Effects Of Cholesterol On Bovine Sperm Survival During Cooling and Freezing

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Damage caused to sperm due to cooling and freezing has long limited the potential of cryopreserved semen. Considering that much of this damage is attributed to injury to the plasma membrane of the sperm cell, methods that stabilize the membrane would greatly improve the prospects of cryopreservation. One such stabilizing factor is cholesterol, which decreases the phase transition of membrane lipids that occurs during the cooling and freezing process. One known method of membrane cholesterol enrichment employs cholesterol loaded cyclodextrin (CD/CHOL). The purpose of this study was to confirm current CD/CHOL results in the bull as well as to examine an alternative method of cholesterol enrichment that utilizes high cholesterol egg yolk. For the cyclodextrin studies, fresh bovine ejaculates were incubated with or without CD/CHOL and cooled and frozen in various concentrations and preparations of egg yolk. Percent motility of the sperm was examined prior to freezing, after cold shock treatment, 0 and 3 hrs after cooling, and post-thaw by phase contrast microscopy. Percent viability was evaluated post-thaw by propidium iodide (PI) and SYBR 14 staining using the LIVE/DEAD Sperm Viability Kit protocol. For the study using high cholesterol yolk, domestic hens were fed one of three diets supplemented with 0.5%, 1.0%, or no cholesterol. Eggs from these hens were used to examine the effects of cooling and freezing with various preparations and levels of yolk. Just as in the CD/CHOL study, fresh ejaculates were extended in their respective treatment, cooled, frozen, and evaluated for percent motility and viability. No treatments yielded significant effects of CD/CHOL incubation on percent sperm motility or viability while studies using high cholesterol yolks demonstrated improved i motility with both the 0.5% and 1.0% diet. The results of this study indicate that treatment with CD/CHOL does not affect motility or viability, while treatment with high cholesterol yolk does. ii

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