Protamine 2 and phospholipase C zeta 1 are possible biomarkers for the diagnosis of male subfertility in frozen-thawed stallion semen.

Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present study aims at assessing the differences in the expression of 10 potential candidate genes in stallions of different fertility. Frozen-thawed semen of 29 stallions was included. Each sample was classified into two groups according to pregnancy rates (PR) achieved with this semen: "good fertility" (GF; n = 17; PR ≥ 30 %) or "poor fertility" (PF; n = 12; PR <20 %). All stallions underwent a breeding soundness examination (BSE) before semen production and were only included into the semen cryopreservation program when raw semen characteristics at BSE met minimal requirements. Semen was cryopreserved following European Union regulations and all stallions met the respective health requirements. Each sample was assessed for concentration (NucleoCounter SP-100), motility (CASA), membrane functionality (SYBR-14/PI), mitochondrial membrane potential (JC-1), morphology (SpermacStain), acrosome integrity (SpermacStain), membrane integrity (HOS test) and chromatin integrity (Aniline blue). Sperm RNAs were extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) and RT-qPCR was performed for each target gene. ACTB and RPL32 were included as reference genes (RGs) for normalization. For each variable of each group, mean, standard deviation and SEM were calculated. The difference in gene expression levels between the GF and PF group were analyzed using the Mann-Whitney U test and Spearman's rank correlation. Significant results were considered with p < 0.05. Sperm quality parameters did not differ significantly between the two groups except for concentration, that was significantly higher in GF (p = 0.043). In GF a positive correlation was identified for PRM1/PRM2 with r = +0.6, while PRM1/ACR (r = -0.495), PRM2/ZPBP (r = -0.645) and CRISP3/ACR (r = -0.551) were inversely correlated. In PF direct correlations were registered for PRM1/PRM2 (r = +0.629), PRM1/PRM3 (r = +0.657), PRM2/SPA17 (r = +0.685), SPA17/PLCZ1 (r = +0.786) and PRM3/ACR (r = +0.627). In the total sample (GF + PF), positive correlations were detected for PRM1/PRM2 (r = +0.625), PRM1/PRM3 (r = +0.368); PRM2/SPA17 (r = +0.465), SPA17/PLCZ1 (r = +0.637) and PLCZ1/ZAN (r = +0.587). Only two of the genes considered were differentially expressed in the 2 groups: PRM2 and PLCZ1, that were significantly (p < 0.05) overexpressed in the GF group. Stallions frozen-thawed semen with higher expression levels of PRM2 and PLCZ1 are more likely to belong to animals with a good pregnancy rate. Further studies are needed to investigate the role of sperm transcripts in male subfertility in stallions.

Selection of frozen-thawed stallion semen by microfluidic technology.

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.

Relationships between Biomarkers of Oxidative Stress in Seminal Plasma and Sperm Motility in Bulls before and after Cryopreservation.

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.