Effects of Cell Concentration During Cryopreservation On the Post-thaw Quality of Santa Inês Ram Sperm

BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe's cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Inês rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G – 400, G – 800, G – 1200, and G – 1600 x 10 6 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5 ºC, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40 ºC/20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G – 400 treatment resulted in samples with the highest linearity and progressive motion (P < 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P < 0.05). Overall, use of the G – 400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G – 800 and G – 1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single rtificial insemination protocol is desirable.