RESEARCH PROPOSAL FOR RAPID GRANT FOR YOUNG …dbtepromis.nic.in/Documents/BudgetDetails... · research proposal for rapid grant for young investigators (rgyi) to department of biotechnology

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RESEARCH PROPOSAL FOR

RAPID GRANT FOR YOUNG INVESTIGATORS (RGYI)

TO

DEPARTMENT OF BIOTECHNOLOGY

NEW DELHI

ANALYSIS OF THE SIGNIFICANCE OF SPERM mRNA AS BIOMARKER FOR

IMPROVEMENT OF FREEZABILITY AND FERTILITY OF BUFFALO BULL

SEMEN

(SUBJECT AREA: ANIMAL SCIENCES)

SUBMITTED BY

BUFFALO PHYSIOLOGY AND REPRODUCTION DIVISION

CENTRAL INSTITUTE FOR RESEARCH ON BUFFALOES

SIRSA ROAD HISAR-125001, HARYANA

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PROFORMA – I

PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH AND

DEVELOPMENT, PROGRAMME SUPPORT

(To be filled by the applicant)

PART I: GENERAL INFORMATION

1. Name of the Institute/University/Organisation submitting the Project Proposal:

Buffalo Physiology and Reproduction Division

Central Institute for Research on Buffaloes, Sirsa Road Hisar

2. State: Haryana

3. Status of the Institute: Institution of ICAR

(Please see Annexure-I)

4. Name and designation of the Executive Authority of the Institute/University

forwarding the application:

Dr R. K. Sethi

Director

Central Institute for Research on Buffaloes

Sirsa Road Hisar, Haryana-125001

5. Project Title:

Analysis of the significance of sperm mRNA as biomarker for improvement of freezability and

fertility of buffalo bull semen

6. Category of the Project (Please tick): √R&D/ Programme Support

7. Specific Area (Please see Annexure - II):

Animal Sciences: any other (Semen Biotechnology)

8. Duration: 3 Years

9. Total Cost (Rs.): 35,70,500/ (` Thirty Five Lakh Seventy Thousand Five Hundred

only)

10. Is the project Single Institutional or Multiple-Institutional (S/M)? : S

11. If the project is multi-institutional, please furnish the following:

Name of Project Coordinator:

Affiliation:

Address:

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12. Scope of application indicating anticipated product and processes:

Buffalo is an integral part of livestock agriculture in Asia since many centuries, but the

buffalo farming industry is under pressure due to lower conception rate around 10-30%

following artificial insemination with frozen semen, reason being buffalo spermatozoa are

more susceptible to hazards during freezing-thawing process. It is well documented that

cryopreservation induces sperm damage owing to the rapid change in low temperature which

leads to mechanical damage to plasma membrane, metabolism alteration, oxidative stress to

phospholipids membrane, DNA damage, and eventually reduced fertilizing capacity.

Traditionally a number of methods are available for evaluation of semen quality in laboratory

like assessment of the integrity of genomic DNA, acrosome, plasma membrane and

mitochondrial as well as sperm-oocyte interactions. But, the development of a reliable method

for routine isolation of high quality RNA from buffalo bull sperm will be an important step to

develop novel non-invasive approaches to evaluate bull fertility. The RNA transcript may have

important roles in sperm development, chromatin repackaging, genomic imprinting,

capacitation, acrosome reaction and early embryonic development. Understanding the make-up

of sperm RNA transcripts may prove to be important in understanding the events surrounding

capacitation, motility, fertilization and ultimately in explaining the male fertility. The

development of new investigative method such as analysis of mRNA profile in sperm and

understanding of the transcripts significance would be helpful as additional diagnostic tool and

be of prognostic value to fertilization and establishment of pregnancy. The addition of RNA

stabilizer which could bring desired alteration in the mRNA profiles may ultimately be useful

to reduce cryo-damages and in improving semen quality.

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13. Project Summary (Not to exceed one page. Please use separate sheet):

The success of freezing relies upon an understanding of internal (inherent

characteristics of spermatozoa) and external (composition of diluents, cryoprotective agents,

rates of cooling and method of freezing and thawing of semen) factors and their interactions

which can influence the capacity of sperm to survive during freezing and thawing. Out of over

2000 to 5000 distinct mRNA expressed in sperm cells, suggests that sperm may contribute

more than simply DNA during fertilization. Further a complex population of mRNA levels of

different transcript molecules may play important roles in nuclear condensation (protamines),

capacitation (NOS), fertility (fertilin) and even in embryo development. Although during

freezing-thawing process of semen, temperature changes from -196 °C to +38 °C in short

period of time triggers a rapid phase transition from solid to liquid and imparts drastic changes

in reactive oxygen species generation, resulting in lipid peroxidation, cytoskeleton

modification and damage to DNA and RNA and ultimately reduce motility and fertility.

Therefore, it is essential to determine the effect of freezing-thawing process on mRNA pool

associated with sperm characteristics and fertility of buffalo bull semen and further maintains

its integrity during freeze-thaw process by addition of suitable supplements to extender for

evaluation of features associated with sperm characteristics and to improve the success of

buffalo sperm cryopreservation and fertility.

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PART II: PARTICULARS OF INVESTIGATORS (One or more co-investigators are preferred in every project. Inclusion of co-investigator(s) is

mandatory for investigators retiring before completion of the project)

Principal Investigator:

14. Name : Dr. Dharmendra Kumar

Date of Birth : 06/07/1977

Sex (M/F) : M

Designation : Scientist

Department : Buffalo Reproduction and Physiology

Institute/University : Central Institute for Research on Buffaloes

Address : Semen Freezing Lab, BPR Division

CIRB, Sirsa Road Hisar

PIN : 125001

Telephone : 09813201414

Fax : 01662275004

E-mail : [email protected]

Number of research projects being handled at present: Two

Co-Investigator

15. Name : Dr. P. S. Yadav

Date of Birth : 10.04.1963

Sex (M/F) : M

Designation : Principal Scientist and Head

Department :


Address : BPR Division, CIRB, Sirsa Road Hisar

PIN : 125001

Telephone : 01662-276631

Fax : 01662275004


Number of research projects being handled at present: Two

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Co-Investigator

16. Name : Dr. Pradeep Kumar


Sex (M/F) : M




Address : Semen Freezing Lab, BPR Division

CIRB, Sirsa Road Hisar

PIN : 125001

Telephone : 01662-278042

Fax : 01662275004


Number of research projects being handled at present: one

Co-Investigator

16. Name : Dr. Sadeesh EM


Sex (M/F) : M




Address : BPR Division, CIRB, Sirsa Road Hisar

PIN : 125001

Telephone : 01662-276631

Fax : 01662275004


Number of research projects being handled at present:

Note: Use separate page, if more investigators are involved

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PART III: TECHNICAL DETAILS OF PROJECT

(Under the following heads on separate sheets)

16. Introduction (not to exceed 2 pages or 1000 words)

16.1 Origin of the proposal

The existence of a complex population of RNA in sperm revealed that there are

between 2000 to 5000 distinct mRNAs expressed in sperm cells, suggesting that sperm may be

contributing more than DNA during fertilization. These RNA transcripts may have important

role in sperm nuclear condensation, sperm function, and capacitation and in zygote

development process. The success of freezing relies upon an understanding of internal

(inherent characteristics of spermatozoa) and external (composition of diluents, cryoprotective

agents, cooling rates, freezing and thawing methods) factors and their interactions during

freezing and thawing. Therefore, investigation of above mentioned features of mRNA may

improve the success of buffalo sperm cryopreservation and fertility.

16.2 (a) Rationale of the study supported by cited literature (b) Hypothesis (c) Key

questions:

Pessot et al (1989) first described the existence of RNA in mature sperm nucleus and

later studied the paternal contribution in fertilization and embryo development. Persistence of a

low but detectable level of transcription in mature sperm cells has also been reported. (Miteva

et al 1995). Naz (1998) has described the existence of transcription and translational activities

in human sperm during capacitation and acrosome reaction, which could also explain the

presence of mRNA in mature sperm. So, the involvement of complex population of mRNA

levels of different transcript molecules in nuclear condensation (protamines), capacitation

(NOS) (Lambard et al 2004), fertility (fertilin) (Depa-martynow et al 2007) and embryo

development can not be denied (Miller et al 2005). Expression of 2000 to 5000 distinct mRNA

in sperm cells, suggests that sperm might be contributing more than simply DNA during

fertilization. Protamines belong to the sperm nuclear specific proteins, which replace somatic

histone and transition proteins during maturation phase of spermatogenesis. After binding of

protamines to the sperm nucleus, chromatin is highly condensed and transcriptionally silent.

Several experiments described an increased correlation between the protamines transcript and

protein concentrations, sperm motility and fertilization ability (Miller et al 1997, Steger et al

2000, Aoki et al 2006). Nitric oxide synthesized by NOS is a potential modulator of sperm

function, especially in the acquisition of motility and capacitation. The high levels of NOS

isoform transcripts in low motile sperm have been related to the excessive production of NOS

responsible for an inhibition of sperm motility (Rosseli et al 1995). Fertilin (also known as

ADAM) is located in the equatorial region of the sperm. The fertilin may play a crucial role in

the process of sperm/egg fusion. Zhang et al (2004) suggested that fertilin is an important

target antigen and has a very promising value in the development of a human

immunocontraceptive vaccine. It was also discovered that sperm with poor motility have more

RNA than sperm with good motility (Roudebush et al 2004). In contrast, morphologically

normal sperm had a greater RNA content compared to morphologically abnormal sperm

(Roudebush et al 2004). It is believed that sperm with abnormal morphology may have

impaired transcriptional and, or translational pathways and therefore cell function is negatively

impacted (Roudebush et al 2004). These studies focused on the general RNA content, but may

be possible that specific transcripts correlate differently with sperm parameters. For example,

there was a decrease in aromatase mRNA levels in sperm with low motility (Lambard et al

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2003) but intense signals for eNOS and nNOS transcripts (Lambard et al 2004). Thus, the

mRNA present in testis could reflect the accurate development of spermatogenesis. Hence, the

heterogenous RNA content of a spermatozoon could be used for genomic analysis to assess

semen quality, in terms of both spermatogenesis and fertility potential. Studies on sperm RNA

are available for humans (Goodrich et al 2007), cattle (D’Amours et al 2010), pig (Jeong et al

2009), stallions (Das et al 2010) and buffalo (Tiwari et al 2008). Therefore, isolation of high

quality RNA from buffalo bull sperm and correlation of its expression with sperm

characteristics will be an important step to develop novel non-invasive approaches to evaluate

sperm quality and bull fertility.

Aoki VW, Liu L, Jones KP, Hatasaka HH, Gibson M, Peterson CM and Carrell DT. 2006.

Sperm protamine 1/protamine 2 ratios are related to in vitro fertilization pregnancy rates

and predictive of fertilization ability. Fertil Steril. 86:1408-1415.

D’Amours O, Frenette G, Fortier M, Leclerc P and Sullivan R. 2010. Proteomic comparison of

detergent-extracted sperm proteins from bulls with different fertility indexes.

Reproduction. 39:545–556.

Das PJ, Paria N, Gustafson-Seabury A, Vishnoi M, Chaki SP, Love CC, Varner DD,

Chowdhary BP and Raudsepp T. 2010. Total RNA isolation from stallion sperm and

testis biopsies. Theriogenology. 74:1099-1106.

Depa-Martynow M, Kempisty B, Lianeri M, Jagodzinski PP and Jedrzejczak P. 2007.

Association between fertilin β, protamines 1 and 2 and spermatid-specific linker histone

H1-like protein mRNA levels, fertilization ability of human spermatozoa, and quality of

preimplantation embryos. Folia Histochemica. 45: 79-85.

Goodrich R, Johnson G and Krawetz SA. 2007. The preparation of human spermatozoal RNA

for clinical analysis. Arch Androl.53: 161-167.

Jeong YJ, Kim MK, Song HJ, Kang EJ, Ock SA, Kumar BM, Balasubramanian S and Rho GJ.

2009. Effect of α-tocopherol supplementation during boar semen cryopreservation on

sperm characteristics and expression of apoptosis related genes. Cryobiology. 58: 181-

189.

Lambard S, Galeraud-Denis I, Bouraima H, Bourguiba S, Chocat A and Carreau S. 2003.

Expression of aromatase in human ejaculated spermatozoa: a putative marker of

motility. Mol Hum Reprod. 9:117-124.

Lambard SL, Galeraud-Denis I, Martin G, Levy R, Chocat A and Carreau S. 2004. Analysis

and significance of mRNA in human ejaculated sperm from normozoospermic donors:

relationship to sperm motility and capacitation. Mol Hum Reprod. 10(7): 535-541.

Miller D, Ostermeier GC and Krawetz SA. 2005. The controversy, potential and roles of

spermatozoal RNA. Trends Mol Med. 11:156-163.

Miller D. 1997. RNA in the ejaculate spermatozoon: a window into molecular events in

spermatogenesis and a record of the unusual requirements of haploid gene expression and

postmeiotic equilibration. Mol Hum Reprod. 3: 669-676.

Miteva K, Valkov N, Goncharova-Peinoval J, Kovachev K, Zlantarev S, Pironcheva G and

Russev G. 1995. Electron microscopic data for the presence of post-meiotic gene

expression in isolated ram sperm chromatin. Cytobios. 88: 7–10.

Naz RK. 1998. Effect of actinomycine D and cycloheximide on human sperm function. Arch

Androl. 41: 135–142.

Pessot CA, Brito M, Figueroa J, Concha II, Yanez A and Burzio LO. 1989. Presence of RNA

in the sperm nucleus. Biochem Biophys Res Commun. 158: 272-278.

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Rosselli M, Dubey RK, Imthurn B, Macas E and Keller PJ. 1995. Effects of nitric oxide on

human spermatozoa: evidence that nitric oxide decreases sperm motility and induces

sperm toxicity. Hum Reprod. 10:1786–1790.

Roudebush WE, Massey JB, Zhu J, Mitchell-Leef DE, Kort HI and Elsner CW. 2004.

Morphologically normal sperm have significantly greater total-RNA content than

abnormal sperm. Proceedings of the 18th World Congress on Fertility and Sterility.

1271:193-196.

Steger K, Pauls K, Klonisch T, Franke FE and Bergmann M.2006. Expression of protamine-1

and -2 mRNA during human spermiogenesis. Mol Hum Reprod. 6: 219-225.

Tiwari A, Singh D, Kumar OS and Sharma MK. 2008. Expression of cytochrome P450

aromatase transcripts in buffalo (Bubalus bubalis)-ejaculated spermatozoa and its

relationship with sperm motility. Domestic Animal Endocrinology. 34: 238–249.

Zhang J, Li Y. 2004. Progress in researches on sperm antigen Fertilin beta. Zhonghua Nan Ke

Xue. 10: 52-54.

16.5 Current status of research and development in the subject (both international

and national status)

International status

The discovery that spermatozoal mRNA are delivered into the oocytes during

fertilization gives us a deeper insight into the molecular basics of embryo development

(Januchowski et al 2004). It has given an impression of spermatozoal mRNA as a complex

component of the ejaculated cell and hopefully dispelled any doubts as to the origin of this

mRNA. However spermatozoal RNA has obvious advantages over more traditional techniques

of fertility/infertility research and clearly the necessary groundwork has now been completed,

leaving the way open for more extensive independent investigations (Miller et al 2005). Most

of the studies have been carried out in human beings for identifying the genes associated with

motility and fertility. Very few reports are available in domestic animals such as pig (Jeong et

al 2009), horse (Das et al 2010) and cattle (D’Amours et al 2010) and only one report in

buffalo sperm RNA (Tiwari et al 2008). Recent studies in boar showed that the sperm

parameters and gene expression significantly decreased following cooling and freezing-

thawing process (Jeong et al 2009, Chanapiwat et al 2011). The success of freezing relies upon

an understanding of internal (inherent characteristics of spermatozoa, and differences between

bulls and ejaculates) and external factors and their interactions which can influence the

capacity of sperm to survive during freezing and thawing (Johnson et al 2000). Moreover, at

the time of thawing of frozen sperm, the temperature changes from -196 to +38 °C in 40-60 sec

triggers a rapid transition from solid to liquid and drastic changes in reactive oxygen species

(ROS) generation, resulting in protein damage, lipid peroxidation, cytoskeleton modification,

inhibition of cellular mechanisms, damage to DNA & RNA and ultimately poor motility and

fertility. For protection of sperm against excessive ROS generation during freezing-thawing

process, α-tocopherol, an antioxidant supplementation in boar semen extender had a positive

effect on post-thaw sperm viability, reducing lipid peroxidation and lowering the expression of

apoptosis genes. (Jeong et al 2009). Recently reported association of CRISP2, CCT8 and

PEBP1 mRNA abundance in sperm of Holstein bulls with sire conception rate, show that

greater CRISP2 and lower CCT8 mRNA expression in sperm had higher probabilities of siring

calves (Arangasamy et al 2011).

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Arangasamy A, Kasimanickam VR, DeJarnette JM and Kasimanickam RK. 2011. Association

of CRISP2, CCT8, PEBP1 mRNA abundance in sperm and sire conception rate in

Holstein bulls Theriogenology. 76: 570–577.

Chanapiwat P, Rho GJ, Tummaruk P and Kaeoket K. 2011. Sperm parameters and gene

expression of boar spermatozoa following cooling and freezing process. Proceedings of

the 5th Asian Pig Veterinary Society Congress 7-9 March 2011, Pattaya, Thailand, 023.

D’Amours O, Frenette G, Fortier M, Leclerc P and Sullivan R. 2010. Proteomic comparison of

detergent-extracted sperm proteins from bulls with different fertility indexes.

Reproduction. 39:545–556.

Das PJ, Paria N, Gustafson-Seabury A, Vishnoi M, Chaki SP, Love CC, Varner DD,

Chowdhary BP and Raudsepp T. 2010. Total RNA isolation from stallion sperm and

testis biopsies. Theriogenology. 74:1099-1106.

Januchowski R, Breborowicz AK, Ofori H, Jedrzejczak P, Pawelczyk L, Jagodzinski PP.

Detection of a short CCR5 messenger RNA isoform in human spermatozoa. J Androl.

2004; 25:757-760.

Jeong YJ, Kim MK, Song HJ, Kang EJ, Ock SA, Kumar BM, Balasubramanian S and Rho GJ.

2009. Effect of α-tocopherol supplementation during boar semen cryopreservation on

sperm characteristics and expression of apoptosis related genes. Cryobiology. 58: 181-

189.

Johnson LA, Weitze KF, Fiser P, Maxwell WM. 2000. Storage of boar semen. Anim Reprod

Sci. 62: 143–172.

Miller D, Ostermeier GC and Krawetz SA. 2005. The controversy, potential and roles of

spermatozoal RNA. Trends Mol Med. 11:156-163.




National status

In RNA isolation feasibility exploration studies our lab isolated RNA from equilibrated

and cryopreserved buffalo semen and checked the expression of housekeeping genes (Kumar et

al 2012). Tiwari et al 2008 reported the expression of cytochrome P450 in ejaculated buffalo

spermatozoa and its relationship with sperm motility of ejaculated spermatozoa through RT-

PCR using total RNA isolated from buffalo ejaculated spermatozoa. They concluded a positive

relation between aromatase transcript and mass motility of buffalo-ejaculated spermatozoa,

which could be a putative marker for the quality of semen in farm animals, particularly the

acquisition of sperm motility.

Kumar D, Mann A, Singh J, Yadav PS, Yadav SP, Singh P, Kumar P and Singh I (2012). Total

RNA isolation and expression of housekeeping genes in equilibrated and cryopreserved

buffalo semen. Indian Veterinary Journal. (Article no 523/11, To be published in

February 2012 issue).




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16.6 The relevance and expected outcome of the proposed study

This study will help in understanding the significance of mRNA in ejaculated and

frozen-thawed sperm and its relationship with motility, capacitation, fertility and possible

functional role during embryonic development. Effect of RNA stabilizer compound or

antioxidant effect on stabilization of mRNA pool during freezing-thawing process will be

evaluated to improve the sperm characteristics and fertility of sperm. It is expected that quality

of frozen semen and conception rate in buffalo will be improved losses due to lower

conception rate will be minimized.

16.7 Preliminary work done so far

This is entirely a new research area at this institute. Though, to explore the feasibility

we have successfully isolated the RNA from buffalo bull semen and amplified it for expression

of housekeeping genes. The semen freezing laboratory of this institute has state-of-the-art

building and instrumentation facilities for cryopreservation and maintenance of buffalo semen.

Studies are going on for improving cryopreservation of buffalo semen by addition of Trahalose

and Sericin and for preventing the backward motility of sperm through proper glycerol addition

in extender. Apart from this, we also evaluate the effect of cryopreservation on membrane and

DNA integrity of spermatozoa. We have also standardized various semen quality evaluation

techniques such as acrosome integrity by FITC-PSA, viability by SYBR-14/Propidium iodide

and sperm kinetics through CASA etc. This institute is the regulator of semen distribution for

all India coordinated project.

17. Specific objectives (should be written in bulleted form, a short paragraph indicating

the methods to be followed for achieving the objective and verifiable indicators of

progress should follow each specific objective):

To determine the effect of freezing-thawing process on mRNA pool of buffalo bull

semen:

The expression and levels of different transcripts coding for molecules involved in nuclear

condensation (Protamines), capacitation (nitric oxide synthase) and fertility (fertilin β) will be

evaluated in fresh and cryopreserved sperm using RT-PCR. The contamination by somatic and

germ cells will be also assessed by looking for specific molecular markers of these cells

respectively CD-45 and E-cadherin for somatic cells and c-kit for germ cells. The sperm

characteristics including sperm motility, kinetic parameters by CASA, sperm viability by

SYBR-14/Propidium iodide, acrosome integrity by FITC-PSA will be determined.

To maintain integrity of mRNA pool during freezing-thawing by addition of

suitable supplement(s) to extender and identify its association with sperm

characteristics and fertility.

Cryopreservation imposes major insult on spermatozoa via osmotic stress at freezing and

thawing. Purified RNA isolated from tissues is stable at -80ºC but thawing can result in its

degradation. For the prevention of degradation of mRNA, RNA stabilizing compounds

(okadain acid/hydroxyl stilbamidine isothionate/RNA later/other suitable compound) will be

supplemented in the extender. Sperm characteristics and fertility related parameters shall be

assessed by comparison of gene expression and CASA parameters.

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To correlate the gene expression and fertilizing ability of supplemented

cryopreserved semen through homologous IVF.

Homologous IVF will be performed to evaluate fertilizing ability and significance of sperm

mRNA in fertilization and early embryonic development.

18. Work Plan: should not exceed 3-4 pages (the section can be divided according to the

specific aims and under each specific aim; the following should be stated clearly as sub

headings)

18.1 Work plan (methodology/experimental design to accomplish the stated aim)

Semen collection, processing and cryopreservation:

Semen samples will be collected with artificial vagina technique from 3–6 years old

Murrah buffalo bulls (n=5) under semen collection and freezing programme at Semen Freezing

Lab., CIRB, Hisar. Semen will be subjected to routine physical examination, determination of

sperm concentration with Accucell photometer and motility assessment under warm-stage

phase-contrast microscope. Semen will then be extended in TRIS-egg yolk-glycerol and frozen

as per the standard procedure at the Semen Freezing Lab of the institute. All the required

parameters will be studied in fresh and after frozen-thawed semen.

Evaluation of sperm characteristics:

Fresh and frozen-thawed samples will be analyzed using a computer assisted semen

analyser (CASA; Hamilton-Thorn IVOS system). CASA also permit the measurement of

velocities, perentages motile sperm and their kinetics parameters in semen. Through CASA a

minimum of 200 spermatozoa per sample will be evaluated. Data collected will include the

percentage of motile spermatozoa (Mot), the percentage of progressively motile spermatozoa

(Prog), the average path velocity (VAP), the straight line velocity (VSL), the curvilinear

velocity (VCL) and the linearity (LIN) of the motile sperm cells.

Assessment of live and dead sperm:

The percentage of live and dead sperm will be assessed using LIVR/DEAD® sperm

viability kit and procedures as described by Koonjaenak et al (2007).

Assessment of acrosomal integrity:

The acrosomal integrity will be evaluated using fluorescently labeled lectin with Pisum

sativum agglutinin (FITC/PSA) or Coomassie Blue G-250 staining method as described by

Mehmood et al. 2008. Sperm possessing an intact acrosome do not get stain when incubated

with PSA, while PSA will be able to stain acrosome of damaged sperm. The PSA binds to α-

mannose and α-galactose moieties of the acrosomal matrix of damaged sperm, causing the

acrosomal region of these sperm to fluoresce.

Gene expression analysis:

Sperm purification:

All sperm samples will be processed by discontinuous gradient centrifugation for

separation of mature sperm from somatic cells, germ cells and immature diploid spermatocytes

as per described by Varner et al 2008. Purified sperm will be used for RNA extraction and

further studies.

RNA isolation and cDNA synthesis through RT-PCR assay:

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Total RNA will be extracted from purified sperm samples of fresh and frozen-thawed

groups using the Trizol/suitable kit method. RNA concentration will be measured by

absorbance at 260 nm in a Picodrop spectrophotometer. Sample that contain low-quality RNA

or contaminated with RNA from somatic cells will be removed from further study. Samples

containing genomic DNA will be treated with DNase. Finally all the genes of interest will be

amplified with specific primers.

Quantitative real-time PCR:

In order to quantify different transcripts level in fresh, frozen-thawed and supplemented

frozen-thawed buffalo bull sperm sample will be evaluated by real-time PCR method.

To maintain integrity of mRNA pool during freeze-thaw process by addition of

suitable supplements to extender

For the prevention of degradation of RNA during freezing-thawing process, RNA stabilizer

compound (okadain acid/RNA later/other suitable compound) or antioxidant (α-tocopherol @

100, 200 or 400µM) will be supplemented in the semen extender. The effect of

supplementation would be assessed by comparison of gene expression and sperm

characteristics of frozen-thawed spermatozoa of buffalo bull.

To correlate the gene expression and fertilizing ability of supplemented

cryopreserved semen through in vitro fertilization:

Cryopreserved semen with various supplements showing improved semen characteristics

will be used for the IVF studies to evaluate its fertilizing ability and significance of sperm

mRNA in fertilization and embryonic development.

Collection of oocytes and in vitro maturation

Buffalo ovaries will be obtained from abattoir and transported to the laboratory in an

insulated container in 0.9% normal saline containing antibiotics 400 IU /ml penicillin and 500

g/ml streptomycin at 32-37°C within 4-5 h. Oocytes will be collected by aspiration of surface

follicles (2-8 mm diameter) with a 19-gauge disposable needle attached to a 10 ml syringe in

the medium consisting of TCM-199 and 0.6% (v/w) bovine serum albumin (BSA). For oocyte

maturation, compact cumulus-oocyte-complexes (COCs) with homogenous evenly granular

ooplasm will be taken. The oocytes will be washed 4 times with the washing medium (TCM-

199 + 10% FBS + 0.81 mM sodium pyruvate + 50 µg/ml gentamycin sulphate) and then twice

with the IVM medium (TCM-199 + 10% FBS + 5 µg/ml porcine FSH + 1µg/ml estradiol-17β

+ 0.81mM sodium pyruvate + 5% buffalo follicular fluid + 50 µg/ml gentamycin sulphate).

The washed COCs will be then placed in 80-μl droplets (15-20 oocytes/droplet) of the IVM

medium, covered with sterile paraffin oil, in a 35 mm Petri dish and cultured for 24 h in a CO2

incubator (5% CO2 in air, 90-95% relative humidity) at 38.5°C. In vitro matured oocytes with

expanded cumulus cells will be subjected to in vitro fertilization (IVF).

Semen preparation and In vitro fertilization

Matured oocytes will be fertilized as described by Kumar et al (2007). Briefly, two

straws of frozen-thawed buffalo semen will be washed twice with washing BO medium (BO

medium containing 10 µg/ml heparin, 137.0 µg/ml sodium pyruvate and 1.942 mg/ml caffeine

sodium benzoate). The pellet will be re-suspended in 0.5 ml of the washing BO medium. The

matured oocytes will be rinsed in washing BO medium and transferred to 50 μl droplets (15-20

oocytes/droplet) of the capacitation and fertilization BO medium (washing BO medium

containing 10 mg/ml fatty acid-free BSA). The spermatozoa in 50 μl of the capacitation and

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fertilization BO medium will then be added to the droplets containing the oocytes, covered

with sterile mineral oil and placed in a CO2 incubator (5% CO2 in air) for 18 h at 38.5°C.

In vitro culture of zygote

After the end of sperm-oocytes incubation, the cumulus cells will be washed off the

oocytes by gentle pipetting. The oocytes will then be washed few times with the IVC medium

i.e. modified Charles Rosenkrans medium with amino acids (mCR2aa), as used earlier by

Kumar et al (2007). The medium will be replaced with 50% of fresh IVC medium every

alternate day. The cleavage rate will be recorded on day 2 post insemination and the

development to morula, blastocyst and hatched blastocyst stages will be recorded on days 5, 8

and 9 respectively post-insemination.

18.2 Connectivity of the participating institutions and investigators (in case of

multi- institutional projects only)

NA 18.3 Alternate strategies (if the proposed experimental design or method does not

work what is the alternate strategy): In the proposed study, we will use RNA stabilizing compound for maintaining

integrity of RNA pool during freezing-thawing process of sperm, if it will not work

than alternatively antioxidant will be used for reducing ROS generated during freezing-

thawing process.

19. Timelines: (Please provide quantifiable outputs)

Period of study Achievable targets

6 Months

Manpower recruitment and procurement of

equipments/chemicals/kits and consumables.

12 Months Processing of semen, sperm purification and evaluation through

CASA and other quality tests

18 Months RNA extraction, measurement of concentration, cDNA

preparation and amplification with specific gene primers and

quantities with real-time PCR.

24 Months Evaluation for difference in the level of transcript,

supplementation of RNA stabilizer compound or antioxidant and

check any difference in expression pattern

30 Months

In vitro maturation, fertilization and culture of oocytes and

embryos

36 Months Data analysis, compilation and submission of report.

20. Name and address of 5 experts in the field SNo Name Designation Address

1

Dr. M S Chauhan Principal Scientist Animal Biotechnology Centre,

NDRI, Karnal

2

Dr. R K Sharma Professor Deptt of Zoology, Kurukshetra

University, Kurukshetra

3

Dr. Pawan Singh Principal Scientist Artificial Breeding Centre, NDRI,

Karnal

4

Dr. Birbal Singh Senior Scientist IVRI, Regional Station, Palampur

5

Dr. S K Jindal Principal Scientist

& Head

Physiology and Reproduction

CIRG, Makhdoom

15

PART IV: BUDGET PARTICULARS

Budget (In Rupees)

A. Non-Recurring (e.g. equipments, accessories, etc.)

S.

No.

Item Year 1

(in Rs)

Year 2 Year 3 Total

(in Rs)

1 Gradient Thermal Cycler 5,50,000 -- -- 5,50,000

2 Electrophoresis apparatus with

accessories and power supply

2,00,000 -- -- 2,00,000

3 UV Tran-illuminator 50,000 -- -- 50,000

4 Refrigerated Centrifuge 3,00,000 -- -- 3,00,000

Sub-Total 11,00,000 11,00,000

Sub-Total (A) = 11,00,000/

B. Recurring

B.1 Manpower (See guidelines at Annexure-III)

S.

No

.

Position

No.

Consolidated

Emolument

Year 1

(in Rs)

Year 2

(in Rs)

Year 3

(in Rs)

Total

(in Rs)

1 Senior

research

fellow (1)

16,000-

18,000+10%

HRA

2,11,200/ 2,11,200/ 2,37,600

/

6,60,000

2 Skilled Project

Assistant (1)

8000/month 96,000/ 96,000/ 96,000/ 2,88,000

Sub-Total Change

accordingly

3,07,200/ 3,07,200/ 3,33,600 9,48,000/

Sub-Total (B.1) = 9,48,000/

B.2 Consumables

S.

No.

Item

Quantity Year 1

(in lakh)

Year 2

(in lakh)

Year 3

(in lakh)

Total

(in lakh)

1 Glass wares/plastic

wares

As per

requirements

1,50,000 1,00,000 50,000 3,00,000

2 Chemicals/kits 2,00,000 3,00,000 1,50,000 6,50,000

3 Miscellaneous-

outsourcing, primer

designing / services

1,00,000 50,000 50,000 2,00,000

Sub-Total 4,50,000 4,50,000 2,50,000 11,50,000

Sub-Total (B.2) =11,50,000/

Other items Consolidated

Emolument

Year 1 Year 2 Year 3 Total

B.3 Travel 50,000/ -- -- -- 50,000/

B.4 Contingency 1,50,000/ -- -- -- 1,00,000/

B.5 Overhead

(If applicable)

1,72,500/ 1,72,500/

Sub-total of B

(B.1+B.2+B.3+B.4+B.

5)

7,57,200 7,57,200 5,83,600 20,98,000

Grand Total (A + B) 3,72,500 18,57,200 7,57,200 5,83,600 35,70,500

Note: Please give justification for each head and sub-head separately mentioned in the above

table.

Financial Year: April - March

In case of multi-institutional project, the budget estimate to be given separately for each institution.

16

PART V: EXISTING FACILITIES

Resources and additional information

1. Laboratory:

a. Manpower

The scientists in the Division of Buffalo Physiology and Reproduction have vast

experience for semen evaluation, processing, freezing and conducting research

on various aspect of semen biology for improving cryopreservation and fertility.

The division also has trained attendants for handling the breeding bulls and

collection of semen. This division also has the expertise in molecular biology

and IVF work.

b. Equipments

Sr.

No. Name Year of

purchase

Manufacturer Status

1. Phase contract micro-scope 1992 Nikon working

2. Auto Filling and sealing

machine

1993 IMV working

3. Autoclave 1993 Yorco working

4. Digital water bath

(Thermostatic)

1993

2010

IMV

Sanco

working

5 Fluorescent microscope 2004 Olympus working

6. Straw Printing machine 2005 IMV working

7. Digital Photometer , IMV 2005 IMV working

8. Cold handling cabinet 2005 IMV working

9 CASA 2005 Hamilton

Thorne-IMV

working

10. AV sterilizer 2008 Yorco working

11. Biological freezer 2009 IMV working

12. Millipore water purification

system

2009 Milipore working

13. Electronic weighing

balance

2009 Citizen working

14. UPS -5KVA 2009 Synergy working

15. Pico drop 2009 Applied biosci working

16. PH Meter 2010 EUTECH working

17. DIC-Microscope 2011 Olympus working

18. Laminar air flow unit 2005, 2009 Atlantis working

2. Other resources such as clinical material, animal house facility, glass house.

Experimental garden, pilot plant facility etc.

CIRB, Hisar has the facilities of bulls and bull sheds and modern freezing laboratory for

conducting research.

17

18

PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS

Provide the following information for the key personnel in the order listed on PART II.

Follow this format for each person. DO NOT EXCEED THREE PAGES

Name : Dr. Dharmendra Kumar


Department : Buffalo Physiology and Reproduction Div.

Institute/University : Central Institute for Research on Buffaloes, Hisar

Date of Birth : 06/07/1977

Sex (M/F) : M SC/ST: NO

Education (Post-Graduation onwards & Professional Career)

Sl

No.

Institution

Place

Degree

Awarded

Year Field of Study

1 WBUAFS, Kolkata B.V.Sc &

A.H

2003 Animal And Veterinary

Science

2 NDRI, Karnal M.V.Sc 2005 Animal Biotechnology

3 NDRI, Karnal Ph.D 2008 Animal Biotechnology

A. Position and Honors

Position and Employment (Starting with the most recent employment)

Sl

No.

Institution

Place

Position From (Date) To (date)

1 NDRI, Karnal Research Associate 18/11/2008 18/04/2009

2 CIRB, Hisar Scientist 21/04/2009 Till date

Honors/Awards

Best poster award from Indian Society for the Study of Reproduction and fertility (ISSRF),

2006 at Karnal during symposium on frontiers in reproduction: concepts and applications in

genomic era.

Best poster award from Indian Society for Buffalo Development (ISBD), 2010 at New Delhi

during International buffalo conference on optimizing buffalo productivity through

conventional and novel technologies.

Best poster award from Society for Conservation of Domestic Animal Biodiversity (SOCDAB),

2010 at Anand during national symposium on “Challenges to domestic animal biodiversity &

action plan for its management and utilization”.

19

Professional Experience and Training relevant to the Project

Got training on ‘application of molecular biology techniques in reproductive biomedicine’

from Department of reproductive biomedicine, National Institute for Health and Family

Welfare, New Delhi, 18-29 October, 2010.

Attend training on ‘computational genome analysis uses ANVAYA’ at Centre for Agricultural

Bioinformatics, IASRI, New Delhi 22-24 June, 2011.

B. Publications (Numbers only) Research Papers: 14 (in peer reviewed journals)

Books: Nil Reports: Nil General articles: 3 (in English), 5 (in Hindi)

Patents: Nil Others (Please specify): Abstracts 6 (published in reviewed journal)

17 (published in symposium

compendium)

Selected peer-reviewed publications (Ten best publications in chronological order)

1. Kumar D, Mann A, Singh J, Yadav PS, Yadav SP, Singh P, Kumar P and Singh I

(2012). Total RNA isolation and expression of housekeeping genes in equilibrated and

cryopreserved buffalo semen. Indian Veterinary Journal. (Article no 523/11, to be

published in February 2012 issue).

2. Kumar D, Anand T, Singh KP, Singh MK, Shah RA, Chauhan MS, Singla SK Palta P,

and Manik RS (2011). Derivation of buffalo embryonic stem-like cells from in vitro-

produced blastocysts on homologous and heterologous feeder cells. J Assisted Reprod

Gen, 28: 679-688.

3. Anand T, Kumar D, Singh MK, Shah RA, Chauhan MS, Manik RS, Singla SK and

Palta P. (2011). Buffalo (Bubalus bubalis) embryonic stem cell-like cells and

preimplantation embryos exhibit comparable expression of pluripotency-related

antigens. Reprod Dom. Ani. 46(1):50-58.

4. Mukherjee A, Kumar D, Singh KP, Chauhan MS, Palta P and Manik RS. (2010).

Assessment of DNA damage during in vitro development of buffalo (Bubalus bubalis)

embryos: effect of cysteamine. Reprod Dom. Ani. 45: 1118-1121.

5. Saugandhika S, Kumar D, Singh MK, Shah R, Anand T, Chauhan MS, Manik RS,

Singla SK and Palta P. (2010). Effect of nitric oxide on in vitro development of buffalo

(Bubalus bubalis) embryos. Reprod Dom. Ani. 45 (5): 931-933. 6. Shah RA, George A, Singh MK, Kumar D, Anand T, Chauhan MS, Manik RS, Palta P and

Singla SK. (2009). Pregnancy Established from Handmade Cloned Blastocysts Reconstructed

using Skin Fibroblasts in Buffalo (Bubalus bubalis). Theriogenology. 71: 1215-1219.

7. RA Shah, George A, Singh MK, Kumar D, Chauhan MS, Manik RS, Palta P and Singla SK.

(2008). Handmade cloned buffalo (Bubalus bubalis) embryos: Comparison of different media

and culture systems. Cloning and Stem cells. 10 (4): 435-442.

8. Anand T, Kumar D, Chauhan MS, manik RS and Palta P. (2008). Cysteamine supplimentation

of in vitro maturation and / or culture media promote in vitro development of buffalo (Bubalus

bubalis) embryos. Reprod. Ferti. Dev. 20:253-257.

9. Kumar D, Anand T and Chauhan MS. (2008). Development of in vitro produced buffalo

embryos using simple media. Indian Veterinary Journal. 85 (8): 819-821.

10. Kumar D, Palta P, Manik RS, Singla SK and Chauhan MS. (2007). Effect of culture media

and serum supplementation on the development of in vitro buffalo embryos. Indian journal of

animal science. Vol-77 (8): 697-701.

20

List maximum of five recent publications relevant to the proposed area of work.

1 Kumar D, Mann A, Singh J, Yadav PS, Yadav SP, Singh P, Kumar P and Singh I


cryopreserved buffalo semen. Indian Veterinary Journal. (Article no 523/11, to be

published in February 2012 issue).

2 Saugandhika S, Kumar D, Singh MK, Shah R, Anand T, Chauhan MS, Manik RS,

Singla SK and Palta P. (2010). Effect of nitric oxide on in vitro development of buffalo

(Bubalus bubalis) embryos. Reprod Dom. Ani. 45 (5): 931-933.

3 Anand T, Kumar D, Chauhan MS, manik RS and Palta P. (2008). Cysteamine

supplimentation of in vitro maturation and / or culture media promote in vitro

development of buffalo (Bubalus bubalis) embryos. Reprod. Ferti. Dev., 20:253-257.

4 Kumar D, Anand T and Chauhan MS. (2008). Development of in vitro produced

buffalo embryos using simple media. Indian Veterinary Journal. 85 (8): 819-821.

5 Kumar D, Palta P, Manik RS, Singla SK and Chauhan MS. (2007). Effect of culture

media and serum supplementation on the development of in vitro buffalo embryos.

Indian journal of animal science. Vol-77 (8): 697-701.

C. Research Support Ongoing Research Projects

Sl

No.

Title of Project Funding

Agency

Amount Date of

sanction and

Duration

1 Effect of cryopreservation on integrity of buffalo

sperm membrane and DNA in relation to fertility ICAR 6.8 lakhs March 2010,

2 years

2 Leptin and its receptor gene polymorphism

and their association with milk production

traits in Murrah breed of buffalo (Bubalus

bubalis)

ICAR 8.0 lakhs March 2010,

3 years

Completed Research Projects (State only major projects of last 3 years)

Sl No. Title of Project Funding Agency Amount Date of

completion

Place: Signature of Investigator

Date:

21




Name : Dr. Prem Singh Yadav

Designation : Principal Scientist

Department : Buffalo Physiology & Reproduction Division


Address : CIRB Sirsa Road Hisar. (Haryana)

Pin : 125001

Telephone : 01662-276631, Fax 01662-275004


Date of Birth : 10.04.1963, Sex: Male, SC/ST: NO

Education (Post-graduation onwards) & Professional Career

Degree/ Certificate Year Institution Division/

Rank

Subject

DAAD Research Stay

2010

Dec.10 -

Feb 2011

Institute of Farm

Animal Genetics,

Germany

- iPS cells in bovines

(DBT Long- overseas

associate ship)

Nov. 2003

-Nov. 04

Institute of Animal

Sciences Germany

- Embryonic Stem Cells

(Animal

Biotechnology)

Doctorate (PhD) 1991 CCS HAU, Hisar

Haryana

First Animal Physiology

Master’s (MSc) 1987 HAU, Hisar

Haryana

First Animal Physiology

25. Research Experience in various institutions (if necessary, attach separate sheets).

Professional Record:

Position Institute Period

From To

Principal Scientist CIRB Hisar 2.2.2008 Continue

Senior Scientist CIRB Hisar 2.2.2000 1.2.2008

Scientist (Sr. Scale) CIRB Hisar 12.4.1998 2.2.2000

Scientist NAARM Hyderabad, IVRI

Izatnagar & CIRB Hisar,

12.4.1993 11.4.1998

AES(Animal Science) KVK, BARA, HPKVV Palampur 16.11.1992 6.4.1993

Training Assoc KVK Munger , R.A.U. Pusa Bihar 29.5.1991 2.11.1992

26. Publications (number only)

Research Papers 20 Research papers presented 23

Books Authored 1 Book Chapter Iowa USA 1

Technical Articles 28 Popular articles 9

Projects DBT as PI 1 Institute funded PI -3, Co- PI-4 Note: Principal Investigator and Co-Investigators should provide their bio0data in this format.

List of other ongoing projects/programmes aiming at rural upliftment/welfare: NIL

22

Honours and Awards: Best poster SOCDAB 2010 at Anand Gujrat, DBT Sponsored project

on Adult stem cells on Buffaloes 2007-2011 for Rs 67.13 lakhs, Book Title: Reproductive

biotechnology in Buffaloes. 2010 Authors: P SYadav B Singh, I Singh & RK Sethi, Course

Director, ICAR Sponsored Short Course on Application of Stem Cells in Livestock December,

10-19, 2007, Outstanding Young Scientist ,ISSAR 1996-1997, ICAR Sr. Fellowship - Ph. D.

Selected publications: 1. Yadav PS, A Mann, V Singh, S Yashveer, RK Sharma and I Singh 2010 Expression of

Pluripotency Genes in Buffalo (Bubalus bubalis) Amniotic Fluid Cells. Reprod. Dom. Anim. doi:

10.1111/j.1439-0531.2010.01733.x

2. Yadav PS et al 2011 Buffalo fetal fibroblastss differentiate in other lineages Submitted- Stem cells

3. Yousef Deneke, Trilok Nanda, P S Yadav, I Singh and G Prasad 2008 Buffalo oocyte vitrification

and their postthaw potential for in vitro fertilization. Indial Vety Journal 85 (8) 816-818.

4. Yadav P S, Inderjeet Singh and RK Sethi (2008). In vitro cleavage rates of homologous oocytes

for fertility evaluation of buffalo bulls Indial Vety Journal 85 (7) 723-25.

5. Yadav P S, M. Mukesh and Inderjeet Singh (2008). Sex ratio of in vitro derived bubaline embryos

determined by PCR based sexing. Indial Vety Journal 85 (7) 707-710.

6. Yadav PS Wilfried A. Kues, D. Herrmann, Joseph W. Carnwath and Heiner Niemann (2005)

Bovine ICM derived cells express the OCT-4 ortholog Mol. Reprod Dev. 72 (2) 182-190

7. Yadav P S, Inderjeet Singh and N N Pathak (2002). Effect of sera and hormones on in vitro

maturation cleavage and development of buffalo embryos. Bubalus Bubalis IV: 57-60.

Place: Hisar

Date: Signature of the Investigator

23




Name : Dr. Pradeep Kumar


Department : Buffalo Physiology and Reproduction

/Institute/University : Central Institute for Research on Buffaloes, Hisar


Sex (M/F) : M SC/ST: NO


Sl

No.

Institution

Place

Degree Awarded Year Field of Study

1. B. V. C.

Patna

M.V.Sc (Veterinary

Gynaecology and Obstetrics)

2006 Semen biology

2. I.V.R.I.

Izatnagar

Ph.D (Veterinary

Gynaecology and Obstetrics)

(Pursuing) Purification and

characterization of

pregnancy associated

glycoproteins.

D. Position and Honors


Sl No. Institution

Place


1 Central Institute for Research

on Buffaloes, Hisar, Haryana

Scientist 17. 05. 2010 continue

Honors/Awards

Professional Experience and Training relevant to the Project

1. M.V.Sc research work on semen biology.

2. Posted in semen freezing lab of CIRB, Hisar.

3. Attended winter School training on “Recent Advances in Endocrine Control of

Livestock Production and Reproduction organized by CAFT in Veterinary Physiology,

Division of Physiology & Climatology, IVRI, Izatnagar

4. Attended winter School training on “Advances in reproductive technologies to

augument fertility in farm animals organised by Animal Reproduction Division, IVRI,

Izatnagar.

24

B. Publications (Numbers only):

Books : 1.. Research Papers 5 Reports : .4 (Abstracts). General articles: .8

Patents : .........................Others (Please specify) :.....

Selected peer-reviewed publications (Ten best publications in chronological order)

1. Kumar Pradeep ( 2009). Applied Veterinary Gynaecology and Obstetrics (Ed).

International Book Distributing Company, Lucknow, ISBN: 81-8129-218-6.

2. Kumar Pradeep, Kumar, D., Singh, I., Yadav, P.S. (2011). Seminal plasma proteome:

promising biomarker for bull fertility. Agricultural Research (Springer)

(communicated).



cryopreserved buffalo semen. Indian Veterinary Journal. (accepted).

4. Kumar Pradeep, Roy G.P., Akhtar M.H., Pandey R.P., and R.K. Nirala. Effect of foot

and mouth disease vaccination on semen quality, quantity and preservabality in

Holstein Friesian Bulls. The Royal Veterinary Journal of India.

5. Kumar Pradeep, Roy G.P., Bharati, P.K.(2009). Influence of thawing temperature on

spermatozoan characteristics of Holstein Friesian bull semen and its conception

following single insemination. RVJI 5: 45-47.

6. Kumar Pradeep, Roy G.P., Akhtar M.H., Pandey R.P. and Kumar Nirbhay. Pseudo

pregnancy its management and therapy in Bitch Pashudhan.

7. Kumar pradeep: Pregnancy diagnosis in small ruminant. Pashudhan

8. Kumar pradeep: Transgenesis animal: their benefits to human welfare. Pashudhan

9. Kumar Pradeep, Bharati, P.K., Nirala, R. K., Shekhar, D., Kumar, R. Early pregnancy diagnosis of cattle in field condition. Livestock international


1. Kumar Pradeep, Kumar, D., Singh, I., Yadav, P.S. (2011). Seminal plasma proteome:

promising biomarker for bull fertility. Agricultural Research (Springer)

(communicated).



cryopreserved buffalo semen. Indian Veterinary Journal. (accepted).

3. Kumar Pradeep, Roy G.P., Akhtar M.H., Pandey R.P., and R.K. Nirala. Effect of foot

and mouth disease vaccination on semen quality, quantity and preservabality in

Holstein Friesian Bulls. The Royal Veterinary Journal of India.

4. Kumar Pradeep, Roy G.P., Bharati, P.K.(2009). Influence of thawing temperature on

spermatozoan characteristics of Holstein Friesian bull semen and its conception

following single insemination. RVJI 5: 45-47.

25

E. Research Support Ongoing Research Projects

Sl No. Title of Project Funding Agency Amount Date of sanction

and Duration

1. Effect of trehelose and sericin

on freezability of buffalo bull

semen

ICAR Sept, 2010

2. Identification of early

pregnancy biomarkers in

buffaloes by proteomics

analysis

ICAR Sept, 2010

Place: Hisar

Date: 11.11.11 Signature of Investigator

26




Name : Dr. Sadeesh.EM


Department : Buffalo Physiology and Reproduction

/Institute/University : Central Institute for Research on Buffaloes, Hisar


Sex (M/F) : M SC/ST: NA


Sl

No

Educational

qualification

Major subject Year of

passing

University

1 BVSc & AH Veterinary and

Animal sciences

2007 KAU, Kerala

2 MVSc Animal

Biochemistry

2009 NDRI, Karnal

3 Ph.D. Animal

Biochemistry

Temporary

dropping

IVRI, Izatnagar

F. Position and Honors


Sl No. Institution

Place


1 CIRB Hisar Scientist Till Date

Honors/Awards

1. ICAR Junior Research Fellowship-2007 (JRF), New Delhi qualified for M.V.Sc in Animal

Biochemistry.

2. CSIR National Eligibility Test-2009 (NET), New Delhi qualified in Life Sciences.

3. ICAR National Eligibility Test-2009(NET), New Delhi qualified in Animal Biochemistry.

Publications (Numbers only): 4

Research Papers: 3

General articles: 1

27

Selected peer-reviewed publications

1. Ramesh.D, Sadeesh.EM, R.Deb, B.Sailo, V.K.Saxena, V.G.Joshi and Yosef.D

(2010), Prediction of MHC-1 binding epitopes in a gene fragment encoding 183

amino acids of Mycobacterium bovis Bacillus Calmette-Guerin (BCG) strain.

Biotechnology International, 3(2&3):29-32.

2. Rajib Deb, Ramesh D, V.K.Saxena, D.Thakuria, Sadeesh.EM (2010). Insilico based

structural prediction and identification of nanomeric CTL epitopes in 34.9kDa PPE

protein of Mycobacterium Avium Para tuberculosis for diagnostic and or subunit

vaccine design. Bioinformatics trends, 5(3&4):88-92

3. Pratheesh.MD, Anandalakshmi.N, Deb.R, Sadeesh.EM, Justin Davis, Harish.C,

Ramesh.D. Transfection of bIFN-t EGFP gene construct in goat fibroblast cell by

nucleofection technique. Indian veterinary journal (in press).


G. Research Support Ongoing Research Projects

Sl No. Title of Project Funding Agency Amount Date of sanction

and Duration

Completed Research Projects (State only major projects of last 3 years)

Sl No. Title of Project Funding Agency Amount Date of

completion

Place: Hisar Signature of Investigator

Date: 11/11/11