycerol (Takahashi et al., 1988; Echlin et al., 1977). These properties of the

polymers make them more efficient cryoprotectant compared to commonly use

penetrating low molecular weight compounds.

PVP was found capable to maintain acceptable post-thaw quality of rooster,

ring-necked pheasant and red-tailed hawks semen (Herrera et al., 2005). The

plasma membrane integrity of bull was found significantly higher in an extender

having 5% PVP replacing glycerol while decreased significantly at higher

concentration of PVP (10%) (Nasr-Esfahani et al., 2008) found to be in agreement

to results of present study. Similar results were obtained on the effect of PVP as a

cryoprotective agent for channel cat fish sperm and only lower concentration of

PVP (5%) was found better to maintain post thaw sperm quality compared to

higher concentrations of PVP (Tiersch et al., 1994).

The study on human sperm showed better motility and condensation

compared to its absence when sperm was preserved in PVP based cryoprotectant

(Irez et al., 2013; Kato and Nagao, 2012). A study on cattle reported significantly

enhanced sperm capacitation status in extender containing 10% PVP while the

fertility rate was decreased (Kato and Nagao, 2009). The remarkable fertility of

39% in rooster and 57% in ring-necked pheasant was recorded with semen frozen

in PVP (Herrera et al., 2005). Findings of the preset study are inline with the earlier

reports that demonstrated PVP equally capable to penetrating cryoprotectant in

extender.

162

It is considered that efficiency of cryoprotectants for animal cells largely

depends on its hydrogen binding capacity (Williams, 1983). The high capacity of

PVP hydrogen binding (Turker et al., 1990) may enhance its potential to protect the

integrity of sperm cell during cryopreservation process. It is believed that PVP

cannot cross the cell membrane; protect the sperm from cryopreservation induced

damages by extracellular actions rather interacting with plasma membrane, very

different from permeable cryoprotectants (Takahashi et al., 1988). The exact

mechanism of PVP through which it protects the biological cells during

cryopreservation is not known. Nevertheless, the higher viability, plasma

membrane integrity and acrosome integrity of IRJF spermatozoa in present study

with 8% PVP can be explained by following assumptions.

The PVP in water has the ability to lower surface energy of the solution

below the surface energy of the biological cells that might result in the formation of

a stable interface at the cellular surface. The interface forming at the cellular

membrane seem hides the membrane defects/disruption and cause hindrance in the

leakages from the cytoplasm (Williams, 1983). The PVP has a great capacity to

form films around the surface of plasma membrane (Nash, 1966; Freshney, 2000).

The use of 8% PVP in extender compared to glycerol might have resulted in low

surface energy of the solution and formation of stable interface at the surface of

IRJF spermatozoa that resulted in higher post-thaw quality in terms of motility,

viability, plasma membrane and acrosome integrity in present study. Egg yolk has

been routinely used as cryoprotectant for buffalo sperm (Akhter et al., 2008), but

for avian species, the data is very limited to few studies on chicken (Wilcox, 1960;

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Bajpai and Brown, 1963). The avian sperm have low cholesterol: phospholipid

ratio and freeze-thawing cause more damage during freeze-thawing. Egg yolk in an

extender makes protective layer outside the sperm membrane and reduce cryo-

damage (White, 1993). The results of the present study are in agreement to the

study on turkey semen that indicated lower post-thaw motility in diluent having

10% egg yolk as cryoprotectant compared to other concentrations of the egg yolk

(Bajpai and Brown, 1963).

They have also shown that all levels of egg yolk were significantly different

to each other at all stages of cryopreservation and resulted in less number of

abnormal spermatozoa production. It was observed that too low (10%) and high

(25%) levels of egg-yolk in extender would cause physical as well as chemical

injuries to the spermatozoa. The excessive amount of egg-yolk in an extender may

slow down the enzyme activity of spermatozoa (Bishop, 1962) while the lower

amount of egg-yolk might not be able to provide sufficient ATPs during the energy

cascades in cryopreservation process (Schindler et al., 1955).

In another study on storage of chicken semen having egg yolk as a

cryoprotectant, higher post-thaw sperm quality and fertility after artificial

insemination was reported (Wilcox, 1960). The fertility results obtained in the

present study showed that glycerol and egg yolk are not significantly different to

each other and have produced the similar results. The results are in agreement to

the study on chicken semen which showed that lowered fertility with egg yolk may

be due to the diluent used (Wilcox, 1960) as the diluent used in the study had low

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buffered capacity. On the average, it has been observed that egg yolk has the

ability to protect post-thaw semen (40-50%) quality (motility) compared to control.

However fertility obtained with the egg yolk is similar to that obtained with

glycerol.


A variety of cryoprotectants viz; Permeable (Glycerol, DMA, DMSO and

DMF) and impermeable (egg yolk and PVP) were tested for their cryoprotectant

ability for IRJF sperm in this chapter. Different levels of cryoprotectants for DMA,

DMSO, DMF and PVP (4%, 6%, 8% and 10% and control 20%). For egg yolk,

10%, 15%, 20% and 25% levels were tested against control 20% glycerol. Among

all the cryoprotectants evaluated in present study, 6% DMA, 8% DMSO, 8% DMF

and 6% PVP maintained higher (P < 0.05) semen quality and fertility compared to

control. Among levels of egg yolk (10, 15, 20 and 25%) evaluated; 15% egg yolk

was found superior (P < 0.05) for semen quality and fertility compared to control.

The higher number of fertilized eggs, fertility %, number of hatched chicks, hatch

% and the hatcability of fertilized eggs were obtained with all of the

cryoprotectants tested compared to control. All of these cryoprotectants are

effective and better than the commonly used cryoprotectants in poultry i.e.

glycerol. One of extender can be used for the cryopreservation of IRJF

spermatozoa according to the availability of the resources with improved recovery

rates compared to glycerol in terms of post-thaw quality (motility, plasma

membrane integrity, viability, acrosome integrity and in vivo fertility rates It is

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concluded that IRJF semen can be cryopreserved with the protocol based on having

DMA (6%), DMSO (8%), DMF (8%), PVP (6%) and egg yolk (15%) in an

extender.

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Chapter 6

GENERAL DISCUSSION

Successful application of reproductive biotechnologies requires prior

knowledge of semen collection, ejaculation frequency, semen characteristics,

identification of a suitable semen extender, a suitable cryo-protectant and the

development of a freezing regime for semen cryopreservation and artificial

insemination (Sontakke et al., 2004; Malik et al., 2013). However little research

has been done on the semen collection and artificial insemination in the pheasant

species closely related to genus Gallus, despite the large number of individuals viz;

cheer pheasant (Jalme et al., 2003); Malaysian red jungle fowl (Malik et al., 2013)

in this genus are threatened in the wild. The present study was conducted to

investigate semen characteristics, frequency of collection, timing of collection,

suitable extender for liquid/cryopreserved semen, permeable/non-permeable cryo-

protectants and its use in artificial insemination for the conservation and

propagation of red jungle fowl.

The sperm concentration, volume and total number of sperm per ejaculate

in IRJF were observed generally lower compared to domestic chicken, Malaysian

red jungle fowl and broiler breed (Malik et al. 2013; broiler breeder (McDanieal,

1995). Moreover, proportion of motile sperm was relatively low compared to

commercial parents. However, sperm viability, acrosome integrity and plasma

membrane integrity was recorded equivalent to those reported in other studies on

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different breeds of chicken (Graham et al., 1990; Froman et al., 1999; Parker et al.,

2000; Snook, 2005; Partyka et al., 2012).

It is interesting that motility, plasma membrane integrity, viability and

acrosomal integrity of IRJF was recorded similar (P > 0.05) in all birds of study. I

found no other reason but to attribute the resemblence to similar age, uniform food

and managemental conditions. The correlation analysis showed positive association

between motility, acrosome integrity and plasma membrane integrity. Similar

trends have been described in Rhode Island red, white breeder cocks (Nwagu et al.,

1996), seven strains of chicken (Peters et al., 2008) and Spanish breeds of chicken

(Prieto et al., 2011).

Sperm motility is an important criterion for evaluation of semen quality and

fertility of the spermatozoa. The records of the sperm motility showed higher

motility at 24, 48 and 72 hours of collection compared to twice daily collections.

The studies are in agreement with the previous study on a Taiwan country chicken

breed (Fan et al., 2004) and Domyati ducks (Egyptian local breed; Ghonim et al.,

2009). While, in Hubbard broiler breeder (Riaz et al., 2004), maximum sperm

motility was observed at twice daily frequency of collection that decreased as the

interval between the collections increased.

It is known that the collection frequency at which semen of optimum quality

can be achieved is highly specific to a species or breeds/strain. The results of the

studies showed uniqueness of the IRJF and specific collection frequency. It is

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revealed that semen ejaculate of optimum quality can be achieved with high

collection frequency in breeds/strains of poultry species especially developed for

intensive farming compared to local breeds of chicken and ducks. IRJF being the

ancestor of the domestic chicken showed optimum quality of the semen ejaculates

with comparatively long collection interval.

The optimum values of semen volume were observed with semen collection

frequency at 48 and 72 hours of intervals. The results showed that ejaculate volume

(146.9 µL) of IRJF is mostly low compared to Nigerian indigenous chicken

(Naked-neck, Frizzle and Normal feathered) that ranged from 0.25 to 0.5mL

(Peters, et al., 2008) and turkey 0.25 to 0.35 mL (Zahraddeen et al., 2005).

Species/breed differences are reported on the ejaculate volume and frequency of

ejaculation (Zhang et al., 2009). Highest ejaculate volume was reported in Domyati

ducks (Egyptian local breed) at once and twice a week collections (Ghonim et al.,

2009) and Hubbard broiler breeder at once a daily collection (Riaz et al., 2004).

A gradual decline in sperm concentration was observed with increasing

collection frequency in IRJF. The results of the present study are in agreement to

the previous studies on domestic chicken (Santayana, 1985) and turkey

(Zaharddeen et al., 2005). Nevertheless, some researchers have reported that semen

collections at any interval have no effect on sperm concentration (Lorenz et al.,

1955; McCartney et al., 1958; Cecil, 1982). These differences in observations

might be due to the difference in time intervals considered for evaluation in

particular studies. On the other hand, percentages of sperm with intact acrosome

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were observed higher with semen collection performed at the intervals of 72 hours

compared to 12 and 24 hours of collection intervals. It is agreement to the previous

studies that maximum sperm with normal acrosome can be obtained after 2 and/or

3 days interval between semen collections (Donoghue et al., 1996).

Interestingly, plasma membrane integrity of IRJF spermatozoa remained

similar with all of experimental collection frequencies. These findings are in

agreement to the previous studies on poultry species (Bilgilli and Renden, 1984;

Bilgilli et al., 1985; Bakst et al., 1991; Donoghue et al., 1995). It is pertinent to

mention that most of the studies reported no change in sperm plasma membrane

and acrosome integrity with change in collection frequency.

The percentage of live sperm in an ejaculate determine its fertility potential,

higher percentage of viability resulted in higher fertility in poultry (Riaz et al.,

2004; Blesbois et al., 2008), Turkey (Zahraddeen et al., 2005) and Tragopan

(Zhang, 2006). Higher percentages of live sperm were observed at collection

interval of 12 hours compared to 24, 48 and 72 hours. These observations are inline

with the previous findings of a study on turkey (Zahraddeen et al., 2005) that

reported maximum percentage of live spermatozoa with colloetion frequency of

twice daily. Nevertheless, another study on turkeys (Sexton, 1981) indicated that

higher number of live sperm can be obtained with a collection frequency of three

times a week due to better management and health conditions of the animals. It is

believed that the increase in collection interval cause changes in spermatozoa

stored storage tubules leading to functional dysfunction (Jalme et al., 2003).

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Extensive studies have been conducted on various avian species for holding

the sperm in different conditions at low temperature in various extenders (Marzoni

et al., 2003). The storage of sperm from certain species needs specific media and

conditions. The storage ability and approach used for the sperm of a specific

species determine its fertilizing ability for hours/days/years after preservation. In

this context, an appropriate semen extender has to provide energy for spermatozoa,

maintain pH and osmolarity levels identical to those of seminal plasma, the natural

medium for sperm (Siudzinska and Lukaszewicz, 2008).

Ensuring proper pH and osmotic pressure of suspension is of great

significance in maintaining the viability of spermatozoa of a certain species. It has

been shown that fowl sperm can tolerate a pH range of 6.0 to 8.0 (Wambeke, 1967;

Siudzinska and Lukaszewicz, 2008; Blesbois et al., 2007). Being one of the

ancestors of the domestic, the extenders are selected for Red jungle fowl

spermatozoa which have pH range from 6.0 to 7.0, similar to chicken sperm. These

extenders are Beltsville Poultry Semen Extender (BPSE) (Sexton, 1977), Diluent

for Turkey/red fowl extender (Previously by Sexton, 1988; modified in the present

study), Lake diluent (Lake, 1978), EK extender (Łukaszewicz, 2002), Poultry

extender (Tselutin et al., 1995) and chicken semen extender (Blanco et al., 2000).

The pH of a diluent can affect the metabolic rate and motility of spermatozoa.

Buffering agents, consisting of a mixture of an acid and its conjugate base are

formulated into a diluent to limit changes in pH. These usually include the mixture

of phosphates, citrates and/or organic zwitter ionic molecules in poultry diluents

(Donoghue and Wishwart, 2000).

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Spermatozoa of different avian species have different physiological and

metabolic requirements that require the development of diluents and storage

systems either in liquid or frozen state (Donoghue and Wishwart, 2000). The liquid

storage is the best option only for short term storage of semen. However, the semen

stored more than 6 hours in avian species have much reduced fertility (Blesbois et

al., 2007). It has been reported that sperm fail to penetrate in the sperm storage

tubules (SSTs) which were stored more than 6 hours in poultry. This might be due

the production of oxygen species and metabolites from the dead spermatozoa

which deteriorate the quality of semen stored in liquid form (Donoghue et al.,

1996).

Various semen extenders when evaluated for the liquid storage of IRJF

spermatozoa, the overall sperm motility, plasma membrane integrity, viability and

acrosome integrity was recorded higher in red fowl extender compared to Lake,

EK, Beltsville poultry, Tselutin poultry and chicken semen extender stored at 5 °C.

However time dependant decrease was observed in the number of motile

spermatozoa in all diluents at all times of storage time from 0, 3, 6, 24 and 48

hours. Similar results have been reported by Siudzinska and Lukaszewics (2008)

that sperm motility decreased significantly after 24 hours of storage which is

independent of diluents used at 5 °C. Blesbois and Reviers (1992) reported that

Lake and Beltsville poultry semen extender are equally efficient in maintaining the

sperm motility at 4 °C.

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The results of the studies by Han et al. (2005) have shown that Beltsville

poultry semen extender has detrimental effect on sperm motility in duck semen

which is in contrast to the previous studies done by Sexton (1981), where Beltsville

poultry semen extender has better maintained the sperm motility in chicken.

However, in another study Blanco et al. (2000) has proved that red fowl extender

has better maintained the sperm quality in free-ranging poultry. The reason may be

the variation in semen extenders in maintaining the sperm motility in every species

and even in every breed/strains.

Dilution of chicken and turkey spermatozoa with Beltsville poultry semen

extender resulted in maintaining better sperm plasma membrane integrity as

compared to other commercially available diluents when semen was stored at 5°C

for 24 hours (Clarke et al., 1984). Sugar contents in an extender either glucose or

fructose facilitates the penetration of sperm in ovum, however absence or less

amount of fructose renders the sperm incapable of fertilization (Lake, 1960;

Sexton, 1976). In the present study, the drastic reduction of live sperm after 0, 3, 6,

24 and 48 hours of storage is in agreement to the previous study on guinea fowl,

where time dependant decrease was observed after 24 hours irrespective of diluents

used (Mohan et al., 2013). The possible explanation about the effect of diluents on

viability of IRJF may be the osmotic pressure of the diluents, as it was observed in

previous studies that extenders having osmolarities ranged from 350-450 mOsm/kg

have less detrimental effect on the fertilizing ability of spermatozoa in chicken

(Harris et al., 1963; Graham and Brown, 1971; Bakst, 1980).

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The red fowl extender has osmolarity of 380 mOsmol/kg, confirms the

findings of this study, that osmotic balance is necessary in the penetration/survival

of spermatozoa in the reproductive tract (Seiki, 1960; Tsukunaga, 1971). The

populations of viable sperm are likely to decrease with the passage of time because

the ejaculate is likely to be a mixture of live cells having lost acrosome or dead

cells with viable acrosome; as dead cells produce reactive oxygen species Nagy et

al. (2003). Previous studies (Lake and McIndoe, 1959; Ogasawara and Ernst, 1975;

Sexton, 1977) have reported that glutamic acid in avian extender is a major

constituent of avian seminal plasma. It may act to maintain the level of sodium

glutamate, which is proved to have very interesting relationship in maintaining the

fertilization potential of the sperm by protecting its acrosome. The red fowl

extender contains higher percentage of sodium glutamate compared to all other

extenders, which might have resulted in better protective ability of red fowl semen

extender to maintain the number of viable spermatozoa at all times of storage.

The extenders were evaluated for cryopreservation of IRJF spermatozoa

and red fowl extender was found superior for protecting motility, viability and

plasma membrane and acrosome integrity and achieved higher recovery rates after

freezing-thawing. The similar observation was made by using absolute livability

index that give some rough indirect clue about the longevity and/or surviving

ability of the sperm at ambient temperature. The composition of this extender is

close to the one used by Blanco et al., 2000, 2011, 2012) in different, avian species

(Poultry, Eagle, Peregrine Falcon, Turkey and crane) Spermatozoa. However, other

diluents such as the BPSE or Lake‟s diluents have been successful for chicken

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semen (Lake 1960; Lake 1966; Sexton, 1981, Lake 1978, Lake and Ravie, 1982).

The BPSE has been reported to have detrimental effects on motility of duck semen

(Han et al., 2005) and higher percentages of live duck spermatozoa were recorded

in EK extender compared to Lake and Tselutin poultry extender (Han et al., 2005).

These different results illustrate well the point that ability of sperm to respond to

huge media changes, temperature and osmotic variations during dilution, cooling,

equilibration and freeze-thawing phases is highly variable from species to species

(Blanco et al., 2009; 2012). The usability of an extender for routine practices is

always questionable until supported with satisfactory outcomes for fertility (Ansari

et al., 2012). The red fowl extender showed percent fertility rates of 86.7 ± 2.2 and

57.8 ± 3.9 percent in IRJF with unfrozen diluted and frozen-thawed semen,

respectively. Although a decrease in fertility after cryopreservation was evident but

the recorded 61% fertility rate support to suggest its potential for IRJF. The

observed fertility rates with frozen semen are comparatively higher than in

domestic chicken, knowing that, here, it was measured after a single insemination

of less than 300 sperm in contrast to the practice in domestic chicken and other

species where successive inseminations are needed to get good fertility results

(Wishart, 1999; Tselutin et al, 1999; Blanco et al 2012; Seigneurin and al., 2013;

Getachew, 2016). This attests the success of the methodology that we have chosen

with the IRJF sperm. The red fowl extender has osmolarity 380 mOsm/kg that

probably offers better protection during cryopreservation as higher osmolarity

(350-450 mOsm/kg) are considered suitable for fertility of chicken sperm (Harris et

al., 1963; Graham and Brown, 1971; Bakst, 1980). The availability of the energy is

highly associated with ability of sperm to reach the site of fertilization (Seiki, 1960;

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Tsukunaga, 1971) and fertilization process itself (Lake, 1960; Sexton, 1976). It is

suggested that higher osmolarity and availability of energy source i.e. fructose both

may had positive contributions in fertility attributes of IRJF spermatozoa.

In the present study, the optimum 6% concentration of DMA was obtained.

The previous work on DMA usage at 6% concentration in Houbara bustard

(Hartley et al., 1999), poultry (Blesbois et al., 2005) would confer our results that

lower concentration of penetrating cryoprotectant is more favorable to sperm

during cryopreservation process (Abouelezz et al., 2015; Tselutin et al., 1999; Han

et al., 2005). It is assumed that cryoprotectant in hand is able to increase

osmolarity, suitably dehydrates cells and thus avoids ice crystal formation during

cryopreservation process (Blanch et al.., 2012). Cryopreservation stages (post-

dilution, post-cooling, post-equilibration and post-thawing) do affect sperm

survival and are highly dependable on the diluent and cryoprotectant used. It has

been observed that fertility rates [66.8% (6% DMA) vs. 56.4% (control) were

significantly higher in the diluent in hand compared to previous studies on chicken

(Blesbois et al., 2008) duck (Han et al., 2005), turkey (Iaffaldano et al., 2016)

which used the same cryoprotectant with different diluents. Sexton (1977) and

Rakha et al. (2016) suggested that the diluent having glutamic acid as a major

constituent would better maintain sperm quality and fertility, which is in favor to

the present study.

In present study, 8% DMSO in extender resulted in highest motility, plasma

membrane integrity, viability and acrosome integrity at post-dilution, cooling,

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equilibration and freeze-thawing compared to 4%, 6%, 10% DMSO and control

(with glycerol). DMSO being permeable to sperm membrane offers extracellular

and intracellular protection to spermatozoa by causing efflux of water molecules

and maintaining cell volume through osmosis (Purdey 2006). The results of the

present study are in agreement and showed that addition of 10% DMSO

significantly decreased the post-thaw semen quality compared to 8% DMSO. The

addition of 8% DMSO in extender might have increased the process of osmosis,

dehydrated the sperm cells and avoided ice crystallization during the process of

cryopreservation. The other reason may be that optimized concentration of DMSO

(8%) have maintain physico-chemical structures of lipid bi-layer of sperm and

provided it greater permeability compared to lower concentrations and glycerol

(control).

The results showed that 8% DMF in extender resulted in highest motility,

plasma membrane integrity, viability and acrosome integrity at pot-dilution,

cooling, equilibration and freeze-thawing compared to 4%, 6%, 10% DMF and

control (with glycerol) and fertility after artificial insemination. Osmotic stress

during the different phases of cryopreservation (post-dilution, cooling,

equilibration and freeze-thawing) not only damages the sperm plasma membrane

but also results in the production of reactive oxygen species (Carvalho et al., 2010;

Bochenek et al., 2005). The use of DMF to reduce DNA damage during

cryopreservation process and production of ROS molecules is an appealing strategy

to improve the post-thaw semen quality and fertility of Indian red jungle

spermatozoa.

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Motility, plasma membrane integrity, viability and acrosome integrity was

recorded higher with 6% PVP compared to 4%, 8% and 10% PVP and control at

post-dilution, cooling, equilibration and freeze-thawing. Similarly, higher no. of

fertile eggs, percent fertility, number of hatched chicks, percent hatch and

hatchability were recorded with 6% PVP and control. It is reported that PVP is

capable to maintain acceptable post-thaw quality of rooster and ring-necked

pheasant and red-tailed hawks semen (Herrera et al., 2005).

The plasma membrane integrity of IRJF sperm was found significantly

higher (P < 0.05) in an extender having PVP 6% replacing glycerol while

decreased significantly at higher concentration of PVP (10%), which is in

agreement to the present study (De Leeuw et al., 1993). Similar results were

obtained on the effect of PVP as a cryoprotective agent on channel cat fish sperm

and where only lower concentration of PVP (5%) better maintained post thaw

sperm quality compared to higher concentrations of PVP (Tiersch et al., 1994). The

study on human sperm showed that sperm preserved in PVP based cryoprotectant

exhibited better sperm motility and sperm condensation compared to its absence

(Irez et al., 2013; Kato and Nagao, 2012). In another study on cattle, the sperm

capacitation status was significantly enhanced in extender containing 10% PVP;

while the fertility rate decreased (Kato and Nagao, 2009). Previously, the

remarkable fertility of 39% in rooster and 57% in ring-necked pheasant was

recorded with frozen semen (Herrera et al., 2005). The results of all mentioned

studies support findings of present studies that IRJF sperm can successfully be

cryopreserved with 8% PVP.

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An advantageous property of the PVP that contributes in cryoprotective

action is exponential rise in osmolarity with increasing concentration which

enhances their ability to prevent ice crystallization (Capaccioli and Ngai, 2011).

The PVP in water is more effective than glycerol (20%) decreasing melting point

and nucleation temperature. Thus, increased PVP concentration has greater

influence in the glass transformation temperature. It was hypothesized that initial

concentration and viscosity of protective polymer solutions reduce the amount and

rate of cell water loss to extracellular ice and limit the injurious osmotic stress

which cells faces during freezing. The glass formation prevents further osmotic

stress after -20°C by isolating cells from extracellular ice crystals, virtually

eliminating cell water loss at lower temperatures. It is suggested that success of

cryopreservation is highly correlated with extracellular fraction of unfrozen water

(Mazur et al., 1981). It was observed that polymer solution with limiting glass

transition temperature ~-20°C is highly related with extent of cryoprotective

properties (Takahashi et al., 1988). Simultaneously, protective polymer solutions

cause diffusion of some water away from cells at temperate above glass transition

temperature. Under ideal conditions, cells can concentrate the cytoplasm during the

initial cooling to glass transition temperature to avoid lethal intracellular freezing

between extracellular glass transition temperature and intracellular glass transition

temperature, both depressed to lower temperatures by that concentration

(Takahashi et al., 1988). It has been suggested that non-penetrating cryoprotectants

including polymers mediated protection during cryopreservation is associated with

its ability in maintaining osmotic stress within narrow limits (McGann, 1978). It is

suggested that physical properties of the PVP in extender were favorable to the

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IRJF spermatozoa that resulted in higher post-thaw motility, plasma membrane

integrity, acrosome integrity and in vivo fertility. On the average, it has been

observed that egg yolk has the ability to protect post-thaw semen (40-50%) quality

compared to control. However fertility rate obtained with the egg yolk is similar to

those with glycerol and can be used effectively for IRJF spermatozoa.

In the present study, IRJF sperm have shown greater adaptability to

freezing temperatures with some modifications in freezing protocols (either adding

DMA 6% or DMSO 8% or DMF 8% or PVP 6% or egg yolk 15% added at 4 °C

and freezing from 37 °C to 4 °C in two hours (0.275 min-1

) and equilibration for 10

min at 4°C) used for poultry and for other avian species. It is well acknowledged

that IRJF is one of the ancestors of domestic chickens; but the reproductive

potential of the roosters has decreased with the process of selection of birds for

meat and eggs.

The reason of these differences could possibly be attributed to

heterogeneity of the semen quality among breeds, strains and species-specificity. It

is clear from the literature (Padhy et al., 2016) that primitive chicken breeds have

better reproductive efficeincy in relation to newly developed breed/strains. The

possible explanation of higher freezaability and fertility outcomes in IRJF could be

that the birds used in this study were the parent stock and they seemed to have well

adapted to the management conditions, nutrition and classical environment of

Pakistan (tropical and temprate). The varying seasonaility in its native evironement

has made IRJF more resistant to temperature changes and may have caused higher

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expression of genes that express the heat shock proteins (HSPs.) as reported by

Oksala et al. (2014) that breeds/strains of tropical environment have higher

expression of HSPs. HSPs are recognized to have positive role in the process of

cryopreservation and fertility. Moreover, the biochemical compostion of semen

ejaculate varies from species to species and is affected by the nutrition and

managemental consitions. IRJF in the process of evoloution and adaptation may

have developed/conserved the composition of such characteristics that made its

sperm more resistant to cryo-induced damages and ultimately enhanced its

freezability and fertility.

CONCLUSION

It is concluded that optimum semen output can be achieved with daily

semen collection frequency practiced preferentially at the evening time. The

present study shows the first success of fertility obtained with IRJF cryopreserved

sperm. This was obtained with the use of RFE diluent. It is to believe that higher

levels of fructose, glutamate, PVP and presence of glycine in red fowl extender

contributed to make it superior extender for liquid storage and cryopreservation of

IRJF spermatozoa compared to Lake, EK, Beltsville poultry, Tselutin poultry and

chicken semen extender.

It is worth mentioning to highlight that data on fertility rate and hatchability

of fertilized eggs also support the results of post-thaw in vitro semen quality

parameters i.e. higher fertility and hatchability of fertilized eggs with 20% glycerol.

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Glycerol (20%) added to diluent at 4°C improved the cryopreservability and

fertility attributes (no. of fertile eggs, fertility, no. of hatched chicks, percent hatch

and hatchability) of IRJF semen. The permeable cryoprotectants; 6% DMA, 8%

DMF, 8% DMSO improved the semen quality and fertility in IRJF. It has been

recorded that 6% PVP maintained better post-thaw quality and fertility of IRJF

spermatozoa and can be used in routine practice avoiding the contraceptive effects

of glycerol. Egg-yolk is being essential constituent of extender for many livestock

species. However, egg yolk (15%) addition in freezing extender can effectively be

used for cryopreservation of IRJF sperm.

RECOMMENDATIONS

It is recommended that the germplasm of IRJF can be conserved in Red

fowl extender in liquid state (for two days) or frozen state (for indefinite period)

with cryopreservation protocol based on DMA or DMF or DMSO developed in

present study, and thereafter efficiently used for artificial breeding program.

Although acceptable recovery rates after cryopreservation were achieved in present

study but further studies are required to reduce damages that occur during the

cryopreservation to the spermatozoa of IRJF. The following further investigations

are suggested to improve the post-thaw quality and fertility after freeze-thawing.

Development of advanced semen quality assessment techniques using

fluorescent probes for selection of superior cocks of high freezability.

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Elucidation of seasonal and age related changes in reproductive

performance of IRJF

Enrichment of extender with suitable substances to reduce oxidative stress

during freeze-thawing process

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SUMMARY

The IRJF (Gallus gallus murghi) is a wild native gallus sub-species

of Southern Asia. The semen characteristics, impact of ejaculate collection

frequencies and timing of collection were evaluated. Mean sperm concentration

800 million/ mL, total sperm per ejaculate 0.015 billion, motility 63.5%, live/total

sperm 92.4%, intact acrosome 75.5% and plasma membrane integrity 89.2% were

recorded. Percentage of abnormal sperm (head, mid-piece and tail) was 8.1% and

included mainly the mid-piece abnormalities. The motile sperm percentage was

positively correlated with intact acrosomes (r=0.34) and plasma membrane

integrity (r=0.41). Total sperm per ejaculate (0.015 billion) was maximum at 72

hours of collection followed by 24 and 48 hours of collection. Daily and weekly

sperm output (billion) was found maximum at 24 hours of collection compared to

12, 48 and 72 hours of collection. Sperm motility was higher at 24, 48 and 72 hours

of collection compared to 12 hours of collection, but the number of live sperm were

higher at 12 hours of collection compared to 24, 48 and 72 hours. Sperm

concentration was better in the morning time, while the values for sperm viability

and plasma membrane integrity were higher in the semen collected at evening time.

The extenders (Beltsville poultry, RFE, Lake, EK, Tselutin poultry and

Chicken semen extender) were evaluated for long-term and short-term storage of

IRJF (Gallus gallus murghi) spermatozoa. Percentages of motility, plasma

membrane integrity, viability and acrosome integrity were higher (P < 0.05) in Red

fowl extender at 0, 2 and 4 hours of incubation post-thaw and liquid semen at 0, 3,

183

184

6, 24 and 48 hours of storage. After cryopreservation and post-thawing at 37°C, the

highest (P < 0.05) recovery rates and absolute livability index was also recorded in

red fowl extender that was thus used for further artificial insemination of cooled-

diluted (Liquid) and cryopreserved sperm. The no. of fertilized eggs (Liquid, 20.6 ±

0.4; Cryopreserved, 12.6 ± 0.5), percent fertility (86.7 ± 2.2; 57.2 ± 3.9), no. of

hatched chicks (18.2 ± 0.8; 10.0 ± 0.3), percent hatch (76.5 ± 2.7; 45.3 ± 2.2) and

hatchability of fertilized eggs (88.3 ± 3.4; 79.6 ± 3.4) were higher with sperm

freshly cooled-diluted or cryopreserved, respectively in red fowl extender.

The different glycerol concentrations (11%, 15% and 20%) were evaluated

for post-thaw quality, recovery rates, absolute livability index and fertility

attributes (number of fertile eggs, percent fertility, no. of hatched chicks, percent

hatch and hatchability of fertilized eggs) of IRJF semen. Percentages of motility,

plasma membrane integrity, viability and acrosome integrity were recorded higher

(P < 0.05) at 0, 2 and 4 hours post-thawing at 37°C with glycerol 20% compared to

15% and 11% glycerol. Likewise, recovery rates (%) of aforementioned parameters

and absolute livability index after cryopreservation were observed highest (P <

0.05) with 20% glycerol. Multivariate regression analysis showed least negative

effect of hours of incubation on semen quality in diluent with 20% glycerol

followed by 15% and 11% glycerol.

The fertility outcomes (number of fertile eggs, fertility (%), no. of hatched

chicks, percent hatch and hatchability of fertilized eggs) were recorded higher (P <

0.05) with 20% glycerol followed by 15% and 11% glycerol. Among

185

cryoprotectants evaluated in present study, 6% DMA, 8% DMSO, 8% DMF and

6% PVP maintained higher (P < 0.05) semen quality and fertility compared to

control. Among levels of egg yolk (10, 15, 20 and 25%) evaluated; 15% egg yolk

was found superior (P < 0.05) for semen quality and fertility compared to control.

It is concluded that IRJF semen with maximum efficiency can be collected once in

a day either in the morning or evening hours. The germplasm of IRJF can be

conserved in liquid (for two days) and in frozen state (for indefinite period) with

cryopreservation protocol based on DMA or DMF or DMSO or PVP and egg yolk

developed in present study, and can efficiently be used in artificial breeding

program for conservation.

186

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APPENDICES

Table 1.

For Table 2.1. Semen characteristics of Indian red jungle fowl.

ANALYSIS OF VARIANCE TABLE

Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-

value

p-

value

Treatment 7,474,637 7 1,067,805 1.32 0.274

Error 25,942,674 32 810,708

Total 33,417,311 39

232

233

Table 2.

For Table 2.5. Effect of collection frequency on weekly sperm output (Mean ± SE) of

Indian red jungle fowl spermatozoa.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-

value

p-

value

Treatment 3.05460 3 1.018198 3.49 .0336

Error 6.11806 21 0.291336

Total 9.17266 24

234

Table 3.

For Table 2.5. Effect of collection frequency on daily sperm output (Mean ± SE) of Indian

red jungle fowl spermatozoa.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 0.05476 3 0.018252 2.90 .0593

Error 0.13238 21 0.006304

Total 0.18714 24

213

235

Table 4.

For Table 2.5. Effect of collection frequency on total sperm output (Mean ± SE) of Indian

red jungle fowl spermatozoa.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 0.71660 3 0.238868 26.67 2.34E-07

Error 0.18809 21 0.008957

Total 0.90470 24

236

Table 5.

For Table 2.5. Effect of collection frequency on sperm concentration (Mean ± SE) of

Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 134.00871 3 44.669569 15.43 4.53E-09

Error 582.03778 201 2.895710

Total 716.04649 204

237

Table 6.

For Table 2.5. Effect of collection frequency on sperm motility (Mean ± SE) of Indian red

jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 16,995.94 3 5,665.313 13.30 6.93E-08

Error 77,092.41 181 425.925

Total 94,088.35 184

238

Table 7.

For Table 2.5. Effect of collection frequency on sperm acrosomal integrity (Mean ± SE) of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 630.825 3 210.2749 0.77 .5133

Error 54,771.087 200 273.8554

Total 55,401.912 203

239

Table 8.

For Table 2.5. Effect of collection frequency on sperm plasma membrane integrity (Mean

± SE) of Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,382.767 3 460.9223 2.49 .0613

Error 36,992.213 200 184.9611

Total 38,374.980 203

240

Table 9.

For Table 2.5. Effect of collection frequency on sperm viability (Mean ± SE) of Indian red

jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,169.782 3 389.9272 4.50 .0044

Error 17,314.791 200 86.5740

Total 18,484.572 203

241

Table 10.

For Table 2.6. Effect of semen collection time (Morning vs. evening) on spermatozoal

quality parameters of Indian red Jungle fowl.

t-TEST BETWEEN TWO INDEPENDENT GROUPS

Df Pooled standard

deviation

Standard

error

t-

Value

P-

Value

Semen volume 98 39.487 7.897 -0.08 0.9336

Sperm motility 98 24.602 4.920 -1.18 0.2413

Sperm concentration 98 1.23 0.25 -2.63 0.01

Sperm Acrosomal

integrity

98 10.85 2.17 8.37 4.17-13

Plasma membrane

integrity

98 16.27 3.25 4.17 0.0001

Sperm viability 98 7.61 1.522 0.42 0.67

242

Table 11.

For Figure 3.6.1.1 Effect of extenders on motility of Indian red jungle fowl spermatozoa

stored at 5 ºC.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Extender 9257.083 5 1851.417 24.5977 0.0000

Post-thaw

hours

100842.292 4 25210.573 334.9447 0.0000

Interaction 1443.958 20 72.198 0.9592 0.001

Error 15806.250 210 75.268

Total 127349.583 239

243

Table 12.

For Figure 3.6.1.2. Effect of extenders on plasma membrane integrity of Indian red jungle

fowl spermatozoa stored at 5 ºC.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Extender 8823.973 5 1764.795 80.9150 0.0000

Post-thaw

hours

93336.370 4 23334.093 1069.8570 0.0000

Interaction 1262.856 20 63.143 2.8951 0.0001

Error 4580.200 210 21.810

Total 108003.399 239

244

Table 13.

For Figure 3.6.1.3. Effect of extenders on viability of Indian red jungle fowl spermatozoa

stored at 5 ºC.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Extender 10576.075 5 2115.215 59.2927 0.0000

Post-thaw

hours

128671.360 4 32167.840 901.7133 0.0000

Interaction 949.122 20 47.456 1.3303 0.1624

Error 7491.568 210 35.674

Total 147688.125 239

245

Table 14.

For Figure 3.6.1.4. Effect of extenders on acrosomal integrity of Indian red jungle fowl

spermatozoa stored at 5 ºC.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Extender 4717.464 5 943.493 49.5991 0.0000

Post-thaw

hours

123074.536 4 30768.634 1617.4954 0.0000

Interaction 1369.539 20 68.477 3.5998 0.0000

Error 3994.703 210 19.022

Total 133156.241 239

246

Table 15.

For Figure 3.6.2.1.1. Effect of extenders on post-thaw motility of Indian red jungle fowl

spermatozoa.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Extender 5985.556 5 1197.111 13.7687 0.0000

Post-thaw

hours

4143.889 2 2071.944 23.8307 0.0000

Interaction 589.444 10 58.944 0.6780 0.61

Error 6260.000 72 86.944

Total 16978.889 89

247

Table 16.

For Figure 3.6.2.1.2. Effect of extenders on post-thaw plasma membrane integrity of

Indian red jungle fowl spermatozoa.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Extender 4813.556 5 962.711 41.8874 0.0000

Post-thaw

hours

2682.022 2 1341.011 58.3471 0.0000

Interaction 241.578 10 24.158 1.0511 0.4109

Error 1654.800 72 22.983

Total 9391.956 89

248

Table 17.

For Figure 3.6.2.1.3. Effect of extenders on post-thaw viability of Indian red jungle fowl

spermatozoa.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Extender 7786.133 5 1557.227 38.7584 0.0000

Post-thaw

hours

1125.000 2 562.500 14.0003 0.0000

Interaction 87.667 10 8.767 0.2182 0.451

Error 2892.800 72 40.178

Total 11891.600 89

249

Table 18.

For Figure 3.6.2.1.4. Effect of extenders on post-thaw acrosomal integrity of Indian red

jungle fowl spermatozoa.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Extender 5925.067 5 1185.013 38.4537 0.0000

Post-thaw

hours

2908.476 2 1454.233 47.1898 0.0000

Interaction 144.067 10 14.407 0.4675 0.32

Error 2218.800 72 30.817

Total 11196.400 89

250

Table 19.

For Table 3.2. Recovery rate of sperm motility of Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 6,034.803 5 1,206.9607 3.70 .0127

Error 7,833.497 24 326.3957

Total 13,868.301 29

251

Table 20.

For Table 3.2. Recovery rate of sperm plasma membrane integrity of Indian red jungle

fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,688.686 5 337.737 3.56 .0150

Error 2,276.772 24 94.8655

Total 3,965.458 29

252

Table 21.

For Table 3.2. Recovery rate of sperm viability of Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,223.974 5 244.7949 2.38 .0693

Error 2,473.213 24 103.0505

Total 3,697.187 29

253

Table 22.

For Table 3.2. Recovery rate of sperm acrosomal integrity of Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,211.422 5 242.284 3.72 .0124

Error 1,563.763 24 65.1568

Total 2,775.185 29

254

Table 23.

For Table 3.2. Absolute livability index of semen quality parameters of Indian red jungle

fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 30,150.000 5 6,030.0000 5.90 .0011

Error 24,520.000 24 1,021.6667

Total 54,670.000 29

255

Table 24.

For Table 3.3. Fertility attributes of Indian red jungle fowl spermatozoa after artificial

insemination of 25 hens with semen stored either in liquid or frozen state in red fowl

semen extender


Df Pooled standard

deviation

Standard

error

t-

Value

P-

Value

No. of laid eggs 8 1.40 0.883 -1.81 0.10

No. of fertilized eggs 8 1.025 0.648 -12.34 1.73-06

Fertility (%) 8 7.1373 4.51 -6.54 0.0002

No. of hatched chicks 8 1.360 0.860 -9.53 1.21-05

Hatch (%) 8 5.52 3.49 -8.94 1.95-05

Hatchability of fertilized

eggs

8 6.5965 4.17 -2.09 0.07

256

Table 25.

For Figure 4.7.1.1. Effect of different levels of glycerol on spermatozoa motility (%) of

Indian red jungle fowl post-tahw at 0 hours of incubation at 37 ºC.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,110.00 2 555.000 20.18 .0001

Error 330.00 12 27.500

Total 1,440.00 14

257

Table 26.


Indian red jungle fowl post-thaw at 2 hours of incubation at 37 ºC.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 520.00 2 260.000 17.33 .0003

Error 180.00 12 15.000

Total 700.00 14

258

Table 27.




Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 763.33 2 381.667 25.44 4.83E-05

Error 180.00 12 15.000

Total 943.33 14

259

Table 28.

For Figure 4.7.1.2. Effect of different levels of glycerol on spermatozoa plasma membrane

integrity (%) of Indian red jungle fowl post-thaw at 0 hours of incubation at 37 ºC.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 714.13 2 357.067 11.81 .0015

Error 362.80 12 30.233

Total 1,076.93 14

260

Table 29.




Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 496.53 2 248.267 22.04 .0001

Error 135.20 12 11.267

Total 631.73 14

261

Table 30.




Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,143.33 2 571.667 34.30 1.09E-05

Error 200.00 12 16.667

Total 1,343.33 14

262

Table 31.

For Figure 4.7.1.3. Effect of different levels of glycerol on spermatozoa Viability (%) of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,663.33 2 831.667 23.21 .0001

Error 430.00 12 35.833

Total 2,093.33 14

263

Table 32.




Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,882.53 2 941.267 41.34 4.14E-06

Error 273.20 12 22.767

Total 2,155.73 14

264

Table 33.




Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 3,050.13 2 1,525.067 60.68 5.31E-07

Error 301.60 12 25.133

Total 3,351.73 14

265

Table 34.

For Figure 4.7.1.4. Effect of different levels of glycerol on spermatozoa acrosomal



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,892.80 2 946.400 55.45 8.66E-07

Error 204.80 12 17.067

Total 2,097.60 14

266

Table 35.




Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2,190.00 2 1,095.000 57.63 7.03E-07

Error 228.00 12 19.000

Total 2,418.00 14

267

Table 36.




Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2,224.53 2 1,112.267 30.53 1.96E-05

Error 437.20 12 36.433

Total 2,661.73 14

268

Table 37.

For Table 4.1. Recovery rate of sperm motility after cryopreservation with different levels

of glycerol


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 5,009.516 2 2,504.7582 16.02 .0004

Error 1,875.772 12 156.3143

Total 6,885.288 14

269

Table 38.

For Table 4.1. Recovery rate of sperm plasma membrane integrity after cryopreservation

with different levels of glycerol


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 5,340.507 2 2,670.2537 26.35 4.07E-05

Error 1,216.198 12 101.3499

Total 6,556.706 14

270

Table 39.

For Table 4.1. Recovery rate of sperm plasma viability after cryopreservation with

different levels of glycerol


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,402.714 2 701.3572 4.59 .0331

Error 1,834.926 12 152.9105

Total 3,237.640 14

271

Table 40.

For Table 4.1. Recovery rate of sperm plasma acrosomal integrity after cryopreservation

with different levels of glycerol


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2,550.360 2 1,275.1802 19.54 .0002

Error 783.034 12 65.2528

Total 3,333.395 14

272

Table 41.

For Figure 4.1. Effect of different levels of glycerol on absolute livability index of Indian

red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 11,103.33 2 5,551.667 38.73 5.82E-06

Error 1,720.00 12 143.333

Total 12,823.33 14

273

Table 42.

For Table 4.3. Effect of different levels of glycerol on number of laid eggs after artificial

insemination in Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 10.533 2 5.2667 0.52 .6065

Error 121.200 12 10.1000

Total 131.733 14

274

Table 43.

For Table 4.3. Effect of different levels of glycerol on number of fertilized eggs after

artificial insemination in Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 827.733 2 413.867 66.04 3.34E-07

Error 75.200 12 6.2667

Total 902.933 14

275

Table 44.

For Table 4.3. Effect of different levels of glycerol on number of hatched chicks after



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 678.7729 2 339.386 7.47 .0056

Error 681.5655 15 45.4377

Total 1,360.338 17

276

Table 45.

For Table 4.3. Effect of different levels of glycerol on hatch (%) after artificial

insemination in Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 3,489.057 2 1,744.53 82.24 9.88E-08

Error 254.542 12 21.2119

Total 3,743.600 14

277

Table 46.

For Table 4.3. Effect of different levels of glycerol on hatchability of fertilized eggs after



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1,485.007 2 742.504 8.44 .0051

Error 1,055.746 12 87.9789

Total 2,540.754 14

278

Table 47.

For Figure 5.4.1.1. Effect of different concentrations (0%, 4%, 6%, 8% and 10%) of

Dimethyleacetamide on the sperm motility (%) at various stages of

cryopreservation in Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2969.075 4 742.269 38.45 0.0000

Stage 9119.650 3 3039.883 157.50 0.0000

Interaction 639.225 12 53.269 2.76 0.0048

Error 1158.000 60 19.300

Total 13885.950 79

279

Table 48.


Dimethyleacetamide on the sperm plasma membrane integrity (%) at various stages

of cryopreservation in Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2762.925 4 690.731 36.6841 0.0000

Stage 5447.537 3 1815.846 96.4379 0.0000

Interaction 237.275 12 19.773 1.0501 0.4173

Error 1129.750 60 18.829

Total 9577.487 79

280

Table 49.


Dimethyleacetamide on the sperm viability (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1471.950 4 367.988 12.84 0.0000

Stage 5779.237 3 1926.412 67.23 0.0000

Interaction 288.950 12 24.079 0.8403 0.62

Error 1719.250 60 28.654

Total 9259.388 79

281

Table 50.


Dimethyleacetamide on the sperm acrosomal integrity (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1505.800 4 376.450 6.8368 0.0001

Stage 6118.138 3 2039.379 37.04 0.00000

Interaction 113.300 12 9.442 0.1715 0.56

Error 3303.75 60 55.063

Total 11040.987 79

282

Table 51.

For Table 5.1. Effect of DMA (6%) on fertility of spermatozoa and hatchability in Indian

red jungle fowl.


Df Pooled standard

deviation

Standard

error

t-

Value

P-

Value

No. of laid eggs 8 2.646 1.673 1.20 0.27

No. of fertilized eggs 8 2.811 1.778 3.71 0.005

Fertility (%) 8 7.96 5.03 2.23 0.05

No. of hatched chicks 8 1.761 1.114 7.36 0.0001

Hatch (%) 8 4.777 3.021 5.10 0.0009

Hatchability of

fertilized eggs

8 5.131 3.245 2.90 0.01

283

Table 52.


Dimethylsulfoxide on the sperm motility (%) at various stages of cryopreservation

in Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 454.375 4 113.594 3.9368 0.0067

Stage 12175.938 3 4058.646 140.6606 0.0000

Interaction 438.125 12 36.510 1.2653 0.2627

Error 1731.250 60 28.854

Total 14799.688 79

284

Table 53.


Dimethylsulfoxide on the sperm plasma membrane integrity (%) at various stages



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1571.575 4 392.894 12.93 0.0000

Stage 5080.838 3 1693.613 55.72 0.0000

Interaction 145.225 12 12.102 0.40 0.72

Error 1823.750 60 30.396

Total 8621.388 79

285

Table 54.


Dimethylsulfoxide on the sperm viability (%) at various stages of cryopreservation

in Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1971.425 4 492.856 12.6902 0.0000

Stage 6482.237 3 2160.746 55.6356 0.0000

Interaction 102.575 12 8.548 0.2201 0.72

Error 2330.250 60 38.838

Total 10886.487 79

286

Table 55.


Dimethylsulfoxide on the sperm acrosomal integrity (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1398.800 4 349.700 6.4258 0.0002

Stage 5703.737 3 1901.246 34.9360 0.0000

Interaction 291.700 12 24.308 0.4467 0.57

Error 3265.250 60 54.421

Total 10659.487 79

287

Table 56.

For Table 5.2. Effect of DMSO (8%) on fertility of spermatozoa and hatchability in Indian

red jungle fowl.


Df Pooled standard

deviation

Standard

error

t-

Value

P-

Value

No. of laid eggs 8 4.43 2.807 1.21 0.26


Fertility (%) 8 9.72 6.16 3.24 0.01


Hatch (%) 8 10.6884 6.75 4.19 0.003

Hatchability of

fertilized eggs

8 12.18 7.70 2.40 0.04

288

Table 57.


Dimethylformamide on the sperm motility (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1779.375 4 444.844 14.7259 0.0000

Stage 14387.500 3 4795.833 158.7586 0.0000

Interaction 990.625 12 82.552 2.7328 0.0051

Error 1812.500 60 30.208

Total 18970.0 79

289

Table 58.


Dimethylformamide on the sperm plasma membrane integrity (%) at various stages



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 3780.657 4 945.169 27.1730 0.0000

Stage 9623.500 3 3207.833 92.23 0.0000

Interaction 653.625 12 54.469 1.5659 0.1266

Error 2087.00 60 34.783

Total 16144.800 79

290

Table 59.


Dimethylformamide on the sperm viability (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2013.800 4 503.450 11.0851 0.0000

Stage 5002.050 3 1667.350 36.7123 0.0000

Interaction 384.700 12 32.058 0.7059 0.45

Error 2725.00 60 45.417

Total 10125.550 79

291

Table 60.


Dimethylformamide on the sperm acrosomal integrity (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 960.625 4 240.156 6.3248 0.0003

Stage 7960.438 3 2653.479 69.8820 0.0000

Interaction 754.875 12 62.906 1.6567 0.1002

Error 2278.250 60 37.971

Total 11954.188 79

292

Table 61.

For Table 5.3. Effect of DMF (8%) on fertility of spermatozoa and hatchability in Indian

red jungle fowl.


Df Pooled standard

deviation

Standard

error

t-

Value

P-

Value

No. of laid eggs 8 4.43 2.80 1.21 0.26


Fertility (%) 8 7.31 4.62 -4.07 0.003


Hatch (%) 8 6.65 4.20 5.05 0.001

Hatchability of

fertilized eggs

8 1.57 0.996 7.68 0.0001

293

Table 62.


Polyvinylpyrrolidone on the sperm motility (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1468.875 4 367.219 16.8160 0.0000

Stage 10353.037 3 3451.012 158.0315 0.0000

Interaction 604.525 12 50.377 2.3069 0.0170

Error 1310.250 60 21.838

Total 13736.688 79

294

Table 63.


Polyvinylpyrrolidone on the sperm plasma membrane integrity (%) at various

stages of cryopreservation in Indian red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2661.500 4 615.375 16.9817 0.0000

Stage 7098.737 3 2366.246 65.2983 0.0000

Interaction 692.200 12 57.683 1.5918 0.1185

Error 2174.250 60 36.238

Total 12426.688 79

295

Table 64.


Polyvinylpyrrolidone on the sperm viability (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1147.450 4 286.863 12.0194 0.0000

Stage 8102.450 3 2700.817 113.1627 0.0000

Interaction 470.050 12 39.171 1.6412 0.1043

Error 1432.00 60 23.867

Total 11151.950 79

296

Table 65.


Polyvinylpyrrolidone on the sperm acrosomal integrity (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 3064.175 4 766.044 31.2140 0.0000

Stage 5821.450 3 1940.483 79.0689 0.0000

Interaction 435.425 12 36.285 1.4785 0.1578

Error 1472.500 60 24.542

Total 10793.550 79

297

Table 66.

For Table 5.4. Effect of PVP (8%) on fertility of spermatozoa and hatchability in Indian

red jungle fowl.


Df Pooled standard

deviation

Standard

error

t-

Value

P-

Value

No. of laid eggs 8 2.30 1.45 0.00 1.000


Fertility (%) 8 6.36 4.022 4.15 0.003


Hatch (%) 8 6.07 3.84 4.44 0.002

Hatchability of

fertilized eggs

8 4.06 2.57 1.23 0.25

298

Table 67.


Egg yolk on the sperm motility (%) at various stages of cryopreservation in Indian

red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2944.325 4 736.081 37.9342 0.0000

Stage 8010.338 3 2670.113 137.6051 0.0000

Interaction 796.975 12 66.415 3.4227 0.0007

Error 1164.250 60 19.404

Total 12915.888 79

299

Table 68.


Egg yolk on the sperm plasma membrane integrity (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2979.125 4 744.781 32.4877 0.0000

Stage 3920.150 3 1306.717 56.9996 0.0000

Interaction 625.975 12 52.165 2.2754 0.0186

Error 1375.500 60 22.925

Total 8900.750 79

300

Table 69.


Egg yolk on the sperm viability (%) at various stages of cryopreservation in Indian

red jungle fowl.


Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 1784.075 4 446.019 19.1733 0.0000

Stage 5422.138 3 1807.379 77.6950 0.0000

Interaction 310.425 12 25.869 1.1120 0.3677

Error 1395.750 60 23.263

Total 8912.388 79

301

Table 70.


Egg yolk on the sperm acrosomal integrity (%) at various stages of



Source Sum of

Squares

Degree of

Freedom

Mean

squares

F-value p-value

Treatment 2213.125 4 553.281 10.0300 0.0000

Stage 3197.339 3 1065.779 19.3207 0.0000

Interaction 259.975 12 21.665 0.3927 0.57

Error 3309.750 60 55.163

Total 8980.188 79

302

Table 71.

For Table 5.5. Effect of Egg yolk (15%) on fertility of spermatozoa and hatchability in



Df Pooled standard

deviation

Standard

error

t-

Value

P-

Value

No. of laid eggs 8 2.449 1.54 0.77 0.46


Fertility (%) 8 8.79 5.56 -0.59 0.57


Hatch (%) 8 6.39 4.04 0.19 0.85

Hatchability of

fertilized eggs

8 6.81 4.31 0.09 0.93

Documents

SEMEN CHARACTERISTICS, PRESERVATION AND ITS USE IN ARTIFICIAL INSEMINATION …prr.hec.gov.pk/jspui/bitstream/123456789/8107/1/Bushra... · 2018-07-23 · 5.3.5.3. Sperm viability