Slow Cooling Is Beneficial for Storage of Frozen-Thawed Equine Spermatozoa

Background: Recently, researchers have reported acceptable motility and pregnancy results with frozen-thawed semen that has been cooled-stored for up to 24 h opening new opportunities for the use of cryopreserved semen. 
Objectives: To compare two cooled-storage protocols for frozen-thawed semen. 
Study design: Motility and viability of frozen-thawed semen stored at 4°C for 8 h or 24 h were compared between two storage protocols. 
Methods: Two straws of frozen-thawed semen from 31 stallions was diluted and divided in 5 aliquots that were either examined immediately (T0); placed immediately after warming in a semen shipping box, resulting in fast cooling to 4°C, and stored for 8 h or 24 h (T8-CB and T24-CB); or placed first in a refrigerator (4°C), resulting in slower cooling, for 30 minutes and subsequently transferred to semen shipping box (T8-REF and T24-REF). Median total and progressive motility were evaluated after 5 minutes (T0, T8, T24) and 30 minutes (T0.5, T8.5, and T24.5) incubation at 37.5°C. 
Results: After 8 h cooled storage, total and progressive motility remained unchanged in all REF-aliquots. Progressive motility remained unchanged in T8-CB but was lower in T8.5-CB (-8.52%) and total motility was lower in T8-CB (-8.12%) and T8.5-CB (-9.96%) (p < 0.05). After 24 h, total and progressive motility were lower in all CB and REF aliquots. Viability was lower at 8 h in CB (-11.87%), REF (-9.65%), and at 24 h in CB (-13.52%), and REF (-12.32%) (p < 0.05). The results demonstrate that sperm motility and viability decrease during cooled storage of frozen- thawed semen but a slow cooling rate before transferring to a semen shipping box reduces the effect of short-term cooled storage (8 h). 
Main limitations: Motility and viability are not directly related to fertility. 
Conclusions: A slower cooling rate to 4°C after thawing/warming the semen to 37.5°C has a beneficial influence on motility after 8 h of cooled storage of frozen-thawed semen. 
Ethical animal research: Research ethics committee oversight not currently required by this conference: material was collected during clinical procedures.
Informed consent: Horse owners consented to excess samples being used for research. 
Competing interests: None declared. 
Sources of funding: None.

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