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Title: Improving the quality of frozen-thawed ram semen
Author: Kafi, Ahmed
ISNI:       0000 0004 8503 5121
Awarding Body: Harper Adams University
Current Institution: Harper Adams University
Date of Award: 2019
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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 by reactive oxygen species (ROS) and changes in heat 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. This study aimed to improve the integrity of frozen-thawed ram sperm, so that an increase in the fertility rates could be achieved. The first experiment evaluated 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 effect on frozen-thawed ejaculated spermatozoa. This reduction could be related to the effect of ROS on sperm integrity. Therefore, the second experiment was designed to assess the effect of the antioxidants cysteine, 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 ROS production. In the third experiment, the effect of oxidative stress induced by 5mM and 15mM hydrogen peroxide (H2O2) on the integrity of fresh ram sperm was assessed. ROS production, lipid peroxidation (LPO) in SP, and the expression of heat shock proteins (70 and90) were determined. H2O2has the capability to eliminate 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 experiment identified the relationship between supplementation with 1.0 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, and effectively contribute to reproductive technologies in the sheep industry.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available