EFFECT OF EXTENDER AND EXTENSION RATE ON ACROSOMAL INTEGRITY OF MALABARI BUCK SPERMATOZOA

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1 Journal of Cell and Tissue Research Vol. 15(3) (2015) (Available online at ISSN: ; E-ISSN: Original Article EFFECT OF EXTENDER AND EXTENSION RATE ON ACROSOMAL INTEGRITY OF MALABARI BUCK SPERMATOZOA? LEKSHMI BHAI, K., 1 METILDA, J., 2 BEHERA, S., 3 HARSHAN, H. M., 4 ARAVINDA GHOSH, K. N. 5 AND RAGHAVAN, K. C. 6 Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur , Kerala. E. mail: bhai04vet@gmail.com, Cell: Dept. of Animal Reproduction, Gynaecology and Obstetrics, CV&AS, Pookode , Wayanad; 2,4,5 Dept. of Animal Reproduction, Gynaecology and Obstetrics; 3 Dept. of Veterinary Gynaecology and Obstetrics, Veterinary College Hebbal, Banglore; 6 Centre for advanced studies in Animal Genetics and breeding Received: July 8, 2015; Accepted: August 10, 2015 Abstract: The objective of the study was to find out the effect of extension rate on the acrosomal integrity of Malabari buck spermatozoa in two different extenders. 48 ejaculates collected from two Malabari bucks were pooled and divided into four groups. Semen in group I and II were extended with egg yolk based extender and Andromed respectively at the rate of 400 million/ml. Extension rate of group III and IV was 800 million/ml with egg yolk based extender and Andromed, respectively. Intactness of acrosome, assessed prior to freezing and 24 hours after freezing showed better results at the extension rate of 400 million/ml with both the extenders. Andromed showed better acrosomal integrity at both the extension rates. Key words: Malabari buck, Acrosomal integrity INTRODUCTION Regarding the longevity and transportability, frozenthawed semen is more advantageous than fresh and refrigerated semen. At the same time, the success of getting good quality frozen thawed semen is possible only by minimising the cryodamage of spermatozoa. Type of extender and mode of extension determine the degree of cryodamage to a certain extent. The low density lipoprotein in the egg yolk prevents loss of membrane phospholipids during freezing process and thereby reduces cryodamage [1,2]. The seminal plasma of buck is said to be containing an egg yolk coagulating enzyme of bulbourethral gland origin [3] which hydrolyses egg yolk lecithin resulting in detrimental effects to the spermatozoa. The plant derived lecithin from soybean which proved its protective role during freezing in different species [4-7] can be used as an alternative of egg yolk. Also, the less microbial contamination reported with soybean lecithin based extender as compared to the extenders containing animal product [8], supports this substitution. The concentration of spermatozoa per dose is also an important factor influencing the freezability [9] and fertility of frozen buck semen [10]. MATERIALS AND METHODS Double ejaculate regime was followed for semen collection thrice weekly from two Malabari bucks, maintained at the Artificial Insemination centre, Dept. of Animal Reproduction, Gynaecology and 5169

2 J. Cell Tissue Research Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, Thrissur. Prior to the semen collection, extenders were prepared in such a way that each 100 ml of egg yolk based extender contained g Tris hydroxy methyl amino methane, g citric acid, 1.25 g fructose, 5ml egg yolk, 6 ml glycerol, 100 mg streptomycin and 1 lakh IU benzyl penicillin. Soybean lecithin based extender, Andromed (Minitub, Germany) working solution was prepared as per the directions from the manufacturer. Immediately after collection, sperm concentration was determined using haemocytometer. After the initial evaluation, semen samples with more than 80% initial motility were divided into 4 groups. Samples of group I and II were extended with egg yolk based and soyabean lecithin based extenders respectively at the rate of 400 million motile spermatozoa per ml whereas those of group III and IV were extended with egg yolk based and soyabean lecithin based extenders respectively at the rate of 800 million/ml. Glycerolisation of semen samples in group I and III was done in 3 steps after attaining 5ºC within the cold handling chamber. Samples in all the groups were filled manually in precooled 0.5 ml sterile French straws and undergone equilibration at 5ºC for 2 hours. Conventional mode of freezing was adopted in which the straws were exposed to liquid nitrogen vapour for 10 minutes and then plunged into liquid nitrogen. Acrosomal integrity of spermatozoa in each group was assessed by Giemsa staining technique [11] at the time of initial evaluation, prior to freezing and 24h after freezing. Giemsa stock solution was already prepared with 1g Giemsa stain powder, 66ml methanol and 60ml glycerol and kept for one week ripening with intermittent mixing. Just before the staining procedure, Sorenson s 0.1M buffer was prepared by mixing 17 ml solution A ( g potassium dihydrogen phosphate/100ml) and 33 ml of solution B ( g disodium hydrogen ortho phosphate/100ml). Hancock s fixative (1g Sodium chloride, 0.05g Sodium bicarbonate and 12.5 ml formalin per 100ml) was used to fix the smears for 15 minutes followed by washing in running tap water for 10 minutes. After staining with Giemsa working solution (6 ml Giemsa stock solution, 4 ml Sorenson s 0.1 M phosphate buffer and 90 ml distilled water) for 150 minutes at 37ºC, the slides were washed with distilled water and air dried. A minimum of 200 sperms were screened for acrosomal intactness from different microscopic fields under an oil immersion objective of a light microscope. SPSS (Statistical package for social studies) software was used for data analysis. Percentage data were transformed using Arcsine prior to analysis. Treatment means were compared using one way ANOVA [12]. Duncan s multiple range test (DMRT) was used for comparing the treatment means wherever needed. RESULTS Mean percentage of spermatozoa with intact acrosomes in fresh semen was ± Spermatozoa with swollen and detached acrosomes were of 4.21 ± 0.83 per cent and 1.13 ± 0.10 per cent, respectively. The status of acrosomal integrity of buck spermatozoa is expressed in Table.1, Fig. 1 and Fig. 2. During prefreeze evaluation, percentage of spermatozoa with intact acrosomes were higher in Malabari buck semen extended with Andromed compared to Tris egg yolk based extender at the dilution rate of 400 (91.60 ± 1.09% vs ± 1.18%) and 800 (89.54 ± 1.38% vs ± 1.99%) million motile spermatozoa per ml. But, there was no significant difference between the groups. Out of the damaged acrosomes, both swollen and detached acrosomes were less in Andromed compared to egg yolk based extender (7.3 ± 1.20% vs 8.2 ± 1.22%; 1.17 ± 0.20% vs 1.93 ± 0.43%) at the sperm concentration of 400 million/ml. Here also, no significant difference was noticed. When the sperm concentration was changed to 800 million, swollen acrosomes showed no significant difference in Andromed (8.88 ± 1.32%) and egg yolk based extender (11.52 ± 1.91%), whereas the percentage of completely detached acrosomes were significantly (p<0.05) higher (1.58 ± 0.27% vs 2.53 ± 0.36%) in egg yolk based extender. Even if concentration of spermatozoa had no significant effect on the percentage of sperms with intact acrosomes, the 5170

3 Lekshmi et al. Acrosomal integrity Prefreeze Post thaw Group I Group II Group III Group IV Fig. 1: Effect of extender and rate of extension on acrosomal integrity of spermatozoa in Malabari buck semen (n=12). Group I- Egg yolk based extender with 400 million sperms/ml, Group II- Andromed with 400 million sperms/ml, Group III- Egg yolk based extender with 800 million sperms/ml, Group IV- Andromed with 800 million sperms/ml. D I Fig. 2: Giemsa stained smear of buck semen showing spermatozoa with intact (I) and detached (D) acrosomes (1000 ) 5171

4 J. Cell Tissue Research Table 1: Effect of extender and sperm concentration per dose on pre-freeze and post- thaw acrosomal integrity of spermatozoa in Malabari buck semen, per cent (n= 12). Stage of evaluation of acrosomal integrity Prefreeze Post- thaw Acrosomal status Group I Group II Group III Group IV (Mean ± SE) (Mean ± SE) (Mean ± SE) (Mean ± SE) F value Intact ± 1.18 a ± 1.09 a ± 1.99 a ± 1.38 a Swollen 8.20 ± 1.22 a 7.30 ± 1.20 a ± 1.91 a 8.88 ± 1.32 a Detached 1.93 ± 0.43 a 1.17 ± 0.20 a 2.53 ± 0.36 b 1.58 ± 0.27 a * Intact ± 1.46 b ± 0.94 a ± 2.12 c ± 1.23 a ** Swollen ± 1.58 b 9.20 ± 0.87 a ± 2.12 c ± 1.24 a ** Detached 2.73 ± 0.32 a 1.68 ± 0.30 a 3.50 ± 0.58 b 3.06 ± 0.37 b * Group I- Egg yolk based extender at 200 million sperms, Group II- Andromed at 200 million sperms, Group III- Egg yolk based extender at 400 million sperms, Group IV- Andromed at 400 million sperms. * Significantly different at 0.01 level (p<0.01), * Significantly different at 0.05 level (p<0.05). Values with different superscripts within row differ significantly acrosomal damages were less at the sperm concentration of 400 million/ml. After freezing and thawing, the semen preserved in andromed showed significantly higher percentage (p<0.01) of spermatozoa with intact acrosomes than in Tris egg yolk based extender at the dilution rate of 400 million (89.12 ± 0.94% vs ± 1.46%) and 800 million (84.98 ± 1.23% vs ± 2.12%) motile spermatozoa per ml. Percentage of spermatozoa with swollen acrosomes differed significantly (p<0.01) in Tris egg yolk based extender and andromed at 400 million (13.64 ± 1.58% and 9.20 ± 0.87%) and 800 million (25.99 ± 2.12% and ± 1.24%) spermatozoa per ml. Percentage of detached acrosomes was not significantly different in Tris egg yolk based extender and Andromed with the sperm concentration of 400 million (2.73 ± 0.32% and 1.68 ± 0.30%) and 800 million (3.50 ± 0.58% and 3.06 ± 0.37%) spermatozoa per ml. There was no significant effect of sperm concentration on percentage of sperms having intact and swollen acrosomes in the Andromed extender. Sperm concentration of 400 million/ml showed significantly lower (p<0.05) percentage of sperms with detached acrosomes in both the extenders. DISCUSSION In the present study, it was observed that percentage of spermatozoa with intact acrosomes were higher in soyabean lecithin based extender compared to egg yolk based extender at the extension rates of 400 million/ml and 800 million/ml. This finding is in agreement with that of Aboagla and Terada findings [13] where it was noticed that combination of egg yolk and glycerol significantly reduced the percentage of sperms with intact acrosomes. Ritar and Salamon [14] also suggested that egg yolk could reduce post- thaw acrosomal integrity of spermatozoa in buck semen. The reason for more acrosomal damage of spermatozoa in Tris egg yolk based extender might be the calcium ions of egg yolk, which rapidly enter the cells when the temperature is below 30ºC [5]. It was also noticed that the artefacts in the Giemsa stained smears of semen extended with egg yolk based extender interfered the evaluation compared to andromed. This may be due to the reaction of egg yolk with formaldehyde in the Hancock s fixative forming particles that adhere to the plasma membrane of acrosome. Even though higher values of sperms with intact acrosomes were observed with the sperm concentration of 400 million/ml, concentration of spermatozoa had no significant effect on the percentage of intact acrosomes in both the extenders. Presence of high content of egg yolk like phospholipids in soyabean lecithin based extender [15] might have protected the acrosome more effectively. CONCLUSION In conclusion, considering the acrosomal integrity of spermatozoa, the soyabean lecithin based extender is better than egg yolk based extender for the cryopreservation of Malabari buck semen. 5172

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