Browsing by Author "Owiny, David Okello"
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- ItemEffects of cryopreservation of bovine gametes on fertilizing potential and in vitro embryo development(Stellenbosch : Stellenbosch University, 2007-03) Owiny, David Okello; Barry, Daniel Malan; Coetzer, W. A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Animal Science.ENGLISH ABSTRACT: Cryopreservation of gametes has significant importance in the advancement of assisted reproductive technologies (ART) in the management of livestock and laboratory animal species, conservation of biodiversity, and treatment of human infertility. Cryopreservation also reduces the cost, genetic drift and diseases associated with maintaining live animals and cell lines. The increasing use of mammalian gametes obtained from the testis, and excurrent ducts, and the ovaries in ART has, therefore, been enhanced by the cryopreservation. There is need to maximize survival of cryopreserved gametes and the ability of cryopreserved gametes to produce embryos. Gametes from bovine ovaries and testes obtained from abattoirs, and culled buffalo testes were used to examine the effects of cryopreservation of gametes on embryo development, and the ability of frozen-thawed African buffalo ( Syncerus caffer caffer) epididymal sperm in in vitro production of cattle x Buffalo hybrid embryos. Effects of a commercial tris-egg yolk-based extender, Biladyl® (BIL) and modified Tyrode's lactate (MTL) on sperm fertilizing potential were compared by evaluating sperm motility, viability, and membrane and acrosome integrity. Tyrode's lactate medium was supplemented with 20 % foetal bovine serum (FBS) and 0.95 M glycerol (GL Y), ethylene glycol (EG) or dimethyl sulfoxide (DMSO). Pre-freezing effects of the extenders and cryoprotectants were minimal in all treatments. Post-thaw parameters were lower (P < 0.05) than the prefreezing parameters and the highest difference (P < 0.0001) was observed after 0 h of equilibration. Post-thaw motility and viability of treatments equilibrated for 2 h and 4 h in MTL, and frozen with GL Y or EG were not different from the control (P > 0.05). Significant differences in post-thaw membrane and acrosomal status occurred between the control and treatments equilibrated for 2 h and 4 h. Using MTL with GL Y or EG, and 2 h or 4 h of equilibration produced results comparable to the control. However, freezing bull epididymal spermatozoa without equilibration is not recommended, and DMSO should only be used in MTL in the absence of GL Y and EG. Bovine epididymal spermatozoa cryopreserved in BIL and MTL after equilibration for 2 h and 4 h was used to inseminate in vitro matured bovine oocytes. In vitro embryo development was assessed to compare the efficacy of BIL and MTL in cryopreserving the fertilizing potential of bovine epididymal spermatozoa. Cleavage rates varied between the treatments, and were lower (P < 0.05) for the treatments than that of the control (BIL). As embryo development progressed, differences between treatments decreased except for sperm cryopreserved in DMSO that maintained a lower (P < 0.0001) development rate than other treatments and the control. Embryo development did not differ (P > 0.05) when sperm equilibrated for 2 h or 4 h was cryopreserved in BIL. However, embryo development was comparable when sperm equilibrated for 2 h or 4 h in MTL was cryopreserved with GL Y or EG. There was no difference in blastomere numbers of embryos of all treatments equilibrated for 4 h. Spermatozoa cryopreserved in MTL containing 0.95 M GL Y or EG, but not DMSO, produced embryos at rates comparable to BIL. Therefore, MTL supplemented with 20% FBS and 0.95 molar concentration of GL Y or EG can be used as a substitute for commercial extender such as BIL for freezing bovine epididymal sperm, and possibly also of other species, for use in ARTs. Frozen-thawed bovine cauda epididymal sperm were subjected to a second freeze-thaw cycle in BIL and MTL, and used for in vitro fertilization (IVF) of in vitro matured bovine oocytes. Cleavage, morula and blastocyst rates were lower (P < 0.05) for oocytes inseminated with sperm that underwent one freeze-thaw cycle in MTL and two freeze-thaw cycles in BIL and MTL (treatments), than the control (one freeze-thaw cycle in BIL). Embryo expansion and/or hatching on days 7-15, were not different (P > 0.05) among the treatments, but embryo expansion and/or hatching on the same days were lower (P < 0.05) when oocytes were inseminated with sperm refrozen in BIL. The blastomere numbers of embryos from sperm of freeze-thaw cycle one in BIL (119 ± 52.8) and MTL (130 ± 43) were not different (P > 0.05) from each other, but were higher (P < 0.05) than that of embryos from sperm refrozen in BIL (58 ± 29). It was concluded that bovine epididymal sperm can be refrozen in BIL, and MTL containing 20 % FBS, EG and used in ARTs. Hybridization between cattle and its closest African wild relative, the African buffalo (Syncerus caffer caffer) was investigated. In an attempt to produce pre-implantation cattle x buffalo hybrid embryos in vitro, matured bovine oocytes were subjected to standard IVF procedure with either homologous bovine (n = 1166 oocytes) or heterologous buffalo (n = 1202 oocytes) frozen-thawed epididymal sperm. After IVF, 67.2% of the oocytes inseminated with homologous sperm cleaved. In contrast, insemination with buffalo sperm resulted in a 4.6% cleavage rate (P < 0.0001). Cleavage was also slower in hybrids than in cattle embryos. Up to 52.2% of the cleaved homologous embryos progressed to the morula stage compared with 12. 7% for the hybrids (P < 0.0001 ). No hybrid embryos developed beyond the 16-cell stage, while 40.1 % of cleaved bovine embryos developed to the blastocyst stage. Developmental anomalies such as polyspermy, uneven cleavage, vacuolization and absence of nuclei in some blastomeres were common in the hybrid embryos. It was concluded that interspecies fertilization of cattle oocytes with African buffalo epididymal sperm occurs in vitro, and that the barrier to hybridization occurs in the early stages of embryonic development. Chromosomal disparity is likely the cause of the fertilization abnormalities, abnormal development and subsequent arrest impairing formation of pre-implantation hybrid embryos. Investigation into the developmental abnormalities including reciprocal hybridization and genetic studies of the hybrid embryos are recommended. The effects of supplementing oocyte maturation medium with 100 μM cysteamine, and the use of a copper-wire cryoloop for vitrification of the in vitro matured bovine oocytes in the production bovine embryos in vitro was examined. Cysteamine did not improved the cleavage rate (P > 0.05), but improved morula and the blastocyst rates (P < 0.05) in one trial. In a second trial, cysteamine did not improve embryo development in the fresh oocytes and the cleavage rate of vitrified oocytes (P > 0.05), but improved the morula and the blastocyst rates (P < 0.05) of vitrified oocytes. Bovine oocytes can be successfully vitrified using a copper-wire cryoloop. Addition of cysteamine in maturation medium improves embryo development of vitrified-thawed mature oocytes. Cysteamine also improves embryo development in non-vitrified oocytes but this effect appears to be influenced by the pre-culture and culture conditions.