116 EFFECT OF INCUBATION OF BOVINE EPIDIDYMAL SPERMATOZOA IN SEMINAL PLASMA PRIOR TO CRYOPRESERVATION

Abstract

Studies examining the influence of seminal plasma on sperm function have shown both beneficial and detrimental effects. However, its effect on pre-frozen bovine epididymal sperm (BES) has not been documented. The objective of this study was to determine the effect of a 30-min incubation of BES in bovine seminal plasma (SEMP) prior to freezing on post-thaw sperm parameters. Paired testes were obtained from mature bulls (n = 10) at a local abattoir and transported to the laboratory (25–28°C) within 3–5 h postmortem. BES were harvested by multiple incisions from the caudae epididymides of each bull, pooled, and split into either Treatment A, 3 mL of egg yolk Tris-based medium (EYT) (No SEMP), or Treatment B, 3 mL of SEMP, each for 30 min in a 37°C water bath. SEMP was extracted from ejaculates of mature bulls (n = 6), and then pooled and stored at −20°C until used. Sperm from both treatments were re-suspended in EYT, adjusted to 70 × 106 sperm mL−1 and cooled to 4°C at 0.1°C min−1. Samples were diluted slowly over a 30-min period with 1:1 EYT medium containing 14% glycerol and loaded into 0.5-mL straws. Straws were frozen in liquid nitrogen vapors. For sperm analyses, straws were thawed in a water bath at 37°C for 40 s. Morphology was determined by staining with eosin-nigrosin. Viability and acrosome integrity were measured simultaneously with the combination of SYBR 14, propidium iodide, and PE-PNA. Mitochondrial activity was measured by the combination of MitoTracker Red and SYBR 14. DNA integrity was determined by acridine orange using the sperm chromatin structure assay (SCSA®; SCSA Diagnostics, Inc., Brookings, SD, USA). All assays were performed by multicolor flow cytometry. Differences between treatments were analyzed using one-way ANOVA (P < 0.05). In summary, all sperm quality parameters decreased significantly after thawing in both treatments (Table 1). A significantly higher overall and progressive motility post-thaw was achieved when sperm were incubated in SEMP prior to freezing. However, no difference was detected in sperm viability, acrosome integrity, mitochondrial activity, and DNA integrity between treatments. Also, SEMP reduced the quantity of distal droplets and broken tails post-thaw over that of no SEMP. Results indicate that incubation of BES in bovine seminal plasma prior to freezing improves post-thaw sperm quality. Table 1. Effect of bovine seminal plasma (SEMP) on quality parameters (mean ± SEM) of post-thaw bovine epididymal sperm