Effects of water- and lipid-soluble antioxidants on turkey sperm viability, membrane integrity, and motility during liquid storage

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Abstract
Aerobic conditions are required to maintain the viability of turkey sperm in vitro. In mammalian sperm, excess oxygen during in vitro storage results in lipid peroxidation, causing membrane damage and reduced sperm motility and subsequent fertility. The effect of adding antioxidants to turkey sperm during liquid storage was studied. Semen was collected and pooled from 20 toms and antioxidants were tested at a minimum of six concentrations, n = 6 observations per concentration. Semen was diluted into Beltsville Poultry Semen Extender. Extended semen served as a control; treatments were extended semen supplemented with tocopherol (vitamin E, 1 to 80 micrograms/mL); butylated hydroxytoluene (BHT, 0.02 to 1.25 mM); Tempo (0.039 to 1.25 microM), or vitamin C (1 to 400 micrograms/mL) and stored at 5 C for 48 h. Sperm viability in extended semen was evaluated after 0, 24, or 48 h storage. Membrane integrity and motility were also measured. Flow cytometric analysis was done using the live/dead stain combination (SYBR-14/propidium iodide) for sperm viability, and membrane integrity was assessed using a hypo-osmotic stress test. Sperm motility was evaluated subjectively. Control sperm viability was reduced almost 50% between 0 and 48 h. However, supplementation with vitamin E, Tempo, and BHT maintained populations of viable sperm similar to the 0 h levels at 48 h. Hypo-osmotic membrane integrity in the control sperm was reduced to approximately 22% (at 24 h, P < or = 0.05) and 5% (at 48 h, P < or = 0.05) of the total sperm population. Similar to controls after 24 h in vitro storage, sperm treated with the antioxidants vitamin E, Tempo, and BHT had reduced hypo-osmotic membrane integrity compared to 0 h samples. However, many of these treated samples maintained hypo-osmotic membrane integrity observed from 24 through 48 h (range, 21.5 to 44.6%), whereas hypo-osmotic membrane integrity fell to 4.6% at 48 h for the control (P < or = 0.05). Vitamin C treatments were similar to controls at all time points. Addition of the antioxidants vitamin E, BHT, and Tempo to extended turkey semen improves sperm survival, membrane integrity, and reduces the loss of motility after 48 h storage.