Effect of seminal plasma on the acrosome status of cryopreserved stallion spermatozoa

Abstract: Today breeding through artificial insemination (AI) with cryopreserved semen is frequently used in several domestic species, but its use is more problematic in horses, partly because of reduced sperm quality and partly because the timing of insemination relative to ovulation is more crucial for frozen semen than for fresh. Therefore, improvements in the freezing technique are needed. In this study, the effect of adding pooled seminal plasma (SP) from stallions with good freezability (Good freezer stallion; GF) and bad freezability (Bad freezer stallion; BF) to spermatozoa selected by single layer centrifugation from GF or BF stallions prior to cryopreservation was investigated. Of particular interest was whether SP can have a positive impact on sperm acrosome status, thereby enhancing fertility. Frozen semen from 12 stallions was available. Aliquots of 5 ml from each ejaculate through normal centrifugation and then frozen were used as controls (C). The remaining parts of the ejaculates were processed by single layer centrifugation (SLC) to remove SP and divided into three aliquots; sample without SP addition (S), sample with SP from GF (S-GF) and sample with SP from BF (S-BF). After thawing, the proportion of live or dead spermatozoa in the sample and the proportion of spermatozoa with an intact or reacted acrosome were evaluated by flow cytometry. The results indicate that the source of the SP (GF or BF) and the origin of the spermatozoa (GF or BF) were important. There was a positive effect of adding SP from GF to SLC-selected spermatozoa (p = 0.0017) compared to the control sample. If the sperm samples were divided according to whether they came from good freezer stallions or bad freezer stallions, the effect of adding SP was most marked when the spermatozoa came from a BF stallion (P = 0.04). In other words, the effect of adding SP to sperm samples is due to both the SP and the spermatozoa themselves. No statistical differences were seen between the different treatments (S, S-GF and S-BF) of SLC-selected samples. To draw more reliable conclusions more studies are needed. The study also confirms SLC as a reliable processing method for increasing sperm survival during freezing.