Individual variation of frozen-thawed sperm from stallions to survive dilution and cooling after thawing

Within the equine industry, there has been interest in the ability to thaw frozen semen in a semen production laboratory, dilute further in extender and ship it as a cooled dose to the farm for insemination. Two previous studies concluded that acceptable retention of motility and fertility can be achieved using this technique, but was only done on a small set of samples. Goal of this experiment was to measure semen quality after thawing and cooling over 48 hours on a large number of stallions and illustrate the variability in the success among individual stallions. We evaluated 22 doses of frozen semen of variable post-thaw quality, all of which previously resulted in pregnancies. Motility analyses were performed using a Hamilton Thorne Ceros II computer assisted sperm analysis system to evaluate at least 400 motile sperm from each sample at each time point. Samples were analyzed for 40 video frames at 60 frames/second and progressive motility (PM) was defined as motile sperm moving with an average path velocity > 50 µm/second and a straightness value of > 75%. A full dose of eight (0.5 ml) straws per sample were thawed at 37C for 30 seconds, then combined in a prewarmed 5 ml tube. Concentration was determined with a NucleoCounter SP-100 and an aliquot of the sample was added to pre-warmed cooling extender to a concentration of approximately 10 million/ml. After incubation for 30 minutes at 37C, initial motility was determined. A second aliquot of thawed sample was incubated in modified Whitens medium with SYBR-14 and propidium iodide at a concentration of 5 million/ml. Membrane integrity analysis was completed on a Guava EasyCyte HT flow cytometer. Frozen-thawed samples were diluted to 30 million/ml in extender previously determined to be optimum for cooling semen of that individual stallion (INRA96 ® or a standard skim milk-glucose extender). Motility and membrane integrity analyses were performed initially (time 0), 6, 10, 24, 32 and 48 hours postthaw. Percent change in motility and membrane integrity was calculated from time 0 (initial) to 6, 24, and 48 hours after cooling. Average percent decrease in PM across all 22 stallions was 23, 41, and 67% at 6, 24, and 48 hours. Average percent decrease in sperm with intact membranes was 16, 16, and 23% at 6, 24, and 48 hours. Individual variation in the success of this technique was substantial. Decrease in semen quality after thawing and cooling for 24 hours, ranged among stallions from 2.5 to 77.6% for PM and 0 to 37% for membrane integrity. Decrease in PM for top 5 stallions at 6, 24, and 48 hours were 5, 13, and 33% respectively whereas for bottom 5 stallions there was a 47%, 71%, and 98% change in PM, respectively. It is concluded that although this technique is a viable option for some stallions, it is not appropriate for all stallions and therefore must be tested on every stallion.

Keywords: Equine sperm, frozen-thawed, cooled, membrane integrity

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.