This study was designed to identify factors, including estrous cycle length, affecting the incidence of double ovulations in dairy cows. The study population comprised 748 primiparous cows undergoing spontaneous estrous cycles that had undergone their first postpartum artificial insemination following the second observed estrus. A subset of 341 cows with inter-estrus intervals (IEIs) of 18 to 30 days were selected to investigate the impacts of IEI, which was classified as normal (18-23 days) or lengthened (24-30 days). The odds ratio (OR) for double ovulations was 0.82 (p < 0.0001) for each unit increase in genomic prediction values for twin pregnancies, 2.3 (p < 0.0001) for cows inseminated during the negative photoperiod, compared to cows inseminated during the positive photoperiod, and 2.8 (p = 0.02) for cows with lengthened cycles, compared to cows with normal cycles. The OR for lengthened IEIs was 0.91 (p = 0.001) for each unit increase in genomic prediction value, and 4.4 (p < 0.0001) for cows inseminated during the negative photoperiod. In conclusion, lengthened estrous cycles were associated with double ovulations, genomic prediction values were able to identify the risk of lengthened cycles, and the negative photoperiod was found to favor both lengthened cycles and double ovulations.

Effective bovine artificial insemination (AI) training requires balancing technical skill development with animal welfare considerations. Commercial simulators typically replicate Bos taurus anatomy, limiting utility in regions where Bos indicus breeds predominate. This study validates the TrAI4Nel simulator, customized for Nelore cattle AI training. Validation employed a multi-dimensional framework encompassing face, physical, content, construct, and concurrent validity, plus usability and training effectiveness assessments. Of the 85 participants in standardized AI technician courses who were randomly allocated to control (abattoir specimen-based) and experimental (simulator-integrated) groups, 61 provided feedback about TrAI4Nel (19 in the control group and 42 in the experimental group). The simulator was also independently evaluated by 14 AI experts. Trainees rated the simulator highly for anatomical realism and procedural consistency. Compared with abattoir specimens, TrAI4Nel significantly enhanced skill transfer to live animals, particularly cervical pipette navigation and semen deposition identification. The simulator increased trainee confidence, reduced anxiety, and improved perceived preparedness. Performance assessments demonstrated simulator-trained participants achieved significantly higher success rates (78.6%) versus controls (52.6%; p = 0.043), without prolonging completion times. Qualitative feedback emphasized the simulator's pedagogical value in enhancing anatomical comprehension, skill acquisition, and learner autonomy while supporting animal welfare. Training sequence analysis indicated biological specimen exposure before simulator use may optimize learning efficiency. These findings validate TrAI4Nel as an effective, ethically sound tool for Bos indicus AI training. Simulator integration into curricula provides scalable improvement of reproductive management in zebu-dependent regions.

Early pregnancy diagnosis in buffaloes enhances reproductive efficiency and dairy production. This study aimed to develop and validate a duplex TaqMan-based RT-qPCR assay using two interferon-stimulated genes (ISGs), ISG15 and LGALS3BP, for early pregnancy diagnosis in buffaloes. Whole blood samples were collected from artificially inseminated buffaloes before (day 0) and after (days 20, 25 and 40) artificial insemination (AI). On 40days, post-insemination ultrasonography was performed to categorize animals as pregnant or non-pregnant. RNA from peripheral blood mononuclear cells (PBMCs) was isolated and converted into cDNA. A duplex TaqMan-based RT-qPCR assay was developed to predict pregnancy based on the expression of ISG15 and LGALS3BP in PBMCs. Gene expression was evaluated in a test dataset at various time points. The assay's performance was validated using two additional datasets, under which blood samples were collected randomly at 20±2days to evaluate its utility for predicting pregnancy. Colour Doppler ultrasonography was utilized to confirm pregnancy at 40days post-insemination for all the animals. Significantly different abundance of transcripts of ISG15 and LGALS3BP, in pregnant buffaloes, was observed on day 20 post-insemination. A statistically significant fold change (p<0.05) of ISG15 and LGALS3BP transcripts was observed between pregnant and non-pregnant buffaloes. The receiver operating characteristic curve of validation datasets demonstrated the AUC=0.95 and AUC=0.90. The negative predictive value and positive predictive value range from 90% to 95% and 75% to 85%. In conclusion, the developed duplex RT-qPCR-based assay demonstrates high sensitivity and specificity in detecting early pregnancy in buffaloes.

Early and accurate assessment of bull fertility is critical for the success of artificial insemination (AI) programs in cattle production. However, current selection tools, including genomic predictions and standard semen evaluations, offer limited reliability in forecasting field fertility outcomes. To address this limitation, we explored the sperm metabolome as a potential source of novel fertility-associated biomarkers. Using high-throughput untargeted metabolomics, we profiled frozen-thawed sperm from Holstein-Friesian bulls with high (n = 12) and low (n = 12) adjusted fertility scores, each with a minimum of 500 AI service records (range from 519 to 99,953 per bull). Raw peak intensities for 615 metabolites were normalized to the total protein concentration of each sample and following data filtration, 547 metabolites were retained for downstream analyses. Unpaired t-tests combined with fold-change thresholding identified 18 differentially abundant metabolites between high fertility and low fertility groups (P<0.1, absolute fold change >1.5), with significant enrichment in pathways relating to lipid and energy metabolism. Further interrogation of these differentially abundant metabolites in the literature revealed possible metabolic differences associated with calcium channel inhibition and reactive oxygen species (ROS) production in the low fertility bulls. Machine learning-based biomarker discovery further identified a subset of 5 metabolites - 3-phosphoglycerate, phenylalanine, ceramide, citrate, and citrulline - capable of distinguishing fertility status with high predictive accuracy (AUROC=0.877; P=0.02). Overall, these data support sperm metabolomics as a promising omics-based approach to enhance bull fertility evaluation and improve selection strategies in AI programs.

The aim of this study was to compare clinical pregnancy rate after IUI-D in heterosexual couples (HC), lesbian couples (LC), and single women (SW). This retrospective multicentric study was conducted in two university-based ART centers in France in all consecutive IUI-D cycles performed after stimulation with gonadotropins in 2021-2023. The primary outcome was clinical pregnancy rate (CPR). Cumulative clinical pregnancy rate was also calculated. Comparisons were made between groups taking HC as the control group and adjusting for age. A total of 962 patients undergoing 2737 IUI-D cycles were included in the analysis, 234 HC women (24.3%), 478 LC women (49.7%), and 250 (30%) single women (SW). SW were significantly older than HC and LC. Clinical pregnancy rate (CPR) per cycle was significantly lower in LC and SW than in HC in univariate analysis. Age was negatively associated with clinical pregnancy. After adjusting for age, no statistically significant relationship was found between category and clinical pregnancy (p = 0.07). Cumulative clinical pregnancy rates (CCPR) and the average number of cycles required to achieve pregnancy were not significantly different between HC, LC and SW. This first study conducted in France after change in law allowing ART access for LC and SW confirms that CPR after IUI-D is not significantly associated with relationship status after adjusting for age.

With estrus confined to three winter months, early pregnancy detection is essential for reproductive management in farmed sika deer. However, the development of reliable non-invasive early pregnancy detection techniques has been hindered by limited understanding of their reproductive physiology. To identify pregnancy-specific biomarkers in sika deer, we performed RNA-sequencing (RNA-Seq) on maternal peripheral blood collected on days 0, 7, 15, and 20 after artificial insemination. Using time-series clustering analysis and weighted gene co-expression network analysis (WGCNA), we identified key genes and pathways at each stage. Notably, maternal-fetal recognition-related interferon-stimulated genes (ISGs; IFNAR1/2, STAT1/2, MX1/2, and RSAD2), anti-apoptotic and immune-regulatory genes (BCL2, XIAP, and IL10), and cysteine metabolism genes (CTH, CBS, GCLC, and GCLM) were upregulated by day 7, suggesting their role in supporting corpus luteum development through immune regulation and redox homeostasis. By days 15-20, upregulated genes were enriched in pathways related to mitochondrial function, cell adhesion, and cell cycle regulation, indicating their involvement in embryo adhesion and syndesmochorial placentation. In conclusion, this study demonstrates that ISGs, immune-regulatory genes and cysteine metabolism genes are detectable as early as day 7 post-insemination, highlighting their promise as early pregnancy biomarkers and providing a molecular basis for non-invasive diagnostic development in sika deer.

The objective of the present study was to evaluate whether the inclusion of a gonadotropin-releasing hormone (GnRH) treatment at a novel time (34 h) after intravaginal progesterone device (IPD) removal improves ovulation synchrony and increases fertility in crossbred Girolando dairy cows (B. taurus × B. indicus). A total of 226 crossbred Girolando lactating cows (predominantly 5/8, ¾, and ½ Holstein blood share) were subjected to an estradiol-progesterone based timed artificial insemination (TAI) protocol. At the IPD removal, all cows were treated with 1 mg i.m estradiol cypionate (EC), and then allocated into two groups to receive either 2.5 mL of saline solution (EC-Control, n = 111) or 10.5 μg i.m. buserelin acetate (GnRH analogue) 34 h after IPD removal (EC-GnRH34, n = 115). A subgroup of 30 cows was examined by ultrasonography at 12 h intervals to determine the time of ovulation after IPD removal. All cows were subjected to TAI 48 h after IPD removal. Cows in the EC-GnRH34 group ovulated earlier (60.4 ± 3·8 h, p = 0.03) and more synchronously (100%; p = 0.006) than EC-Control cows (69.8 ± 4·0 h and 60%). Females submitted to the EC-GnRH34 protocol had 42.6% pregnancy per AI (P/AI) compared with 35.1% for the ones treated with EC only (EC-Control, p = 0.24). In conclusion, the present study demonstrated that the ovulation synchrony is optimized with the use of GnRH 34 h after IPD removal. Fertility to the TAI protocol, however, was not significantly improved in comparison with cows treated with a single ovulation inducer, EC.

&amp;lt;i&amp;gt;Background:&amp;lt;/i&amp;gt; The dairy sub-sector plays a pivotal role in Kenya’s economy, contributing to employment creation, food security, household incomes, and rural development. Dairy cooperatives emerge when members come together democratically and voluntarily to pool resources with the aim of meeting their socioeconomic needs. These cooperatives serve as vital platforms for the production, processing, and marketing of raw milk and its byproducts. Beyond marketing, they extend technical support to members through services such as artificial insemination, improved animal husbandry practices, and the provision of essential farm inputs, including animal feeds and fertilizer. As such, dairy cooperatives not only enhance productivity but also foster community empowerment and economic stability. &amp;lt;i&amp;gt;Methodology:&amp;lt;/i&amp;gt; The study was grounded in the Theory of Planned Behavior and employed an explanatory research design to establish causal relationships. The target population consisted of 216 registered dairy cooperatives, drawn from 11 counties in Kenya. A sample size of 140 cooperatives was selected using stratified sampling to ensure representativeness across the counties. Structured questionnaires were administered to gather primary data from management committees and members. &amp;lt;i&amp;gt;Findings:&amp;lt;/i&amp;gt; The results revealed a positive and significant relationship between management committee diversity and sustainable financing of dairy cooperatives. Diversity in age, gender, educational qualifications, and professional experience within management committees was shown to enhance decision-making and resource mobilization, thereby contributing to the long-term financial stability of cooperatives. &amp;lt;i&amp;gt;Conclusion:&amp;lt;/i&amp;gt; The study concluded that diversity in management committees fosters financial sustainability in dairy cooperatives. &amp;lt;i&amp;gt;Recommendations:&amp;lt;/i&amp;gt; The study recommends that dairy cooperatives in Kenya should intentionally promote diversity in their management committees by ensuring inclusive representation across demographic and professional dimensions.

This study investigated the effectiveness of a bacteriostatic-coated blister in preserving swine semen quality and its impact on reproductive performance. Two experiments were conducted: an in vitro assessment of the blister’s bacteriostatic efficacy and semen quality during three days of storage (Experiment 1), and a seven-day commercial farm trial evaluating its effect on reproductive outcomes in artificially inseminated gilts and sows (Experiment 2). In Experiment 1, the bacteriostatic blister effectively controlled bacterial proliferation, maintaining counts below 2 log10, comparable to controls with added antibiotics. Sperm quality parameters, including total and progressive motility, consistently exceeded the critical threshold for artificial insemination. Experiment 2 demonstrated that the bacteriostatic coating did not negatively affect key reproductive performance indicators, such as farrowing rate, total piglets born, or live piglets under commercial conditions. These findings suggest that the bacteriostatic-coated blister offers a viable, potentially antibiotic-free, alternative for semen preservation, extending storage viability for up to seven days. This technology supports sustainable reproductive practices, representing a significant advancement in commercial swine production.

Intrauterine insemination (IUI) is a popular method for the treatment of infertility, however, its success rate ranges from 7 to 13% in a cycle. The present study was carried out to assess the IUI success rate and its predictors. A prospective observational study was conducted, and 167 women (aged 21-49 years) with primary or secondary infertility were enrolled to undergo intrauterine insemination. The induction of ovulation for IUI was performed according to the standard protocols. Stimulation was done with letrozole/clomiphene/ FSH/HMG or a combination of these drugs. Human Chorionic Gonadotropin (hCG) was administered at achievement of follicular size 17-18 mm. IUI was done 36-40 hours after the hCG trigger. The luteal phase was supplemented wherever needed. A positive urine pregnancy test (UPT) done 2 weeks after IUI was considered as the primary outcome. Predictors of UPT success were evaluated using Chi-square and Independent Samples t-tests. The mean age of women was 30.58±(SD)4.43 years. The mean duration of infertility was 4±2.65 years. Majority (77.6%) had primary infertility. UPT positivity rate was 18.2% in <30 years and 21.3% in 30 to 45 years and no positive pregnancy in > 45 years. No significant association of age, BMI, level of physical activity, fertility type, ovulation induction frequency, AMH, sperm count, and HSG was seen with outcome (p>0.05). A significant association of outcome with endometrial thickness was observed( p-value 0.001). The IUI success rate was 18.8%. The endometrial thickness was significant predictors of IUI.

Simple SummaryCopper (Cu) and zinc (Zn) are essential trace minerals for growth, reproduction, and immune function in cattle, but their supplementation is often challenging in grazing systems. We conducted two experiments to evaluate whether a single injection of a CuZn solution given before artificial insemination could improve the performance of beef heifers. In a small-scale experiment, heifers receiving CuZn tended to gain more weight and showed signs of enhanced antioxidant activity and ovarian function. In a larger experiment designed to test reproductive outcomes, heifers injected with CuZn were heavier, had greater blood Cu concentrations, and those with a low body condition score showed improved estrus activity and tended to have greater pregnancy rates. These findings suggest that CuZn injection may be a practical strategy to support the growth and reproductive success of beef heifers, particularly when their body condition is suboptimal.Two experiments evaluated the effects of copper (Cu) and zinc (Zn) injection on body weight (BW), body condition score (BCS), pregnancy rate, ovarian traits, and antioxidant and inflammatory responses of beef heifers. In Exp. 1, 29 heifers were assigned to having saline or CuZn (a solution containing 15 and 50 mg/mL of Cu and Zn) subcutaneously injected (5 mL/heifer) 9 days before artificial insemination. Exp. 2 was conducted to increase the statistical power to evaluate pregnancy rate, and 283 heifers were assigned to either the saline or CuZn group. In Exp. 1, CuZn heifers tended (p ≤ 0.10) to gain more BW and to have greater corpus luteum size and plasma concentration of glutathione peroxidase. No effects of treatment were detected (p ≥ 0.18) for BCS; estrus score; serum concentration of Cu, Zn, and cortisol; and plasma concentration of haptoglobin, ceruloplasmin, and superoxide dismutase. In Exp. 2, CuZn heifers had greater (p < 0.01) BW and serum Cu. The CuZn heifers with low BCS had greater (p ≤ 0.05) estrus scores and tended (p = 0.10) to have greater pregnancy rates. Thus, injecting CuZn may be an effective strategy to enhance growth, reproductive performance, and antioxidant responses in heifers, especially when their BCS is below 5.

ABSTRACTArtificial insemination (AI) is a technique in which semen is artificially collected from a selected male animal, processed, stored and artificially introduced into the selected female reproductive tract for genetic improvement. The study was conducted from December 2022 to November 2023 to determine the efficiency, opportunities and challenges of AI in the West Wallaga Zone in Oromia, Ethiopia. The zone is located 441 km away from Addis Ababa. It is located at 9°10′N–9°17′N latitude and 35°44′E–36°09′E longitude. The three selected districts were Gimbi, Nadjo and Ayira and six representative kebeles from each district (Lalisa Yesus, Chuta Gochi), (Eba waqayo, Humna waqayo) and (Wayu kolli, Jarso Abayani), respectively. Purposive sampling methods were used to select the study area districts, kebeles and households based on the number of livestock population, the potential of AI implementation, accessibility and households who have one or more artificially inseminated dairy cows. Methods of data collection are questionnaire surveys, field monitoring, focal group discussions and retrospective data from the record book of each kebele and district office of agriculture from 2019 to 2023, which were used for the study. Both primary and secondary sources of information were used to obtain the data. In the three representative districts, the total target population (N = 900) of households’ owners of artificially inseminated cows was determined using the Yamane formula (n = 277). The sample size of the households of owners of artificially inseminated cows for each of the three selected districts was 92, and the households of owners of artificially inseminated cows for each kebele were surveyed 46. The collected data were statistically analysed using the Statistics Package for the Social Sciences (SPSS) (version 25). The efficiency/conception and calving rate in 2019–2023 from the total 2565 inseminated cows were 1285 (50%) and 1254 (97%), respectively. The overall average level of education was 53.8% (elementary school). The top opportunities and challenges were farmer interest and heat detection, respectively. Therefore, there should be an extension on AI, the AI technicians should be trained to improve their skills and increase the efficiency of AI, and the owners of dairy cows should be trained on heat detection.

Simple SummarySwine Inflammation and Necrosis Syndrome (SINS) can be detected in different body regions of pigs, such as the tail, ears, teats, or claws, and is an important indicator for health and welfare. Besides environmental influences, genetic factors play a major role in the development of SINS. In this study, we examined boars from different breeds kept at an artificial insemination (AI) station and compared their visible signs of SINS with those of their offspring. We found clear differences between breeds and lines, and a strong association between the severity of SINS in boars and in their piglets. Offspring of boars with the highest SINS scores also showed the most pronounced signs of SINS. However, some changes in heels and claws in the boars were mainly caused by environmental factors. Our findings suggest that pre-selecting AI boars with fewer signs of SINS could help reduce the occurrence of SINS in piglets and thereby improve their health and welfare.Animal-based measures, such as detecting inflammation in areas like the tail, ears, teats, coronary band, heels and claws (Swine Inflammation and Necrosis Syndrome, SINS), are used to monitor animal health and welfare. When parameters deviate from the established range, these measures enable prompt action to adjust husbandry practices, feeding regimens and management strategies. In addition to environmental factors, genetics have been shown to play a key role in inflammation and necrosis processes, and selection can reduce the severity of the disease. This study examined whether different breeds of AI boar exhibit different signs of SINS and how these signs are associated with SINS in their offspring when they are suckling piglets and weaners. Initially, 286 AI boars of 7 breeds from a German artificial insemination center were evaluated for SINS. The following parameters were assessed: tail base, tail tip, ears, skin, scrotum, coronary bands, heels and claws. Subsequently, 23 Pietrain and Duroc boars were used in combination with a Topigs DL sow line. The progeny of the AI boars was evaluated as suckling and weaned piglets, with the assessment framework encompassing SINS traits. The results revealed significant differences between the breeds and lines, as well as a strong correlation between the SINS phenotypes of the AI boars and the SINS scores of their offspring. The offspring of the 25% most extreme boars exhibited a 17% variation in SINS scores. This association was particularly evident when comparing the boars’ tail base. However, the development of the boars’ heels and claws was found to be significantly influenced by mechanical environmental factors and not associated with the piglets’ scores. These findings imply that heritable, endogenous processes, as proposed for SINS, also visibly impact the phenotype of the AI boar. This study’s fundamental premise suggests that pre-selecting AI boars could mitigate the occurrence of SINS and enhance piglet health and welfare.

The article examines the practices implemented by Italian lesbian mother couples confronted with the ban on assisted reproductive technology (ART) in their country, a prohibition that compels them to undertake reproductive journeys abroad. Drawing on ethnographic research conducted between 2023 and 2024, the author analyzes the strategies—multiple and sometimes paradoxical—adopted by these couples to circumvent legal norms and obtain recognition of their parenthood. By resorting to practices such as the ROPA method or artificial insemination, these families challenge heteronormative and biogenetic frameworks of kinship, while at times reintegrating biological elements in order to strengthen their legitimacy. This study shows how these trajectories, situated at the intersection of legal, economic, and cultural constraints, progressively contribute to transforming kinship norms in Italy and interrogate the relationships between law, culture, and parenthood within the broader struggle for the recognition of lesbian families.

Abstract Management practices induce stress, resulting in impaired growth and reproduction. Therefore, techniques to mitigate stress and its effects must be investigated. One strategy includes administration of a synthetic maternal bovine appeasing pheromone studied to have calming effects on cattle. The aim of this study was to evaluate the effects of a bovine appeasing substance (BAS) applied at estrous synchronization on reproductive stress and pregnancy rates. Crossbred beef cows (n = 131) were stratified by body weight (BW, 385 kg ± 0.53 kg), body condition score (BCS), and post-partum interval, then allocated to 1 of 2 pens. Pens were assigned randomly to treatment 1) administered BAS topically to the nuchal skin (5 mL) and poll (5 mL) at estrous synchronization (BAS; n = 71), or 2) control where BAS was not administered (CON; n = 60). BW was recorded on d 0, 7, breeding (d 9 and 10), and 106 to calculate change in BW. BCS was recorded on d 0 and d 106. Estrous synchronization and breeding protocols followed a 7-day-CO-Synch + CIDR combined with heat detection and timed artificial insemination (AI). Blood subsamples (BAS, n = 20; CON, n = 20) for serum cortisol analysis were collected on d 0, 7, breeding, and 106. Pregnancy was determined on d 106 via transrectal ultrasonography. Cortisol and change in BW were analyzed using the GLIMMIX procedures of SAS while GENMOD was used for AI and pregnancy variables. Significance was declared at P ≤ 0.05. There were no effects on BW for treatment by day interaction, however, BAS tended to be heavier compared to CON (P = 0.08) at breeding. There was an effect for treatment (P = 0.03), day (P ˂ 0.01), and their interaction (P = 0.03) for serum cortisol. At breeding, animals experienced greater cortisol concentrations of 11.68 ng/mL compared to d 0, 7, and 106; however, there were no differences in cortisol concentrations between treatments at breeding (P = 0.28). Overall, 51.18% of animals were open while 48.82% were bred, of those, 28.71% were bred via AI and 61.29% were bred via NS. In CON, 18 were bred via AI compared to 6 in BAS while 25 in BAS were bred via NS compared to 13 in CON (P ˂ 0.01). Previous research has reported a reduction in stress when a bovine appeasing substance was applied. In our study, applying a bovine appeasing substance at estrous synchronization did not reduce stress at the time of AI and did not impact reproductive success.

Abstract The bovine reproductive tract harbors a diverse microbiome that may influence pregnancy outcomes. Recently, we characterized the vaginal and uterine microbiota of virgin yearling heifers and cows at the time of artificial insemination (AI) using both 16S rRNA gene sequencing and culturing approaches. We identified distinct microbial taxa associated with pregnancy success and observed differential abundance between pregnant and non-pregnant groups, however most taxa remained unclassified at the genus level. Therefore, in this study we used shotgun metagenomic sequencing for higher taxonomic resolution and deeper functional insights into the bovine reproductive microbiome. The objective of the present study was to characterize the vaginal and uterine microbiomes of beef cattle that became pregnant compared to those that remained open following AI using shotgun metagenomic sequencing. The vaginal (7 open; 54 pregnant) and uterine (9 open; 41 pregnant) swabs were collected from two different cohorts of Angus-crossbred cattle consisting of mature cows (vaginal and uterine swabs) and heifers (only vaginal swabs) prior to AI. Genomic DNA were extracted from these samples and the microbiomes were profiled using shotgun metagenomic sequencing. We observed that the uterine and vaginal microbiomes had distinct compositions (PERMANOVA: R2 = 0.102 and P &amp;lt; 0.001). The composition (PERMANOVA: R2 = 0.0075, P = 0.7935), as well as the richness and diversity (P &amp;gt; 0.05) of the vaginal microbiome did not differ between open and pregnant cattle. A total of 422 different genera were detected from the vaginal samples, with Negativicutes-UBA1444, Streptococcus, Mycobacterium, and Ureaplasma being the most relatively abundant. Twenty-five of these genera including Aphodosoma, Egerieisoma, Alitiscatomonas, Lentihominibacter, Enterocola, Akkermansia, Ruminococcus, and Faecousia were more abundant (P &amp;lt; 0.05) in the vaginal microbiome of non-pregnant cattle. A significant difference in the composition of the uterine microbiome was observed between pregnant and open cattle (R2 = 0.049 and P = 0.042). Furthermore, microbial richness (P = 0.035) and diversity [(Shannon diversity: P = 0.014), (inverse Simpson diversity: P = 0.011)], as well as evenness (Pielou’s index: P = 0.047) were greater in the uterine microbiome of open than pregnant cattle. Overall, we profiled 329 bacterial genera across uterine samples, with Negativicutes-UBA1444, Cutibacterium, Streptomyces, and Acinetobacter being the most predominant genera. At species level, the vaginal microbiome had 1161 species, including Streptococcus pluranimalium, Ureaplasma diversum, Facklamia hominis, Histophilus somni, and Enterococcus faecalis, whereas the uterine microbiome was dominated by Negativicutes-UBA1444 sp012798135, Cutibacterium acnes, Giesbergeria lacusdiani, Bacillus_J hisashii, Thiopseudomonas sp012518175, and Acinetobacter idrijaensis. While the results of this metagenomic sequencing were negatively impacted by contaminating host DNA and consequently, low microbial sequencing depth, our results suggest that the uterine microbiome may have implications in AI pregnancy success rate.

Abstract The in vitro culture system is a powerful tool to study pre-implantation development of embryos. In contrast, studying the peri-implantation period is limited because of the lack of suitable in vitro models reflecting embryonic changes in vivo. Consequently, less is known about molecular cues governing conceptus elongation, attachment, and placentation during this stage, especially in large animal models such as pigs. Previous works have highlighted the role of small non-coding RNA (sncRNA) during peri-implantation. They primarily function to induce post-transcriptional gene silencing but appear to be critical for embryo-maternal communication. Global changes in embryonic sncRNA expression occur throughout this period and are implicated to regulate a variety of cellular functions. However, differences in sncRNA between embryonic lineages have not been investigated. In this study, we performed sncRNA sequencing to characterize lineage-specific sncRNA expression profiles during the period of conceptus attachment in pigs. Multiparous sows were artificially inseminated with semen from a boar constitutively expressing GFP. Conceptuses were collected 14 and 16 days after artificial insemination. Transverse dissections were made to separate the embryonic layers: inner cell mass (ICM), extraembryonic mesoderm (MESO), and trophectoderm (TE). A portion of TE was used to sex the conceptuses through genomic PCR. Total RNA was extracted (n=8) and NEBNext Small RNA Library Prep Set was used for cDNA library preparation. Sequencing was performed following standard Illumina procedures. Raw data was demultiplexed, cleaned, and trimmed then mapped to Sscrofa11.1 genome assembly using STAR. An average 7,859,877 reads passed quality filtering per sample with 62% being uniquely mapped and 30% being multi-mapped to reference genome. Raw count matrix was generated followed by differential expression analysis with DESeq2. Differences in gene expression were assessed using likelihood ratio test or Wald test for pairwise comparisons with significance determined by genes with Padj&amp;lt; 0.05 or |log2fc| &amp;gt;1, respectively. On day 14, we found 970 differentially expressed sncRNA between the embryonic layers. At least 44 sncRNAs were most abundant in ICM, 3 are most abundant in MESO, and 134 are abundant in TE. On day 16, we found 869 differentially expressed sncRNA between ICM and TE. Several sncRNAs were exclusive to ICM during these days, such as ssc-mir-105-1, MIR135B, and MIR219A2, etc. Others were exclusive to TE but only ssc-mir-129b has been annotated. We identified 4,221 differentially expressed sncRNA between conceptuses from day 14 and day 16. As the conceptus aged, sncRNAs such as ssc-mir-217-1, ssc-mir-216-1, ssc-mir-181a-1, etc. dramatically increased in expression while ssc-mir-1224, ssc-mir-582, etc. decreased. This study identifies lineage-specific sncRNA profiles during the peri-implantation period, and the sncRNA data sets are being analyzed to identify genes that are regulated by the sncRNAs. This work is supported in part by USDA-NIFA 2022-67015-36299.

Abstract The objective of the present study was to determine whether administration of a non-specific immunostimulant prior to timed artificial insemination (TAI) could improve pregnancy per artificial insemination (P/AI). Mature beef cows [59.2 ± 0.77 days postpartum (mean ± SEM)] consisting of a stabilized composite (1/2 Red Angus, 1/4 Charolais, and 1/4 Tarentaise) were submitted to synchronization and TAI during two successive breeding seasons (Year 1: n = 297; Year 2: n = 248). For synchronization, cows received the 7-d CO-Synch + CIDR protocol [100 μg of gonadotropin-releasing hormone (GnRH), i.m., and a controlled internal drug releasing device (CIDR) was inserted intravaginally on d -9; CIDR removal plus prostaglandin F2α (25 mg, i.m.) on d -2 with TAI on d 0]. On d 0, approximately 50 ± 2 h following CIDR removal, cows were randomly treated with 5 ml, i.m., of either sterile saline or mycobacterium cell wall fraction (MCWF; Amplimune, Novavive) and cows that had not exhibited estrus were administered a second dose of GnRH (100 μg, i.m.). All cows were submitted to TAI at approximately 56 ± 4 h following CIDR removal using frozen-thawed semen from 1 of 16 randomly selected bulls (n = 8 different bulls within breeding season). Pregnancy was initially diagnosed at d 35-60 (PREG1) and subsequently confirmed at d 105-110 (PREG2) of gestation. Data was analyzed using the Proc Glimmix procedure of SAS with year, treatment, estrus, sire(year), AI technician and all two-way interactions considered as independent variables. Overall, there was no effect of MCWF treatment prior to TAI on PREG1 (Saline: 130/267 = 48.7% vs. MCWF: 132/274 = 48.2%; P = 0.87) or PREG2 (Saline: 129/269 = 47.8% vs. MCWF: 130/274 = 47.2%; P = 0.80) and there was no interaction between year and MCWF treatment for PREG1 (P = 0.32) or PREG2 (P = 0.26). While there was no overall effect of sire, PREG1 and PREG2 were affected by a sire x treatment interaction (P = 0.04). Additionally, P/AI was affected by estrus response. Specifically, cows that exhibited estrus prior to TAI had greater P/AI at both PREG1 (135/236 = 57.2% vs. 127/308 = 41.2%; P = 0.0001) and PREG2 (136/235 = 57.9% vs. 123/308 = 39.9 %; P = 0.0001) than for cows that did not exhibit estrus. There was no interaction between treatment and exhibition of estrus prior to TAI for either PREG1 (P = 0.83) or PREG2 (P = 0.87). Pregnancy loss between PREG1 and PREG2 was not affected by any of the independent variables analyzed. Results indicate that treatment with a non-specific immunostimulant prior to TAI does not impact P/AI or pregnancy loss in mature beef cows.

Abstract We recently validated an automated monitoring device (i.e., accelerometers) for estrous detection in grazing beef cows with 91% of accuracy, but whether this methodology predicts pregnancy by artificial insemination (AI) still unclear. We hypothesized that the variables (rumination and activity) measured by the accelerometer software are associated positively with the probability of pregnancy/AI in grazing beef cows. Beef cows (n = 100) were fitted with an ear-mounted accelerometer tag (Sense Hub Cow Calf; Merck Animal Health, USA) in the middle third of the right ear on the first day of a 14-day estrous synchronization protocol. On the last day of the synchronization, cows were fitted with an estrous detection patch (ESTROTECTTM, Rockway Inc, USA). A split-time AI was performed 66 or 90 hours later based on estrus by the ESTROTECT, and the diameter of the largest follicle was measured. During estrus the following variables from the accelerometer software were collected: peak heat value (highest heat value), heat duration (hours between estrus onset and end), heat index (index value from the software), proestrus duration (hours from CIDR removal to estrus onset), daily eating (average eating value), raw activity (highest activity value), and peak activity (value of activity at the moment peak heat). Pregnancy diagnosis was performed 30 days post-AI. The relationship between the accelerometer variables and pregnancy/AI was analyzed using mixed-effects models (PROC MIXED; SAS). 92% of cows displayed estrus. Peak heat value (94.8 ± 1.32 vs 99.4 ± 1.17; P = 0.011), heat duration (20.31 ± 0.56 vs 21.9 ± 0.62 hours; P = 0.052), heat index (90.83 ± 1.37 vs. 97.55 ± 1.18; P = 0.0004), and proestrus duration (66.70 ± 3.53 vs. 73.67 ± 3.47 hours P= 0.001) were reduced in pregnant than to non-pregnant cows. Daily eating tended to be greater in pregnant cows (280.7 ± 32.36 vs. 236.44 ± 31.53; P=0.077). No significant association between raw activity or peak activity and pregnancy/AI was observed (P &amp;gt;0.1). For each unit of increase in the follicular diameter, heat index increases 0.92 (P=0.049). There was a quadratic association between follicular diameter and the percentage of increase in activity (P=0.041). ROC curve analysis testing the ability of the accelerometer variables to predict pregnancy/AI harvested accuracies from 0.6055 to 0.7211. In conclusion, accelerometers show potential for pregnancy prediction in grazing beef cows, but more calibration might be necessary to increase the accuracy. The reduction in activity-related variables in pregnant cows suggests that when displaying estrus, the control of its intensity is important for the pregnancy. Perhaps cows that display estrus that are not of very high intensity save more energy which helps with the establishment of pregnancy.

Abstract Approximately 50% of beef heifers do not become pregnant after artificial insemination (AI) which affects the profitability of beef operations. Anogenital distance (the vertical distance from the center of the anus to the top of the clitoris; AGD) is programmed during the fetal life and was associated negatively with pregnancy/AI in dairy heifers. We hypothesized that, yearling heifers with a shorter AGD during the pre-breeding examination have a greater probability of pregnancy/AI than heifers with a longer AGD. Heifers (n = 554) from 3 different farms, were submitted to an estrous synchronization protocol using an intravaginal progesterone device (CIDR; Eazi-Breed™ Cattle insert, Zoetis Animal Health, NJ, USA). At CIDR insertion, body weight and age were collected, heifers were submitted to transrectal palpation for reproductive tract score (ranging 1-5; 1-3 pre pubertal and 4-5 pubertal) and using a rice pelvimeter the pelvic area was calculated. Concomitant, AGD from the center of the anus to the top of the clitoris (AGD-C) and AGD from the center of the anus to the dorsal commissure of the vulva (AGD-T) were measured. Each farm used the following estrous synchronization protocol: Farm 1 (7-, 8-, or 9-day CIDR and AI), Farm 2 (14-day CIDR and timed-AI), and Farm 3 (7- or 8-day CIDR and timed-AI). On the day of CIDR withdraw heifers were fitted with an estrus patch (ESTROTECT™, Rockway Inc., USA), and the diameter of the largest follicle (n = 308) was measured. AI was performed using the semen of 5 bulls. Pregnancy/AI was measured 35 days after AI. Statistical analysis was performed using a linear mixed-effects model and logistic regression considering protocol as a block. 85% (471/554) of heifers were pubertal, 62% (344/554) displayed estrus, and 59% (167/283) were pregnant/AI. Pregnancy/AI on Farm 3 was 19.5% (53/271) and eliminated from statistical analysis. ADG-C and AGD-T were associated positively (P &amp;lt; 0.001, R2 = 0.1982). For each unit of increase in body weight, AGD-C increased 0.0064 units (P = 0.0003). AGD-C was associated positively with the probability of puberty during the pre-breeding examination (P = 0.0019) and probability of pregnancy in heifers without a corpus luteum (CL) at pre-breeding examination (Interaction AGD*CL P= 0.10). No association between pelvic area and follicular diameter with AGD-C or -T were observed (P &amp;gt; 0.05). ROC curve analysis found a poor area under the curve for both AGDs (AUC= 0.59) to predict pregnancy/AI. The association with AGD and reproductive measurements were contrary to our hypothesis and what is found in dairy heifers. Perhaps the prenatal effects on the AGD are diluted overtime and are not as influential to the reproductive outcome when the calf is an adult.