PhD / The aim of this thesis was to investigate the possibility of integrating frozen-thawed boar semen into reproductive technologies and into commercial production of pigs in Australia. This was to be achieved by establishing a semen freezing and AI regime that was of a standard acceptable to industry, and integrating the resultant frozen-thawed sperm into other reproductive technologies, such as flow cytometric sperm sorting and IVF. Initially, a protocol for freezing and thawing boar semen was established, based on the method described by Westendorf et al. (1975) and attempts were made to modify this protocol to improve the post-thaw sperm quality, as determined by in vitro assessment of motility, acrosome integrity and longevity. First, the egg yolk used in the freezing extenders was investigated, and the chicken yolk was replaced with either duck or quail yolk. It was shown that there was no benefit in substituting yolk from duck or quail for the chicken yolk traditionally used in freezing extender. Second, the effect of seminal plasma addition to the freezing extender, or seminal plasma addition to resuspension medium post-thaw was tested. Incorporating whole seminal plasma into the freezing extender at levels above 50% was found to be detrimental to post-thaw sperm quality. Reducing levels to 20% of the final volume improved acrosome integrity, but adversely affected motility of sperm. However, adding 20% seminal plasma to the resuspension medium used after thawing of boar semen had no significant influence on sperm quality compared with resuspension in medium without seminal plasma. The antioxidant catalase, and the iron chelator desferal added to the freezing extender, did not improve post-thaw sperm quality, nor was any benefit seen with addition of these substrates to the resuspension medium post-thaw. However, the bioactive phospholipid PAF and its regulating enzyme PAF:AH appeared to enhance post-thaw motility and acrosome integrity of sperm, respectively, when added to the semen pre-freezing. Unfortunately, due to the restrictions imposed on rPAF:AH as a research drug, it was not possible to test the in vivo effects at this time. After the in vitro experiments were completed, the in vivo fertility of frozen-thawed sperm was tested using the optimal freezing protocol and a novel technology, enabling non-surgical deep intrauterine insemination of sows. The aim was to establish the lowest possible dose of frozen-thawed sperm that could be used, without compromising fertility. Successful pregnancies were achieved with doses as low as 62.5 x 106 frozen-thawed sperm but the farrowing rates were too low to be practicable on a commercial scale. This is the first report of litters born after insemination of such a low dose of frozen-thawed sperm and using the novel DIU insemination technique. However, it was concluded that a double dose of 250 x 106 frozen-thawed sperm was the minimum dose required for maintaining acceptable fertility. Reduction in sperm numbers to such an extent made it possible to consider non-surgical insemination of sex-sorted, frozen-thawed semen. Previously, pregnancies had been achieved only after surgical insemination of sex-sorted boar sperm, or with DIU insemination of unfrozen sperm, immediately after sex-sorting. The low numbers of sex-sorted sperm available restricted the inseminate dose used here to 50 x106 motile sperm. A litter of 5 piglets was born after a low-dose, DIU insemination of sex-sorted, frozen-thawed sperm. This is the first report of piglets born after insemination with sex-sorted frozen-thawed sperm and non-surgical insemination. The low farrowing rate achieved in this experiment prompted the investigation of integrating sex-sorted, frozen-thawed boar sperm into IVF. Morulae were produced after IVF with sex-sorted, frozen-thawed sperm and successfully transferred using non-surgical techniques. This is the first report of pregnancy achieved with non-surgical transfer of embryos produced after IVF and IVC of IVM oocytes with sex-sorted, frozen-thawed boar sperm. Unfortunately, the pregnancy did not hold, and the embryos were lost prior to Day 32, but PCR of non-transferred embryos confirmed successful pre-selection of sex. Overall, this thesis demonstrated that it is still not economically feasible to incorporate frozen-thawed boar semen into the commercial production of pigs although it has considerable application in breeding programmes. However, the development of novel techniques enabling reduction in sperm dose, and for non-surgical transfer of embryos into recipient sows and incorporation of frozen-thawed semen into these technologies means that progress is being made with the integration of reproductive technologies and frozen-thawed semen into the pig industry.
| Identifer | oai:union.ndltd.org:ADTP/216064 |
| Date | January 2004 |
| Creators | Bathgate, Roslyn Anne |
| Publisher | Faculty of Veterinary Science, The University of Sydney. |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_AU |
| Detected Language | English |
| Rights | The author retains copyright of this thesis., http://www.library.usyd.edu.au/copyright.html |
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