Stallion spermatozoa: Reassessing the role of penetrating cryoprotectants

Factors influencing post-thaw quality of stallion spermatozoa: Reassessing the role of penetrating cryoprotectants (CPAs)

Anim Reprod Sci. 2026 Nov:294:108321. doi: 10.1016/j.anireprosci.2026.108321. Epub 2026 Aug 31.
Authors
Camilo Hernández-Avilés 1 , Jarred B Crowley 2 , Victor Zhidkov 3 , Lisa A Jolley 4 , Charles C Love 3 , Luisa Ramírez-Agámez 3
Affiliations

1 Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA. Electronic address: chernandez@cvm.tamu.edu.
2 Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA; Veterinary Medical Teaching Hospital, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
3 Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
4 Veterinary Medical Teaching Hospital, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.

PMID: 42679715
DOI: 10.1016/j.anireprosci.2026.108321

Free article
Abstract

This study examined interactions between cryoprotectant (CPA) composition and processing factors (i.e., pre-freeze cooling rate, egg yolk concentration, cryopreservation step, final sperm concentration) that may influence post-thaw quality of stallion sperm. In Experiment 1, 24 ejaculates were cryopreserved in extenders with either 20% egg yolk (EY) or 2% egg yolk with added milk proteins (SMEY) and containing either glycerol (Gly) and methyl formamide (MF; Gly + MF), methyl formamide (MF), or dimethylformamide (DF). Post-thaw sperm quality was assessed via computer-assisted motion analysis and flow cytometry. An interaction between egg yolk concentration (EY vs. SMEY) and CPA (Gly+MF vs. MF vs. DF) was observed, whereas an effect of pre-freeze cooling rate (-0.1 vs. -1 °C/min) was not detected. Sperm cryopreserved in EY-Gly + MF exhibited the highest sperm motility parameters and plasma membrane integrity values, as well as the lowest levels of apoptotic and oxidative stress-associated changes. In Experiment 2 (n = 24 ejaculates), sperm quality was not affected immediately after CPA addition (Gly vs. MF vs. Gly+MF) or pre-freeze cooling but decreased significantly after freezing and thawing. Post-thaw motility and viability were similar between Gly and Gly + MF, both higher than for MF. Exposure to hyperosmotic (CPA-containing) or isosmotic (CPA-free) media during analysis at every step of the cryopreservation process had no impact on sperm quality. In Experiment 3 (n = 24 ejaculates), sperm cryopreserved at 200, 400, or 800 × 106 sperm/mL exhibited higher post-thaw motility and viability than sperm cryopreserved at 50 or 100 × 106 sperm/mL, regardless of CPA.

Keywords: Apoptosis; Cryopreservation; Cryoprotectant; Oxidative stress; Post-thaw quality; Stallion sperm.

Published by Elsevier B.V.
Conflict of interest statement

Declaration of Competing Interest The authors declare no conflict of interest.