Influence of extender, temperature, and equilibration time on postthaw sperm motility in ram semen

Objective was to investigate effects of extender, loading temperature, and equilibration time on postthaw sperm motility. Ejaculates were collected with an artificial vagina from 4 rams from May - July. Each ejaculate was split into 3 aliquots and extended in a 1 step protocol to a concentration of 200 x 106  sperm/ml with a liposome-based extender (OPTIXcell™), an egg yolk-based extender (Triladyl® ), and a soy lecithin-based extender (AndroMed ). Half of each extended aliquot was loaded into straws at ambient temperature (23C) and left to equilibrate for either 2, 4, or 12 hours at 4C before cryopreservation in liquid nitrogen. Remaining half was loaded at 4C after equilibration before freezing. Concentration and motility were measured with iSperm ® Semen Analyzer. Linear mixed models were used for statistical analysis. Results are expressed as least square means + SEM. Postthaw sperm motility differed (p < 0.0001) among OPTIXcell™ (37.1 + 1.5%), AndroMed  (26.3 + 1.8%), and Triladyl®  (30 + 0.8%) extenders. Ambient loading temperature (32.6 + 1.5%) also had appreciable positive effects (p < 0.0147) on postthaw motility compared to 4C (29 + 1.1%). Samples with an equilibration time of 2 hours had lowest (p < 0.0001) postthaw sperm motility (23 + 1.2%) compared to 4 hours (33.8 + 2.3%) and 12 hours (35.6 + 1.5%) equilibration. However, sperm postthaw sperm motility between 4 hours and 12 hours equilibration was not different (p < 0.4270). In conclusion, it is optimal to extend ram semen with OPTIXcell™ to avoid risk of microbial contamination that comes with animal-based extenders, load at ambient temperature which is less labor intensive and may limit possibility for temperature fluctuations, and to equilibrate at 4C for 4 hours which could lengthen the window of AI postthaw. This protocol is ideal for efficiency and attractive to those without experience or cuttingedge equipment to freeze semen.

Keywords: Extender, cryopreservation, ram semen, motility, iSperm, temperature

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.