This study aimed at evaluating the relationship between biomarkers of oxidative stress (OS) in seminal plasma and sperm motility in bulls before and after cryopreservation. Three ejaculates per bull were collected from 20 young bulls. Each ejaculate was analyzed for motility before and after cryopreservation (by CASA), and the SP concentration of Advanced Oxidation Protein Products (AOPP), thiols, and carbonyl groups (CT) were examined. Then, based on their motility, the ejaculates were grouped into: high motility fresh (HMF), low motility fresh (LMF), high motility thawed (HMT), and low motility thawed (LMT) groups. Higher AOPP and thiol concentrations on SP were related (p < 0.05) to the higher LIN and BCF and lower ALH of fresh semen. In addition, AOPP and thiols were significantly higher in HMF than LMF. As a confirmation of this, the Receiver Operating Characteristic (ROC) curve analysis showed that AOPP and thiol concentrations in SP were able to discriminate between HMF and LMF ejaculates (Area Under the Curve of 71.67% and 72.04%, respectively). These observations give an alternative perspective on the relationship between sperm motility and the OS parameters of SP, which need further investigations.

The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 μl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 μl) for stallion semen. Frozen–thawed semen from 15 stallions that were previously classified as being of ‘good fertility’ (GF; n = 8; pregnancy rate ≥ 40%) and ‘poor fertility’ (PF; n = 7; pregnancy rate < 20%), respectively, was used. Each ejaculate was assessed before and after microfluid recovery for kinetics (CASA), membrane integrity (MI) (SYBR14/PI), membrane functionality (MF) (HOS test), acrosome integrity (Spermac Stain Kit), morphology (Spermac stain kit), mitochondrial membrane potential (MMP) (JC‐1) and chromatin integrity (aniline blue staining). Sperm concentration was reduced after sperm recovery in both groups, but more markedly in frozen–thawed semen of PF stallions (p < .05). Microfluid recovery increased total motility, MI, MF and MMP. While there was a significant increase in the percentage of progressively motile sperm after sperm microfluid recovery, there was a decrease in DAP, DSL, VAP, VSL, LIN, WOB and ALH (p < .05). A slight increase (p < .05) was detected in beat‐cross frequency. The present results suggest that the ZyMot Multi (850 μl) device selects a specific sperm population from any stallion ejaculate with motile sperm and could therefore be a valid tool for in vitro testing with the aim to predict the fertility of frozen–thawed stallion semen.

The aim of this study was to evaluate the effects of three methods for seminal plasma removal - standard centrifugation (SC), gradient centrifugation (GC), and membrane filtration (MF) - on sperm recovery and maintenance of sperm characteristics during liquid storage of stallion semen. Semen from each of 5 stallions collected over three consecutive weeks (n=15 ejaculates) was included. Half of the ejaculate was diluted at a 1:1 (v:v) ratio with prewarmed Botusemen and SpermSafe extender, respectively. Semen aliquots were i) left unprocessed as control (CON) or ii) underwent SC (700 x g for 12 min), iii) GC (Equipure; 800 x g for 20 min) or iv) MF (SpermFilter), were subsequently diluted with the respective extenders (50 x 106 sperm/mL) and maintained at 5 °C (Botusemen) or 17 °C (SpermSafe). Sperm morphology was analyzed after processing. Semen motility characteristics and membrane integrity were assessed by CASA after 0, 24, 48, and 72 hours of storage. Data were evaluated by repeated measures GLM Anova. Gradient centrifugation provided the lowest recovery rate (p<0.001) and percentage of morphological aberrations (p<0.01). It was most effective in maintaining motility especially when stored in SpermSafe (time x method p<0.001; time x extender p<0.001). Motility and membrane integrity were less well preserved after MF compared with GC. In conclusion, the method of seminal plasma removal and the choice of extender influence stallion sperm characteristics during liquid storage. Gradient centrifugation, particularly when combined with storage at 17 °C, offers a superior processing method for liquid storage of stallion sperm.