INFLUENCE OF BULL SEMINAL PLASMA ON POST-THAW RAM SEMEN PARAMETERS AND FERTILITY

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1 Bull Vet Inst Pulawy 50, , 2006 INFLUENCE OF BULL SEMINAL PLASMA ON POST-THAW RAM SEMEN PARAMETERS AND FERTILITY ULGEN GUNAY, IBRAHIM DOGAN, ZEKARIYA NUR, IVAN MANOLOV 1, HAKAN SAGIRKAYA, MUSTAFA KEMAL SOYLU, CUNEYT KAPTAN 2 AND LATIF AKPINAR 2 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Uludag University, 16059, Gorukle, Bursa, Turkey 1 Department of Animal Reproduction, Agricultural Faculty, Thracian University, 6000, Stara Zagora, Bulgaria 2 Bandirma Research Institute, 10200, Balikesir, Turkey ugunay@uludag.edu.tr Received for publication March 24, Abstract The aim of the study was to evaluate the effect of the addition of 20% bull seminal plasma to semen extenders on post-thaw sperm motility, morphology, and fertility. Two Merino rams at 3-5 years of age, 43 ewes, and one Holstein bull were used. Fourteen ejaculates were collected by the use of an artificial vagina, twice a day, at 60 min interval. Six semen ejaculates were extended with Tris-egg yolk extender containing 20% bull seminal plasma (TEY-BSP group), and 8 ejaculates were extended with Tris-egg yolk (TEY group), at a rate to obtain 150x10 6 spermatozoa/straw. The ewes in oestrous were divided at random into two groups. Twenty-two ewes were inseminated with TEY-BSP and 21 ewes were inseminated with TEY. The mean percentage of motility, defected acrosome, and total morphological defect for the equilibrated and post-thaw semen parameters of the TEY, were 69.4%, 8.6%, and 17.0% and 42.8%, 49.9%, and 51.2%, respectively. The mean percentage of non-return rate (NRR) (n=43) was 30.2% and for the semen frozen with TEY-BSP (n=22) and TEY (n=21) %, and 28.6%, respectively. There were no significant differences between TEY-BSP and TEY for NRR. In conclusion, the addition of the bull seminal plasma to freezing extender has a supportive effect on extended, equilibrated, and thawed semen. Moreover, the addition of bull seminal plasma to ram semen extender improved fertility. Key words: ram, frozen semen, bull seminal plasma, fertility. Cervical insemination with frozen-thawed semen has not been widely applied for commercial artificial insemination (AI) of sheep (32). The cryopreservation process results in reduced motility and fertilizing capacity compared with fresh semen (1, 8, 34). Unlike other farm animals, ram spermatozoa are more susceptible to cryopreservation (1, 8, 19), and the cervix of ewes restricts the transcervical passage of insemination instruments, and consequently AI (17, 18, 20, 29, 30). The success of AI with frozen ram semen depends on the physiology of sperm transport (21, 24, 30, 31, 33), and on the survival of spermatozoa in the female reproductive tract (21). Also, cooling and freezing rates (8, 26), type of cryoprotective agents and their concentration, extender composition, dilution rates, temperature at which glycerol is added to the semen, equilibration time, and thawing rate interact with the success of AI with frozen semen (1-4, 25-27, 32). The extenders composition assists in stabilizing the cells during the freezing and thawing process (4, 6, 10, 13, 14). Several studies (1, 2, 6-9, 13, 15, 34) have been conducted to determine the optimal extender to enhance post-thaw motility, morphology, and fertility of ram semen, including addition of egg yolk, bull seminal plasma, milk, and sodium sulphate (6, 19, 21-23, 25, 28). In various studies (4, 6) the effects of seminal plasma on the post-thaw motility, morphology, and fertility abnormalities were examined. Some researchers (7, 28, 32) agreed that dialysed egg yolk, bull seminal plasma, milk, and sodium sulphate could prevent spermatozoa from the post-thaw side effect of lipid peroxidation The aim of the present study was to evaluate effect of addition of bull seminal plasma semen extenders on post-thaw sperm motility, morphology, and fertility. Material and Methods Animals. Two Merino rams at 3-5 years of age, 43 Merino ewes housed at Bandirma Research Institute, and one Holstein bull housed at Veterinary Faculty Farm of Uludag University were used in the study. Semen collection and freezing. Bull semen was collected by an artificial vagina and centrifuged at rpm for 10 min to separate its seminal plasma. Fourteen ejaculates from rams (2x7) were collected by

2 504 the use of an artificial vagina, in the presence on oestrous ewe, twice a day, at 60 min intervals. Semen samples were placed in a water bath at 37 C. Soon after the collection, volume, wave activity, motility, concentration, and percentage of spermatozoa, exhibiting morphologic abnormalities were evaluated according to standard procedures (16). Only ejaculates with a volume higher than 0.5 ml, and wave motility higher than (+++) were used. The volume was recorded from the collection tube, which was graduated in 0.1 ml divisions. Sperm concentration was assessed using haematocytometer. To assess the wave activity, a drop of semen deposited on a glass slide and placed on a warm stage (40 C) was examined under the light microscope (x100). The semen sample was scored using a scale ranging from 0 (no wave movement), to 5 (extreme wave movement). Sperm motility was assessed subjectively using a phase-contrast microscope (x400) with a warm slide (40 C). Defected acrosome () and total morphological defect (TMD) were evaluated using Giemsa staining procedure (16). A total of 200 spermatozoa were examined under a light microscope (x1000), to determine the percentages of spermatozoa with and. Semen evaluation (motility, and TMD) was carried out at 3 stages (after dilution, equilibration, and thawing). Six ejaculates were extended with Tris-egg yolk extender containing 20% bull seminal plasma (TEY- BSP group), and 8 ejaculates were extended with Trisegg yolk (TEY group), at a rate to obtain 150x10 6 spermatozoa/straw. Extended semen was cooled to 5 C for 120 min. Cooled semen was glycerolized in 5 steps, at 5 min intervals with Tris-egg yolk extender containing 10% glycerol. Final concentration of glycerol was 5%. After glycerolisation, it was subjected to equilibration for 2 h. Semen in 0.25 ml straws was frozen horizontally on a rack, about 7 cm above liquid nitrogen vapour for 10 min, and stored in liquid nitrogen containers at -196 C. At least 3 straws from each ejaculate were thawed at 50 C for 15 s, in a water bath to evaluate post-thaw semen motility and morphology (14x3=42 straws). Artificial insemination. Intra-cervical insemination was performed on ewes during the transition period from non-breeding to natural breeding season in the region, under natural lighting. Oestrous was detected with a teaser ram every day. The ewes in oestrous were divided at random into two groups. Twenty-two ewes were inseminated with TEY-BSP and 21 ewes were inseminated with TEY. Fertility. To detect ewes returning to oestrous, all the ewes were checked from days after insemination using teaser rams. Ewes which did not return to oestrous were considered pregnant and recorded as non-return rate 30 d (NRR-30) (28). The mean semen parameters of motility,, and TMD calculated for the 14 ejaculates obtained from the rams, were subjected to analysis of variance (oneway NOVA), and the differences among means were tested for significance by the Fisher s PLSD. Pregnancy rates were analysed using the chi-square test. The SPSS 10.0 software was used for all statistical analyses (Instat, ). Results The results of the study are summarised in Table 1. The mean of volume, wave activity, and concentration for the diluted semen of the TEY-BSP group were 0.7 ml, 3.5, and 2.8X10 9 /ml, and in the TEY group they were 1.0 ml, 3.6, and 3.2X10 9 /ml, respectively. The mean percentage of motility,, and TMD for the equilibrated and post-thaw semen parameters of the TEY-BSP was 68.3%, 9.6%, and 12.5% and 48.3%, 47.3%, and 48.3%, respectively. The mean percentage of motility,, and TMD for the equilibrated and post-thaw semen parameters of the TEY was 69.4%, 8.6%, and 17.0% and 42.8%, 49.9%, and 51.2%, respectively (Table 1). No significant differences in all the studied parameters for diluted, equilibrated, and post-thaw semen were found between rams (P>0.05). The NRR-30 according to extenders and rams was summarised in Table 2. The mean percentage of NRR (n=43) was 30.2% and for the frozen semen with TEY-BSP (n=22) and TEY (n=21) was 31.8% and 28.6%, respectively (Table 2). These differences were not statistically significant (P>0.05). After the use of both extenders, similar results were obtained. Discussion The cryopreservation cycle for semen samples, includes the entire process from sperm collection, preparation, and dilution through to post-thawing maintenance of fertilizing ability. Many extenders have been used for freezing ram semen (1, 2, 4, 6, 12). The present study was aimed at investigating effects of bull seminal plasma (BSP) on post-thaw ram sperm motility, morphology and fertilizing ability. At the mean volume, wave activity, and concentration for the diluted semen of the TEY-BSP and TEY groups were in accordance with previous reports (15, 16). For the extended and equilibrated semen, the addition of bull seminal plasma to freezing extender had supportive effect on motility, acrosome, and morphological integrity. The mean percentage of motility,, and TMD for the diluted and equilibrated semen parameters of the TEY-BSP and TEY groups, were in agreement with previous studies (6, 15). Furthermore, the addition of bull seminal plasma to freezing ram semen extender protects spermatozoa from the damaging effect of low temperatures, and can also support post-thaw motility (48.3% vs. 42.8%), (47.3% vs. 49.9%), and TMD (48.3% vs. 51.2%). TEY-BSP provided higher post-thaw motility, acrosomal, and morphological integrity than TEY extender, there were no significant differences between post-thaw motility, acrosomal, and morphological integrity in the two groups (P>0.05).

3 505 Table 1 Mean parameters of diluted, equilibrated, and thawed sperm for the individual ram, according to extenders Diluted Equilibrated Post-thaw Extender Ram No Thawed straw (n) Volume (ml) Wave activity (1-5) Concentration (X10 9 /ml) TEY-BSP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.4 General mean ± ± ± ± ± ± ± ± ± ± ± ±1.5 TEY ± ± ±0.7 80± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.3 General mean ± ± ± ± ± ± ± ± ± ± ± ±2.9 ± SD, -defected acrosome; TMD-total morphological defects; TEY-BSP-Tris-egg yolk-bull seminal plasma; TEY-Tris-egg yolk Table 2 Fertility according to TEY-BSP and TEY extenders Extender Ram No Number of inseminated ewes (n) NRR /8 (27.3) TEY-BSP /7 (36.4) mean 22 7/15 (31.8) /7 (36.4) TEY /8 (20.0) mean 21 6/15 (28.6) /15 (31,8) General mean /15 (28.6) mean 43 13/30 (30.2) NRR-30-non return rate 30 days; TEY-BSP-Tris-egg yolk-bull seminal plasma; TEY-Tris-egg yolk 505

4 506 The post-thaw motility observed in the present study for the semen extended with TEY, is in accordance with the results of researchers (1, 13, 15) who used different glycerol and egg yolks rates, in various extenders. The post-thaw motility of the TEY-BSP was higher than that reported by Baran et al. (6), who used bull seminal plasma in different rates (7.5% and 15%). The reason for this difference is thought to be that semen samples were centrifuged by the last authors. (6). Moreover, it might be associated with beneficial effect of different rates of bull seminal plasma for ram semen. and for the two groups were similar to findings (40-60%) demonstrated by some researchers (13, 15). However, our results were not similar to those reported by Baran et al. (6). The physiological status of the ewe, lambing- AI interval, its age and breed (3, 11, 12), insemination technique (9, 11, 27), inseminator (27), and farming conditions, have an effect on fertility rates. Other important factors affecting fertility are, post-thaw sperm motility and morphology (3, 8, 21, 28, 29, 34). Fertility was slightly higher in the TEY-BSP than TEY group for NRR-30 (31.8% vs. 28.6%), although these differences were not statistically significant. The NRR-30 in the present study, though lower than results reported by Anel et al. (3) and Paulenz et al. (28), was in agreement with results reported by Danovan et al. (11), King et al. (20), and Ataman and Coyan (5) who used different NRR, lambing rate, extender, and insemination technique. Maxwell et al. (21) observed that the improvement in the post-thaw motility reflected in fertilizing capacity. Although the ram fertility results, including both extenders, were higher for ram 60 than 570, these differences were not significant (P>0.05). This result was not surprising, because post-thaw spermatological findings of ram 60 were better than of ram 570 for TEY-BSP and TEY, generally. 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