MicroRNAs in the Pathogenesis of Preeclampsia-A Case-Control In Silico Analysis.

Preeclampsia (PE) occurs in 5% to 7% of all pregnancies, and the PE that results from abnormal placentation acts as a primary cause of maternal and neonatal morbidity and mortality. The objective of this secondary analysis was to elucidate the pathogenesis of PE by probing protein-protein interactions from in silico analysis of transcriptomes between PE and normal placenta from Gene Expression Omnibus (GSE149812). The pathogenesis of PE is apparently determined by associations of miRNA molecules and their target genes and the degree of changes in their expressions with irregularities in the functions of hemostasis, vascular systems, and inflammatory processes at the fetal-maternal interface. These irregularities ultimately lead to impaired placental growth and hypoxic injuries, generally manifesting as placental insufficiency. These differentially expressed miRNAs or genes in placental tissue and/or in blood can serve as novel diagnostic and therapeutic biomarkers.

MicroRNAs and Their Associated Genes Regulating the Acrosome Reaction in Sperm of High- versus Low-Fertility Holstein Bulls.

Bioinformatics envisage experimental data as illustrated biological networks, exploring roles of individual proteins and their interactions with other proteins in regulation of biological functions. The objective was to identify differentially expressed miRNAs and their associated genes regulating the acrosome reaction in capacitated sperm of high- compared to low-fertility dairy bulls and to elucidate biological functional pathways using a systems biology approach, featuring miRNA-mRNA cluster analysis. Categorized bovine-specific miRNAs (n = 84) were analyzed by RT-PCR; 19 were differentially expressed in high- compared to low-fertility sperm (p ≤ 0.05, fold regulation ≥ 2 magnitudes). Six miRNAs (bta-miR-129-5p, bta-miR-193a-3p, bta-miR-217, bta-mir-296-5p, bta-miR-27a, and bta-miR-320a) were highly upregulated (p < 0.05; fold regulation ≥ 5 magnitudes) in high- compared to low-fertility sperm. Highly scored integrated genes of differentially expressed miRNAs predicted associations with pathways regulating acrosome vesicle exocytosis, acrosome reaction, and sperm-oocyte binding. The mRNA expressions of genes associated with the acrosome reaction (including hub genes) were greater, ranging from 2.0 to 9.1-fold (p < 0.05) in high- compared to low-fertility capacitated bull sperm. In conclusion, differentially expressed miRNAs in high-fertility bovine sperm regulating acrosome functions have potential for predicting bull fertility.

Differentially Expressed Candidate miRNAs of Day 16 Bovine Embryos on the Regulation of Pregnancy Establishment in Dairy Cows.

Recent advances in high-throughput in silico techniques translate experimental data into meaningful biological networks through which the role of individual proteins, interactions, and their biological functions are comprehended. The study objective was to identify differentially expressed (DE) miRNAs between the day 16 competent, elongated embryo from normal cows and the day 16 noncompetent, tubular embryos from repeat breeder cows, assimilate DE-miRNAs to their target genes, and group target genes based on biological function using in silico methods. The 84 prioritized bovine-specific miRNAs were investigated by RT-PCR, and the results showed that 19 were differentially expressed (11 up- and 8 down-regulated) in the competent embryos compared to noncompetent ones (p ≤ 0.05; fold regulation ≥ 2 magnitudes). Top-ranked integrated genes of DE-miRNAs predicted various biological and molecular functions, cellular processes, and signaling pathways. Further, analysis of the categorized groups of genes showed association with signaling pathways, turning on or off key genes and transcription factors regulating the development of embryo, placenta, and various organs. In conclusion, highly DE-miRNAs in day 16 bovine conceptus regulated the embryogenesis and pregnancy establishment. The elucidated miRNA-mRNA interactions in this study were mostly based on predictions from public databases. Therefore, the causal regulations of these interactions and mechanisms require further functional characterization.

In Silico Analysis of miRNA-Mediated Genes in the Regulation of Dog Testes Development from Immature to Adult Form.

High-throughput in-silico techniques help us understand the role of individual proteins, protein-protein interaction, and their biological functions by corroborating experimental data as epitomized biological networks. The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. Differentially expressed (DE) canine testis miRNAs between healthy immature (2.2 ± 0.13 months; n = 4) and mature (11 ± 1.0 months; n = 4) dogs were utilized in this investigation. In silico analysis was performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The results showed protein-protein interaction for the upregulated miRNAs, which revealed 978 enriched biological processes GO terms and 127 KEGG enrichment pathways, and for the down-regulated miRNAs revealed 405 significantly enriched biological processes GO terms and 72 significant KEGG enrichment pathways (False Recovery Rate, p < 0.05). The in-silico analysis of DE-miRNA's associated genes revealed their involvement in the governing of several key biological functions (cell cycle, cell proliferation, growth, maturation, survival, and apoptosis) in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways (ErbB, p53, PI3K-Akt, VEGF and JAK-STAT), cytokines and hormones (estrogen, GnRH, relaxin, thyroid hormone, and prolactin). Elucidation of DE-miRNA predicted genes' specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control.

Investigation of Sperm and Seminal Plasma Candidate MicroRNAs of Bulls with Differing Fertility and In Silico Prediction of miRNA-mRNA Interaction Network of Reproductive Function.

Recent advances in high-throughput in silico techniques portray experimental data as exemplified biological networks and help us understand the role of individual proteins, interactions, and their biological functions. The objective of this study was to identify differentially expressed (DE) sperm and seminal plasma microRNAs (miRNAs) in high- and low-fertile Holstein bulls (four bulls per group), integrate miRNAs to their target genes, and categorize the target genes based on biological process predictions. Out of 84 bovine-specific, prioritized miRNAs analyzed by RT-PCR, 30 were differentially expressed in high-fertile sperm and seminal plasma compared to low-fertile sperm and seminal plasma, respectively (p ≤ 0.05, fold regulation ≥ 5 magnitudes). The expression levels of DE-miRNAs in sperm and seminal plasma followed a similar pattern. Highly scored integrated genes of DE-miRNAs predicted various biological and molecular functions, cellular process, and pathways. Further, analysis of the categorized genes showed association with pathways regulating sperm structure and function, fertilization, and embryo and placental development. In conclusion, highly DE-miRNAs in bovine sperm and seminal plasma could be used as a tool for predicting reproductive functions. Since the identified miRNA-mRNA interactions were mostly based on predictions from public databases, the causal regulations of miRNA-mRNA and the underlying mechanisms require further functional characterization in future studies.

Sertoli, Leydig, and Spermatogonial Cells' Specific Gene and Protein Expressions as Dog Testes Evolve from Immature into Mature States.

Sertoli, Leydig, and spermatogonial cells proliferate and differentiate from birth to puberty and then stay stable in adulthood. We hypothesized that expressions of spermatogenesis-associated genes are not enhanced with a mere increase of these cells' numbers. To accept this postulation, we investigated the abundances of Sertoli cell-specific FSHR and AMH, Leydig cell-specific LHR and INSL3, and spermatogonia-specific THY1 and CDH1 markers in immature and mature canine testis. Four biological replicates of immature and mature testes were processed, and RT-PCR was performed to elucidate the cells' specific markers. The data were analyzed by ANOVA, using the 2-∆∆Ct method to ascertain differences in mRNA expressions. In addition, Western blot and IHC were performed. Gene expressions of all the studied cells' specific markers were down-regulated (p < 0.05) in adult testis compared with immature testis. Western blot and immunohistochemistry showed the presence of these proteins in the testis. Protein expressions were greater in immature testis compared with mature testis (p < 0.05). Despite the obvious expansion of these cells' numbers from immature to adult testis, the cells' specific markers were not enriched in mature testis compared with immature dog testis. The results support the postulation that the gene expressions do not directly correlate with the increase of the cell numbers during post-natal development but changes in gene expressions show functional significance.

Difference in Body Weight at Breeding Affects Reproductive Performance in Replacement Beef Heifers and Carries Consequences to Next Generation Heifers.

Nutrition imprinting carries consequences across generations. The effect of 55% vs. 65% of mature cow body weight (MBW; 545 kg) at breeding on the reproductive performance of heifers and their offspring was investigated. Angus-cross dam heifers were randomly fed to attain 55% (n = 1622) vs. 65% (n = 1578) of MBW, and offspring (F1) heifers born to dam heifers [55% (n = 1285) vs. 65% (n = 1324)] were fed to attain 65% of MBW. Bodyweight and reproductive indices were recorded throughout the study. In dam heifers, puberty (44% vs. 53%), breeding season pregnancy (86.4% vs. 90.6%) and 21-day calving rates (55.2% vs. 65.4%) did vary, but dystocia rate (8.7% vs. 9.0%) did not differ between 55% and 65% MBW groups. Puberty (49.2% vs. 58.2%), breeding season pregnancy (87.2% vs. 92.8%) and 21-day calving rates (53.8% vs. 64.1%) did differ (p < 0.05), but dystocia rate (8.4 vs. 9.2%) did not differ between F1 heifer groups. In conclusion, 55% of MBW at breeding negatively affected the reproductive performance of heifers and its offspring heifers. The recommendation is to feed heifers a balanced diet to reach 65% of MBW at breeding with consideration of production traits.

Pregnancy and offspring sex ratio following insemination with SexedULTRA and conventional semen in cows in a commercial beef operation.

Pregnancy rate per AI (PR/AI) and breeding season pregnancy rates between insemination with sexed semen (SS; at 18 hr after the onset of oestrus) and conventional semen (CS; at 12 hr after the onset of oestrus,) and offspring gender ratio between two groups were compared. Angus cross cows (n = 686, during 2019 and 2020 breeding seasons) were oestrus-synchronized using Select-Synch + CIDR protocol and were observed thrice daily for oestrus until 72 hr after PGF2α administration. Cows expressed oestrus (n = 513) were inseminated with either SS (n = 246; SexedULTRA 4M™; y chromosome-bearing sperm) or CS (n = 267). Cows (n = 173) that failed to express oestrus at 72 hr after PGF2α received 100 µg of GnRH and CS insemination concomitantly. Two weeks later, cows were penned with natural service sires (bull:cow ratio 1:25) for 45 days. Pregnancy was diagnosed 30 days after bull removal. Calves' gender was determined at birth. For cows that expressed oestrus, PR/AI did not differ (p > .1) between SS (65.0%) and CS (66.7%) groups. The overall PR/AI differed (p < .05) between SS (65.0%) and CS (56.4%) groups. The natural service PR differed (p < .001) but breeding season PR (p > .05) did not differ between SS vs. CS groups. Bull:heifer gender ratio following AI was 88:12 and 52:48 for SS and CS groups, respectively, with an overall 66:34 ratio. Bull:heifer gender ratio for the two breeding seasons was 79:21 and 52:48 for SS and CS groups, respectively, with an overall 62:38 ratio. In conclusion, the fertility of SS insemination at 18 hr after onset of oestrus was 97% of CS insemination at 12 hr after onset of oestrus. Though breeding season pregnancy did not differ between SS and groups, preferred calf gender was 25 percentage points greater for SS over CS application. The gender accuracy was 88%.

Impact of heat stress on embryonic development during first 16 days of gestation in dairy cows.

Objective was to elucidate the effects of heat stress (HS) on embryo development during first 16 gestational days (GD) and circulating hormone concentrations on GD-16 in lactating Holstein cows. Cows in HS and control (CON) groups were exposed to temperature humidity index (THI) of ≥ 73 and < 73, respectively, for 3 weeks before the experiment. GD-7 (67 vs 49%) and GD-16 (52 vs. 31%) conception rates following single insemination were greater (P < 0.01) for CON compared with HS cows. Control cows produced more GD-7 transferrable embryos following superovulation compared with HS cows (84.8 vs 53.1%; P < 0.001). Mean (± SEM) length (45.2 ± 10.6 vs. 59.2 ± 9.1 mm) and weight (31.4 ± 4.3 vs. 42.4 ± 6.2 mg) of GD-16 conceptus were greater for CON compared with HS cows (P < 0.05). Control cows yielded more filamentous conceptus (≥ 25 mm) compared with HS cows (71 vs 45%; P < 0.05). Progesterone (2.09-fold) was higher, and cortisol (1.86-fold), prolactin (1.60-fold), substance-P (1.55-fold), Isoprostane-8 (1.34-fold) and prostaglandin F metabolites (1.97-fold) were lower in CON compared with HS cows (P < 0.05). Progesterone positively, and substance-P, isoprostane-8 and the THI negatively were associated with GD-16 conceptus length (P < 0.05). In conclusion, altered hormones concentrations in heat-stressed cows plausibly resulted in lower GD-7 and GD-16 conception rates, fewer GD-7 transferable embryos, and stunted GD-16 conceptus elongation.

Association of gastrointestinal parasite burden, serum cytokines and hormones concentrations, and pregnancy in Angus-cross beef cows.

The objective was to elucidate the relationships among gastrointestinal (GI) parasite load, serum cytokines (Th 1 - Interleukin (IL) 2, Interferon (IFN) γ and Tumor necrosis factor (TNF) α; Th 2- IL4, IL6, and IL10) levels, hormones (progesterone, cortisol, 8-epi-prostaglandin F2 alpha (isoprostane), prolactin, substance-p, and prostaglandin F metabolites) concentrations, and pregnancy in beef cattle. Angus-cross beef cows (n = 700; age, 3-8 y) were blocked by age and body condition score (BCS, 1-9), and were randomly assigned to treatment (n = 350, TRT, 50 mg of eprinomectin/50 kg BW, im) or control (n = 350, CON, no treatment) on Day -30. Cows were synchronized using Controlled Internal Drug Release insert (CIDR) + CO-Synch protocol and artificially inseminated at a fixed time on Day 0 (66 h after CIDR removal). Fecal samples were collected to determine fecal egg count per gram (FEG, McMaster method) on Days -30, -23, -16, -7, 7, 0, 16 and 23, and blood samples were collected on Days -7, 0, 7, 16 and 23. Serum cytokines were determined on Days -7, 0, 7, 16 and 23, and circulating hormones were measured on Day 16. BCS were recorded on Day 16 following artificial insemination (AI), and pregnancy status was diagnosed on Day 30 and 60. Pregnancy/AI varied among treatment groups on Day 30 [TRT, 62.0% (217/350); CON, 54.9% (192/350) (P = 0.05)] and Day 60 [TRT, 60.9% (213/350); CON, 51.7% (181/350) (P < 0.05)]. Pregnancy loss between 30 and 60 days for TRT and CON groups were 1.8% (4/217) and 5.7% (11/192), respectively (P < 0.05). The BCS on Day 16 did not differ among treatment groups (P> 0.1). Four groups of 40 cows were selected based on their pregnancy status and treatment: pregnant, TRT; non-pregnant, TRT; pregnant, CON; and non-pregnant, CON to compare the mean FEG, cytokines, and hormones levels. The FEG and cytokine concentrations were significantly (P < 0.05) influenced by treatment, pregnancy status, day, treatment by pregnancy status, and treatment by day. Day 16 hormone concentrations were considerably influenced by treatment, pregnancy status, and treatment by pregnancy. Although FEG on Day -30 did not differ among the groups (P> 0.1), it was lower in treated, pregnant cows compared with cows in other three groups from Day -23 onwards (P < 0.05). Overall and pairwise comparisons showed that serum concentrations of Type 1 cytokines, IL2, IFNγ, and TNFα were lower (P < 0.05) from gestational Day 7 onwards in treated, pregnant cows compared with cows in other three groups. In contrast, serum concentrations of Type 2 cytokines, IL4, IL6 and IL10 were greater (P < 0.05) from gestational Day 7 onwards in treated, pregnant cows compared with cows in other groups. Serum concentrations of progesterone was greater and other hormones were lower for pregnant cows in TRT group compared to cows in other groups on gestational Day 16. In conclusion, GI parasite load was reduced; Th 1 cytokines levels were decreased; Th 2 cytokines concentrations were increased; progesterone level was increased; and cortisol, substance-P, prolactin, isoprostane, and PGFM were decreased in pregnant, TRT cows. These changes also resulted in an increase in P/AI. It is plausible that direct and bidirectional host-parasite interactions mediated by cytokines and hormones may have promoted maternal tolerance of an immunologically diverse conceptus and the establishment of pregnancy.

mRNA Expressions of Candidate Genes in Gestational Day 16 Conceptus and Corresponding Endometrium in Repeat Breeder Dairy Cows with Suboptimal Uterine Environment Following Transfer of Different Quality Day 7 Embryos.

Effect of the gestational day (GD) 7 embryo quality grade (QG) and subclinical endometritis (SCE) on mRNA and protein expressions of candidate genes [Interferon-τ (IFNT), IFN stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), Peroxisome proliferator activated receptors (PPARA, D, and G), Retinoid X receptors (RXRA, B, and G), and Mucin-1 (MUC1)] in GD16 conceptus and corresponding endometrium were evaluated. After screening of performance records (n = 2389) and selection of repeat breeders (n = 681), cows with SCE (≥6% polymorphonuclear neutrophils-PMN; n = 180) and no-SCE (<6%PMN; n = 180) received GD7 embryos of different QGs. Based on GD16 conceptus recovery, cows with SCE (n = 30) and No- SCE (n = 30) that received GD7 embryos QG1 (good, n = 20), 2 (fair, n = 20), and 3 (poor, n = 20) were included for gene analysis. mRNA and protein expressions (IFNT, ISG15, CXCL10, PPARG, RXRG, SLC2A1, and SLC27A6) differed between SCE and embryo QG groups. All genes but MUC1 and all proteins but MUC1 expression was greater in filamentous conceptus and corresponding endometrium vs. tubular conceptus and matching endometrium in SCE and embryo QG groups. In conclusion, disrupted embryo-uterine communication by altered expression of candidate genes in SCE cows, and in cows following the transfer of poor embryo negatively programs the conceptus development and plausibly affects conceptus survival.

Antibiotics Use in Food Animal Production: Escalation of Antimicrobial Resistance: Where Are We Now in Combating AMR?

The use of antibiotics has been very beneficial to human health, animal wellbeing, and food production, however, there are no alternatives to antimicrobials in treating infectious diseases. Their use can contribute to the development of antimicrobial resistance, but the world has realized the need to combat antimicrobial resistance in recent decades due to the continued escalation of the problem jeopardizing human and veterinary medicine and food and environmental safety. Understanding the AMR and judicious use of antimicrobials are critical, and one health approach involving several sectors and multiple disciplines is important to tackle the problem. National, regional, and global action plans have been instigated to tackle the escalation of AMR. Antimicrobials are frequently used in food animal production. Therefore, food animal producers are important participants to prevent overuse and misuse of antimicrobials. Recent regulations to address the challenges have not been perceived well in animal farming communities. More awareness regarding these action plans and understanding the impact of AMR are needed. A nationwide survey of perceptions of food animal producers regarding AMR mitigation approaches should be conducted to evaluate the effectiveness of the current policies regarding antibiotics use and AMR. These outcomes should be incorporated in future policies and awareness campaigns targeting food animal producers.

Day 7 embryo quality and suboptimal uterine environment influence morphometry of Day 16 conceptus in dairy cows.

The objectives of this study were to investigate the effect of Day 7 embryo quality and subclinical endometritis (SCE) in repeat breeder recipient cows on morphometry of Day 16 embryo and to determine the association of %PMN, serum progesterone and Day 16 conceptus length. Holstein dairy cows that failed to conceive at least 3 times, (parity, 3 and 4; body condition score, 3 to 3.5 out of 5) with subclinical endometritis (n = 180; SCE, >6% PMN on endometrial cytology) or without subclinical endometritis (n = 180; No-SCE, ≤ 6% PMN) were selected. Cows in each group received single, frozen-thawed, quality 1 (n = 60), 2 (n = 60) and 3 (n = 60) embryos (compact morula or early blastocyst) on Day 7 post estrus in the uterine horn ipsilateral to the ovary containing a corpus luteum, using standard nonsurgical techniques. Only cows that expressed estrus (Select-Synch protocol) and with acceptable corpus luteum (≥1.5 cm in size) were included. Conceptuses were collected on Day 16 from all recipient cows by standard non-surgical uterine flushing technique, using an 18-g embryo collection catheter with Phosphate Buffered Saline (pH 7.4). Blood samples were collected on Day 16 to determine serum progesterone concentrations. After collection, conceptuses were weighed and measured, and were categorized as tubular (underdeveloped, 10-20 mm) or filamentous (normal, >25 mm). Between cows with SCE and No-SCE, mean (±SEM) width (1.68 ± 0.13 mm vs. 1.84 ± 0.16 mm), length (34.4 ± 9.6 mm vs. 55.8 ± 13.4 mm) and weight (22.3 ± 3.7 vs. 40.6 ± 6.4 mg) of Day 16 conceptuses differed (P < 0.05). The mean width (1.75 ± 0.19 mm vs. 1.81 ± 0.22 mm), length (57.7 ± 11.2 vs. 51.1 ± 13.6 mm) and weight (34.3 ± 6.4 vs. 38.5 ± 8.2 mg) of Day 16 embryo following transfer of Day 7 embryo quality grade 1 and grade 2 embryos were not different (P > 0.1), but both differed from the mean width (1.59 ± 0.11 mm), length (28.9 ± 9.7 mm) and weight (25.3 ± 4.6 mg) of Day 16 embryo from Day 7 embryo quality grade 3 (P < 0.05). Total percentage of embryos recovered differed between SCE and No-SCE groups (P < 0.05; 36.1 vs 48.9%). Total percentage of embryos recovered on Day 16 following transfer of grade 1 (53.3%) and 2 (44.2%) Day 7 embryos were greater (P < 0.05) compared with transfer of grade 3 embryos (29.2%) (P < 0.001). Total percentage of filamentous embryos recovered was lower for SCE cows compared with No-SCE cows (P < 0.01; 15.0 vs. 25.6%). Total percentage of tubular embryos recovered did not differ between SCE and No-SCE cows (P > 0.1; 21.1% vs. 22.8%). Filamentous embryo recovered for grade 3 was lower (P < 0.05) compared with grade 1 in both SCE (8.3 vs. 21.7%) and No-SCE groups (15.0 vs. 33.3%). The mean (±SEM) CL volume (cm3; 11.8 ± 0.29 vs. 15.9 ± 0.31) and progesterone concentrations (ng/mL; 5.17 ± 1.8 vs. 8.2 ± 1.2) on Day 16 differed between SCE and No-SCE groups (P < 0.05) but not among Day 7 embryo grade groups (P > 0.1). The mean (±SEM) CL volume (cm3; 15.6 ± 0.28 vs 12.1 ± 3.9) and serum progesterone concentrations (ng/mL; 8.6 ± 1.4 vs. 4.9 ± 1.9) on Day 16 differed (P < 0.05) between cows yielded filamentous and tubular embryos. When all cows were considered, multiple regression analysis showed that the %PMN (P < 0.0001), progesterone concentrations (P < 0.0001), embryo qulaity (P < 0.05) and %PMN by progesterone interactions (P < 0.0001) influenced the length of Day 16 conceptus. Among cows without subclinical endometritis, only progesterone concentrations (P < 0.0001) and among cows with subclinical endometritis, only %PMN (P < 0.04) influenced the length of Day 16 conceptus. Progesterone concentrations (P < 0.0001) influenced the length of Day 16 conceptus in cows that received embryo quality 1 and 2. Progesterone concentration by %PMN interaction (P < 0.05) also influenced the length of Day 16 conceptus in cows that received embryo quality 2. The %PMN (P = 0.05) influenced the length of Day 16 conceptus in cows that received embryo quality 3. In conclusion, poor quality Day 7 embryo and presence of SCE negatively influenced early embryo development between Days 7 and 16 of gestation probably by dysregulated embryo-maternal interactions due to lower progesterone, prompting loss of the conceptus in sub-optimal uterine environment.

Transcript abundance of anti-Mullérian hormone and follicle-stimulating hormone receptor predicted superstimulatory response in embryo donor Holstein cows.

The goal was to investigate the relationship among mRNA expressions of anti-Mullérian hormone (AMH), follicle-stimulating hormone receptor (FSHR) and responses to superovulation (SO) in embryo donor dairy cows. Holstein cows (n = 19) were submitted to a standard SO protocol, with twice daily FSH treatments, and artificially inseminated. Prior to SO (Day 0), relative mRNA expressions of AMH and FSHR in blood were determined for all cows. Day 7 embryos were collected and were graded to determine superovulatory response for each donor. Results showed that relative mRNA expressions of AMH and FSHR were positively correlated (R2  = 0.94). Relative mRNA expressions of both AMH and FSHR were positively correlated with total embryos (R2  = 0.68 and 0.69, respectively), total transferable embryos (R2  = 0.92 and 0.97, respectively) and total grade 1 embryos (R2  = 0.54 and 0.59, respectively). Further, transcript abundances of AMH and FSHR positively associated with milk production of donor cows, and meanwhile, they were negatively associated with days in milk (DIM) at submission of cows to SO (p < .05) protocol. The relative mRNA expression of AMH was higher (p < .05) in donor cows <5 years of age. However, age of donor at superovulation did not influence mRNA expression of FSHR. Collectively, we infer that the mRNA expressions of AMH and FSHR prior to superovulation can predict donor cows' positive response to superovulation.

Reduced gastrointestinal worm burden following long term parasite control improves body condition and fertility in beef cows.

The objective of this study was to compare effects of gastrointestinal parasite control over a long vs short term (PC-LT vs. PC-ST) on fecal parasite load, body condition and pregnancy in beef cows. On Day 0, fecal samples were collected from Angus cross cows (n = 1462) and they were assigned a body condition score (BCS: 1, emaciated; 9, Obese) and randomly divided into two groups (within location) to receive extended-release eprinomectin [PC-LT; n = 749; 50 mg/50 kg body weight (BW)] or pour-on ivermectin (PC-ST; n = 713; 25 mg/50 kg BW). All cows were synchronized with CO-Synch + CIDR [100 µg GnRH + progesterone vaginal insert (CIDR) application on Day 20, CIDR removal +25 mg PGF2a on Day 27, and artificial insemination +100 µg GnRH on Day 30 (66 h after CIDR removal)] protocol, artificially inseminated (AI; Day 30) and on Day 44, exposed to breeding bulls (1:40 bull to cow ratio) for the remainder of the 85 day breeding season. On Day 90, a second fecal sample was collected from all cows and the cows were examined to determine pregnancy/AI (P/AI). All cows were assigned a BCS on Day 180 and re-examined to determine pregnancy/breeding season (P/BS). Worm egg count per gram of feces (FEG) was determined by McMaster method. There were no differences (P > 0.1) between PC-LT and PC-ST groups on Day 0 for FEG (46.9 ±â€¯13.1 vs 42.6 ±â€¯15.2, respectively; mean ±â€¯SEM) or BCS (5.95 ±â€¯0.12 vs 6.00 ±â€¯0.20). The mean FEG (PC-LT, 12.3 ±â€¯4.7 vs. PC-ST, 131.3 ±â€¯10.9) on Day 90 and BCS (PC-LT, 6.04 ±â€¯0.07 vs. PC-ST, 5.79 ±â€¯0.13) on Day 180 differed (P < 0.05) between the two groups. Mean P/AI [PC-LT, 62.9 %; (471/749) vs PC-ST, 57.4 %; (409/713)] and P/BS [PC-LT, 92.9 % (696/749) vs PC-ST, 90.0 (642/713)] also differed (P < 0.05). Lower FEG at Day 90 resulted in moderate to good body condition at Day 180 and cows with moderate to good body condition at Day 180 had higher P/BS. In conclusion, lower worm burden with long-term parasite control reduced FEG and improved BCS, P/AI and P/BS.

Estrous response and pregnancy percentages following use of a progesterone-based, split-time estrous synchronization treatment regimens in beef heifers.

Mean estrous response rate (%ERR) and pregnancy/AI percentages (%P/AI) were determined after imposing split-time AI (STAI) and fixed time AI (FTAI) following 14-d controlled internal drug release (CIDR)+PGF2α or 5-d Select Synch + CIDR regimens. In Experiment 1, 1152 heifers (five locations) were randomly assigned to 14- or 5-d and to 54 + 74- or 64 + 84-h STAI treatment combinations. Estrous detection patches were affixed at PGF2α administration (19 day after- and on day 5 at- CIDR removal for 14- and 5-d regimens, respectively), assessed at 54- or 64-h and again at 74- or 84-h after PGF2α. Heifers determined to be in estrus at respective times were inseminated and non-estrous heifers at 74- or 84-h were given GnRH and inseminated concomitantly. The %ERR between 54 + 74- and 64 + 84-h STAI combinations differed (73.2 % and 78.8 %, respectively; P < 0.05), but %P/AI did not. In Experiment 2, 2014 heifers (eight locations) were randomly assigned to 14- or 5-d regimens and were inseminated split-time (64+84-h combination, similar to Experiment 1) or at fixed time (72- or 56-h after PGF2α for 14- or 5-d regimens, respectively). There were differences (P < 0.01) between STAI and FTAI treatments for %ERR (81.3 % and 64.4 %) and %P/AI (61.2 % and 55.4 %). Estrous synchronization regimen by AI treatment interaction (P < 0.05) showed that the %ERR were 79.8 %, 82.6 %, 66.2 % and 62.8 % and the %P/AI were 58.9 %, 63.4 %, 56.5 % and 56.5 % (for 14-d/STAI, 5-d/STAI, 14-d/FTAI and 5-d/FTAI, respectively). In conclusion, the 5-d CIDR with 64+84-h STAI combination was the most effective because of the greater %P/AI when this regimen was imposed.

Effects of twice daily compared with split-time estrous detection on pregnancy percentage in recipient beef cows.

Objectives were to compare pregnancy percentages per embryo transfer (P/ET) in recipient beef cows following twice daily compared with split-time estrous detection and to determine associations of dominant follicle diameter, CL volume and progesterone concentrations. All cows (n = 695) were treated to synchronize time of estrus among cows using a CIDR + Select-Synch treatment regimen and randomly assigned to twice-daily or split-time estrous detection (CS-DEET or CS-SEET, respectively). Cows in the CS-DEET group were observed twice daily (eight times) for estrus until 96 h after the time of PGF2α administration, whereas cows in the CS-SEET group were observed twice (64 and 84 h after PGF2α). In 280 recipient cows, blood sampling (for progesterone) and ultrasonographic assessment of dominant follicle diameter were conducted 48 h after the time of PGF2α administration. At 7 d after estrus, the CL was imaged and there was transfer of a frozen-thawed embryo into cows with a CL ≥ 1.5 cm. There were positive correlations between follicle diameter and CL volume (rs = 0.827; P < 0.001) and CL volume and progesterone concentration (rs = 0.680; P < 0.001). Progesterone and CL volume differed between cows in CS-SEET and CS-DEET groups P < 0.05), however, percentage P/ET for cows in the CS-SEET and CS-DEET groups did not differ (P > 0.1). Dominant follicle diameter, CL volume and progesterone concentrations were greater in pregnant compared with nonpregnant cows. In conclusion, percentage P/ET did not differ when there was twice daily and split-time estrous detection highlighting the value of this approach in beef enterprises.

Presynchronization with CIDR, with or without GnRH, prior to CO-Synch in beef heifers.

Objectives were to compare ovarian responses and pregnancy per AI (P/AI) in Angus-cross beef heifers (n = 521; 4 locations) synchronized with CIDR-CO-Synch (CCOS) versus CIDR-GnRH-CO-Synch (CGCOS) protocols. Heifers were assigned a reproductive tract score (RTS: 1, immature, acyclic; 5, mature, cyclic), body condition score (BCS: 1, emaciated; 9, obese) and temperament score (0, calm, 1, excitable). Heifers in the CCOS (n = 261) group received a CIDR on Day -20 (removed on Day -13), 100 µg GnRH on Day -10, 25 mg PGF2α on Day -3 and were timed inseminated 60 h later, with concomitant GnRH (Day 0). Heifers in the CGCOS (n = 260) group received a CIDR on Day -26 (removed on Day -19), 100 µg of GnRH on days -16 and -10, 25 mg of PGF2α on Day -3 and were timed inseminated 60 h later, with concomitant GnRH (Day 0). Ovarian ultrasonography was done in a subset of heifers (n = 60; 30 in each group) to determine number and size of ovarian follicles and presence of corpus luteum (CL). There was increased (P < 0.05) percentage of heifers with CL in CGCOS group compared to heifers in CCOS group on Day -10 (82.3 vs 68.2%) and on Day -3 (88.3 vs 75.1%). Average size of the largest ovarian follicle on Day 0 was greater for heifers in CGCOS group compared to CCOS group (P < 0.05). However, P/AI did not differ between CCOS and CGCOS groups, 55.0% (143/260) and 59.8% (156/261), respectively (P > 0.1). In conclusion, CIDR presynchronization with or without GnRH (CCOS and CGCOS protocols) in beef heifers resulted in similar P/AI. Adding GnRH to presynchronization with CIDR resulted in more heifers with a CL at PGF2α and increased preovulatory follicle diameter at AI. Future studies are needed with bigger sample size and CIDR + CO-Synch treatment as control to determine economic benefit.

A Method to Isolate CD34+ Mononuclear Cells from Canine Peripheral Blood.

Embryonic stem cells are pluripotent whereas adult stem cells are multipotent in nature. In recent years, evidence suggests that adult stem cells not only differentiate into specific cell lineages but also transdifferentiate into multiple cell lineages. Progenitor cells are found in adult bone marrow, blood, and other organs and differentiate into numerous cell lineages regardless of origin. Identifying a subset that can differentiate into mature endothelial cells is essential. This article describes peripheral blood collection in adult beagle dogs, isolation of peripheral blood mononuclear cells (PBMNCs) from the cell fraction, separation of a subset of CD34+ cells using immunomagnetic principles, characterization of PBMNCs and CD34+ cells using flow cytometry, and evaluation of gene expression of CD34, KDR, and CD133 in CD34+ fractions. Efficient methods of isolation of endothelial progenitor cells (EPCs) will promote the ex vivo expansion and transplantation of EPCs in ischemic injury to enable neovascularization. © 2019 by John Wiley & Sons, Inc.

Patterns of expression of sperm and seminal plasma microRNAs in boar semen.

Although sperm and seminal plasma differ in their origin, biophysical and biochemical properties of seminal plasma influence the sperm function. Seminal plasma is a fluid medium containing substances from testes, epididymides and accessory glands. Composition of seminal plasma varies among animal species and in boars, prostate and bulbourethral glands are major contributors to the volume and contents. While the origin of some components of seminal plasma are known, the source of recently discovered seminal plasma microRNAs remains unknown, in part due to the difficulty of recovering and characterizing RNA from porcine sperm and seminal plasma. To test the hypothesis that seminal plasma miRNAs interact with sperm, the first objective was to validate protocols for recovering RNAs from porcine seminal plasma and sperm, whereas the second objective was to characterize expression patterns of 84 prioritized microRNAs employing real time PCR methodology. The study identified a relationship between sperm and seminal plasma microRNAs, based on the normalized threshold cycle of amplifying cDNA in sperm and seminal plasma from the same semen of Landrace boars. Therefore, it was concluded that seminal plasma miRNAs may originate from sperm or these miRNAs may shuttle between sperm and seminal plasma in order to facilitate cell-to-cell communication.