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In vitro maturation of sheep oocytes in different concentrations of mare serum
Abstract
The aim of the study was to determine the optimum concentration of the mare serum (MS) for sheep in vitro oocyte maturation. Sheep ovaries were collected from a local abattoir and transported within 1 h to
the laboratory in a warm saline solution (30 – 35oC), supplemented with 100 IU penicillin G and 100 g streptomycin sulfate/ml. Cumulus-oocyte complexes (COC’s) were obtained by slicing of follicles, washed in TCM-199 modification with NaHCO3 and supplemented with 50 g/ml gentamycin, and 0.25 mM sodium pyruvate without any serum supplementation. The COC’s were randomly divided into four groups. Group 1 (n = 105) COC’s were fresh control and cultured in TCM-199 medium without serum supplementation. Group 2 (n = 108) COC’s were washed five times and cultured in TCM-199 medium supplemented with 10% MS. Group 3 (n = 112) COC’s were washed five times and cultured in TCM-199
medium supplemented with 15% MS. Group 4 (n = 114) COC’s were washed five times and cultured in TCM-199 medium supplemented with 20% MS. After 38 - 42 h of IVM, oocytes were denuded with the aid
of 0.1% hyaluronidase and passing them through a fine pipette, fixed for 24 – 48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for the evidence of different stages of maturation. Significantly higher (p < 0.05) maturation rates of oocytes (69 – 72%) were observed in all concentrations of mare serum compared to
those without serum supplementation. However, no significant difference was observed between the 10, 15 and 20% serum supplemented group.
the laboratory in a warm saline solution (30 – 35oC), supplemented with 100 IU penicillin G and 100 g streptomycin sulfate/ml. Cumulus-oocyte complexes (COC’s) were obtained by slicing of follicles, washed in TCM-199 modification with NaHCO3 and supplemented with 50 g/ml gentamycin, and 0.25 mM sodium pyruvate without any serum supplementation. The COC’s were randomly divided into four groups. Group 1 (n = 105) COC’s were fresh control and cultured in TCM-199 medium without serum supplementation. Group 2 (n = 108) COC’s were washed five times and cultured in TCM-199 medium supplemented with 10% MS. Group 3 (n = 112) COC’s were washed five times and cultured in TCM-199
medium supplemented with 15% MS. Group 4 (n = 114) COC’s were washed five times and cultured in TCM-199 medium supplemented with 20% MS. After 38 - 42 h of IVM, oocytes were denuded with the aid
of 0.1% hyaluronidase and passing them through a fine pipette, fixed for 24 – 48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for the evidence of different stages of maturation. Significantly higher (p < 0.05) maturation rates of oocytes (69 – 72%) were observed in all concentrations of mare serum compared to
those without serum supplementation. However, no significant difference was observed between the 10, 15 and 20% serum supplemented group.
