21 EFFECTS OF HETEROLOGOUS SEMEN PLASMA AND SEMEN EXTENDERS ON PROGRESSIVE MOTILITY OF FROZEN-THAWED RAM SPERM

Abstract

Frozen-thawed ram semen crosses the cervix poorly, necessitating laparoscopic insemination. Acceptable fertility can be achieved with frozen-thawed ram semen deposited at the external cervical opening if ram semen plasma (SP) is added (McPhie et al. 2000 14th ICAR 2, 78 abst). Homologous SP improves the fertility of frozen-thawed sperm of boars and dogs. Heterologous SP may have effects as well; the addition of bovine SP increased the ability of buffalo sperm (Syncerus caffer) to fertilize bovine oocytes in vitro (de Haas et al. 2003 Theriogenology 59, 392). The aim of the current study was to compare the effects of SP of rams (SPR), bulls (SPB), and dogs (SPD), protein-free TALP, Triladyl (Minitüb, Tiefenbach, Germany), and skim milk upon longevity and percentage of progressively motile frozen-thawed ram sperm. Three ejaculates from each of six rams (2 Dorpers, 2 Döhne merinos, and 2 merinos), aged 2–4 years, were extended in Triladyl, pooled and frozen as a single batch per ram at 200 × 10⁶/mL in 0.25-mL straws. SPR was obtained from the same rams and SPB from 5 bulls by centrifugation, while the post-sperm fractions were collected from 5 dogs (SPD). Within a species, the SP from different donors was pooled and frozen in aliquots at −18°C. The 108 straws (6 rams, 6 diluents, 3 replicates) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85 mL of the appropriate fluid at 37°C and kept for 6 h. Percentage of progressively motile sperm was estimated at ×200 magnification immediately and 2, 4 and 6 h after thawing. One person thawed the semen and prepared motility specimens, while another performed all motility evaluations. Data were evaluated by means of repeated-measures ANOVA, with rams as subjects and time and fluid as fixed effects. Non-significant interactions were removed from the model. Means were compared by means of Bonferroni's test (P < 0.05). The model included ram, time, fluid, and ram × fluid, and time × fluid interactions, which were all significant (P < 0.01). Mean motility decreased from each time to the next and were 39.0% (0 h), 26.0% (2 h), 19.6% (4 h) and 12.6% (6 h), SEM 1.38%, n = 108. Mean motility was higher for skim milk (39.9%) than for all other fluids except Triladyl (27.7%), which was better than SPB (13.0%), whereas TALP (20.5%) and SPR (21.9%) were similar to Triladyl and SPB (n = 72, SEM 2.85%). The interactions (ram × fluid or time × fluid) were mainly due to SPD, SPR, Triladyl, and TALP, while milk resulted in the best and SPB in the lowest motility. This study shows that heat-treated skim milk maintains progressive motility of frozen-thawed ram sperm better than the SP of various species and protein-free TALP. In contrast to SPR, skim milk is known to result in poor fertility of frozen-thawed ram semen after cervical insemination. It would thus appear that maintenance of progressive motility in vitro may be a poor indicator of fertility after cervical insemination.