Functional assessment of diluent choice for semen cryopreservation from stallions with high and low freezability

Abstract

Background: Fertility using horse frozen-thawed semen remains lower than in other livestock species. This fact suggests that horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is substantial evidence of genetic factors upon horse cryopreservation outcome. Nonetheless, diluent and cryoprotectant choice for horse semen cryopreservation are under intense research. Thus these factors could be explored to identify conditions that may increase semen viability after thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®, Lactose-EDTA®, and INRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA). Materials, Methods & Results: Frozen-thawed semen was evaluated for motility and membrane integrity using computer-assisted semen analysis (CASA), and also inferred for sperm DNA fragmentation by sperm chromatin structure assay during the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA and LFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger (P < 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® was greater (P < 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P > 0.05) at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h. Discussion: Artificial insemination in horses using frozen-thawed semen is gaining wider acceptance under commercial settings, although its current limited outreach due to low semen viability after thawing. Therefore, several efforts were made toward modifying horse semen cryopreservation methodologies to meet the market demand, particularly for breeds that display low semen freezability under a regular basis. Frozen-thawed semen from stallions of high freezing ability showed greater motility at all analysis, irrespectively of diluent choice, suggesting a strong influence of genetic factors on cryopreservation outcome under the experimental conditions described in the text. Nonetheless, the diluent choice did affect motility for LFA stallions, and cryopreservation protocols including lower glycerol concentrations seem more promising. Membrane integrity was similar immediately after thawing but did differ later on other TRT time-points, irrespectively of diluent choice. As observed for motility, it was expected that sperm cells of stallions of HFA would show higher membrane integrity than their LFA counterparts. Hence, the diluent choice is relevant for semen cryopreservation from LFA stallions. This fact is supported by the well-described correlation between sperm membrane viability and motility. Sperm DNA fragmentation was quite low for both groups, as generally described in horses. Surprisingly, sperm DNA fragmentation incidence was constant throughout the analysis for both HFA and LFA. It was initially envisioned that increased DNA fragmentation would be found in semen from LFA stallions since it is caused by multiple origins such as genetic factors. Therefore, it can be suggested that sperm DNA integrity was more influenced by the balance of genetic and environmental factors during gametogenesis than the TRT assay. In conclusion, the semen diluent affects horse sperm motility after thawing, particularly from stallions with lower semen freezability.