STALLION SEMEN PRESERVED USING 1% GLYCEROL AND 1% DIMETHYL FORMAMIDE AS CRYOPROTECT

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1 Indian J. Anim. Res., 45 (4) : , 2011 AGRICULTURAL RESEARCH COMMUNICATION CENTRE ccjournals.com / indianjournals.com nals.com EFFECT OF DIFFERENT SEASONS AND THAWING PROTOCOLS ON CERTAIN SEMINAL ATTRIBUTES OF INDIAN STANDARD BRED STALLION SEMEN PRESERVED USING 1% GLYCEROL AND 1% DIMETHYL FORMAMIDE AS CRYOPROTECT OPROTECTANT Devender Kumar,, Dinesh Jhamb* and Atul tul Saxena Equine Fertility Research Center. Equine Breeding Stud, Babugarh, Ghaziabad , India Received : Accepted : ABSTRACT Standard bred stallion semen was collected during the breeding (n=5) (March-October) and non breeding (n=5) (November-January) seasons using artificial vagina for seven occasions on alternate days from each stallion for both seasons. Semen was evaluated for physical attributes, motility and membrane integrity,, diluted using INRA 82 extender to which 2% egg yolk, 1% glycerol and 1% dimethyl formamide were added filled in french medium straws and cryopreserved by instant freezing over LN 2 Vapors. After 24 hours of storage, semen was thawed using three ee different ent protocols Treatment 1 (T1) 37 O C for 30 secs, Treatment 2 (T2) 60 O C for 8secs and Treatment 3 (T3) 75 O C for 7 secs. The post thaw motility,, post thaw viability and post thaw HOS positive sperms ms were significantly higher during the non breeding season. The thawing protocol T2 was found to be the best during the breeding as well was non-breeding seasons. Key words: Cryopreservation, Dimethyl formamide, Glycerol, Stallion semen parameters, Thawing. INTRODUCTION The use of Artificial Insemination in equine breeding has become increasingly popular in horse industry. Semen cryopreservation has the potential of adding a new dimension to the horse breeding industry by allowing long-term preservation of spermatozoa from superior stallions and permitting distribution of this semen to breeding establishments worldwide. If properly stored, frozen semen may maintain its viability for centuries or even millennia. One of the important factors in stallion semen freezing is that not all stallions have sperm that will survive the freezing and thawing processes. Roughly 30% of stallions have sperm that will result in *Corresponding authors equineage@rediffmail.com good quality thawed semen; a further 40% will have acceptable post-thaw results, and only 30% would be considered possessors of sub fertile frozen semen. In other words, 70% of stallions have semen that is capable of achieving pregnancies once frozen and thawed. (Samper et al., 1991). Frozen semen is a comparatively new technology in equine breeding as is with any new technology, until its practices are standradised, there is need to do further research. The current study was aimed to compare the post-thaw motility, sperm viability and membrane integrity after freezing equine semen using cryoprotective agents combination and optimization of thawing protocol suitable to Indian conditions.

2 248 INDIAN JOURNAL OF ANIMAL RESEARCH MATERIALS AND METHOD Experimental animals : Five standard bred stallion (between 5-10 years of age) maintained at Equine Breeding Stud, Babugarh (Distt Ghaziabad, UP) were used for the study. The stallions were the crosses between imported thoroughbred stallions and Indian mares having 50 per cent inheritance of thoroughbred and Indian blood. Stallions were kept under ideal managemental conditions as applicable to Military Stud Farm. Semen collection, dilution and freezing : Semen samples were collected in two seasons i.e. breeding season (March October) and non breeding season (November January). First five ejaculates of the stallions in each season were discarded to minimize the extra gonadal sperm reserve and were not included in the study. In all seventy (70) ejaculates were collected from five stallions (fourteen ejaculates from each stallion i.e. seven in breeding season and seven in non breeding season) using artificial vagina (INRA model) after stimulating stallion on estrous mare. The semen was collected on alternate days between 9.00 to AM in summer as well as in winter months. The temperature of artificial vagina (AV) was maintained at 42 C by filling it with warm water. The lubrication was done with non spermicidal jelly (sterilized Vaseline) applied on inner surface of artificial vagina. The inner pressure of AV was maintained by filling water in outer and inner jacket. The semen was collected in clean, dry, heat sterilized graduated semen collecting bottles attached with inner latex lining. After collection and removal of gel fraction (in line gel filter) the semen was subsequently maintained at 37 C in a water bath. Semen evaluation, extension and freezing : The ejaculated semen was evaluated for volume, colour, mass motility, concentration, viability (live: dead count) and membrane integrity as per standard methods described previously, (Dott and Foster, 1972 and Pant et al., 2002). The remaining semen was extended up to 200 ml in INRA 82 (primary extender) equine semen extender (Davis Morel, 1999) and centrifuged at 22 C in 50 ml test tubes at 600 g for 10 min to make the pellet of sperms. The supernatant was discarded and pellets obtained after centrifugation were re-suspended in freezing extender (secondary extender) making the final concentration of sperms as 100 million/ml. Composition of primary extenders {INRA 82} i) salt solution (for 250 ml) : Glucose 12.5 gm; Lactose 750 mg; Raffinose 750 mg; Tri Sodium Citrate 2H 2 O. 150 mg; Tri Potassium Citrate 200 mg; Hepes 2380 mg; Pencillin 5000 IU/100 ml; Gentamycin 5 mg /100 ml; Millipore Water To add upto 250 ml. ii) UHT Skimmed Milk : 250 ml INRA 82 was prepared fresh by mixing of solution A & B in equal proportions on the morning of freezing. Skimmed milk was prepared by dissolving skimmed milk powder (Hi media) 100gms in 1000 ml of Millipore water and sterilized by autoclaving at 15 lbs pressure (121 o C) for 5 minutes. Water and Buffer for INRA 82 were autoclaved at 15 lbs pressure (121 o C) for 15 minutes. The ph was adjusted to 6.8 using N/10 NaOH and/or HCl. Composition of secondary extender INRA % Egg Yolk + 1% Glycerol +1% Dimethyl Formamide 1% Dimethyl formamide (Sigma Aldrich) and 1%glycerol (Sigma Aldrich) were used as cryoprotectants. Egg yolk was separated from freshly brought eggs from a near by poultry farm under strict aseptic conditions. Equilibration, Filling and storage semen in LN 2 : The semen diluted in freezing extenders were kept in cold handling unit for 1 hour and 15 minutes for equilibration and gradual cooling from water bath temperature to 4 C. Usually cooling to 4 C occurs in this time. Following equilibration, the semen was filled in French medium straws of 0.5 ml capacity (IMV, France) as per standard method, arranged on a pre cooled stainless steel trey for vapour freezing.the filled straws arranged on a stainless steel tray were placed in modified freezing chamber (thermacol box) for vapour freezing. The tray was

3 held in vapours 4 cm above Liquid Nitrogen Gas (- 140 C) level for 12 min and were subsequently plunged into goblets filled with LN 2 and stored in canisters in LN 2 containers. Thawing : After 24 hours of freezing, semen straws were thawed adopting three different protocol. T C for 30sec, T C for 8sec, T C for 7 sec. For post freeze examination of the semen, the straws were taken out from storage canisters (taking all precautions) with the help of forceps and dipped in water maintained at required temperature for the particular thawing protocols. In all the three thawing protocols, the semen was analysed for motility, viability and membrane integrity in a similar way as described previously. Statistical analysis : Statistically analyses of the data were done as per the standard procedures (Snedecor and Cochran, 1971). The results were expressed in terms of means and Vol. 45, No. 4, their standard error and were compared using students t test. RESULTS TS AND DISCUSSION: Average volume of semen during breeding season (B.S.) was ml ( ) and during non breeding season (N.B.S.) was ml ( ). Concentration was ( ) and ( ) during B.S and N.B.S. respectively. Colour of semen was milky white irrespective of season.the comparison of relevant data for sperm motility (%),viability (%) and per cent positive sperms to HOS in different thawing protocols in freezing extender containing 1% glycerol and 1% dimethyl formamide, during both seasons has been presented in Table 1. A successful cryopreservation depends upon many factors that involve proper mixing of semen with extenders, concentrating sperm cells using Table 1.: Comparison of pre freeze and post thaw motility (%), viability (%) and HOS positive sperms (%) between breeding(b.s.) and non breeding season(n.b.s.) at different thawing protocols (T1, T2 and T3) of semen cryopreserved in freezing extender containing 1% glycerol and 1% dimethyl formamide, Breeding season(b.s.) Non-breeding season (N.B.S.) Pre freeze and Post thaw motility (%) Pre freeze k p2n4vgv9n0v k p1n5x GThawing protocol T1G2w.v9 ± 0q.x6 a j s9n7r ñ p1n9p b GThawing protocol T2G2x.w4 ± 0q.s8 c * ± d Thawing protocol T ± e * ± f Overall ± g * ± h Pre freeze and Post thaw viability (%) Pre freeze Thawing protocol T ± NS ± Thawing protocol T ± k ** ± l Thawing protocol T ± m ** ± n Overall ± 0.85 NS ± Pre freeze and Post thaw HOS positive sperms (%) Pre freeze Thawing protocol T ± q * ± r Thawing protocol T ± s * ± t Thawing protocol T ± u * ± v Overall ± w * ± x Data with in a row with different superscripted letters significantly different *(P<0.01), **(P<0.05), NS= Non significant.

4 250 INDIAN JOURNAL OF ANIMAL RESEARCH Table 2 : Performance of different stallions (Indian Standard bred) based on their total sperm production in the neat semen during breeding season (March October). Mean + S. E. = 35 Seminal attributes {Fresh semen} Per cent motility Per cent viability Per cent HOS positive sperms Breeding season Non breeding season Breeding season Non breeding season Breeding season Non breeding season ( ) ( ) ( ) ( ) ( ) ( ) centrifugation, resuspension of the concentrated cells into extenders containing cryoprotectants, interaction between cryoprotectants, extenders, cooling rate, warming rate, packaging and above all the individual stallion variations (Loomis et al., 1983, Cristanelli et al., 1984 and Graham, 1996). Even in the ideal conditions some damage to spermatozoa occurs du ring freezing process, which leads to low post thaw parameters as compared to pre freeze parameters as shown in Table 1. The main damage to sperm cells occurs due to formation of internal ice crystals, which alters the sperm structure and secondly due to internal solute concentration. (Mazur, 1984 and Graham, 1996). Comparison (paired t test) of post thaw motility and positive sperms to HOST between B.S and N.B.S. of the semen cryopreserved in and thawed in different thawing protocols (T1 to T3) revealed a significant (P <0.01) difference. For viability difference was significant for T 2 and T 3 only (P <0.05). The values for post thaw motility, viability and positive sperms to HOST were found to be higher for all the thawing protocols during N.B.S. (Table 1). Irrespective of thawing protocols, the overall per cent motility during B.S was per cent whereas in N.B.S. it was found as per cent. The overall per cent viability during B.S was per cent whereas in Motility (%) Breeding Season Non Breeding Season T1 T2 T3 Over all Fig. 1.0 : Comparison of post thaw motility (%) in semen of Indian Standard bred stallions cryopreserved in freezing extender containing 1% glycerol and 1% dimethyl formamide,following thawing in different thawing protocols in breeding and non breeding season.

5 N.B.S. it was found as per cent. The overall per cent positive sperms to HOST during B.S were per cent whereas in N.B.S it was found as per cent. Thawing of stallion semen has been done between 4 to 75 C. Cochran et al., (1984) compared thawing at 75 C for 7 sec and 37 C for 30 sec and reported that thawing at 75 C for 7 sec (motility: 38%) was superior to thawing at 37 C for 30 sec (motility: 30%). Miragaya et al., (2001) carried out thawing of equine semen in 75 0 C for 7 seconds followed by at least 5 seconds at 37 0 C and found post thaw motility equal to or greater than 30%. Cristanelli et al., (1984) thawed semen by immersion in 75C for 10 sec and reported pregnancy rate of per cent. Magistrini et al., (2001) reported that thawing semen in 50 C water bath for 40, 50 or 60 sec or in 60 C water for 30, 40 or 50 sec had no significant influence on percentage of progressive motile spermatozoa after thawing.. Papa et al., (2001) used 46 C for 20 sec protocol for thawing frozen equine semen and reported average post thaw motility to be per cent. Metcalf (2005) used thawing temperature of 37 C for 30 Vol. 45, No. 4, seconds and found post thaw motility and percent viable sperm ranged per cent and per cent respectively. Squires et al., (2004) thawed frozen semen in 37 C water bath for 30 seconds and embryo recovery was done after 7-9 days of inseminating the mares. The average embryo recovery was per cent Our results in the present study are comparable to other studies reported in literature. Thawing exposes sperms to same type of stress as it happens during freezing, but in a reverse fashion. Cells rehydrates as water moves back across plasma membrane to balance the osmotic imbalance because of melting of intracellular ice crystals formed during freezing. Improper thawing (Too fast or too slow) of the semen had detrimental effect on viability of spermatozoa and decreases the chances of obtaining a pregnancy (Loomis and Squires, 2005). In this study when three thawing protocols were compared, thawing protocol T2 was found to be best despite of the fact that it preserve a significantly lower per cent viable sperm compare to other thawing protocols, however, it preserves maximum number of HOS positive spermatozoa. Viability (%) Breeding Season Non Breeding Season T1 T2 T3 Over all Fig. 2.0 : Comparison of post thaw viability (%) in semen of Indian Standard bred stallions cryopreserved in freezing extender containing 1% glycerol and 1% dimethyl formamide, following thawing in different thawing protocols in breeding and non breeding season.

6 252 INDIAN JOURNAL OF ANIMAL RESEARCH In the N.B.S. although no significant difference amongst thawing protocols was observed, However, thawing protocol T2 was found best as it preserves higher percentage of HOS positive spermatozoa. Thus thawing protocol T2 was found best both for B.S and N.B.S. Henry et al., (2002) observed that most frequent damage to sperm cells is the loss of membrane integrity and this directly reflects the freezing ability. These workers further reported obtaining per cent HOS positive sperms using a freezing extender containing 1% glycerol and 4% ethylene glycol. Various other workers have supported the view that maintenance of normal function of sperm membrane is a crucial pre requisite for sperm viability as well as reactivity at the site of fertilization. Increased proportion of spermatozoa with damaged membranes are indicative of reduced fertility (Jeyendran et al., 1984, Zhang et al., 1990 and Grondahl et al., 1994). For most of hypo osmotic swelling test (HOST) various workers used hypo osmotic solution of 100 mosm (Nield et al., 2000, Samper et al., 1991, Coiza et al., 1997, Cabrita et al., 1999 and Nie and Wenzel, 2001). In the present study also hypo osmotic solution of 100 mosm was used. Rate of thawing has a significant effect on post thaw results. It has been observed that faster thaw rates significantly increases the percentage of progressively motile and viable sperms compared to slow thaw rates. Further it has been observed that using a thawing protocol of 75 C for 7 sec apparently causes less membrane damage on thawing resulting in higher post thaw motility and viability and reduced premature capacitation (Bradfort and Buhr, 2002). In our study we could observe that this effect was visible in semen cryopreserved during B.S and not in the N.B.S. Also, the effect held true only with motility and viability but not with HOS positive sperms. The temperature had a significant effect on spermatogenesis. Elevation of testicular temperature due to rise in body temperature or due to an increase in environmental temperature would result in an elevation of the temperature of germinal epithelium and hence had direct adverse effect on spermatogenesis. It has been reported that summer temperature, if increased Positive sperms to HOS (%) Breeding Season Non Breeding Season T1 T2 T3 Over all Fig. 3.0 : Comparison of post thaw per cent positive sperms to HOS in semen of Indian Standard bred stallions cryopreserved in freezing extender containing 1% glycerol and 1% dimethyl formamide, following thawing in different thawing protocols in breeding and non breeding season.

7 Vol. 45, No. 4, Table 3 : Performance of different stallions (Indian Standard bred) based on their total sperm production in the neat semen during non breeding season (November to January). Mean + S. E. = 35 Seminal attributes {Fresh semen} Volume(ml) Concentration(millions / ml) Total Sperm Production(millions) Breeding season Non Breeding Season Breeding Season Non Breeding Season Breeding Season Non Breeding Season ( ) ( ) ( ) ( ) ( ) ( above 41.5º C for only two hours may be responsible for a depression in spermatogenesis in horses (Johnson, 1991). In northern India, ambient temperature in breeding season especially in May and June months is more than 41.5º C and hence the quantity of sperm production was decreased in the months leading to comparatively higher sperm production in N.B.S. Maximum of previous studies reported in literature have been carried out in cold climate, where there is no summer stress and hence there is an increased total sperm production in breeding season in their study. Our results for different thawing protocols further suggests thawing protocol 60 0 C for 8 sec (T2) serves best in terms of preserving maximum number of HOS positive spermatozoa both in B.S as well as in N.B.S.. Thus while thawing protocol 60 0 C for 8 sec (T2) as best thawing protocol, we obtained 28, per cent motility, per cent viability and per cent HOS positive sperms during B.S as supported by nearly similar finding by Dillaqua et al., (2001) who analyzed post thawed semen by using different temperatures for 0.5 ml straws (38 C/40 sec; 46 C/20 sec, 65 C/10 sec) and for 0.25 ml straws (38 C/ 30 sec, 46 C/15 sec; 65 C/6 sec). They found that 65 C/6 sec is best thawing temperature for both straws Various other sssaworkers have reported nearly similar results with extenders containing combination of cryoprotectants and using T2 as thawing protocol. With same thawing protocol during N.B.S., we were able to obtain per cent motility, per cent viability and per cent HOS positive spermatozoa. CONCLUSION Irreversible damage to sperms during freezing and thawing results in low post thaw parameters as compared to prefreeze parameters. While comparing different thawing protocol for frozen thawed semen, it can be broadly concluded that all the seminal attributes studied were significantly better in N.B.S. as compared to B.S. So freezing semen during N.B.S.semen gives better post thaw values than during B.S. REFERENCE Bradford, L. L. and Buhr, M. M. (2002). Function of cryopreserved horse semen is improved by optimal thawing rates. Equine Vet. Sci.., 7: Cabrita, E., Alveraj, R., Anel, E. and Heeraez, M. P. (1999). The hypo osmotic swelling test performed with coulter counter. A method to assay functional integrity of sperm membrane in rainbow trout. Animal Reproduction Sci.., 55 (3-4): Cristanelli, M. J., Squires E. L., Amann, R. P. and Pickett, E. W. (1984). Fertility of stallion semen processed frozen and thawed by a new procedure. Theriogenology., 22: Cochran, J. D., Amann, R. P., Froman, D. P and Pickett, B. W. (1984). Effect of centrifugation, glycerol level, cooling to 5 C, freezing rate and thawing rate on the post thaw motility of equine spermatozoa. Theriogenology., 22:

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