Associations of the Single Bovine Embryo Growth Media Metabolome with Successful Pregnancy.

This study investigated whether metabolomic fingerprints of bovine embryo growth media improve the prediction of successful embryo implantation. In this prospective cohort study, the metabolome from in vitro-produced day 7 blastocysts with successful implantation (n = 11), blastocysts with failed implantation (n = 10), and plain culture media without embryos (n = 5) were included. Samples were analyzed using an AbsoluteIDQ® p180 Targeted Metabolomics Kit with LC-MS/MS, and a total of 189 metabolites were analyzed from each sample. Blastocysts that resulted in successful embryo implantation had significantly higher levels of methionine sulfoxide (p < 0.001), DOPA (p < 0.05), spermidine (p < 0.001), acetylcarnitine-to-free-carnitine ratio (p < 0.05), C2 + C3-to-free-carnitine ratio (p < 0.05), and lower levels of threonine (nep < 0.001) and phosphatidylcholine PC ae C30:0 (p < 0.001) compared to control media. However, when compared to embryos that failed to implant, only DOPA, spermidine, C2/C0, (C2 + C3)/C0, and PC ae C30:0 levels differentiated significantly. In summary, our study identifies a panel of differential metabolites in the culture media of bovine blastocysts that could act as potential biomarkers for the selection of viable blastocysts before embryo transfer.

Uterine Fluid Extracellular Vesicles Proteome Is Altered During the Estrous Cycle.

Uterine environment is tightly and finely regulated via various signaling pathways mediated through endocrine, exocrine, autocrine, juxtacrine, and paracrine mechanisms. In utero signaling processes are paramount for normal and abnormal physiology which involves cell to cell, cells to gametes, cells to embryo, and even interkingdom communications due to presence of uterine microbiota. Extracellular vesicles (EVs) in the uterine fluid (UF) and their cargo components are known to be mediators of in utero signaling and communications. Interestingly, the changes in UF-EV proteome during the bovine estrous cycle and the effects of these differentially enriched proteins on embryo development are yet to be fully discovered. In this study, shotgun quantitative proteomics-based mass spectrometry was employed to compare UF-EV proteomes at day 0, 7, and 16 of the estrous cycle to understand the estrous cycle-dependent dynamics. Furthermore, different phase UF-EVs were supplemented in embryo cultures to evaluate their impact on embryo development. One hundred fifty-nine UF-EV proteins were differentially enriched at different time points indicating the UF-EV proteome is cycle-dependent. Overall, many identified pathways are important for normal uterine functions, early embryo development, and its nutritional needs, such as antioxidant activity, cell morphology and cycle, cellular homeostasis, cell adhesion, and carbohydrate metabolic process. Furthermore, the luteal phase UF-EVs supplementation increased in vitro blastocyst rates from 25.0 ± 5.9% to 41.0 ± 4.0% (p ≤ 0.05). Our findings highlight the importance of bovine UF-EV in uterine communications throughout the estrous cycle. Interestingly, comparison of hormone-synchronized EV proteomes to natural cycle UF-EVs indicated shift of signaling. Finally, UF-EVs can be used to improve embryo production in vitro.

Characterization of bovine uterine fluid extracellular vesicles proteomic profiles at follicular and luteal phases of the oestrous cycle.

Extracellular vesicles (EV) have been identified in uterine fluid (UF), however the bovine UF-EV profile during different phases of the oestrous cycle has not yet been established. Therefore, we compared the UF-EV, and their protein profile at follicular and luteal phases of the oestrous cycle. UF samples were collected from healthy uteri of six live and six slaughtered cows at follicular or luteal phases. Isolation of EV was performed using tangential flow filtration followed by size exclusion chromatography. EV were characterized by nanoparticle tracking analysis (NTA), fluorescence NTA, zeta potential, and transmission electron microscopy. Mass-spectrometry was used to evaluate EV protein profile from live cows. Particle concentrations (mean ± SD) were higher (P < 0.05) at follicular than at luteal phase in both live (1.01 × 108 ± 1.66 × 107 vs 7.56 × 107 ± 1.80 × 107, respectively) and slaughtered cows (1.17 × 108 ± 2.34 × 107 vs 9.12 × 107 ± 9.77 × 106, respectively). The proportion of fluorescently labelled EV varied significantly between follicular and luteal phases across live (28.9 ± 1.9% vs 19.3 ± 2.8%, respectively) and slaughtered cows (26.5 ± 6.3% vs 27.3 ± 2 .7%, respectively). In total, 41 EV proteins were differentially expressed between the phases. Some of the proteins were involved in reproductive processes, cell adhesion and proliferation, and cellular metabolic processes. The results indicated differences in bovine UF-EV concentration and protein profile at follicular and luteal phases, which would suggest that EV modulate uterine microenvironment across the oestrous cycle. Further research is needed to understand the effect of EV changes throughout the oestrous cycle.

Bovine colostrum-derived antibodies against SARS-CoV-2 show great potential to serve as prophylactic agents.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to impose a serious burden on health systems globally. Despite worldwide vaccination, social distancing and wearing masks, the spread of the virus is ongoing. One of the mechanisms by which neutralizing antibodies (NAbs) block virus entry into cells encompasses interaction inhibition between the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and the spike (S) protein of SARS-CoV-2. SARS-CoV-2-specific NAb development can be induced in the blood of cattle. Pregnant cows produce NAbs upon immunization, and antibodies move into the colostrum immediately before calving. Here, we immunized cows with SARS-CoV-2 S1 receptor binding domain (RBD) protein in proper adjuvant solutions, followed by one boost with SARS-CoV-2 trimeric S protein and purified immunoglobulins from colostrum. We demonstrate that this preparation indeed blocks the interaction between the trimeric S protein and ACE2 in different in vitro assays. Moreover, we describe the formulation of purified immunoglobulin preparation into a nasal spray. When administered to human subjects, the formulation persisted on the nasal mucosa for at least 4 hours, as determined by a clinical study. Therefore, we are presenting a solution that shows great potential to serve as a prophylactic agent against SARS-CoV-2 infection as an additional measure to vaccination and wearing masks. Moreover, our technology allows for rapid and versatile adaptation for preparing prophylactic treatments against other diseases using the defined characteristics of antibody movement into the colostrum.

A dual colour FISH method for routine validation of sexed Bos taurus semen.

BACKGROUND: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. RESULTS: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). CONCLUSIONS: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.

Male mice with deleted Wolframin (Wfs1) gene have reduced fertility.

BACKGROUND: Wolfram Syndrome (WS) is an autosomal recessive disorder characterised by non-autoimmune diabetes mellitus, optic atrophy, cranial diabetes insipidus and sensorineural deafness. Some reports have described hypogonadism in male WS patients. The aim of our study was to find out whether Wfs1 deficient (Wfs1KO) male mice have reduced fertility and, if so, to examine possible causes. METHODS: Wfs1KO mice were generated by homologous recombination. Both Wfs1KO and wild type (wt) male mice were mated with wt female mice. The number of litters and the number of pups were counted and pregnancy rates calculated. The motility and morphology of the sperm and the histology of testes were analysed. Serum testosterone and FSH concentrations were also measured. RESULTS: The pregnancy rate in wt females mated with Wfs1KO males was significantly lower than in the control group (15% vs. 32%; p < 0.05), but there was no significant difference in litter size. Analysis of male fertility showed that, in the Wfs1KO group, eight males out of 13 had pups whereas in the control group all 13 males had at least one litter. Sperm motility was not affected in Wfs1KO mice, but Wfs1KO males had less proximal bent tails (14.4 +/- 1.2% vs. 21.5 +/- 1.3 p < 0.05) and less abnormal sperm heads (22.8 +/- 1.8 vs. 31.5 +/- 3.5, p < 0.05) than wt males. Testes histology revealed significantly reduced number of spermatogonia (23.9 +/- 4.9 vs. 38.1 +/- 2.8; p < 0.05) and Sertoli cells (6.4 +/- 0.5 vs. 9.2 +/- 1.0; p < 0.05) in Wfs1KO mice. Serum testosterone and FSH concentrations did not differ between the two groups. CONCLUSION: The impaired fertility of Wfs1KO male mice is most likely due to changes in sperm morphology and reduced number of spermatogenic cells. The exact mechanism through which the Wfs1 gene influences sperm morphology needs to be clarified in further studies.