The Effects of Different Cryoprotectants on Stallion Epididymal Spermatozoa
Beattie, C. (2011). The Effects of Different Cryoprotectants on Stallion Epididymal Spermatozoa (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/5597
Permanent Research Commons link: http://hdl.handle.net/10289/5597
Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from a region adjacent to the testicles called the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. This thesis investigated the effects of different cryoprotectants and dilution after thawing with SW3 media (SW3, JEL Media, NZ), which contains complex amino acids, sugars and no protein, on the fertility and sperm characteristics of frozen-thawed epididymal sperm. Epididymal sperm were obtained from 5 colts at the time of castration and each collected sample was split into different treatment groups and frozen with different cryoprotectants (glycerol, formamides or a combination) in the same base extender. The sperm were frozen in liquid Nitrogen until the time of insemination or post-thaw analysis. The effects of different cryoprotectants and the addition of the post-thaw dilution medium on motility parameters, morphology, acrosome status, plasma membrane integrity and DNA integrity were evaluated after thawing. The reproductive tract of mares were examined by ultrasound during oestrous and the mares were inseminated at 4 hours prior to ovulation with epididymal sperm frozen in either glycerol or a glycerol and formamide combination. Fertility was determined at either 8 days after ovulation when the uterus of each mare was flushed for an embryo or at 14 days after ovulation when the mare was scanned to determine if there was a pregnancy. The post-thaw dilution medium (SW3) was found to have a significant beneficial effect on the motility of frozen-thawed epididymal sperm frozen with glycerol in vitro. Pregnancy data revealed that the glycerol and formamide combination cryoprotectant was superior to glycerol alone, with 50% versus 10% pregnancy rates recorded respectively. These results suggest that the higher pregnancy rates are the effect of the cryoprotectant and not the complex amino acids of the base extender (which was the same for all samples) which contribute to the improved fertility of the frozen epididymal sperm. Furthermore, because good pregnancy rates without the addition of SW3 were obtained prior to insemination, this supports the conclusion that the cryoprotectant is a key factor altering the fertilizing ability of epididymal sperm. Investigations into the fertility of epididymal sperm will enhance our understanding of factors which affect sperm fertility whilst in storage in the cauda epididymius. Furthermore, the opportunity to preserve the fertility of these gametes at the time of gelding or death of a stallion will have long-lasting beneficial effects on the size of the gene pool for selection. This is not only important for the domestic horse but for endangered equine species also. Ultimately, the fertility of epididymal sperm should be optimised to ensure that the maximum numbers of doses are available of this limited resource.
University of Waikato
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- Masters Degree Theses