Quality of frozen equine semen stored in different shipping containers

Abstract

The use of cryopreserved semen in horses requires sophisticated equipment, including containers maintaining the temperature of liquid nitrogen (LN). Simpler and less expensive devices for transport of frozen semen may help breeding programs in developing countries and reduce the costly return of empty containers. Veterinarians sometimes question the quality of frozen-thawed shipped semen because of potential fluctuations in shipping temperatures. This study aimed to investigate the effects of shipping duration and temperature on post-thaw quality of frozen semen stored either in a dry shipper (≤ -150°C) or on dry ice (≤ -78°C) for 14 days. Straws (n=165) containing cryopreserved semen from five stallions stored in liquid nitrogen were transferred (D0) to a dry shipper (DS) and to a styrofoam box with dry ice (DI). Straws from DS and DI were returned to LN on D1, D3, D7, D10, and D14. All semen doses were evaluated at each time point by CASA for total (TMot) and progressive motility (PMot), and kinematic parameters such as average path velocity (VAP), curve-linear velocity (VCL) and straight-line velocity (VSL), by fluorescence microscopy for the percentage of membrane-intact spermatozoa (SYBR14/PI stain), and high mitochondrial membrane potential (HMMP; JC-1 stain). Data were analyzed by repeated measures ANOVA with both storage time and storage device as within subject factors. Thetemperature inside the DS and DI containers was monitored continuously with a Tempod 200XB data logger. There were no temperature fluctuations during the 14-day storage in both containers. Until D7 of storage, no changes were observed in TMot, PMot, and membrane-intact spermatozoa (%). Thereafter all three parameters decreased in semen stored in DI but not in DS (storage device at least p<0.05, time p<0.01, time x storage device p<0.001; e.g., TMot D7 on DI and in the DS both 59±14 versus 34±10 and 60±13%, respectively, on D14; membrane-intact spermatozoa D7 on DI 50±13 and in the DS 52±12 versus 28±7 and 53±19% on D14). The HMMP percent decreased continuously during storage in both containers (p<0.001) and this decrease was more pronounced for DI compared to DS (time x storage device p<0.05; DI from 43±4 on D1 to 26±3% on D14, DS from 43±5 on D1 to 30±4% on D14). All velocity parameters remained constant with storage in both devices. In conclusion, cryopreserved frozen stallion semen can be stored for up to 7 days on dry ice without negative effects on post-thaw characteristics. Dry ice may thus be an alternative to liquid nitrogen containers for the short-term transport of frozen stallion semen. Controlled insemination trials to confirm the fertility of frozen stallion semen shipped on dry ice are justified.