Improving the quality of frozen-thawed ram semen

Kafi, A. (2019) Improving the quality of frozen-thawed ram semen. Doctoral thesis, Harper Adams University.

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Cryopreserved semen is widely used for assisted reproduction in livestock, however, in sheep its use is still limited. Freezing and thawing cause biochemical and physiological alterations into the sperm. These alterations such as oxidative damage caused byreactive oxygen species (ROS) and changes inheat shock proteins (HSP) appear to determine the fertilizing ability of the sperm. Seminal plasma (SP) and antioxidants have the ability to improve sperm function and reduce the effect of cryopresrvation. Thisstudy aimed to improve the integrity of frozen-thawed ram sperm, so that an increase the fertility rates could be achieved. The first experimentevaluated the effect of the post-thaw addition of 1.5 mg/ml of different seminal plasma (SP) protein fractions(Whole SP, >100, 30-100, and <30kDa) on the quality of frozen-thawed ejaculated and epididymal ram sperm. SP proteins fractions,particularly <30kDa improved the integrity of fresh and frozen-thawed epididymal spermatozoa and fresh ejaculated spermatozoa (P< 0.001), but there was no effecton frozen-thawed ejaculated spermatozoa. This reductioncould be related to the effect of ROSon sperm integrity. Therefore, the second experimentwas designed to assess the effect of the antioxidantscysteine, taurine,and vitamin C on frozen-thawed ram semen. Semen samples were treated pre-freeze (PF) and post-thaw (PT) to evaluate the optimal timing and concentration of antioxidant supplementation on frozen-thawed ram semen to improve sperm function and reduce ROS production. The addition of 0.5 and 1.0 mg/ml cysteine or taurine (PF + PT) improved the integrity of frozen-thaw ram sperm. There was no effect of vitamin C supplementation on frozen-thawed ram sperm, however, it improved penetrability and reduced ROSproduction. In the third experiment, the effect of oxidative stress induced by 5mM and 15mM hydrogen peroxide (H2O2) on the integrity of fresh ram spermwas assessed.ROS production,lipid peroxidation (LPO) in SP, and the expression of heat shock proteins (70 and90) were determined.H2O2has the capability toeliminate sperm functions significantly at 15μM. This effect could indicate the importance of HSP70 and HSP90 to protect sperm membrane functions, thus there is a need to maintain the function of these proteins. The fourth experimentidentified the relationship between supplementation with1.0 mg/ml (PF + PT) of taurine or cysteine and the expression level of HSP90 and HSP70 on frozen-thawed ram sperm. The results showed that PF or PT supplementation of antioxidants (1.0 mg/ml of cysteine or taurine) improved post-thaw ram sperm integrity and maintained the expression of HSP70 and HSP90. There was a positive relationship between the level of expression of HSP70 and HSP90 and sperm parameters such as motility, acrosome integrity, viability, penetrability, ROS concentration and the level of LPO in SP and sperm. Therefore, the findings of this thesis collectively may assist to improve the quality of cryopreserved ram semen, andeffectively contribute to reproductive technologies in the sheep industry.

Item Type: Thesis (Doctoral)
Divisions: Animal Production, Welfare and Veterinary Sciences
Depositing User: Ms Kath Osborn
Date Deposited: 15 Nov 2019 14:58
Last Modified: 15 Nov 2019 14:58

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