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

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1 Vol. 11, No SHORT COMMUNICATION 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 higher (p 0.05) in 0.25 ml than 0.5 ml straw, thawed at 37 C either for 30 or 60 sec. In conclusion, cryopreservation of buffalo semen in 0.25 ml straw resulted in a higher post-thaw quality. Reproductive Biology : Key words: buffalo bull spermatozoa, straw size, thawing time, cryopreservation 1 Corresponding autor: Animal Physiology Laboratory, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rwalpindi-46300, Pakistan; sashraf1993@gmail.com Copyright 2011 by the Society for Biology of Reproduction

2 50 Straw size and semen quality 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 and fertility of semen, the artificial insemination industry continuously modified semen packaging methods from bottles, bags, tubes, ampules to straws of different sizes. It is known that the cryopreservation of semen in mini-straws (0.25 ml) increases the number of doses and storage efficiency in liquid nitrogen and decreases extender and antibiotics costs [4]. Information 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 of straw size (0.25 vs. 0.5 ml) and thawing time (30 vs. 60 sec at 37 C) on 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 of known fertility and similar age (7 8 years) with an artificial vagina (42 C) at weekly intervals for three weeks (replicates). Ejaculates were transferred to the laboratory immediately for initial evaluation (volume, motility, concentration). This took six hours and 40 minutes from semen collection to final freezing and storage. This included semen collection, initial evaluation (volume, motility and concentration), dilution, post-dilution motility evaluation, 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. The same operator assessed sperm motility. Qualified semen ejaculates

3 Ansari et al. 51 were diluted with the extender. The extender consisted of 1.56% citric acid (Fisher Scientific, UK), 3% tris (hydroxymethyl)-aminomethane (Research Organics, USA), 0.2% fructose (Scharlau, Spain), 7% glycerol (Riedel-deHaen, Germany) and 20% egg yolk in distilled water (ph 7.0). The antibiotics streptomycin sulphate (1mg/ml), procaine penicillin (300 IU/ ml), benzyl penicillin (100 IU/ml) were added to the extender [1]. At least one ejaculate from each bull per week qualified [motility (>60%), volume (>1 ml) and concentration ( spermatozoa/ml)] for further processing. Semen aliquots were diluted with extender ( motile spermatozoa/ ml; 37 C), cooled to 4 C in 2 h and equilibrated for 4 h at 4 C. After equilibration, semen was filled in 0.5 ml and 0.25 ml French straws with a suction pump at 4 C in a cold cabinet unit and kept on liquid nitrogen vapors (5 cm) for 10 min. Straws were then plunged into liquid nitrogen (-196 C) for storage. After 24 h, three straws of each size were thawed at 37 C for 30 or 60 sec and assessed for motility, plasma membrane integrity and viability. A drop of semen sample was placed on a glass slide (37 C), cover slipped 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 solution, 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 least five different fields. Clear heads and tails as well as swollen tails were 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 trypanblue and semen were placed on glass slides, mixed quickly and air-dried before the fixation in formaldehyde-neutral red for 5 min. Then the slides were rinsed in running distilled water before Giemsa stain (7.5%) was applied for 4 h. The slides were rinsed, air-dried and mounted with Balsam

4 52 Straw size and semen quality of Canada. Trypan-blue penetrated non-viable, dead spermatozoa with a disrupted membrane, which appeared stained in blue, whereas live and intact spermatozoa appeared unstained. Giemsa stain accumulated in spermatozoa with an intact acrosome (staining the acrosome region in purple). One hundred spermatozoa were evaluated in at least five different fields in each smear under phase contrast microscope at The data is presented as means ±SD. The effects of straw size and thawing time on motility, plasma membrane integrity and viability were analyzed by one-way analysis of variance (ANOVA). When the F ratio was found significant (p<0.05), Duncan s Multiple Range test was used to compare different treatment means (MINITAB Release 12.22, 1998). 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 be attributed to slower cooling in the straw centre [8]. 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 combination with the hypo-osmotic swelling test, indicates the functional as well as structural status of the plasma membrane and is highly related to fertility [7]. In the present study, higher (p<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 effective way to predict the fertilizing ability of buffalo bull spermatozoa [7].

5 Ansari et al. 53 Table 1. Effect of straw size and thawing time on motility, plasma membrane integrity and viability of cryopreserved buffalo bull semen. Straw size (ml) Thawing time (s) Motility (%) Plasma membrane integrity (%) Viability (%) ± 2.9 a 59.0 ± 2.6 a 79.3 ± 3.2 a ± 3.0 a 57.0 ± 1.0 a 77.0 ± 1.0 a ± 3.0 b 44.3 ± 2.1 b 65.3 ± 3.1 b ± 2.9 b 45.7 ± 1.5 b 64.3 ± 3.2 b The values with different superscripts differ significantly (p 0.05) in the same column 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 Andrabi SMH 2009 Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reproduction in Domestic Animals Anzar M, Rasul Z, Ahmed TA, Ahmad N 2010 Response of buffalo spermatozoa to low temperatures during cryopreservation. Reproduction, Fertility and Development

6 54 Straw size and semen quality 4. Johnson MS, Senger PL, Allen CH, Hancock DD, Alexander BM, Sasser RG 1995 Fertility of bull semen packaged in.25 and.5-milliliter French straws. Journal of Animal Science Kovacs A, Foote RH 1992 Viability and acrosome staining of bull, boar and rabbit spermatozoa. Biotechnica and Histochemistry Senger PL, Mitchell JR, Almquist JO 1983 Influence of cooling rates and extenders upon post-thaw viability of bovine spermatozoa packaged in.25-ml and.5-ml French straws. Journal of Animal Science Tartaglione CM, Ritta MN 2004 Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen. Theriogenology Weitze KF, Rath D, Baron G 1987 Deep freezing of boar semen in plastic straws. (in German). Deustche Tierärtzliche Wochenschrift