Capacitation of Nguni semen for in vitro fertilization of bovine oocytes.
Durrans, Lesley Jane.
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In vitro maturation, fertilization and culture is a technique which is increasingly utilised in the animal science industry for embryo production. However, optimal conditions for in vitro maturation, fertilization and culture have not been clearly defined and much research is being done to improve this situation. If these techniques are to be valuable in the production of large numbers of animals in short periods, they must be efficient and reliable. The first part of this investigation aimed to produce comparable rates of fertilization from each of 5 Nguni bulls tested in an in vitro fertilization trial. It was hypothesised that each bull would respond favourably to one or more of the treatments tested. A complete randomised block design was set up to account for block and day effects. A total of 2422 cumulus-oocyte complexes were matured and fertilized with swim-up separated frozen-thawed Nguni semen incubated for 1 min in Ca-ionophore A23187 (0.1, 0.2 or 0.4 µM) or for 15 min in heparin (0.05, 10 or 25 µg/ml). Controls for parthenogenesis and capacitation or acrosome reaction in the fertilization medium were included. Presumptive zygotes were transferred to CR1aa medium and supplemented with 10% and S% FCS on days 4 and 7 post-insemination, respectively. Bull performance was assessed using four criteria: embryo cleavage, a score based on a cleavage index (Score 1; minimum -1, maximum 6) and one which gave greater weight to morulae and blastocysts (Score 2; minimum -1, maximum 10) and blastocyst production. Day effects were highly significant (P<0.001). Parthenogenetic activation of 31.6% of oocytes occurred. Depending on the bull used, cleavage rates (%±s.e.m.) varied from 29.S±2.2 to 40.6±2.6, Score 1 from 0.30±0.07 to 0.6S±0.07, Score 2 from 0.30±0.07 to 0.72±0.08 and % blastocysts from 0.6±0.6 to 4.8± 1.1. Treatment did not significantly affect performance and there were no bull x treatment interactions. However, bull differences in performance were observed. The IVF system employed was not stable and did not produce repeatable results. It was concluded that treatment concentrations tested may have been too low or, alternatively, that treatment effects were being masked by a factor influencing the IVF system more strongly than the treatments tested. In the second part of this investigation, laboratory tests to determine semen quality were examined. It was hypothesised that semen quality would correlate with performance determined during the in vitro trial and, thus, provide predictors for bull fertility in vitro. A dual staining procedure which detects live/dead or acrosome reacted/not acrosome reacted sperm was also utilised to determine whether bull x treatment interactions may have existed in the in vitro trial but were obscured by other factors influencing the IVF system. Sperm motility and abnormal morphology were assessed using light microscopy on whole frozen-thawed semen samples. Statistical analysis could not be done because of lack of replication. Percentages of immotile sperm were high (60.16% to 78.51%), with considerable variation in progressive motility (13.90% to 34.35%) between bulls. A large variation in numbers of morphologically normal sperm was observed between bulls (66% to 90%), with major deformities (9% to 19%) accounting for most of the abnormalities. High negative correlations with performance (as defined in the in vitro trial) were found between % normal sperm and % proximal droplets (r=-0.66 to -0.88). Percentage of minor abnormalities, distal droplets and coiled tails correlated positively with fertility (r=0.67 to 0.91). Motility did not correlate highly with any of the criteria used to assess bull performance. Correlations of semen quality to performance were contradictory to expected results and this may have been due to swim-up separation of sperm for the in vitro trial which was not carried out for semen quality assessment. At present, these semen quality tests do not allow prediction of bull fertility in vitro. Assessment of sperm stained for evaluation of live/dead and acrosome reacted/not acrosome reacted was a lengthy procedure. Again, statistical analysis was not possible due to the lack of replication. High percentages of sperm were characterised as dead (52.4% to 100%). Bulls did respond differently to the various treatments, as determined by the proportion of acrosome reacted sperm and live acrosome reacted sperm. Thus, bull x treatment interactions were apparent, suggesting that the IVF system was more strongly influenced by other factor(s) which reduced sensitivity to the treatments tested. In summary, more research is needed to stabilise the IVF system if production of large numbers of embryos is to become economically viable.