35 The effect of different cooled storage protocols on frozen-thawed equine semen
M. van Heule A , M. H. Verstraete A , Z. Blockx A , P. De Blende B , P. Dini C and P. Daels AA Department of Veterinary Medical Imaging and Small Animal Orthopaedics, Faculty of Veterinary Medicine, UGent, Merelbeke, Belgium
B Equine Reproduction Center De Morette, Asse, Belgium
C Department of Population Health and Reproduction (PHR), School of Veterinary Medicine, UCDavis, Davis, CA, USA
Reproduction, Fertility and Development 34(2) 252-253 https://doi.org/10.1071/RDv34n2Ab35
Published: 7 December 2021
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
The use of frozen-thawed semen is an essential part of equine breeding. However, the logistics associated with this technique, including transport and thawing of frozen semen, can be challenging. Thawing the semen in a specialised centre and transporting the thawed-cooled semen to the place of insemination could mitigate these challenges. Consequently, the goals of this study were (1) to examine motility and viability of frozen-thawed semen after 24 h of cooled storage, and (2) to compare two cooling protocols for storage of frozen-thawed semen. Semen from 31 stallions, frozen using one of three commercial freezing extenders (INRA Freeze®, Gent Freezing Extender, or BotuCryo®), was thawed, diluted 1:5 in INRA96® to a concentration of 355 ± 123 × 106 spermatozoa mL−1, and either stored immediately in a passive cooling box (CB; transport box for chilled stallion semen; ref. 17229/0002, Minitube) or placed first in a refrigerator for 30 min and then transferred to the cooling box (FRDG). Viability and total and progressive motility were evaluated at 0 (T0), 8 (T8), and 24 (T24) h, using NucleoCounter® SP-100TM and computer-assisted sperm motion analysis (AndroVision® software). Data description, descriptive statistics, plots, and statistical tests were performed in JMP® Pro 15 (SAS Institute Inc.). All data were tested for normal distribution and equal variances. Non-normally distributed data were transformed using normal quantiles. Data were analysed with a mixed model, and post hoc analyses were performed using a Tukey test. Significance was set to P < 0.05. There was a significant reduction in total motility (−12.2%) and progressive motility (−11.5%) between T0 and T24-CB (P < 0.05), when semen was stored immediately in the cooling box. However, we did not observe any significant reduction in the total and progressive motility of samples that were placed for 30 min in the refrigerator before transferring to the cooling box, implicating that for long-term, cooled transport, storing samples in a refrigerator can be advantageous. In comparison to T0, viability decreased ∼11% and ∼13% in both groups at T8 and T24, respectively. Our data demonstrated that the storage protocol influences sperm motility, and storing the samples in a refrigerator for 30 min before cooled storage can be advantageous. Our data suggest that the faster cooling rate in the samples that were placed in the refrigerator (FRDG) compared with those placed directly in the passive cooling device (CB) could result in a better preservation and protection of the frozen-thawed semen. Therefore, the rapid cooling step (FRDG) before the use of a passive cooling device could mitigate the effect of cooled storage on the quality of frozen-thawed semen. A further fertility trial to examine the fertilising capacity of cooled-stored, frozen-thawed semen is required to compare the pregnancy rates with those of frozen-thawed semen.
