Within 2 wk postpartum approximately 20% of dairy cows develop metritis, a uterine infection associated with reduced fertility and increased risk of reproductive culling. Antibiotics improve clinical cure and milk production following metritis but do not consistently increase subsequent fertility postpartum compared with untreated control cows. To better understand why antibiotic treatment improves clinical recovery but not fertility, we tracked changes over time in blood leukocyte gene expression to examine circulating immune cell function during the resolution of metritis in antibiotic-treated and untreated cows. Twenty-three primiparous Holstein cows diagnosed with metritis (n = 11) or classified as healthy controls (n = 12) between 7 and 10 d postpartum were enrolled. Cows were assigned to receive systemic ceftiofur (1.25 g/d for 3 d) or remain untreated in a 2 × 2 factorial design. Blood samples were collected at diagnosis (wk 1), and at 1 and 3 wk after treatment (wk 2 and wk 4) postpartum. The blood leukocyte RNA was isolated and sequenced, and differentially expressed genes were analyzed for functional enrichment and immune cell type. Healthy cows exhibited enrichment of B cell activation and receptor signaling pathways, whereas metritis cows showed upregulation of inflammatory and innate immune responses, including monocyte and neutrophil markers. In untreated cows (healthy versus metritis), transcriptomic differences in blood leukocytes were resolved by wk 4 postpartum. In contrast, antibiotic-treated metritis cows displayed a distinct immune trajectory. At wk 2 postpartum (1 wk after antibiotic treatment), cytotoxic T cell and natural killer cell gene expression signatures increased, differing clearly from untreated metritis cows. By wk 4, a second wave of inflammatory cell-associated differentially expressed genes emerged exclusively in antibiotic-treated metritis cows, suggesting prolonged immune cell activation. These findings demonstrate that antibiotic treatment reshapes systemic immune dynamics during recovery from metritis. The altered sequence (e.g., early cytotoxic activation followed by extended inflammation) may explain why antibiotic-induced clinical cure in metritis cows does not consistently translate into improved fertility.

Abstract Postpartum fertility complications in lactating dairy cows challenge the economic value of most dairy herds, restricting the industry’s efforts to extend herd survivability. Impaired fertility is strongly associated with high prevalence of postpartum uterine disease, resulting from invasion of pathogenic bacteria into the uterus during parturition. Sustained inflammation hinders the reestablishment of the uterine glandular epithelium (GE) by suppressing cellular proliferation at the base of the endometrium (stratum basalis), resulting in incomplete epithelial repair. Previously, our group identified an epithelial population at the basalis endometrium of the adult bovine uterus that exhibits unique stem/progenitor transcriptional signatures. These findings led us to hypothesize that decreased fertility may be attributed to the effect of postpartum uterine disease on a population of long-lived epithelial stem/progenitor cells (eS/P), which become established at the basalis endometrium postnatally, persist into adulthood, and play a key role in coordinating epithelial regeneration following parturition. To test this, we combined spatial transcriptomic datasets generated from bovine uterine cross-sections collected during critical developmental and regenerative states (n = 2 per time point), including 1) onset of postnatal GE branching morphogenesis [postnatal day (PND) 28], 2) completion of postnatal adenogenesis (gland development; PND 56) and 3) completion of postpartum repair [postpartum day (PPD) 30]. Here, we characterized the signatures of stem cell activity unique to basalis GE throughout development and repair, to elucidate the identity of this putative eS/P cell population. Integrative UMAP analysis revealed two GE clusters consistently mapped to the basalis endometrium of postnatal and postpartum uteri, which expressed N-cadherin (CDH2), a marker associated with human endometrial eS/P cells. The CDH2+ clusters displayed transcriptional hallmarks of eS/P activity, including retinoic acid synthesis (CDH2+ALDH1A1+) and canonical Wnt signaling (CDH2+RSPO3+). Differential expression analysis showed dynamic shifts in cell activity between the two CDH2+ populations across time points, including enrichment for proliferation markers at PND28 and PPD30 within the CDH2+ALDH1A1+ population, consistent with a state of eS/P activation. Conversely, at PND56, signatures of cell cycle arrest and telomere maintenance suggested a predominantly quiescent state, indicative of a regenerative program that may alternate between activation and dormancy in the adult endometrium. Using flow cytometry, we quantified CDH2+ and ALDHbright (ALDH activity by Aldefluor Assay) endometrial epithelial cells (EPCAM+) at the designated time points. We observed a significant decrease in the CDH2+ and ALDHbright epithelia from branching morphogenesis (PND28, CDH2+ = 59.7%; ALDHbright = 3.6%) to completion of adenogenesis (PND56, CDH2 + = 2.86%; ALDHbright = 1.25%). Notably, during postpartum GE regeneration (PPD 30), CDH2+ and ALDH bright epithelia expanded comprising 65.5% and 5.25% of the epithelial population, respectively. In summary, these data support the existence of a long-lived, putative stem/progenitor population within the GE of the bovine endometrium that is established postnatally and maintained throughout adulthood.

The hypothesis was that early postpartum uterine disease would reduce the developmental capacity of oocytes thus contributing to the reduced fertility of dairy cows with uterine disease. Dairy cows were diagnosed healthy or with metritis at 7-10 d postpartum. The reproductive tract was collected at approximately 1 mo (Exp. 1) or approximately 80 or 165 d (Exp. 2) postpartum for the collection of cumulus-oocyte complexes (COC). The COC were matured, co-incubated with sperm for fertilization, and cultured to the blastocyst stage (8 d) in vitro. For Exp.1, the disease diagnosis (healthy or metritis) did not affect the number of collected COC or the subsequent embryo development to the blastocyst stage. The presence of purulent material in the uterine lumen (endometritis) at time of oocyte collection, however, was associated with a reduced cleavage rate evaluated 3 d following fertilization. For Exp. 2, there was no effect of disease diagnosis (healthy or metritis) on the number of COC or their subsequent development. Reduced cleavage rates were observed in COC retrieved from cows slaughtered at 80 d postpartum, but not at 165 d postpartum, and this reduction was associated with a vaginal microbiome indicative of uterine disease at 4-5 wk postpartum. Regression analyses that included plasma haptoglobin or energy metabolite concentrations or uterine bacterial genera abundance did not explain a large percentage of the variation in oocyte development in vitro. We conclude that there is an effect of uterine disease at one month postpartum on the oocyte and its capacity for development (Exp. 1) and this effect may be present at 80 d postpartum (Exp. 2). In later postpartum cows (165 d postpartum; Exp. 2) there was no effect of uterine disease on in vitro oocyte development.

Optimal fertility after calving in lactating dairy cows is dependent upon the successful completion of uterine involution. Invasion of pathogenic bacteria into the uterine environment within the first week postpartum can lead to uterine disease (metritis). Metritis is associated with decreased fertility and a failure or delay in establishing pregnancy. We hypothesized that early postpartum metritis would be associated with long-term changes in uterine morphology that begin within the first 30 d postpartum (DPP) and are present during a typical breeding window (2-6 mo postpartum). First parity Holstein cows were diagnosed with metritis (M) or deemed healthy (H) at 7 to 10 DPP and uterine tissues were collected and analyzed postmortem at 30 (experiment [Exp.] 1; M = 10, H = 10), or 80 and 165 (Exp. 2; M = 9, H = 10) DPP for the presence of abnormal morphology, including abnormal invasion of endometrial glands and stroma into the myometrium (adenomyosis) and endometrial fibrosis. Glands were identified using immunohistochemistry for FOXA2 (uterine gland specific marker) and fibrosis was identified using Masson's trichrome stain (MTS). The severity of adenomyosis was assessed by the number and size of adenomyotic foci and the distance of foci from the endometrium-myometrium interface (EMI). The degree of fibrosis was defined by MTS intensity. Data were analyzed using a 2-way ANOVA procedure including the effect of metritis and DPP on the dependent variables foci size, distance from EMI, and MTS intensity. A negative binomial regression model was used for the dependent variable of foci number. The presence, size, and distance from the EMI of adenomyotic foci were greater for later postpartum (Exp. 2, 80 and 165 DPP) and early postpartum cows (Exp. 1, 30 DPP) that were previously diagnosed with metritis, suggesting greater severity of adenomyosis. Endometrial fibrosis was greater at the stratum basalis (near EMI) compared with the stratum compactum endometrium (near uterine lumen) for all Exp. 2 (80 and 165 DPP) cows. Greater fibrosis (regardless of endometrial region) was observed in cows diagnosed with metritis compared with healthy controls. Taken together, these data indicate that early postpartum metritis is associated with long-term changes to postpartum uterine morphology, including increased pathological fibrogenesis, leading to the presence of late postpartum endometrial fibrosis (scar tissue), and aberrant endometrial invasion into the myometrium (adenomyosis). Additionally, increased collagen fiber at the EMI suggests a correlation between the development of adenomyosis and fibrosis, which could result from sustained endometrial inflammation caused by uterine disease.

An experiment was designed to evaluate modifications to the 7 & 7 Synch protocol, with the hypothesis that tandem administration of prostaglandin F2α (PG) and gonadotropin-releasing hormone (GnRH) during the presynchronization portion of the protocol would generate a convergent ovarian response among anestrous and estrous cycling females. Nulliparous, primiparous, and multiparous Angus and Angus-cross females were blocked based on parity and pre-treatment luteal status and, within a block, randomly assigned to one of three treatments. The females assigned to the 7 & 7 Synch treatment (n = 765) received an intravaginal progesterone-releasing insert (CIDR®) and an administration of PG on Day 0, GnRH on Day 7, and PG concurrent with CIDR removal on Day 14. Estrotect™ estrus detection aids were applied on Day 14 at the time of CIDR removal and PG administration. The females assigned to the 7 & 7 + G treatment (n = 769) received the same treatment schedule, with the modification that GnRH was administered in tandem with PG on Day 0. The females assigned to the 7 & 7 + G + P treatment (n = 786) received the same treatment schedule, with the modification that GnRH was administered in tandem with PG on both Days 0 and 7. Fixed-time artificial insemination (FTAI) was performed at 54 ± 2 h (nulliparous) or 66 ± 2 h (primiparous and multiparous) after CIDR removal. Transrectal ovarian ultrasonography was performed on Days 7 and 14 to characterize the largest follicle diameter (LFD) and the presence of corpora lutea (CL). Estrus prior to FTAI was characterized using estrus detection aids. Pregnancy status was determined via transrectal ultrasonography approximately 60–100 days after FTAI. The proportion of multiparous females with CL on Day 14 tended to be greater following treatment with 7 & 7 Synch or 7 & 7 + G (90% and 87%, respectively), as compared to females treated with 7 & 7 + G + P (82%; p = 0.09). A greater proportion of multiparous females expressed behavioral estrus prior to FTAI when treated with 7 & 7 + G + P (90%) as compared to multiparous females treated with 7 & 7 + G (79%; p < 0.002) or 7 & 7 Synch (78%; p = 0.0004); however, pregnancy rates resulting from FTAI did not differ based on treatment. In summary, tandem administration of GnRH and PG on Days 0 and 7 of the 7 & 7 Synch protocol effectively generated a convergent ovarian response among females with variable pretreatment luteal status but failed to enhance pregnancy rates to FTAI.

Ceftiofur treatment in cows with metritis enhances the likelihood of clinical cure but does not consistently improve subsequent pregnancy rates compared with healthy (nonmetritis) cows. We tested the hypothesis that ceftiofur treatment has no effect on uterine microbiota or endometrial inflammation at 1 mo postpartum in cows that were either healthy or diagnosed with metritis at 5 to 10 d postpartum. Cows diagnosed with metritis were matched with healthy cows in a 2 × 2 factorial design where they were either treated with an antibiotic (ceftiofur hydrochloride) or left untreated. Cows were slaughtered to collect the uterus at 1 mo postpartum, and the uterine microbiota was measured using bacteriology and 16S rRNA gene sequencing. The lumen of the uterus was examined visually for the presence of purulent material, and inflammation within the endometrium was assessed histologically. When bacteria were cultured from the lumen of the uterus at 1 mo postpartum, a greater number of species was isolated and a greater number of colonies formed from cows that were previously diagnosed with metritis compared with healthy cows. Antibiotic treatment administered at the time of disease diagnosis (5 to 10 d postpartum) had no effects on the abundance of culturable bacteria at 1 mo postpartum in healthy cows but reduced the number of culturable bacteria from the uterus of cows previously diagnosed with metritis. Based on 16S rRNA gene sequencing, cows originally diagnosed with metritis had an increased number of sequencing reads 1 mo postpartum than cows initially deemed healthy. Furthermore, antimicrobial treatment 5 to 10 d postpartum decreased the number of sequencing reads 1 mo postpartum irrespective of the initial disease diagnosis. The presence of purulent or metritis-like discharge in the uterine lumen 1 mo postpartum had a major effect on the uterine microbiota. Histopathological analysis revealed that the presence of purulent material in the uterus or signs of acute infection at 1 mo postpartum was associated with greater uterine inflammation compared with a clear uterine flush but minimally affected by treatment. Furthermore, antibiotic treatment had no main effect on α-diversity (Faith's phylogenetic diversity or Pielou's evenness), and no differentially abundant taxa were detected by analysis of composition of microbes. However, β-diversity analyses (unweighted UniFrac) showed a small but significant effect of treatment, with treated cows having lower within-group variability than untreated cows, suggesting a modest homogenizing effect on the uterine microbiota. A multinomial regression analysis revealed that the relative abundance of Fusobacterium was 145.45 times lower in ceftiofur-treated cows compared to untreated cows. This shift suggests a partial restoration of the microbial community in cows with metritis toward a profile resembling that of their healthy counterparts. Additionally, the analysis indicated that the uterine microbiota at 1 mo postpartum was collectively influenced by visual detection of purulent material, occurrence of metritis postpartum, antibiotic treatment, and estrus cyclicity. We rejected the hypothesis that early postpartum ceftiofur treatment did not affect uterine microbiota at 1 mo postpartum and concluded that ceftiofur treatment had a measurable effect on uterine microbiota and inflammation.

Postpartum uterine involution in cows restores the endometrium to its pre-pregnancy status to support subsequent pregnancy. Endometrial cells in the cow may differ from those in the heifer because cow endometrium arises from a regenerative process that occurs in the presence of microbial infection and tissue inflammation. The objective, therefore, was to determine if the quality and proliferative potential of bovine endometrial epithelial cells (bEECs) differed for heifers compared with postpartum cows. Postpartum Holstein cows (n=11) and virgin Holstein-Jersey heifers (n=5) were used. Sampling was performed for 5 consecutive weeks, either at 7, 14, 21, 28, and 35±3 days postpartum (cows) or starting at 365±7 days of age (heifers). Blood samples were collected for plasma progesterone (P4) and β-hydroxybutyrate (BHB) analyses. On day 35, the estrous cycles of all animals were synchronized using an injection of PGF2α. Seven days after injection, bEECs were collected in vivo by using a transcervical approach and Cytobrush Plus Cell collector (Cooper Surgical; Trumbull CT). Primary cell populations of bEECs were established according to the procedures of Rocha et al. (2022; JDS Communications 3:217-221) and grown to 80% confluency. Confluent cells were then dissociated with trypsin/EDTA, counted and analyzed for viability using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific; Waltham, MA). The effects of status (cow versus heifer), cyclicity (P4), and metabolic activity (BHB) on the bEEC were evaluated by using PROC GLM in SAS. The results are presented as LSMEAN±SEM. Based on vaginal discharge score, none of the cows or heifers had uterine disease. The Cytobrush collection tended (P< 0.0904) to yield more cells from heifers compared with cows (2.16×107 and 9.22×106 fresh cells, respectively). Days to confluency after the initial plating (P0) was less (P< 0.0174) for heifers compared with cows (5.2±1.8 and 10.9±1.2 days, respectively). The total number (P< 0.0457; 5.15×105 and 1.47×105) and percentage of live cells (P< 0.0337; 50.5±7.5 and 29.3±5.0) was greater for heifers compared with cows (respectively) at the first passage (P1). There was no effect of cyclicity or subclinical ketosis (BHB >1.2 mmol/L) in cows on the number of cells collected or their growth in culture, but more cells were collected from cows with >5 ng/mL versus < 5ng/mL plasma P4 (2.6×107 versus 3.5×106 fresh cells, respectively; P< 0.0047). In conclusion, the bEEC isolated from cows differed from those isolated from heifers for their proliferative capacity (time to confluence) and viability following routine cell culture procedures (passaging). Cyclicity and metabolic disease in cows did not affect the development of cells in culture. Unique developmental properties of bEEC isolated from cows versus heifers may indicate that the regenerated uterine endometrium differs from the virgin endometrium. These differences may explain, in part, reduced fertility in postpartum cows compared with heifers.

IntroductionThe concept of a sterile uterus was challenged by recent studies that have described the microbiome of the virgin and pregnant uterus for species including humans and cattle. We designed two studies that tested whether the microbiome is introduced into the uterus when the virgin heifer is first inseminated and whether the origin of the microbiome is the vagina/cervix.MethodsThe uterine microbiome was measured immediately before and after an artificial insemination (AI; Study 1; n = 7 AI and n = 6 control) and 14 d after insemination (Study 2; n = 12 AI and n = 12 control) in AI and non-AI (control) Holstein heifers. A third study (Study 3; n = 5 Holstein heifers) that included additional negative controls was subsequently conducted to support the presence of a unique microbiome within the uterus despite the low microbial biomass and regardless of insemination. Traditional bacteriological culture was performed in addition to 16S rRNA gene sequencing on the same samples to determine whether there were viable organisms in addition to those detected based on DNA sequencing (16S rRNA gene sequence).Results and discussionInseminating a heifer did not lead to a large change in the microbiome when assessed by traditional methods of bacterial culture or metataxonomic (16S rRNA gene) sequencing (results of Studies 1 and 2). Very few bacteria were cultured from the body or horn of the uterus regardless of whether an AI was or was not (negative control) performed. The cultured bacterial genera (e.g., Bacillus, Corynebacterium, Cutibacterium, Micrococcus, Staphylococcus, and Streptococcus) were typical of those found in the soil, environment, skin, mucous membranes, and urogenital tract of animals. Metataxonomic sequencing of 16S rRNA gene generated a large number of amplicon sequence variants (ASV), but these larger datasets that were based on DNA sequencing did not consistently demonstrate an effect of AI on the abundance of ASVs across all uterine locations compared with the external surface of the tract (e.g., perimetrium; positive control samples for environment contamination during slaughter and collection). Major genera identified by 16S rRNA gene sequencing overlapped with those identified with bacterial culture and included Cutibacterium, Staphylococcus, and Streptococcus.

Optimal reproductive success following parturition in lactating dairy cows is dependent upon adequate completion of uterine involution. Failure to resolve pathogenic bacterial contamination within the first week postpartum can lead to uterine disease (metritis). Metritis is associated with decreased fertility and a failure or delay to establish pregnancy. We hypothesized that the inflammation resulting from early postpartum metritis would be associated with long-term changes in uterine morphology due to impaired uterine involution within the first 30 days postpartum (dpp). First parity Holstein cows were diagnosed with or without metritis at 7-10 dpp and uterine tissue were analyzed at 30 (Exp. 1), or 80 and 165 (Exp. 2) dpp for the presence of abnormal morphology, including abnormal invasion of endometrial glands and stroma into the myometrium (adenomyosis) using immunohistochemistry for FOXA2 (uterine gland specific marker) and presence of late postpartum endometrial fibrosis using mason’s trichrome stain (MTS). Severity of adenomyosis was determined by the number and size of adenomyotic foci, distance of foci from the endometrium-myometrium interface (EMI), and degree of fibrosis (MTS stain intensity). The presence, size, and distance from the EMI of adenomyotic foci were greater later postpartum and in cows with early postpartum diagnosis of metritis. Endometrial fibrosis was greater at the stratum basalis (at EMI) compared to the stratum compactum endometrium (near lumen) for all Exp. 2 cows, but greater endometrial fibrosis (regardless of endometrial region) was observed in cows that were diagnosed with metritis. Taken together, these data indicate that early postpartum metritis is associated with long-term modifications to the postpartum uterine morphology, including aberrant endometrial invasion into the myometrium (adenomyosis) and increased pathological fibrogenesis, leading to the presence of late postpartum endometrial fibrosis (scar tissue). Additionally, increased collagen fiber at the EMI suggests a correlation between the development of adenomyosis and fibrosis, which could possibly result from sustained endometrial inflammation caused by uterine disease. ### Competing Interest Statement The authors have declared no competing interest.

IntroductionThe 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).MethodsThe 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.ResultsThere 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.ConclusionThere 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.

Two experiments were designed to evaluate later timepoints for fixed-time artificial insemination (FTAI) of beef heifers and cows, with the hypothesis that use of a later timepoint would allow a greater proportion of animals to express estrus prior to FTAI and result in greater conception rates among estrous females inseminated with sex -sorted semen. In Experiment 1, estrus was synchronized for 1640 heifers using the 14 d CIDR-PG protocol: insertion of an intravaginal progesterone-releasing insert (CIDR; 1.38 g progesterone) on Day-33 and removal on Day-19, and administration of prostaglandin F2 alpha (PG; 500 mu g cloprostenol sodium) on Day-3. Heifers were inseminated at one of three FTAI timepoints: 66 h, 70 h, or 74 h after PG administration. In Experiment 2, estrus was synchronized for 414 beef cows using the 7 & 7 Synch protocol: administration of PG coincident with CIDR insertion on Day-17, gonadotropin-releasing hormone (GnRH; 100 mu g gonadorelin) on Day-10, and PG coincident with CIDR removal on Day-3. Cows were inseminated at one of two FTAI timepoints: 66 h or 72 h after PG administration. In both experiments, only animals that expressed estrus prior to FTAI were inseminated with sex-sorted semen. In Experiment 1, the proportion of heifers that expressed estrus prior to FTAI (66 h: 62 %; 70 h: 67 %; 74 h: 71 %) was greater when FTAI was performed at 74 h versus 66 h (P = 0.0097); however, conception rate of heifers that expressed estrus and were serviced with sex-sorted semen did not differ among treatments (P = 0.67; 66 h: 56 %; 70 h: 53 %; 74 h: 53 %). In Experiment 2, the proportion of cows expressing estrus prior to FTAI did not differ between treatments (P = 0.30; 66 h: 71 %; 72 h: 76 %). Additionally, conception rate of estrous cows inseminated with sex-sorted semen did not differ between treatments (P = 0.24; 66 h: 45 %; 72 h: 40 %). These results indicate that performing FTAI later following the 14 d CIDR-PG protocol increases the proportion of heifers that express estrus and are serviced with sex-sorted semen but does not improve conception rates. Later timing of FTAI following the 7 & 7 Synch protocol was not observed to increase the proportion of cows expressing estrus prior to FTAI or improve conception rates among estrous cows inseminated with sex-sorted semen. Together, these results provide further insight into optimal timing of FTAI when using sex-sorted semen.

Cows with metritis (uterine disease) during the first 1 to 2 weeks postpartum have lower pregnancy rates when inseminated later postpartum (typically >10 weeks). 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 weeks postpartum to either metritis (clinical disease; n = 10) or healthy (control; n = 10) groups and randomly assigned to be slaughtered at approximately 80 and 165 dpp (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.

Background: Postpartum uterine disease (metritis) is common in dairy cows. The disease develops within 1 week after calving and is associated with microbial dysbiosis, fever, and fetid uterine discharge. Cows with metritis have a greater likelihood of developing endometritis and infertility later postpartum. Antibiotic treatment is used to relieve symptoms of metritis but the capacity of antibiotic treatment to improve fertility later postpartum is inconsistent across published studies. We hypothesized that an antibiotic has only a short-term effect on the uterine microbiome and does not change the progression of disease from metritis to endometritis. To test this hypothesis, we studied the effects of systemic antibiotic given to cows diagnosed with metritis and healthy cows early postpartum on the development of endometritis and the uterine microbiome at 1 month postpartum. Results: Cows diagnosed with metritis were compared to healthy ones in a 2 x 2 factorial design, where they were either treated with an antibiotic (ceftiofur hydrochloride) at 7 to 10 days postpartum or left untreated. Cows were slaughtered at one month postpartum and the uterus was assessed for endometritis (presence of purulent material in the uterine lumen and inflammation in the endometrium) and uterine samples were collected for bacteriology and metagenomics (16S rRNA gene sequencing). As expected, the uterine microbiome at disease diagnosis had dysbiosis of typical metritis pathogens (e.g., Fusobacterium , Bacteroides , and Porphyromonas ) in diseased compared with healthy cows. At one month postpartum, there was a tendency for more endometritis in metritis cows compared with healthy but antibiotic treatment had no effect on endometritis prevalence regardless of the original disease diagnosis. Likewise, when bacteria were cultured or sequenced, there were a greater number of species (culture) or amplicon sequence variants (ASV; sequencing) in the uterine lumen of cows with metritis. However, antibiotic treatment had no effect on the prevalence of cultured species or the composition of the detected ASV. The uterine microbiome at 1 month postpartum was associated with the clinical observation of the uterus (endometritis or healthy). Conclusions: Early postpartum antibiotic treatment only provides temporary resolution of uterine dysbiosis that is not sustained long-term. Failure to resolve the dysbiosis is associated with a greater prevalence of endometritis in cows with metritis, and the occurrence of endometritis significantly impacts fertility later postpartum.

An experiment was designed to evaluate modifications to the 7 & 7 synch protocol with the hypothesis that tandem administration of prostaglandin F2α (PG) and gonadotropin-releasing hormone (GnRH) during the presynchronization portion of the protocol would generate a convergent ovarian response among anestrous and estrous cycling females. Nulliparous (n = 153), primiparous (n = 92), and multiparous (n = 413) Angus-cross females across four locations were blocked based on parity and pre-treatment cyclicity status (presence or absence of a corpus luteum) as assessed via transrectal ovarian ultrasonography. Within block, females were randomly assigned to one of three treatments. Females assigned to the 7 & 7 Synch treatment received an intravaginal progesterone-releasing insert (CIDR) and administration of PG on day 0, administration of GnRH on day 7, and administration of PG coincident with CIDR removal on day 14. Females assigned to 7 & 7 Synch + G received the same treatment schedule with the modification that GnRH was administered in tandem with the PG on day 0. Females assigned to 7 & 7 Synch + G + P received GnRH in tandem with PG on both day 0 and day 7 of the treatment schedule. Fixed-time artificial insemination (FTAI) was performed at 54 h after CIDR removal for nulliparous females and at 66 h for primiparous and multiparous females. Transrectal ovarian ultrasonography was performed on day 7 and day 14 to characterize the largest follicle diameter (LFD) as well as the presence/absence and number of corpora lutea (CL). Expression of estrus before FTAI was characterized in three locations using estrus detection patches (Estrotect. Approximately 75 to 90 days after FTAI, pregnancy status was determined via transrectal ultrasonography. Day 7 CL status was affected by treatment (P = 0.002), with a greater proportion of females presenting with CL on Day 7 when treated with the modified treatments (64%) as compared with the 7 & 7 Synch treatment (31%). Day 14 LFD was affected by treatment (P = 0.002), with a larger average LFD among females treated with 7 & 7 Synch + G + P (12.0 mm) as compared with 7 & 7 Synch (11.4 mm) or 7 & 7 + G (10.7 mm). Pregnancy rate to FTAI was affected by pre-treatment cyclicity status (P < 0.0001) and semen type (P = 0.0003) and tended to be affected by treatment (P = 0.09) with 7 & 7 Synch + G + P tending to result in a greater proportion pregnant (58.86%) as compared with 7 & 7 Synch (49.17%) and 7 & 7 Synch + G (48.79%). Tandem administration of GnRH and PG on days 0 and 7 of the 7 & 7 Synch protocol effectively generated a convergent ovarian response among anestrous and estrous cycling beef females and tended to result in increased pregnancy rates to FTAI.

There is limited data available regarding pathogens causing intramammary infections (IMI) in Jersey cows. The objectives of this study were to characterize the prevalence of IMI caused by different microorganisms in lactating Jersey cattle and evaluate the associations among microbes and somatic cell count (SCC) and persistence of IMI.This prospective, observational, longitudinal study included lactating Jersey cows (n = 753) from 4 farms within a 250-mile radius of Columbia, Missouri. Quarter foremilk samples were aseptically collected monthly for 3 consecutive months. Microorganisms were identified using aerobic milk culture and MALDI-TOF mass spectrometry. A commercial laboratory measured SCC using flow cytometry. Milk culture results were used to classify single microorganism infections as persistent (same microorganism species identified at first sampling and one other sampling) or non-persistent infection. Mixed models were built to evaluate the associations between IMI status and lnSCC as well as persistence and lnSCC.Overall, staphylococci were the most commonly isolated microorganisms among the 7,370 quarter-level milk samples collected. Median prevalence (using all 3 samplings) of specific microbes varied among farms; however, Staphylococcus chromogenes was a common species found at all farms. The most common microbial species that persisted were Staph. chromogenes, Staphylococcus aureus, Staphylococcus simulans, and Streptococcus uberis. Streptococcus dysgalactiae and Staph. aureus were the IMI associated with the most inflammation based on lnSCC. The small number of herds included in this study with the large variation in herd type limits the generalizability of the data. However, results of this study seem to be similar to those of previous studies in other breeds, suggesting management factors are more important than breed-specific differences when evaluating causes of IMI and associated subclinical mastitis.

The postpartum uterus involutes to its pre-pregnant and fully functional state within approximately 60 d after calving. Uterine glands are essential for fertility but little is known about their regeneration postpartum. Likewise, the effect of uterine disease (metritis) on gland regeneration is unknown. We hypothesized that uterine glands would be regenerated early postpartum and that metritis would be associated with slower gland regeneration to affect their numbers later postpartum during the breeding period. Postpartum dairy cows were diagnosed as healthy (n = 17 and 9 for experiment [Exp.] 1 and 2) or metritis (n = 17 and 10 for Exp. 1 and 2, respectively) at 7 to 10 d postpartum. Cows were slaughtered at approximately 1 mo (Exp. 1) or approximately 80 or 165 d (Exp. 2) postpartum for the collection of the uterus. Uterine tissue was sectioned and the number of glandular cross-sections per unit area was counted and cross-sectional area measured. Cellular proliferation within the luminal epithelium (LE) and glandular epithelium (GE) was quantified by MKI67 (marker of cellular proliferation) immunohistochemistry. In early postpartum cows (Exp. 1), the greatest amount of MKI67 staining was found in the deep endometrium (cells closest to the myometrium). Cows with purulent material in the uterine lumen at d 30 slaughter (Exp. 1) had fewer endometrial glands per unit area in the deep and middle endometrium when compared with nonpurulent cows. The MKI67 staining was less in the deep endometrial GE and LE for purulent compared with nonpurulent cows. Estrus cyclicity was associated with a greater number of gland cross-sections in the deep and middle endometrium. Later postpartum (80 and 165 d; Exp. 2), there was greater glandular development compared with Exp. 1 and a tendency for a lesser number of gland cross-sections per unit area in diseased cows without an effect on MKI67 staining in the GE or LE. We conclude that uterine disease slows the development of uterine glands early postpartum (by 1 mo) through a mechanism that involves cellular proliferation within the GE. The impact of the early postpartum disease on glandular development later postpartum (Exp. 2) appeared to be less. Additional time, therefore, may allow recovery of the GE in later postpartum cows.

Crossbreeding dairy cows with beef sires has increased in the U.S. dairy industry. This beef x dairy mating results in dairy-beef which has altered the U.S. fed beef supply. The predominant mating has been Holstein x Angus, thus research was conducted to determine the effects of Holstein x SimAngus breeding on growth performance, carcass characteristics, and fresh meat quality. Starting on d 1, Holstein steers (n = 9) and Holstein x SimAngus (HolSim) steers (n = 12) were fed once daily at 0830 h, until an end weight of 635 kg was reached. Holstein steers averaged 421 d and HolSim steers averaged 432 days on feed. Daily feed offered was delivered into a semi-autonomous feed monitoring system designed to measure individual animal intake and feeding behavior (GrowSafe System Ltd). Steer body weight was recorded every 28 d. Data collected were used to calculate dry matter intake (DMI), average daily gain (ADG), and gain to feed ratio (G:F). Steers were humanely slaughtered under USDA inspection, immediately following slaughter hot carcass weight (HCW) was recorded and 48 h following slaughter, carcass length, body width, backfat thickness and area of the longissimus muscle (LM) was recorded. Strip loins were collected from each carcass and aged for 14 d. After aging, Warner-Bratzler shear force (WBSF) and slice shear force (SSF) were determined. Remaining steaks were placed under retail display for 7 d. Objective color was measured after 0, 1, 3, 5, and 7 d in retail display. No differences were observed for ADG and G:F between Holstein and HolSim steers (P > 0.05). A tendency was observed for Holstein steers to have a greater DMI compared with HolSim steers (P = 0.0646). The HolSim steers had greater HCW and dressing percentages compared with Holstein steers (P < 0.0001). The HolSim carcasses had shorter carcass lengths and body widths compared with Holstein carcasses (P = 0.0005). Holstein carcasses had less backfat compared with HolSim carcasses (P = 0.0034). The LM area was larger in HolSim carcasses (P < 0.0001). No differences were observed for WBSF or SSF (P > 0.05). Strip steaks from HolSim steers had less deoxymyoglobin and greater oxymyoglobin values on d 0 of retail (P < 0.05). Strip steaks from HolSim steers had greater a* and saturation index values on d 7 in retail (P < 0.05). Strip steaks from HolSim steers had decreased metmyoglobin values and greater oxymyoglobin values on d 7 in retail (P < 0.05). Thus, indicating strip steaks from HolSim steers had increased red color. Growth performance data in this study confirm previous literature reported when comparing dairy x dairy to beef x dairy steers. Carcass and fresh meat data suggest that Holstein x SimAngus steers had improved yield production compared with Holstein steers without a loss in quality when steers were fed to a weight constant end point.