Effects of cell concentration during cryopreservation on the post-thaw quality of Santa Inês ram sperm

Authors: DE OLIVEIRA, MATHEUS - Pio Decimo College; MOLINA, JULIO - Universidade Federal De Sergipe; SANTOS DA SILVA, REBECA - Universidade Federal De Sergipe; RAMOS, ALEXANDRE - Brazilian Agricultural Research Corporation (EMBRAPA); Purdy, Phil; AZEVEDO, HYMERSON - Brazilian Agricultural Research Corporation (EMBRAPA)

Submitted to: CryoLetters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/23/2022
Publication Date: 11/1/2022
Citation: De Oliveira, M.B., Molina, J.C., Santos Da Silva, R., Ramos, A.F., Purdy, P.H., Azevedo, H.C. 2022. Effects of cell concentration during cryopreservation on the post-thaw quality of Santa Inês ram sperm. CryoLetters. 43(6):357-367. https://doi.org/10.54680/fr22610110812.
DOI: https://doi.org/10.54680/fr22610110812

Interpretive Summary: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose due to the challenges of traversing the ewe’s reproductive anatomy and because greater numbers of sperm in a single insemination dose may preclude the need for a second insemination per estrus while still resulting in acceptable fertility rates. Therefore, the aim of this work was to compare a range of sperm concentrations when freezing semen and determine the effects of this on post-thaw quality. Overall, samples with lower sperm concentrations resulted in higher proportions of high quality, motile sperm following freezing, but will require use of two semen straws for artificial insemination to ensure sufficient numbers of sperm are inseminated. However, freezing at some higher sperm concentrations produces samples with a greater total number of motile sperm with acceptable quality and a simultaneous loss of a large number of sperm due to damage during freezing compared to samples with lower sperm concentrations. Although freezing sperm samples at higher concentrations results in the loss of a large number of sperm compared to lower sperm concentrations, this method may be advantageous because it enables use of a single artificial insemination per ewe which decreases labor and supply costs.

Technical Abstract: 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. Greater numbers of sperm in a single insemination dose may preclude the need for a second insemination per estrus and result in acceptable fertility rates. Therefore, the aim of this work was to compare a range of sperm concentrations when cryopreserving semen from Santa Inês rams and determine the effects of this on post-thaw quality. 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 106 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 submitted to functional (computerized automated sperm analysis, and capacitation and acrosome integrity using chlortetracycline) and structural assessment (plasma membrane integrity using SYBR14/propidium iodide). 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, which may be advantageous if a single artificial insemination protocol is desirable.