|Keywords (Extracted from title, table of contents and abstract of thesis)
maturation, fertilization, sahiwal cow (bos indicus), preimplantation emberyos, ovarian oocytes, in-vitro maturation (ivm), in-vitro fertilization (ivf), sperm concentration, serum
This project was planned to study various factors that affects harvest of ovarian oocytes, their in-vitro maturation (IVM) and fertilization (IVF) and subsequent development to morula and blastocyst stages.
The experiments were carried out to investigate the effect of factors like: animal age, presence or absence of CL on the ovary and oocyte recovery methods (scoring and aspiration) were evaluated on the yield of total and good quality oocytes recovered. Culture media (BSFF, TCM-199, Ham’s F-10 and MEM) with the addition of cow scra (Collected during proestrus, estrus and postestrus phases of cycle) were evaluated for their effect on IVM of oocytes. Sperm preparation protocol (percoll gradient, simple washing in SP-TL, 29$ sodium citrate and in IVF-TL) were compared for sperm recovery, their motility percentage, livability, absolute index of livability and their capacity to fertilizer IVM oocyte in vitro.
The effect of sperm concentration (1x10-6 and 2x10-6), heparin level (0, 20 and 40 ug/ml) and sperm-oocyte co-incubation times (5, 12 & 24 h) on cleavage rate of IVM oocytes was also compared. Three culture media (CZB, TCM-199 and MEM) were tested for their efficacy to support cleavage and subsequent development of IVM-IVF oocytes to morula and bastocyst stages. The data thus obtained were analyzed using appropriate experimental designs and means were tested for significance.
The statistical analysis of the data revealed that the scoring method of oocytes retrieval yielded significantly higher number of total (8.28=0.36 vs 5.98=0.23) as well as good quality oocytes per overy (6.55=0.20 vs 3.13=0.24) compared to aspiration method. The difference between heifer and cow ovaries for the yield of total and good quality oocytes per ovary was non significamnt. The kovaries without a CL yielded greater number of good quality oocytes than the ovaries having CL (7.33=0.10 vs 6.18=0.15), while the total number of oocytes recovered per ovary differed non significantly.
Culture media, BSFF, TCM-199, Ham’s F-10 and MEM with serum supplementation were found equipotent with their means differing non significantly (P>0.05) for maturing oocytes in vitro. However, oocyte mean maturation rates obtained from supplementing proestrus and estrus cow serum (81.63 and 84.84%) were different (P<0.50) than the mean rate (64.21%) observed with postestrus cow serum. In this study, the percoll method of sperm preparation yielded significantly mean values of sperm recovery, their motility percentage, livability, absolute index of livability. Mean cleavage rate in respect of the sperms recovered form percoll method (52.63%) were also lower (P<0.05) than the means (From 68.75 to 72.15%) observed for other simple sperm washing protocols. However, the other three methods differed non significantly among themselves for these parameters. Non significant difference (P>0.05) were observed in the mean cleavage rates for the two sperm concentrations and three sperm oocyte co-incubation times employed for IVF of IVM oocytes. However, cleavage rate means obtained in respect of three heparin levels used for IVF, differed significantly (9.63=1.06 vs 79.44=2.02 and 77.85=1.78, respectively for 0, 20 and 40 ug heparin/ml).
Three serum free culture media employed for culturing IVM-IVF oocytes differed (P<0.05) for the mean cleavage rates (80.59=3.00 vs 68.59=2.24 and 65.53=2.93% respectively for CZBe, TCM-199 and MEM). They also differed significantly in supporing subsequent embryonic development to morula and blastocyst stages (40.87=1.73 vs 30.00=2.03 and 26.17=2.82% respectively for CZBg, TCM-199 and MEM).