Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A > G (SNP1), CRISP3c.+566C > A (SNP2), CRISP3c.+622G > A (SNP3) and CRISP3c.+716A > G (SNP4). CRISP-3 protein content in seminal plasma was determined by enzyme-linked immunosorbent assay. Semen from 30 stallions was cryopreserved and post-thaw motility, kinetics, abnormal morphology (AM), sperm vitality (SV) and membrane integrity (MI) were evaluated. Generalized linear models were fitted and means were compared using Tukey’s test. Correlation and regression analyses were performed. For SNP1 and SNP3, the AA genotype had the highest results for motility and MI; for SNP2, the best results for motility and AM were obtained with the CC genotype. For SNP4, the GG genotype had the lowest results, except for MI. A high level of CRISP-3 protein in seminal plasma had the best results for motility, kinetics, SV and AM. In conclusion, there was a relationship between CRISP-3 genotype and seminal plasma protein and post-thawing semen quality in stallions.

Sperm undergo oxidative stress due to excessive production of reactive oxygen species (ROS) during cryopreservation. Some unconventional natural antioxidants can reduce ROS-induced changes in cryopreserved canine sperm. This study aimed to identify the cryoprotective effects of ergothioneine and isoespintanol on the quality of thawed canine semen. Twelve ejaculates from six dogs were cryopreserved in a tris-yolk extender without (control) or with 50 (E50), 100 (E100), or 150 (E150) µM ergothioneine or 20 (I20), 40 (I40), or 60 (I60) µM isoespintanol. We evaluated the motility and kinetics of thawed sperm using computerized analysis; determined morphology by eosin-nigrosin staining; functional membrane integrity using hypoosmotic tests, and structural membrane and acrosome integrity; mitochondrial membrane potential by fluorescence microscopy; and ROS production by spectrophotometry. Data were statistically analyzed using mixed models and Tukey tests. E100 increased total (60.6% vs. 49.6%) and progressive (26.4% vs. 20.1%) motility, straight line velocity (41.3 vs. 35.9 µm/s), and rapid sperm (17.6% vs. 12.3%) compared with controls. However, E150 reduced the numbers of hyperactive sperm. E100, I40, and I60 reduced the abnormal morphology and ROS production, and all concentrations of both antioxidants increased acrosomal integrity. We concluded that ergothioneine and isoespintanol reduce deleterious sperm alterations and oxidative stress in thawed canine semen.