Metritis and the uterine disease microbiome are associated with long-term changes in the endometrium of dairy cows†.

Cows with metritis (uterine disease) during the first 1 to 2 wk postpartum have lower pregnancy rates when inseminated later postpartum (typically >10 wk). We hypothesized that metritis and the disease-associated uterine microbiome have a long-term effect on endometrial gene expression. Changes in gene expression may inform a mechanism through which disease lowers pregnancy rates. A total of 20 cows were enrolled at 1 to 2 wk postpartum to either metritis (clinical disease; n = 10) or healthy (control; n = 10) groups and randomly assigned to be slaughtered at approximately 80 d and 165 d postpartum (mid-lactation). The microbiome of the reproductive tract was sampled to confirm the presence of pathogens that are typical of metritis. In addition to the original clinical diagnosis, study cows were retrospectively assigned to uterine-disease and control groups based on the composition of their microbiome. There was no effect of early postpartum uterine disease on the uterine microbiome at mid-lactation (time of slaughter). Nonetheless, early postpartum metritis and the disease microbiome were associated with a large number of differentially-expressed genes at mid-lactation primarily in the caruncular compared with the inter-caruncular endometrium. Gene enrichment analysis identified oxidative phosphorylation as the primary pathway increased in caruncular endometrium of diseased cows whereas growth factor signaling pathways were reduced. The current study demonstrated that metritis and a uterine disease microbiome leave a sustained imprint on gene expression in the caruncular endometrium that may explain lower fertility in cows with postpartum uterine disease.

The microbiome of the pregnant uterus in Holstein dairy heifers and cows assessed by bacterial culture and 16S ribosomal RNA gene sequencing.

Introduction: The possibility that there is a resident and stable commensal microbiome within the pregnant uterus has been supported and refuted by a series of recent studies. One element of most of the initial studies was that they were based primarily on 16S rRNA gene sequencing from bacteria. To account for this limitation, the current study performed both bacterial culture and 16S rRNA gene sequencing in a side-by-side manner (e.g., same tissues isolated from the same animal). Methods: The uteruses of 10 mid-pregnant (156 ± 5 d of gestation) Holstein heifers and cows were collected following slaughter. The external surface of the reproductive tract (positive control for contamination during tissue collection) as well as tissues within the pregnant uterus (placentome, inter-cotyledonary placenta, inter-caruncular endometrium, amnionic fluid, allantoic fluid, fetal abomasum content, and fetal meconium) were sampled for bacterial culture and 16S rRNA gene sequencing. Results: There were 87 unique bacterial species cultured from the external surface of the pregnant reproductive tract (contamination control) and 12 bacterial species cultured from pregnancy tissues. Six out of 10 cattle (60%) exhibited bacterial growth in at least one location within the pregnant uterus. For the metataxonomic results (16S rRNA gene sequencing), a low targeted microbial biomass was identified. Analyses of the detected amplicon sequence variants (ASV) revealed that there were: (1) genera that were prevalent on both the external surface and within the pregnant uterus; (2) genera that were prevalent on the external surface but either not detected or had very low prevalence within the pregnant uterus; and (3) genera that were either not detected or had low prevalence on the external surface but found with relatively high prevalence within the pregnant uterus. Conclusion: There are a small number of viable bacteria in the pregnant uterus. The 16S rRNA gene sequencing detected a microbial community within the pregnant uterus but with a low biomass. These results are consistent with recent studies of the pregnant bovine uterus and leave open the question of whether there is adequate microbial mass to significantly affect the biology of the normal healthy bovine pregnancy.

Sexually Dimorphic Transcriptomic Changes of Developing Fetal Brain Reveal Signaling Pathways and Marker Genes of Brain Cells in Domestic Pigs.

In this study, transcriptomic changes of the developing brain of pig fetuses of both sexes were investigated on gestation days (GD) 45, 60 and 90. Pig fetal brain grows rapidly around GD60. Consequently, gene expression of the fetal brain was distinctly different on GD90 compared to that of GD45 and GD60. In addition, varying numbers of differentially expressed genes (DEGs) were identified in the male brain compared to the female brain during development. The sex of adjacent fetuses also influenced gene expression of the fetal brain. Extensive changes in gene expression at the exon-level were observed during brain development. Pathway enrichment analysis showed that the ionotropic glutamate receptor pathway and p53 pathway were enriched in the female brain, whereas specific receptor-mediated signaling pathways were enriched in the male brain. Marker genes of neurons and astrocytes were significantly differentially expressed between male and female brains during development. Furthermore, comparative analysis of gene expression patterns between fetal brain and placenta suggested that genes related to ion transportation may play a key role in the regulation of the brain-placental axis in pig. Collectively, the study suggests potential application of pig models to better understand influence of fetal sex on brain development.

Identification of Loci and Pathways Associated with Heifer Conception Rate in U.S. Holsteins.

Heifer conception rate (HCR) is defined as the percentage of inseminated heifers that become pregnant at each service. The genome-wide association analyses in this study focused on identifying the loci associated with Holstein heifer (n = 2013) conception rate at first service (HCR1) and the number of times bred (TBRD) to achieve a pregnancy. There were 348 unique loci associated (p < 5 × 10-8) with HCR1 and 615 unique loci associated (p < 5 × 10-8) with TBRD. The two phenotypes shared 302 loci, and 56 loci were validated in independent cattle populations. There were 52 transcription factor binding sites (TFBS) and 552 positional candidate genes identified in the HCR1- and TBRD-associated loci. The positional candidate genes and the TFBS associated with HCR1 and TBRD were used in the ingenuity pathway analysis (IPA). In the IPA, 11 pathways, 207 master regulators and 11 upstream regulators were associated (p < 1.23 × 10-5) with HCR1 and TBRD. The validated loci associated with both HCR1 and TBRD make good candidates for genomic selection and further investigations to elucidate the mechanisms associated with subfertility and infertility.

Analysis of the uterine lumen in fertility-classified heifers: II. Proteins and metabolites†.

Survival and growth of the bovine conceptus is dependent on endometrial secretions or histotroph. Previously, serial blastocyst transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components (proteins and metabolites) in the uterine lumen of day 17 fertility-classified heifers. Interferon tau (IFNT) was more abundant in uterine lumenal fluid (ULF) of pregnant HF than SF animals as the conceptus was longer in HF heifers. However, no differences in endometrial expression of selected classical and nonclassical interferon-stimulated genes (ISGs) were observed, suggesting that IFNT signaling in the endometrium of pregnant HF and SF heifers was similar. Pregnancy significantly increased the abundance of several proteins in ULF. Based on functional annotation, the abundance of a number of proteins involved in energy metabolism, oxidative stress, amino acid metabolism, and cell proliferation and differentiation were greater in the ULF of pregnant HF than SF heifers. Metabolomics analysis found that pregnancy only changed the metabolome composition of ULF from HF heifers. The majority of the metabolites that increased in the ULF of pregnant HF as compared to SF heifers were associated with energy and amino acid metabolism. The observed differences in ULF proteome and metabolome are hypothesized to influence uterine receptivity with consequences on conceptus development and survival in fertility-classified heifers.

Analysis of the uterine lumen in fertility-classified heifers: I. Glucose, prostaglandins, and lipids†.

Survival and growth of the bovine conceptus (embryo and associated extraembryonic membranes) are dependent on endometrial secretions or histotroph found in the uterine lumen. Previously, serial embryo transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components [glucose, prostaglandins (PGs), and lipids] in the uterine lumen of day 17 pregnant and open fertility-classified heifers. Concentrations of glucose in the uterine lumen were increased by pregnancy but did not differ among fertility-classified heifers. Differences in expression of genes encoding glucose transporters and involved with glycolysis and gluconeogenesis were observed between conceptuses collected from HF and SF heifers. In the uterine lumen, PGE2 and PGF2α were increased by pregnancy, and HF heifers had higher concentrations of PGE2, PGF2α, and 6-keto-PFG1α than SF heifers. Differences were found in expression of genes regulating PG signaling, arachidonic acid metabolism, and peroxisome proliferator-activated receptor signaling among conceptuses and endometrium from fertility-classified heifers. Lipidomics was conducted exclusively in samples from HF heifers, and phosphatidylcholine was the main lipid class that increased in the uterine lumen by pregnancy. Expression of several lipid metabolism genes differed between HF and SF conceptuses, and a number of fatty acids were differentially abundant in the uterine lumen of pregnant HF and SF heifers. These results support the ideas that uterine luminal histotroph impacts conceptus survival and programs its development and is a facet of dysregulated conceptus-endometrial interactions that result in loss of the conceptus in SF cattle during the implantation period of pregnancy establishment.

Physiological, health, lactation, and reproductive traits of cooled dairy cows classified as having high or low core body temperature during the dry period1.

Primary objectives of this study were to compare concentrations of pregnancy-associated glycoprotein (PAG) before calving, prolactin (PRL) after calving, and energy balance indicators before and after calving in cooled cows classified as having high (HT) or low (LT) core body temperature (CBT) during the dry period. Secondary objectives were to investigate associations between dry-period CBT and likelihood of cows developing health disorders, and compare health, productive and reproductive traits of HT and LT cows. Dry Holstein cows (n = 260) with 250 to 260 d of gestation from three herds were enrolled in the study during summer. Cows were provided evaporative cooling during the dry and lactating period. The vaginal temperature was recorded in 5-min intervals during 7 consecutive days and cows were classified as HT or LT. Blood samples were collected weekly from enrollment until 14 ± 3 d in milk (DIM). Additional blood samples were collected within 12 h postpartum from a subgroup of cows (n = 25) to determine PRL concentration. Cows were monitored for health disorders, productive, and reproductive performance until 13 wk of the subsequent lactation. High temperature cows had shorter (P < 0.01) gestation length (273.9 ± 0.9 vs. 278.2 ± 0.9 d) and greater (P < 0.01) incidence of twinning (19.7 vs. 4.2%) than LT cows. Cows classified as HT had greater (P = 0.02) PAG concentration (134.1 ± 4.9 vs. 117.4 ± 4.9 ng/mL), but postpartum PRL concentration did not (P = 0.55) differ between HT and LT cows. Primiparous HT cows had greater (P = 0.05) prepartum nonesterified fatty acids concentration (135, 95% CI = 102 to 178 vs. 104, 95% CI = 75 to 144 mmol/dL) than primiparous LT cows, but no differences (P = 0.72) were observed between CBT group in multiparous cows. The concentration of ß-hydroxybutyrate was greater (P = 0.04) for LT compared with HT cows at 7 ± 3 DIM. The quadratic effect of CBT tended (P = 0.09) to be associated with risk of health disorders within 60 DIM. Milk yield tended (P = 0.10) to be greater for LT compared with HT cows (49.3 ± 1.9 vs. 46.2 ± 1.6 kg). Pregnancy per AI at first service did not (P = 0.64) differ between HT and LT cows. In conclusion, HT cows have distinct concentrations of PAG in late gestation and energy balance indicators during the transition period. In addition, CBT assessment during the dry period may be a useful tool to identify cows expected to have impaired health and milk yield in the subsequent lactation.

Genomic Analysis of Spontaneous Abortion in Holstein Heifers and Primiparous Cows.

BACKGROUND: The objectives of this study were to identify loci, positional candidate genes, gene-sets, and pathways associated with spontaneous abortion (SA) in cattle and compare these results with previous human SA studies to determine if cattle are a good SA model for humans. Pregnancy was determined at gestation day 35 for Holstein heifers and cows. Genotypes from 43,984 SNPs of 499 pregnant heifers and 498 pregnant cows that calved at full term (FT) were compared to 62 heifers and 28 cows experiencing SA. A genome-wide association analysis, gene-set enrichment analysis-single nucleotide polymorphism, and ingenuity pathway analysis were used to identify regions, pathways, and master regulators associated with SA in heifers, cows, and a combined population. RESULTS: Twenty-three loci and 21 positional candidate genes were associated (p < 1 × 10-5) with SA and one of these (KIR3DS1) has been associated with SA in humans. Eight gene-sets (NES > 3.0) were enriched in SA and one was previously reported as enriched in human SA. Four master regulators (p < 0.01) were associated with SA within two populations. CONCLUSIONS: One locus associated with SA was validated and 39 positional candidate and leading-edge genes and 2 gene-sets were enriched in SA in cattle and in humans.

Identification of loci associated with conception rate in primiparous Holstein cows.

BACKGROUND: Subfertility is a major issue facing the dairy industry as the average US Holstein cow conception rate (CCR) is approximately 35%. The genetics underlying the physiological processes responsible for CCR, the proportion of cows able to conceive and maintain a pregnancy at each breeding, are not well characterized. The objectives of this study were to identify loci, positional candidate genes, and transcription factor binding sites (TFBS) associated with CCR and determine if there was a genetic correlation between CCR and milk production in primiparous Holstein cows. Cows were bred via artificial insemination (AI) at either observed estrus or timed AI and pregnancy status was determined at day 35 post-insemination. Additive, dominant, and recessive efficient mixed model association expedited (EMMAX) models were used in two genome-wide association analyses (GWAA). One GWAA focused on CCR at first service (CCR1) comparing cows that conceived and maintained pregnancy to day 35 after the first AI (n = 494) to those that were open after the first AI (n = 538). The second GWAA investigated loci associated with the number of times bred (TBRD) required for conception in cows that either conceived after the first AI (n = 494) or repeated services (n = 472). RESULTS: The CCR1 GWAA identified 123, 198, and 76 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. The TBRD GWAA identified 66, 95, and 33 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. Four of the top five loci were shared in CCR1 and TBRD for each GWAA model. Many of the associated loci harbored positional candidate genes and TFBS with putative functional relevance to fertility. Thirty-six of the loci were validated in previous GWAA studies across multiple breeds. None of the CCR1 or TBRD associated loci were associated with milk production, nor was their significance with phenotypic and genetic correlations to 305-day milk production. CONCLUSIONS: The identification and validation of loci, positional candidate genes, and TFBS associated with CCR1 and TBRD can be utilized to improve, and further characterize the processes involved in cattle fertility.

Validation of 46 loci associated with female fertility traits in cattle.

BACKGROUND: Subfertility is one challenge facing the dairy industry as the average Holstein heifer conception rate (HCR), the proportion of heifers that conceive and maintain a pregnancy per breeding, is estimated at 55-60%. Of the loci associated with HCR, few have been validated in an independent cattle population, limiting their usefulness for selection or furthering our understanding of the mechanisms involved in successful pregnancy. Therefore, the objectives here were to identify loci associated with HCR: 1) to the first artificial insemination (AI) service (HCR1), 2) to repeated AI services required for a heifer to conceive (TBRD) and 3) to validate loci previously associated with fertility. Breeding and health records from 3359 Holstein heifers were obtained after heifers were bred by AI at observed estrus, with pregnancy determined at day 35 via palpation. Heifer DNA was genotyped using the Illumina BovineHD BeadChip, and genome-wide association analyses (GWAA) were performed with additive, dominant and recessive models using the Efficient Mixed Model Association eXpedited (EMMAX) method with a relationship matrix for two phenotypes. The HCR1 GWAA compared heifers that were pregnant after the first AI service (n = 497) to heifers that were open following the first AI service (n = 405), which included those that never conceived. The TBRD GWAA compared only those heifers which did conceive, across variable numbers of AI service (n = 712). Comparison of loci previously associated with fertility, HCR1 or TBRD were considered the same locus for validation when in linkage disequilibrium (D' > 0.7). RESULTS: The HCR1 GWAA identified 116, 187 and 28 loci associated (P < 5 × 10- 8) in additive, dominant and recessive models, respectively. The TBRD GWAA identified 235, 362, and 69 QTL associated (P < 5 × 10- 8) with additive, dominant and recessive models, respectively. Loci previously associated with fertility were in linkage disequilibrium with 22 loci shared with HCR1 and TBRD, 5 HCR1 and 19 TBRD loci. CONCLUSIONS: Loci associated with HCR1 and TBRD that have been identified and validated can be used to improve HCR through genomic selection, and to better understand possible mechanisms associated with subfertility.

Influences of sire conception rate on pregnancy establishment in dairy cattle.

Establishment of pregnancy in cattle is complex and encompasses ovulation, fertilization, blastocyst formation and growth into an elongated conceptus, pregnancy recognition signaling, and development of the embryo and placenta. The objective here was to investigate sire influences on pregnancy establishment in cattle. First, 10 Holstein bulls were classified as high or low fertility based on their sire conception rate (SCR) value. In a field trial, pregnancy at first timed insemination was not different between high and low SCR bulls. Next, 5 of the 10 sires were phenotyped using in vitro and in vivo embryo production. There was no effect of SCR classification on in vitro embryo cleavage rate, but low SCR sires produced fewer day 8 blastocysts. In superovulated heifers, high SCR bulls produced a lower percentage of unfertilized oocytes and fewer degenerated embryos compared to low SCR bulls. Recipient heifers received three to five in vivo produced embryos from either high or low SCR sires on day 7 postestrus. Day 16 conceptus recovery and length were not different between SCR groups, and the conceptus transcriptome was not appreciably different between high and low SCR sires. The reduced ability of embryos from low SCR bulls to establish pregnancy is multifactorial and encompasses sperm fertilizing ability, preimplantation embryonic development, and development of the embryo and placenta after conceptus elongation and pregnancy recognition. These studies highlight the importance of understanding genetic contributions of the sire to pregnancy establishment that is crucial to increase reproductive efficiency in dairy cattle.

Uterine influences on conceptus development in fertility-classified animals.

A major unresolved issue is how the uterus influences infertility and subfertility in cattle. Serial embryo transfer was previously used to classify heifers as high-fertile (HF), subfertile (SF), or infertile (IF). To assess pregnancy loss, two in vivo-produced embryos were transferred into HF, SF, and IF heifers on day 7, and pregnancy outcome was assessed on day 17. Pregnancy rate was substantially higher in HF (71%) and SF (90%) than IF (20%) heifers. Elongating conceptuses were about twofold longer in HF than SF heifers. Transcriptional profiling detected relatively few differences in the endometrium of nonpregnant HF, SF, and IF heifers. In contrast, there was a substantial difference in the transcriptome response of the endometrium to pregnancy between HF and SF heifers. Considerable deficiencies in pregnancy-dependent biological pathways associated with extracellular matrix structure and organization as well as cell adhesion were found in the endometrium of SF animals. Distinct gene expression differences were also observed in conceptuses from HF and SF animals, with many of the genes decreased in SF conceptuses known to be embryonic lethal in mice due to defects in embryo and/or placental development. Analyses of biological pathways, key players, and ligand-receptor interactions based on transcriptome data divulged substantial evidence for dysregulation of conceptus-endometrial interactions in SF animals. These results support the ideas that the uterus impacts conceptus survival and programs conceptus development, and ripple effects of dysregulated conceptus-endometrial interactions elicit loss of the postelongation conceptus in SF cattle during the implantation period of pregnancy.

Effects of intrauterine infusion of Escherichia coli lipopolysaccharide on uterine mRNA gene expression and peripheral polymorphonuclear leukocytes in Jersey cows diagnosed with purulent vaginal discharge.

The objectives of the current experiment were to investigate the effects of intrauterine treatment of cows with purulent vaginal discharge (PVD) using lipopolysaccharide (LPS) from Escherichia coli on uterine mRNA expression of genes related to inflammatory responses, peripheral polymorphonuclear leukocyte (PMN) function, hematological parameters, and blood concentrations of cortisol, haptoglobin, and progesterone (P4). Jersey cows (n = 3,084) were examined for PVD at 35 ± 6 d postpartum using the Metricheck device (Simcro, Hamilton, New Zealand). At examination, 310 cows had PVD (10.1%), but to ensure proper collection and processing of samples, 267 cows were used in this experiment. Cows were balanced for lactation number, body condition score, and milk yield, and randomly assigned to the control treatment [intrauterine infusion of 20 mL of phosphate-buffered saline (PBS); n = 87] or to receive intrauterine infusion of 20 mL of PBS containing 150 µg (LPS150; n = 91) or 300 µg (LPS300; n = 89) of E. coli LPS. Uteri were biopsied in a subgroup of cows at 6 h after infusion and in another subgroup of cows at 24 h after infusion. Peripheral PMN expression of adhesion molecules (L-selectin and MAC-1) and phagocytosis and oxidative burst were evaluated at 0, 2, and 6 h after infusion. Blood sampled 0, 2, 6, 24, 48, and 168 h after infusion was used for complete hemogram and to determine concentrations of cortisol, haptoglobin, and P4. Treatment did not affect uterine mRNA expression of adhesion molecules [endothelial leukocyte adhesion molecule (E-selectin), intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)], cytokines (tumor necrosis factor-α, IL-1ß, IL-6, IL-8, and IL-10), and toll-like receptor-4. Treatment did not affect PMN expression of L-selectin, but intensity of expression of MAC-1 was higher for LPS150 cows than PBS cows, and tended to be higher in LPS150 than LPS300 cows. Furthermore, a greater percentage of PMN from LPS300 cows were positive for phagocytosis and oxidative burst compared with PBS and LPS150 cows. No effects were observed of treatment on hematological parameters and concentrations of cortisol, haptoglobin, and P4. These observations suggest that intrauterine infusion of E. coli LPS moderately stimulates peripheral PMN function, but further research is needed to better understand the immunomodulatory effects of LPS in the uterus of cows with PVD.

Effects of intrauterine infusion of Escherichia coli lipopolysaccharide on uterine health, resolution of purulent vaginal discharge, and reproductive performance of lactating dairy cows.

The objectives of the current experiment were to evaluate the effects of intrauterine infusion of Escherichia coli lipopolysaccharide (LPS) in cows diagnosed with purulent vaginal discharge (PVD) on intrauterine cell population, resolution of PVD, uterine health, and reproductive performance. Jersey cows (n = 3,084) were examined using the Metricheck device to diagnose PVD at 35 ± 6 d postpartum. Purulent vaginal discharge was defined as the presence of purulent (≥50% pus) discharge detectable in the vagina. Of the 310 cows positive for PVD, 267 cows were enrolled in the current experiment. To ensure proper timing of treatment and collection of samples, only 9 PVD-positive cows were treated per day. Selected cows were balanced at 35 ± 6 d postpartum for lactation number, body condition score, and milk yield and were randomly assigned to receive an intrauterine infusion of 20 mL of phosphate-buffered saline (PBS; control, n = 87), 20 mL of PBS with 150 µg LPS (LPS150, n = 91), or 20 mL of PBS with 300 µg of LPS (LPS300, n = 89). Uterine cytology was performed immediately before treatment and 1, 2, and 7 d after treatment to evaluate the effect of LPS treatment on intrauterine cell population. Cows were examined with the Metricheck device at 7 and 28 d after treatment to evaluate the effects of treatment on resolution of PVD. Reproductive status was recorded up to 200 d postpartum. Cows diagnosed with PVD had greater incidence of twinning, dystocia, retained placenta, and metritis after calving than cows without PVD. Count of polymorphonuclear leukocytes (PMNL) in uterine cytology 1, 2, and 7 d after intrauterine infusion was not statistically different among treatments. From d 0 to 1, however, PMNL count in uterine cytology of PBS cows increased by 5%, whereas the PMNL count in uterine cytology of LPS150 and LPS300 cows increased by 54 and 48%, respectively. Treatment did not affect the likelihood of cows being diagnosed with PVD 7 and 28 d after intrauterine infusion. Cows without PVD and LPS150 cows were more likely to be pregnant after the first postpartum AI than PBS cows. After the second postpartum AI, cows without PVD were more likely to be pregnant than PBS and LPS300 cows. Hazard of pregnancy up to 200 d postpartum was decreased for PBS and LPS300 cows compared with cows without PVD, and it tended to be decreased for LPS150 cows compared with cows without PVD. Intrauterine treatment with 150 µg of E. coli LPS of cows diagnosed with PVD improved likelihood of pregnancy after the first postpartum AI, but further research is needed to elucidate the mechanism by which LPS treatment improved fertility.

Analysis of the Uterine Epithelial and Conceptus Transcriptome and Luminal Fluid Proteome During the Peri-Implantation Period of Pregnancy in Sheep.

Studies support the idea that uterine epithelia and their secretions have important biological roles in conceptus survival, elongation, and implantation in sheep. The present study evaluated the transcriptome of the uterine luminal epithelium (LE) and glandular epithelium (GE) and the conceptus and proteome of uterine luminal fluid (ULF) during the peri-implantation period of pregnancy. Transcriptome (RNA-sequencing) analysis was conducted in LE and GE isolated from uteri of Day 10, 12, 14, 16, and 20 pregnant sheep by laser capture microdissection. In the LE, the total number of expressed genes increased between Days 10 and 20, whereas expressed genes in the GE increased from Days 10 to 14 and then decreased to Day 20. Most of the expressed genes in LE and GE from Days 10 to 14 are involved in cell survival and growth, whereas genes involved in cell organization and protein synthesis were most abundant on Days 16 and 20. Total expressed genes in the conceptus was greatest on Day 12, decreased to Day 16, and then increased to Day 20. Genes abundantly expressed in the elongating conceptus included IFNT, PTGS2, MGST1, FADS1, and FADS2, whereas SERPINA1, CSH1, and PLET1 were most abundant in the Day 20 conceptus. Proteins, identified by mass spectrometry, increased in the ULF from Days 10 to 16 and are involved in cellular reorganization or are proteases or chaperone proteins. These results support the idea that conceptus elongation and implantation is regulated by both extrinsic and intrinsic factors. This study provides critical information that serves as a foundation to discover new regulatory pathways governing uterine receptivity, conceptus elongation, trophectoderm differentiation, conceptus-endometrial interactions, and pregnancy establishment in ruminants.

Identification of Beef Heifers with Superior Uterine Capacity for Pregnancy.

Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In study one, predominantly Angus heifers were classified based on fertility using serial embryo transfer to select animals with intrinsic differences in pregnancy loss. In each of the four rounds, a single in vitro-produced, high-quality embryo was transferred into heifers on Day 7 postestrus and pregnancy was determined on Days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In study two, fertility-classified heifers were resynchronized and bred with semen from a single high-fertility bull. Blood samples were collected every other day from Days 0 to 36 postmating. Pregnancy rate was determined on Day 28 by ultrasound and was higher in HF (70.4%) than in heifers with low fertility (36.8%; SF and IF). Progesterone concentrations in serum during the first 20 days postestrus were not different in nonpregnant heifers and also not different in pregnant heifers among fertility groups. In study three, a single in vivo-produced embryo was transferred into fertility-classified heifers on Day 7 postestrus. The uteri were flushed on Day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the Day 14 endometrial biopsies of pregnant HF, SF, and IF heifers (n = 5 per group) and analyzed by edgeR-robust analysis. There were 26 differentially expressed genes (DEGs) in the HF compared to SF endometrium, 12 DEGs for SF compared to IF endometrium, and three DEGs between the HF and IF endometrium. Several of the DEG-encoded proteins are involved in immune responses and are expressed in B cells. Results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between Days 14 and 28 when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy.

Short communication: Plasma progesterone concentration and ovarian dynamics of lactating Jersey cows treated with 1 or 2 intravaginal progesterone inserts.

The objectives of the current experiment were to determine circulating progesterone concentrations and ovarian follicle development of lactating Jersey cows treated with 1 or 2 controlled internal drug release (CIDR) insert containing 1.38 g of progesterone during proestrus. Cows were enrolled in the experiment at 34 ± 3 d in milk and were paired by parity, body condition score, body weight, and milk yield. Estrous cycles were presynchronized with an injection of GnRH concurrent with a new CIDR insert (study d -7) and 2 injections of PGF2α given 5 and 6 d after the GnRH injection (study d -2 and -1, respectively). Cows assigned to the 1CIDR treatment (n=30) or 2CIDR treatment (n=30) received 1 and 2 CIDR inserts, respectively, from study d 0 through 7. Control cows (n=10) did not receive further treatment. On study d -2 and daily from study d 0 through 7, ovaries were examined by transrectal ultrasound and blood samples were collected for determination of progesterone. On study d 7, CIDR inserts were removed after ultrasound exam and blood sample collection. Progesterone concentration from study d 0 through 7 was greatest for 2CIDR cows (2.17 ± 0.09 ng/mL), followed by 1CIDR cows (1.37 ± 0.10 ng/mL) and control cows (0.62 ± 0.21 ng/mL). The interaction between treatment and study day affected progesterone concentration from study d 0 through 7. The average increase in progesterone concentration from study d 1 through 7 was 0.80 ng/mL for 1CIDR and 1.72 ng/mL for 2CIDR cows compared with control cows. The percentage of cows that ovulated between study d 0 and 7 was greatest for control cows (80%), but it did not differ between 1CIDR (12%) and 2CIDR (3.7%) cows. Growth of class III follicles (10-17 mm) identified on study d 0 was affected by treatment because 1CIDR cows had larger class III follicles than 2CIDR cows on study d 5, 6 and 7. A larger proportion of control cows developed a new follicular wave between study d 0 and 7 (control=60.0%, 1CIDR=12.0%, 2CIDR=7.4%). Treatment of lactating Jersey cows with 1 and 2 CIDR inserts resulted in a 1 and 2 fold increase, respectively, in circulating progesterone concentration. Growth class III follicles from cows treated with 2 CIDR inserts was reduced compared with untreated cows and cows treated with 1 CIDR insert.