Regional abundances of binder of sperm (BSP) proteins are negatively associated with the quality of frozen-thawed bovine spermatozoa.

In brief: The localization and abundance of the sperm BSP proteins correlate with in vitro fertility in domestic bulls used in artificial insemination service. Abstract: Binder of sperm (BSP) proteins, secreted mainly by the accessory sex glands, are the major protein family present in bovine seminal plasma and on the sperm surface after ejaculation. In vivo, BSP proteins facilitate sperm capacitation and sperm reservoir formation; however, their impact on sperm function within the in vitro systems is less clear. Therefore, this biomarker-based study aimed to characterize the localization and abundance of BSP proteins from in vitro processed frozen-thawed bovine spermatozoa. Using image-based flow cytometry and Western blotting, BSP protein localization, abundance, membrane and acrosomal integrity were investigated in the supernatant (nonmotile) and pellet (motile) fractions of gradient-separated bull spermatozoa. Spermatozoa from the supernatant fraction had high enrichment of all BSP proteins investigated (BSP1, BSP3, BSP5; P < 0.05) when compared to the pellet fraction. In the pellet fraction, BSP1 and BSP3 bound predominately to the acrosomal region, whereas BSP5 had a high affinity for the midpiece. However, in the supernatant fraction, BSP proteins predominately coated the entire sperm surface resulting in the loss of regional specificity. High BSP protein abundance in the spermatozoa also correlated with acrosome and membrane damage. Whereas a high abundance of BSP5 correlated with low embryo cleavage rates, high abundance of BSP1 on the sperm head coincided with a high blastocyst rate. Therefore, changes in the quantity and localization of specific BSP proteins could act as potential biomarkers of sperm quality and fertility.

The development of new biomarkers of spermatozoa quality in cattle.

There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high (n = 4) or low (n = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater (p < 0.05) incidence of aggresome defects than post-gradient samples from high-performing sires. Additionally, IBFC was used to evaluate spermatozoa viability following gradient purification. Distinct populations of sperm cells were identified. High-performing sires had more spermatozoa in the population deemed most viable than low-performing sires. This study demonstrated that spermatozoa defects vary in populations before and following gradient purification, indicating that it may be beneficial to separately evaluate semen for in vivo and in vitro use. Furthermore, a prevalent defect in low-performing sires that could explain a discrepancy between successful fertilization and embryo development was identified. Therefore, elucidating a malfunction regulated by sire, that could potentially affect early embryo development.

Calving date as a potential predictor for the probability of approval in the first breeding soundness evaluation of Nellore bulls.

Beef production systems primarily use natural service (NS) for breeding. However, a significant number of bulls used for NS are subfertile, limiting the profitability of the cow-calf operations. Therefore, producers should select bulls based on breeding soundness evolutions (BSE) to ensure higher pregnancy rates. Several factors can affect the bull ability to pass a BSE. We hypothesize that calving date would be a factor that affects the bull probability of approval at the first BSE. For this purpose, a multivariate logistic regression in a dataset of 14,737 BSEs from young Nellore bulls was used. Correlations between calving date, biometrics, and semen traits were evaluated using Pearson's correlation coefficient. Our results demonstrated that the calving date affected the probability of approval at the first BSE (p < .05). Indeed, the variable that added more information to our model was the calving date, far more than the age group of the bulls according to Akaike's information criterion. Hence, bulls born on day 0 of the calving season have 1.26 more chances to be approved at the first BSE than bulls born 21 days later. This result highlights the importance of getting the dams of future bulls pregnant as soon as possible in the breeding season. In addition, the calving season should be no longer than 47 days to achieve 80% BSE approval in 20-22 months old Nellore bulls. The strongest correlation was with SC, which decreased as the calving date increased. Therefore, the calving date may be used as a predictor of the outcome of the first BSE in young bulls. In that way, the calving date can help seedstock producers to maximize efficiency in making crucial management decisions during the breeding and calving season including nutrition, reproductive, and culling.

Branched germline cysts and female-specific cyst fragmentation facilitate oocyte determination in mice.

During mouse gametogenesis, germ cells derived from the same progenitor are connected via intercellular bridges forming germline cysts, within which asymmetrical or symmetrical cell fate occurs in female and male germ cells, respectively. Here, we have identified branched cyst structures in mice, and investigated their formation and function in oocyte determination. In fetal female cysts, 16.8% of the germ cells are connected by three or four bridges, namely branching germ cells. These germ cells are preferentially protected from cell death and cyst fragmentation and accumulate cytoplasm and organelles from sister germ cells to become primary oocytes. Changes in cyst structure and differential cell volumes among cyst germ cells suggest that cytoplasmic transport in germline cysts is conducted in a directional manner, in which cellular content is first transported locally between peripheral germ cells and further enriched in branching germ cells, a process causing selective germ cell loss in cysts. Cyst fragmentation occurs extensively in female cysts, but not in male cysts. Male cysts in fetal and adult testes have branched cyst structures, without differential cell fates between germ cells. During fetal cyst formation, E-cadherin (E-cad) junctions between germ cells position intercellular bridges to form branched cysts. Disrupted junction formation in E-cad-depleted cysts led to an altered ratio in branched cysts. Germ cell-specific E-cad knockout resulted in reductions in primary oocyte number and oocyte size. These findings shed light on how oocyte fate is determined within mouse germline cysts.

Interaction of Mycobacterium avium subsp. paratuberculosis with bovine sperm.

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Paratuberculosis mainly affecting domestic ruminants. The interaction between MAP and sperm and/or germ cells has not yet been established, however the adherence between MAP and the host cell surface is associated to the 85 complex proteins that bind to the host cell's fibronectin. Therefore, this study aimed to evaluate the binding of MAP to bovine sperm and to verify changes in these cells by the presence of MAP before and after sperm cryopreservation. Polyclonal antibodies to MAP 85 complex proteins were produced and utilized in the analyzes. Two Nelore bulls were used for semen collection and MAP dilutions (103-108 CFU/mL) were inoculated in the samples; sperm motility and vigor were evaluated using light microscopy at different times before and after cryopreservation and in the presence and absence of the antibodies 85A and 85B. Interaction of MAP and sperm, interaction of MAP and sperm in the presence of Ab 85A and in the presence of Ab 85B were analyzed by scanning electron microscopy. The viability of MAP after sperm cryopreservation were evaluated by plating the samples after thawing. It was observed that sperm in the presence of MAP shows a decrease in motility and vigor, and that the higher the MAP concentration, the lower the sperm performance. It was possible to determine the viability of MAP after cryopreservation in samples of higher concentrations, which demonstrates the potential of transmission of this pathogen through artificial insemination. The interaction of MAP with bovine sperm occurs mainly in the midpiece and may be linked to the proteins 85A and 85B present in the MAP membrane.

In vitro evaluation of cryopreserved bovine sperm and its relation to field fertility in fixed-time artificial insemination.

This study aimed to assess characteristics of bovine cryopreserved sperm and evaluate its relation to field fertility in fixed-time artificial insemination (FTAI). Semen samples of 16 bulls were used to inseminate 811 Nellore cows, and four of these bulls were also used to inseminate 101 Nellore heifers. Samples of the same ejaculate used for FTAI from each bull were analysed in the laboratory after thawing. Sperm motility and vigour were subjectively assessed by light microscope, and integrity of the plasma and acrosome membranes, and H2 O2 production were evaluated by flow cytometer. Relation among sperm characteristics and pregnancy rate of cows and heifers were evaluated by univariate and multivariate logistic regression. Subjective sperm motility and vigour did not affect the probability of pregnancy in cows or heifers. In univariate analysis for pregnancy in cows, sperm traits related to acrosome injury positively affected probability of pregnancy mainly when associated with plasma membrane integrity; H2 O2 production seems to be less important than plasma membrane integrity in affecting probability of pregnancy. In multivariate analysis, sperm traits related to injured acrosome positively affected probability of cow and heifer pregnancies while intact acrosome was negatively related to cow pregnancy. Intact plasma membrane and high H2 O2 production were positively related to cow pregnancy but negatively related to heifer pregnancy. Results suggest that a capacitation-like status of the acrosome may benefit probability of pregnancy in cows.