Characterization of the semen microbiota of healthy stud dogs using 16S RNA sequencing.

The reproductive microbiota of male dogs has never been investigated using culture-independent sequencing techniques. The purpose of the present study was to get seminal knowledge on the microbiota of the ejaculate. Specifically, factors as the fraction of the ejaculate, the sperm quality (normospermia, teratozoospermia), and the living environment were evaluated. The sperm-rich and the prostatic fractions of the ejaculate were collected from healthy stud dogs. Following the sperm analysis, samples from twenty animals (normospermic n = 10 and teratozoospermic n = 10) were stored at - 80 °C until further processing including DNA extraction and 16S rRNA sequencing. Alpha- (Shannon index) and beta- (Bray-Curtis, Unweighted UniFrac) diversities were assessed and compared (PERMANOVA) based on the group of samples (biological samples from the ejaculate and controls), the fraction of the ejaculate (sperm-rich and prostatic fractions), the animal group (normospermia and teratozoospermia), and the living environment of the animal (kennel or pet living in-house). The most abundant bacterial phyla in canine semen samples were Proteobacteria, Firmicutes, and Actinobacteria. Overall, the dominant bacterial family was that of Pasteurellaceae The genus Mycoplasma was never detected. No differences in terms of bacterial composition were found based on the fraction of the ejaculate and based on the animal group (P > 0.05). On the other hand, differences in alpha and beta diversities were highlighted based on the living environment (P = 0.001). Overall, the results of the present study provide preliminary insights on dog semen microbiota, opening a new chapter in the field of canine andrology. Our results suggest that the environment may play a role in influencing the reproductive microbiota of male dogs and that the prostatic fraction of the ejaculate can be used for further research as a representative of the semen microbiota.

Differential gene expression patterns in spermatozoa from teratospermic and normospermic domestic cats.

Large percentages of abnormal sperm, termed teratospermia, are associated with poor fertility in cats, many of which are threatened with extinction from their natural habitats. Even normal appearing spermatozoa from felids with teratospermia may have a compromised capacity for motility and fertilization indicating there are factors affecting the fertilization capacity of all sperm regardless of morphology. There was a comparative study conducted using the RNA-Seq approach to identify differentially expressed genes between morphologically normal and abnormal sperm from domestic cates with normospermia and teratospermia to elucidate genes and pathways associated with abnormal sperm function. Normal sperm from cats with teratospermia have a gene expression profile similar to abnormal sperm from males with teratospermia. There was also downregulation of cGMP pathways which may be associated with a lesser sperm motility in ejaculates from males with teratospermia. Kinase phosphorylation pathways also were downregulated in normal spermatozoa from ejaculates of males with teratospermia. Results indicate that analysis of sperm gene expression provides for a more precise assessment of sperm function in semen of cats with teratospermia and facilitates identification of molecular abnormalities that may lead to compromised fertilization capacity.

Novel Proteomic Profiling of Epididymal Extracellular Vesicles in the Domestic Cat Reveals Proteins Related to Sequential Sperm Maturation with Differences Observed between Normospermic and Teratospermic Individuals.

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals.

Relationship between morphological abnormalities in commercial bull frozen semen doses and conception rate.

Commercial doses of frozen bull semen for artificial insemination may have a certain percentage of morphological defects, despite being subject to prior selection. The aims of this study were to determine the prevalence of morphological abnormalities in commercial doses (n = 55, r = 2) of dairy and beef bulls, from AI Centers and to determine the possible existence of differences between them, regarding the percentage of abnormal spermatozoa. At least 200 spermatozoa per sample were evaluated using Bengal Rose stain (3% m/v) and light microscopy (×1000 magnification). The mean percentage of abnormal sperm samples from dairy breeds was 7.19% ± 4.91% and from beef breeds was 15.83% ± 9.28%. Significant differences between biotypes were found in the proportion of abnormal spermatozoa, abnormal heads and abnormal midpieces; it could be due to different selection pressure. It was observed that the percentage of abnormal spermatozoa was not a good fertility level predictor for the commercial samples of frozen bovine semen used in this study. In both biotypes, the midpiece abnormalities were the most frequent, mainly its distal flexion (compensable defect). This could be as a result of the effects of freezing and thawing on spermatozoa.