Optimized total RNA isolation from bovine sperm with enhanced sperm head lysis.

Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells due to the compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using the TRIzol reagent. This study critically investigated the effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents 1,4-dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP)). Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total sperm RNA. Furthermore, RNA integrity was confirmed based on the electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of the sperm head using TCEP with mechanical treatment, in total RNA isolation and presented a bovine-specific sperm RNA isolation method to reduce experimental variabilities.

Colostrum-derived extracellular vesicles: potential multifunctional nanomedicine for alleviating mastitis.

Bovine mastitis is an infectious disease that causes substantial economic losses to the dairy industry worldwide. Current antibiotic therapy faces issues of antibiotic misuse and antimicrobial resistance, which has aroused concerns for both veterinary and human medicine. Thus, this study explored the potential of Colo EVs (bovine colostrum-derived extracellular vesicles) to address mastitis. Using LPS-induced murine mammary epithelial cells (HC11), mouse monocyte macrophages (RAW 264.7), and a murine mastitis model with BALB/C mice, we evaluated the safety and efficacy of Colo EVs, in vivo and in vitro. Colo EVs had favorable biosafety profiles, promoting cell proliferation and migration without inducing pathological changes after injection into murine mammary glands. In LPS-induced murine mastitis, Colo EVs significantly reduced inflammation, improved inflammatory scores, and preserved tight junction proteins while protecting milk production. Additionally, in vitro experiments demonstrated that Colo EVs downregulated inflammatory cytokine expression, reduced inflammatory markers, and attenuated NF-κB pathway activation. In summary, we inferred that Colo EVs have promise as a therapeutic approach for mastitis treatment, owing to their anti-inflammatory properties, potentially mediated through the NF-κB signaling pathway modulation.

Landscape transcriptomic analysis of bovine follicular cells during key phases of ovarian follicular development.

BACKGROUND: There are many gaps in our understanding of the mechanisms involved in ovarian follicular development in cattle, particularly regarding follicular deviation, acquisition of ovulatory capacity, and preovulatory changes. Molecular evaluations of ovarian follicular cells during follicular development in cattle, especially serial transcriptomic analyses across key growth phases, have not been reported. This study aims to address this gap by analyzing gene expression using RNA-seq in granulosa and antral cells recovered from ovarian follicular fluid during critical phases of ovarian follicular development in Holstein cows. RESULTS: Integrated analysis of gene ontology (GO), gene set enrichment (GSEA), protein-protein interaction (PPI), and gene topology identified that differentially expressed genes (DEGs) in the largest ovarian follicles at deviation (Dev) were primarily involved in FSH-negative feedback, steroidogenesis, cell proliferation, apoptosis, and the prevention of early follicle rupture. In contrast, DEGs in the second largest follicles (DevF2) were mainly related to loss of cell viability, apoptosis, and immune cell invasion. In the dominant (PostDev) and preovulatory (PreOv) follicles, DEGs were associated with vascular changes and inflammatory responses. CONCLUSIONS: The transcriptome of ovarian follicular fluid cells had a predominance of granulosa cells in the dominant follicle at deviation, with upregulation of genes involved in cell viability, steroidogenesis, and apoptosis prevention, whereas in the non-selected follicle there was upregulation of cell death-related transcripts. Immune cell transcripts increased significantly after deviation, particularly in preovulatory follicles, indicating strong intrafollicular chemotactic activity. We inferred that immune cell invasion occurred despite an intact basal lamina, contributing to follicular maturation.

Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China.

Klebsiella pneumoniae can cause severe clinical mastitis in dairy cows, with K. pneumoniae type K57 (K57-KP) being the most common capsular serotype. To identify virulence factors and antimicrobial-resistance (AMR) genes of K57-KP with varying virulence, Galleria mellonella (greater wax moth) larvae were infected as a screening model to characterize virulence of 90 K57-KP strains, with 10 and 11 strains defined as virulent or attenuated, respectively, based on larval survival rates. Next, virulence of these 21 isolates was subsequently confirmed in adhesion and lactate dehydrogenase release assays, using bovine mammary epithelial cells cultured in vitro. Finally, genes associated with virulence and AMR were characterize with whole-genome sequencing. These 21 K57-KP strains were designated into 16 sequence types based on multi-locus sequence typing and allocated in phylogenetic analysis based on single nucleotide polymorphisms. We found great genetic diversity among isolates. In addition, adhesion-associated genes (e.g., fimA, sfaA, and focA) aminoglycoside-resistance genes (aph(6)-Id, strAB) were associated with virulence. This study provided new knowledge regarding virulence of K57-KP associated with bovine mastitis, which may inform development of novel diagnostic tools and prevention strategies for bovine mastitis.

Herd-level prevalence of bovine leukemia virus, Salmonella Dublin, and Neospora caninum in Alberta, Canada, dairy herds using ELISA on bulk tank milk samples.

Endemic infectious diseases remain a major challenge for dairy producers worldwide. For effective disease control programs, up-to-date prevalence estimates are of utmost importance. The objective of this study was to estimate the herd-level prevalence of bovine leukemia virus (BLV), Salmonella enterica ssp. enterica serovar Dublin (Salmonella Dublin), and Neospora caninum in dairy herds in Alberta, Canada, using a serial cross-sectional study design. Bulk tank milk samples from all Alberta dairy farms were collected 4 times, in December 2021 (n = 489), April 2022 (n = 487), July 2022 (n = 487), and October 2022 (n = 480), and tested for antibodies against BLV, Salmonella Dublin, and N. caninum using ELISA. Herd-level apparent prevalence was calculated as positive herds divided by total tested herds at each time point. A mixed-effect modified Poisson regression model was employed to assess the association of prevalence with region, herd size, herd type, and type of milking system. Apparent prevalence of BLV was 89.4%, 88.7%, 86.9%, and 86.9% in December, April, July, and October, respectively, whereas for Salmonella Dublin apparent prevalence was 11.2%, 6.6%, 8.6%, and 8.5%, and for N. caninum apparent prevalence was 18.2%, 7.4%, 7.8%, and 15.0%. For BLV, Salmonella Dublin, and N. caninum, a total of 91.7%, 15.6%, and 28.1% of herds, respectively, were positive at least once, whereas 82.5%, 3.6%, and 3.0% of herds were ELISA positive at all 4 times. Compared with the north region, central Alberta had a high prevalence (prevalence ratio [PR] = 1.13) of BLV antibody-positive herds, whereas south Alberta had a high prevalence (PR = 2.56) of herds positive for Salmonella Dublin antibodies. Furthermore, central (PR = 0.52) and south regions (PR = 0.46) had low prevalence of N. caninum-positive herds compared with the north. Hutterite colony herds were more frequently BLV positive (PR = 1.13) but less frequently N. caninum-positive (PR = 0.47). Large herds (>7,200 L/d milk delivered ∼>250 cows) were 1.1 times more often BLV positive, whereas small herds (≤3,600 L/d milk delivered ∼≤125 cows) were 3.2 times more often N. caninum positive. For Salmonella Dublin, Hutterite colony herds were less frequently (PR = 0.07) positive than non-colony herds only in medium and large strata but not in small stratum. Moreover, larger herds were more frequently (PR = 2.20) Salmonella Dublin-positive than smaller herds only in non-colony stratum but not in colony stratum. Moreover, N. caninum prevalence was 1.6 times higher on farms with conventional milking systems compared with farms with an automated milking system. These results provide up-to-date information of the prevalence of these infections that will inform investigations of within-herd prevalence of these infections and help in devising evidence-based disease control strategies.

Invited Review: Improved control of Johne's disease in dairy cattle through advancements in diagnostics, testing and management of young stock.

Johne's disease (JD; paratuberculosis) control programs have been regionally implemented across the globe, but few have successfully eradicated the pathogen (Mycobacterium avium ssp. paratuberculosis (MAP)) causing this disease. The limited success may partly be attributed to excluding young stock (calves and replacement heifers or bulls) from testing strategies aimed at identifying MAP-infected cattle. Young stock can shed MAP in feces and can have detectable MAP-specific antibodies in blood, as confirmed in experimentally and naturally infected cattle. Furthermore, MAP transmission causes new infections in young stock. Calves and heifers are often included in JD management strategies on dairy farms but excluded from conventional diagnostic tests due to a presumed lag between infection and detection of MAP shedding and/or MAP-specific serum antibodies. We summarize evidence of MAP shedding early in the course of infection and discuss promising diagnostics, testing and management strategies to support inclusion of young stock in JD control programs. Improvements in fecal Polymerase Chain Reaction, interferon-gamma release assay (IGRA), and enzyme-linked immunosorbent assay (ELISA) enable earlier detection of MAP and specific early immune responses. Studies on IGRA and ELISA have focused on evaluation of new antigens and optimal age of testing. There are new diagnostics, including phage-based tests to detect viable MAP, and gene expression patterns and metabolomics to detect MAP-infected young stock. In addition, refinements in testing and management of calves and heifers may enable reductions in MAP prevalence. We provide recommendations for dairy farmers, researchers, veterinarians, and other stakeholders that may improve JD control programs with an objective to control and potentially eradicate JD. Additionally, we have identified the most pressing gaps in knowledge that currently hamper inclusion of young stock in JD prevention and control programs. In summary, transmission among young stock may cause new MAP infections, and appropriate use of new diagnostic tests, testing and management strategies for young stock may improve the efficacy of JD control programs.

Selenomethionine Mitigates Effects of Nocardia cyriacigeorgica-Induced Inflammation, Oxidative Stress, and Apoptosis in Bovine Mammary Epithelial Cells.

Nocardia cyriacigeorgica causes bovine mastitis, reduces milk quantity and quality, and is often resistant to antimicrobials. Selenomethionine (SeMet) is a form of selenium, which reduces reactive oxygen species (ROS)-mediated apoptosis and intramammary infections. However, the protective effects of SeMet on N. cyriacigeorgica-infected bovine mammary epithelial cells (bMECs) are unclear. The objective of this study was to evaluate whether SeMet mitigated N. cyriacigeorgica-induced inflammatory injury, oxidative damage and apoptosis in bMECs. Cells were cultured with or without being pretreated with 40 µM of SeMet for 12 h, then challenged with N. cyriacigeorgica (multiplicity of infection = 5:1) for 6 h. Although N. cyriacigeorgica was resistant to lincomycin, erythromycin, enrofloxacin, penicillin, amoxicillin, cephalonium, cephalexin, and ceftriaxone, 40 µM SeMet increased cell viability and inhibited lactate dehydrogenase release in infected bMECs. Furthermore, N. cyriacigeorgica significantly induced mRNA production and protein expression of TNF-α, IL-1ß, IL-6, and IL-8 at 6 h. Cell membrane rupture, cristae degeneration and mitochondria swelling were evident with transmission electron microscopy. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activities were down-regulated after 3, 6, or 12 h, whereas malondialdehyde (MDA) and ROS contents were significantly upregulated, with cell damage and apoptosis rapidly evident (the latter increased significantly in a time-dependent manner). In contrast, bMECs pretreated with 40 µM SeMet before infection, SOD, and GSH-px activities were upregulated (p < 0.05); MDA and ROS concentrations were downregulated (p < 0.05), and apoptosis was reduced (p < 0.05). In conclusion, 40 µM SeMet alleviated inflammation, oxidative stress and apoptosis induced by N. cyriacigeorgica in bMECs cultured in vitro.

Effective of phage cocktail against Klebsiella pneumoniae infection of murine mammary glands.

Phage therapy has potential to combat antibiotic-resistant bacteria causing bovine mastitis. Our objective was to use 3 Klebsiella lytic phages to create a phage cocktail, and to compare bactericidal activity of this phage cocktail versus an individual phage, both in vitro and in vivo. Based on transmission electron microscopy, phage CM_Kpn_HB154724 belonged to Podoviridae and on double agar plates, it formed translucent plaques on the bacterial lawn of Klebsiella pneumoniae KPHB154724. In one-step growth curves, this phage had a latent period of 40 min, an outbreak period of 40 min, a burst size of 1.2 × 107 PFU/mL, and an optimal multiplicity of infection (MOI) of 1. Furthermore, it was inactivated under extreme conditions (pH ≤ 3.0 or ≥ 12.0 and temperatures of 60 or 70 °C). It had a host range of 90% and had 146 predicted genes (Illumine NovaSeq). Based on histopathology and expression of inflammatory factors interleukin-1ß, tumor necrosis factor-α, interleukin-6, and prostaglandin, phage cocktail therapy had better efficiency than an individual phage in K. pneumoniae-infected murine mammary glands. In conclusion, we used 3 Klebsiella lytic phages to create a phage cocktail and confirmed its effectiveness against K. pneumoniae both in vitro (bacterial lawn) and in vivo (infected murine mammary glands).

MicroRNA miR-223 modulates NLRP3 and Keap1, mitigating lipopolysaccharide-induced inflammation and oxidative stress in bovine mammary epithelial cells and murine mammary glands.

Bovine mastitis, the most prevalent and costly disease in dairy cows worldwide, decreases milk quality and quantity, and increases cow culling. However, involvement of microRNAs (miRNAs) in mastitis is not well characterized. The objective was to determine the role of microRNA-223 (miR-223) in regulation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and kelch like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) oxidative stress pathway in mastitis models induced by lipopolysaccharide (LPS) treatment of immortalized bovine mammary epithelial cells (bMECs) and murine mammary glands. In bMECs cultured in vitro, LPS-induced inflammation downregulated bta-miR-223; the latter interacted directly with the 3' untranslated region (3' UTR) of NLRP3 and Keap1. Overexpression of bta-miR-223 in bMECs decreased LPS and Adenosine 5'-triphosphate (ATP)-induced NLRP3 and its mediation of caspase 1 and IL-1ß, and inhibited LPS-induced Keap1 and Nrf2 mediated oxidative stress, whereas inhibition of bta-miR-223 had opposite effects. In an in vivo murine model of LPS-induced mastitis, increased miR-223 mitigated pathology in the murine mammary gland, whereas decreased miR-223 increased inflammatory changes and oxidative stress. In conclusion, bta-miR-223 mitigated inflammation and oxidative injury by downregulating the NLRP3 inflammasome and Keap1/Nrf2 signaling pathway. This study implicated bta-miR-223 in regulation of inflammatory responses, with potential as a novel target for treating bovine mastitis and other diseases.

Wound inflammation post-orchiectomy affects the social dynamic of Nelore bulls.

BACKGROUND: Confinement of cattle imposes spatial restrictions and predisposes to aversive social encounters that can lead to contusions, wounds, pain, stress, fright, and reduced productivity. Although endogenous testosterone concentrations are linked to agonistic dominance behaviors in males, it is unknown whether decreased blood testosterone concentrations after castration alter social hierarchy rank in Nelore bulls. Therefore, in this study, we investigated the impact of the surgical would inflammation post-orchiectomy on social dynamics in a group of Nelore bulls (Bos indicus). Fourteen Nelore (Bos indicus) bulls were castrated and assessed pre- and post-surgically. Parameters evaluated were agonistic (mounting, headbutting, and fighting) and affiliative (head-play) behavior, plasma testosterone concentrations, average daily weight gain (ADG), and a score for severity of post-surgical infection. Exploratory statistics included social network analysis (SNA), hierarchy rank delta (Δ), and principal component analysis (PCA). Furthermore, statistical inferences included the Wilcoxon test, multiple logistic regression models, and Spearman's correlation. RESULTS: The social dynamic of Nelore bulls was modified after castration based on the findings of the SNA and the PCA. The moderate correlation between the postoperative inflammation level with the Δ, and the significant effect of this level in the logistic model post-castration were partially attributed to effects of pain on social relations. CONCLUSIONS: Our findings suggest the severity of post-surgical inflammation, which has an association with pain intensity, was closely associated with changes in the social hierarchy.

Contextualized understandings of dairy farmers' perspectives on antimicrobial use and regulation in Alberta, Canada.

Antimicrobial resistance (AMR) has been largely attributed to antimicrobial use (AMU). To achieve judicious AMU, much research and many policies focus on knowledge translation and behavioral change mechanisms. To address knowledge gaps in contextual drivers of decisions made by dairy farmers concerning AMU, we conducted ethnographic fieldwork to investigate one community's understanding of AMU, AMR, and associated regulations in the dairy industry in Alberta, Canada. This included participation in on-farm activities and observations of relevant interactions on dairy farms in central Alberta for 4 mo. Interviews were conducted with 25 dairy farmers. The interviews were analyzed using thematic analysis and yielded several key findings. Many dairy farmers in this sample: (1) value their autonomy and hope to maintain agency regarding AMU; (2) have shared cultural and immigrant identities which may inform their perspectives of future AMU regulation as it relates to their farming autonomy; (3) feel that certain AMU policies implemented in other contexts would be impractical in Alberta and would constrain their freedom to make what they perceive to be the best animal welfare decisions; (4) believe that their knowledge and experience are undervalued by consumers and policy makers; (5) are concerned that the public does not have a complex understanding of dairy farming and, consequently, worry that AMU policy will be based on misguided consumer concerns; and (6) are variably skeptical of a link between AMU in dairy cattle and AMR in humans due to their strict adherence to milk safety protocols that is driven by their genuine care for the integrity of the product. A better understanding of the sociocultural and political-economic infrastructure that supports such perceptions is warranted and should inform efforts to improve AMU stewardship and future policies regarding AMU.

Invited review: Selective treatment of clinical mastitis in dairy cattle.

Treatment of clinical mastitis (CM) and use of antimicrobials for dry cow therapy are responsible for the majority of animal-defined daily doses of antimicrobial use (AMU) on dairy farms. However, advancements made in the last decade have enabled excluding nonsevere CM cases from antimicrobial treatment that have a high probability of cure without antimicrobials (no bacterial causes or gram-negative, excluding Klebsiella spp.) and cases with a low bacteriological cure rate (chronic cases). These advancements include availability of rapid diagnostic tests and improved udder health management practices, which reduced the incidence and infection pressure of contagious CM pathogens. This review informed an evidence-based protocol for selective CM treatment decisions based on a combination of rapid diagnostic test results, review of somatic cell count and CM records, and elucidated consequences in terms of udder health, AMU, and farm economics. Relatively fast identification of the causative agent is the most important factor in selective CM treatment protocols. Many reported studies did not indicate detrimental udder health consequences (e.g., reduced clinical or bacteriological cures, increased somatic cell count, increased culling rate, or increased recurrence of CM later in lactation) after initiating selective CM treatment protocols using on-farm testing. The magnitude of AMU reduction following a selective CM treatment protocol implementation depended on the causal pathogen distribution and protocol characteristics. Uptake of selective treatment of nonsevere CM cases differs across regions and is dependent on management systems and adoption of udder health programs. No economic losses or animal welfare issues are expected when adopting a selective versus blanket CM treatment protocol. Therefore, selective CM treatment of nonsevere cases can be a practical tool to aid AMU reduction on dairy farms.

Selective treatment of nonsevere clinical mastitis does not adversely affect cure, somatic cell count, milk yield, recurrence, or culling: A systematic review and meta-analysis.

Treatment of clinical mastitis (CM) contributes to antimicrobial use on dairy farms. Selective treatment of CM based on bacterial diagnosis can reduce antimicrobial use, as not all cases of CM will benefit from antimicrobial treatment, e.g., mild and moderate gram-negative infections. However, impacts of selective CM treatment on udder health and culling are not fully understood. A systematic search identified 13 studies that compared selective versus blanket CM treatment protocols. Reported outcomes were synthesized with random-effects models and presented as risk ratios or mean differences. Selective CM treatment protocol was not inferior to blanket CM treatment protocol for the outcome bacteriological cure. Noninferiority margins could not be established for the outcomes clinical cure, new intramammary infection, somatic cell count, milk yield, recurrence, or culling. However, no differences were detected between selective and blanket CM treatment protocols using traditional analyses, apart from a not clinically relevant increase in interval from treatment to clinical cure (0.4 d) in the selective group and higher proportion of clinical cure at 14 d in the selective group. The latter occurred in studies co-administering nonsteroidal anti-inflammatories only in the selective group. Bias could not be ruled out in most studies due to suboptimal randomization, although this would likely only affect subjective outcomes such as clinical cure. Hence, findings were supported by a high or moderate certainty of evidence for all outcome measures except clinical cure. In conclusion, this review supported the assertion that a selective CM treatment protocol can be adopted without adversely influencing bacteriological and clinical cure, somatic cell count, milk yield, and incidence of recurrence or culling.

Melatonin improves post-thaw sperm quality after mild testicular heat stress in rams.

The objective was to determine effects of slow-release melatonin on post-thaw sperm quality in rams exposed to mild testicular heat stress (HS; scrotal neck insulation). Twelve yearling Dorset rams were randomly and equally allocated to receive either 36 mg melatonin in 1 ml corn oil or 1 ml corn oil injected subcutaneously (SQ); 15 day later, all rams had HS for 96 h (start of HS = start of Week 0). Semen was collected before HS and once weekly from Weeks 1 to 7, extended in Steridyl CSS One Step, held at 5°C for ~3 h, loaded into 0.5 ml straws, held 5 cm above liquid nitrogen for 10 min and then plunged. Computer assisted semen analysis (CASA) was conducted on frozen-thawed sperm. There were group and week effects for total and progressive motility (p < .001), plus group and week effects and group*week interactions (p < .001) for post-thaw total abnormalities, acrosome integrity, post-thaw sperm DNA fragmentation index (DFI) and high mitochondrial membrane potential (HMMP). Post-thaw sperm total and progressive motility, acrosome integrity and HMMP were higher (p < .05) in melatonin versus control groups from Weeks 1 to 7, and the melatonin group reached baseline level (pre-heat stress) at Week 7 (75.79 ± 0.96, 65.48 ± 1.51, 75.00 ± 0.89 and 67.00 ± 1.06, respectively; mean ± SEM). Conversely, post-thaw sperm total abnormalities and DFI were lower (p < .05) in melatonin versus control, and both reached baseline at Week 7 in the melatonin group (26.00 ± 0.57 and 5.66 ± 0.17, respectively). Coiled tails, distal midpiece reflexes, distal cytoplasmic droplets, ruffled acrosomes, bowed midpieces, pyriform heads and knobbed acrosomes were the most common abnormalities in both groups, with lower percentages in melatonin-treated rams. Results supported our hypothesis that HS reduces post-thaw sperm quality, and that melatonin lessens those reductions, manifested by significantly better total and progressive motility, acrosome integrity and HMMP, and fewer sperm total abnormalities and DFI.

Rumen-protected glucose hastens uterine involution and increases numbers of ovarian follicles in early post-partum dairy cows.

The primary objectives were to investigate the effects of feeding a new rumen-protected glucose (RPG) on uterine involution and ovarian follicular dynamics in recently calved dairy cattle. From 4 to 30 days after calving, 16 Holsteins (first to third lactation, mean parity 1.75) were randomly assigned to be fed either a basal diet top-dressed with either 600 g RPG (RPG group) or 600 g of the coating material and glucose (CONT group). Based on transrectal ultrasonography, conducted every 3 days starting 20 days after calving, the interval from calving to complete uterine involution was shorter in RPG versus CONT (27.1 vs. 30.4 days, p < .01). Furthermore, based on transrectal ultrasonography conducted every 2 days, cattle fed RPG had smaller (3.0-4.9 mm) ovarian follicles (2.96 vs. 0.9, p < .001) and more total follicles (5.26 vs. 2.85, p < .01). Feeding RPG had increased serum insulin concentrations (4.59 ± 0.54 vs. 3.13 ± 0.57, p < .05), but had no significant effects on serum glucose concentrations, dry matter intake or milk yield. In conclusion, we inferred that cattle fed RPG had increased glucose turnover that was responsible for higher insulin concentrations, faster uterine involution, and more ovarian follicles.

Characterization of the Testis-Specific Angiotensin Converting Enzyme (tACE)-Interactome during Bovine Sperm Capacitation.

A comprehensive understanding of molecular and biochemical changes during sperm capacitation is critical to the success of assisted reproductive technologies. We reported involvement of the testis-specific isoform of Angiotensin Converting Enzyme (tACE) in bovine sperm capacitation. The objective of this study was to characterize the tACE interactome in fresh and heparin-capacitated bovine sperm through immunoprecipitation coupled with mass spectrometry. These interactions were validated by co-localization of tACE with beta-tubulin as an identified interactome constituent. Although interactions between tACE and several proteins remained unchanged in fresh and capacitated sperm, mitochondrial aldehyde dehydrogenase 2 (ALDH2), inactive serine/threonine protein-kinase 3 (VRK3), tubulin-beta-4B chain (TUBB4B), and tubulin-alpha-8 chain (TUBA8) were recruited during capacitation, with implications for cytoskeletal and membrane reorganization, vesicle-mediated transport, GTP-binding, and redox regulation. A proposed tACE interactional network with identified interactome constituents was generated. Despite tACE function being integral to capacitation, the relevance of interactions with its binding partners during capacitation and subsequent events leading to fertilization remains to be elucidated.

Prepubertal nutritional modulation in the bull and its impact on sperm DNA methylation.

Enhanced pre-pubertal nutrition in Holstein bulls increased reproductive hormone production and sperm production potential with no negative effects on sperm quality. However, recent trends in human epigenetic research have identified pre-pubertal period to be critical for epigenetic reprogramming in males. Our objective was to evaluate the methylation changes in sperm of bulls exposed to different pre-pubertal diets. One-week-old Holstein bull calves (n = 9), randomly allocated to 3 groups, were fed either a high, medium or low diet (20%, 17% or 12.2% crude protein and 67.9%, 66% or 62.9% total digestible nutrients, respectively) from 2 to 32 weeks of age, followed by medium nutrition. Semen collected from bulls at two specific time points, i.e. 55-59 and 69-71 weeks, was diluted, cryopreserved and used for reduced representation bisulfite sequencing. Differential methylation was detected for dietary treatment, but minimal differences were detected with age. The gene ontology term, "regulation of Rho protein signal transduction", implicated in sperm motility and acrosome reaction, was enriched in both low-vs-high and low-vs-medium datasets. Furthermore, several genes implicated in early embryo and foetal development showed differential methylation for diet. Our results therefore suggest that sperm epigenome keeps the memory of diet during pre-pubertal period in genes important for spermatogenesis, sperm function and early embryo development.

Invited review: Selective use of antimicrobials in dairy cattle at drying-off.

Administering intramammary antimicrobials to all mammary quarters of dairy cows at drying-off [i.e., blanket dry cow therapy (BDCT)] has been a mainstay of mastitis prevention and control. However, as udder health has considerably improved over recent decades with reductions in intramammary infection prevalence at drying-off and the introduction of teat sealants, BDCT may no longer be necessary on all dairy farms, thereby supporting antimicrobial stewardship efforts. This narrative review summarizes available literature regarding current dry cow therapy practices and associated impacts of selective dry cow therapy (SDCT) on udder health, milk production, economics, antimicrobial use, and antimicrobial resistance. Various methods to identify infections at drying-off that could benefit from antimicrobial treatment are described for selecting cows or mammary quarters for treatment, including utilizing somatic cell count thresholds, pathogen identification, previous clinical mastitis history, or a combination of criteria. Selection methods may be enacted at the herd, cow, or quarter levels. Producers' and veterinarians' motivations for antimicrobial use are discussed. Based on review findings, SDCT can be adopted without negative consequences for udder health and milk production, and concurrent teat sealant use is recommended, especially in udder quarters receiving no intramammary antimicrobials. Furthermore, herd selection should be considered for SDCT implementation in addition to cow or quarter selection, as BDCT may still be temporarily necessary in some herds for optimal mastitis control. Costs and benefits of SDCT vary among herds, whereas impacts on antimicrobial resistance remain unclear. In summary, SDCT is a viable management option for maintaining udder health and milk production while improving antimicrobial stewardship in the dairy industry.

Supplementation of freezing medium with encapsulated or free glutathione during cryopreservation of bull sperm.

The objective was to compare effects of encapsulated or free glutathione (GSH) on the quality of frozen-thawed bull sperm. Ejaculates were collected via artificial vagina from six mature Holstein bulls once weekly for 6 weeks. All ejaculates had motility ≥70%, sperm concentration ≥1.0 × 109 /ml and ≤15% morphologically abnormal sperm. Each week, semen was pooled and diluted with lecithin-based extenders containing various concentrations of encapsulated (E0, E1, E2.5 and E5 mM) or free (F0, F1, F2.5 and F5 mM) GSH, with total glutathione content determined before and after cryopreservation. Total GSH in fresh semen was (mean+SEM) 4.8 ± 0.2 nmol/108 sperm, whereas in frozen-thawed semen of group F0 (control), it decreased to 1.4 ± 0.2 nmol/108 sperm, a 70.8% reduction (p < .05). In addition, total GSH in frozen-thawed semen from groups E2.5, E5 and F5 were 2.4 ± 0.2, 2.8 ± 0.2 and 1.8 ± 0.2 nmol/108 sperm, respectively (E5 versus. F0, p < .05). Compared to group F0, frozen-thawed sperm from group E2.5 had greater (p < .05) percentages of sperm that were viable (Annexin-V) (61.1 ± 1.8 versus. 71.1 ± 1.8) and that had cell membrane integrity (eosin-nigrosin) (64.5 ± 3.1 versus. 80.0 ± 3.1). Furthermore, frozen-thawed sperm from group E2.5 had the numerically highest total and progressive motility (CASA) and cell membrane functionality (HOS) and the lowest percentage of early apoptotic sperm (Annexin-V). However, acrosome membrane integrity (PSA) of E5 had the lowest mean (p < .05), whereas E2.5 caused a small nonsignificant decrease (69.1 ± 1.4%) compared to E0 and F0. In conclusion, 2.5 mM encapsulated GSH in semen extender significantly improved the quality of frozen-thawed bull sperm.

Testis-Specific Isoform of Na+-K+ ATPase and Regulation of Bull Fertility.

An advanced understanding of sperm function is relevant for evidence-based male fertility prediction and addressing male infertility. A standard breeding soundness evaluation (BSE) merely identifies gross abnormalities in bulls, whereas selection based on single nucleotide polymorphisms and genomic estimated breeding values overlooks sub-microscopic differences in sperm. Molecular tools are important for validating genomic selection and advancing knowledge on the regulation of male fertility at an interdisciplinary level. Therefore, research in this field is now focused on developing a combination of in vitro sperm function tests and identifying biomarkers such as sperm proteins with critical roles in fertility. The Na+-K+ ATPase is a ubiquitous transmembrane protein and its α4 isoform (ATP1A4) is exclusively expressed in germ cells and sperm. Furthermore, ATP1A4 is essential for male fertility, as it interacts with signaling molecules in both raft and non-raft fractions of the sperm plasma membrane to regulate capacitation-associated signaling, hyperactivation, sperm-oocyte interactions, and activation. Interestingly, ATP1A4 activity and expression increase during capacitation, challenging the widely accepted dogma of sperm translational quiescence. This review discusses the literature on the role of ATP1A4 during capacitation and fertilization events and its prospective use in improving male fertility prediction.