Exploring associations between the teat apex metagenome and Staphylococcus aureus intramammary infections in primiparous cows under organic directives.

The primary objective of this study was to identify associations between the prepartum teat apex microbiome and the presence of Staphylococcus aureus intramammary infections (IMI) in primiparous cows during the first 5 weeks after calving. We performed a case-control study using shotgun metagenomics of the teat apex and culture-based milk data collected longitudinally from 710 primiparous cows on five organic dairy farms. Cases had higher odds of having S. aureus metagenomic DNA on the teat apex prior to parturition compared to controls (OR = 38.9, 95% CI: 14.84-102.21). Differential abundance analysis confirmed this association, with cases having a 23.8 higher log fold change (LFC) in the abundance of S. aureus in their samples compared to controls. Of the most prevalent microorganisms in controls, those associated with a lower risk of post-calving S. aureus IMI included Microbacterium phage Min 1 (OR = 0.37, 95% CI: 0.25-0.53), Corynebacterium efficiens (OR = 0.53, 95% CI: 0.30-0.94), Kocuria polaris (OR = 0.54, 95% CI: 0.35-0.82), Micrococcus terreus (OR = 0.64, 95% CI: 0.44-0.93), and Dietzia alimentaria (OR = 0.45, 95% CI: 0.26-0.75). Genes encoding for Microcin B17 AMPs were the most prevalent on the teat apex of cases and controls (99.7% in both groups). The predicted abundance of genes encoding for Microcin B17 was also higher in cases compared to controls (LFC 0.26). IMPORTANCE: Intramammary infections (IMI) caused by Staphylococcus aureus remain an important problem for the dairy industry. The microbiome on the external skin of the teat apex may play a role in mitigating S. aureus IMI risk, in particular the production of antimicrobial peptides (AMPs) by commensal microbes. However, current studies of the teat apex microbiome utilize a 16S approach, which precludes the detection of genomic features such as genes that encode for AMPs. Therefore, further research using a shotgun metagenomic approach is needed to understand what role prepartum teat apex microbiome dynamics play in IMI risk.

The effect of selective dry cow therapies based on two different algorithms on antimicrobial use, udder health, milk production, and culling in the absence of internal teat sealant use at dry-off.

The objective of this study was to evaluate the effect of selective dry cow therapy (SDCT) strategies based on 2 different algorithms as compared with blanket dry-cow therapy for measures of udder health, milk yield, and culling in herds not using internal teat sealant. Cows from 2 commercial farms in West Texas were randomized into 3 different groups: SDCT Algorithm 1 (ALG1; n = 455) cows treated with an intramammary antimicrobial at dry-off if somatic cell count (SCC) > 200,000 cells/mL at any Dairy Herd Improvement Association (DHIA) test date or if the cow had 2 or more cases of clinical mastitis during the enrollment lactation; SDCT Algorithm 2 (ALG2; n = 458) cows treated with an intramammary antimicrobial at dry-off if SCC >200,000 cell/mL at last test date or any case of clinical mastitis during the enrollment lactation; Control cows (CON = 447) received blanket dry cow therapy. All cows enrolled in the study did not receive an internal or external teat sealant. Data related to milk and somatic cell count linear score (LSCC) was collected monthly. Milk yield and LSCC during the first 6 mo of lactation were analyzed using repeated measures ANOVA models, while Cox's Proportional Hazards models were fitted to culling and clinical mastitis data. The farm was fitted as a random effect in all models. The percentage of cows receiving antimicrobials at dry cow was 51.3, 24.7, and 100% for ALG1, ALG2, and CON, respectively. Treatment did not influence the IMI dynamics during the dry period. Additionally, no statistical differences related to treatment were observed for LSCC and milk yield. The LSCC for ALG1, ALG2, and CON was 2.44, 2.41, and 2.26, respectively. The average milk yield for ALG1, ALG2, and CON cows was 43.2, 43.2, and 44.0 kg/d, respectively. Treatment did not affect clinical mastitis incidence and culling. The cumulative incidence of clinical mastitis was 19.6%, 19.4%, and 21.4% for ALG1, ALG2, and CON cows respectively. Additionally, the cumulative risk of death or culling was 18.5%, 17.1%, and 19.5% for ALG1, ALG2, and CON cows respectively. In conclusion, SDCT strategies led to a decrease in antimicrobial drug use at dry-off, without significantly impacting the incidence of clinical mastitis, the risk of culling, LSCC and milk yield of dairy cows. However, numerical differences in LSCC and milk yield were observed between treatment groups.

Predictive models for metritis cure using farm-collected data, metabolic and inflammation biomarkers, and hemogram variables measured at diagnosis.

Our objective was to evaluate the accuracy of predictive models for metritis spontaneous cure (SC) and cure among ceftiofur-treated cows using farm-collected data only, and with the addition of hemogram variables and circulating concentration of metabolites, minerals, and biomarkers (BM) of inflammation measured at time of diagnosis. Data related to parity, calving-related issues, BCS, rectal temperature, and DIM at metritis diagnosis were collected from a randomized clinical trial that included 422 metritic cows from 4 herds in Texas, California, and Florida. Metritis was defined as the presence of red-brownish, watery, and fetid vaginal discharge, and cure was defined as the absence of metritis 14 d after initial diagnosis. Cows were randomly allocated to receive systemic ceftiofur therapy (2 subcutaneous doses of 6.6 mg/kg of ceftiofur crystalline-free acid on the day of diagnosis and 3 d later; CEF) or to remain untreated (control). At enrollment (day of metritis diagnosis), blood samples were collected and submitted to complete blood count (CBC) and processed for the measurement of 13 minerals and BM of metabolism and inflammation. Univariable analysis to evaluate the association of farm-collected data and blood-assessed variables with metritis cure were performed, and variables with P ≤ 0.20 were offered to multivariable logistic regression models and retained if P ≤ 0.15. The areas under the curve for models predicting SC using farm data only and farm + BM were 0.70 and 0.76, respectively. Complete blood count variables were not retained in the models for SC. For models predicting cure among CEF cows, the area under the curve was 0.75, 0.77, 0.80, and 0.80 for models using farm data only, farm + CBC, farm + BM, and farm + CBC + BM, respectively. Predictive models of metritis cure had fair accuracy, with SC models being less accurate than models predictive of cure among CEF cows. Additionally, adding BM variables marginally improved the accuracy of models using farm collected data, and CBC data did not improve the accuracy of predictive models.

A randomized controlled trial evaluating the efficacy of systemic ceftiofur administration for metritis therapy in dairy cows and the effect of metritis cure on economically important outcomes.

The main objective of this study was to evaluate the effect of ceftiofur on metritis cure, milk yield, reproductive performance, and culling up to 300 DIM. The secondary objective was to evaluate the effect of metritis cure at 5 (ECURE) and 14 (LCURE) d after diagnosis on milk production, reproduction, and culling. A total of 422 Holstein cows diagnosed with metritis from 4 herds located in TX, CA, and FL were enrolled in a randomized clinical trial. Cows diagnosed with metritis (fetid, watery, reddish/brownish uterine discharge) were blocked by herd and parity and were randomly allocated to receive systemic administration of ceftiofur (CEF) or to remain untreated (CON). In addition, 399 non-metritic cows (NMET) were included for comparison purposes. Metritis cure was evaluated at 5 and 14 d after diagnosis and was defined as the absence of metritis clinical signs. Logistic regression models were fitted to the data to assess the effect of treatment on metritis cure. Milk yield was analyzed using a mixed linear model, while logistic regression, Cox proportional hazard and Kaplan-Meier survival analysis models were fitted to culling and reproduction data. Cows treated with CEF had 1.86 (95% CI: 1.22 - 2.81) and 1.68 (95% CI: 1.02 - 2.75) greater odds of being cured than CON cows at 5 and 14 d after diagnosis, respectively. No effect of CEF was observed for milk yield; however, NMET cows had greater milk yield compared with metritic cows (CEF = 36.0, 95% CI = 33.8 - 38.1; CON = 36.1, 95% CI = 33.9 - 38.2; NMET = 36.9 kg/d, 95% CI = 34.8 - 39.4). Likewise, no effect of CEF was observed on reproductive performance and culling. Nonetheless, the likelihood of conceiving for NMET cows was 1.72 (95% CI = 1.41 - 2.12) and 1.64 (95% CI = 1.33 - 2.00) times greater than for CEF and CON cows, respectively. Ceftiofur-treated and CON cows had 2.93 (95% CI = 1.90 - 4.51) and 2.37 (95% CI = 1.51 - 3.71) greater hazard of culling compared with NMET, respectively. Regardless of treatment, no differences between ECURE and LCURE were observed on milk yield, reproduction, and culling throughout the entire lactation, but cows that cured at 5 or 14 d after diagnosis had greater milk production in the first 60 DIM compared with cows that did not cure (NCURE). Cows in ECURE and LCURE also had a 1.59 (95% CI = 1.16 - 2.16) and 1.49 (95% CI = 1.08 - 2.05) greater hazard of pregnancy and 0.43 (95% CI = 0.26-0.71) and 0.56 (95% CI = 0.34-0.92) hazard of culling compared with NCURE. Ceftiofur therapy increased metritis cure, but benefits to productivity and longevity were not observed. Also, cows that fail to cure have impaired lactation performance, but no differences regarding timing of cure were observed.

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.

Association of time to metritis diagnosis with circulating concentration of metabolites, minerals, and haptoglobin in Jersey cows.

Our objective was to investigate the association of early metritis [EMET, diagnosed at <5 d in milk (DIM)] and late metritis (LMET, diagnosed at ≥5 DIM) with circulating concentrations of energy metabolites, minerals, and haptoglobin (Hp) throughout the first 14 d postpartum. A total of 379 purebred Jersey cows were enrolled in a prospective cohort study from a single herd in west Texas. Cows were examined for metritis using the Metricheck device (Simcro Ltd.) at 4, 7, and 10 DIM. Cows identified by farm employees as possible metritis cases were also evaluated for metritis. Blood samples were collected for analysis of concentrations of Ca, Mg, and glucose at DIM 1 through 5, 7, 10, and 14. Albumin, urea, fructosamine, free fatty acids (FFA), creatinine, and ß-hydroxybutyrate (BHB) were analyzed at DIM 3, 5, 7, 10, and 14, and Hp at DIM 1 through 5 and 7. Data were analyzed using the MIXED and PHREG procedures of SAS (SAS Institute Inc.). A series of mixed general linear models accounting for repeated measures were fitted to the data. The independent variables metritis [no metritis (NMET), EMET, and LMET], DIM of analyte assessment, and parity were forced in all models. Multivariable Cox proportional hazard models were built to assess the risk of pregnancy and culling within 150 DIM. The overall metritis incidence was 26.9% (EMET = 49; LMET = 53; NMET = 277). Average concentrations of glucose, Mg, and urea were not associated with metritis. The associations of Ca, creatinine, BHB, and fructosamine with metritis were dependent on the DIM of analyte assessment. Cows categorized as EMET and LMET had, on average, lower albumin and fructosamine compared with NMET cows. Both EMET and LMET cows had, on average, greater BHB than NMET cows. A greater FFA concentration was only observed in cows diagnosed with EMET compared with NMET cows (EMET = 0.58, LMET = 0.52, NMET = 0.48 mmol/L). Additionally, circulating Hp concentration was greater for LMET and EMET compared with NMET cows, and EMET cows had greater Hp compared with LMET cows (EMET = 1.15; LMET = 1.00; NMET = 0.84). In conclusion, several blood biomarkers were temporally associated with early versus late metritis diagnosis in postpartum Jersey cows. No meaningful differences were observed in production, reproduction, or culling between EMET and LMET cows. These results suggest that cows with EMET undergo a more severe degree of inflammation and negative energy balance compared with NMET cows.

Feeding an acetate-based oral electrolyte reduces the ex vivo Escherichia coli growth potential in the abomasum of calves fed oral electrolytes alone or 30 minutes following a milk feeding compared with feeding a bicarbonate-based oral electrolyte.

Oral electrolyte solutions (OES) are a common, on-farm therapy to reestablish hydration and electrolyte balances in scouring and stressed calves. The objectives were to determine the effects of OES alkalinizing agent and the presence of a milk replacer feeding before OES administration on the abomasal environment in healthy Holstein calves. Abomasum cannulation was performed on 16 Holstein bull calves at 5 d of age. One calf was removed from the study before the calves were randomly assigned to treatments at 9 d of age. Treatments were arranged as a 2-by-2 factorial, with the following factors: oral electrolyte alkalinizing agent [acetate (A) or bicarbonate (B)] and liquid meal type milk replacer (MR) + OES (MR-A, MR-B), or OES only (OES-A, OES-B)]. The OES differed only by alkalinizing agent. On d 9, calves assigned to MR-A (n = 4) or MR-B (n = 4) received their morning MR aliquot 0.5 h before feeding 2 L of OES; the OES-A (n = 3) and OES-B (n = 4) treatment groups were fed 2 L of OES only. Peripheral blood samples and postprandial abomasal fluid samples were collected to assess abomasal pH, abomasal emptying rate (AER), and ex vivo abomasal Escherichia coli growth potential. Postprandial pH was greater in calves fed MR or B-based OES. Abomasal emptying rate was slower in calves receiving MR + OES, regardless of the alkalinizing agent. Ex vivo E. coli colony-forming unit counts were greater in calves fed either MR + OES or bicarbonate-based OES. Supplementing bicarbonate OES in addition to MR alters abomasal dynamics and may promote E. coli growth in postprandial abomasal fluid, partially due to sustained elevations in gastric pH and delayed gastric emptying rates. The OES containing sodium acetate limited ex vivo E. coli growth potential in abomasal fluid, thereby potentially reducing the risk of additional enteric bacterial complications associated with OES therapy.

Heat stress during the transition period is associated with impaired production, reproduction, and survival in dairy cows.

Objectives of this retrospective cohort study were to determine the association of heat stress (HS) exposure during the periparturient period with production, health, reproduction, and survival during the first 90 d postpartum in dairy cows. A total of 5,722 Holstein cows (2,324 nulliparous and 3,397 parous) were categorized into environmental condition groups based on average temperature-humidity index (THI) exposure as thermoneutral (TN) or HS during the prepartum (PRE) and early postpartum (POST) periods into TN-TN (THI: PRE = 57.9 and POST = 63.7), TN-HS (THI: PRE = 68.2 and POST = 72.0), HS-TN (THI: PRE = 71.4 and POST = 65.6), and HS-HS (THI PRE = 72.2 and POST = 72.7). Nulliparous and parous cows were analyzed separately. In nulliparous cows, exposure to HS during the PRE, POST, or both PRE and POST periods was associated with a 1.7 kg/cow per day reduction in milk yield compared with TN-TN. Postpartum HS was associated with increases of 4.4 percentage points in incidence of retained placenta, 18.1 percentage points in incidence of metritis, and 2.0 percentage points in incidence of mastitis, but a reduction of 5.3 percentage points in pregnancy at first AI, and an increase of 4.5 percentage points in pregnancy loss compared with POST TN. Exposure to HS during PRE and POST periods was associated with increased removal from the herd. In parous cows, exposure to HS during PRE, POST, or both PRE and POST was associated with a 2.4-kg/cow per day reduction in milk yield when compared with TN-TN. There was an interaction between HS exposure PRE and POST and incidence of retained placenta, because POST HS was associated with an increase in incidence of 5.8 percentage points within PRE HS cows, whereas no difference was found within PRE TN cows. Postpartum HS was associated with an increase of 6.3 percentage points in incidence of metritis and tended to be associated with an increase of 1.9 percentage points in mastitis incidence compared with POST TN. There was an interaction between HS exposure PRE and POST and pregnancy per AI because POST HS was associated with reduced pregnancy incidence by 10.6 percentage points within PRE TN cows, whereas no difference was found within PRE HS cows. Removal from the herd increased in cows exposed to HS during the PRE or POST or PRE and POST. These data suggest that POST HS is associated with performance losses to a greater extent than prepartum HS and that nulliparous and parous cows are prompt to losses associated with exposure to HS during the transition period. The results corroborate findings from manipulative experiments that showed improved milk production and reproduction in cows under HS provided with heat abatement but adds insights to the associations between HS and health.

Effect of injectable trace mineral supplementation on peripheral polymorphonuclear leukocyte function, antioxidant enzymes, health, and performance in dairy cows in semi-arid conditions.

The objective of this study was to evaluate the effect of subcutaneous injections of 15 mg/mL Cu, 5 mg/mL Se, 60 mg/mL Zn, and 10 mg/mL Mn on health, performance, polymorphonuclear leukocyte (PMNL) function, circulating glutathione peroxidase (GPx) and superoxide dismutase (SOD) concentrations, and inflammation of dairy cows undergoing the transition period in high temperature-humidity index. A total of 923 multiparous cows from 2 commercial dairy farms were randomly allocated into 1 of 2 treatment groups as follows: control and injectable trace mineral supplementation (ITMS). Cows in the ITMS group received 7 mL of subcutaneous injections at dry-off (208 ± 3 d of gestation), 260 ± 3 d of gestation, and at 35 ± 3 d in milk (DIM). Data regarding health traits, reproductive performance, milk yield, and survivability were extracted from farm database software, and animals were followed-up until 300 DIM. For a subset of 142 cows from one herd, blood samples were collected at enrollment, and at 3 ± 1, 7 ± 1, 10 ± 1, and 35 ± 3 DIM to evaluate hematology, PMNL function, GPx and SOD concentrations, and circulating haptoglobin. Logistic regression was used to assess health and pregnancy per artificial insemination at first service. Cox proportional hazards models were used to evaluate hazard of pregnancy and culling. Mixed linear regression models accounting for repeated measures were used to assess all continuous variables collected over time. Parity, twinning, and previous gestation length were considered as potential confounders. Farm was included as a random effect. The ITMS cows tended to have lower incidence of metritis and stillbirth compared with control group. However, ITMS treatment did not influence the incidence of other diseases (e.g., mastitis, retained placenta), milk yield, reproductive performance, culling, and leukocyte count. Neutrophil-to-lymphocyte ratio, PMNL phagocytosis, and oxidative burst as well as intensity of the oxidative burst were greater for ITMS-treated cows in comparison to control cows. The ITMS cows had decreased expression of the adhesion molecule L-selectin on PMNL surface. The serum concentration of GPx and SOD were not affected by ITMS treatment. In conclusion, ITMS tended to reduce the incidence of metritis and stillbirth parturition, improved PMNL function, and improved the inflammatory status of dairy cows undergoing the transition period in high temperature-humidity index conditions. However, these findings did not translate into improved milk yield, reproductive performance, and survivability.

Characterization of metabolic profile, health, milk production, and reproductive outcomes of dairy cows diagnosed with concurrent hyperketonemia and hypoglycemia.

The objective of this prospective cohort study was to characterize the metabolic profile, health, milk production, and reproductive outcomes of cows diagnosed with hyperketonemia (HK; ß-hydroxybutyrate ≥1.2 mmol/L), hypoglycemia (HG; glucose ≤2.2 mmol/L), or concurrent HK and HG (HKHG). Glucose and ß-hydroxybutyrate concentrations in whole blood were assessed using a handheld device (Precision Xtra, Abbott Laboratories) in lactating dairy cows (n = 2,418) between 3 and 9 d postpartum. Cows were categorized into 4 groups: no HK or HG (healthy; Norm = 1,821), HK only (HK = 232), HG only (HG = 161), and concurrent HK and HG (HKHG = 204). Subsequent milk production, along with health and reproductive outcomes, as recorded by farm personnel, were analyzed according to metabolic category. Serum collected on the day of cow-side diagnosis of hyperketonemia and hypoglycemia was evaluated for total calcium (tCa), magnesium (Mg), nonesterified fatty acids (NEFA), triglycerides (TG), and urea using an automated chemistry analyzer (Randox Daytona; Randox Laboratories Ltd.). Statistical analysis was carried out using SAS version 9.4 (SAS Institute Inc.). Hyperketonemia in multiparous cows was associated with greater incidence of metabolic abnormalities (hypomagnesemia, hypocalcemia, and elevated NEFA and urea). Hyperketonemia in primiparous and multiparous cows led to increased adverse health events (culling rate, retained fetal membranes, puerperal metritis, clinical ketosis, left displaced abomasum) relative to Norm cows. Multiparous cows with HKHG had fewer metabolic disturbances (hypomagnesemia, hypocalcemia, elevated NEFA) than HK cows. Cows with HKHG had an increased incidence of clinical ketosis and left displaced abomasum relative to Norm cows. Cows with HG had similar metabolic profiles to Norm cows and had lower incidence of retained fetal membranes and puerperal metritis than cows with HK. Multiparous cows with HG produced more milk than Norm cows from wk 10 to 20, whereas multiparous cows with HK produced less milk than Norm cows. For primiparous cows, HK did not have a negative effect on milk production compared with Norm cows.

Effect of subclinical mastitis detected in the first month of lactation on somatic cell count linear scores, milk yield, fertility, and culling of dairy cows in certified organic herds.

It is well established that subclinical mastitis (SCM), characterized by somatic cell count (SCC) >200,000 cells/mL, has a negative effect on the productivity, reproductive performance, and survivability of cows from conventional dairy herds. However, in organic herds, where the use of antimicrobial drugs is restricted for the treatment and control of intramammary infections (IMI) in dairy cows, little is known about the effect of SCM on performance and survivability. The objective of this study was to evaluate whether SCM diagnosed during the first month of lactation was associated with SCC linear score dynamics, milk production, fertility, and culling of dairy cows in USDA-certified organic herds. We collected data from 2 organic herds in New Mexico and Texas. A total of 1,511 cows that calved between June 2018 and May 2019 were included in the study and were followed until month 10 of the current lactation. Cows with SCC >200,000 cells/mL in the first month of lactation were considered to have SCM. We used mixed linear regression models accounting for repeated measures to assess the effect of SCM on monthly milk production and SCC linear scores. We used Cox proportional hazards models to evaluate the effect of SCM on the risk of pregnancy and culling. We considered parity, farm, previous gestation length, stillbirth, twinning, dystocia, and 2- and 3-way interactions as potential confounders. Cows diagnosed with SCM during the first month of lactation produced less milk than cows without SCM. Cows with SCM had elevated SCC linear scores during their previous lactation and throughout the subsequent months of lactation compared to cows without SCM. The effect of SCM on SCC linear scores was more pronounced in multiparous than primiparous cows. Subclinical mastitis during the first month of lactation did not affect the likelihood of pregnancy during the first 300 d in milk. Cows with SCM in the first month were more likely to die or be culled during the 300 d of lactation than cows without SCM. We observed that elevated SCC in the first month of lactation had detrimental effects on the milk yield and survivability of dairy cows in USDA organic herds, but it did not affect reproductive performance. We demonstrated that cows with SCM diagnosed in the first month of lactation continued to have elevated SCC linear scores throughout their entire lactation, and that elevated SCC was carried over from the previous lactation.

Prolonged, low-grade inflammation in the first week of lactation: Associations with mineral, protein, and energy balance markers, and milk yield, in a clinically healthy Jersey cow cohort.

Our objectives were to perform a proof-of-concept study to assess the association of a prolonged inflammatory state (based on a continually elevated haptoglobin concentration at the end of the first week after parturition) with mineral, protein, and energy balance markers in the first 2 wk after parturition, and milk production in the first 60 d of lactation in clinically healthy cows. We conducted a cohort study in 1 herd in west Texas that was milking Jersey and Jersey-Holstein crosses. Only multiparous Jersey cows were eligible for enrollment. Cows were classified as having or not having elevated haptoglobin concentrations based on plasma concentrations evaluated on d 4 and 7 postpartum. We used median concentrations of haptoglobin in the reference population (i.e., before the exclusion of cows diagnosed with clinical diseases) as the limits for categorizing cows into 2 groups: cows with plasma haptoglobin concentrations greater than the median values on both d 4 (0.49 g/L) and 7 (0.35 g/L) had continually elevated haptoglobin (with eHp); and cows with plasma haptoglobin concentrations lower than or equal to the median values of the reference population on d 4 or 7 did not have continually elevated haptoglobin (without eHp). Next, cows with clinical diseases in the first 2 wk of the postpartum period were excluded, so that 233 cows remained for the final analyses. Evaluated outcomes were average daily milk production across the first 60 d of lactation, plasma Ca, Mg, and glucose concentrations on d 1, 2, 3, 4, 5, 7, 10, and 14 postpartum, and ß-hydroxybutyrate (BHB), nonesterified fatty acids (NEFA), fructosamine, albumin, urea, and creatinine concentrations on d 3, 5, 7, 10, and 14 postpartum. Rectal temperatures measured on d 4, 7, and 10 postpartum were also analyzed. We performed statistical analyses using linear mixed models while accounting for the repeated effect of sampling time (plasma analytes and rectal temperature models) and weekly milk test (milk production model). Cows with eHp had lower plasma Ca concentrations in the first 2 wk after calving, but no differences in Mg, BHB, NEFA, glucose, or rectal temperatures compared to cows without eHp. Cows with eHp had lower plasma fructosamine, albumin, and urea concentrations in a time-dependent manner. They also had lower milk production (2.3 kg/d less than cows without eHp in the first 60 DIM). Our study demonstrated that 25% of cows without clinical disorders in the first 2 wk after parturition had continually elevated haptoglobin concentrations at d 7 after parturition relative to d 4, suggestive of a prolonged, low-grade systemic inflammatory state. More observational studies are needed to more fully characterize the duration of prolonged postpartum subclinical inflammation in cows without clinical diseases, as well as its long-term effects, and to evaluate the use of other potential markers of systemic inflammation to describe this disorder.

Short communication: Associations of serum biomarkers of stress and inflammation measured at arrival with health, mortality, and growth of calves transported within the first 4 days of life.

The objective of this study was to assess the association between serum concentrations of haptoglobin (Hp), cortisol, and l-lactate following transport of dairy heifer calves within their first week of life with health, mortality, and growth during the preweaning period. Blood samples were collected at arrival and included a cohort of 168 Jersey and Jersey-cross calves from 8 different sources located in Minnesota. All calves were raised at a single facility in New Mexico. Incidence of respiratory disease (BRD) and mortality during the preweaning period (60 d of life) were extracted from the farm's software database. Individual body weight was measured at birth and at weaning to estimate average daily gain. No simple linear correlations were found between the biomarkers. The incidence of BRD during the preweaning period was 7.7%. While cortisol and l-lactate serum concentrations were not associated with BRD, Hp was negatively associated with BRD. Receiving operating characteristic curve analysis was performed to determine the optimal threshold value for Hp [threshold ≤0.63 µmol/L (63 µg/mL), area under the curve = 0.65]. A Cox's proportional hazards model revealed that calves with Hp concentration ≤0.63 µmol/L were more likely to be diagnosed with BRD (hazard ratio = 5.0, 95% confidence interval = 1.3-19.0). A mixed linear model showed that calves with Hp concentration ≤0.63 µmol/L tended to have lower average daily gain (454.4 vs. 479.9 g/d) during the preweaning period than calves with Hp >0.63 µmol/L at arrival. Overall mortality of the cohort was 3.5%, and Hp was not associated with mortality. Although circulating concentrations of l-lactate and cortisol measured at arrival were not associated with BRD incidence during the preweaning period of heifer calves transported within the first 4 d of life, calves with serum Hp concentration >0.63 µmol/L were less likely to be diagnosed with BRD and tended to grow more than calves with Hp ≤0.63 µmol/L. Further research is needed to replicate these results in a larger cohort and to better understand the possible influence of greater inflammatory status at arrival on health and growth of calves transported to a calf-raising facility.

Association of blood calcium concentration in the first 3 days after parturition and energy balance metabolites at day 3 in milk with disease and production outcomes in multiparous Jersey cows.

Research exploring specific associations of markers of negative energy balance and Ca in postpartum Jersey cows with lactation performance is lacking. Our objectives were to evaluate the associations of total Ca concentration (tCa) measured at 1 through 3 d in milk (DIM) and free fatty acids (FFA), ß-hydroxybutyrate (BHB), and glucose measured at 3 DIM with (1) the risk of multiparous Jersey cows being diagnosed with early-lactation diseases and culling, (2) milk production in the first 9 wk of lactation, and (3) the risk of pregnancy in the first 150 DIM. A cohort study was performed in 1 dairy herd in Texas. Multivariable Poisson regression models were built to evaluate the association of the analytes of interest with the risks of early-lactation diseases and culling in the first 60 DIM (i.e., binary outcomes). Linear mixed models were used to evaluate the association of the analytes of interest with milk production within the first 9 wk of lactation, and a Cox proportional hazard model was built to assess the risk of pregnancy within 150 DIM. A total of 380 cows were used in the final analyses. Total Ca measured at 1 through 3 DIM was not associated with the risk of metritis. Cows with increased FFA and BHB had an increased risk of being diagnosed with metritis and clinical mastitis, respectively. Increased concentrations of glucose and FFA and decreased tCa at 3 DIM were associated with an increased risk of culling. Reduced tCa concentrations at 1 DIM (≤1.84 mmol/L) and 2 DIM (≤2.04 mmol/L) were associated with increased milk production across the first 9 wk of lactation compared with tCa concentrations above those thresholds. Total Ca was not associated with milk production when assessed at 3 DIM, whereas increased FFA (≥0.37 mmol/L) and decreased glucose (≤2.96 mmol/L) at 3 DIM were associated with increased milk production. None of the metabolites measured were associated with the risk of pregnancy in the first 150 DIM. Our results demonstrate that tCa concentration assessed in the first 3 DIM show temporary associations with milk production and culling in multiparous Jersey cows. Although increased concentration of FFA assessed at 3 DIM was associated with greater milk yield, it was a detrimental factor for the risk of metritis. This study attempted to better elucidate the relationship of tCa, FFA, BHB, and glucose assessed in early postpartum with health and performance of Jersey cows. Based on this study, assessments performed at 3 DIM using tCa concentration ≤1.99 mmol/L for increased risk of early-lactation culling and FFA ≥0.43 mmol/L for increased risk of metritis could be used as starting points. More studies evaluating the dynamics of energy balance markers and tCa in postpartum Jersey cows using a greater number of herds are needed to better inform dairy consultants on critical levels for exacerbated postpartum negative energy balance and subclinical hypocalcemia for the Jersey breed.

Integration of statistical inferences and machine learning algorithms for prediction of metritis cure in dairy cows.

The study's objectives were to identify cow-level and environmental factors associated with metritis cure to predict metritis cure using traditional statistics and machine learning algorithms. The data set used was from a previous study comparing the efficacy of different therapies and self-cure for metritis. Metritis was defined as fetid, watery, reddish-brownish discharge, with or without fever. Cure was defined as an absence of metritis signs 12 d after diagnosis. Cows were randomly allocated to receive a subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid (Excede, Zoetis) at the day of diagnosis and 3 d later (n = 275); and no treatment at the time of metritis diagnosis (n = 275). The variables days in milk (DIM) at metritis diagnosis, treatment, season of the metritis diagnosis, month of metritis diagnostic, number of lactation, parity, calving score, dystocia, retained fetal membranes, body condition score at d 5 postpartum, vulvovaginal laceration score, the rectal temperature at the metritis diagnosis, fever at diagnosis, milk production from the day before to metritis diagnosis, and milk production slope up to 5, 7, and 9 DIM were offered to univariate logistic regression. Variables included in the multivariable logistic regression model were selected from the univariate analysis according to P-value. Variables were offered to the model to assess the association between these factors and metritis cure. Additionally, the univariate logistic regression variables were offered to a recursive feature elimination to find the optimal subset of features for a machine learning algorithms analysis. Cows without vulvovaginal laceration had 1.91 higher odds of curing of metritis than cows with vulvovaginal laceration. Cows that developed metritis at >7 DIM had 2.09 higher odds of being cured than cows that developed metritis at ≤7 DIM. For rectal temperature, each degree Celsius above 39.4°C led to lower odds to be cured than cows with rectal temperature ≤39.4°C. Furthermore, milk production slope and milk production difference from the day before to the metritis diagnosis were essential variables to predict metritis cure. Cows that had reduced milk production from the day before to the metritis diagnosis had lower odds to be cured than cows with moderate milk production increase. The results from the multivariable logistic regression and receiver operating characteristic analysis indicated that cows developing metritis at >7 DIM, with increase in milk production, and with a rectal temperature ≤39.40°C had increased likelihood of cure of metritis with an accuracy of 75%. The machine learning analysis showed that in addition to these variables, calving-related disorders, season, and month of metritis event were needed to predict whether the cow will cure or not from metritis with an accuracy ≥70% and F1 score (harmonic mean between precision and recall) ≥0.78. Although machine learning algorithms are acknowledged as powerful tools for predictive classification, the current study was unable to replicate its potential benefits. More research is needed to optimize predictive models of metritis cure.

Effects of oral calcium bolus supplementation on intracellular polymorphonuclear leukocyte calcium levels and functionality in primiparous and multiparous dairy cows.

The objectives of this study were (1) to characterize Ca levels and polymorphonuclear leukocyte (PMN) function in primiparous and multiparous animals following oral Ca bolus supplementation, and (2) to determine differential responses of boluses containing a lower dose of Ca than traditionally used in primiparous animals on Ca levels and PMN function. Jersey × Holstein crossbred animals (n = 104) were enrolled within 24 h of parturition. All animals were blocked by time relative to calving and randomly assigned to treatment. The Ca boluses were composed of a mixture of Ca chloride, Ca sulfate, and Ca propionate. For objective 1, animals were assigned to control (CON; no Ca supplementation), or a series of 2 Ca boluses given 24 h apart for a total of 50 g of Ca. Objective 2 treatments included control (CON; no Ca supplementation), a series of 2 Ca boluses given 24 h apart containing 50 g of Ca, or a series of 2 Ca boluses given 24 h apart containing 25 g of Ca. Blood samples were collected on d 1 (<24 h), 2, 3, 5, and 7 relative to parturition. Total serum Ca, serum haptoglobin, PMN intracellular Ca, PMN intracellular Ca after stimulation with an environmental Escherichia coli, PMN L-selectin surface expression, and PMN phagocytic and oxidative burst activities were analyzed. For objective 1 a tendency was detected for a treatment difference on basal intracellular PMN Ca and a treatment difference on E. coli-stimulated intracellular PMN Ca. We detected a parity × DIM effect for PMN oxidative burst intensity. However, no other interactions or parity effects on other functional PMN variables were detectable. In primiparous animals, we found a treatment difference for E. coli-stimulated intracellular PMN Ca among animals given 50 g of Ca but no treatment difference on basal intracellular PMN Ca. The 50 g of Ca treatment increased both PMN phagocytosis and oxidative burst intensities. Supplementing animals with 50 g of oral Ca increased intracellular PMN Ca and influenced PMN function.

The association of cow-related factors assessed at metritis diagnosis with metritis cure risk, reproductive performance, milk yield, and culling for untreated and ceftiofur-treated dairy cows.

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, ß-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.

Using chitosan microparticles to treat metritis in lactating dairy cows.

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, ß-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.

Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health, metabolism, and lactation performance in Holstein cattle I: Production and functional characterization of rbIL-8 in vitro and in vivo.

In the present study, we standardized processes of cloning and purification of recombinant bovine interleukin-8 (rbIL-8) from bacterial culture and assessed its biological activity in Holstein cattle. Plasmid containing a subclone of bovine IL-8 was expressed using Escherichia coli BL21 and cell lysate was purified by chromatography. The presence of rbIL-8 was assessed by Western blot analyses and function was confirmed in vitro using a chemotaxis chamber. Based on optical density values, chemoattractant properties of rbIL-8 were 10-fold greater compared with control wells. Two in vivo studies were conducted to assess the biological activity of rbIL8. For study 1, one-year-old Holstein heifers (n = 20) were randomly allocated to receive a single intravaginal administration containing 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 10) or a single intravaginal administration of 20 mL of saline solution (control, n = 10). For study 2, nonpregnant lactating Holstein cows (n = 31) were randomly allocated to receive an intrauterine administration with 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 11), a positive control consisting of resin-purified lysate of E. coli BL21 not transfected with the plasmid coding for rbIL-8 diluted in 20 mL of saline solution (E. coli, n = 10), and a negative control administered with 20 mL of saline solution (control, n = 10). An increase in vaginal neutrophils was observed in heifers treated with rbIL-8 within 3 h of treatment, but not in control heifers. Additionally, intrauterine administration of rbIL-8 increased the proportion of PMN cells in uterine cytological samples from 3.5% before treatment to 75.8% 24 h later-an increase that was not observed in the negative control group and cows treated with resin-purified lysate of E. coli. To further evaluate the effect of local and systemic rbIL-8 stimulation on the dynamics of circulating white blood cells, a third study was conducted. In study 3, nonpregnant 8-mo-old Holstein heifers (n = 30) were randomly allocated into 1 of 3 treatment groups: intravenous rbIL-8 (1,125 µg of rbIL-8 diluted in 5 mL of saline solution, n = 10); intravaginal rbIL-8 (1,125 µg of rbIL-8 diluted in 20 mL of saline solution; n = 10); or intravaginal saline (20 mL of saline solution, n = 10). Intravenous injection of rbIL-8 resulted in a transient increase in rectal temperature, which was greater at 2 h after treatment compared with cows treated intravaginally with rbIL-8 or heifers treated with saline solution. Heifers treated with rbIL-8 intravenously displayed a marked reduction in neutrophils, basophils, lymphocytes, and monocytes within the first 4 h posttreatment compared with heifers treated intravaginally. However, at 6 h after treatment, heifers treated with rbIL-8 intravenously displayed a rebound in white blood cell counts caused by an increase in neutrophil counts. These results show that the presented purification method is effective and results in biologically active rbIL-8 that can be used safely to modulate immune responses in cattle.

Prepartum application of internal teat sealant or intramammary amoxicillin on dairy heifers: Effect on udder health, survival, and performance.

Intramammary infusion of antimicrobials (IA) and application of an internal teat sealant (ITS) at the end of the lactation is a common strategy to prevent and treat intramammary infections during the dry period. In heifers, precalving IA has been reported to improve udder health and milk quality; however, the effect of ITS in heifers housed in freestall barns is still unknown. The objective of this study was to investigate the effect of prepartum application of ITS or IA on udder health of dairy heifers. A total of 886 heifers were randomly allocated into 1 of 4 treatment groups when they were 245 ± 3 d pregnant. Control heifers (CON) did not receive any treatment. Heifers in the ITS group received the application of ITS containing 2.6 g of bismuth subnitrate. Animals in the IA group received intramammary administration of 62.5 mg of amoxicillin. Heifers in IA+ITS group received intramammary administration of amoxicillin followed by ITS application. The follow-up period was 270 d in milk (DIM). Clinical mastitis was diagnosed and treated by trained farm personnel throughout the study period. Composite milk somatic cell counts (SCC) were determined monthly for the first 9 mo of lactation by Dairy Herd Improvement Association. Subclinical mastitis was defined as a cow having a SCC >200,000 cells/mL for at least 1 test day and not diagnosed with clinical mastitis during the study period. At 7 ± 3 DIM, composite milk samples were collected for bacteriological analysis for a subset of the study animals. The presence of pathogens in milk at 7 ± 3 DIM was more frequently detected in the CON heifers compared with animals enrolled in the treatment groups. Heifers in IA+ITS group had a decreased incidence of clinical mastitis compared with CON heifers (12.9 vs. 21.4%). Additionally, the incidence of subclinical mastitis was decreased for IA+ITS heifers compared with CON counterparts (20.1 vs. 43.8%). During the first 9 mo of lactation, IA and IA+ITS heifers had lower SCC linear scores compared with ITS and CON counterparts. Although IA+ITS treatment successfully improved udder health, it did not result in better milk yield, fertility, and survivability. In conclusion, ITS alone was not effective in improving udder health; however, the combination of ITS and IA decreased the incidence of clinical and subclinical mastitis during the first 9 mo of lactation. Additionally, IA+ITS had a long-term effect on the SCC linear score throughout the study period.