Assessing different cross-validation schemes for predicting novel traits using sensor data: an application to dry matter intake and residual feed intake using milk spectral data.

Feed efficiency is important for economic profitability of dairy farms; however, recording daily dry matter intakes (DMI) is expensive. Our objective was to investigate the potential use of milk mid-infrared (MIR) spectral data to predict proxy phenotypes for DMI based on different cross-validation schemes. We were specifically interested in comparisons between a model that included only MIR data (Model M1), a model that incorporated different energy sink predictors, such as body weight, body weight change, and milk energy (Model M2), and an extended model that incorporated both energy sinks and MIR data (Model M3). Models M2 and M3 also included various cow level variables (stage of lactation, age at calving, parity) such that any improvement in model performance from M2 to M3, whether through a smaller root mean squared error (RMSE) or a greater squared predictive correlation (R2), could indicate a potential benefit of MIR to predict residual feed intake. The data used in our study originated from a multi-institutional project on the genetics of feed efficiency in US Holsteins. Analyses were conducted on 2 different trait definitions based on different period lengths: averaged across weeks vs. averaged across 28-d. Specifically, there were 19,942 weekly records on 1,812 cows across 46 experiments or cohorts and 3,724 28-d records on 1,700 cows across 43 different cohorts. The cross-validation analyses involved 3 different k-fold schemes. First, a 10-fold cow-independent cross-validation was conducted whereby all records from any one cow were kept together in either training or test sets. Similarly, a 10-fold experiment-independent cross-validation kept entire experiments together whereas a 4-fold herd-independent cross-validation kept entire herds together in either training or test sets. Based on cow-independent cross-validation for both weekly and 28-d DMI, adding MIR predictors to energy sinks (Models M3 vs M2) significantly (P < 10-10) reduced average RMSE to 1.59 kg and increased average R2 to 0.89. However, adding MIR to energy sinks (M3) to predict DMI either within an experiment-independent or herd-independent cross-validation scheme seemed to demonstrate no merit (P > 0.05) compared with an energy sink model (M2) for either R2 or RMSE (respectively, 0.68 and 2.55 kg for M2 in herd-independent scheme). We further noted that with broader cross-validation schemes, i.e., from cow-independent to experiment-independent to herd-independent schemes, the mean and slope bias increased. Given that proxy DMI phenotypes for cows would need to be almost entirely generated in herds having no DMI or training data of their own, herd-independent cross-validation assessments of predictive performance should be emphasized. Hence, more research on predictive algorithms suitable for broader cross-validation schemes and a more earnest effort on calibration of spectrophotometers against each other should be considered.

Dietary supplementation of rumen native microbes improves lactation performance and feed efficiency in dairy cows.

Objectives were to determine the effects of 2 dietary microbial additives supplemented to diets of Holstein cows on productive performance and feed efficiency. One-hundred and 17 Holstein cows were enrolled at 61 d (31 to 87 d) postpartum in a randomized complete block design experiment. Cows were blocked by parity group, as nulliparous or multiparous cows and, within parity, by pre-treatment energy-corrected milk yield. Within block, cows were randomly assigned to one of 3 treatments administered as top-dress for 140 d. Treatments consisted of either 100 g of corn meal containing no microbial additive (CON; 15 primiparous and 25 multiparous), 100 g of corn meal containing 5 g of a mixture of Clostridium beijerinckii and Pichia kudriavzevii (G1; 4 × 107 cfu of C. beijerinckii and 1 × 109 cfu of P. kudriavzevii; 14 primiparous and 24 multiparous), or 100 g of corn meal containing 5 g of a mixture of C. beijerinckii, P. kudriavzevii, Butyrivibrio fibrisolvens, and Ruminococcus bovis (G2; 4 × 107 cfu of C. beijerinckii, 1 × 109 cfu of P. kudriavzevii, 1 × 108 cfu of B. fibrisolvens, and 1 × 108 cfu of R. bovis; 15 primiparous and 24 multiparous). Intake of DM, milk yield, and BW were measured daily, whereas milk composition was analyzed at each milking 2 d a week, and body condition was scored twice weekly. Milk samples were collected on d 60 and 62 in the experiment and analyzed for individual fatty acids. The data were analyzed with mixed-effects models with orthogonal contrast to determine the impact of microbial additive (MA; CON vs. 1/2 G1 + 1/2 G2) and type of microbial additive (TMA; G1 vs. G2). Results are described in sequence as CON, G1, and G2. Intake of DM (22.2 vs. 22.4 vs. 22.4 kg/d), BW (685 vs. 685 vs. 685 kg) and the daily BW change (0.40 vs. 0.39 vs. 0.39 kg/d) did not differ among treatments; however, feeding MA tended to increase BCS (3.28 vs. 3.33 vs. 3.36). Supplementing MA increased yields of milk (39.9 vs. 41.3 vs. 41.5 kg/d), ECM (37.9 vs. 39.3 vs. 39.9 kg/d), fat (1.31 vs. 1.37 vs. 1.40 kg/d), total solids (4.59 vs. 4.75 vs. 4.79 kg/d), and ECM per kg of DMI (1.72 vs. 1.76 vs. 1.80 kg/kg). Furthermore, cows fed MA increased yields of pre-formed fatty acids in milk fat (>16C; 435 vs. 463 vs. 488 g/d), particularly unsaturated fatty acids (367 vs. 387 vs. 410 g/d), such as linoleic (C18:2 cis-9, cis-12; 30.9 vs. 33.5 vs. 35.4 g/d) and α-linolenic acids (C18:3 cis-9, cis-12, cis-15; 2.46 vs. 2.68 vs. 2.82 g/d) on d 60 and 62 in the experiment. Collectively, supplementing G1 and G2 improved productive performance of cows with no differences between the 2 MA.

Integrating uterine microbiome and metabolome to advance the understanding of the uterine environment in dairy cows with metritis.

BACKGROUND: Metritis is a prevalent uterine disease that affects the welfare, fertility, and survival of dairy cows. The uterine microbiome from cows that develop metritis and those that remain healthy do not differ from calving until 2 days postpartum, after which there is a dysbiosis of the uterine microbiome characterized by a shift towards opportunistic pathogens such as Fusobacteriota and Bacteroidota. Whether these opportunistic pathogens proliferate and overtake the uterine commensals could be determined by the type of substrates present in the uterus. The objective of this study was to integrate uterine microbiome and metabolome data to advance the understanding of the uterine environment in dairy cows that develop metritis. Holstein cows (n = 104) had uterine fluid collected at calving and at the day of metritis diagnosis. Cows with metritis (n = 52) were paired with cows without metritis (n = 52) based on days after calving. First, the uterine microbiome and metabolome were evaluated individually, and then integrated using network analyses. RESULTS: The uterine microbiome did not differ at calving but differed on the day of metritis diagnosis between cows with and without metritis. The uterine metabolome differed both at calving and on the day of metritis diagnosis between cows that did and did not develop metritis. Omics integration was performed between 6 significant bacteria genera and 153 significant metabolites on the day of metritis diagnosis. Integration was not performed at calving because there were no significant differences in the uterine microbiome. A total of 3 bacteria genera (i.e. Fusobacterium, Porphyromonas, and Bacteroides) were strongly correlated with 49 metabolites on the day of metritis diagnosis. Seven of the significant metabolites at calving were among the 49 metabolites strongly correlated with opportunistic pathogenic bacteria on the day of metritis diagnosis. The main metabolites have been associated with attenuation of biofilm formation by commensal bacteria, opportunistic pathogenic bacteria overgrowth, tissue damage and inflammation, immune evasion, and immune dysregulation. CONCLUSIONS: The data integration presented herein helps advance the understanding of the uterine environment in dairy cows with metritis. The identified metabolites may provide a competitive advantage to the main uterine pathogens Fusobacterium, Porphyromonas and Bacteroides, and may be promising targets for future interventions aiming to reduce opportunistic pathogenic bacteria growth in the uterus.

Evaluating differences in milk production, reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows.

The objective was to assess differences in productive and reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows located in Western and Southern states of the U.S.A. This retrospective cohort study included data from 3 experiments conducted in 9 dairies. Vaginal discharge was evaluated twice within 12 DIM and scored on a 5-point scale. The highest score observed for each cow was used for group assignment (VD group) as follows: VD 1 and 2 (VD 1/2; n = 1,174) = clear mucus/lochia with or without flecks of pus; VD 3 (n = 1,802) = mucopurulent with < 50% pus; VD 4 (n = 1,643) = mucopurulent with ≥50% of pus or non-fetid reddish/brownish mucous, n = 1,643; VD 5 = fetid, watery, and reddish/brownish, n = 1,800. All VD 5 cows received treatment according to each herd's protocol. Rectal temperature was assessed in a subset of VD 5 cows, and subsequently divided into Fever (rectal temperature ≥39.5°C; n = 334) and NoFever (n = 558) groups. A smaller proportion of cows with VD 5 (67.6%) resumed ovarian cyclicity compared with VD 1/2 (76.2%) and VD 4 (72.9%) cows; however, a similar proportion of VD5 and VD 3 (72.6%) cows resumed ovarian cyclicity. A smaller proportion of VD 5 (85.8%) cows received at least one artificial insemination (AI) compared with VD 1/2 (91.5%), VD 3 (91.0%), or VD 4 (91.6%) cows. Although we did not detect differences in pregnancy at first AI according to VD, fewer cows with VD 5 (64.4%) were pregnant at 300 DIM than cows with VD 1/2 (76.5%), VD 3 (76.2%), or VD 4 (74.7%). Hazard of pregnancy by 300 DIM was smaller for VD 5 compared with VD 1/2, VD 3, or VD 4 cows. A greater proportion of VD 5 cows were removed from the herd within 300 DIM compared with other VD groups. There was 760 kg lesser milk production within 300 DIM for VD 5 compared with VD 2, VD 3, and VD 4, whereas VD 2, VD 3, and VD 4 had similar milk production. We did not detect an association between fever at diagnosis of VD 5 and reproductive performance or milk production. A greater proportion of VD 5 cows without fever were removed from the herd by 300 DIM compared with VD 5 cows with fever. Differences in productive and reproductive performance, and removal of the herd were restricted to fetid, watery, and reddish/brownish vaginal discharge, which was independent of fever.

Graduate Student Literature Review: Exploring choline's important roles as a nutrient for transition dairy cows.

In late gestation and in the first weeks postpartum, lipid droplets accumulate in the hepatic tissue resulting in approximately 40% to 50% of the dairy cows developing hepatic lipidosis in the first weeks of lactation. Elevated concentrations of triacylglycerol in the hepatic tissue are associated with increased risk of peripartum diseases and impaired productive performance. Cows with hepatic lipidosis need to dispose the excess of hepatic triacylglycerol, but this is a slow process in the bovine liver and relies on primary mechanisms such as complete oxidation and ketogenesis because of the limited export of triacylglycerols as lipoproteins. Choline is a lipotropic compound because, among other functions, it facilitates the export of lipids from the liver. Supplementing choline as rumen-protected choline (RPC) to diets of feed-restricted dairy cows reduces the degree of triacylglycerol infiltration into the hepatic parenchyma in part by enhancing export of triacylglycerol as nascent lipoprotein. The reduced accumulation of triacylglycerol in hepatic tissue in feed-restricted cows fed RPC might affect secondary pathways involved in hepatic disposal of fatty acids such as increased cellular autophagy and lipophagy and minimize endoplasmic reticulum stress response and hepatocyte inflammation. Collectively, these effects on secondary pathways might further reduce the severity of hepatic lipidosis in cows. One of the benefits of supplementing RPC is improved fat digestibility, perhaps because choline, through phosphatidylcholines, facilitates lipid transport within the enterocyte by increasing the synthesis of chylomicrons. Finally, when supplemented during the transition period, RPC improves productive performance of cows, irrespective of their body condition, that extends well beyond the period of supplementation. This review summarizes the current understanding of hepatic lipidosis in early lactation, recapitulates the absorption, transport and metabolism of choline, and discusses its role on hepatic metabolism and gastrointestinal functions, which collectively results in improved performance in dairy cows.

Application of behavior data to predictive exploratory models of metritis self-cure and treatment failure in dairy cows.

The objective was to evaluate the performance of exploratory models containing routinely available on-farm data, behavior data, and the combination of both to predict metritis self-cure (SC) and treatment failure (TF). Holstein cows (n = 1,061) were fitted with a collar-mounted automated-health monitoring device (AHMD) from -21 ± 3 to 60 ± 3 d relative to calving to monitor rumination time and activity. Cows were examined for diagnosis of metritis at 4 ± 1, 7 ± 1, and 9 ± 1 d in milk (DIM). Cows diagnosed with metritis (n = 132), characterized by watery, fetid, reddish/brownish vaginal discharge (VD), were randomly allocated to 1 of 2 treatments: control (CON; n = 62), no treatment at the time of metritis diagnosis (d 0); or ceftiofur (CEF; n = 70), subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid on d 0 and 3 relative to diagnosis. Cure was determined 12 d after diagnosis and was considered when VD became mucoid and not fetid. Cows in CON were used to determine SC, and cows in CEF were used to determine TF. Univariable analyses were performed using farm-collected data (parity, calving season, calving-related disorders, body condition score, rectal temperature, and DIM at metritis diagnosis) and behavior data (i.e., daily averages of rumination time, activity generated by AHMD, and derived variables) to assess their association with metritis SC or TF. Variables with P-values ≤0.20 were included in the multivariable logistic regression exploratory models. To predict SC, the area under the curve (AUC) for the exploratory model containing only data routinely available on-farm was 0.75. The final exploratory model to predict SC combining routinely available on-farm data and behavior data increased the AUC to 0.87, with sensitivity (Se) of 89% and specificity (Sp) of 77%. To predict TF, the AUC for the exploratory model containing only data routinely available on-farm was 0.90. The final exploratory model combining routinely available on-farm data and behavior data increased the AUC to 0.93, with Se of 93% and Sp of 87%. Cross-validation analysis revealed that generalizability of the exploratory models was poor, which indicates that the findings are applicable to the conditions of the present exploratory study. In summary, the addition of behavior data contributed to increasing the prediction of SC and TF. Developing and validating accurate prediction models for SC could lead to a reduction in antimicrobial use, whereas accurate prediction of cows that would have TF may allow for better management decisions.

Association between prepartum body condition score and prepartum and postpartum dry matter intake and energy balance in multiparous Holstein cows.

The objectives of this retrospective observational study were to investigate the association between BCS at 21 d before calving with prepartum and postpartum DMI, energy balance (EB), and milk yield. Data from 427 multigravid cows from 11 different experiments conducted at the University of Florida (Gainesville, FL) were used. Cows were classified according to their BCS at 21 d before calving as fat (BCS ≥ 4.00; n = 83), moderate (BCS = 3.25-3.75; n = 287), and thin (BCS ≤ 3.00; n = 57). Daily DMI from -21 to -1 and from +1 to +28 DIM was individually recorded. Energy balance was calculated as the difference between net energy for lactation consumed and required. Dry matter intake in fat cows was lower than that in moderate and thin cows both prepartum (fat = 9.97 ± 0.21, moderate = 11.15 ± 0.14, and thin = 11.92 ± 0.22 kg/d) and postpartum (fat = 14.35 ± 0.49, moderate = 15.47 ± 0.38, and thin = 16.09 ± 0.47 kg/d). Dry matter intake was also lower for moderate cows compared with thin cows prepartum, but not postpartum. Energy balance in fat cows was lower than in moderate and thin cows both prepartum (fat = -4.16 ± 0.61, moderate = -1.20 ± 0.56, and thin = 0.88 ± 0.62 Mcal/d) and postpartum (fat = -12.77 ± 0.50, moderate = -10.13 ± 0.29, and thin = -6.14 ± 0.51 Mcal/d). Energy balance was also lower for moderate cows compared with thin cows both prepartum and postpartum. There was a quadratic association between BCS at 21 d before calving and milk yield. Increasing BCS from 2.5 to 3.5 was associated with an increase in daily milk yield of 6.0 kg and 28 d cumulative milk of 147 kg. Increasing BCS from 3.5 to 4.5 was associated with a decrease in daily milk yield of 4.4 kg and 28 d cumulative milk of 116 kg. In summary, a moderate BCS at 21 d before calving was associated with intermediate DMI and EB pre- and postpartum but greater milk yield compared with thinner and fatter cows. Our findings indicate that a moderate BCS is ideal for ensuring a successful lactation.

Characterization of a model to induce hyperlipidemia in feed-restricted dairy cows.

Hepatic lipidosis is a prevalent metabolic disorder, and in vivo models to study intermediary lipid metabolism are needed in dairy cows. Objectives were to apply a method to induce hyperlipidemia and characterize the responses and safety of the intervention in feed-restricted dry Holstein cows at 8 mo of gestation. It was hypothesized that infusion of tyloxapol would induce hyperlipidemia without deleterious effects on health of dairy cows. Pregnant, nonlactating parous Holstein cows (n = 33) at a mean (± standard deviation) of 234 ± 2.2 d of gestation were fed for ad libitum intake on d 1 to 5 and restricted to 41% of the required NEL from d 6 to 13. On d 14, when cows were 247 ± 2.2 d of gestation, cows were kept off feed, and received i.v. a 10% solution of tyloxapol at 120 mg/kg body weight to block hydrolysis of triacylglycerols in very-low-density lipoprotein (VLDL) particles. Blood was sampled for 720 min and analyzed for concentrations of triacylglycerol, VLDL cholesterol, and total cholesterol in serum to reflect hepatic secretion or reduced clearance of such metabolites from blood. Rectal temperature, respiration and heart rates, and clinical signs related to potential anaphylaxis were monitored for the first 30 min relative to tyloxapol infusion, and for any abnormal behavior in the subsequent 24 h. Infusion of tyloxapol progressively increased the concentrations of triacylglycerol, VLDL cholesterol, and total cholesterol in serum. Tyloxapol increased rectal temperature by 0.19°C at 30 min after infusion and increased respiration and heart rates in the first 10 min after infusion by 29% and 40%, respectively. Tyloxapol induced tachycardia (heart rate >80 beats/min) in 66.7% (n = 22), frothy salivation in 39.4% (n = 13), muzzle twitching in 15.2% (n = 5), eyes twitching in 12.1% (n = 4), muscle twitching in 48.5% (n = 16), nystagmus in 6.1% (n = 2), signs of hyperexcitement in 18.2% (n = 6), staggering gait in 18.2% (n = 6), and anaphylaxis in 12.1% (n = 4) of the cows; however, all these signs were transient, and cows returned to normal after 20 min of infusion. No other abnormal behavior was observed past 20 min of tyloxapol infusion. None of the cows aborted and gestation length, calf birth weight, and risk of diseases in the first 21 d postpartum did not differ between cows receiving tyloxapol and a companion group that did not receive tyloxapol. Infusion of tyloxapol induced hyperlipidemia in cows with some animals showing transient reactions to the treatment, but without complications to the cow and the offspring. Application of this model can be useful to study intermediary lipid metabolism in dairy cows.

Estimation of genetic parameters for feed efficiency traits using random regression models in dairy cattle.

Feed efficiency has become an increasingly important research topic in recent years. As feed costs rise and the environmental impacts of agriculture become more apparent, improving the efficiency with which dairy cows convert feed to milk is increasingly important. However, feed intake is expensive to measure accurately on large populations, making the inclusion of this trait in breeding programs difficult. Understanding how the genetic parameters of feed efficiency and traits related to feed efficiency vary throughout the lactation period is valuable to gain understanding into the genetic nature of feed efficiency. This study used 121,226 dry matter intake (DMI) records, 120,500 energy-corrected milk (ECM) records, and 98,975 metabolic body weight (MBW) records, collected on 7,440 first-lactation Holstein cows from 6 countries (Canada, Denmark, Germany, Spain, Switzerland, and the United States), from January 2003 to February 2022. Genetic parameters were estimated using a multiple-trait random regression model with a fourth-order Legendre polynomial for all traits. Weekly phenotypes for DMI were re-parameterized using linear regressions of DMI on ECM and MBW, creating a measure of feed efficiency that was genetically corrected for ECM and MBW, referred to as genomic residual feed intake (gRFI). Heritability (SE) estimates varied from 0.15 (0.03) to 0.29 (0.02) for DMI, 0.24 (0.01) to 0.29 (0.03) for ECM, 0.55 (0.03) to 0.83 (0.05) for MBW, and 0.12 (0.03) to 0.22 (0.06) for gRFI. In general, heritability estimates were lower in the first stage of lactation compared with the later stages of lactation. Additive genetic correlations between weeks of lactation varied, with stronger correlations between weeks of lactation that were close together. The results of this study contribute to a better understanding of the change in genetic parameters across the first lactation, providing insight into potential selection strategies to include feed efficiency in breeding programs.

Novel genetic parameters for genetic residual feed intake in dairy cattle using time series data from multiple parities and countries in North America and Europe.

Residual feed intake is viewed as an important trait in breeding programs that could be used to enhance genetic progress in feed efficiency. In particular, improving feed efficiency could improve both economic and environmental sustainability in the dairy cattle industry. However, data remain sparse, limiting the development of reliable genomic evaluations across lactation and parity for residual feed intake. Here, we estimated novel genetic parameters for genetic residual feed intake (gRFI) across the first, second, and third parity, using a random regression model. Research data on the measured feed intake, milk production, and body weight of 7,379 cows (271,080 records) from 6 countries in 2 continents were shared through the Horizon 2020 project Genomic Management Tools to Optimise Resilience and Efficiency, and the Resilient Dairy Genome Project. The countries included Canada (1,053 cows with 47,130 weekly records), Denmark (1,045 cows with 72,760 weekly records), France (329 cows with 16,888 weekly records), Germany (938 cows with 32,614 weekly records), the Netherlands (2,051 cows with 57,830 weekly records), and United States (1,963 cows with 43,858 weekly records). Each trait had variance components estimated from first to third parity, using a random regression model across countries. Genetic residual feed intake was found to be heritable in all 3 parities, with first parity being predominant (range: 22-34%). Genetic residual feed intake was highly correlated across parities for mid- to late lactation; however, genetic correlation across parities was lower during early lactation, especially when comparing first and third parity. We estimated a genetic correlation of 0.77 ± 0.37 between North America and Europe for dry matter intake at first parity. Published literature on genetic correlations between high input countries/continents for dry matter intake support a high genetic correlation for dry matter intake. In conclusion, our results demonstrate the feasibility of estimating variance components for gRFI across parities, and the value of sharing data on scarce phenotypes across countries. These results can potentially be implemented in genetic evaluations for gRFI in dairy cattle.

Modeling the relationship between heat stress, feed intake, and day relative to calving in nonlactating dairy cows.

Heat stress (HS) during the dry period can affect animal welfare, health, dry matter intake (DMI), and milk production in the subsequent lactation, which will negatively affect the profitability of dairy farms. In this study, the objective was to model the changes in DMI in pregnant nonlactating heat-stressed dairy cows with or without access to evaporative cooling systems. A database was built, composed of individual DMI records from 244 pregnant nonlactating dairy cows from an average -29.3 d (range: -42 to -21 d; SD: ±7.54 d) to -1 d relative to calving (DRC) and housed in environmental conditions in which temperature-humidity index (THI) ranged from 58.4 to 83.3, with or without access to evaporative cooling systems. Generalized additive mixed-effects models were used to describe the relationships of DMI with HS and DRC. Changes in DMI with the increase in THI and the progression of pregnancy in cows with or without evaporative cooling systems were estimated using differential equations. On average, cows housed in barns without evaporative cooling systems had a reduction in DMI of 1.30 kg/d and increased rectal temperature in 0.22°C in relation to those housed in barns with evaporative cooling systems. Dry matter intake decreased as THI increased, but the reduction was greater for noncooled cows as THI values increased. In addition, regardless of the THI, DMI started to decrease at -14 DRC for cooled cows, whereas for noncooled cows it already started at -30 DRC, relative to the previous days evaluated. The intensity of the reduction was lesser for cows that had access to evaporative cooling systems or were in the dry period in May to June as compared with those that were in the dry period in July to August or September to October. The models generated in this study, which include environmental variables, should lead to more accurate predictions of DMI during HS that can be used to formulate diets to meet the needs of the late pregnant cow because it is possible to predict changes in DMI as the heat load and DRC change. Such models are also expected to help dairy nutritionists to decide when and how to apply the dietary strategies available to attenuate the reductions in DMI with the intensity of HS and progression of pregnancy.

Rumen-protected choline reduces hepatic lipidosis by increasing hepatic triacylglycerol-rich lipoprotein secretion in dairy cows.

Objectives were to determine the effects of supplementing rumen-protected choline (RPC) on hepatic composition and secretion of triacylglycerol-rich lipoprotein when cows were subjected to feed restriction to develop fatty liver. It was hypothesized that RPC reduces hepatic triacylglycerol by enhancing secretion of hepatic lipoprotein. Pregnant, nonlactating parous Holstein cows (n = 33) at mean (± standard deviation) 234 ± 2.2 d of gestation were blocked by body condition (3.79 ± 0.49) and assigned to receive 0 g/d (CON), 25.8 g/d choline ion from a RPC product containing 28.8% choline chloride (CC; treatment L25.8), or 25.8 g/d of choline ion from a RPC product containing 60.0% CC (H25.8). Cows were fed for ad libitum intake for the first 5 d and restricted to 41% of the net energy for lactation required for maintenance and pregnancy from d 6 to 13. Intake of metabolizable methionine was maintained at 18 g/d during feed restriction by supplying rumen-protected methionine. Hepatic tissue was sampled on d 6 and 13 and analyzed for triacylglycerol and glycogen, and mRNA expression of hepatic tissue was investigated. On d 14, cows were not fed and received a 10% solution of tyloxapol intravenously at 120 mg/kg of body weight to block hydrolysis of triacylglycerols in very low density lipoprotein (VLDL). Blood was sampled sequentially for 720 min and analyzed for concentration of triacylglycerol and total cholesterol. Lymph was sampled 6 h after tyloxapol infusion, and analyzed for concentrations of fatty acids, ß-hydroxybutyrate, glucose, triacylglycerol, and total cholesterol. A sample of serum collected at 720 min after tyloxapol was assayed for the metabolome composition. The area under the curve (AUC) of serum triacylglycerol, VLDL cholesterol, and total cholesterol were calculated. Orthogonal contrasts evaluated the effect of supplementing RPC (CON vs. [1/2 L25.8 + 1/2 H25.8]) and source of RPC (L25.8 vs. H25.8). Least squares means and standard errors of the means are presented in sequence as CON, L25.8, H25.8. During feed restriction, supplementation of RPC reduced hepatic triacylglycerol (9.0 vs. 4.1 vs. 4.5 ± 0.6%) and increased glycogen contents (1.9 vs. 3.5 vs. 4.1 ± 0.2%). Similarly, supplementation of RPC increased the expression of transcripts involved in the synthesis and assembly of lipoproteins (MTTP), cellular autophagy (ATG3), and inflammation (TNFA), and reduced the expression of transcripts associated with mitochondrial oxidation of fatty acids (HADHA, MLYCD) and stabilization of lipid droplets (PLIN2). After infusion of tyloxapol, RPC increased the AUC for serum triacylglycerol (21,741 vs. 32,323 vs. 28,699 ± 3,706 mg/dL × min) and VLDL cholesterol (4,348 vs. 6,465 vs. 5,740 ± 741 mg/dL × min) but tended to reduce the concentrations of triacylglycerol in lymph (16.7 vs. 13.8 vs. 11.9 ± 1.9 mg/dL). Feeding RPC tended to increase the concentrations of 89 metabolites in serum, after adjusting for false discovery, including 3 acylcarnitines, 1 AA-related metabolite, 11 bile acids, 1 ceramide, 6 diacylglycerols, 2 dihydroceramides, 1 glycerophospholipid, and 64 triacylglycerols compared with CON. Feeding 25.8 g/d of choline ion as RPC mediated increased hepatic triacylglycerol secretion to promote lipotropic effects that reduced hepatic lipidosis in dairy cows.

Transcriptome changes associated with elongation of bovine conceptuses I: Differentially expressed transcripts in the conceptus on day 17 after insemination.

The objective was to characterize transcriptome changes associated with elongation in bovine conceptuses during preimplantation stages. Nonlactating Holstein cows were euthanized 17 d after artificial insemination (AI) and the uterine horn ipsilateral to the CL was flushed with saline solution. Recovered conceptuses were classified as small (1.2 to 6.9 cm; n = 9), medium (10.5 to 16.0 cm; n = 9), or large (18.0 to 26.4 cm; n = 10). Total mRNA was extracted and subjected to transcriptome analyses using the Affymetrix Gene Chip Bovine array. Data were normalized using the GCRMA method and analyzed by robust regression using the Linear Models for Microarray library within Bioconductor in R. Transcripts with P ≤ 0.05 after adjustment for false discovery rate and fold change ≥1.5 were considered differentially expressed. Functional analyses were conducted using the Ingenuity Pathway Analysis platform. Comparisons between large versus small (LvsS), large versus medium (LvsM), and medium versus small (MvsS) conceptuses yielded a total of 634, 240, and 63 differentially expressed transcripts, respectively. Top canonical pathways of known involvement with embryo growth that were upregulated in large conceptuses included actin cytoskeleton (LvsS), integrin signaling (LvsS and LvsM), ephrin receptor (LvsS), mesenchymal transition by growth factor (LvsM), and regulation of calpain protease (LvsS). Transcripts involved with lipid metabolism pathways (LXR/RXR, FXR/RXR, hepatic fibrosis) were associated with the LvsS and LvsM, and some transcripts such as APOC2, APOH, APOM, RARA, RBP4, and PPARGC1A, were involved in these pathways. An overall network summary associated biological downstream effects of invasion of cells, proliferation of embryonic cells, and inhibition of organismal death in the LvsS. In conclusion, differently expressed transcripts in the LvsS comparison were associated with the cell growth, adhesion, and organismal development, although part of these findings could be attributed to differences in circulatory concentrations of progesterone of the cows that bore large and small conceptuses. The large and medium conceptuses developed under similar concentrations of progesterone and presented 240 differently expressed transcripts, associated with cell differentiation, metabolite regulation, and other biological processes.

Transcriptome changes associated with elongation of bovine conceptuses II: Differentially expressed transcripts in the endometrium on day 17 after insemination.

The objective was to characterize endometrial transcriptome on d 17 of gestation in dairy cows according to conceptus length. Nonlactating Holstein cows (n = 48) were slaughtered 17 d after AI and the uterine horn ipsilateral to the corpus luteum (CL) was flushed with saline solution. Recovered conceptuses were classified as small (1.2-6.9 cm; n = 9), medium (10.5-16.0 cm; n = 9), or large (18.0-26.4 cm; n = 10). Samples of intercaruncular endometrium dissected from the caudal, intermediate, and cranial portions of the uterine horn ipsilateral to the pregnancy were pooled for analyses. Total mRNA was extracted from endometrial tissue and subjected to transcriptome analyses using the Affymetrix Gene Chip Bovine array. Data were normalized using the GCRMA method and analyzed by robust regression using the Linear Models for Microarray library within Bioconductor in R. Transcripts with P ≤ 0.05 after adjustment for false discovery rate and fold change ≥1.5 were considered differentially expressed. Functional analyses were conducted using the Ingenuity Pathway Analysis platform. Comparisons between endometria of cows carrying large versus small (LvsS), large versus medium (LvsM), and medium versus small (MvsS) conceptuses yielded a total of 235, 21, and 94 differentially expressed transcripts, respectively. Top canonical pathways included the antigen presentation pathway and Th1/Th2 activation pathways, both for LvsS and MvsS. Interferon-α and -γ were identified as activated upstream regulators, primarily based on differently expressed transcripts such as IDO1, ISG20, WARS, LGALS9, IFI44, and PSMB9 (LvsS and MvsS). For LvsS, regulator analyses revealed predicted activation of FOXO1, IFN, NFACTC2, IL-12, IL-6, and IL-18, whereas it depicted inhibition of IL10RA and ZBTB1. Changes in these regulators were associated with a downstream activation of leukocytes, as well as quantity and expansion of T lymphocytes. Canonical pathways associated with the comparison LvsM included cell cycle G2/M DNA damage checkpoint regulation, cell cycle control of chromosomal replication. Moreover, tretinoin was predicted, as activated in upstream analysis for the same comparison. In conclusion, most of the differently expressed transcripts in the endometrium on d 17 of gestation were identified between cows carrying small conceptuses compared with counterparts carrying medium and large conceptuses and were involved with pathways associated with modulation of the immune response.

Blood metabolomics and impacted cellular mechanisms during transition into lactation in dairy cows that develop metritis.

The objective of this study was to identify metabolites associated with metritis and use them for identification of cellular mechanisms affected during transition into lactation. Holstein cows (n = 104) had blood collected in the prepartum period (d -14 ± 6 relative to calving), at calving (d 0), and at the day of metritis diagnosis (d 7 ± 2 after calving). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 52). Cows with metritis were paired with herdmates without metritis (n = 52) based on days in milk. The metabolome of plasma samples was evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Univariate analyses included t-tests and fold change analyses. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were used for partial least squares discriminant analysis coupled with permutational analysis using 2,000 permutations. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were also used for enriched pathway analyses and identification of cellular processes. Cows that developed metritis had affected cellular processes associated with lower amino acid metabolism in the prepartum period, greater lipolysis, cell death, and oxidative stress at calving and at metritis diagnosis, and greater leukocyte activation at calving, but lower immune cell activation at metritis diagnosis. In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.

Unraveling the immune and metabolic changes associated with metritis in dairy cows.

The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at -14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at -14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1ß. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

Modeling the effects of heat stress in animal performance and enteric methane emissions in lactating dairy cows.

Heat stress (HS) negatively affects dry matter intake (DMI), milk yield (MY), feed efficiency (FE), and free water intake (FWI) in dairy cows, with detrimental consequences to animal welfare, health, and profitability of dairy farms. Absolute enteric methane (CH4) emission, yield (CH4/DMI), and intensity (CH4/MY) may also be affected. Therefore, the goal of this study was to model the changes in dairy cow productivity, water intake, and absolute CH4 emissions, yield, and intensity with the progression (days of exposure) of a cyclical HS period in lactating dairy cows. Heat stress was induced by increasing the average temperature by 15°C (from 19°C in the thermoneutral period to 34°C) while keeping relative humidity constant at 20% (temperature-humidity index peaks of approximately 83) in climate-controlled chambers for up to 20 d. A database composed of individual records (n = 1,675) of DMI and MY from 82 heat-stressed lactating dairy cows housed in environmental chambers from 6 studies was used. Free water intake was also estimated based on DMI, dry matter, crude protein, sodium, and potassium content of the diets, and ambient temperature. Absolute CH4 emissions was estimated based on DMI, fatty acids, and dietary digestible neutral detergent fiber content of the diets. Generalized additive mixed-effects models were used to describe the relationships of DMI, MY, FE, and absolute CH4 emissions, yield, and intensity with HS. Dry matter intake and absolute CH4 emissions and yield reduced with the progression of HS up to 9 d, when it started to increase again up to 20 d. Milk yield and FE reduced with the progression of HS up to 20 d. Free water intake (kg/d) decreased during the exposure to HS mainly because of a reduction in DMI; however, when expressed in kg/kg of DMI it increased modestly. Methane intensity also reduced initially up to d 5 during HS exposure but then started to increase again following the DMI and MY pattern up to d 20. However, the reductions in CH4 emissions (absolute, yield, and intensity) occurred at the expense of decreases in DMI, MY, and FE, which are not desirable. This study provides quantitative predictions of the changes in animal performance (DMI, MY, FE, FWI) and CH4 emissions (absolute, yield, and intensity) with the progression of HS in lactating dairy cows. The models developed in this study could be used as a tool to help dairy nutritionists to decide when and how to adopt strategies to mitigate the negative effects of HS on animal health and performance and related environmental costs. Thus, more precise and accurate on-farm management decisions could be taken with the use of these models. However, application of the developed models outside of the ranges of temperature-humidity index and period of HS exposure included in this study is not recommended. Also, validation of predictive capacity of the models to predict CH4 emissions and FWI using data from in vivo studies where these variables are measured in heat-stressed lactating dairy cows is required before these models can be used.

Effect of source and amount of rumen-protected choline on hepatic metabolism during induction of fatty liver in dairy cows.

Objectives were to determine the effect of supplementing increased amounts of rumen-protected choline (RPC) from sources with low (L, 28.8%) or high (H, 60.0%) concentration of choline chloride on hepatic metabolism when cows were subjected to feed restriction to develop fatty liver. It was hypothesized that increased supplementation of RPC reduces hepatic triacylglycerol and enhances glycogen concentrations. Pregnant, nonlactating multiparous Holstein cows (n = 110) at mean (± standard deviation) 232 ± 3.9 d of gestation were blocked by body condition (4.01 ± 0.52) and assigned to receive 0 (CON), 12.9 (L12.9 or H12.9), or 25.8 (L25.8 or H25.8) g/d of choline ion. Cows were fed for ad libitum intake on d 1 to 5 and restricted to 50% of the NEL required for maintenance and pregnancy from d 6 to 13. Intake of metabolizable methionine was maintained at 19 g/d during the feed restriction period by supplying rumen-protected methionine. Hepatic tissue was sampled on d 6 and 13 and analyzed for triacylglycerol, glycogen, and mRNA expression of genes involved in choline, glucose, and fatty acids metabolism, cell signaling, inflammation, autophagy, lipid droplet dynamics, lipophagy, and endoplasmic reticulum stress response. Blood was sampled and analyzed for concentrations of fatty acids, ß-hydroxybutyrate (BHB), glucose, triacylglycerol, total cholesterol, and haptoglobin. Orthogonal contrasts evaluated the effect of supplementing RPC [CON vs. (1/4·L12.9 + 1/4·L25.8 + 1/4·H12.9 + 1/4·H25.8)], source of RPC [(1/2·L12.9 + 1/2·L25.8) vs. (1/2·H12.9 + 1/2·H25.8)], amount of RPC [(1/2·L12.9 + 1/2·H12.9) vs. (1/2·L25.8 + 1/2·H25.8)], and interaction between source and amount [(1/2·L12.9 + 1/2·H25.8) vs. (1/2·H12.9 + 1/2·L25.8)]. Least squares means and standard error of the means are presented in sequence as CON, L12.9, L25.8, H12.9, H25.8. Supplementation of RPC reduced hepatic triacylglycerol (9.3 vs. 6.6 vs. 5.1 vs. 6.6 vs. 6.0 ± 0.6% as-is) and increased glycogen contents (1.8 vs. 2.6 vs. 3.6 vs. 3.1 vs. 4.1 ± 0.2% as-is) on d 13 of the experiment. Feeding RPC reduced serum haptoglobin (136.6 vs. 85.6 vs. 80.6 vs. 82.8 vs. 81.2 ± 4.6 µg/mL) during the feed restriction period; however, blood concentrations of fatty acids, BHB, glucose, triacylglycerol, and total cholesterol did not differ among treatments. During feed restriction, supplementation of RPC enhanced the mRNA expression of genes related to choline metabolism (BHMT), uptake of fatty acids (CD36), and autophagy (ATG3), and reduced the expression of a transcript associated with endoplasmic reticulum stress response (ERN1). An increase in the amount of choline ion from 12.9 to 25.8 g/d enhanced the mRNA expression of genes associated with synthesis and assembly of lipoproteins (APOB100), and inflammation (TNFA), whereas it reduced the expression of genes linked to gluconeogenesis (PC), oxidation of fatty acids (ACADM, MMUT), ketogenesis (ACAT1), and synthesis of antioxidants (SOD1) on d 13 of the experiment. Feeding RPC, independent of the product used, promoted lipotropic effects that reduced hepatic lipidosis in dairy cows.

Induced endometritis in early lactation compromises production and reproduction in dairy cows.

Objectives of this experiment were to study the effect of infusing utero-pathogenic bacteria to induce endometrial inflammation on productive performance in early lactation and subsequent reproduction. Although endometritis is associated with perturbed reproduction, numerous factors may contribute to the observed association. It was hypothesized that induced endometrial inflammation, resulting in localized and systemic inflammatory responses, compromises production and reproduction. Holstein cows without clinical disease and with less than 18% polymorphonuclear leukocytes (PMN) in endometrial cytology on d 31 ± 3 postpartum had their estrous cycle synchronized. Cows were blocked by parity and genomic breeding value for cow conception rate and, within block, assigned randomly to remain as untreated controls (CON; n = 37) or to receive an intrauterine infusion of 5.19 × 108 cfu Escherichia coli and 4.34 × 108 cfu Trueperella pyogenes during the luteal phase to induce endometrial inflammation (INF; n = 48). Endometrial cytology was taken on d 2 and 7 after treatment to evaluate the proportion of PMN. Rectal temperature, dry matter intake, and yields of milk and components were measured in the first 7 d after treatment. Blood serum was analyzed for concentration of haptoglobin. Leukocytes were isolated from blood on d 2 and 7 after treatment and on d 19 after artificial insemination (AI) and mRNA was quantified for a select group of genes. Cows received AI and reproduction was followed for 300 d postpartum. Bacterial infusion induced endometrial inflammation with increased proportions of PMN in the endometrial cytology on d 2 (4.4 ± 0.7 vs. 26.3 ± 2.8%) and 7 (10.9 ± 1.7 vs. 17.4 ± 2.1%) after treatment, resulting in increased mean prevalence of subclinical endometritis (>10% PMN; 23.3 ± 6.3 vs. 80.9 ± 5.1%). Rectal temperature did not differ between CON and INF, but the concentration of haptoglobin in serum tended to increase in INF compared with CON (113 ± 14 vs. 150 ± 16 µg/mL). Induced endometrial inflammation reduced yields of milk (44.9 ± 0.8 vs. 41.6 ± 0.8 kg/d), protein (1.19 ± 0.03 vs. 1.12 ± 0.03 kg/d), and lactose (2.17 ± 0.04 vs. 2.03 ± 0.04 kg/d) and tended to reduce dry matter intake (20.7 ± 0.5 vs. 19.4 ± 0.6 kg/d) in the first 7 d after treatment. Indeed, the reduction in milk yield lasted 4 wk. However, treatment did not affect yields of energy-corrected milk or fat because treatment with INF increased the concentration of fat in milk (3.54 ± 0.10 vs. 3.84 ± 0.10%). Induced endometrial inflammation reduced pregnancy per AI at all inseminations (33.4 ± 5.1 vs. 21.6 ± 3.7%) and the hazard of pregnancy (0.61; 95% CI = 0.36-1.04), which extended the median days open by 24 d. Blood leukocytes from INF cows had increased mRNA expression of the pro-inflammatory gene IL1B on d 2 and 7 after treatment, but reduced expression of the IFN-stimulated genes ISG15 and MX2 on d 19 after AI. Induced endometrial inflammation depressed production and caused long-term negative effects on reproduction in lactating dairy cows.