Effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram sperm

Abstract

Frozen-thawed ram semen crosses the cervix poorly, necessitating laparoscopic insemination. Acceptable fertility can be achieved with frozenthawed ram semen deposited at the external cervical opening if ram seminal plasma is added. Homologous seminal plasma improves the fertility of frozen-thawed sperm of boars and dogs. Heterologous seminal plasma may have effects as well; the addition of bovine seminal plasma increases the ability of buffalo sperm (Syncerus caffer) to fertilize bovine oocytes in vitro. The aim of the current study was to compare the effects of seminal plasma of rams and bulls, dog prostatic fluid, protein-free TALP, TrilEq (Triladyl with 0.5 ml of Equex STM paste added to each 100 ml) and skim milk upon longevity and percentages of progressively and aberrantly motile frozenthawed ram sperm. Three ejaculates from each of 6 rams (2 Dorpers, 2 Döhne merinos, and 2 merinos), aged 2 to 4 years, were extended in TrilEq, pooled and frozen as a single batch per ram at 200 × 106/ml in 0.25 ml straws. Seminal plasma of rams was obtained from the same rams, while seminal plasma of five bulls were obtained by centrifugation of their ejaculates and dog prostatic fluid consisted of the post-sperm fractions of the ejaculates of 5 dogs. Within a 10 species, the seminal plasma or prostatic fluid 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 medium at 37 °C and kept at that temperature for 6 h. The percentage of progressively motile sperm was estimated at ×200 magnification immediately (time zero) 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 medium as fixed effects. Non-significant interactions were removed from the model. Pairwise comparison of means was done by means of Bonferroni's test (P < 0.05). The model included Ram, Time, Medium, and Ram × Medium, and Time × Medium interactions, which were all significant (P < 0.01). Mean progressive 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 media except TrilEq (27.7%), which was better than bull seminal plasma (13.0%), whereas TALP (20.5%) and ram seminal plasma (21.9%) were similar to TrilEq and bull seminal plasma (SEM 2.85%, n = 72). The interactions (Ram × Medium or Time × Medium) were mainly due to dog prostatic fluid, ram seminal plasma, TrilEq, and TALP, while milk resulted in the best and bull seminal plasma in the lowest motility. This study shows that heat-treated skim milk maintains progressive motility of frozen-thawed ram sperm better than dog prostatic fluid and seminal plasma of bulls and rams, TrilEq and protein-free TALP. In contrast to ram seminal plasma, skim milk is known to result in poor fertility of frozenthawed 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.