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.

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.

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.

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.

An Efficient Approach for RNA Extraction from Boar Sperm and Seminal Plasma.

Despite transcriptional silencing in mature sperm and cytoplasmic expulsion of RNA during the final sperm maturation process, thousands of RNAs have been successfully identified in ejaculated sperm. Although most of RNAs' function is still unknown, it is suggested that sperm RNAs have a vital biological role in fertilization and post-fertilization events. Nevertheless, the lack of accurate RNA isolation techniques and the resultant good quality sperm RNA has hampered the exploration of sperm RNAs function. Additionally, small non-coding RNAs are found in extracellular fluids including seminal plasma. These small RNAs may participate in cell to cell communication or intracellular and extracellular message transmission. Developing precise protocols to extract RNA from sperm and seminal plasma is critical to elucidate sperm physiology and paternal contributions to fertilization and post-fertilization events. A detailed procedure consisting of semen collection, separation of sperm and seminal plasma, extracting RNA from sperm and seminal plasma, and determining the quantity and quality of RNA for boar semen is presented here. This efficient protocol can be extrapolated to isolate RNAs from sperm and seminal plasma across mammalian species.

Aggressive attempted escape behavior during head-lock restraint reduced reproductive performances in Holstein heifers.

The objective was to determine associations between behavior during head-lock restraint and reproductive performance in dairy heifers. Behavior of 817 Holstein heifers from four farms was evaluated at feeding (Days 0 and 7) while restrained in a self-locking stanchion. All heifers were assigned a body condition score (BCS; 1, emaciated to 5, obese) on Day 0. Heifers were timed-inseminated on a maximum of three occasions to determine impact of behavior for first service pregnancy per AI (FS-P/AI) and cumulative P/AI (C-P/AI). Ovulation was synchronized with an Ovsynch synchronization protocol for first service and thereafter either Ovsynch and/or prostaglandin F2α-based protocols. More heifers displayed calm escape behavior (P < 0.05) compared with mild or aggressive escape behaviors (45.2, 28.2 and 26.6%, respectively). Adjusting for BCS (P < 0.05), FS-P/AI was greater (P < 0.05) for calm heifers compared with aggressive escape behavior, 58.0% (214/369) vs 48.2% (105/218), with FS-P/AI of heifers with mild aggressive behavior [53.5% (123/230)] intermediate and did not differ from other means. Adjusting for BCS (P < 0.0001), C-P/AI was greater (P < 0.0001) for heifers with calm compared with mild or aggressive escape behaviors [84.8% (313/369), 71.3% (164/230) and 64.7% (141/218), respectively]. Serum cortisol concentrations were not different among behavior categories, but serum substance P concentrations were greater (P < 0.05) in aggressive heifers compared with mild or calm heifers, 97.1 ±â€¯4.9, 58.4 ±â€¯2.9 and 52.3 ±â€¯2.6 ng/mL, respectively. In conclusion, Holstein heifers with aggressive escape behavior during head-lock restraint had significantly reduced reproductive performance.

Subclinical Pregnancy Toxemia-Induced Gene Expression Changes in Ovine Placenta and Uterus.

The objective was to elucidate gene expression differences in uterus, caruncle, and cotyledon of ewes with subclinical pregnancy toxemia (SCPT) and healthy ewes, and to identify associated biological functions and pathways involved in pregnancy toxemia. On Day 136 (±1 day) post-breeding, ewes (n = 18) had body condition score (BCS; 1-5; 1, emaciated; 5, obese) assessed, and blood samples were collected for plasma glucose and ß-hydroxybutyrate (BHBA) analyses. The ewes were euthanized, and tissue samples were collected from the gravid uterus and placentomes. Based on BCS (2.0 ± 0.02), glucose (2.4 ± 0.33), and BHBA (0.97 ± 0.06) concentrations, ewes (n = 10) were grouped as healthy (n = 5) and subclinical SCPT (n = 5) ewes. The mRNA expressions were determined by quantitative PCR method, and prediction of miRNA partners and target genes for the predicted miRNA were identified using miRDB (http://mirdb.org/miRDB/). Top ranked target genes were used to identify associated biological functions and pathways in response to SPCT using PANTHER. The angiogenesis genes VEGF and PlGF, and AdipoQ, AdipoR2, PPARG, LEP, IGF1, IGF2, IL1b, and TNFα mRNA expressions were lower in abundances, whereas hypoxia genes eNOS, HIF1a, and HIF 2a, and sFlt1 and KDR mRNA expressions were greater in abundances in uterus and placenta of SCPT ewes compared to healthy ewes (P < 0.05). The predicted miRNA and associated target genes contributed to several biological processes, including apoptosis, biological adhesion, biological regulation, cellular component biogenesis, cellular process, developmental process, immune system process, localization, metabolic process, multicellular organismal process, reproduction, and response to stimulus. The target genes were involved in several pathways including angiogenesis, cytoskeletal regulation, hypoxia response via HIF activation, interleukin signaling, ubiquitin proteasome, and VEGF signaling pathway. In conclusion, genes associated with blood vessel remodeling were lower in abundances and that the genes associated with hypoxic conditions were greater in abundances in the uteroplacental compartment of SCPT ewes. It is obvious that the factors that influence placental vascular development and angiogenesis as noted in this study set the course for hemodynamic changes and hence have a major impact on the rate of transplacental nutrient exchange, fetal growth, and health of the dam.

Fertility after two doses of PGF2α concurrently or at 6-hour interval on the day of CIDR removal in 5-day CO-Synch progesterone-based synchronization protocols in beef heifers.

Timed artificial insemination protocols in beef cattle are designed to synchronize ovulation in a greater proportion of females while simultaneously achieving acceptable pregnancy rates and a concise calving season. Protocols achieving such goals reduce time and labor associated with estrus detection and make advanced reproductive technologies implementable for beef producers. The objective of the study was to determine the effect of three different PGF2α (PGF) dosage schemes on artificial insemination (AI) pregnancy rates in beef heifers. We hypothesized that two doses of PGF administered concurrently at the time of controlled internal drug release (CIDR) removal would attain similar pregnancy rates compared with two doses given 6-hours apart-one at CIDR removal and the next 6 hours later in the 5-day CO-Synch progesterone-based synchronization protocol. Angus heifers (n = 875) at six locations in Washington, Idaho, and Oregon states were included in this study. Heifers within locations were assigned a body condition score (BCS). All heifers received a CIDR (1.38 g of progesterone) and 100 µg IM of GnRH on Day 0. The CIDRs were removed on Day 5, heifers were randomly allocated to one of three protocol groups: 1PGF (n = 291), received 25 mg IM of dinoprost (PGF); 2CO-PGF (n = 291), received 50 mg IM of dinoprost at CIDR removal, 2PGF (n = 293), received 25 mg IM of dinoprost at CIDR removal, and an additional 25 mg IM of dinoprost 6 hours later. Each heifer was given GnRH (100 µg, IM) and artificially inseminated at 56 hours after CIDR removal. Heifers were examined for pregnancy status between 50 and 70 days after AI to determine time of conception. A mixed-model procedure (PROC GLIMMIX of SAS) was used to evaluate the effect of treatments (1PGF, 2CO-PGF, and 2PGF) on AI pregnancy rates. Models included were treatments, BCS categories (≤5 and >5), and treatment by BCS category interaction. Location (state), handling facilities, handlers, inseminators, and AI sires were included as a random effect in the model. The 2PGF group had greater AI pregnancy rate of 63.6% (185/291), compared with the 2CO-PGF group at 51.9% (151/291) and 1PGF group at 54.9% (161/293; P < 0.001). An AI pregnancy rate of 50% (104/208) was observed for heifers with BCS less than or equal to 5 versus 58.9% (393/667) for heifers with BCS greater than 5 (P < 0.05). Location did not influence the AI pregnancy rate (P > 0.1). In conclusion, beef heifers received two 25-mg doses of PGF at 6-hour interval on Day 5 at CIDR insert removal in a 5-day CO-Synch + CIDR synchronization protocols achieved greater pregnancy compared with heifers received 50 mg of PGF concurrently at CIDR removal.

Estrous synchronization strategies to optimize beef heifer reproductive performance after reproductive tract scoring.

Three experiments comparing four estrous synchronization protocols were conducted to determine estrous expression rate and artificial insemination pregnancy rate (AI-PR) in heifers with a range (1-5) of reproductive tract scores (RTSs). At enrollment (Day 0), 1783 Angus cross beef heifers from six locations were given body condition score and RTS. The four protocols were: (1) HRTS-DPGF group-heifers with RTS 5 received prostaglandin F2α (PGF; Dinoprost 25 mg; im) on Days 0 and 14; (2) HRTS-CIDR-PGF group-heifers with RTS 5 received a CIDR (1.3-g progesterone) insert on Day 7, followed by CIDR removal and PGF on Day 14; (3) LRTS-CIDR-PGF group-heifers with RTS 4 or less received a CIDR insert on Day 7, followed by CIDR removal and PGF on Day 14; and (4) HRTS-Select-Synch group-heifers with RTS 5 received 100 µg of gonadorelin diacetate tetrahydrate (gonadotropin releasing homone; im) on Day 7 and PGF on Day 14. In all groups, heifers observed in estrus were artificially inseminated (within 120 hours after PGF) using the AM-PM rule. In Experiment 1, estrus expression rates were 82.2% (282/343) and 88.5% (184/208) for HRTS-DPGF and LRTS-CIDR-PGF, respectively (P < 0.05), whereas AI-PR were 51.3% (176/343) and 59.1% (123/208; P < 0.1). In Experiment 2, estrus expression rates were 79.6 (168/211), 86.9 (186/214) and 84.2% (176/209) for HRTS-DPGF, HRTS-CIDR-PGF, and LRTS-CIDR-PGF groups (P > 0.1) and AI-PR were 52.1 (110/211), 60.3 (129/214), and 58.4% (122/209; P > 0.05). In Experiment 3, estrus expression rates were 77.5 (131/169), 85.5 (142/166), and 83.3% (219/263) for HRTS-DPGF, HRTS-Select-Synch and LRTS-CIDR-PGF (P > 0.05) and AI-PR were 53.3 (90/169), 60.2 (100/166), and 58.6% (154/263; P > 0.1). Overall, estrus expression rates for HRTS-DPGF, HRTS-Select-Synch, LRTS-CIDR-PGF, and HRTS-CIDR-PGF groups were 80.4 (581/723), 85.5 (142/166), 85.1 (579/680), and 86.9% (186/214), respectively; higher for heifers in LRTS-CIDR-PGF and HRTS-CIDR-PGF groups compared to heifers in HRTS-DPGF group (P < 0.05). The AI-PR for heifers in HRTS-DPGF was lower (52.0 [376/723]) compared with HRTS-Select-Synch (60.2 [100/166]), LRTS-CIDR-PGF (58.7 [399/680]), and HRTS-CIDR-PGF (60.3 [129/214]); P < 0.05). In conclusion, heifers achieved greater AI-PR after CIDR-PGF or HRTS-Select-Synch estrous synchronization protocols. Even though acceptable AI-PRs achieved in heifers with RTS 5 that were subjected to a double PGF protocol, the reproductive performance was reduced compared with other protocols used in this study.

Differential expression of microRNAs in sexually immature and mature canine testes.

Mammalian testis exhibits spatiotemporal gene expression patterns that are essential for successful and continuous spermatogenesis. Although microRNAs (miRNAs) modify gene expression through translational repression and degradation of target messenger RNAs, the precise molecular mechanisms of these regulatory processes are unclear. We used canine miScript miRNA polymerase chain reaction (PCR) Array technology to elucidate the repertoire of canine testis miRNAs and compared their expression patterns between sexually immature (prepubertal) and mature (adult) dog testes. Eighty-four well-characterized canine miRNAs were customized in this study. The data were analyzed by RT(2) Profiler PCR Array Data Analysis (version 3.5). Results identified upregulation of 32 and considerable downregulation of 12 miRNAs in adult dog testis. In conclusion, the two developmental stages had significantly different miRNAs expression patterns. The finding provides fundamental information of miRNAs which may help to elucidate their role in spermatogenesis and male infertility in this species. To the best of our knowledge, this study is the first to offer comparative profile of the miRNA transcriptome in prepubertal and adult canine testes using miRNA PCR array approach.

Dysregulated microRNA clusters in response to retinoic acid and CYP26B1 inhibitor induced testicular function in dogs.

Spermatogenesis is a multistep synchronized process. Diploid spermatogonia differentiate into haploid spermatozoa following mitosis, meiosis and spermiogenesis. Division and differentiation of male germ cells is achieved through the sequential expression of several genes. Numerous mRNAs in the differentiating germ cells undergo post-transcriptional and translational regulation. MiRNAs are powerful negative regulators of mRNA transcription, stability, and translation and recognize their mRNA targets through base-pairing. Retinoic acid (RA) signaling is essential for spermatogenesis and testicular function. Testicular RA level is critical for RA signal transduction. This study investigated the miRNAs modulation in an RA- induced testicular environment following the administration of all-trans RA (2 µM) and CYP26B1- inhibitor (1 µM) compared to control. Eighty four canine mature miRNAs were analyzed and their expression signatures were distinguished using real-time PCR based array technology. Of the miRNAs analyzed, miRNA families such as miR-200 (cfa-miR-200a, cfa-miR-200b and cfa-miR-200c), Mirlet-7 (cfa-let-7a, cfa-let-7b, cfa-let-7c, cfa-let-7g and cfa-let-7f), miR-125 (cfa-miR-125a and cfa-miR-125b), miR-146 (cfa-miR-146a and cfa-miR-146b), miR-34 (cfa-miR-34a, cfa-miR-34b and cfa-miR-34c), miR-23 (cfa-miR-23a and cfa-miR-23b), cfa-miR-184, cfa-miR-214 and cfa-miR-141 were significantly up-regulated with testicular RA intervention via administration of CYP26B1 inhibitor and all-trans-RA and species of miRNA such as cfa-miR-19a, cfa-miR-29b, cfa-miR-29c, cfa-miR-101 and cfa-miR-137 were significantly down-regulated. This study explored information regarding chromosome distribution, human orthologous sequences and the interaction of target genes of miRNA families significantly distinguished in this study using prediction algorithms. This study importantly identified dysregulated miRNA species resulting from RA-induced spermatogenesis. The present contribution serves as a useful resource for further elucidation of the regulatory role of individual miRNA in RA synchronized canine spermatogenesis.

Surveillance, response systems, and evidence updates on emerging zoonoses: the role of one health.

Globally, emerging zoonotic diseases are increasing. Existing surveillance systems for zoonoses have substantial gaps, especially in developing countries, and the systems in place in the developed world require improvements. Resources and updates on evidence-based practice (EBP) for zoonoses are sparser in the veterinary literature as compared to the medical literature. Evidence updates for emerging zoonoses are either absent or rudimentary in both human and veterinary medicine. A 'one-health' concept, including a global signaling surveillance system for emerging zoonoses, will be essential for correct diagnoses, interventions, and public health strategies. An open access EBP platform supported by builders of EBP resources is urgently needed to counter emerging zoonoses.

Associations among serum pro- and anti-inflammatory cytokines, metabolic mediators, body condition, and uterine disease in postpartum dairy cows.

BACKGROUND: Adipose tissue is an active endocrine organ which secretes a wide range of hormones and protein factors, collectively termed adipokines. Adipokines affect appetite and satiety, glucose and lipid metabolism, inflammation and immune functions. The objectives were to evaluate serum concentrations of adipokines (adiponectin, leptin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6) in lactating dairy cows with postpartum uterine inflammatory conditions (metritis, clinical endometritis or subclinical endometritis) and in cows experiencing loss of body condition, and to assess the relationship of adipokines and body condition loss in the establishment of persistent uterine inflammatory conditions. METHODS: Lactating multiparous Holstein cows (N = 40), with body condition scores (BCS) from 2 to 4 (eight cows for each 0.5 score increment) were enrolled. Body condition was monitored for all cows weekly for 7 weeks post calving; cows with uterine inflammatory conditions were also re-evaluated 2 weeks later. Blood samples were collected from 1 week prior to calving to 7 weeks after calving for determination of serum concentrations of adipokines, insulin and insulin like growth factor (IGF)-1. RESULTS: Cows with metritis or clinical endometritis had higher serum concentrations of adiponectin, leptin, TNF-alpha, IL-1beta and IL-6 compared to normal cows (P < 0.05). Furthermore, serum leptin, TNF-alpha, IL-1beta and IL-6 were higher in cows with subclinical endometritis compared to normal cows (P < 0.05), and insulin and IGF-1 concentrations were lower in cows with metritis or clinical endometritis. Cows with low BCS (2 and 2.5) had significantly higher adiponectin, TNF-alpha, IL-1beta and IL-6 than those with high BCS (3 to 4). Cows with persistent uterine inflammatory conditions had higher adiponectin, leptin TNF-alpha, IL-1beta and IL-6 and insulin compared to normal and spontaneously recovered cows, except for IGF-1 (P < 0.05). CONCLUSIONS: Serum concentrations of adipokines, insulin, and IGF-1 had significant associations with BCS categories (low vs. high) and postpartum uterine inflammatory conditions. Perhaps loss of body condition mediated increases in anti- and pro-inflammatory cytokines, whereas increased pro- and anti-inflammatory cytokines concentrations mediated body condition loss and thereby prolonged persistence of uterine inflammation in dairy cows.

Fertility in Angus cross beef cows following 5-day CO-Synch + CIDR or 7-day CO-Synch + CIDR estrus synchronization and timed artificial insemination.

The present study determined whether a 5-day CO-Synch + controlled internal drug release (CIDR) protocol with two doses of PGF2α would improve timed artificial insemination (AI) pregnancy rate compared with 7-day CO-Synch + CIDR protocol in beef cows. Angus cross beef cows (N = 1817) at 12 locations were randomly assigned to 5-day CO-Synch + CIDR or 7-day CO-Synch + CIDR groups. All cows received 100 µg of GnRH and a CIDR insert on Day 0. Cows (n = 911) in the 5-day CO-Synch + CIDR group received two doses of 25 mg PGF, the first dose given on Day 5 at CIDR removal and the second dose 6 hours later, and 100 µg GnRH on Day 8 and were inseminated concurrently, 72 hours after CIDR removal. Cows (n = 906) in 7-day CO-Synch + CIDR group received 25 mg of PGF at CIDR removal on Day 7, and 100 µg GnRH on Day 10 and were inseminated concurrently, 66 to 72 hours after CIDR removal. All cows were fitted with a heat detector aid at CIDR removal and were observed twice daily until insemination for estrus and heat detector aid status. Accounting for estrus expression at or before AI (P < 0.0001) and body condition score (P < 0.01), cows in the 5-day CO-Synch + CIDR group had greater AI pregnancy rate compared with cows in the 7-day CO-Synch + CIDR group (58.1% vs. 55.1%; P = 0.04). More cows that exhibited estrus at or before AI became pregnant compared with cows that did not [65.7% (681/1037) vs. 44.5% (347/780); P < 0.0001]. The AI pregnancy rate was lesser for cows with body condition ≤4 [≤4 - 49.3% (101/219), 5-6 - 57.9%; >6 - 55.8%]. The mean AI pregnancy rate difference between treatment groups and projected economic outcome varied among locations. In conclusion, cows synchronized with the 5-day CO-Synch + CIDR protocol had greater AI pregnancy rate than those that received the 7-day CO-Synch + CIDR protocol.

Prevention and treatment of biofilms by hybrid- and nanotechnologies.

Bacteria growing as adherent biofilms are difficult to treat and frequently develop resistance to antimicrobial agents. To counter biofilms, various approaches, including prevention of bacterial surface adherence, application of device applicators, and assimilation of antimicrobials in targeted drug delivery machinery, have been utilized. These methods are also combined to achieve synergistic bacterial killing. This review discusses various multimodal technologies, presents general concepts, and describes therapies relying on the principles of electrical energy, ultrasound, photodynamics, and targeted drug delivery for prevention and treatment of biofilms.