This experiment was conducted with the aim to study the effect of replacing egg yolk with soybean lecithin (SL) for cryopreservation of Black Bengal buck semen. Sexually matured Black Bengal buck (n = 5) were used and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in Tris extender with 5% Glycerol containing either 15% egg yolk (control group) or SL at different concentrations (1% SL, 1.5% SL and 2% SL). The semen samples were filled in straws and cooled gradually to 5 °C. Semen straws were equilibrated for 3 hours at 5°C and were frozen in static liquid nitrogen vapor and stored in liquid nitrogen. Semen samples were evaluated after initial dilution, after completion of equilibration and after freeze thawing for in vitro sperm characters such as sperm motility, functional membrane integrity and malondioldehyde (MDA) concentration. Semen samples preserved in extender containing 1% SL was able to maintain in vitro sperm characters similar to the extender containing egg yolk. However, significant (P<0.05) reduction in all semen parameters was observed as the concentration of soybean lecithin increased above 1% level. It is concluded that an extender containing soybean lecithin @ 1% with 5% Glycerol can be used for replacing egg yolk for cryopreservation of Black Bengal buck semen.

The experiment was conducted to study the effect of trehalose an impermeant extra cellular cryoprotectant supplementation on cryopreservation of Black Bengal buck semen. Semen ejaculates (n = 24) were diluted in Tris-soybean lecithinglycerol extender having trehalose @ 0, 50,100and 150 mM. Samples were equilibrated at 5°C for 3 hrs, frozen and stored in liquid nitrogen. Semen samples were evaluated for sperm motility,functional membrane integrityand concentration of lipid peroxide compound malondialdehyde at three stages such as 10 minsafter dilution with extender, after completion of equilibration period and after freeze thawing. It was found that the control group (0mM trehalose)had significantly better in vitro sperm characters when compared to extenders supplemented with trehalose (50 mM , 100 mM and 150 mM) during different stages of cryopreservation. It can be concluded that supplementation of trehaloseas a cryoprotectant did not improve the cryopreservability of Black Bengal buck semen.

The aim of the present study was to produce goat embryo in different culture media through in vitro fertilization using cryopreserved black Bengal buck semen. Total 1265 fresh cumulus oocyte complexes (COCs) were collected by aspiration method with a 19 gauge hypodermic needle, washed 5-6 times and cultured in maturation media maintaining 5% CO2 level at 38.5ºC with maximum humidity in a incubator. After 27 h of incubation cumulus cells were stripped off from matured oocytes and transferred to acidified Tyrode’s medium for zona thinning and co-incubated with in vitro capacitated sperms for fertilization in Fert-BO media. In the experiment I, fresh buck semen and in experiment II, frozen buck semen was used for in vitro fertilization after in vitro processing. After 5 h of co-incubation, presumptive zygotes were washed and co-incubated with oviductal cells in three different culture media (RVCL, mSOF, KSOM) for further development. In fresh group cleavage (%) were 37.76 ± 2.98, 39.60 ± 1.75, 29.01 ± 1.74, and morula formation (%) were 7.72 ± 3.38, 6.03 ± 1.29, and 3.00 ± 3.00 in RVCL, mSOF and KSOM media respectively. However, in frozen group cleavage (%) were 29.17 ± 2.56, 27.70 ± 2.31, 24.17 ± 1.44 in RVCL, mSOF and KSOM media respectively and morula formation (%) was 2.93 ± 0.97 only in RVCL media. These results indicate that cryopreserved black Bengal buck semen have competence to produce embryos and could be used for embryo development in RVCL media through in vitro fertilization.

The purpose of this study was to check the competence of cryopreserved black Bengal buck semen to produce goat embryo through IVF. So far, cryopreserved black Bengal buck semen has not been used to produce goat embryos by IVF. For the study, fresh goat oviducts and ovaries were collected from slaughterhouse in a thermo flask containing 0.9% saline solution supplemented with antibiotic (400 IU mL−1 penicillin and 500 mg mL−1 streptomycin) at 30–35°C and transported to laboratory within 2–3 h of slaughter. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, washed 5–6 times, and cultured in maturation media (TCM-199 + 10% FBS + 10 mg mL−1 FSH-P + 0.81 mM sodium pyruvate + 5% follicular fluid + 50 mg mL−1 gentamicin sulfate + 1 μg mL−1 oestradiol + 100 μM cysteamine) for 27 h in a 5% CO2 incubator at 38.5°C with maximum humidity. After 27 h of culture, cumulus cells were separated from matured oocytes by repeated pipetting using a fine pipette in fertilization Bracket and Oliphant’s (BO) medium. After removal of cumulus cells, the oocytes were transferred to acidified Tyrode’s medium for zona thinning for 52 s and were co-incubated with capacitated sperms for fertilization in fertilization BO medium at 38.5°C in 5% CO2 in air with maximum humidity. In the experiment I, freshly collected buck semen was used for IVF after processing for capacitation. In experiment II, cryopreserved buck semen straws were thawed and sperm were capacitated in vitro and used for fertilization. After 5 h of co-incubation, presumptive zygotes were washed thoroughly and cultured in embryo development medium for cleavage. Three different in vitro development media (RVCL, mSOF + 2.5% BSA, and KSOM + 0.5% BSA) were used. After 40 to 42 h, cleavage was observed and embryos were co-incubated with oviducal cells in replacement media for further development. In the fresh group, overall cleavage rates (%) were 37.76 ± 2.98, 39.60 ± 1.75, 29.01 ± 1.74 and morula formation rates (%) were 7.72 ± 3.38, 6.03 ± 1.29, 3.00 ± 3.00 in RVCL, mSOF, and KSOM media, respectively. However, in the cryopreserved group the overall cleavage rates (%) were 29.17 ± 2.56, 27.70 ± 2.31, and 24.17 ± 1.44 in RVCL, mSOF, and KSOM media, respectively, and morula formation (%) was achieved 2.93 ± 0.97 in RVCL media. These results indicate that cryopreserved black Bengal buck semen have competence to produce embryos and could be used for embryo development through IVF.