Supplementation of rumen-protected lysine during the close-up period improves vaginal discharge clearance in Holstein dairy cows.

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.

Exploring vaginal discharge scoring to assess clinical metritis severity: Comparison between intrauterine dextrose and systemic antibiotics treatments.

The objectives of this study were to assess: 1) differences in the metabolic status, systemic inflammation, daily milk yield, and daily rumination time between Holstein dairy cows with different vaginal discharge scores (VDS) in the first 7±3 DIM, and 2) effects of intrauterine dextrose infusion on metabolic status, systemic inflammation, daily milk yield and daily rumination time in dairy cows with VDS4 and VDS5. Cows (n=641) from a farm located in central Pennsylvania were screened at 7±3 DIM (study d 0) to assess vaginal discharge scores. Vaginal discharge was scored using a five-point scale (i.e., 1- clear fluid, 2- <50% white purulent fluid, 3- >50% white purulent fluid, 4- red-brownish fluid without fetid smell, and 5- fetid red-brownish watery fluid). Cows with VDS4 and VDS5 were blocked by parity and randomly assigned to one of two treatment groups: 1) CONV (VDS4 n=15; VDS5 n= 23): two injections of ceftiofur (per label; 6.6 mg/Kg) 72 h apart; and 2) DEX (VDS4 n=15; VDS5 n=22): three intrauterine infusions of a 50% dextrose solution (1 L/cow) every 24 h. Cows that presented a VDS 1, 2, and 3 were categorized as normal vaginal discharge animals (NOMVDS; n=35) and were randomly selected and matched by parity to CONV and DEX cows. Daily milk yield and rumination time for the first 150 DIM were collected from on-farm computer records. Blood samples were collected to assess haptoglobin (HP) and ß-hydroxybutyrate (BHB) concentrations at study d 0, d 7, and d 14 relative to enrollment. Subclinical ketosis was defined as having a BHB concentration >1.2 mmol/dL at any of the sampling points. The data were analyzed using the MIXED and GLIMMIX procedures of SAS as a randomized complete block design. When comparing cows with different VDS (i.e., NOMVDS, VDS4, VDS5) separately, cows with VDS5 had the highest concentration of HP at enrollment compared to cows with VDS4 and NOMVDS; however, cows with VDS4 had higher concentrations of HP compared to cows with NOMVDS. Cows with VDS4 or VDS5 had a higher incidence of subclinical ketosis compared to cows with NOMVDS (p=0.005; VDS4= 62.08±9.16%; VDS5=74.44±6.74%; NOMVDS=34.36±8.53%). Similarly, daily milk yield (p<.0001; VDS4=30.17±1.32 kg/d; VDS5=27.40±1.27 kg/d; NOMVDS=35.14±1.35 kg/d) and daily rumination time (p=0.001; VDS4=490.77±19.44 min; VDS5=465±16.67 min; NOMVDS=558.29±18.80 min) was lower for cows with VDS4 and VDS5 compared to cows with NOMVDS at 7±3 days in milk. When analyzing HP concentration between treatment groups in cows with VDS4 (p=0.70), VDS5 (p=0.25), or VDS4 and VDS5 combined (p=0.31), there was no difference in HP concentration by study d 14 between treatment groups. Interestingly, when only cows with VDS4 were considered for treatment, both treatments, DEX and CONV, increased the daily milk yield to the levels of NOMVDS cows by 14 days in milk. On the other hand, when only cows with VDS5 were considered for treatment, cows treated with DEX produced, on average, 4.48 kg/d less milk in the first 150 days in milk compared to cows treated with CONV or cows that had NOMVDS. Similarly, when cows with either VDS4 or VDS5 were considered for treatment, DEX treatment also impaired milk yield. These results suggest that cows with either VDS 4 or 5 have an altered inflammatory status, and decreased milk yield and rumination compared to cows with NOMVDS. Furthermore, DEX treatment may have similar effects on daily milk yield and metabolic status compared to CONV in cows with VDS4, while DEX is not recommended for cows with VDS5.

Progesterone-independent endometrial mRNA expression in dairy cows with clinical or subclinical endometritis.

Up to 50 % of dairy cows fail to resolve uterine involution and develop chronic clinical (CE) or subclinical endometritis (SE) 21 days after calving. Clinical endometritis is associated with purulent discharge, while SE is not associated with overt clinical signs. Along with numerous knowledge gaps related to its pathogenesis, SE does not allow for a straightforward and effective therapy. Therefore, it is crucial to unravel differences in the expression of genes among healthy, CE, and SE cows. This might contribute to the discovery of new drug candidates and, in consequence, a potentially effective treatment. In the present study, cows between 21 and 28 days postpartum (PP) were examined using vaginoscopy for the presence of vaginal discharge and endometrial cytology for the determination of the endometrial polymorphonuclear cell (PMN) percentage. Next, an endometrial biopsy sample was taken to investigate the expression of 13 selected candidate genes by qPCR. Uterine health status was assigned to healthy (absence of abnormal vaginal discharge and ≤5 % PMN, n = 13), SE (absence of abnormal vaginal discharge and >5 % PMN, n = 30), and CE (mucopurulent or purulent vaginal discharge and >5 % PMN, n = 9). At the same time, a blood sample was collected to assess serum progesterone concentration and to categorize cows as low (≤1 ng/mL) or high (>1 ng/mL) in progesterone. High expression of IL1B, IL6, IL17A, CXCL8, PTGES, PTGS1, PTGS2, and INHBA genes and low expression of FST was noted in the endometrium of CE compared to healthy cows. Increased endometrial INHBA expression was observed in both SE and CE compared to healthy cows. Interestingly, greater expression of PTGES and PRXL2B genes and lower expression of PTGS2 were characteristic of SE versus CE or healthy. Among cows with no overt clinical symptoms of uterine disease (healthy and SE), the endometrial expression of IL1 B, CXCL8, and PTGES was greater in cows with high versus low serum progesterone. Several genes were differentially expressed among healthy, SE, and CE cows indicating different pathways for the development of different uterine diseases. In conclusion, we found progesterone-independent SE markers, which suggests that low endometrial PTGS2 expression may be indicative of an inadequate immune response and thus contribute to the pathogenesis of SE.

Effect of rumen-protected choline on dairy cow metabolism, immunity, lactation performance, and vaginal discharge microbiome.

Rumen-protected choline (RPC) promotes benefits in milk production, immunity, and health in dairy cows by optimizing lipid metabolism during transition period management and early lactation. However, the RPC success in dairy cows depends on choline bioavailability, which is affected by the type of protection used in rumen-protected choline. Therefore, our objectives were to determine the effects of a novel RPC on dry matter intake (DMI), identify markers of metabolism and immunity, and evaluate lactation performance. Dry Holstein (n = 48) cows at 245 ± 3 d of gestation were blocked by parity and assigned to control or RPC treatment within each block. Cows enrolled in the RPC treatment received 15 g/d of CholiGEM (Kemin Industries, Cavriago RE, Italy) from 21 d prepartum and 30 g/d of CholiGEM from calving to 21 d postpartum. During the transition period, DMI was measured daily, and blood was sampled weekly for energy-related metabolites such as ß-hydroxybutyrate (BHB), glucose, and nonesterified fatty acids (NEFA), as well as immune function markers such as haptoglobin (Hp) and lipopolysaccharide-binding protein (LPB). Vaginal discharge samples were collected at the calving and 7 d postpartum and stored in microcentrifuge tubes at -80°C until 16S rRNA sequencing. The main responses of body condition score, body weight, DMI, milk yield, milk components, and immune function markers were analyzed using the GLIMMIX procedure of SAS with the effects of treatment, time, parity, and relevant covariates added to the models. The relative abundance of microbiome α-diversity was evaluated by 3 indexes (Chao1, Shannon, and Simpson) and ß-diversity by principal coordinate analysis and permutational multivariate ANOVA. We found no differences in DMI in the pre- and postpartum periods. Cows fed RPC increased the yields of energy- and 3.5% fat-corrected milk and fat yield in primiparous and multiparous cows, with an interaction between treatment and parity for these lactation variables. However, we found no differences in milk protein and lactose up to 150 DIM between treatments. Glucose, NEFA, and BHB had no differences between the treatments. However, RPC decreased BHB numerically (control = 1.07 ± 0.13 vs. RPC = 0.63 ± 0.13) in multiparous on the third week postpartum and tended to reduce the incidence of subclinical ketosis (12.7% vs. 4.2%). No effects for Hp and LPB were found in cows fed RPC. Chao1, Shannon, and Simpson indexes were lower at calving in the RPC treatment than in the Control. However, no differences were found 7 d later for Chao1, Shannon, and Simpson indexes. The vaginal discharge microbiome was altered in cows fed RPC at 7 d postpartum. Fusobacterium, a common pathogen associated with metritis, was reduced in cows fed RPC. Rumen-protected choline enhanced lactation performance and health and altered the vaginal discharge microbiome which is a potential proxy for uterine healthy in dairy cows. The current study's findings corroborate that RPC is a tool to support adaptation to lactation and shed light on opportunities for further research in reproductive health.

Postpartum vaginal discharge score is associated with genetic traits, postpartum fertility phenotypes, metabolic status, and overall reproductive performance in seasonal-calving pasture-based dairy cows.

The aims of this study were (1) to evaluate potential associations between genetic traits, postpartum phenotypes, cow factors, and postpartum vaginal discharge score (VDS); and (2) to investigate possible associations between postpartum VDS, plasma progesterone (P4) after first service, and reproductive performance. First- and second-parity (n = 2,842) spring-calving lactating dairy cows from 35 dairy herds were enrolled. Farm visits were performed every 2 wk during the postpartum period and weekly during the breeding period. Cows that were at wk 3 and wk 7 postpartum and between 7 and 13 d after first artificial insemination (AI) were examined. Body condition score (BCS) was measured on all farm visits using a 1-to-5 scale (low ≤2.5; target ≥2.75). Transrectal ultrasound examinations were conducted to determine the presence or absence of a corpus luteum (CL). Vaginal discharge score was determined at wk 3 and wk 7 using a Metricheck device (Simcro) and a 1-to-4 scale (1 = clear mucus; 4 = mucopurulent with >50% purulent material ± odor). At wk 3, cows having a VDS ≤2 were considered to have normal reproductive tract health status (RTHS). At wk 7, cows having VDS = 1 were considered to have normal RTHS. Blood samples were collected at each visit, and plasma concentrations of glucose, ß-hydroxybutyrate, fatty acids, and progesterone (only during breeding visit) were analyzed. Animals with target BCS at wk 3 and wk 7 had greater odds of having normal RTHS at wk 3 and wk 7, respectively, than cows with low BCS. Cows with a CL at wk 3 and wk 7 had greater prevalence of normal RTHS at wk 3 and wk 7, respectively, compared with cows without a CL. Cows with normal RTHS had a lesser plasma concentration of ß-hydroxybutyrate at wk 3 and wk 7 and greater plasma concentration of glucose at wk 3 compared with animals with abnormal RTHS. More cows in the greatest quartiles for the fertility subindex of the Economic Breeding Index and genetic merit for milk production traits (milk kg and milk protein [%]) had normal RTHS at wk 3 and wk 7 compared with the other quartiles. Cows with VDS = 4 at wk 7 postpartum had lesser plasma P4 concentration after first AI (-1.2, -1.1, and -1.0 ng/mL compared with cows with VDS = 1, 2, and 3, respectively). Similarly, cows with VDS = 4 at both wk 3 and wk 7 had lesser pregnancy at first service, lesser cumulative pregnancy rates at wk 3, 6, and 12 during the breeding period, and longer interval from mating start date to conception (+3 d if VDS = 4 at wk 3; +5 d if VDS = 4 at wk 7), compared with cows having other VDS. In conclusion, cows with superior genetic merit for fertility traits and milk production traits, and favorable fertility phenotypes at wk 3 and wk 7, were all associated with greater likelihood of having normal RTHS. In turn, abnormal postpartum RTHS and greater postpartum VDS (score = 4) were associated with lesser odds of successful pregnancy establishment.

Association of dry matter intake, milk yield, and days to first ovulation with cytological endometritis in Holstein cows.

Dry matter intake (DMI, kg/d) is closely related to the magnitude of negative energy and protein balance during the transition period, and the metabolic adaptations to support lactation in dairy cows. Thus, DMI might affect the development of cytological endometritis in the early postpartum period. Difficulty to adapt to these metabolic changes is related to impaired immune function and increased occurrence of reproductive disorders. We aimed to examine the association of pre- and postpartum DMI, body weight (BW), body condition score, milk yield and milk composition, and days to first ovulation with cytological endometritis at 15 (CYT15) and 30 DIM (CYT30). A second objective was to understand the association of vaginal discharge with CYT15 and CYT30 and performance. We conducted a pooled statistical analysis of 5 studies, including data from 280 multiparous Holstein cows. Based on the cutoffs for the percentage of uterine polymorphonuclear cells (PMN), determined by taking the median value of the data set for 15 and 30 DIM, cows were categorized as follows: LOW15 (PMN % at 15 DIM ≤24%; n = 125), HIGH15 (PMN % at 15 DIM >24%; n = 125), LOW30 (PMN % at 30DIM ≤7%; n = 141); and HIGH30 (PMN % at 30DIM >7%; n = 139). Cows in HIGH15 consumed an average of 1.97 ± 0.5 kg/d less DM than cows in LOW15 during prepartum, and 3.01 ± 0.5 kg/d less DM during postpartum. Dry matter intake (as a percentage of BW) was higher for cows in LOW15 during pre- and postpartum than for cows in HIGH15. Moreover, cows in HIGH15 tended to have lower milk yield than cows in LOW15 from the third until the fifth week postpartum. Although DMI was not associated with CYT30, DMI (as a percentage of BW) was lower for cows in LOW30 pre- and postpartum than for cows in HIGH30. There was no association between CYT30 and milk yield. Cows in LOW15 had greater days to first ovulation than cows in HIGH15, while cows in LOW30 also had greater days to first ovulation than cows in HIGH30. Simple regression analyses demonstrated linear associations of increased DMI, particularly postpartum, with decreased uterine PMN percentage and lower vaginal discharge score. Additionally, increased units of vaginal discharge score and increased percentage units of uterine PMN were linearly associated with decreased milk yield. Corroborating with the notion of the ovarian function being associated with uterine inflammatory status, cows in HIGH15 and HIGH30 ovulated on average 3 d before than cows in LOW15 and LOW30, respectively. Cytological endometritis at 15 DIM was associated with lower DMI from 4 wk before calving until 4 wk postpartum and was associated with lower milk yield. The association of vaginal discharge with cytological endometritis was variable and dependent on the day of evaluation.

Review: Postpartum reproductive disease and fertility in dairy cows.

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.

Use of intravaginal lactic acid bacteria prepartum as an approach for preventing uterine disease and its association with fertility of lactating dairy cows.

The objective of this study was to evaluate the effects of the use of intravaginal probiotics prepartum on the incidence risk of metritis postpartum and conception risk after first artificial insemination (AI). A total of 606 Holstein cows were enrolled 3 wk before their expected calving date from 2 farms. Cows were randomly assigned to either receive a 2-mL dose of a combination of 3 lactic acid bacteria (probiotic treatment) washed with approximately 2 mL of a sterile saline solution, into the vaginal canal twice weekly until parturition, or no intervention (control). Metritis diagnoses were carried out on 6 and 12 d postpartum. Vaginal discharge and rectal temperature were assessed, and vaginal discharge was scored on a scale from 1 to 4, where 1 = clear and 4 = fetid, purulent discharge. Metritis was defined as cows having a vaginal discharge score of 4 with or without fever (rectal temperature ≥39.5°C) on either 6 or 12 d postpartum, or both. Cows were bred after a 60-d voluntary waiting period primarily via the detection of estrus using automated activity monitors; cows not found in estrus were enrolled onto timed AI protocols to receive first breeding before 100 DIM. Pregnancy diagnosis was carried out at d 35 ± 7 post-AI on both farms. Data were analyzed via ANOVA using linear mixed regression models and survival analysis using a Cox proportional hazard model. Total incidence risk of metritis was 23.7% and 34.4% on farm A and farm B, respectively. Overall, the incidence of metritis was not different between treatment groups (control: 41.6 ± 3.8%; probiotic: 38.6 ± 4.0%); however, an interaction by farm was detected, where the probiotic treatment reduced metritis on one farm but not on the other. Conception risk after first AI was not affected by treatment. However, we detected an interaction between parity and treatment, where multiparous cows receiving the probiotic treatment were more likely to become pregnant compared with multiparous cows within the control group (hazard ratio: 1.33; 95% confidence interval: 1.10-1.60); no effect of probiotic treatment was found on the hazard of pregnancy for primiparous cows. In addition, the probiotic treatment was associated with an increased proportion of cows being detected in estrus for the first AI postpartum. In conclusion, vaginal probiotic treatment applied during the 3 wk prepartum was associated with a decreased incidence of metritis on one farm but not the other, suggesting that farm management may be a key player influencing treatment efficacy. Overall, probiotic treatment was found to have only limited effects on fertility in the current study.

Albumin and other metabolic parameters as potential indicators of purulent vaginal discharge in dairy cows during the transition period.

The aims of this study were to evaluate metabolic profiles obtained at -14, 14, and 28 days in milk (DIM), and to identify potential predictive biomarkers of Holstein dairy cows with purulent vaginal discharge (PVD) at 28 DIM. The body condition score (BCS) and hematocrit (Hct) were evaluated, and a metabolic profile test (MPT) was performed at -14, 14, and 28 DIM using serum samples. Cows at 28 DIM were classified using a vaginoscopy and divided into groups of healthy cows (n=89) and cows with PVD (n=31). Albumin (Alb), total cholesterol (TCho), calcium (Ca) and, magnesium (Mg) levels were lower in cows with PVD than in healthy cows at 14 DIM. At 28 DIM, levels of Alb, TCho, Ca, blood urea nitrogen (BUN), Mg, and Hct were lower in cows with PVD. A multivariate stepwise logistic regression analysis showed that higher non-esterified fatty acids (NEFA; odds ratios; OR=4.47; P<0.01), lower Alb (OR=0.07; P<0.01) and lower TCho (OR=0.99; P=0.08) at 14 DIM, and lower Hct (OR=0.83; P=0.05), lower Alb (OR=0.12; P<0.01), and lower BUN (OR=0.74; P=0.02) at 28 DIM were significantly associated with PVD. In conclusion, serum Alb levels was a potential indicator associated with PVD, reflecting dietary protein deficiency preceding disease. Our findings suggest that MPT should be considered to monitor health status during the postpartum period for early diagnosis of PVD.

Viability and function dynamics of circulating versus endometrial polymorphonuclear leukocytes in postpartum dairy cows with subclinical or clinical endometritis.

We aimed to compare the viability of circulating polymorphonuclear leukocyte (cPMN) and endometrial PMN (ePMN) and their function dynamics in postpartum dairy cows with subclinical (SCE) or clinical endometritis (CE). To do so, blood samples from 38 Holstein cows were collected at -7, 9, 21, and 36 d relative to calving, and endometrial cytology samples from 32 Holstein cows were harvested at 9, 21, and 36 d postpartum. Uterine health status was assessed at 36 d postpartum, and cows were classified as healthy (absence of abnormal vaginal discharge and ≤5% ePMN), SCE (absence of abnormal vaginal discharge and >5% ePMN), or CE (mucopurulent or purulent vaginal discharge and >5% ePMN). Viability (viable, apoptotic, and necrotic) and function parameters phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation were evaluated for cPMN via flow cytometry. For ePMN, only viability and PC were evaluated. The association of cPMN and ePMN viability and functional parameters with reproductive tract health classification were fitted in mixed linear regression models, accounting for repeated measures, sampling day, and interactions of reproductive tract status and day. Cows with CE had a lower proportion of cPMN viability (84.5 ± 2.1%; least squares means ± standard error) and a higher proportion of apoptosis (14.4 ± 2.0%) than healthy (92.4 ± 1.3 and 6.7 ± 1.3%, respectively) or SCE (95.3 ± 2.4 and 3.8 ± 2.3%, respectively) at 9 d postpartum. Interestingly, cPMN intracellular proteolytic degradation was lower [6.2 ± 0.1 median fluorescence intensity (MFI)] in SCE compared with healthy (6.7 ± 0.08 MFI) or CE (6.8 ± 0.1 MFI) at d 9 postpartum. No other differences in cPMN function were found among experimental groups. The proportion of necrotic ePMN was higher for healthy (49.6 ± 5.1%) than SCE (27.4 ± 7.3%) and CE (27.7 ± 7.3%) cows at 36 d postpartum. Also, at 36 d postpartum, the proportion of ePMN performing PC was higher in CE (47.0 ± 8.6%) than in healthy (18.4 ± 7.6%) cows, but did not differ from SCE cows (25.9 ± 8.7%). Results of the present study suggest that cPMN viability and function at 9 d postpartum are associated with the development of uterine disease. Furthermore, ePMN at 36 d postpartum are mostly necrotic in healthy cows but viable and functional in cows with CE, probably due to active uterine inflammation. Remarkably, ePMN in cows with SCE at 36 d postpartum are also mostly viable but seem to display a numerically lower proportion of PC compared with ePMN in CE cows.

Differences in uterine and serum metabolome associated with metritis in dairy cows.

Objectives were to evaluate differences in the uterine and serum metabolomes associated with metritis in dairy cows. Vaginal discharge was evaluated using a Metricheck device (Simcro) at 5, 7, and 11 d in milk (DIM; herd 1) or 4, 6, 8, 10, and 12 DIM (herd 2). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 24). Cows with metritis were paired with herdmates without metritis (i.e., clear mucous vaginal discharge or clear lochia with ≤50% of pus) based on DIM and parity (n = 24). Day of metritis diagnosis was considered study d 0. All cows diagnosed with metritis received antimicrobial therapy. The metabolome of uterine lavage collected on d 0 and 5, and serum samples collected on d 0 were evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Normalized data were subjected to multivariate canonical analysis of population using the MultBiplotR and MixOmics packages in R Studio. Univariate analyses including t-test, principal component analyses, partial least squares discriminant analyses, and pathway analyses were conducted using Metaboanalyst. The uterine metabolome differed between cows with and without metritis on d 0. Differences in the uterine metabolome associated with metritis on d 0 were related to the metabolism of butanoate, amino acids (i.e., glycine, serine, threonine, alanine, aspartate, and glutamate), glycolysis and gluconeogenesis, and the tricarboxylic acid cycle. No differences in the serum metabolome were observed between cows diagnosed with metritis and counterparts without metritis on d 0. Similarly, no differences in uterine metabolome were observed between cows with metritis and counterparts not diagnosed with metritis on d 5. These results indicate that the establishment of metritis in dairy cows is associated with local disturbances in amino acid, lipid, and carbohydrate metabolism in the uterus. The lack of differences in the uterine metabolome on d 5 indicates that processes implicated with the disease are reestablished by d 5 after diagnosis and treatment.

Effects of a single transdermal administration of flunixin meglumine in early postpartum Holstein Friesian dairy cows: Part 1. Inflammatory and metabolic markers, uterine health, and indicators of pain.

The objectives of this study were to assess the effects of a single transdermal administration of flunixin meglumine (FM) in early postpartum Holstein Friesian dairy cows on serum concentrations of inflammatory and metabolic markers, uterine health, and indicators of pain. The hypothesis was that the anti-inflammatory, antipyretic, and analgetic effects of the pharmaceutic agent would reduce systemic inflammation, resulting in improved metabolic and inflammatory profile, diminished incidence of metritis, and reduced expression of pain. A total of 500 cows (153 primiparous, 347 multiparous) from 3 different commercial dairy farms in the northeast of Germany were included in a randomized controlled clinical trial. Farms were preselected based on high haptoglobin concentrations in their fresh lactating cows. Cows were excluded if they had experienced dystocia, stillbirth, or twin birth, or if they showed any signs of milk fever, retained fetal membranes, or fever (>40°C). The cows were treated once with either FM (3.33 mg/kg) or a placebo as control (CON) through transdermal administration between 24 to 36 h postpartum (d 2). General health examinations were performed (daily from d 2-8 and additionally on d 15 postpartum), vaginal discharge was assessed using the Metricheck device (d 8 and 15 postpartum) and serum samples were analyzed for inflammatory and metabolic markers (d 2, 4, and 6 postpartum). Effects of treatment, parity, sampling day, and their interactions were evaluated using mixed effects models. Primiparous cows treated with FM showed lower serum haptoglobin concentrations (0.90 ± 0.08 vs. 1.17 ± 0.07 g/L; ± standard error of the mean) and higher serum albumin concentrations (35.5 ± 0.31 vs. 34.8 ± 0.31 g/L) on d 6 postpartum. They also had a lower risk for purulent vaginal discharge with or without a fever compared with CON cows on d 15 postpartum (odds ratio for CON vs. FM: 1.63, 95% CI: 1.26-2.00), and body temperature was lower throughout the first 15 d in milk (39.1 ± 0.11 vs. 39.2 ± 0.11°C). Multiparous cows treated with FM had lower serum ß-hydroxybutyrate concentrations on d 4 postpartum (0.71 ± 0.05 vs. 0.78 ± 0.05 mmol/L) and d 6 postpartum (0.74 ± 0.05 vs. 0.80 ± 0.05 mmol/L). Regardless of parity, FM-treated cows were significantly less likely to abduct their tail from their body (14.3 vs. 23.6%) and show an arched back (27.9 vs. 39.7%) on the day after treatment compared with CON cows. It can be concluded that FM treatment slightly reduced inflammation and diminished the risk for metritis in primiparous cows, improved metabolic profile in multiparous cows, and reduced expressions of pain in all cows.

Effect of dose and frequency of prostaglandin F2α treatments during a 7-day Ovsynch protocol with an intravaginal progesterone releasing device on luteal regression and pregnancy outcomes in lactating Holstein cows.

Our objective was to evaluate the effect of 3 different Ovsynch protocols on progesterone (P4) and pregnancies per artificial insemination (P/AI), where all cows received a P4 releasing intravaginal device (PRID) from d 0 until d 8. We hypothesized that (1) both modified PGF2α treatments lead to decreased P4 at the second GnRH treatment (G2), resulting in greater P/AI, (2) the treatment effect is influenced by the presence of a corpus luteum (CL) at the beginning of the protocol, and (3) potential vaginal discharge caused by the PRID does not have a negative influence on fertility. Lactating Holstein cows (n = 1,056) were randomly assigned to 1 of 3 treatment groups on a weekly basis (n = 356; control: d 0, 100 µg of GnRH + PRID; d 7, 25 mg of dinoprost; d 8, PRID removal; d 9, 100 µg of GnRH). Cows in the second group (n = 353) received an Ovsynch protocol with a double dose of PGF2α (DoubleDose: d 0, 100 µg of GnRH + PRID; d 7, 50 mg of dinoprost; d 8, PRID removal; d 9, 100 µg of GnRH). Cows in the third group (n = 347) received an Ovsynch protocol with a second PGF2α treatment 24 h after the first one (2PGF: d 0, 100 µg of GnRH + PRID; d 7, 25 of mg dinoprost; d 8, 25 mg of dinoprost and PRID removal; d 9, 100 µg of GnRH). All cows had their ovaries scanned to determine the presence of a CL at the beginning of the Ovsynch protocol. Vaginal discharge score (VS) was evaluated at PRID removal. All cows received timed artificial insemination approximately 16 h after G2. Pregnancy diagnosis was performed via transrectal ultrasonography (d 38 ± 3 after timed artificial insemination) and rechecked on d 80 ± 7 after timed artificial insemination. Blood samples were collected on d 0, 7, and 9 of the protocol to determine P4 concentrations. Treatment affected P4 at G2. Progesterone was lower for 2PGF and DoubleDose cows compared with cows in the control group (control 0.35 ± 0.02 ng/mL; DoubleDose 0.29 ± 0.02 ng/mL; 2PGF 0.30 ± 0.02 ng/mL). Overall, P/AI did not differ among treatments. We found, however, an interaction between treatment and CL at the first GnRH treatment. Cows lacking a CL at the first GnRH treatment in the 2PGF group had greater P/AI (47.9%) compared with the same type of cows in the DoubleDose group (32.7%). We observed an effect of VS on P4 concentration at d 7. We found an increase in P4 with greater VS. Vaginal discharge score at PRID removal tended to have a positive effect on P/AI at d 38 (VS0: 36.5%; VS1: 41.3%; VS2: 49.7%). In conclusion, the addition of a second PGF treatment on d 7 and 8 of a 7-d Ovsynch protocol increased luteal regression and decreased mean P4 at G2. Cows treated with PGF2α 2 times 24 h apart showed greater P/AI, compared with cows treated with an increased dose of PGF2α.

Intrauterine infusion of a pathogenic bacterial cocktail is associated with the development of clinical metritis in postpartum multiparous Holstein cows.

Metritis is a uterine disorder common in dairy cattle caused by bacterial infection, with greater incidence in the early postpartum period. The disease causes delayed uterine involution, with a fetid, watery, red-brown discharge, with animals presenting different clinical signs including fever, dullness, inappetence, decreased milk yield, and dehydration. We developed an in vivo model of clinical metritis in Holstein multiparous cows using a pathogenic cocktail of Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes. A total of 36 multiparous cows were randomly allocated to 1 of 3 treatment groups of 12 animals each. Cows assigned to the control group received an intrauterine administration of sterile saline solution, those in the low-dose group received a bacterial inoculum containing 106 cfu of Escherichia coli, Trueperella pyogenes, and Fusobacterium necrophorum; and those in the high-dose group received 109 cfu of these same organisms, all within 24 h of parturition. Clinical signs; milk yield; dry matter intake; serum concentration of acute phase proteins, metabolites, and cytokines; and bacterial counts in vaginal discharge were measured from parturition until 14 d in milk. Based on clinical signs, cows in the low-dose group had a greater incidence of metritis (83.3%) compared with the control (9%) or high-dose (25%) group. The low-dose group also had lesser dry matter intake compared with controls and an acute phase protein profile that typified metritis. The low-dose group presented greater relative abundance of bacteria from the genus Fusobacterium in the vaginal discharge compared with control cows, and the high-dose group was similar to the control group. Unexpectedly, intrauterine infusion of cows with a lower dose of bacteria was more effective than a higher dose, indicating that the bacterial load may affect metritis incidence in a nonlinear manner. These findings demonstrate that clinical metritis can be experimentally induced in postpartum multiparous Holstein cows, creating a relevant model for the study of uterine diseases.

Prevalence of subclinical endometritis in dairy cows that recovered after treatment of clinical endometritis with cephapirin and PGF2α.

The aims of our study were to evaluate (1) the prevalence of subclinical endometritis (SE) in clinically cured cows after treatment of clinical endometritis (CE) with uterine cephapirin infusion or with systemic application of PGF2α and after self-healing, (2) the prevalence of SE in clinically cured cows in relation to the type of vaginal discharge. The study was conducted on 222 Polish Holstein Friesian cows with CE diagnosed by vaginoscopy and ultrasound. The animals were randomly allocated to one of three groups: Group 1 (n = 72): intrauterine cephapirin infusion; Group 2 (n = 73): single intramuscular injection of dinoprost; and Group 3 (n = 77): left untreated as a control. All cows were reexamined two weeks later (Exam 1). If there was still CE at this examination, these cows were treated using the same method as previously and were examined 2 weeks later (Exam 2). Cows still having CE at the second examination were examined two weeks later (Exam 3). Cows without signs of CE at Exams 1, 2 and 3 were considered cured, and endometrial samples from the uteri were collected by cytobrush to diagnose SE using cytological evaluation of PMN percentage. The threshold for SE was set at ≥ 5% PMNs. Two cows each from Groups 1 and 3 and three cows from Group 2 were not clinically recovered at Exam 3 and were excluded from the analysis. In total, SE was diagnosed in an average of 40.9% of clinically cured cows. The prevalence of SE in Group 1, 2 and 3 was 35.0%, 47.1% and 40.0%, respectively (p > 0.05). There were significantly higher (p < 0.05) SE cases after treatment of CE associated with purulent vaginal discharge than with mucopurulent discharge. In relation to the method of treatment, the prevalence of SE was significantly higher (p < 0.05) in cows treated with cephapirin with purulent vaginal discharge than with mucopurulent vaginal discharge. In conclusion, the study showed a high prevalence of SE in cows clinically cured after treatment of clinical endometritis with cephapirin or PGF2α and after self-healing. The prevalence of SE was significantly higher in cows with purulent vaginal discharge than with mucopurulent vaginal discharge. Therefore, in cows clinically recovered from clinical endometritis, the prevalence of SE should always be considered a negative consequence, and the resulting management of this uterine disease should be implemented at the herd level.

Effect of feeding rumen-protected lysine through the transition period on postpartum uterine health of dairy cows.

Feeding rumen-protected methionine as an indispensable amino acid source has been shown to improve reproductive performance in dairy cows, but the effect of feeding rumen-protected lysine (RPL) during the peripartum period on reproductive performance is not well explored. Therefore, we aimed to determine the effects of feeding RPL (AjiPro-L Generation 3, Ajinomoto Heartland Inc.) prepartum, postpartum, or both on follicular dynamics, uterine health, and mRNA gene expression of the endometrium. Seventy-five multiparous Holstein cows were assigned to 1 of 2 dietary treatments with or without RPL in a randomized, complete block design. A 2 × 2 factorial arrangement of treatments was used. Prepartum (-28 d to calving), animals were fed a diet (68% of dietary DM from forage) with RPL [PRE-L; 0.54% RPL of dietary dry matter intake] or without RPL (PRE-C). After calving, half of the cows from each prepartum treatment group were assigned to a diet (56% forage) with RPL (PRE-L POST-L; PRE-C POST-L; 0.40% RPL of dietary dry matter intake) or without RPL (PRE-C POST-C; PRE-L POST-C) until 28 d in milk (DIM). Vaginal discharge was detected with a Metricheck device (Simcro) to detect metritis, and at 28 DIM polymorphonuclear leukocytes were evaluated as a percentage of the epithelial cells using a cytology brush (Andwin Scientific) and an endometrial tissue biopsy was collected for mRNA expression and histology. The first postpartum follicular growth cycle was monitored at 7, 10, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 28 DIM via transrectal ultrasonography. Time to first ovulation did not differ between treatments and averaged 18 ± 1.6 DIM. Follicular diameter at first ovulation was not affected by the treatments, but the growth rate of dominant follicle before first ovulation tended to be lower for cows in POST-L in comparison with cows in POST-C. Prevalence of fetid vaginal discharge and metritis did not differ between treatments. Cows in PRE-L POST-L had lower polymorphonuclear leukocytes percentage at 15 and 28 DIM than cows in PRE-L POST-C, PRE-C POST-L, and PRE-C POST-C. Feeding RPL prepartum downregulates the expression of TLR4, SLC7A6, EHMT2, and tends to downregulate the expression of PTGES3 in uterine tissues at 28 DIM. Additionally, it upregulates the expression of APOL3 and NFKB1, and tends to upregulate the expression of AHCY and MAT2A. In conclusion, feeding RPL pre- and postpartum improved indicators of uterine immune status, but did not change days to first ovulation postpartum.

Reproductive tract disease in Irish grazing dairy cows: Retrospective observational study examining its association with reproductive performance and accuracy of 2 diagnostic tests.

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.

A pilot study on bacterial isolates associated with purulent vaginal discharge in dairy cows in the south-west region of Western Australia.

This study aimed to determine the bacterial isolates associated with postpartum endometritis among dairy cows in Western Australia and their antimicrobial susceptibility profiles. A cross-sectional study was conducted between June-October 2020. Endometritis was defined as evidence of mucopurulent to purulent vaginal discharge 60-100 days postpartum. Vaginal discharge samples were obtained, cultured, identified and tested for antimicrobial susceptibility. A total of 118 bacterial isolates were grown from 46 animals, representing 36 species. The bacteria isolated from both aerobic and anaerobic cultures included Bacillus (60.2%), Streptococcus (12.7%), Trueperella (10.1%), Escherichia (6.7%) and Staphylococcus (5.9%). The remaining genera <5% were Histophilus, Aeroccocus, Enterococcus and Moraxella. Resistance was variable between isolates, but the highest resistance levels were observed in Streptococcal and Bacillus isolates to enrofloxacin, clindamycin and erythromycin, respectively. All Streptococcal isolates exhibited 100% resistance to enrofloxacin, and the greatest resistance levels were found in Streptococcus luteinises to trimethoprim-sulfamethoxazole 83%, clindamycin 66% and 33% quinupristin-dalfopristin. There was 84.5% resistance to clindamycin and 35.2% to erythromycin in the Bacillus isolates, with the highest resistance found in Bacillus licheniformis and Bacillus subtilis. Escherichia coli exhibited 12.5% resistance to gentamycin, ceftiofur, whereas amoxicillin-clavulanic acid exhibited 37.5%. Within the Staphylococcal isolates, 28.5%, 28.5%, 42.8% and 14.2% resistance to ceftiofur, erythromycin, cefoxitin, penicillin and tetracycline were observed, respectively. The presence of resistance to important antimicrobials for human use, such as cephalosporins, macrolides and fluoroquinolones, highlights the need for judicious use of antimicrobials in dairy cattle.

Defining clinical diagnosis and treatment of puerperal metritis in dairy cows: A scoping review.

Although puerperal metritis (PM) is a common infectious disease in dairy cattle, there are currently discrepancies between clinical case definitions within and between available peer-reviewed literature and on-farms practices. The inconsistent use of PM criteria across studies and on-farms practices can result in disparities related to recommendations for treating cows, affecting judicious use of antimicrobials. The objective of this study was to systematically review the peer-reviewed literature for clinical signs used for case definition of PM. The criteria used included local (e.g., vaginal discharge) and systemic clinical signs of infection (e.g., fever, drop in milk). The Preferred Reporting Items for Systematic Review and Meta-Analysis extension for scoping reviews protocols were used to screen commonly used databases. Following this protocol, one reviewer screened title and abstract for eligibility (n = 2,096), followed by full-text screening of selected articles (n = 396) by 2 reviewers to confirm eligible articles (n = 174). The most frequently cited reference article (37.5%) for the definition of PM was published in 2006, followed by articles published between 1998 and 2009 (13%). In 40.2% of articles, no reference was provided for definition of PM; vaginal discharge was described in terms of color, odor, and viscosity when related to the PM definition. Terms used for description of vaginal discharge color were red-brown (61.4%), red (5.1%), brown (8.6%), chocolate (4%), white (1.7%), yellow (0.5%), pink (5.7%), or gray (0.5%); vaginal discharge color was not reported in 24.1% articles. The vaginal discharge odor was described as fetid (75.8%), putrid (5.1%), foul (10.3%), or other (5.7%; e.g., abnormal, malodorous, odoriferous); odor was not mentioned in 7.4% of articles. The vaginal discharge viscosity was described as watery (74.1%), purulent (27%), mucopurulent (8.6%), thin (4%), serous (2.8%), or abnormal (2.3%) and was not mentioned in 11.5% of articles. Fever was included in 59.7% of articles as a criterion for PM diagnosis. The most used rectal temperature threshold was ≥39.5°C (56.8%), followed by ≥39.2°C (2.8%). Approaches used for vaginal discharge evaluation included rectal palpation (37.3%), intravaginal exploration with a gloved hand (18.4%), Metricheck (9.8%), or speculum (5.7%); and in 28.7% of articles, diagnostic tools used were not mentioned. Many of the color and odor vaginal discharge descriptions observed in the literature, used synonymous words to describe the same vaginal discharge sample, highlighting a lack of terminology consensus that could result in disagreements, especially due to the subjective character of these clinical evaluations of vaginal discharge color and odor. Although select consensus articles are available, it is common for studies to disregard a reference when defining PM cases. Furthermore, our findings highlight the need for a robust and clear consensus on criteria and terminology used to diagnose PM.

Risk factors for purulent vaginal discharge and its association with reproductive performance of lactating Jersey cows.

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 ß-hydroxybutyrate. In the multivariable analysis that considered data collected for the subgroup of cows, peripartum nonesterified fatty acids, postpartum ß-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.