Processing poor quality ram semen by colloid centrifugation prior to freezing: a proof-of-concept study

Introduction The sheep breeding season is short in some breeds, restricting the availability of rams for semen collection as they are required for natural mating. Therefore, cryopreservation may need to be done before the start of the breeding season, when semen quality may be sub-optimal. Poor quality samples do not show good cryosurvival. Colloid centrifugation, particularly Single Layer Centrifugation (SLC), was shown previously to select the most robust spermatozoa from an ejaculate. The objective of the present study was to prepare sub-optimal ram ejaculates by scaled-up SLC to determine if sperm quality could be improved sufficiently for cryopreservation, in a proof-of-concept trial. Methods Semen was collected from a ram during the month preceding the breeding season. The first ten ejaculates were discarded because of sub-optimal motility (< 70% motile in raw semen). Five further sub-optimal ejaculates were processed by SLC. The semen was extended in warm Andromed (30 °C) to 100 x 10 6 spermatozoa/ml; 20 ml of extended semen were layered over 15 ml Ovicoll in a 50 ml tube and centrifuged for 20 min at 300 g. The sperm pellet was resuspended in Andromed (18 °C) at 500 x 10 6 spermatozoa/ml before loading into plastic straws. The straws were cooled for 2 h at 6 °C and were then frozen in a programmable freezing machine. After storing for 24 h in liquid nitrogen, post-thaw motility was evaluated subjectively. Samples with post thaw motility ≥50% were used for artificial insemination. Results Initial motility in the 5 ejaculates was 20-50%. After SLC, motility was 80%−90%, while post-thaw motility was 50% in all samples. Thawed semen was deposited vaginally in oestrous ewes (22), producing 46 lambs in total, of which 4 were still-born. Conclusion Ram semen can be processed by SLC with Ovicoll to improve sperm cryosurvival.