6
Vol. 11, No. 1 49 Copyright © 2011 by the Society for Biology of Reproduction Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen Muhammad S Ansari, Bushra A. Rakha, Syed M. H. Andrabi, Shamim Akhter 1 Animal Physiology Laboratory, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan Received: 10 September 2010; accepted: 5 January 2011 SUMMARY This study was designed to compare the effect of straw size (0.25 vs. 0.5 ml) and thawing time (30 vs. 60 sec) on the quality of cryopreserved buffalo bull semen. Sperm motility, plasma membrane integrity and viability were 30 or 60 sec. In conclusion, cryopreservation of buffalo semen in 0.25 ml straw resulted in a higher post-thaw quality. Reproductive Biology 2011 11 1: 49–54. Key words : buffalo bull spermatozoa, straw size, thawing time, cryopreser- vation 1 Corresponding autor: Animal Physiology Laboratory, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rwalpindi-46300, Pakistan; e-mail: [email protected] SHORT COMMUNICATION

Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen

  • Upload
    shamim

  • View
    216

  • Download
    1

Embed Size (px)

Citation preview

Page 1: Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen

Vol. 11, No. 1 49

Copyright © 2011 by the Society for Biology of Reproduction

Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus

bubalis) semenMuhammad S Ansari, Bushra A. Rakha, Syed M. H. Andrabi,

Shamim Akhter1

Animal Physiology Laboratory, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan

Received: 10 September 2010; accepted: 5 January 2011

SUMMARY

This study was designed to compare the effect of straw size (0.25 vs. 0.5 ml) and thawing time (30 vs. 60 sec) on the quality of cryopreserved buffalo bull semen. Sperm motility, plasma membrane integrity and viability were '��' ����¿�#�#F$����#�"F�����'���#�F���������%��'�� �����X\��� ��' �����30 or 60 sec. In conclusion, cryopreservation of buffalo semen in 0.25 ml straw resulted in a higher post-thaw quality. Reproductive Biology 2011 11 1: 49–54.Key words: buffalo bull spermatozoa, straw size, thawing time, cryopreser-vation

1Corresponding autor: Animal Physiology Laboratory, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rwalpindi-46300, Pakistan; e-mail: [email protected]

SHORT COMMUNICATION

Page 2: Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen

Straw size and semen quality50

INTRODUCTION

There are numerous factors that may affect the motility, plasma membrane integrity and viability of buffalo bull semen during the storage e.g. type of extender, permeable and non-permeable cryoprotectants, packaging system or freezing and thawing time [2]. Considering cost effectiveness and saving storage space without compromising the post-thaw quality ����� ����������� � �%��' �������������� ����������������������������� ��� � ������������� �'����������� �%�����%��� �%����� ����straws of different sizes. It is known that the cryopreservation of semen �������=��������#�"F���$����� �� ���' ���� ������ ����������� � ���� ����in liquid nitrogen and decreases extender and antibiotics costs [4]. Informa-tion on the impact of mini vs. medium size straw and different thawing time on post-thaw semen quality of buffalo bull semen is lacking and needs to be investigated. Therefore, this study was designed to compare the effect ����������� ��#�"F�����#�F���$������'��������� ��X#�����>#�� �����X\��$���post-thaw motility, plasma membrane integrity and viability of Nili-Ravi buffalo bull spermatozoa.

MATERIALS AND METHODS

Semen was collected in the winter from three Nili-Ravi buffalo bulls �������� ���������������������� ��\�?�� ���$����'����������������������["��$�at weekly intervals for three weeks (replicates). Ejaculates were transferred to the laboratory immediately for initial evaluation (volume, motility, con-centration). This took six hours and 40 minutes from semen collection to ������� �������������� ��Z'�������� ��� � ����� ����%��������� ��������(volume, motility and concentration), dilution, post-dilution motility evalu-ation, cooling, equilibration, packaging, pre-freezing motility evaluation, deep freezing and storege.

Sperm motility (%) was assessed with phase contrast microscope at 200× and sperm concentration was measured with Neubauer hemocytometer. Z' ���� �� �������� �� ���� ������������À���� ��� � �� ������ ��

Page 3: Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen

Ansari et al. 51

were diluted with the extender. The extender consisted of 1.56% citric acid (Fisher Scientific, UK), 3% tris��'������ �'��$=����� �'�� ��� � ���'���������%����$%�#�"K������ ����'����%������$%�\K����� ������ � �=� �� �%�� �����$�����"#K� ����������������� ����� ������\�#$��The antibiotics streptomycin sulphate (1mg/ml), procaine penicillin (300 IU/ml), benzyl penicillin (100 IU/ml) were added to the extender [1]. At least � � ������ ����� ��'������ ��� ��*���� ��}���������º>#K$%���� ��º!���$�������� ���������#�F�!#9 spermatozoa/ml)] for further processing.

Semen aliquots were diluted with extender (50×106 motile spermatozoa/����X\��$%��� ����[������"�'����� *������� �����[�'����[������� �� *���-������%�� � ��������� �����#�F��������#�"F������ ��'�����������'����������������[����������������� ����������� �������*�������� ���������F���$����!#�������������� � ��' ������ ��������*�������� ���=!Y>��$��������� ����� ��"[�'%��'� ���������� ��'���� �� � ��'�� �����X\������X#���60 sec and assessed for motility, plasma membrane integrity and viability. ��������� � ������� ��������� ���������������� ��X\��$%��� ������� ��and assessed for sperm motility under phase contrast microscope (400×).

Sperm plasma membrane integrity was evaluated using the supravital hypo-osmotic swelling (HOS) test. After the semen incubation in HOS solu-tion, equal drops of the HOS solution and eosin [0.5% (w/v); sodium citrate 2.92%] were placed on a warm slide, mixed for 10 sec and cover slipped before the evaluation for plasma membrane integrity under phase contrast microscope at 400×. A total of one hundred spermatozoa were observed in at � ������ ����� � ���� ������� ���' ������������������ ���������� ��������� � �considered as intact with biochemically active sperm membranes, while pink heads and tails and unswollen tails were considered as disrupted, inactive sperm membranes. Sperm viability (live sperm with intact acrosome) was assessed by dual staining procedure [5]. Supravital stain trypan-blue was used to distinguish live and dead spermatozoa while Giemsa stain was used to evaluate the integrity of the acrosome membrane. Equal drops of trypan-blue and semen were placed on glass slides, mixed quickly and air-dried � �� ��' ����������������� '�� =� ������ �����F������Z' ���' ����� ��� � ����� ������������������� ����� ��� �� ��� �����������\�FK$�������-plied for 4 h. The slides were rinsed, air-dried and mounted with Balsam

Page 4: Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen

Straw size and semen quality52

of Canada. Trypan-blue penetrated non-viable, dead spermatozoa with a dis-rupted membrane, which appeared stained in blue, whereas live and intact spermatozoa appeared unstained. Giemsa stain accumulated in spermato-zoa with an intact acrosome (staining the acrosome region in purple). One '��� ���� �������� � � ����� ��������� ������ ����� � ���� ������� ��'�smear under phase contrast microscope at 1000×.

The data is presented as means ±SD. The effects of straw size and thaw-ing time on motility, plasma membrane integrity and viability were analyzed ���� =���������������������� �����{�$���' ���' �����������������������������<0.05), Duncan’s Multiple Range test was used to compare different treatment means (MINITAB®�� � �� �!"�""%�!YY?$�

RESULTS AND DISCUSSION

In the present study, the motility of buffalo bull semen cryopreserved in 0.25 ml straw was higher (p<0.05) than that of 0.5 ml straw, independently of thawing time (30 or 60 sec; tab. 1). Similar thawing times did not affect the motility of buffalo bull spermatozoa cryopreserved in 0.5 ml straw [2]. As in boars, a large volume of the straw resulted in poor motilities that may � �������� ������� ������������' �������� ��� �}?~�

It is known that a structurally and biochemically active plasma membrane is required to accomplish the processes of capacitation, acrosome reaction and the oocyte penetration. In this study the sperm plasma membrane integrity was assessed through the HOS test that has been recognized as a reliable procedure for the evaluation of the functional status of the sperm plasma membrane. In this technique supravital eosin stain used in combina-tion with the hypo-osmotic swelling test, indicates the functional as well as structural status of the plasma membrane and is highly related to fertility }\~��̀ ���' ��� � �������%�'��' ����<0.05) sperm plasma membrane integrity (tab. 1) was recorded in 0.25 ml straw compared to 0.5 ml straw thawed either for 30 or 60 sec.

Sperm viability assay (live sperm with intact acrosome) is an effec-��� ��������� ������' �� �������������������������������� �������}\~��

Page 5: Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen

Ansari et al. 53

It is well documented that cold-shock reduces the percentage of buffalo sperm with intact acrosome after cryopreservation [3]. It is believed that for bull spermatozoa a higher post-thaw recovery of viable spermatozoa may be obtained in 0.25 ml straw by optimizing cooling procedures, rapid thawing and handling techniques compared to 0.5 ml straw [6]. Similarly, we observed in a buffalo bull higher (p<0.05) sperm viability in 0.25 ml straw compared to 0.5 ml straw independently of thawing time (tab. 1). It should be emphasized that data on viability is supported by plasma membrane integrity data. In conclusion, cryopreservation of Nili-Ravi buffalo bull semen in 0.25 ml straw resulted in a higher post-thaw quality of spermatozoa than that of 0.5 ml straw, independently of the examined thawing times.

REFERENCES

1. Akhter S, Ansari MS, Andrabi SMH, Ullah N, Qayyum M 2008 Effect of antibiotics in extender on bacterial and spermatozoal quality of cooled buffalo (Bubalus bubalis) bull semen. Reproduction in Domestic Animals 43�"\"�"\?�

2. Andrabi SMH 2009 Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reproduction in Domestic Animals 44�FF"�F>Y�

3. Anzar M, Rasul Z, Ahmed TA, Ahmad N 2010 Response of buffalo spermatozoa to low temperatures during cryopreservation. Reproduction, Fertility and Development 22�?\!�??#�

Table 1. Effect of straw size and thawing time on motility, plasma membrane integrity and viability of cryopreserved buffalo bull semen.

Straw size(ml)

Thawingtime (s)

Motility(%)

Plasma membraneintegrity (%)

Viability(%)

0.25 30 >!�\�­�"�Ya 59.0 ± 2.6a \Y�X�­�X�"a

60 >!�\�­�X�#a F\�#�­�!�#a \\�#�­�!�#a

0.5 30 [?�X�­�X�#b 44.3 ± 2.1b 65.3 ± 3.1b

60 [>�\�­�"�Yb [F�\�­�!�Fb 64.3 ± 3.2b

Z' ���� �����'����� � ����� ������������� �����������������¿#�#F$�����' ���� �����

Page 6: Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen

Straw size and semen quality54

4. Johnson MS, Senger PL, Allen CH, Hancock DD, Alexander BM, Sasser RG 1995 � ��������������� � �������� ������"F��������F=�������� ���� ��'���������Journal of Animal Science 73�!Y![�!Y!Y�

5. Kovacs A, Foote RH 1992 Viability and acrosome staining of bull, boar and rabbit spermatozoa. Biotechnica and Histochemistry 67�!!Y�!"[�

6. Senger PL, Mitchell JR, Almquist JO 1983�`�� �� ������������ ������ �� �� ���upon post-thaw viability of bovine spermatozoa packaged in .25-ml and .5-ml French straws. Journal of Animal Science 56 1261.

\�� Z�������� ��|%�������|��2004 Prognostic value of spermatological parameters as pre-dictors of in vitro fertility of frozen-thawed bull semen. Theriogenology 62�!"[F�!"F"�

?�� � ��� �¥�%����'��%��������1987 Deep freezing of boar semen in plastic straws. (in German). Deustche Tierärtzliche Wochenschrift 94�[?F�[??�