Abnormal centriolar biomarker ratios correlate with unexplained bull artificial insemination subfertility: a pilot study.

The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) correlate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group's high-quality sperm population), whereas all of the subfertile bulls (6/6, 100%) had positive FRAC tests (P = 0.00008). The most sensitive biomarker was acetylated tubulin, which had a novel labeling pattern between the DC and Ax. These data suggest that FRAC and acetylated tubulin labeling can identify bull subfertility that remains undetected by current methods and may provide insight into a novel mechanism of subfertility.

Angus sire field fertility and in vitro sperm characteristics following use of different sperm insemination doses in Brazilian beef cattle.

The primary objective was to determine if Angus bull fertility varied by number of sperm inseminated. A secondary objective was to characterize the potential impact of random variation on fertility using two identical sperm per dose treatments, which differed only in straw color. Computer-assisted sperm analysis (CASA) and flow cytometry (FC) were used to identify post-thaw sperm characteristics associated with field fertility differences between bulls. Ejaculates from five Angus bulls were collected, extended, and cryopreserved at 10, 20, 20 or 40 × 106 sperm per dose in color-coded 0.5-mL French straws. Multiparous cows (n = 4866) from ten Brazilian farms were synchronized for first-service timed artificial insemination (TAI). Bull identification and straw color were recorded at TAI. Pregnancy per TAI (P/TAI) did not differ between sperm doses (43.8, 45.3, 43.8 and 47.1% for 10, 20, 20 or 40 × 106 sperm respectively; P = 0.31) nor was there an interaction between bull and dose (P = 0.53). The P/TAI differed between bulls and ranged from 40.7 to 48.1% (P < 0.01). The overall P/TAI between the two control groups were not different (45.3 vs 43.8%); however, the numerical variation within bull ranged from 0.5 to 4.9 percentage points. Numerous CASA and FC post-thaw sperm characteristics differed among bulls (P < 0.05), but these characteristics did not explain the fertility difference between bulls. Principal component analysis provided a multivariate description of the CASA and FC data, where three principal components (Prin1, Prin2, and Prin3) accounted for a combined total of 88.7% of the data variability. The primary components of each PCA axis were flow cytometric measures of sperm viability and DNA fragmentation (Prin1), and CASA-derived sperm movement patterns (Prin2) and motility (Prin3); however, the relative influence of these characteristics varied by bull. Although fertility differences between bulls were detected, neither sperm per dose nor post-thaw in vitro sperm analyses (CASA and FC) were able to explain the observed differences in field fertility between bulls, further illustrating the difficulties in predicting bull fertility.

Effect of seminal plasma or transforming growth factor on bovine endometrial cells.

Semen induces post-coital inflammation of the endometrium in several species. Post-coital inflammation is proposed to alter the endometrial environment of early pregnancy, mediate embryonic development and modulate the maternal immune response to pregnancy. In cattle, it is common for pregnancies to occur in the absence of whole semen due to the high utilization of artificial insemination. Here, we have utilized a cell culture system to characterize semen-induced expression of inflammatory mediators in bovine endometrial cells and test the efficacy of transforming growth factor beta as the active agent in mediating any such change. We hypothesize that seminal plasma-derived transforming growth factor beta increases the expression of inflammatory mediators in bovine endometrial cells. Initially, we describe a heat-labile cytotoxic effect of seminal plasma on BEND cells, and a moderate increase in IL1B and IL6 expression. In addition, we show that transforming growth factor beta is present in bovine semen and can increase the expression of endometrial IL6, whereas blocking transforming growth factor beta in semen ameliorates this effect. However, intra-uterine infusion of seminal plasma, sperm or transforming growth factor beta did not alter the endometrial expression of inflammatory mediators. We conclude that bovine semen can modulate endometrial gene expression in vitro, which is partially due to the presence of transforming growth factor beta. It is likely that additional, unidentified, bioactive molecules in semen can alter the endometrial environment. Characterizing bioactive molecules in bovine semen may lead to the development of additives to improve artificial insemination in domestic species.