Establishment of the uterine microbiome following artificial insemination in virgin heifers.

Introduction: The 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. Methods: The 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 discussion: Inseminating 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.

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.

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.

Systemic antibiotic treatment of cows with metritis early postpartum does not change the progression of uterine disease or the uterine microbiome at 1 month postpartum.

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 × 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.

Changing Demographics of the Commercial Dairy Calf Industry: Why Use Beef on Dairy?

The use of beef bulls on dairy cattle has increased in the last 6 years. In fact, beef semen sales have more than doubled. Dairyman needs to capture real value for the beef on dairy cross calf, selection of beef sires that produce offspring that complement dairy cattle are needed, not simply the cheapest black bull in the tank. Those beef sires should be selected on calving ease, ribeye area, marbling, feed efficiency, fertility, polled, and an industry preference for black hided. Even more important, the use of beef on dairy has allowed dairy producers to manage replacement animal inventories.

Evaluation of cervical and uterine size, at 4 weeks postpartum, as a predictor of subsequent fertility in Jersey cattle.

Uterine and cervical size of Holstein dairy cows is reported among reasons for a decline in dairy cow fertility. Therefore, the objectives of this study were to (a) determine whether size of the cervix and uterus at 4 weeks postpartum impacted subsequent fertility at first service in Jersey cattle, (b) determine whether progesterone level at 4 weeks postpartum impacted cyclicity and (c) the association of the presence of corpus luteum and uterus and cervix size. Body condition scores at calving, presence of postpartum diseases, parity number and milk weights were taken from lactating Jersey dairy cows (N = 147) for 28 days postpartum. During the fourth week postpartum, a blood sample was obtained for progesterone concentration, and transrectal ultrasonography was performed by a high-resolution ultrasound machine to determine cervical and uterine horn diameter, as well as ovarian structures measurements. Correcting for parity number, BCS at calving, presence of diseases and milk yield, cows with a cervix >2.54 ± 0.63 cm and uterine horn >2.25 ± 0.59 cm were less likely to become pregnant at first service (p = .04 and p = .003, respectively). The cows with larger cervix had a trend to be less likely to have a corpus luteum present at the 4th week of lactation (p = .067). Cows with larger uterine horn size were less likely to have a corpus luteum present at the 4th week of lactation (p = .015). It is concluded that a larger cervix and/or uterus during the postpartum was associated negatively with fertility and cyclicity in Jersey cows.

Concurrent and long-term associations between the endometrial microbiota and endometrial transcriptome in postpartum dairy cows.

BACKGROUND: Fertility in dairy cows depends on ovarian cyclicity and on uterine involution. Ovarian cyclicity and uterine involution are delayed when there is uterine dysbiosis (overgrowth of pathogenic bacteria). Fertility in dairy cows may involve a mechanism through which the uterine microbiota affects ovarian cyclicity as well as the transcriptome of the endometrium within the involuting uterus. The hypothesis was that the transcriptome of the endometrium in postpartum cows would be associated with the cyclicity status of the cow as well as the microbiota during uterine involution. The endometrium of first lactation dairy cows was sampled at 1, 5, and 9 weeks postpartum. All cows were allowed to return to cyclicity without intervention until week 5 and treated with an ovulation synchronization protocol so that sampling at week 9 was on day 13 of the estrous cycle. The endometrial microbiota was measured by 16S rRNA gene sequencing and principal component analysis. The endometrial transcriptome was measured by mRNA sequencing, differential gene expression analysis, and Ingenuity Pathway Analysis. RESULTS: The endometrial microbiota changed from week 1 to week 5 but the week 5 and week 9 microbiota were similar. The endometrial transcriptome differed for cows that were either cycling or not cycling at week 5 and cyclicity status depended in part on the endometrial microbiota. Compared with cows cycling at week 5, there were large changes in the transcriptome of cows that progressed from non-cycling at week 5 to cycling at week 9. There was evidence for concurrent and longer-term associations between the endometrial microbiota and transcriptome. The week 1 endometrial microbiota had the greatest effect on the subsequent endometrial transcriptome and this effect was greatest at week 5 and diminished by week 9. CONCLUSIONS: The cumulative response of the endometrial transcriptome to the microbiota represented the combination of past microbial exposure and current microbial exposure. The endometrial transcriptome in postpartum cows, therefore, depended on the immediate and longer-term effects of the uterine microbiota that acted directly on the uterus. There may also be an indirect mechanism through which the microbiome affects the transcriptome through the restoration of ovarian cyclicity postpartum.

Longitudinal microbiological evaluation of subclinical non-aureus staphylococcal intramammary infections in a lentivirus-infected dairy goat herd.

The objectives of this study were to 1) correlate pre-partum teat skin colonization with non-aureus staphylococcal (NAS) intramammary infection (IMI) in early lactation, and 2) evaluate infection dynamics of subclinical NAS IMI in goats during lactation in a small ruminant lentivirus-infected herd. Pre-partum teat skin swabs (41 goats, 82 halves) and post-partum half-level milk samples (106 goats, 203 halves) were collected at various intervals starting at ≤10 days in milk (DIM) until ≥120 DIM. Teat skin colonization and IMI were defined by culture and strain-typing. The association between the pre-kidding udder-half teat skin sample status and early lactation IMI status for a given species was investigated using McNemar's exact test or logistic regression. Time to IMI elimination and time to new IMI were evaluated by discrete-time survival analysis. Halves with S. caprae isolated from teat skin prior to kidding had increased odds of S. caprae IMI ≤ 10 DIM. Time to IMI elimination varied as a function of NAS species. Intramammary infections detected >10 DIM had a higher hazard of elimination (hazard ratio [HR] 5.6, 95% CI 2.8-11.2) than IMI detected ≤10 DIM. The presence of an IMI in the contralateral half was associated with a higher hazard of new IMI (HR 2.1, 95% CI 1.3-3.4) in an uninfected half. Further studies on interventional strategies targeting early IMI and IMI caused by persistent species are warranted.

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.

A Dairy Herd Case Investigation with Very Low Dietary Cation-Anion Difference in Prepartum Dairy Cows.

During the periparturient period, subclinical hypocalcemia (total plasma Ca concentration <2.0 mmol/l) is a potential problem for the dairy cow; consequently, its prevention is essential for success of fertility and productive performance. Dietary cation-anion difference (DCAD) has been defined as the difference in milliequivalents of cations (Na, K) and anions (Cl, S) per kilogram of dry matter (DM) and has a direct impact on blood acid-base metabolism. Diets rich in K and Na induce metabolic alkalosis, interfering with tissue sensitivity to parathyroid hormone, and diets rich in Cl and S (anionic salts) cause metabolic acidosis, reducing the risk of hypocalcemia. Consequently, the use of anionic salts has become a popular method to prevent hypocalcemia in dairy cattle. Monitoring diets with anionic salts can be done by measuring urine pH, with optimal values between 6.2 and 6.8 for Holstein cows. The objective of this report is to present a herd case investigation involving a dairy farm feeding a very low DCAD (-143 mEq/kg DM), expecting improved Ca homeostasis. The diet of -143 mEq/kg (urine pH 5.2-5.8) was changed to a diet with -53 mEq/kg DM (urine pH 6.2-6.8). Blood samples were taken at the time of calving for 10 cows that calved before and then for 10 cows that calved after changing the diet. Cows with extremely low DCAD had Ca concentrations of 2.11 ± 0.22 mmol/l and cows with a more moderated DCAD, 2.11 ± 0.16 mmol/l (P > 0.05). Several other blood metabolites (P, Mg, Na, K, Cl, albumin, globulins, blood urea nitrogen, creatinine, and GGT) were also similar between groups. This very low DCAD during the prepartum period may severely compromise animal physiology unnecessarily, with little advantage over normal calcium concentrations at parturition, when compared with a less negative DCAD (-53 mEq/kg DM). Feeding a less negative DCAD ration (-53 mEq/kg DM) did not decrease plasma Ca levels right after parturition compared to a DCAD ration of -143 mEq/kg DM, reinforcing the lack of benefit of a more negative DCAD.

Setting the stage for long-term reproductive health.

This article discusses some of the aspects of heifer development that contribute to long-term health and productivity, such as disease prevention and control. Nutrition is also an important component of long-term health, and body condition score is discussed as a way to determine whether the nutrient demands of heifers are being met.

Incorporating reproductive management of beef heifers into a veterinary practice.

Veterinarians play an important role in reproductive management of dairy herds across the United States; however, in many cases, their involvement in reproductive management of beef herds has been limited. The reasons for this vary; however, there are ways for veterinarians to become more actively involved in reproductive management of US beef herds. Veterinarians can have an impact on producers' profits by implementing their skills and knowledge to beef heifer development programs. This article provides an overview of the services veterinarians can provide to beef cattle producers that pertain to reproductive management of replacement beef heifers.

Management strategies for adding value to replacement beef heifers: a working model.

This article provides an overview of the Missouri Show-Me-Select Replacement Heifer Program, and is included to provide an example of a working model for the collective considerations that relate to beef heifer development presented in this issue. This program expanded services provided by veterinarians to cow-calf producers, making veterinary practitioners a more integral part of the overall reproductive management of beef herds in their practice areas.

Luteal function, largest follicle, and fertility in postpartum dairy cows treated with 14dCIDR-PGF2α versus 2xPGF2α-Ovsynch for timed AI.

A method for timed artificial insemination (AI) that is used for beef cows, beef heifers, and dairy heifers employs progesterone-releasing inserts, such as the controlled internal drug release (CIDR; Zoetis, New York, NY, USA) that are left in place for 14 days. The 14-day CIDR treatment is a method of presynchronization that ensures that cattle are in the late luteal phase of the estrous cycle when PGF2α is administered before timed AI. The objective of this study was to test the effectiveness of the 14dCIDR-PGF2α program in postpartum dairy cows by comparing it with the traditional "Presynch-Ovsynch" (2xPGF2α-Ovsynch) program. The 14dCIDR-PGF2α cows (n = 132) were treated with a CIDR insert on Day 0 for 14 days. At 19 days after CIDR removal (Day 33), the cows were treated with a luteolytic dose of PGF2α, 56 hours later were treated with an ovulatory dose of GnRH (Day 35), and 16 hours later were inseminated. The 2xPGF2α-Ovsynch cows were treated with a luteolytic dose of PGF2α on Day 0 and again on Day 14. At 12 days after the second PGF2α treatment (Day 26), the cows were treated with GnRH. At 7 days after GnRH, the cows were treated with PGF2α (Day 33), then 56 hours later treated with GnRH (Day 35), and then 16 hours later were inseminated. There was no effect of treatment or treatment by parity interaction on pregnancies per AI (P/AI) when pregnancy diagnosis was performed on Day 32 (115/263; 43.7%) or Days 60 to 90 (99/263; 37.6%) after insemination. There was an effect of parity (P < 0.05) on P/AI because primiparous cows had lesser P/AI (35/98; 35.7%) than multiparous cows (80/165; 48.5%) on Day 32. Cows observed in estrus after the presynchronization step (within 5 days after CIDR removal or within 5 days after the second PGF2α treatment) had greater P/AI than those not observed in estrus (55/103; 53.4% vs. 60/160; 37.5%; observed vs. not observed; P < 0.01; d 32 pregnancy diagnosis). When progesterone data were examined in a subset of cows (n = 208), 55.3% of cows had a "prototypical" response to treatment (i.e., the cow had an estrous cycle that was synchronized by the presynchronization treatment and then the cow responded appropriately to the subsequent PGF2α and GnRH treatments before timed AI). Collectively, cows with a prototypical response to either treatment had 52.2% P/AI that was greater (P < 0.001) than the P/AI for cows that had a nonprototypical response (19%) (P/AI determined at 60-90 days of pregnancy). In conclusion, we did not detect a difference in P/AI when postpartum dairy cows were treated with 14dCIDR-PGF2α or 2xPGF2α-Ovsynch before timed AI. The primary limitation to the success of either program was the failure of the cow to respond appropriately to the sequence of treatments.

Efficacy of feeding a lacteal-derived colostrum replacer or pooled maternal colostrum with a low IgG concentration for prevention of failure of passive transfer in dairy calves.

OBJECTIVE: To compare the efficacy of a lacteal-derived colostrum replacer (LDCR) for the prevention of failure of passive transfer of immunity (FPT) in calves with that of pooled maternal colostrum (MC). DESIGN: Randomized field trial. ANIMALS: 568 heifer calves from 1 California dairy. PROCEDURES: Calves were randomly allocated to 1 of 2 treatment groups and fed 2 doses (200 g of IgG) of an LDCR or 3.8 L of pooled MC. From each calf, blood samples were collected before and approximately 24 hours after treatment. Serum IgG and total protein (TP) concentrations were quantified with standard methods, and the apparent efficiency of IgG absorption was calculated. RESULTS: At 24 hours after treatment, mean serum TP and IgG concentrations were significantly lower for calves fed pooled MC (TP, 4.77 g/dL; IgG, 7.50 g/L), compared with those for calves fed the LDCR (TP, 5.50 g/dL; IgG, 15.15 g/L). Calves fed the LDCR were 95% less likely to develop FPT (OR, 0.05; 95% confidence interval, 0.03 to 0.08) than were calves fed pooled MC. However, the mean IgG concentration in the pooled MC fed during the study (21.1 g/L) was substantially lower than that (64.3 g/L) determined for representative samples of pooled MC from other southwestern US dairies during a national survey. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that, on this particular dairy, calves fed an LDCR were at less risk of developing FPT than were calves fed pooled MC. The LDCR evaluated was a viable alternative for the prevention of FPT in calves.