Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792).

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Effects of Nerve Growth Factor-β From Bull Seminal Plasma on Steroidogenesis and Angiogenic Markers of the Bovine Pre-ovulatory Follicle Wall Cell Culture

Nerve growth factor-β (NGF) is critical for ovulation in the mammalian ovary and is luteotrophic when administered systemically to camelids and cattle. This study aimed to assess the direct effects of purified bovine NGF on steroidogenesis and angiogenic markers in the bovine pre-ovulatory follicle. Holstein heifers (n = 2) were synchronized with a standard protocol, and heifers with a preovulatory follicle (≥ 12 mm) had the ovary containing the dominant follicle removed via colpotomy. Pre-ovulatory follicles were dissected into 24 pieces containing theca and granulosa cells that were randomly allocated into culture media supplemented with either purified bovine NGF (100 ng/mL) or untreated (control) for 72 h. The supernatant media was harvested for quantification of progesterone, testosterone, and estradiol concentrations, whereas explants were subjected to mRNA analyses to assess expression of steroidogenic and angiogenic markers. Treatment of follicle wall pieces with NGF upregulated gene expression of steroidogenic enzyme HDS17B (P = 0.04) and increased testosterone production (P < 0.01). However, NGF treatment did not alter production of progesterone (P = 0.81) or estradiol (P = 0.14). Consistently, gene expression of steroidogenic enzymes responsible for producing these hormones (STAR, CYP11A1, HSD3B, CYP17A1, CYP19A1) were unaffected by NGF treatment (P ≥ 0.31). Treatment with NGF downregulated gene expression of the angiogenic enzyme FGF2 (P = 0.02) but did not alter PGES (P = 0.63), VEGFA (P = 0.44), and ESR1 (P = 0.77). Collectively, these results demonstrate that NGF from seminal plasma may interact directly on the theca and granulosa cells of the bovine pre-ovulatory follicle to stimulate testosterone production, which may be secondary to theca cell proliferation. Additionally, decreased FGF2 expression in NGF-treated follicle wall cells suggests hastened onset of follicle wall cellular remodeling that occurs during early luteal development.

Proteomic analysis of spermatozoa reveals caseins play a pivotal role in preventing short-term periods of subfertility in stallions

Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days post-breeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations, and to determine the biological mechanisms governing such differences. Using LC–MS/MS, we compared the proteomic profile of semen samples collected from commercially “fertile” stallions, during high- and low-fertility periods. A total of 1702 proteins were identified, of which, 38 showed a significant change in abundance (p ≤ 0.05). Assessment of intra- and inter-stallion variability revealed that caseins (namely κ-, α-S1-, and α-S2-casein), were significantly more abundant during “high-fertility” periods, while several epididymal, and seminal plasma proteins (chiefly, epididymal sperm binding protein 1 [ELSPbP1], horse seminal plasma protein 1 [HSP-1] and clusterin), were significantly more abundant during “low-fertility” periods. We hypothesised that an increased abundance of caseins offers greater protection from potentially harmful seminal plasma proteins, thereby preserving cell functionality and fertility. In vitro exposure of spermatozoa to casein resulted in decreased levels of lipid scrambling (Merocyanine 540), higher abundance of sperm-bound caseins (α-S1-, α-S2-, and κ-casein), and lower abundance of sperm-bound HSP-1 (p ≤ 0.05). This study demonstrates key pathways governing short-term fertility fluctuations in the stallion, thereby providing a platform to develop robust, fertility assessment strategies into the future.

Application and Progress of Raman Spectroscopy in Male Reproductive System

Raman spectroscopy is a fast-developing, unmarked, non-invasive, non-destructive technique which allows for real-time scanning and sampling of biological samples in situ, reflecting the subtle biochemical composition alterations of tissues and cells through the variations of spectra. It has great potential to identify pathological tissue and provide intraoperative assistance in clinic. Raman spectroscopy has made many exciting achievements in the study of male reproductive system. In this review, we summarized literatures about the application and progress of Raman spectroscopy in male reproductive system from PubMed and Ovid databases, using MeSH terms associated to Raman spectroscopy, prostate, testis, seminal plasma and sperm. The existing challenges and development opportunities were also discussed and prospected.

Deep Metabolomic Profiling Reveals Alterations in Fatty Acid Synthesis and Ketone Body Degradations in Spermatozoa and Seminal Plasma of Astheno-Oligozoospermic Bulls

Male fertility is extremely important in dairy animals because semen from a single bull is used to inseminate several thousand females. Asthenozoospermia (reduced sperm motility) and oligozoospermia (reduced sperm concentration) are the two important reasons cited for idiopathic infertility in crossbred bulls; however, the etiology remains elusive. In this study, using a non-targeted liquid chromatography with tandem mass spectrometry-based approach, we carried out a deep metabolomic analysis of spermatozoa and seminal plasma derived from normozoospermic and astheno-oligozoospermic bulls. Using bioinformatics tools, alterations in metabolites and metabolic pathways between normozoospermia and astheno-oligozoospermia were elucidated. A total of 299 and 167 metabolites in spermatozoa and 183 and 147 metabolites in seminal plasma were detected in astheno-oligozoospermic and normozoospermic bulls, respectively. Among the mapped metabolites, 75 sperm metabolites were common to both the groups, whereas 166 and 50 sperm metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Similarly, 86 metabolites were common to both the groups, whereas 45 and 37 seminal plasma metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Among the differentially expressed metabolites, 62 sperm metabolites and 56 seminal plasma metabolites were significantly dysregulated in astheno-oligozoospermic bulls. In spermatozoa, selenocysteine, deoxyuridine triphosphate, and nitroprusside showed significant enrichment in astheno-oligozoospermic bulls. In seminal plasma, malonic acid, 5-diphosphoinositol pentakisphosphate, D-cysteine, and nicotinamide adenine dinucleotide phosphate were significantly upregulated, whereas tetradecanoyl-CoA was significantly downregulated in the astheno-oligozoospermia. Spermatozoa from astheno-oligozoospermic bulls showed alterations in the metabolism of fatty acid and fatty acid elongation in mitochondria pathways, whereas seminal plasma from astheno-oligozoospermic bulls showed alterations in synthesis and degradation of ketone bodies, pyruvate metabolism, and inositol phosphate metabolism pathways. The present study revealed vital information related to semen metabolomic differences between astheno-oligozoospermic and normospermic crossbred breeding bulls. It is inferred that fatty acid synthesis and ketone body degradations are altered in the spermatozoa and seminal plasma of astheno-oligozoospermic crossbred bulls. These results open up new avenues for further research, and current findings can be applied for the modulation of identified pathways to restore sperm motility and concentration in astheno-oligozoospermic bulls.

Sperm exposure to accessory gland secretions alters the transcriptomic response of the endometrium in cattle

In a recent study from our group, mating to intact, but not vasectomised, bulls modified the endometrial transcriptome, suggesting an important role of sperm in the modulation of the uterine environment in this species. However, it is not clear whether these changes are driven by intrinsic sperm factors, or by factors of accessory gland (AG) origin that bind to sperm at ejaculation. Thus, the aim of the present study was to determine whether ejaculated sperm, which are suspended in the secretions of the AGs, elicit a different endometrial transcriptomic response than epididymal sperm, which have never been exposed to AG factors. To this end, bovine endometrial explants collected from heifers in oestrus were incubated alone (control), or with epididymal or ejaculated sperm. RNA-sequencing revealed 1912 differentially expressed genes (DEGs) between in endometrial explants exposed to epididymal sperm compared with control explants, whereas 115 DEGs genes detected between endometrial explants exposed to ejaculated sperm in comparison to control explants. In both cases, the top pathways associated with these genes included T cell regulation and NF-KB and IL17 signalling. To confirm whether AG factors were directly responsible for the dampening of the endometrial response elicited by ejaculated sperm, endometrial explants were incubated with epididymal sperm previously exposed, or not, to seminal plasma (SP). Exposure to SP abrogated the downregulation of SQSTM1 by epididymal sperm, and partially inhibited the upregulation of MYL4 and CHRM3 and downregulation of SCRIB. These data indicate that factors of AG origin modulate the interaction between sperm and the endometrium in cattle.