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Review: Postpartum reproductive disease and fertility in dairy cows
Abstract
This paper reviews recent data and concepts on metritis, purulent vaginal discharge (PVD), and endometritis in dairy cows and the ways in which these diseases affect reproductive performance. Metritis is characterized by fetid discharge from the uterus, with or without fever. Purulent vaginal discharge describes exudate that is >50% pus that may be attributable to uterine infection or cervicitis. Endometritis is inflammation of the uterus diagnosed by endometrial cytology with a proportion of neutrophils (typically ≥5%) that is associated with impaired fertility. Metritis and PVD are associated with uterine bacterial dysbiosis: changes in the microbiota to lesser diversity and greater abundance of pathogens, especially Gram-negative anaerobic bacteria, and Trueperella pyogenes in the case of PVD. Metritis is justifiably treated with approved antibiotics but criteria for more selective treatment without loss of performance are emerging. Purulent vaginal discharge is not synonymous with clinical endometritis, and greater precision in terminology is warranted. PVD is likely under-diagnosed and represents an opportunity for improved management in many herds. Endometritis seems in many cases to reflect persistent, dysregulated inflammation, for which the inciting cause is unclear. Postpartum uterine infection and inflammation have harmful effects on oocytes, embryo development, and the endometrium for at least three months, even if the disease is apparently resolved. Emerging concepts of the resolution and regulation of inflammation are promising for the improvement of prevention and therapy of endometritis.
... This phenomenon is particularly pertinent for primigravid females, as this is their first ever parturition. In dairy cows, postpartum uterine contamination has been documented in nearly 100% of animals [6][7][8], and the same was suggested in humans and other mammals [1,[9][10][11]. Moreover, in dairy cows and women, postpartum uterine inflammatory diseases are more prevalent among primiparous females than multiparous females [12][13][14]. ...
... Moreover, in dairy cows and women, postpartum uterine inflammatory diseases are more prevalent among primiparous females than multiparous females [12][13][14]. In dairy cows, traditional culture-dependent microbiological analyses have identified several microbial species in the postpartum uteri, including Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides spp., Pseudomonas spp., Streptococcus spp., and Staphylococcus spp., some of which have been associated with metritis [6,7,[15][16][17]. However, recent years have witnessed advancements in culture-independent investigations of bacterial communities within postpartum bovine uteri, mainly next-generation sequencing (NGS) of 16S-rDNA amplicon libraries [2,7,8,18]. ...
... In dairy cows, traditional culture-dependent microbiological analyses have identified several microbial species in the postpartum uteri, including Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides spp., Pseudomonas spp., Streptococcus spp., and Staphylococcus spp., some of which have been associated with metritis [6,7,[15][16][17]. However, recent years have witnessed advancements in culture-independent investigations of bacterial communities within postpartum bovine uteri, mainly next-generation sequencing (NGS) of 16S-rDNA amplicon libraries [2,7,8,18]. These approaches have yielded findings that differ from culture-based studies and have enabled the identification of bacteria that are challenging to cultivate through traditional methods. ...
Studies in recent years indicate that reproductive tract microbial communities are crucial for shaping mammals’ health and reproductive outcomes. Following parturition, uterine bacterial contamination often occurs due to the open cervix, which may lead to postpartum uterine inflammatory diseases, especially in primiparous individuals. However, investigations into spatio-temporal microbial transitions in the reproductive tract of primigravid females remain limited. Our objective was to describe and compare the microbial community compositions in the vagina at late gestation and in the vagina and uterus at early postpartum in first-pregnancy heifers. Three swab samples were collected from 33 first-pregnancy Holstein Friesian heifers: one vaginal sample at gestation day 258 ± 4, and vaginal and uterine samples at postpartum day 7 ± 2. Each sample underwent 16S rRNA V4 region metagenetic analysis via Illumina MiSeq, with bioinformatics following Mothur MiSeq SOP. The reproductive tract bacterial communities were assigned to 1255 genus-level OTUs across 30 phyla. Dominant phyla, accounting for approximately 90% of the communities, included Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria. However, the results revealed distinct shifts in microbial composition between the prepartum vagina (Vag-pre), postpartum vagina (Vag-post), and postpartum uterus (Utr-post). The Vag-pre and Utr-post microbial profiles were the most distinct. The Utr-post group had lower relative abundances of Proteobacteria but higher abundances of Bacteroidetes, Fusobacteria, and Tenericutes compared to Vag-pre, while Vag-post displayed intermediate values for these phyla, suggesting a transitional profile. Additionally, the Utr-post group exhibited lower bacterial richness and diversity compared to both Vag-pre and Vag-post. The unsupervised probabilistic Dirichlet Multinomial Mixtures model identified two distinct community types: most Vag-pre samples clustered into one type and Utr-post samples into another, while Vag-post samples were distributed evenly between the two. LEfSe analysis revealed distinct microbial profiles at the genus level. Overall, specific microbial markers were associated with anatomical and temporal transitions, revealing a dynamic microbial landscape during the first pregnancy and parturition. These differences highlight the complexity of these ecosystems and open new avenues for research in reproductive biology and microbial ecology.
... This phenomenon is particularly pertinent for primigravid females, as this is their first-ever parturition. In dairy cows, postpartum uterine contamination has been documented in nearly 100% of animals [6][7][8], and the same was suggested in humans and other mammals [1,[9][10][11]. Moreover, in dairy cows and women, postpartum uterine inflammatory diseases are more prevalent among primiparous females than multiparous females [12][13][14]. ...
... In dairy cows, traditional culture-dependent microbiological analyses have identified several microbial species in the postpartum uteri, including Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides spp., Pseudomonas spp., Streptococcus spp., and Staphylococcus spp., some of which have been associated with metritis [6,7,[15][16][17]. However, recent years have witnessed advancements in culture-independent investigations of bacterial communities within postpartum bovine uteri, mainly next-generation sequencing (NGS) of 16S-rDNA amplicon libraries [2,7,8,18]. ...
... In dairy cows, traditional culture-dependent microbiological analyses have identified several microbial species in the postpartum uteri, including Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides spp., Pseudomonas spp., Streptococcus spp., and Staphylococcus spp., some of which have been associated with metritis [6,7,[15][16][17]. However, recent years have witnessed advancements in culture-independent investigations of bacterial communities within postpartum bovine uteri, mainly next-generation sequencing (NGS) of 16S-rDNA amplicon libraries [2,7,8,18]. These approaches have yielded findings that differ from culture-based studies and have enabled the identification of bacteria that are challenging to cultivate through traditional methods. ...
... This phenomenon is particularly pertinent for primigravid females, as this is their first-ever parturition. In dairy cows, postpartum uterine contamination has been documented in nearly 100% of animals [7][8][9], and the same was suggested in humans and other mammals [2,[10][11][12]. Moreover, in dairy cows and women, postpartum uterine inflammatory diseases are more prevalent among primiparous than multiparous females [13][14][15]. ...
... Moreover, in dairy cows and women, postpartum uterine inflammatory diseases are more prevalent among primiparous than multiparous females [13][14][15]. In dairy cows, traditional culture-dependent microbiological analyses have identified several microbial species in the postpartum uteri, encompassing Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides spp., Pseudomonas spp., Streptococcus spp., and Staphylococcus spp., which some have been associated with metritis [7,8,[16][17][18]. However, recent years have witnessed advancements in culture-independent investigations of bacterial communities within postpartum bovine uteri, mainly next-generation sequencing (NGS) of 16S-rDNA amplicon libraries [3,8,9,19]. ...
... In dairy cows, traditional culture-dependent microbiological analyses have identified several microbial species in the postpartum uteri, encompassing Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides spp., Pseudomonas spp., Streptococcus spp., and Staphylococcus spp., which some have been associated with metritis [7,8,[16][17][18]. However, recent years have witnessed advancements in culture-independent investigations of bacterial communities within postpartum bovine uteri, mainly next-generation sequencing (NGS) of 16S-rDNA amplicon libraries [3,8,9,19]. These approaches have yielded findings that differ from culture-based studies and have enabled the identification of bacteria that are challenging to cultivate through traditional methods. ...
Studies in recent years indicate that microbial communities residing in the reproductive tract are crucial for shaping mammals' health and reproductive outcomes. Following parturition, female mammals often exhibit uterine bacterial contamination due to the open cervix allowing microbes from the vaginal microbiota or the environment to enter. This contamination may be associated with postpartum uterine inflammatory diseases, which are more prevalent among primiparous than multiparous individuals, as reported in dairy cows and women. However, investigations into the spatio-temporal microbial transitions in the reproductive tract of primigravid heifers and women remain limited. Our objective was to describe and compare the community compositions in the vagina at late gestation and in the vagina and uterus at early postpartum, in first-pregnancy heifers. Three swab samples were collected from 33 first-pregnancy Holstein-Friesian heifers: vaginal samples at gestation day 258±4, and vaginal and uterine samples at postpartum day 7±2. Each sample underwent 16S rRNA V4 region metagenetic analysis via Illumina MiSeq, with bioinformatics following Mothur MiSeq SOP. Results revealed distinct shifts in microbial composition between the prepartum vagina, postpartum vagina, and postpartum uterus. The late gestation vaginal and early postpartum uterine microbial profiles were the most distinct, with postpartum vaginal samples serving as intermediaries. The postpartum uterus exhibited lower richness and diversity compared to the prepartum and postpartum vagina. Specific microbial markers were associated with anatomical and temporal transitions, indicating a dynamic microbial landscape associated with parturition. The observed differences in phyla and genera across reproductive sites highlight the complexity of these ecosystems, expending avenues for future research in reproductive biology, microbial ecology, and reproductive tract diseases.
... lactation is characterized by drastic physiological and metabolic changes that start before parturition. Some of these challenges include immune activation, energy, protein and mineral imbalance, and uterine and systemic inflammation [3][4][5]. Adaptations related to Ca and lipid metabolism, immune function, and inflammation often occur concurrently in the early postpartum period when cows are exposed to endotoxins from one or more of the uterus, gut, or mammary glands, increasing the risk of developing disease and reproductive disorders. ...
... We recently conducted a study testing the hypothesis that systemic and uterine inflammation can be triggered reciprocally in clinically healthy cows using intravenous or intrauterine LPS challenge models at 5 or 40 DIM [56]. We expected differences in immune dysfunction, metabolic stress [75], and systemic and uterine inflammation [5,6] at 5 vs. 40 DIM. At 5 DIM, systemic LPS challenge resulted in subtle and inconsistent alterations in markers of systemic inflammation, likely due to a high variation in baseline inflammatory status of cows that are exposed to endotoxins naturally following parturition. ...
In the early postpartum period, dairy cows undergo significant adaptations in Ca and lipid metabolism, immune function, and inflammatory processes. Concurrent exposure to endotoxins from the uterus, gastrointestinal tract, or mammary gland increases the risk of disease and reproductive problems. Metabolic and inflammatory imbalances during this phase can have both immediate and long-term effects on reproductive health. Associations between metabolic disorders and reproductive outcomes are often confounded by immune activation and systemic inflammation. However, optimal markers, thresholds, and durations for identifying maladaptation and predicting adverse health or reproductive outcomes remain unclear. This narrative review examines key physiological changes during the transition period, including hypocalcemia, lipid mobilization, immune activation, systemic inflammation, and uterine disease. We discuss how these events may affect the dominant follicle, corpus luteum, oocyte, and uterus, potentially leading to prolonged anovulation, reduced estrus expression, impaired response to synchronization protocols, lower progesterone concentrations, and compromised fertility. Understanding these mechanisms will support the development of strategies to monitor, prevent, and mitigate the impacts of transition-related maladaptation on reproductive performance. Such advancements can enhance the health and fertility of high-producing dairy cows.
... It is well established that problems with reproductive health make cows less productive, which costs the dairy industry money. This includes lower milk production, lower calf harvest, early depreciation of potentially useful cows, longer inter-conception and calving interval, reduced reproductive rate, high medication costs, and increased risk of culling Sheldon et al., 2020;Chebel, 2021;LeBlanc, 2023). ...
... These findings are consistent with research conducted in other countries (Mahnani et al., 2020;Boakari and Ali, 2021;Chebel, 2021). Several factors for RFM have been identified, and it is stated that risk factors differ depending on region, country, environment, and management strategies (LeBlanc, 2023;Aiello and Moses, 2016). Future research is needed to substantiate the lack of relationship between RFM and the other factors studied. ...
Reproductive disorders are the leading cause of economic loss in the cattle industry, as they reduce the reproductive efficiency and lifetime productivity of cows. In Ethiopian dairy, without proper preventive measures, these disorders can have a severe impact on productivity. Therefore, it is crucial to identify the extent of these issues and the factors contributing to them in order to improve the health and productivity of dairy cows. This study aimed to assess the prevalence, type, and potential risk factors for reproductive health problems in dairy farms in Hawassa city and Wolaita Sodo town. We observed 391 pregnant and postpartum cows for reproductive disorders and used a logistic regression model to identify risk factors. The study found that 165 cows (41.6%; 95% CI: 36.8-46.5%) had at least one reproductive issue. The most common disorders included retained fetal membrane (11.6%), dystocia (10.5%), metritis (7.2%), abortion (5.6%), stillbirth (2.6%), uterine/vaginal prolapse (2.3%), endometritis (2.1%), and pyometra (0.5%). The risk of dystocia significantly increased with male calf deliveries (OR=2.9; p=0.004) and larger farm sizes (OR=2.1; p=0.039). Cows experiencing dystocia were 5.3 times more likely to have retained fetal membrane (p<0.001). Heifers were significantly more likely to develop metritis (OR=5.1; p=0.001), as were cows with retained fetal membrane (OR=11.6; p<0.001). The incidence of abortion was significantly elevated in cows from semi-intensive farms (OR= 5.3; p<0.001). The study concludes that these reproductive health issues can significantly impact future uterine health, resulting in prolonged uterine involution. It is crucial to raise awareness among dairy farmers about effective herd health management practices to minimize losses.
... These findings suggest that some bacteria typically associated with disease are normal inhabitants of the reproductive tract of mares. However, under certain conditions, such as inflammation associated with insemination, specific reproductive strains could colonize the uterus and cause bacterial endometritis [51][52][53][54][55]. ...
... Additionally, the rationality of sampling this group of mares during estrus was based on the uterine immune response [66]. During estrogen dominance (estrus), the uterine immune response is more effective, contrary to when the uterus is under the influence of progesterone (diestrus), increasing the risk of infection [52,66] and potentially modifying the microbiota present during sampling. Although most high-throughput studies use healthy mares, selecting mares that have never been bred from or treated (maiden) is relevant to defining normal and abnormal reproductive microbiota in horses. ...
Simple Summary
This study explored the different types of bacteria found in the reproductive tract of healthy maiden mares during their estrus cycle. Vaginal, uterine, clitoral fossa, and perineal skin swabs were collected from 12 mares, and the bacterial communities were characterized using a high-throughput sequencing methodology. The types and number of bacteria detected in the uterus and vagina were similar to each other but different from those in the clitoral area and skin. Specific bacteria, such as those from the families Streptococcaceae and Staphylococcaceae, were common in the uterus and vagina, but Lactobacillaceae were rare. Some bacteria associated with reproductive diseases were also found in these healthy mares, suggesting that these bacteria are naturally present even in healthy animals.
Abstract
This descriptive cross-sectional study compared the microbiota of the uterus, vagina, clitoral fossa (CF), and perineal skin in healthy maiden mares during estrus. Twelve synchronized, healthy maiden mares (3–4 years old) from one single recipient mare herd were included. Microbial communities were characterized by amplifying the V3–V4 region of the 16S rRNA gene using the Illumina MiSeq platform. The uterine and vaginal microbiota had significantly lower richness (Chao-1) than the skin (p < 0.05). The uterine and vagina bacterial composition was similar in presence and abundance and could be differentiated from that of the CF and perineal skin. The microbial composition (Jaccard and Bray–Curtis distances) significantly differed across body-site locations (p < 0.05), which explained approximately 14% and 19% of the variation in microbial composition for Jaccard and Bray–Curtis distances, respectively. Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes were the dominant taxa in the uterus and vagina, with higher proportions of Proteobacteria in the vaginal samples compared to the uterine samples. Streptococcaceae and Staphylococcaceae were present in high abundance in the uterine and vaginal samples, while Lactobacillaceae were not (<10%). We demonstrate that the uterine and vaginal microbiota of healthy maiden mares during estrus is similar but both distinct from that of the CF and perineal skin.
... Cows with metritis may fail to completely recover from the disease and develop chronic in ammation of the uterus (clinical or subclinical endometritis) that can last for several months [7,9,10]. If left untreated, endometritis places the cow at risk for failure to establish and maintain pregnancy when she is inseminated two to three months postpartum [1,[11][12][13]. The underlying mechanisms through which metritis can cause infertility or how the microbiome (both commensal and pathogenic organisms) interacts with normal processes of uterine tissue regeneration and repair in cows are not understood. ...
... Although ceftiofur will effectively relieve the symptoms of metritis [17,19], the drug is not labeled for improving fertility postpartum when cows are inseminated (several months after cows are treated and the symptoms have subsided). Additional studies that followed the regulatory approval were poorly controlled with respect to testing the e cacy of ceftiofur for improving fertility [12,20]. Antibiotic treatment failed to increase fertility of cows with metritis to a level found in healthy cows in two studies [21,22] whereas a third study showed some improvement [23]. ...
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.
... There is consensus that diseases harm productivity, given that diseased dairy cows may experience a reduction in milk yield, are less likely to become pregnant, and are more likely to be culled [16,17,20]. Postpartum diseases (e.g., metritis) cause economic losses due to the cost of treatment, decreased milk production, lower reproductive efficiency, and increased mortality rate [21,22] and impair animal welfare [23]. It is well known that pathogens invade the uterus at parturition, increasing the risk of developing uterine infections like metritis [24]. ...
This study aimed to evaluate the association between colostrum quality, as indicative of the immune status of the cows, and peripartum disease risk occurrence in a herd of grazing dairy cows. A prospective cohort study was conducted on a grazing dairy farm with 3000 Holstein milking cows from 15 March 2022 to 15 March 2023. Multivariable binary logistic models evaluated the association between colostrum quality (expressed in Brix grades as a continuous predictor) and the risk of peripartum diseases (including dystocia, stillbirth, retention of fetal membranes, metritis, and clinical endometritis). We found that colostrum quality was negatively associated with the risk of dystocia (p = 0.02), stillbirth (p < 0.01), clinical endometritis (p = 0.02), and total peripartum diseases (p < 0.01). Conversely, colostrum quality was not related to the risk of retention of the placenta (p = 0.25) or metritis (p = 0.76). Additionally, we found that the calving season affected colostrum quality (p < 0.01). Conversely, parity number, dry period length, and milk yield in previous lactation did not affect it (p > 0.10). We concluded that colostrum quality, expressed in Brix grades, is negatively associated with peripartum disease risk in dairy cows and could be a useful indicator of the immune status of the cow.
... Endometritis in cattle during the delivery or postpartum periods is typically attributed to microbial infections, predominantly involving purulent and Gram-negative anaerobic bacteria contamination of the genital tract [22]. The clinical signs include heightened vaginal discharge and swelling and congestion of the cervix, leading to adverse impacts on the well-being and reproductive efficiency of cattle, resulting in substantial financial repercussions [23,24]. Inflammation is the hallmark response of the innate immune system when foreign substances or pathogens are detected [25]. ...
The aim of this study is to investigate the therapeutic effect of Poria cocos polysaccharide (PCP) on bovine endometritis. Initially, an inflammation model was induced using LPS-treated bovine endometrial epithelial cells (BEND) to identify the differentially expressed genes (DEGs) between the control and LPS groups by transcriptome sequencing, and GO functional annotation and KEGG enrichment analysis were performed. Subsequently, the mechanism of PCP treatment for endometritis was further evaluated using protein immunoblotting and real-time fluorescence quantitative analysis. Finally, the efficacy of PCP in treating endometritis was evaluated using a rat model of endometritis established with a mixed bacterial infection. The results show that transcriptome sequencing identified 4367 DEGs, with enrichment analysis highlighting the primary influences on the cell cycle and apoptosis signaling pathways. Following treatment of BEND with LPS resulted in cell apoptosis and inflammatory response. However, the introduction of PCP intervention significantly inhibited the progression of apoptosis and inflammation. Animal test results indicate that PCP significantly decreases the levels of serum inflammatory in rats suffering from endometritis and enhances antioxidant capacity. Furthermore, it effectively improved uterine swelling and tissue vacuolization caused by bacterial infection. These findings suggest that PCP could alleviate endometritis by modulating the inflammatory response and suppressing cell apoptosis. Poria cocos polysaccharides demonstrate significant potential for applications in immune modulation, anti-inflammatory responses, and antioxidant activities. Their high safety profile makes them suitable candidates as alternative therapeutic agents for the treatment of endometritis in the veterinary field.
... Reproductive challenges of cows experiencing retained fetal membranes, puerperal metritis, and delayed involution have been well documented. 3,[15][16][17][18][19] Effects of postpartum uterine diseases on fertility and cyclicity have been attributed to bacterial uterine contamination leading to systemic inflammation and endotoxins in follicular fluid and systemically. The inflammatory process may lead to a decrease in LH secretion, slower follicular growth and reduced size of the dominant follicle, and reduced steroidogenesis within the follicle, all of which negatively impact return to cyclicity. ...
Reproductive performance is a primary driver of herd profitability and reproductive failure or infertility is one of the main causes of culling in USA dairy herds. Nearly ⅓ of dairy cows in USA experience anovulation in early lactation leading to increases in days open and reductions in first service conception rates. Anestrus and anovulation in dairy cows can be attributed to many causes including parity, postpartum uterine or systemic diseases, nutritional deficiencies, body condition loss, and stress. Identification and management of anovular cows, including strategies to reduce the incidence of anovulation, are critical to the overall reproductive performance of the herd. The aim of this review is to highlight causes, recent research on anovulatory conditions, and clinical approaches to management to improve dairy herd reproductive outcomes.
... U terine disease during the first week postpartum (metritis) affects 10 to 25% of cows after calving (Bromfield et al., 2015;Galvão et al., 2019;Sheldon et al., 2020). Metritis is an important disease during the transition period because cows that develop metritis require treatment, have reduced milk production, and reduced fertility later postpartum (LeBlanc, 2023). Dairy cows suffer from a variety of illnesses postpartum (mastitis, ketosis, lameness, etc.) all of which have increased incidence as the cow ages (i.e., progresses through successive parities) (Lean et al., 2023b;a). ...
... The reproductive performance of dairy cows largely depends on the health status of the uterus after calving (Barragan et al., 2021;Moraes et al., 2017). One of the most common uterine diseases affecting the reproductive efficiency of dairy herds is endometritis (LeBlanc, 2023). Endometritis is inflammation of the endometrium that can impair the fertility of cows without general symptoms (Várhidi et al., 2024). ...
Clinical endometritis is one of the most common causes of declining reproductive performance in dairy cows. The purpose of this study was to investigate the effects of some factors including cow’s parity, milk yield, dystocia, retained placenta, calf sex, calving season, abortion, and stillbirth on the incidence of clinical endometritis and the effect of clinical endometritis on open days in an Iranian dairy farm. Uterine health of 557 dairy cows was evaluated by rectal palpation and ultrasonography during 30-35 days after calving. The presence of mucopurulent secretions in the vagina and vulva as well as the ultrasonographic observation of echogenic secretions in the uterine lumen were considered endometritis. The effect of the mentioned factors on the incidence of clinical endometritis was then analyzed in univariate and multivariate logistic regression models. The chance of developing clinical endometritis in the low-producing cows was 27.2% higher than that of the high-producing cows (p=0.06). Other parameters had no significant effect on the incidence of endometritis. The incidence of clinical endometritis in the studied herd was 49.5%. Based on the results of this study, the low milk yield may be a risk factor for developing clinical endometritis in the studied herd's condition, while parameters of the parity, dystocia, retained placenta, calf sex, calving season, abortion, and stillbirth was not effective in the occurrence rate of endometritis. There was no association between clinical endometritis and days open in the studied herd.
... The postpartum reproductive disorders of dairy cows seriously affect their production performance and cause economic losses [1]. Due to the cervical laxity and the damage of endometrial epithelium after calving, cows are prone to uterine infections, such as metritis and endometritis [2,3], which can impair the bovine fertility [4]. The tight junction between endometrial epithelial cells prevents pathogens from penetrating into the stroma. ...
Background
An elevated endogenous cortisol level due to the peripartum stress is one of the risk factors of postpartum bovine uterine infections. Selenium is a trace element that elicits anti-inflammation and antioxidation properties. This study aimed to reveal the modulatory effect of selenium on the inflammatory response of primary bovine endometrial stromal cells in the presence of high-level cortisol. The cells were subjected to lipopolysaccharide to establish cellular inflammation. The mRNA expression of toll-like receptor 4 (TLR4), proinflammatory factors, and selenoproteins was measured with qPCR. The activation of NF-κB and MAPK signalling pathways was detected with Western blot and immunofluorescence.
Results
The pretreatment with sodium selenite (2 and 4 µΜ) resulted in a down-regulation of TLR4 and genes encoding proinflammatory factors, including interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor α, cyclooxygenase 2, and inducible nitric oxide synthase. Selenium inhibited the activation of NF-κB and the phosphorylation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38MAPK and c-Jun N-terminal kinase/stress-activated protein kinase. The suppression of those genes and pathways by selenium was more significant in the presence of high cortisol level (30 ng/mL). Meanwhile the gene expression of glutathione peroxidase 1 and 4 was promoted by selenium, and was even higher in the presence of cortisol and selenium.
Conclusions
The anti-inflammatory action of selenium is probably mediated through NF-κB and MAPK, and is augmented by cortisol in primary bovine endometrial stromal cells.
... Importantly, the use of exogenous GM-CSF drugs reduces the incidence of inflammationinduced postpartum endometritis (34). Recombinant GM-CSF lactobacillus, with the dual immunomodulatory effects of lactobacillus and GM-CSF, can reduce the inflammatory response and release of inflammatory mediators in postpartum endometritis by inhibiting the activation and aggregation of neutrophils (35). For example, recombinant GM-CSF lactobacillus may reduce the production of MPO by regulating the activation state of neutrophils (36). ...
Introduction
Postpartum endometritis is a prevalent reproductive disorder in bovines, leading to a prolonged open period, infertility, and other complications. While Lactobacillus strains can mitigate these conditions by reducing uterine inflammation, their effectiveness is limited due to a lack of direct anti microbial action and extended treatment duration. This study aimed to construct a recombinant Lactobacillus johnsonii strain expressing bovine Granulocyte-macrophage colony-stimulating factor (GM-CSF) to evaluate its potential in reducing postpartum uterine inflammation.
Methods
The recombinant Lactobacillus johnsonii strain was engineered to express bovine GM-CSF and administered to pregnant mice via vaginal perfusion. Postpartum endometritis was induced using E. coli infection, and the protective effects of the engineered strain were assessed. Inflammatory markers (IL-6, IL-1β, TNF-α), myeloperoxidase (MPO) activity, and nitric oxide (NO) concentration were measured. Histological examination was performed to evaluate uterine morphology and pathological damage.
Results
The recombinant L. johnsonii strain expressing GM-CSF significantly reduced inflammation levels induced by E. coli infection in the uterus. This reduction was evidenced by decreased expression of IL-6, IL-1β, TNF-α, as well as reduced MPO activity and NO concentration. Histological examination revealed improved uterine morphology and reduced pathological damage in mice treated with the recombinant GM-CSF strain. Crucially, the recombinant strain also exerts beneficial effects on bovine endometritis by reducing levels of inflammatory cytokines, suggesting a beneficial effect on clinical bovine endometritis.
Conclusion
The recombinant Lactobacillus johnsonii expressing GM-CSF demonstrated protective effects against postpartum endometritis in bovines by reducing inflammatory cytokines. The findings indicate the potential clinical application of this engineered strain in preventing postpartum uterine inflammation, offering a novel and effective protective option for related disorders and improving bovine reproductive efficiency.
... Long-lasting damage to the uterine wall associated with PM in the first lactation might be one reason for greater susceptibility for uterine disease in the second lactation. One concept is that intact epithelial barriers allow for commensal colonization, but that breach of this barrier with the invasion of epithelial or deeper cells triggers a substantial inflammatory response (LeBlanc, 2023). It has also been shown that cows affected by metritis had less glandular development and less epithelial proliferation within the deep endometrium up to 80 DIM (Sellmer Ramos et al., 2023), suggesting long-term changes in the tissue architecture in cows with PM. ...
The objective of this study was to evaluate the effects of puerperal metritis (PM) diagnosed and treated during the early postpartum period of the first lactation on transition cow health, milk production, reproduction, and culling of dairy cows in their second lactation. Diagnosis of PM was based on fetid watery red-brown uterine discharge and rectal temperature above 39.5°C. Two farms were enrolled in this retrospective observational cohort study (Farm A and B). In both farms, the following diseases were recorded during the first 30 DIM in lactation 1 and 2: clinical hypocalcemia (CH), retained fetal membrane (RFM), PM, hyperketonemia (KET), left displaced ab-omasum (LDA) and clinical mastitis (MAST). Statistical analyses were performed using SPSS for Windows separately for each farm. Linear and logistic regression models were used for continuous (e.g., milk yield) and binary (e.g., disease, pregnancy per AI, pregnancy loss) outcomes, respectively. Cox proportional hazard regression models were calculated to model the time to event outcomes for culling or death during the first 60 DIM and for pregnancy within 250 d of the second lactation. The initial models contained the following variables: year of calving, month of calving, calving ease, stillbirth, twins, days open in lactation 1, 305 d milk yield in lactation 1, PM in lactation 1, and PM in lactation 2 as explanatory variables. A total of 4,834 cows (Farm A) and 4,238 cows (Farm B) in the second lactation were considered for statistical analyses. On farm A, the incidence of PM in lactation 1 and 2 was 20.1% and 11.2%, respectively. On farm B, the incidence of PM in lactation 1 and 2 was 14.4% and 8.5%, respectively. On both farms, cows with PM in their first lactation had greater odds for RFM and PM in their second lactation, while there was no association of PM in the first lactation with any other non-uterine diseases (i.e., CH, KET, LDA, and MAST) in the second lactation. Cows with PM in lactation 2 had reduced milk yield. The reduction in milk yield in second lactation was greater for cows that already experienced PM in lactation 1. On Farm A, cows with PM in their first lactation had a greater hazard for culling within 60 DIM of the second lactation; however, the same association was not present on Farm B. Cows with PM in lactation 1 had reduced pregnancy per AI at first service in the second lactation only on farm B. Cows with PM in lactation 2 had reduced pregnancy per AI at first service in the second lactation on both farms. Pregnancy loss in lactation 2 was only associated with PM in lactation 2 but not with PM in lactation 1. On both farms, cows had a reduced hazard for pregnancy in their second lactation within 250 DIM when they experienced PM in either lactation. In conclusion , PM in the first lactation had long-lasting negative consequences (i.e., risk of uterine disease and lower reproductive performance) for cows in their next lactation.
... Also, the occurrence of retained placenta was more frequent in surgical cases [18]. There has been little research interest in post-partal ovine uterine pathology, but clinical and subclinical endometritis, often caused by intrauterine contamination or retained placenta, have been extensively studied in cattle, and long-lasting negative effects on fertility have been described in this species, as recently reviewed by LeBlanc [26]. For instance, inflammation leads to the creation of unfavourable conditions for sperm survival, zygote development, implantation, and placentation, as well as negatively affecting ovarian and endocrine functions [27][28][29][30]. ...
Simple Summary
In cases of birth difficulties in sheep, caesarean sections can be life-saving. It is, however, important for farmers to know whether the ewe is likely to conceive again. In field cases following birth difficulties, many factors such as the problematic delivery itself or any underlying health problems will also influence these outcomes. In order to assess a potential direct effect of surgery, it is important to study healthy animals undergoing pre-planned operations. Animals from a university sheep flock were, therefore, studied retrospectively by evaluating their breeding records. Caesarean sections were performed by veterinary students as part of their training, and the subsequent breeding outcomes were compared to animals which had only lambed naturally. There was no negative effect of caesarean surgery on the subsequent pregnancy rate or on lamb viability or birth weights. However, in the pregnancy immediately following a surgical delivery, a smaller number of lambs was born per ewe, and a higher number of mating attempts was necessary to achieve pregnancy. The number of lambs born returned to pre-caesarean levels in further subsequent pregnancies. We, therefore, conclude that caesarean sections in sheep have very little influence on their long-term reproductive performance; this procedure is, therefore, worthwhile.
Abstract
Post-surgical reproductive performance following ovine caesarean section has not been well studied. To assess any direct effects of surgical delivery in the absence of confounders such as dystocia or underlying diseases, we studied elective surgery performed in healthy animals for teaching purposes. Four hundred and eleven paired breeding records following vaginal delivery (n = 233), elective caesarean section (n = 122), and subsequent further vaginal deliveries in animals with a history of one prior elective caesarean operation (n = 56) were evaluated retrospectively. The overall subsequent pregnancy rate was 95%. Multivariable statistical analyses did not reveal any significant influence of planned caesarean surgery on subsequent conception, stillbirth, perinatal lamb mortality, lamb birth weights, or the incidence of premature foetal death (mummification and abortion). A significantly higher number of mating attempts was, however, necessary. Also, a significant reduction in litter size was seen in the first pregnancy immediately following a surgical delivery in comparison to animals which had previously only delivered vaginally (p = 0.001), but litter size returned to pre-caesarean levels in further follow-up pregnancies in animals with a history of one elective caesarean section (p = 0.436). Subsequent long-term reproductive performance of sheep following elective caesarean section is thus excellent, and the results encourage retention for breeding.
... Because this study only reported data from ewes with positive reproductive outcomes, the increase in the ewe vaginal microbial diversity over the course of gestation presumably is associated with successfully established pregnancies. In addition, more diverse communities may add more resiliency to the vaginal microbiota, allowing the microbes to respond to external factors while avoiding deficits in host health (LeBlanc, 2023). ...
Introduction
Numerous factors are known to influence reproductive efficiency in ewes, but few studies have investigated the potential role of vaginal microbiota in sheep reproductive success. The objective of this study was to thoroughly characterize the ewe vaginal microbiota throughout the course of pregnancy.
Methods
Vaginal samples were collected from 31 pregnant Hampshire and Hampshire X Suffolk crossbred ewes on a weekly basis from pre-breeding to pregnancy testing and then biweekly until just after lambing. To characterize the vaginal microbial communities, DNA was extracted and 16S rRNA gene Illumina MiSeq amplicon sequencing was performed.
Results and Discussion
Alpha diversity metrics indicated an increase in species richness, evenness, and overall diversity throughout gestation. Distinct shifts in the bacterial communities were observed during gestation and were segregated into three periods: early gestation, a transitional period and mid/late gestation. During early gestation, Actinobacillus, Histophilus, and unclassified Leptotrichiaceae were found in greater relative abundance. During the transitional period, a population shift occurred characterized by increasing relative abundance of Streptococcus and Staphylococcus. During mid/late gestation, Staphylococcus, Streptococcus, and Ureaplasma had the greatest relative abundance. These shifts in the microbial population throughout the ewe’s gestation are likely related to hormonal changes triggered by the growing conceptus, specifically increasing blood concentration of progesterone. The transitional period shift in vaginal microbial communities potentially aligns with the placental take-over of progesterone production from the corpus luteum at approximately day 50 after conception (gestational week 7). Understanding the observed variability of the vaginal microbiota throughout pregnancy will allow for future comparison of ewes that did not become pregnant or had abnormal pregnancies, which could lead to the discovery of potential bacterial biomarkers for pregnancy outcome; this understanding could also lead to development of probiotics to improve sheep reproductive success.
Післяотельні захворювання матки є серйозною проблемою молочних корів, оскільки вони можуть вражати значну частку поголів’я і мати серйозні наслідки щодо репродуктивної здатності самок і виробництва молока. Тому профілактику та своєчасне й ефективне лікування пуерперальних ускладнень необхідно розглядати як важливу ланку в системі заходів із ліквідації неплідності та яловості корів. У роботі наведені результати клініко-експериментальних досліджень із реґіонарного застосування антибіотиків і стимулюючих препаратів у комплексних схемах лікування корів за післяотельного ендометриту. В основу раціональної етіотропної та патогенетичної терапії було покладено реґіонарне введення лікарських препаратів: у внутрішню здухвинну артерію, паравагінально та внутрішньопіхвово. Були науково обґрунтовані, розроблені й апробовані комплексно-послідовні схеми лікування хворих на ендометрит корів. Клінічними дослідженнями встановлено, що комбіноване реґіонарне введення енрофлоксацину та метранідазолу в поєднанні із простагландином і утеротонічним препаратом виявилося більш ефективним методом лікування корів за гнійного ендометриту, ніж їх внутрішньом’язові ін’єкції. Ін’єктування антимікробних препаратів у магістральну судину, яка живить матку, дозволяє значною мірою сконцентрувати їхню фармакологічну дію в патологічному вогнищі. Водночас зросла запліднюваність корів, зменшилась частка рецидиву захворювання, скоротився термін лікування. Такий раціональний підхід до вдосконалення протоколів терапії дозволяє зменшити витрати на препарати, що забезпечує його оптимальні фармакоекономічні показники. Установлено, що поєднане введення «броестрофану» та «доцитолу» завдяки синергічності їх дії забезпечило активну евакуацію ексудату з матки та лютеоліз, що позитивно вплинуло на результати лікування та відновлення відтворної функції корів.
Die vorliegende Übersichtsarbeit fasst den Stand der Wissenschaft hinsichtlich verschiedener Operationsverfahren und der kurz-und langfristigen Erfolgschancen von Schafkaiserschnitten zusammen und soll damit eine Entscheidungshilfe für die Praxis bieten. Material und Methoden: Es erfolgte eine umfangreiche Recherche der veterinärmedizinischen Literatur und eine Zusammenfassung der zentralen Erkenntnisse. Ergebnisse: Grundsätzlich ist die Operation in der linken Flanke, median oder paramedian möglich. Unter Praxisbedingungen überwiegen die Vorteile des Zugangs in der linken Flanke, da der ventrale Zugang eine Vollnarkose erfordert und aufgrund der Rückenlage mit einem erhöhten Narkoserisiko einhergeht. Publizierte Überlebensraten der Mutterschafe mit dystokiebedingten Kaiserschnitten reichen von 81 % zu 95 %. Nach geplanten, zu Lehr-oder Forschungszwecken durchgeführten Kaiserschnitten überlebten zwischen 98,8 % und 100 %. Für klinisch gesunde Tiere ohne Begleiterkrankungen oder Vorschäden konnten in der Praxis jedoch auch bei dystokiebedingten Operationen ähnlich hohe Überlebensraten erreicht werden. Hinsichtlich der Mortalität der Lämmer spielen vor allem Verzögerungen bis zur Vorstellung zur tierärztlichen Behandlung eine wichtige Rolle. Die Fruchtbarkeitsaussichten nach Kaiserschnitt sind bei Schafen sehr gut und es konnten keine Unterschiede in der nachfolgenden Trächtigkeitsrate zwischen Schafen nach Vaginalgeburt und Schafen nach Kaiserschnitt nachgewiesen werden. Schlussfolgerung: Die kurz-und langfristigen Erfolgschancen von Kaiserschnitten beim Schaf sind sehr gut. Eine längerfristige weitere Zuchtnutzung und ein rechtzeitiges Eingreifen, das die Chancen für das Überleben der Lämmer erhöht, sichern die Wirtschaftlichkeit des Eingriffs. Praktische Relevanz: Die wirtschaftliche und tierschutzrelevante Bedeutung von Mutterschaf-oder Lämmerverlusten infolge Schwergeburten ist nicht zu unterschätzen. Der Kaiserschnitt sollte daher auch in Kleinwiederkäuerbeständen vermehrt zum Einsatz kommen.
Objective: This review summarises the current scientific evidence regarding different surgical approaches to ovine caesarean section, and short and long term outcomes. It aims to serve as a guidance for treatment decisions in veterinary practice. Material and methods: A thorough literature review was performed, and the relevant results were summarised. Results: Caesarean section can be performed via a left flank, median or paramedian incision. Under on-farm conditions, the left flank approach is advantageous over the ventral mid-line, since the latter method requires general anaesthesia, and the supine position is associated with an increased anaesthetic risk. Published survival rates of ewes after dystocia-related caesarean sections range from 81% to 95%. Following planned caesarean sections performed for teaching or research, survival rates ranged from 98.8% to 100%. Similarly high survival rates were also achieved for dystocia-related operations in healthy animals without any concomitant diseases or birth injuries. Delays until treatment is sought play a particularly important role for lamb mortality. The fertility prospects after ovine caesarean sections are very good, and no differences in subsequent pregnancy rates have been demonstrated between sheep that underwent vaginal delivery or caesarean section. Conclusion: The short and long-term chances for a positive outcome of ovine caesarean sections are excellent. Retention for breeding and timely intervention, which increases the chances of lamb survival, ensure the cost-effectiveness of the procedure. Clinical relevance: The economic and animal welfare significance of dystocia-related ewe or lamb mortality should not be underestimated. Caesarean sections should therefore be encouraged in small ruminant flocks.
We explored the biochemistry and morphology of the uterus at approximately one month postpartum for cows that were diagnosed with metritis (uterine disease; n=18) or healthy (n=17) at 7 to 10 d postpartum and either treated or untreated with ceftiofur hydrochloride (2x2 factorial). Cows were slaughtered at one month postpartum and the uterine horn was flushed to identify cows with a purulent or clear (non-purulent) content. RNA was isolated from caruncular (CAR) and inter-caruncular (ICAR) endometrium for RNA sequencing. Light microscopy was used to quantify inflammation and scanning electron microscopy (SEM) was used to assess the closure of the luminal surface of the uterus and the morphology of uterine glands. Results were that postpartum antibiotic treatment did not affect the endpoints that we measured including gene expression and morphology of the uterus. The primary biological driver of uterine function and morphology was inflammation at one month postpartum (endometritis; purulent material in the uterine lumen). Inflammation decreased epithelial growth factor signaling pathway expression (Wnt, Hippo, and Hedgehog) and biological processes involved in the formation of ciliated cells. Based on morphology, epithelial cells closed open areas of a denuded uterine surface using a process that involved depolarization and cell movement (planar cell polarity). Non-canonical Wnt signaling drives planar cell polarity and the loss of Wnt signaling may explain the slower tissue repair in cows with endometritis. Future studies and therapeutic treatments should consider uterine involution as a process of wound repair and tissue regeneration in the face of bacterial infection.
Postpartum uterine disease in dairy cows affects ovarian function, but it is unclear how the type and timing of
disease relates to ovarian function. To explore associations between uterine disease and contemporaneous
ovarian function, postpartum uterine health was evaluated in 17 lactating primiparous Holstein cows that
ovulated. Ovarian function was assessed by measuring daily hormone concentrations and the diameter of the
largest follicle and subsequent corpus luteum. Metritis (≤14 d postpartum) was associated with reduced follicle
diameter (P = 0.002) and tended to reduce plasma estradiol concentrations (P = 0.062) from d 7 to 16 postpartum
compared to cows without metritis. Metritis was associated with reduced plasma progesterone (P =
0.026) but not corpus luteum diameter from d 17 to 26 postpartum. Endometritis (≥15 d postpartum) was
associated with reduced corpus luteum diameter (P = 0.005) but did not alter plasma progesterone from d 17 to
26 postpartum compared to cows without endometritis. To explore longer term effects of uterine infection on
luteal function, non-lactating primiparous Holstein cows received an intrauterine infusion of vehicle control (n
= 11) or pathogenic Escherichia coli and Trueperella pyogenes to induce endometritis (n = 12) and corpora lutea
were recovered on d 16 of the estrous cycle, 146 d after intrauterine infusion. Intrauterine infusion of bacteria
had no effect on plasma progesterone or luteal diameter compared to control, and only altered the expression of 2
of 94 candidate genes (NCF1 and TLR9). Taken together, these studies imply that uterine diseases are principally
associated with changes to ovarian function at a time contemporaneous with disease.
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.
Background
Innovative therapies against bacterial infections are needed. One approach is to focus on host-directed immunotherapy (HDT), with treatments that exploit natural processes of the host immune system. The goals of this type of therapy are to stimulate protective immunity while minimizing inflammation-induced tissue damage. We use non-traditional large animal models to explore the potential of the mammosphere-derived epithelial cell (MDEC) secretome, consisting of all bioactive factors released by the cells, to modulate host immune functions. MDEC cultures are enriched for mammary stem and progenitor cells and can be generated from virtually any mammal. We previously demonstrated that the bovine MDEC secretome, collected and delivered as conditioned medium (CM), inhibits the growth of bacteria in vitro and stimulates functions related to tissue repair in cultured endothelial and epithelial cells.
Methods
The immunomodulatory effects of the bovine MDEC secretome on bovine neutrophils, an innate immune cell type critical for resolving bacterial infections, were determined in vitro using functional assays. The effects of MDEC CM on neutrophil molecular pathways were explored by evaluating the production of specific cytokines by neutrophils and examining global gene expression patterns in MDEC CM-treated neutrophils. Enzyme linked immunosorbent assays were used to determine the concentrations of select proteins in MDEC CM and siRNAs were used to reduce the expression of specific MDEC-secreted proteins, allowing for the identification of bioactive factors modulating neutrophil functions.
Results
Neutrophils exposed to MDEC secretome exhibited increased chemotaxis and phagocytosis and decreased intracellular reactive oxygen species and extracellular trap formation, when compared to neutrophils exposed to control medium. C-X-C motif chemokine 6, superoxide dismutase, peroxiredoxin-2, and catalase, each present in the bovine MDEC secretome, were found to modulate neutrophil functions.
Conclusion
The MDEC secretome administered to treat bacterial infections may increase neutrophil recruitment to the site of infection, stimulate pathogen phagocytosis by neutrophils, and reduce neutrophil-produced ROS accumulation. As a result, pathogen clearance might be improved and local inflammation and tissue damage reduced.
We aimed to evaluate the effects of rumen‐protected lysine (RPL) supplementation during the close‐up period on uterine involution and the resumption of ovarian function in dairy cows. Fifty‐two multiparous Holstein cows were categorized based on parity and expected calving date and randomly assigned to the RPL or control (CON) groups. The RPL group received 80 g of RPL daily from day 21 before the expected calving date until parturition. Blood samples were obtained twice weekly from pre‐supplementation to 6 weeks postpartum. The onset of luteal activity postpartum was determined via ultrasonography twice weekly for up to 6 weeks postpartum. Uterine involution was tracked at 3 and 5 weeks postpartum through the vaginal discharge score, percentage of polymorphonuclear cells (PMN) in endometrial cytology samples, presence of intrauterine fluid, and gravid horn diameter via ultrasonography. Before supplementation, the RPL group showed amino acid imbalance, which was improved by RPL supplementation. There were no significant differences in the onset of luteal activity, percentage of PMN, intrauterine fluid, or the diameter of the uterine horn between the two groups. The vaginal discharge score in the RPL group decreased from 3 to 5 weeks postpartum, whereas that in the CON groups did not decrease. The number of cows with clinical endometritis was lower in the RPL group. Overall, RPL supplementation during the close‐up period enhanced vaginal discharge clearance, potentially averting clinical endometritis, but did not affect the first ovulation in dairy cows.
Dairy cattle are AI at approximately 2 mo after calving to maintain a yearly calving interval. Before first AI, the uterus must undergo involu- tion, a process that includes the restoration of functional uterine glands (UG). The process that gives rise to UG within the glandular endo- metrium (GE) is unclear and the impact of postpartum uterine disease (metritis; M) and antibiotic (AB) treatment is unknown. We studied UG development by using histological analysis of uterine cross sections. At d 7 postpartum, primiparous cows were diagnosed with M (fetid red-brown watery vaginal discharge; n = 18) or were deemed Healthy (H; normal discharge; n = 17) and were either AB-treated (H, n = 9 and M, n = 9) with ceftiofur hydrochloride (i.m.; 1.25 g/d for 3 d) or not treated (H, n = 8 and M, n = 9). Cows were slaughtered at 29.1 ± 1.7 d postpartum. A cross section of the uterus was collected ipsilateral to the previous pregnancy and sectioned (5 μm) for histological analysis. The number of UG cross sections per microscopic field (0.33 mm2) was counted and the area of the UG in cross section (× 103 μm2) was measured in the deep, middle, and superficial GE using ImageJ (2 fields per section). Data were analyzed using PROC MIXED of SAS (Cary, NC). UG were smaller (P < 0.01) but more numerous (P < 0.001) from deep to superficial GE [UG area: 19.7, 22.9, and 38.9 × 103 μm2 (SEM = 4.5) and UG number: 17.3, 13.7, and 7.5 UG per field (SEM = 1.0) for deep, middle, and superficial GE, respectively]. Cows previously diagnosed with M had lesser (P < 0.022) cross-sectional UG area compared with Hcows(21.2±3.6vs.33.2±3.7×103 μm2;Mvs.H).Therewasan interaction (P < 0.01) for number of UG per field because, compared with H cows, the M cows had fewer UG in the deep (13.5 ± 1.4 vs. 21.1 ± 1.4) and middle GE (11.6 ± 1.4 vs. 15.8 ± 1.4) but similar numbers of UG in the superficial GE (8.1 ± 1.4 vs. 6.8 ± 1.4) (M vs. H). There was no effect of AB on the number of UG. We conclude that uterine disease early postpartum may delay fertility in postpartum dairy cows by slowing the regeneration of UG. Supported by the NICHD of the National Institutes of Health under award no. R01HD092254.
Despite differences in gut physiology and morphology, both humans and cattle require a functional gut microbiome in early life. Evidence suggests that both species acquire gut microbes prior to birth, likely from a maternal source, indicating the use of similar mechanisms and timing for fetal gut colonization. Unlike mouse models, cattle share a similar gestation length, parity, and placental microbiome characteristics to humans. The large size of calves allow for contamination-protected sampling of the gut, vagina, and uterus, which would typically require invasive procedures in human cohorts. The ruminant placenta also exhibits a larger degree of separation between maternal and fetal physiology, necessitating a direct and explicit route by which microbes may access the fetal gut. These and other features permit cattle to act as a translational model for early gut colonization. However, cattle do not share similar placental morphology, gut function, or early immune system interactions with humans, creating barriers to their use as a biomedical model. Identifying similarities and differences between humans and cattle may outline the most important functions of the placental and fetal gut microbiomes, indicate the source of these microbes, and highlight the role of maternal or environmental influences upon fetal health across species.
Pregnancy induces changes in the transcriptome of the bovine endometrium from 15 days after insemination. However, pregnancy is less likely to occur if cows had a postpartum bacterial infection of the uterus, even after the resolution of disease. We hypothesized that uterine bacterial infection alters the endometrial transcriptomic signature of pregnancy after the resolution of disease. To examine the endometrial transcriptomic signature of pregnancy, cows were inseminated 130 days after intrauterine infusion of pathogenic Escherichia coli and Trueperella pyogenes, subsequently endometrium was collected 16 days after insemination for RNA sequencing. We found 171 pregnancy regulated genes in cows 146 days after bacterial infection. When comparing our findings with previous studies that described the endometrial transcriptomic signature of pregnancy in healthy cows, 24 genes were consistently differentially expressed in pregnancy, including MX1, MX2 and STAT1. However, 12 pregnancy regulated genes were found only in the endometrium of healthy cows, including ISG15 and TRANK1. Furthermore, 28 pregnancy regulated genes were found only in the endometrium of cows following bacterial infection and these were associated with altered iNOS, TLR, and IL-7 signaling pathways. Although 94 predicted upstream regulators were conserved amongst the studies, 14 were found only in the endometrium of pregnant healthy cows, and 5 were found only in cows following bacterial infection, including AIRE, NFKBIA, and DUSP1. In conclusion, there were both consistent and discordant features of the endometrial transcriptomic signature of pregnancy 146 days after intrauterine bacterial infusion. These findings imply that there is an essential transcriptomic signature of pregnancy, but that infection induces long-term changes in the endometrium that affect the transcriptomic response to pregnancy.
Many species of bacteria produce toxins such as cholesterol-dependent cytolysins that form pores in cell membranes. Membrane pores facilitate infection by releasing nutrients, delivering virulence factors, and causing lytic cell damage - cytolysis. Oxysterols are oxidized forms of cholesterol that regulate cellular cholesterol and alter immune responses to bacteria. Whether oxysterols also influence the protection of cells against pore-forming toxins is unresolved. Here we tested the hypothesis that oxysterols stimulate the intrinsic protection of epithelial cells against damage caused by cholesterol-dependent cytolysins. We treated epithelial cells with oxysterols and then challenged them with the cholesterol-dependent cytolysin, pyolysin. Treating HeLa cells with 27-hydroxycholesterol, 25-hydroxycholesterol, 7α-hydroxycholesterol, or 7β-hydroxycholesterol reduced pyolysin-induced leakage of lactate dehydrogenase and reduced pyolysin-induced cytolysis. Specifically, treatment with 10 ng/ml 27-hydroxycholesterol for 24 h reduced pyolysin-induced lactate dehydrogenase leakage by 88%, and reduced cytolysis from 74% to 1%. Treating HeLa cells with 27-hydroxycholesterol also reduced pyolysin-induced leakage of potassium ions, prevented mitogen-activated protein kinase cell stress responses, and limited alterations in the cytoskeleton. Furthermore, 27-hydroxycholesterol reduced pyolysin-induced damage in lung and liver epithelial cells, and protected against the cytolysins streptolysin O and Staphylococcus aureus α-hemolysin. Although oxysterols regulate cellular cholesterol by activating liver X receptors, cytoprotection did not depend on liver X receptors or changes in total cellular cholesterol. However, oxysterol cytoprotection was partially dependent on acyl-CoA:cholesterol acyltransferase (ACAT) reducing accessible cholesterol in cell membranes. Collectively, these findings imply that oxysterols stimulate the intrinsic protection of epithelial cells against pore-forming toxins and may help protect tissues against pathogenic bacteria.
Simple Summary
An important concern about conducting endometrial sampling procedures prior to breeding is the potential adverse effect on subsequent fertility of sampled cattle. Traumatic injury to the cervix and uterus and risk of introduction of infection caused by these sampling procedures may adversely affect the fertility of cattle sampled prior to breeding. To investigate this, a systematic review of publications assessing the impact of endometrial sampling on subsequent pregnancy rates was conducted. Analysis within the studies selected demonstrated that pregnancy rates were similar between sampled and non-sampled animals when procedures were performed before or around the time of breeding. Unfortunately, it was not possible to compare each sampling procedure between studies due to variation in sampling conditions (i.e., type of breed, age, reproductive status, and the sampling to breeding interval). We concluded that conducting these endometrial sampling procedures does not adversely affect subsequent fertility; therefore they could be used to more accurately identify cattle with a normal endometrium prior to conducting procedures such as embryo transfer. However, further studies with a much larger number of cattle are needed to verify the effects of endometrial sampling on pregnancy rates.
Abstract
Endometrial infections are a common cause of reproductive loss in cattle. Accurate diagnosis is important to reduce the economic losses caused by endometritis. A range of sampling procedures have been developed which enable collection of endometrial tissue or luminal cells or uterine fluid. However, as these are all invasive procedures, there is a risk that sampling around the time of breeding may adversely affect subsequent pregnancy rate. This systematic review compared the pregnancy rates (PR) of cattle which underwent uterine lavage (UL), cotton swab (CS), cytobrush (CB), cytotape (CT), or endometrial biopsy (EB) sampling procedures with those that were not sampled. Using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol, relevant databases, including Pubmed, Web of Science, CAB Abstracts, VetMed Resource–Ruminants, and Scopus, were searched. The outcome measured was the pregnancy rate after the collection of endometrial sample(s). Seven studies, involving a total of 3693 cows, fulfilled the inclusion criteria for the systematic review and allowed the comparison of PR between sampled (n = 1254) and non-sampled cows (n = 2409). The results of the systematic review showed that endometrial sampling procedures can be performed before breeding or shortly after insemination without adversely affecting pregnancy rates in cattle. However, further studies are needed to validate this information.
The objective of this randomized, double-blind, controlled trial was to evaluate the effect of pegbovigrastim (pegylated recombinant bovine granulocyte colony-stimulating factor) on early-lactation disease, milk yield, and reproduction on commercial dairy farms. A total of 1,607 Holstein cows from 6 farms in Ontario and Québec, Canada, were randomly assigned to receive two 2.7-mL subcutaneous injections of either 15 mg of pegbovigrastim (n = 798; Imrestor, Elanco) or sterile physiological saline (placebo; n = 809). The first injection was administered by investigators 1 wk before expected calving, and the second by farm personnel within 24 h after calving, according to the product label. Producers inspected cows daily and using standardized disease definitions, recorded cases of retained placenta, metritis, displaced abomasum, and clinical mastitis until 63 d in milk. Progesterone concentration was measured in serum at wk 3, 5, 7, and 9 postpartum. Cows were examined for purulent vaginal discharge using the Metricheck (Simcro) device and endometritis using the cytobrush method at wk 5 postpartum. Milk production and reproduction data were obtained from farm management software and the national milk recording database. Disease and culling outcomes were assessed with logistic regression, milk production with linear regression, and time-to-event outcomes with proportional hazards regression. All analyses considered parity and pre-treatment body condition score and their interaction with treatment, and accounted for clustering of cows within farm. In a subset of 246 cows, the effect of treatment on metabolic markers (serum concentrations of glucose, β-hydroxybutyrate, nonesterified fatty acids, cholesterol, haptoglobin, albumin, and calcium) was assessed in wk 1 and 2 postpartum. Pegbovigrastim had no significant effects on the incidence of retained placenta, metritis, displaced abomasum, clinical or subclinical mastitis, purulent vaginal discharge, or endometritis. Treatment reduced the serum concentration of glucose, slightly reduced the concentration of albumin, and slightly increased concentrations of β-hydroxybutyrate and nonesterified fatty acids, with no effect on the other markers. There were no differences between treatments in culling risk, time to first insemination, pregnancy at first insemination, or time to pregnancy. Milk yield over the first 3 test days of lactation was 1.0 kg per day lower in the pegbovigrastim group, although a mechanism for that effect could not be explained through analysis of our data.
The objective of this study was to quantify the efficacy of a second intrauterine cephapirin treatment administered 14 d after the initial one on subsequent reproductive performance of postpartum dairy cows affected by purulent vaginal discharge (PVD) or endometritis (ENDO). In total, 4,140 Holstein cows from 30 commercial herds were enrolled in a randomized clinical trial. At 36 (±7) d in milk, cows were examined using the Metricheck device to diagnose PVD. An endometrial cytology sample was also collected from each cow to perform a leukocyte esterase test for diagnosing ENDO. Diagnosis of PVD and ENDO was done cow-side. Cows diagnosed with PVD or ENDO were assigned to receive 1 of 2 treatments: (1) a single intrauterine cephapirin infusion (500 mg of cephapirin benzathin; Metricure, Merck Animal Health, Kirkland, QC, Canada) at the time of initial examination or (2) a single intrauterine infusion at the time of initial examination and a second one 14 d later. Subsequent reproductive and culling events were collected until 200 d in milk. Statistical analyses were performed using univariable and multivariable mixed logistic regression models. In cows affected by PVD, a second intrauterine cephapirin infusion increased the pregnancy risk at first insemination in comparison with cows that only received one treatment (28.0 vs. 38.8%). In cows affected by ENDO, a second treatment also increased the pregnancy risk at first insemination compared with cows that only received one treatment (30.3 vs. 39.2%). Overall, these results demonstrate that administering a second intrauterine cephapirin infusion 14 d after the initial treatment in postpartum cows affected by PVD or ENDO did improve their subsequent reproductive performance.
The objective of this study was to characterize incidences of health disorders during early lactation in a large population of Holstein cows calving in 2 seasons across multiple US dairy herds. In addition, cumulative effects of combinations of health-related events on fertility and survival by season of calving and parity number were tested. Data were prospectively collected from a total of 11,729 cows in 16 herds located in 2 regions in the United States [north (7,820 cows in 10 herds) and south (3,909 cows in 6 herds)]. Cows were enrolled at parturition and monitored weekly for disease occurrence, reproductive events, and survival. Health-related events were grouped into reproductive disorders (REP; dystocia, twins, retained fetal membranes, metritis, and clinical endometritis) and other disorders (OTH; subclinical ketosis, mastitis, displaced abomasum, and pneumonia). Counts of health events within 50 d postpartum were added into each of the groups and categorized as 0, 1, 2, 3, and ≥4 for REP and 0, 1, 2, and ≥3 for OTH. Multivariable logistic regression was used for testing potential associations between categories of disease occurrence and outcome variables, including resumption of ovarian cyclicity, pregnancy per artificial insemination (AI), pregnancy loss, and survival up to and after 50 DIM. The incidence of disease varied with season of calving and parity, and these 2 variables were associated with the reproductive and survival outcomes. The size of the detrimental effect of disease incidence on reproduction and survival depended on disease group and varied for each specific outcome. Resumption of ovarian cyclicity decreased as incidences of disorders increased in both REP and OTH categories. Pregnancy at first AI also was smaller in greater number of REP categories, but the effect of number of OTH categories on pregnancy at first AI was not consistent. Similarly, pregnancy loss at first AI was not affected consistently by REP or OTH. Survival was reduced by REP and OTH. The magnitude of these negative effects was variable, depending on season of calving and parity, but consistently increased with the number of health events during early lactation.
Our objective was to assess the association of cow-related factors with metritis cure risk and economically important outcomes. In this prospective cohort study nested inside a randomized clinical trial, cows enrolled in a clinical trial that aimed to evaluate an alternative metritis therapy that had available plasma samples collected at metritis diagnosis were included. Metritis was defined as fetid, watery, reddish-brownish discharge with or without fever, and cure was defined as the absence of metritis signs 12 d after diagnosis. Cows were randomly allocated to remain untreated (CON; n = 147) or receive subcutaneous injections of 6.6 mg/kg of ceftiofur crystalline-free acid at enrollment and 72 h later (CEF, n = 168). Additionally, a random subset of 150 nonmetritic cows (NMET) was also included to compare milk production, reproductive performance, and culling responses. Cow-related factors evaluated include plasma concentrations of nonesterified fatty acids, β-hydroxybutyrate, and haptoglobin (Hp), parity, rectal temperature, and days in milk (DIM) at metritis diagnosis, vulvovaginal laceration (VL), BCS, dystocia, twins, and retained placenta. Among CON cows, DIM at metritis diagnosis was positively associated with metritis cure [threshold = 8, area under the curve (AUC) = 0.67], whereas plasma Hp concentration tended to be negatively associated with cure of metritis (threshold = 0.54 mg/mL, AUC = 0.64). Among CEF cows, DIM at metritis diagnosis (threshold = 5, AUC = 0.67) and dystocia were positively associated with metritis cure, whereas VL and Hp (threshold = 0.78 mg/mL, AUC = 0.76) were negatively associated with cure. For CON cows that were diagnosed with metritis after 8 DIM or had plasma Hp concentration ≤0.54 mg/mL, milk production, pregnancy, and culling risk were comparable to NMET cows. However, performance was impaired when cows that developed metritis at ≤8 DIM or had Hp >0.54 mg/mL were left untreated. Among CEF cows, Hp, DIM at metritis diagnosis, dystocia, and VL were associated with metritis cure. Milk yield, reproductive performance, and culling losses are more pronounced among CEF cows when metritis was diagnosed at ≤5 DIM, Hp >0.78 mg/mL, or if they had VL or dystocia. In conclusion, these data indicate that timing of the onset of metritis and inflammatory biomarkers could be used for the development of a selective therapy strategy for metritis, but more research is needed to identify more accurate predictors of metritis spontaneous cure and treatment failure.
Our objectives were to describe and compare the uterine bacterial composition of postpartum Holstein cows diagnosed as healthy (n = 8), subclinical endometritis (SCE; n = 8), or clinical endometritis (CE; n = 5) in the fifth week postpartum. We did metagenomic analyses of 16S rRNA gene sequences from endometrial cytobrush samples at 10, 21, and 35 days in milk (DIM), and endometrial bacterial culture at 35 DIM. Uterine bacterial composition in healthy, SCE, and CE was stable at 10, 21, and 35 DIM. Alpha and beta diversities showed a different uterine microbiome from CE compared to healthy or SCE, but no differences were found between healthy and SCE cows. At the phylum level, the relative abundance of Bacteroidetes and Fusobacteria, and at genera level, of Trueperella was greater in CE than healthy or SCE cows. Trueperella pyogenes was the predominant bacteria cultured in cows with CE, and a wide variety of bacterial growth was found in healthy and SCE cows. Bacteria that grew in culture were represented within the most abundant bacterial genera based on metagenomic sequencing. The uterine microbiota was similar between SCE and healthy, but the microbiome in cows with CE had a loss of bacterial diversity.
Postpartum uterine infection reduces fertility in dairy cattle; however, the mechanisms of uterine infection‐mediated infertility are unknown. Paradoxically, infection‐induced infertility persists after the resolution of disease. Oocytes are a finite resource, which are present at various stages of development during uterine infection. It is likely that oocytes development is influenced by uterine infection‐induced changes to the follicular microenvironment. To better understand the impact of infection on oocyte quality we employed global transcriptomics of oocytes collected from heifers after receiving intrauterine infusion of pathogenic Escherichia coli and Trueperella pyogenes. We hypothesized that the oocyte transcriptome would be altered in response to intrauterine infection. A total of 452 differentially expressed genes were identified in oocytes collected from heifers 4 days after bacteria infusion compared to vehicle infusion, while 539 differentially expressed genes were identified in oocytes collected from heifers 60 days after bacteria infusion. Only 42 genes were differentially expressed in bacteria‐infused heifers at both day 4 and day 60. Interferon, HMGB1, ILK, IL‐6 and TGF‐beta signaling pathways were downregulated in oocytes collected at day 4 from bacteria‐infused heifers, while interferon, ILK and IL‐6 signaling were upregulated in oocytes collected at day 60 from bacteria‐infused heifers. These data suggest that bacterial infusion alters the oocyte transcriptome differently at day 4 and day 60, suggesting different follicle stages are susceptible to damage. Characterizing the long‐term impacts of uterine infection on the oocyte transcriptome aids in our understanding of how infection causes infertility in dairy cattle.
The objective of this study was to evaluate the efficacy of 3 vaccine formulations containing proteins (FimH, leukotoxin, and pyolysin), inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes), or both, in the prevention of postpartum uterine diseases. A randomized clinical trial was conducted at a commercial dairy farm; 800 heifers were assigned into 1 of 4 different treatment groups: control, vaccine 1 (bacterin and subunit proteins), vaccine 2 (bacterin), and vaccine 3 (recombinant subunit proteins), and each heifer received a subcutaneous injection of its respective treatment at 240 ± 3 and 270 ± 3 d of gestation. Vaccination significantly reduced the incidence of puerperal metritis when compared with control (9.1% vs. 14.9%, respectively; odds ratio 0.51). Additionally, vaccine 3 was found to reduce the incidence of puerperal metritis when compared with the control (8.0% vs. 14.9%, respectively; odds ratio 0.46). Reproduction was improved for metritic cows that were vaccinated, and the effect was stronger for cows that were treated with vaccine 3. In general, vaccination decreased the total vaginal bacterial load and decreased the vaginal load of F. necrophorum by 9 d in milk. Vaccination reduced the prevalence of puerperal metritis in the first lactation of dairy cows, leading to less metritic disease and improved reproduction.
The cow-side diagnosis of reproductive tract disease (RTD) involves identifying the presence of purulent vaginal discharge (PVD) and ultrasonographic endometritis (UE). The objectives of our study were to obtain prevalence estimates for RTD diagnosed by 2 methods (PVD and UE scoring) and to investigate the risk factors for increased probability of RTD if these methods are used in isolation or in combination. Our retrospective observational cohort study tested the hypothesis that RTD assessed by 2 methods would have similar risk factors, and that those would be mainly cow- and calving-related factors. We analyzed data from 5,049 pre-breeding examinations (PBE) from 2,460 spring-calved cows on 8 farms between 2014 and 2018. Cow-related details assessed were days in milk at PBE, breed, lactation number, dry period length, body condition score at calving and PBE, 305-d milk yield, predicted transmitting ability for production and fertility, the presence of a corpus luteum at PBE, and positive diagnosis the previous year. Calving details assessed were type of sire, calf sex, twinning, stillbirth, calving difficulty score, and retained fetal membranes. We conducted statistical analyses using 4 multivariable logistic regression models to identify the risk of RTD diagnosed by (1) PVD in isolation, (2) UE in isolation, (3) the presence of either PVD or UE; and (4) the presence of both PVD and UE. We accounted for herd, cow, and year as random effects in all 4 models. The overall prevalence of RTD in models 1, 2, 3, and 4 were 7.5, 6.7, 11.6, and 2.6%, respectively. Days in milk at PBE, the interaction between days in milk and retained fetal membranes, twinning, and the predicted transmitting ability for calving interval were consistently significant risk factors for positive scores in all 4 models. Considerable calving difficulty [adjusted odds ratio (AOR) = 13.64], Holstein Friesian dam breed (AOR = 2.58), first lactation (AOR = 2.39), and body condition score at PBE (AOR = 1.64) were risk factors for a positive PVD score but not for a positive UE score. Fifth lactation (AOR = 1.69), a beef-sired calf (AOR = 1.46), and the absence of a corpus luteum at PBE (AOR = 1.57) were risk factors for a positive UE score but not for a positive PVD score. These results support the hypothesis that most of the risk factors for PVD and UE are the same but some are distinctly different, implying that in some instances the 2 methods diagnose separate components of the RTD complex.
Prolonged calving-to-conception interval (CCI) can increase economic loss in cattle. We investigated the effect of post-calving abnormal reproductive tract discharge (ARTD) on CCI in dairy cows and quantified the relationship of ARTD and associated risk factors with CCI. The source population was dairy cows that calved in the study period on three pasture-based, year-round calving farms in the Riverina, NSW, Australia. Farm records and records from veterinarians' visits were analyzed. ARTD was defined as the presence of reproductive tract discharge according to the following classification: per vaginum purulent discharge ≥21 days post-calving or mucopurulent discharge >26 days post-calving. The incidence of ARTD was calculated. A Kaplan-Meier survivor function was used to estimate median time to conception post-calving dependent on the presence or absence of ARTD. Mixed effects Cox-proportional hazard models were used to estimate the direct and indirect effects of ARTD, and other potential risk factors on CCI such as body condition score (BCS), ambient temperature, and milk yield. Model structures were guided by a directed acyclic graph of potential risk factors for ARTD. The incidence of ARTD in lactations was 16% (95% CI 13.8–18.5%) and did not differ significantly between the three farms (P > 0.05). The median CCI was 176 and 118 days for lactations with and without ARTD, respectively (P < 0.01). The rate of pregnancy following calving in cows with ARTD was significantly decreased relative to the rate of pregnancy in cows without (total effect hazard ratio = 0.62, se = 0.18, P = 0.01). High peak milk yield (>32 L) and parity >2 also significantly extended CCI. We did not observe an effect of BCS or ambient temperature on CCI. The incidence of ARTD in the current study was consistent with clinical endometritis (considered a major source of ARTD) reported in other studies. In contrast—and despite regular veterinary assessment and treatment of on the farms in this study—ARTD extended CCI. Evaluation of the economic impact of ARTD on dairy farms in this region is warranted, and methods to identify high risk cows and develop effective interventions are required.
Two retrospective studies examining data of 7,500 lactating cows from a single herd were performed with the objective of evaluating the long-term effects of clinical disease during the early postpartum period on milk production, reproduction, and culling of dairy cows through 305 days in milk (DIM). In the first study, data regarding health, milk production, reproduction, and culling of 5,085 cows were summarized. Cows were classified according to incidence of clinical problem (metritis, mastitis, lameness, digestive problem, or respiratory problem) during the first 21 DIM (ClinD21). During 305 d of lactation, cows that had ClinD21 produced, on average, 410 kg less milk, 17 kg less fat, and 12 kg less protein compared with cows that did not have ClinD21 (NoClinD21). Although the interval to first breeding was not different between groups of interest, pregnancy rate through 305 DIM was lower in cows that had ClinD21 [adjusted hazard ratio (AHR) = 0.81]. When individual breedings were analyzed, cows that had ClinD21 presented lower rates of pregnancy per breeding for breedings performed before 150 DIM, reduced numbers of calving per breeding for breedings performed before 200 DIM, and greater number of pregnancy losses for all breedings performed through 305 DIM. The rate of culling from calving through 305 DIM was higher in cows that had a single ClinD21 (AHR = 1.79) and in cows that had multiple ClinD21 (AHR = 3.06), which resulted in a greater proportion of cows leaving the herd by 305 DIM (NoClinD21 = 22.6%; single ClinD21 = 35.7%; multiple ClinD21 = 53.8%). In the second study, data regarding postpartum health and 305-d yields of milk, fat, and protein were collected from 2,415 primiparous cows that had genomic testing information. Genomic estimated breeding values (EBV) were used to predict 305-d yields of milk, fat, and protein. Genomic EBV and predicted yields of milk, fat, and protein did not differ between cows that had ClinD21 and those that did not have ClinD21. In contrast, the observed 305-d yields of milk, fat, and protein were reduced by 345, 10, and 10 kg, respectively, in cows that had ClinD21 compared with cows that did not have ClinD21. We conclude that clinical disease diagnosed and treated during the first 21 DIM has long-term effects on lactation performance, reproduction, and culling of dairy cows, which contribute to detrimental consequences of health problems on sustainability of dairy herds. Replication of our studies in multiple herds will be important to confirm our findings in a larger population.
Uterine inflammation negatively affects reproductive performance and is an important cause of infertility and subfertility in dairy cows. Several studies have investigated the use of gene expression in endometrial samples collected by biopsy or cytology to evaluate the inflammatory response of the cow uterus. This study aimed to compare the expression of the CCL5, CXCL8, IL6, and IL1B genes in the bovine endometrium according to the site of sample collection [caruncular (C) or intercaruncular (IC)], the collection method (biopsy or cytology), and the category of inflammation based on endometrial cytology (zero, medium, or high) in subclinical endometritis. The reproductive tracts of dairy cows were collected from a slaughterhouse, and punch-biopsy samples of endometrial tissues were obtained from both regions (C and IC). Endometrial cells from these regions were collected with the cytobrush technique and then used for the analysis of mRNA expression by quantitative PCR. After counting polymorphonuclear cells (PMN) by endometrial cytology, 20 uteri with an ovary at stage I (d 1–4 of estrous cycle) were categorized into 3 groups. Uteri with 0% PMN (n = 10) were assigned to group zero, uteri with 5 to 15% PMN (n = 5) to group medium (12.2 ± 1.6% PMN), and uteri with >15% PMN (n = 5) to group high (53.8 ± 32.9% PMN). All data were analyzed with 2-way ANOVA with Bonferroni multiple comparison post test. The results from gene transcripts demonstrated that the region (C or IC) of the endometrial biopsy had no influence on any of the degrees of inflammatory reaction observed. However, gene expression was more elevated in the endometrium of cows with greater inflammation compared with those without inflammation (CCL5, CXCL8, IL6, IL1B) and those with medium inflammation (CCL5, IL6). Expression of the genes evaluated did not differ between the endometrium without inflammation and with medium inflammation. However, in the high inflammation group, all genes were comparatively more expressed in samples collected by cytology relative to those derived from biopsies for both anatomical regions. In conclusion, gene expression did not differ between the C and IC tissue. Samples collected from animals with greater inflammation had greater gene expression than those with zero or medium inflammation. In addition, cytology samples had greater gene expression than biopsy samples in the high inflammation group.
Bacterial overgrowth in the uterus is a normal event after parturition. In contrast to the healthy cow, animals unable to control the infection within 21 days after calving develop postpartum endometritis. Studies on the Microbial Ecology of the bovine reproductive tract have focused on either vaginal or uterine microbiomes. This is the first study that compares both microbiomes in the same animals. Terminal Restriction Fragment Length Polymorphism of the 16S rRNA gene showed that despite large differences associated to individuals, a shared community exist in vagina and uterus during the postpartum period. The largest changes associated with development of endometritis were observed at 7 days postpartum, a time when vaginal and uterine microbiomes were most similar. 16S rRNA pyrosequencing of the vaginal microbiome at 7 days postpartum showed at least three different microbiome types that were associated with later development of postpartum endometritis. All three microbiome types featured reduced bacterial diversity. Taken together, the above findings support a scenario where disruption of the compartmentalization of the reproductive tract during parturition results in the dispersal and mixing of the vaginal and uterine microbiomes, which subsequently are subject to differentiation. This differentiation was observed early postpartum in the healthy cow. In contrast, loss of bacterial diversity and dominance of the microbiome by few bacterial taxa were related to a delayed succession at 7DPP in cows that at 21 DPP or later were diagnosed with endometritis.
Metritis, the inflammation of the uterus caused by polymicrobial infections, is a prevalent and costly disease to the dairy industry as it decreases milk yield, survival, and the welfare of dairy cows. Although the antibiotic ceftiofur is widely used for the treatment of metritis, endometrium and ovary function is compromised, resulting in subfertility and infertility. According to culture-dependent studies, uterine pathogens include Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, and Prevotella melaninogenica. Recent studies using high-throughput sequencing claimed that metritis is a microbiota-associated disease. Herein, we propose that metritis is associated with uterine microbiota with high abundance of Bacteroides, Porphyromonas, and Fusobacterium, but rare bacteria such as Escherichia coli and Helcococcus ovis cannot be ignored.
The objectives of this study were: (i) to assess the reproducibility of polymorphonuclear neutrophil (PMN) cell‐counts at five pre‐defined endometrial sites (corpus uteri, left horn base, right horn base, left horn tip, and right horn tip) and (ii) to determine the agreement for the diagnosis of subclinical endometritis (SE) between the different endometrial sites. Forty milking cows between 28 to 34 days postpartum were enrolled for endometrial sampling using cytobrush‐technique. Intraclass correlation coefficient (ICC) was calculated to evaluate the reproducibility of PMN‐counts at different sites. The right horn base was found to have the greatest agreement of PMN‐counts with the other endometrial sites (ICC = 0.66 to 0.85). Twenty‐eight of 40 cows showed no signs of clinical endometritis and were used for evaluation of agreement for the diagnosis of SE, analyzed by using Cohen′s kappa (κ) statistics. Agreement for SE diagnosis with PMN cut‐off ≥ 5% was greatest between the right horn base and the right horn tip (κ = 0.84), and with PMN cut‐off ≥ 18% between the right horn base and left horn tip (κ = 1.0), respectively. The results indicate that the right horn base can be regarded as suitable for cytobrush‐sampling. The probability to detect an animal positive for SE (PMN ≥ 5%) with a single cytobrush sampling was 51.0%, thus, a second sampling is recommended to improve the accuracy.
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The objectives of this study were (1) to assess the effect of purulent vaginal discharge (PVD) on reproductive performance using a 4-point vaginal discharge score (VDS) and (2) to evaluate the effect of PGF2α treatment in cows with PVD on reproductive performance. Holstein cows (n = 2,473) from 1 commercial dairy farm had their vaginal discharge scored at 32 ± 3 d in milk (DIM) using a 4-point scale. Cows were also scored for body condition score and had their ovaries examined using transrectal ultrasound. A VDS-2 or greater was used to define PVD. Cows with PVD were assigned to receive 2 doses of 500 µg of PGF2α analogue (n = 277; Cloprostenol; PGF Veyx forte, Veyx Pharma Ltd., Schwarzenborn, Germany) 14 d apart (odd ear tag number) or remained untreated (n = 272; even ear tag number). All cows not detected in estrus at 90 ± 3 DIM were enrolled into a timed artificial insemination (AI) protocol. The effect of VDS and the effect of PGF2α in cows with PVD on reproductive performance were evaluated separately. First service conception risk (FSCR), time to first AI, and time to pregnancy were used as indicators for reproductive performance. Data were analyzed with Cox's regression and mixed logistic models. The prevalence of PVD was 22.2% (548/2,473). We detected an interaction of parity and VDS for FSCR and time to pregnancy. In primiparous cows, VDS had no effect on FSCR and time to pregnancy. In multiparous cows, VDS had an effect on FSCR and time to pregnancy. Multiparous cows having a VDS-2 or a VDS-3 had decreased odds of pregnancy at first AI compared with cows having a VDS-0. Multiparous cows with VDS-1 or VDS-3 had a decreased hazard of conceiving within 200 DIM. The treatment effect of PGF2α was conditional on parity. In primiparous animals with PVD without a corpus luteum present at 32 DIM, treatment with PGF2α reduced FSCR. Irrespective of the presence of a corpus luteum, treatment with PGF2α decreased the hazard of conceiving within 200 DIM in primiparous cows. Although multiparous cows with PVD treated with PGF2α had increased odds of pregnancy at first AI, no treatment effect was observed on the hazard of conceiving within 200 DIM. Our results do not support the blanket use of PGF2α as a treatment option in postpartum dairy cows having PVD.
Post-partum metritis is among the most prevalent disease in dairy cows affecting animal welfare and inflicting considerable economic loses. While post-partum contamination of the uterus is rife in dairy cows, only a fraction of these animals will develop metritis. Our main objective was to compare the bacterial communities and the inflammatory response in the endometrium of healthy and metritic dairy cows. Holstein-Friesian cows (n = 35) were sampled immediately following clinical classification as healthy (n = 21), suffering from metritis (n = 13) or septic metritis (n = 1), based on veterinary examination at 5-10 days post-partum. Polymorphonuclear cells (PMN) percentage in endometrial cytology was significantly higher in cows with metritis. Full-thickness uterine biopsy analysis revealed that the luminal epithelium in inter-caruncle areas was preserved in healthy cows, but in metritis it was compromised, with marked PMN infiltration particularly in the apical endometrium. Gram staining revealed that bacterial load and spatial distribution was associated with disease severity. 16S-rDNA bacterial community analysis revealed unique endometrial bacterial community composition in metritic cows, as compared to more diverse communities among healthy cows. The most abundant phyla in healthy cows were Proteobacteria (31.8 ± 9.3%), Firmicutes (27.9 ± 8.4%) and Bacteroidetes (19.7 ± 7.2%), while Bacteroidetes (60.3 ± 10.3%), Fusobacteria (13.4 ± 5.9%) and Firmicutes (10.5 ± 3.3%) were most abundant in the endometrial mucosa of metritic cows. Relative abundance of Bacteroidetes (19.7 ± 7.2% vs. 60.3 ± 10.3%), Fusobacteria (7.5 ± 5.2% vs. 13.4 ± 5.9%) and Proteobacteria (31.8 ± 9.3% vs. 7.3 ± 5.6%) phyla differed significantly between healthy and metritic cows. In summary, endometrial PMN abundance, spatial distribution and bacterial communities differed between healthy and metritic dairy cows at early post-partum.
The objective of this study was to determine the effects of uterine and systemic inflammatory responses to uterine bacterial contamination at calving in dairy cows on the growth and ovulatory outcomes of the first dominant follicle postpartum. Ovulatory capability of the first dominant follicle postpartum was predicted in 53 multiparous cows by using a combination of follicle growth characteristics and circulating estradiol concentrations. Endotoxin levels were assayed in follicular fluid samples that were aspirated the day after ovulatory outcome prediction. Plasma levels of haptoglobin, a proinflammatory acute phase protein, and paraoxonase, a negative acute phase protein were determined. Uterine bacteria and inflammation were evaluated in three uterine fluid samples from each cow collected on the day of calving, the day after follicle aspiration, and at 35 days postpartum. Cows that had a strong initial uterine inflammatory response (robust recruitment of polymorphonuclear leukocytes of ≥ 35% and cows with uterine pH < 8.5 on the day of calving) were more likely to have an ovulatory first dominant follicle. Follicular fluid endotoxin levels were higher in non-ovulatory cows compared with ovulatory cows. Endotoxin levels in circulation were not different between ovulatory groups but were higher prepartum than on day 7 and 14 postpartum. Systemic inflammation characterized by elevated haptoglobin concentrations was higher in non-ovulatory cows despite similar bacterial contamination and circulating endotoxin levels. Paraoxonase activity in follicular fluid was significantly associated with the paraoxonase activity in plasma, however, plasma paraoxonase concentrations were not different between non-ovulatory and ovulatory cows. Cows with a higher uterine bacterial load on the day of calving had slower ovarian follicle growth. In summary, a robust uterine inflammatory response on the day of calving was positively associated with ovarian function while elevated systemic inflammation during the early postpartum period was negatively associated with the ovulatory status of the first dominant follicle postpartum.
We tested the hypothesis that the uterus of virgin heifers and pregnant cows possessed a resident microbiome by 16S rRNA gene sequencing of the virgin and pregnant bovine uterus. The endometrium of 10 virgin heifers in estrus and the amniotic fluid, placentome, intercotyledonary placenta, cervical lumen, and external cervix surface (control) of 5 pregnant cows were sampled using aseptic techniques. The DNA was extracted, the V4 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced using Illumina MiSeq technology (Illumina Inc., San Diego, CA). Operational taxonomic units (OTU) were generated from the sequences using Qiime v1.8 software, and taxonomy was assigned using the Greengenes database. The effect of tissue on the microbial composition within the pregnant uterus was tested using univariate (mixed model) and multivariate (permutational multivariate ANOVA) procedures. Amplicons of 16S rRNA gene were generated in all samples, supporting the contention that the uterus of virgin heifers and pregnant cows contained a microbiome. On average, 53, 199, 380, 382, 525, and 13,589 reads annotated as 16, 35, 43, 63, 48, and 176 OTU in the placentome, virgin endometrium, amniotic fluid, cervical lumen, intercotyledonary placenta, and external surface of the cervix, respectively, were generated. The 3 most abundant phyla in the uterus of the virgin heifers and pregnant cows were Firmicutes, Bacteroidetes, and Proteobacteria, and they accounted for approximately 40, 35, and 10% of the sequences, respectively. Phyla abundance was similar between the tissues of the pregnant uterus. Principal component analysis, one-way PERMANOVA analysis of the Bray-Curtis similarity index, and mixed model analysis of the Shannon diversity index and Chao1 index demonstrated that the microbiome of the control tissue (external surface of the cervix) was significantly different from that of the amniotic fluid, intercotyledonary placenta, and placentome tissues. Interestingly, many bacterial species associated with postpartum uterine disease (i.e., Trueperella spp., Acinetobacter spp., Fusobacteria spp., Proteus spp., Prevotella spp., and Peptostreptococcus spp.) were also present in the uterus of virgin heifers and of pregnant cows. The presence of 16S rRNA gene sequence reads in the samples from the current study suggests that the uterine microbiome is established by the time a female reaches reproductive maturity, and that pregnancies are established and maintained in the presence of a uterine microbiome.
Subclinical endometritis (SCE) is highly prevalent in dairy cows, causing negative effects on reproductive outcomes and the producer economy. Genetic selection for animals with better resilience against uterine disease should be prioritized due to both sustainability and animal welfare. Therefore, the aim of the present study was to estimate the heritability of SCE in the Norwegian Red (NR) population. Moreover, future perspectives of the condition as a fertility phenotype for breeding are discussed. A total of 1,642 NR cows were sampled for SCE at the time of artificial insemination, using cytotape. The percentage of polymorphonuclear cells (PMN) in each sample was established by cytology, through the counting of 300 PMN and epithelial cells. The mean percentage of PMN was 5%. Different trait definitions were examined, and SCE was defined as binary traits, based on the following cut-off levels of PMN: Cyto0 = PMN >0, Cyto3 = PMN >3%, Cyto5 = PMN >5%, Cyto10 = PMN >10%, and Cyto20 = PMN >20%. The mean ranged from 0.07 (Cyto20) to 0.59 (Cyto0). We also analyzed PMN as a continuous variable using percent PMN. Information on the animals and herds was obtained from the Norwegian Dairy Herd Recording System. The pedigree of cows with data included a total of 24,066 animals. A linear animal model was used to estimate the heritability. The only trait definition that had an estimated genetic variance larger than the standard error was Cyto5, with an estimated heritability of 0.04. For all other definitions, the genetic variance was not significantly different from zero. A cut-off level of 5% PMN has been established as a general threshold for the definition of SCE in earlier literature. The standard errors of the estimated variance components were relatively large, and results should be interpreted with caution. However, the current study indicates that SCE is heritable at a similar level to that of clinical endometritis and metritis, and has potential as a future fertility phenotype to be used for breeding purposes. A more feasible method to diagnose SCE is needed to establish larger data sets.
The detection of reproductive tract disease (RTD) 3 wk postpartum is important because of its effect on subsequent reproductive outcomes. Numerous methods for the diagnosis of RTD are described, some of which are more practical and instantaneous in terms of diagnosis. Two of these methods involve identification of purulent vaginal discharge (PVD) and evidence of ultrasonographic uterine changes indicative of endometritis (UE). The objectives of our retrospective observational study were (1) to assess the association of PVD or UE score at the prebreeding examination (PBE) with the hazard of pregnancy within the subsequent breeding season; (2) to determine the test sensitivity (Se) and specificity (Sp) at the point of sampling of both tests using a Bayesian latent class model; and (3) to determine the effect of varying positivity thresholds on test accuracy. To achieve these objectives, we analyzed an initial data set of 5,049 PBE from 2,460 spring-calved cows in 8 herds between 2014 and 2018. Each PBE was conducted once between 25 and 86 d in milk. At each PBE, vaginal discharge was obtained with a Metricheck device (Simcro) whereas uterine contents were assessed using transrectal ultrasonography. Purulent vaginal discharge was scored on a scale of 0 to 3 depending on discharge character, and UE was scored on a scale of 0 to 4 depending on the presence and consistency of intraluminal fluid. Cows with scores of ≥2 in either test had received treatment. Fertility data were available from 4,756 PBE after data exclusion. The association between PVD or UE score at the PBE and subsequent hazard of pregnancy was analyzed using a Cox proportional hazards model. Cows with a PVD score of 2 or 3 were less likely to conceive than cows with a PVD score 0 [score 2 hazard ratio (HR) = 0.74; 95% confidence interval (CI): 0.59–0.94; score 3 HR = 0.65; 95% CI: 0.51–0.84]. Cows with a UE score of 1, 2, 3, or 4 were less likely to conceive than cows with a UE score of 0 (score 1 HR = 0.82; 95% CI: 0.73–0.93; score 2 HR = 0.79; 95% CI: 0.62–1.00; score 3 HR = 0.43; 95% CI: 0.43–0.90; score 4 HR = 0.39; 95% CI: 0.26–0.58). To determine the Se and Sp of PVD or UE score for diagnosis of RTD at the time of PBE, a Bayesian latent class model was fitted on 2,460 individual cow PBE. Flat priors were used for the Se and Sp of UE, whereas informative priors were used for PVD Se (mode = 65%, 5th percentile = 45%) and Sp (mode = 90%, 5th percentile = 80%) and RTD prevalence (mode = 20%, 5th percentile = 10%). Posterior estimates (median and 95% Bayesian probability intervals; BPI) were obtained using ‘rjags' (R Studio). The optimal test thresholds (PVD and UE score ≥1) were selected by assessing the effect of different thresholds on test estimates and using a misclassification cost analysis. Based on these, median (95% BPI) Se for PVD and UE score ≥1 were 44% (29–60%) and 67% (33–100%), respectively. Median Sp for PVD and UE score ≥1 were 90% (86–93%) and 91% (86–93%), respectively. Higher scores in both tests were associated with impaired fertility, and UE scoring with a threshold of ≥1 had the highest test Se and Sp estimates although test Se was conditional on days in milk when the PBE occurred.
David Schneider explains the concept of disease tolerance and describes the first observations of this phenomenon in animal models.
Oxidized phospholipids that result from tissue injury operate as immunomodulatory signals that, depending on the context, lead to proinflammatory or anti-inflammatory responses. In this Perspective, we posit that cells of the innate immune system use the presence of oxidized lipids as a generic indicator of threat to the host. Similarly to how pathogen-associated molecular patterns represent general indicators of microbial encounters, oxidized lipids may be the most common molecular feature of an injured tissue. Therefore, microbial detection in the absence of oxidized lipids may indicate encounters with avirulent microorganisms. By contrast, microbial detection and detection of oxidized lipids would indicate encounters with replicating microorganisms, thereby inducing a heightened inflammatory and defensive response. Here we review recent studies supporting this idea. We focus on the biology of oxidized phosphocholines, which have emerged as context-dependent regulators of immunity. We highlight emerging functions of oxidized phosphocholines in dendritic cells and macrophages that drive unique inflammasome and migratory activities and hypermetabolic states. We describe how these lipids hyperactivate dendritic cells to stimulate antitumour CD8+ T cell immunity and discuss the potential implications of the newly described activities of oxidized phosphocholines in host defence. In this Perspective, the authors propose that innate immune detection of oxidized phospholipids, which result from tissue injury, allows the immune system to assess the degree of danger; the detection of oxidized phosphocholines in the presence of pathogen-associated molecular patterns or damage-associated molecular patterns triggers a heightened immune response.
Despite the rapid growth in popularity of the Jersey breed, most research on dairy cows in the United States has been done with the Holstein breed. Postpartum uterine diseases negatively influence reproductive performance of dairy cows and limited data are available regarding predisposing factors for uterine diseases in Jersey cows. Our objectives were to determine the prevalence and risk factors for purulent vaginal discharge (PVD) and its effect on fertility of lactating Jersey cows. This was a retrospective observational study with data collected from 3,822 Jersey cows. The Metricheck device was used for PVD diagnosis, and positive cases (≥50% of pus in exudate) were further classified for severity using the following 4 categories based on the amount of pus observed: 50 to 60% pus in exudate, 60 to 90% pus in exudate, 90 to 100% pus in exudate, and 90 to 100% pus in exudate + uterine fluid detected by palpation per rectum. Univariable and multivariable regression analyzes were conducted to dissect the risk factors for PVD and severity of PVD in Jerseys cows. The major risk factors for PVD were calving-related problems, retained fetal membrane, metritis, and days in the close-up diet. A subgroup of cows (n = 740) was scored for body condition and locomotion scores and had blood sampled in the peripartum for determination of plasma concentrations of nonesterified fatty acids and β-hydroxybutyrate. In the multivariable analysis that considered data collected for the subgroup of cows, peripartum nonesterified fatty acids, postpartum β-hydroxybutyrate, and peripartum locomotion and body condition scores were not retained in the reduced model of predictors of PVD. Not surprisingly, pregnancy per artificial insemination following the first and second services was reduced in cows diagnosed with PVD compared with cows without PVD. In addition, PVD was associated with increased odds of pregnancy loss after the first service and reduced hazard of pregnancy by 305 d in milk. The stratification of PVD severity according to the amount of pus observed and the consistency of the uterus was meaningful, as observed by the differences in reproductive outcomes between cows with diverging amounts of pus in the retrieved exudate. Risk factors for PVD in Jersey cows was similar to previously reported for Holstein cows, and a strong detrimental effect of PVD on fertility was also observed in Jersey cows.
The objective was to investigate the economic effect of treating dairy cows with metritis using ceftiofur-free acid or leaving them untreated at the time of diagnosis. Cows with a fetid, watery, red-brownish vaginal discharge were diagnosed with metritis (d 0). Data from 875 dairy cows (506 primiparous and 369 multiparous) from 1 herd in northern Florida that had been part of a larger study evaluating different treatments for metritis were used for the economic analysis. Holstein cows with metritis had been randomly assigned to: Ceftiofur (CEF, n = 239) = subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid in the base of the ear at d 0 and d 3; Untreated (UNT, n = 233) = no treatment applied at metritis diagnosis. Both groups could receive escape therapy if condition worsened. A group of nonmetritic healthy cows (NMET; n = 403) from the same cohort was randomly selected for comparison. Continuous outcomes such as 300-d milk production (kg/cow), milk sales (/cow), treatment cost by 60 days in milk (/cow), replacement cost (/cow), and gross profit per cow (112 vs. 2,969 vs. 3,219). The stochastic analysis showed that the mean difference in gross profit between UNT and NMET was -207; saleable milk (82%) and initial metritis treatment cost (9%) accounted for most of the variation. The mean difference in gross profit between the UNT and the CEF group was -$250; replacement cost (41%) and cow sales (31%) accounted for most of the variation. In summary, metritis caused large economic losses when left untreated, and CEF reduced those losses by improving fertility, reducing culling and replacement cost, and reducing milk yield losses.
The main objective of this study was to evaluate the efficacy of intrauterine administration of chitosan microparticles (CM) in curing metritis in dairy cows. A secondary objective was to evaluate the effects of metritis treatments on milk yield, survival, and reproductive performance. Cows with a fetid, watery, red-brownish vaginal discharge were diagnosed with metritis. Holstein cows (n = 826) with metritis from 3 dairies located in northern Florida were blocked by parity (primiparous or multiparous) and, within each block, randomly assigned to one of 3 treatments: CM (n = 276) = intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water at the time of metritis diagnosis (d 0), 2 (d 2), and 4 (d 4) d later; ceftiofur (CEF; n = 275) = subcutaneous injection of 6.6 mg/kg ceftiofur crystalline-free acid in the base of the ear at d 0 and d 3; Control (CON; n = 275) = no treatment applied at metritis diagnosis. All groups could receive escape therapy if condition worsened. Cure was considered when vaginal discharge became mucoid and not fetid. A group of nonmetritic (NMET; n = 2,436) cows was used for comparison. Data were analyzed by generalized linear mixed and Cox's proportional hazard models. Cows in CM and CON had lesser risk of metritis cure on d 12 than cows in CEF (58.6 ± 5.0 vs. 61.9 ± 4.9% vs. 77.9 ± 3.9, respectively). The proportion of cows culled within 60 days in milk (DIM) was greater for cows in CM than for cows in CEF and CON (21.5 ± 2.7 vs. 9.7 ± 1.9 vs. 11.3 ± 2.0%, respectively). Treatment did not affect rectal temperature or plasma nonesterified fatty acids, β-hydroxybutyrate, and haptoglobin concentrations. Milk yield in the first 60 DIM differed for all treatments, and it was lowest for CM (35.8 ± 0.3 kg/d), followed by CON (36.8 ± 0.3 kg/d) and CEF (37.9 ± 0.3 kg/d). The hazard of pregnancy up to 300 DIM was lesser for CM than CEF (hazard ratio = 0.62; 95% CI: 0.50–0.76), for CM than CON (hazard ratio = 0.77; 95% CI: 0.62–0.95) and for CON than CEF (hazard ratio = 0.80; 95% CI: 0.65–0.99). Culling was greater, and milk yield and fertility were lesser for CEF than NMET. In summary, CM did not improve the cure of metritis, and was detrimental to milk yield, survival, and fertility compared with CON. In contrast, CEF increased the cure of metritis, milk yield, and fertility compared with CM and CON. Finally, the negative effects of metritis on milk yield culling and fertility could not be completely reversed by CEF.
Endometritis, diagnosed either by assessing the proportion of nucleated cells that are neutrophils (PMN%) following cytology of the endometrium or by assessing the degree of purulent material within the vagina (purulent vaginal discharge or PVD score), is prevalent among dairy cows. However, limited data exist as to the degree of variation among herds in the prevalence of endometritis diagnosed by these 2 methods. Thus, we undertook a study involving uterine cytological and vaginal sampling at a median of 41 d in milk of 1,807 cows from 100 seasonally breeding dairy herds in New Zealand. The optimal cut-point for PMN% was determined by receiver operator characteristic (ROC) curve analysis using conception to first artificial insemination (AI) as the outcome variable. The prevalence of disease was then calculated at the cow and herd levels, and an estimate of the effect of clustering of cow within a herd was calculated. Reproductive outcomes were collated and associations between endometritis and reproductive outcomes assessed using multivariable models. The optimal PMN% cut-point was ≥2%. The correlation of results for cows within a herd (the intraclass correlation) was 0.03, which was significant; hence, subsequent modeling accounted for this clustering. The cow-level prevalence of PMN% ≥2% was 27.0% [95% confidence interval (CI): 25.0 to 29.1%], whereas the mean within-herd prevalence of PMN% ≥2% was 27.1% (95% CI: 24.7 to 29.6%; range: 5.0 to 63.6%), and the prevalence among herds varied significantly. An elevated PMN% (≥2%) was significantly associated with a reduction in the proportion of cows conceiving to first AI (45.8 vs. 54.5%), a reduced proportion of cows submitted for AI in the first 3 wk of the seasonal breeding program (83.7 vs. 89.3%), and a lower proportion pregnant in the first 3 (44.4 vs. 55.4%) and 6 wk (67.5 vs. 76.4%) of the breeding program relative to cows with a low PMN% (i.e., <2%). A total of 24.6% of cows had a PVD score ≥2. The herd mean prevalence of PVD score ≥2 was 25.1% (95% CI: 22.5 to 27.7%; range: 5.0 to 65.0%) and varied significantly among herds. The level of agreement (kappa) between the PVD score and PMN% was low (16.8%) and nonsignificant. The effects of PVD score and PMN% on reproductive outcomes were independent. The within-herd median prevalence of endometritis based on combining both diagnostic tests and using a Bayesian latent class model was 22.9% (Bayesian 95% CI: 10.4 to 40.1%). We conclude that more than one-fifth of dairy cows have endometritis diagnosed either by PMN% or PVD in seasonal breeding herds when assessed at an average of 41 DIM, which was, on average, 30 d before the start of the seasonal breeding program. There is large and unexplained variation in prevalence of endometritis among herds. The 2 diagnostic methods were both associated with reproductive outcomes but have low levels of agreement between them and their effects appear to be independent.
Infection of the postpartum uterus with pathogenic bacteria is associated with infertility months later in dairy cattle. However, it is unclear whether these bacterial infections lead to long-term changes in the reproductive tract that might help explain this infertility. Here we tested the hypothesis that infusion of pathogenic bacteria into the uterus leads to changes in the transcriptome of the reproductive tract three months later. We used virgin Holstein heifers to avoid potential confounding effects of periparturient problems, lactation, and negative energy balance. Animals were infused intrauterine with endometrial pathogenic bacteria Escherichia coli and Trueperella pyogenes (n = 4) and compared with control animals (n = 6). Three months after infusion, caruncular and intercaruncular endometrium, isthmus and ampulla of the oviduct, and granulosa cells from ovarian follicles > 8 mm diameter were profiled by RNA-sequencing. Bacterial infusion altered the transcriptome of all the tissues when compared with control. Most differentially expressed genes were tissue-specific, with 109 differentially expressed genes unique to caruncular endometrium, 57 in intercaruncular endometrium, 65 in isthmus, 298 in ampulla, and 83 in granulosa cells. Surprisingly, despite infusing bacteria into the uterus, granulosa cells had more predicted upstream regulators of differentially expressed genes than all the other tissues combined. In conclusion, there were changes in the transcriptome of the endometrium, oviduct and even granulosa cells, three months after intrauterine infusion of pathogenic bacteria. These findings imply that long-term changes throughout the reproductive tract could contribute to infertility after bacterial infections of the uterus.
Up to forty percent of dairy cows develop metritis or endometritis when pathogenic bacteria infect the uterus after parturition. However, resilient cows remain healthy even when exposed to the same pathogens. Here, we provide a perspective on the mechanisms that dairy cows use to prevent postpartum uterine disease. We suggest that resilient cows prevent the development of uterine disease using the three complementary defensive strategies of avoiding, tolerating and resisting infection with pathogenic bacteria. Avoidance maintains health by limiting the exposure to pathogens. Avoidance mechanisms include intrinsic behaviors to reduce the risk of infection by avoiding pathogens or infected animals, perhaps signaled by the fetid odor of uterine disease. Tolerance improves health by limiting the tissue damage caused by the pathogens. Tolerance mechanisms include neutralizing bacterial toxins, protecting cells against damage, enhancing tissue repair, and reprogramming metabolism. Resistance improves health by limiting the pathogen burden. Resistance mechanisms include inflammation driven by innate immunity and adaptive immunity, with the aim of killing and eliminating pathogenic bacteria. Farmers can also help cows prevent the development of postpartum uterine disease by avoiding trauma to the genital tract, reducing stress, and feeding animals appropriately during the transition period. Understanding the mechanisms of avoidance, tolerance and resistance to pathogens will inform strategies to generate resilient animals and prevent uterine disease.
Until 2010, our knowledge of the uterine microbiome in cows that developed uterine disease relied almost exclusively on culture-dependent studies and mostly included cows with clinical endometritis (i.e., with purulent uterine discharge). Those studies consistently found a strong positive correlation between Trueperella pyogenes and clinical endometritis, whereas other pathogens such as Escherichia coli, Fusobacterium necrophorum, Prevotella melaninogenica, and Bacteroides spp. were also commonly cocultured. In contrast, Streptococcus spp., Staphylococcus spp., and Bacillus spp. were usually isolated from healthy cows. Starting in 2010, culture-independent studies using PCR explored the microbiome of cows with metritis and clinical endometritis, and observed that E. coli was a pioneer pathogen that predisposed cows to infection with F. necrophorum, which was strongly associated with metritis, and to infection with T. pyogenes, which was strongly associated with clinical endometritis. Starting in 2011, culture-independent studies using metagenomic sequencing expanded our knowledge of the uterine microbiome. It has been shown that cows have bacteria in the uterus even before calving, they have an established uterine microbiome within 20 min of calving, and that the microbiome structure is identical between cows that develop metritis and healthy cows until 2 d postpartum, after which the bacterial structure of cows that developed metritis deviates in favor of greater relative abundance of Bacteroidetes and Fusobacteria and lesser relative abundance of Proteobacteria and Tenericutes. The shift in the uterine microbiome in cows that develop metritis is characterized by a loss of heterogeneity and a decrease in bacterial richness. At the genus level, Bacteroides, Porphyromonas, and Fusobacterium have the strongest association with metritis. At the species level, we observed that Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis were potential emerging uterine pathogens. Finally, we have shown that the hematogenous route is a viable route of uterine infection with uterine pathogens. Herein, we propose that metritis is associated with a dysbiosis of the uterine microbiota characterized by decreased richness, and an increase in Bacteroidetes and Fusobacteria, particularly Bacteroides, Porphyromonas, and Fusobacterium.
Most dairy cows experience a transient decrease in feed intake in the 1 to 2 wk before calving, which has been associated with systemic inflammation (SI), indicated by increased blood haptoglobin (Hp) concentration. We aimed to characterize the association between prepartum decrease in feed intake and the onset of SI and, if present, the ability of meloxicam (MEL), a non-steroidal anti-inflammatory drug, to mitigate SI. Holstein cows (n = 45) were assigned to control (n = 13), feed restriction (FR) untreated (FR-U; n = 15), and FR treated with MEL (FR-T; n = 17) groups. Daily feed intake was measured from -22 d from expected parturition until 35 d postpartum. Control cows were fed ad libitum, whereas FR-U and FR-T cows were reduced to 60% of their average intake for 4 consecutive days (-15 to -12 d from expected calving). The FR-T cows received MEL (0.5 mg/kg of body weight) once daily for 4 consecutive days (-13 to -10 d from expected calving). Blood samples were collected -22, -15, -14, -13, -12, -10, -7, -5, -3, 0, 1, 3, 5, 7, 15, 22, and 35 d relative to calving to measure serum concentrations of total calcium, total protein, albumin, globulin, cholesterol, urea, glucose, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, β-hydroxybutyrate, nonesterified fatty acids, Hp, and insulin-like growth factor-1. Serum concentrations of lipopolysaccharide-binding protein were measured -22, -15, -14, -13, -12, and -10 d from expected calving. Simplified glucose tolerance tests were performed on -15, -12, -5, 1, and 5 d relative to calving. Mixed linear regression models were used to assess the effects of FR and MEL on each metabolite. The interaction between treatment group and blood sampling day was forced into each model. All models accounted for body condition score, parity, and the cow as a random effect. Nonesterified fatty acids concentrations in both the FR-U and FR-T groups significantly increased from the second until the last day of FR. Feed restriction increased urea concentrations compared with the control group on -14 d but decreased urea concentrations on -10 d from expected calving. Control cows had greater β-hydroxybutyrate concentrations compared with FR cows on 15, 21, and 35 d postpartum. For all other metabolites, no differences were found. This model of FR produced substantial fat mobilization but based on serum Hp and lipopolysaccharide-binding protein concentrations did not generate measurable SI; therefore, we were unable to evaluate the ability of MEL to mitigate SI.
We sought to determine whether pre-eclampsia, spontaneous preterm birth or the delivery of infants who are small for gestational age were associated with the presence of bacterial DNA in the human placenta. Here we show that there was no evidence for the presence of bacteria in the large majority of placental samples, from both complicated and uncomplicated pregnancies. Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA. The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour. We conclude that bacterial infection of the placenta is not a common cause of adverse pregnancy outcome and that the human placenta does not have a microbiome, but it does represent a potential site of perinatal acquisition of S. agalactiae, a major cause of neonatal sepsis.
Metritis is a prevalent disease with effects on production, reproduction, and survival, thereby affecting dairy farm profitability. A component of the cost of metritis is antimicrobial therapy. Some antimicrobials result in milk withhold that adds to the cost of disease. The objectives were (1) to determine cost of metritis for 2 antimicrobial treatments using a herd budget that includes costs associated with incidence of concurrent diseases, milk production and reproduction losses, and removal from the herd and (2) to apply sensitivity analysis to determine the cost of different scenarios. Cows with metritis from a previous study assigned randomly to receive ampicillin (AMP, n = 259), an antimicrobial that requires milk withhold, or ceftiofur (CEFT, n = 269), an antimicrobial with no milk withhold, were used for the economic analysis. A group of cows with no metritis (NOMET, n = 268), matched by parity and calving day, served as the baseline for comparison. The incidence of other diseases in the first 60 d postpartum, culling and death, reproductive performance, discarded milk, milk yield, total milk sold per cow, and residual cow value were used as responses. The economic analysis considered the costs associated with therapy, reproductive management, discarded milk, estimated DM consumed, income from saleable milk, and the residual cow value at 300 d postpartum or earlier if the cow was removed from the herd. Sensitivity analyses were performed considering 3 scenarios for milk and feed prices. The incidence of diseases other than metritis (NOMET, 30.4%; AMP, 45.4%; CEFT, 34.0%) and days in the hospital (NOMET, 2.7 d; AMP, 8.6 d; CEFT, 3.5 d) were greater for cows treated with AMP than CEFT. Treatment did not affect the risk of leaving the herd (NOMET, 15.5%; AMP, 15.0%; CEFT, 19.1%). The 21-d pregnancy rate was lower for cows with metritis but did not differ between AMP and CEFT (NOMET, 24.9%; AMP, 18.9%; CEFT, 17.0%). Milk yield was greater for cows without metritis than those with metritis and greater for AMP than CEFT (NOMET, 33.7 kg/d; AMP, 32.5 kg/d; CEFT, 31.2 kg/d). Cost of metritis did not differ with choice of therapy, but it increased as milk price increased. When both milk and feed prices were the average values considered (0.26/kg, respectively), the costs of a case of metritis for AMP and CEFT were, respectively, 410 when milk was discarded and 406 when milk was fed to calves. Cost of therapy for AMP and CEFT represented 16.6 and 24.6% of the total cost of metritis when milk was discarded and 21.5 and 24.8% of the total cost of metritis when milk was fed to calves. The largest component of cost of metritis for both therapies was the reduced income from milk minus feed cost, ranging from 40.0 to 56.7%. Collectively, metritis is an expensive disease, and choice of antimicrobial therapy did not influence survival, reproduction, or cost of the disease.
Metritis is associated with reduced fertility in dairy cows, but the mechanisms are unclear because the disease resolves several weeks before insemination. One hypothesis is that metritis causes persistent changes in granulosa cells during follicle development, which might be evident in the transcriptome of granulosa cells from dominant follicles weeks after parturition. To test this hypothesis we collected follicular fluid and granulosa cells from dominant follicles 63 days post partum from cows previously diagnosed with metritis, at least 6 weeks after resolution of the disease, and from cows not diagnosed with metritis (control cows). Bacterial lipopolysaccharide was detected in follicular fluid, and concentrations were associated with follicular fluid IL-8 and glucose concentrations. Transcriptome analysis using RNAseq revealed 177 differentially expressed genes in granulosa cells collected from cows that had metritis compared with control cows. The most upregulated genes were ITLN1 , NCF2 , CLRN3 , FSIP2 and ANKRD17 , and the most downregulated genes were ACSM1 , NR4A2 , GHITM , CBARP and NR1I3 . Pathway analysis indicated that the differentially expressed genes were involved with immune function, cell-cell communication, cell cycle and cellular metabolism. Predicted upstream regulators of the differentially expressed genes included NFκB, IL-21 and lipopolysaccharide, which are associated with infection and immunity. Our data provide evidence for a persistent effect of metritis on the transcriptome of granulosa cells in ovarian follicles after the resolution of disease.
Diseases of postpartum dairy cows impair reproductive processes, resulting in prolonged anestrus, reduced conception, and increased pregnancy attrition, regardless of whether the initial disease precedes insemination (even by many weeks), occurs close to insemination, or follows fertilization. Bacteria and their products activate pattern recognition receptors that respond to pathogen-associated molecular patterns (PAMP). These receptors include toll-like receptors (TLR), nucleotide-binding oligomerization domain (NOD)-like receptors and others, and their activation culminates in upregulation of proinflammatory cytokines such as IL-1β, IL-18, and tumor necrosis factor-α. These may have direct effects on the uterus and conceptus. Importantly, however, these inflammatory mediators, as well as the bacterial products, make their way to the ovary via the general circulation (even from distant sites) or possibly by using the countercurrent vascular mechanism that normally transports endometrial prostaglandin to the ipsilateral ovary. Endotoxin reaches concentrations in follicular fluid that exceed those found in the circulation or even in the infected uterus. Ovarian follicular cells also express TLR and can respond directly to bacterial products including endotoxin, impairing their function. Inflammation is accompanied by increased oxidative stress. The process of oocyte development from activation of primordial oocytes to potential ovulation spans 4 mo. Competence by an oocyte encompasses the ability to undergo not only fertilization but also a complex cytoplasmic maturation that lays the foundation for completion of meiosis at the appropriate time, the transition to mitosis in the zygote, and further development of the conceptus. Oocyte maturation relies on intimate association between cumulus cells and the oocyte, characterized by gap junctions through which molecules of various sizes pass. Signaling also occurs in the oocyte-to-cumulus cell direction. Because both granulosa and theca interna cells are capable of responding to inflammatory mediators, with observed alterations in some functions, it seems likely that disturbed ovarian follicular function may contribute to failure of oocytes to become fully competent, even if the insult occurs well before ovulation. Therefore, interruption of normal fertility by uterine infections may be mediated at the level of the uterine environment but the effect on the ovary and oocyte is likely to be even more important.
Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows.
The objectives of this retrospective study were: 1) to determine the sensitivity (Se) and specificity (Sp) of leukocyte esterase test (LE), endometrial cytology (CYTO) and vaginal discharge score (VDS) for diagnosis of reproductive tract diseases in dairy cows at the moment of sampling; 2) to assess the impact of different thresholds on test accuracy and misclassification costs; and 3) to quantify herd prevalence of reproductive tract diseases in dairy farms from Québec, Canada. Data from 2092 cows (39 herds) enrolled in two randomized control trials were included. Cows were examined at 35 (± 7) days in milk using LE, CYTO, and VDS to determine their reproductive tract health status. A latent class model assuming conditional dependence of CYTO and LE was fit within a Bayesian framework. Non-informative priors were used for the Se and Sp of LE, CYTO, and VDS, while prior information for disease prevalence was obtained from expert opinions (mode = 20%, 5th percentile = 10%). Posterior inferences (median and 95% Bayesian credible intervals; BCI) were obtained using the WinBUGS statistical freeware. An initial model was built using thresholds of ≥ 1 (small amount of leukocytes), of ≥ 6%, and of ≥ 3 (mucopurulent discharge) for the LE, CYTO, and VDS, respectively. Then, the impact on tests accuracy and misclassification costs of using different thresholds was explored. Optimal thresholds balancing the need for good antimicrobial stewardship and animal health considerations were proposed. The optimal thresholds obtained in the final model were: LE ≥ 2 (moderate amount of leukocytes), CYTO ≥ 6%, and VDS ≥ 2 (mucus with flecks of pus). In the final model, median (95% BCI) Se for LE, CYTO and VDS were 42.6% (38.8–47.0), 45.9% (41.9–50.7), and 64.2% (59.1–70.3), respectively. Median Sp was 90.9% (88.0–93.6) for LE, 92.2% (89.2–94.9) for CYTO and 96.6% (91.3–99.8) for VDS. Median within-herd prevalence of reproductive tract disease was estimated at 47.9% (39.0–56.7). At a threshold ≥ 2 (mucus with flecks of pus), VDS had the highest Se and Sp among the tests evaluated. The LE is an interesting diagnostic alternative for detecting reproductive tract disease in dairy cows. At a threshold ≥ 2 (moderate amount of leukocytes), LE Se and Sp were comparable to those of CYTO. This is the first study reporting the accuracy of LE, CYTO, and VDS for diagnosing reproductive tract diseases in dairy cows at the moment of sampling.
Bacteria are ubiquitous in the uterus of cattle after parturition, but 50 years ago, cows tolerated these bacteria and few animals developed uterine disease. Now, up to 40% of dairy cattle develop postpartum uterine disease. Uterine disease causes infertility by compromising not only the function of the endometrium but also the ovary. Animals defend themselves against pathogens using tolerance and resistance mechanisms. Tolerance is the ability to limit the disease severity induced by a given pathogen burden. Resistance is the ability to limit the pathogen burden, and is usually the function of immunity. Cellular barriers, mucus, and tissue repair contribute to tolerance in the endometrium. However, once tolerance is overcome, endometrial cells also have roles in resistance to pathogens, with innate immunity driving the production of cytokines, chemokines and prostaglandins. We propose that failures in endometrial tolerance to pathogenic bacteria and the subsequent innate immune response shape postpartum uterine disease.
Milk constituent concentrations in samples taken during early lactation are often used to generate proxy measures for energy balance in dairy herds. This study aimed to explore associations between these and other measures routinely recorded by dairy herd improvement schemes and insemination outcome, with an emphasis on the likely predictiveness of such measures for conception risk (the proportion of inseminations that are successful) at herd level. Data from 312 United Kingdom (UK) dairy herds were restructured so that each unit of data represented an insemination at less than 100 DIM. Milk constituent concentrations from first and second test day (corrected for the effects of season and DIM at sampling) were used as potential predictors of insemination outcome in a logistic regression model. Other predictors included representations of milk yield and other information routinely collected by DHIAs; random effects were used to account for clustering at cow and herd level. The final model included a large number of predictors, with a number of interaction and non-linear terms. The relative effect sizes of the measures of early lactation milk constituent concentrations were small. The full model predicted just under 64% of observed variation in herd-year conception risk (i.e. the proportion of inseminations that were successful in each herd in each calendar year): however, around 40% was accounted for by the herd-level random effect. The predictors based on early lactation milk constituent concentrations accounted for less than 0.5% of observed variation, representations of milk yield (both overall level of yield and early lactation curve shape) for around 7%, with the remaining 15% accounted for by DIM at insemination, parity, inter-service interval, year and month. These results suggest that early lactation milk constituent information is unlikely to predict herd conception risk to a useful extent. The large proportion of observed variation explained by the herd-level random effect suggests that there are unmeasured (in this study) or unmeasurable factors which are consistent within herd and are highly influential in determining herd conception risk.
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Aims were to (i) compare specific transcript abundance between endometrial samples collected by transcervical biopsy and cytobrush and (ii) measure the abundance of endometrial transcripts involved in PGF 2α synthesis in samples collected by cytobrush. In Experiment 1, endometrial samples were taken transcervically by cytobrush and biopsy 10 days after ovulation. Compared to biopsy samples, abundance of transcripts for MSTN , AKR 1C4 and PGR was similar, VIM , FLT 1 and PTGES was lower ( p < .05) and KRT 18 and CD 3D was greater in cytobrush samples ( p < .05). Thus, there was an enrichment of epithelial and immune cells in the cytobrush samples. In Experiment 2, endometrial samples were collected by cytobrush on days 10, 13, 16 and 19 after ovulation. Abundance of PGR 2 mRNA was maximum on day 10 then decreased ( p < .05). Abundance of ESR 1 decreased gradually from day 10 to day 16 then increased again on day 19. The greatest abundance of OXTR was noted on day 19. The sequential alterations in abundance of these transcripts are consistent with the release of PGF 2α associated with luteolysis. In summary, cytobrush sampling provides representative, physiologically relevant samples of the luminal epithelium in cattle.
Bacterial invasion of the bovine uterus during the postpartum period occurs in most cows, but the general consensus is that these bacteria are eliminated before the next pregnancy. The pregnant uterus has therefore hitherto been considered a sterile environment, but this assumption has now been challenged by recent studies in humans, which indicate that bacteria can be present in the placenta of term pregnancies without causing abortion. The aim of the present study was therefore to investigate whether bacteria are present in the uterus of pregnant cows. Specimens were taken from the inter-caruncular endometrium and from placentomes of slaughtered pregnant cows (n = 43) and subjected to histology, fluorescence in situ hybridization and massive parallel sequencing. Bacteria were observed in the tissue from 90.7% (39/43) of the cows by fluorescence in situ hybridization. Fusobacterium necrophorum, Porphyromonas levii and Trueperella pyogenes were located within the endometrium, on the endometrial surface and in the caruncular stroma, but their presence was not associated with inflammation. Data from massive parallel sequencing of the 16S rRNA gene from a subset of 15 cows indicated that the most abundant bacteria were the families Porphyromonadaceae, followed by Ruminococcaceae and Lachnospiraceae. Our results indicate that the bovine uterus is not a sterile environment during pregnancy as previously assumed and that a cow can carry a pregnancy despite the presence of a few potentially pathogenic bacteria in the uterus.
In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria such as Escherichia coli causes uterine inflammation resulting in low fertility. The aim of this study was to determine the effect of LPS on the developmental competence of bovine oocytes in vitro. LPS perturbed the nuclear maturation of bovine oocytes by inhibiting meiotic progression. Although LPS did not affect the copy number of mitochondrial DNA, it decreased mitochondrial membrane potential in matured oocytes. LPS inhibited mitochondrial redistribution throughout the cytoplasm. Oocytes matured under LPS treatment showed decreased development to the blastocyst stage. Moreover, the trophoblast cell number of blastocysts was significantly lower when the oocytes were matured in the presence of LPS. Our findings suggest that LPS might impair the nuclear and cytoplasmic maturation of oocytes and obstruct subsequent embryonic development in dairy cows.
In the last decade, several new aspects of the inflammation of the bovine endometrium have been investigated and described, including a new definition of subclinical endometritis. This review summarizes the recent discussion about the definition, diagnosis and pathomechanism of subclinical endometritis. Subclinical endometritis also referred to as cytological endometritis is defined by findings of endometrial cytology, which is usually performed with the cytobrush-technique or by low-volume flushing of the uterus. The sampling procedure is minimally invasive and has no negative impact on subsequent conception rate. The suggested threshold value for polymorphonuclear cells (PMN) as diagnostic for subclinical endometritis depends on the time postpartum and varies from 5 to 18%. It has also been shown that a general threshold of 5% PMN is eligible for all cows between 21 and 62 days postpartum. Accuracy and repeatability of counting PMN under the microscope have been evaluated and can be regarded as reliable. The impact of subclinical endometritis on reproductive performance is characterized by decreased conception rates, and prolonged days to first service and days open. In addition, it has been demonstrated that subclinical endometritis has an impact on survival and quality of the embryo. Some studies, however, did not confirm this negative effect of subclinical endometritis on fertility. More detailed analyses of the cytobrush samples revealed higher mRNA expression of several cytokines in cows with subclinical endometritis compared with healthy cows, and contributed to the understanding of detrimental effects of subclinical endometritis on fertility. In contrast to clinical endometritis, there are no predominant bacteria related to subclinical endometritis, but associations between the presence of α-hemolytic streptococci and Trueperella pyogenes and subclinical endometritis have been found. For the treatment of subclinical endometritis, intrauterine infusions with cephapirin as well as the administration of PGF2α have been recommended. Other studies, however, did not confirm the efficiency of these treatments.
The objectives of this study were to quantify the herd-level prevalence of postpartum diseases in a large number of dairy farms, and to identify prevalence alarm levels of these diseases based on association with a low prevalence of success at first service, with a high prevalence of pregnancy loss following pregnancy diagnosis at first service, and with a high prevalence of postpartum culling. A total of 126 commercial dairy herds were enrolled in this cohort study, and the herd was the unit of interest. Twenty cows from every herd were enrolled during the study period (a total of 2,520 lactating cows in the study). Cows were diagnosed with hyperketonemia, retained placenta, displaced abomasum, purulent vaginal discharge, cytological endometritis, leukocyte esterase endometritis, and prolonged anovulation. The prevalence of each of these diseases was computed for every herd. The study outcomes were the prevalence of success at first service, the prevalence of pregnancy loss following pregnancy diagnosis at first service, and the prevalence of postpartum culling (≤60 d in milk). Descriptive statistics of disease and outcome prevalence were computed. Logistic regression models were used to identify prevalence alarm levels associated with poor outcome prevalence. Median herd prevalence for hyperketonemia, retained placenta, displaced abomasum, purulent vaginal discharge, cytological endometritis, leukocyte esterase endometritis, and prolonged anovulation were 18.8, 4.9, 4.0, 5.0, 29.4, 43.8, and 35.2%, respectively. Herds were defined as having low prevalence of success at first service if <40.0%, as having a high prevalence of pregnancy loss if ≥6.3%, and as having a high prevalence of postpartum culling if ≥13.3%. Risk factors for herds having a low prevalence of success at first service were ≥11.8% hyperketonemia, ≥5.0% purulent vaginal discharge, ≥18.8% cytological endometritis, ≥35.3% leukocyte esterase endometritis, ≥21.0% prolonged anovulation, and ≥4.0% of displaced abomasum. Risk factors for herds having a high prevalence of pregnancy loss were ≥5.0% purulent vaginal discharge and ≥4.9% retained placenta. Risk factors for herds having a high prevalence of postpartum culling were ≥23.1% hyperketonemia, ≥4.9% retained placenta, and ≥4.0% displaced abomasum. Overall, postpartum diseases were prevalent in these dairy herds and alarm levels were identified as risk factors for poor reproductive performance and increased culling.