Indian Journal of Small Ruminants (The)

A study was undertaken with the aim to investigate the role of addition of, oviductal cells granulosa cells or follicular fluid to in vitro fertilization medium on cleavage and embryonic development of sheep oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries. Oocytes were cultured for in vitro maturation in tissue culture medium199 (TCM-199) + 10% foetal calf serum (FCS) for 24 h in CO₂ incubator. Oocytes were fertilized with fresh-diluted ram semen capacitated in Brackett and Oliphant (BO) medium containing heparin and caffeine. After 5 h of co-incubation, sperm-attached matured oocytes were transferred to culture media and co-culture as Gr 1: without co-culture (control), Gr 2: sheep oviductal epithelial cells (SOECs) and Gr 3: Granulosa cells (GCs). Matured oocytes were fertilized with fresh diluted semen. In another experiment, matured oocytes were fertilized with frozen-thawed semen capacitated in BO medium containing 20% sheep follicular fluid (SFF), then transferred to culture media for further development. Oocytes were observed for cleavage after 48 h of fertilization. The cleavage rate and frequency of morula and blastocyst were recorded. Addition of SOECs to the culture media significantly (P<0.05) produced higher cleavage (40 vs. 20%) and embryonic developmental rates of morula and blastocyst compared to control (35.0, 15.0 vs. 10.0, 10.0%, respectively). Morula and blastocyst production rates were significantly (P<0.05) higher in TCM-199+10% FCS+GCs than TCM-199+10%FCS (33.34, 13.34 vs. 10.00, 0.0%, respectively). The fertilization of oocytes with semen capacitated in BO medium containing 20% SFF resulted in significantly (P<0.05) higher embryo developmental rates of morula and blastocyst compared with the use of heparin and caffeine in the same medium (22.23, 13.34 vs. 10.0, 0.0%, respectively). In conclusion, oviductal or granulosa cell co-culture had marked effect on cleavage and embryonic development of sheep oocytes. Addition of sheep follicular fluid to BO medium could increase the percentage of morula and blastocyst production.