Milk yield residuals and their link with the metabolic status of dairy cows in the transition period.

The transition period is one of the most challenging periods in the lactation cycle of high-yielding dairy cows. It is commonly known to be associated with diminished animal welfare and economic performance of dairy farms. The development of data-driven health monitoring tools based on on-farm available milk yield development has shown potential in identifying health-perturbing events. As proof of principle, we explored the association of these milk yield residuals with the metabolic status of cows during the transition period. Over 2 yr, 117 transition periods from 99 multiparous Holstein-Friesian cows were monitored intensively. Pre- and postpartum dry matter intake was measured and blood samples were taken at regular intervals to determine ß-hydroxybutyrate, nonesterified fatty acids (NEFA), insulin, glucose, fructosamine, and IGF1 concentrations. The expected milk yield in the current transition period was predicted with 2 previously developed models (nextMILK and SLMYP) using low-frequency test-day (TD) data and high-frequency milk meter (MM) data from the animal's previous lactation, respectively. The expected milk yield was subtracted from the actual production to calculate the milk yield residuals in the transition period (MRT) for both TD and MM data, yielding MRTTD and MRTMM. When the MRT is negative, the realized milk yield is lower than the predicted milk yield, in contrast, when positive, the realized milk yield exceeded the predicted milk yield. First, blood plasma analytes, dry matter intake, and MRT were compared between clinically diseased and nonclinically diseased transitions. MRTTD and MRTMM, postpartum dry matter intake and IGF1 were significantly lower for clinically diseased versus nonclinically diseased transitions, whereas ß-hydroxybutyrate and NEFA concentrations were significantly higher. Next, linear models were used to link the MRTTD and MRTMM of the nonclinically diseased cows with the dry matter intake measurements and blood plasma analytes. After variable selection, a final model was constructed for MRTTD and MRTMM, resulting in an adjusted R2 of 0.47 and 0.73, respectively. While both final models were not identical the retained variables were similar and yielded comparable importance and direction. In summary, the most informative variables in these linear models were the dry matter intake postpartum and the lactation number. Moreover, in both models, lower and thus also more negative MRT were linked with lower dry matter intake and increasing lactation number. In the case of an increasing dry matter intake, MRTTD was positively associated with NEFA concentrations. Furthermore, IGF1, glucose, and insulin explained a significant part of the MRT. Results of the present study suggest that milk yield residuals at the start of a new lactation are indicative of the health and metabolic status of transitioning dairy cows in support of the development of a health monitoring tool. Future field studies including a higher number of cows from multiple herds are needed to validate these findings.

Association between days post-conception and lactation persistency in dairy cattle.

Determining the optimal insemination moment for individual cows is complex, particularly when considering the effects of pregnancy on milk production. The effect of pregnancy on the absolute milk yield has already been reported in several studies. Currently, there is limited quantitative knowledge about the association between days post-conception (DPC) and lactation persistency, based on a lactation curve model, and, specifically, how persistency changes during pregnancy and relates to the days in milk at conception (DIMc). Understanding this association might provide valuable insights to determine the optimal insemination moment. This study, therefore, aimed to investigate the association between DPC and lactation persistency, with an additional focus on the influence of DIMc. Available milk production data from 2005 to 2022 were available for 23,908 cows from 87 herds located throughout the Netherlands and Belgium. Persistency was measured by a lactation curve characteristic decay, representing the time taken to halve milk production after peak yield. Decay was calculated for 8 DPC (0, 30, 60, 90, 120, 150, 180, and 210 d after DIMc) and served as the dependent variable. Independent variables included DPC, DIMc (≤60, 61-90, 91-120, 121-150, 151-180, 181-210, >210), parity group, DPC × parity group, DPC × DIMc, and variables from 30 d before DIMc as covariates. The results showed an increase in decay, which is to say, a decrease in persistency, during pregnancy for both parity groups, albeit in different ways. Specifically, from DPC 150 to DPC 210, multiparous cows showed a greater decline in persistency compared with primiparous cows. Furthermore, a later DIMc (cows conceiving later) was associated with higher persistency. Except for the early DIMc groups (DIMc <90), DIMc does not affect the change in persistency by gestation. The findings from this study contribute to a better understanding of how DPC and DIMc during lactation influence lactation persistency, enabling more informed decision-making by farmers who wish to take persistency into account in their reproduction management.

Heat stress in a temperate climate leads to adapted sensor-based behavioral patterns of dairy cows.

Most research on heat stress has focused on (sub)tropical climates. The effects of higher ambient temperatures on the daily behavior of dairy cows in a maritime and temperate climate are less studied. With this retrospective observational study, we address that gap by associating the daily time budgets of dairy cows in the Netherlands with daily temperature and temperature-humidity index (THI) variables. During a period of 4 years, cows on 8 commercial dairy farms in the Netherlands were equipped with neck and leg sensors to collect data from 4,345 cow lactations regarding their daily time budget. The time spent eating, ruminating, lying, standing, and walking was recorded. Individual cow data were divided into 3 data sets: (1) lactating cows from 5 farms with a conventional milking system (CMS) and pasture access, (2) lactating cows from 3 farms with an automatic milking system (AMS) without pasture access, and (3) dry cows from all 8 farms. Hourly environment temperature and relative humidity data from the nearest weather station of the Dutch National Weather Service was used for THI calculation for each farm. Based on heat stress thresholds from previous studies, daily mean temperatures were grouped into 7 categories: 0 = (<0°C), 1 = (0-12°C, reference category), 2 = (12-16°C), 3 = (16-20°C), 4 = (20-24°C), 5 = (24-28°C), and 6 = (≥28°C). Temperature-humidity index values were grouped as follows: 0 = (THI <30), 1 = (THI 30-56, reference category), 2 = (THI 56-60), 3 = (THI 60-64), 4 = (THI 64-68), 5 = (THI 68-72) and 6 = (THI ≥72). To associate daily mean temperature and THI with sensor-based behavioral parameters of dry cows and of lactating cows from AMS and CMS farms, we used generalized linear mixed models. In addition, associations between sensor data and other climate variables, such as daily maximum and minimum temperature, and THI were analyzed. On the warmest days, eating time decreased in the CMS group by 92 min/d, in the AMS group by 87 min/d, and in the dry group by 75 min/d compared with the reference category. Lying time decreased in the CMS group by 36 min/d, in the AMS group by 56 min/d, and in the dry group by 33 min/d. Adaptation to daily temperature and THI was already noticeable from a mean temperature of 12°C or a mean THI of 56 or above, when dairy cows started spending less time lying and eating and spent more time standing. Further, rumination time decreased, although only in dry cows and cows on AMS farms. With higher values for daily mean THI and temperature, walking time decreased as well. These patterns were very similar for temperature and THI variables. These results show that dairy cows in temperate climates begin to adapt their behavior at a relatively low mean environmental temperature or THI. In the temperate maritime climate of the Netherlands, our results indicate that daily mean temperature suffices to study the effects of behavioral adaptation to heat stress in dairy cows.

Associations between body condition score, locomotion score, and sensor-based time budgets of dairy cattle during the dry period and early lactation.

Lameness, one of the most important disorders in the dairy industry, is related to postpartum diseases and has an effect on dairy cow welfare, leading to changes in cows' daily behavioral variables. This study quantified the effect of lameness on the daily time budget of dairy cows in the transition period. In total, 784 multiparous dairy cows from 8 commercial Dutch dairy farms were visually scored on their locomotion (score of 1-5) and body condition (score of 1-5). Each cow was scored in the early and late dry period as well as in wk 4 and 8 postpartum. Cows with locomotion scores 1 and 2 were grouped together as nonlame, cows with score 3 were considered moderately lame, and cows with scores 4 and 5 were grouped together as severely lame. Cows were equipped with 2 types of sensors that measured behavioral parameters. The leg sensor provided number of steps, number of stand-ups (moving from lying to standing), lying time, number of lying bouts, and lying bout length. The neck sensor provided eating time, number of eating bouts, eating bout length, rumination time, number of rumination bouts, and rumination bout length. Sensor data for each behavioral parameter were averaged between 2 d before and 2 d after locomotion scoring. The percentage of nonlame cows decreased from 63% in the early dry period to 46% at 8 wk in lactation; this decrease was more severe for cows with higher parity. Cows that calved in autumn had the highest odds for lameness. Body condition score loss of >0.75 point in early lactation was associated with lameness in wk 4 postpartum. Moderately lame cows had a reduction of daily eating time of around 20 min, whereas severely lame cows had a reduction of almost 40 min. Similarly, moderately and severely lame dry cows showed a reduction of 200 steps/d, and severely lame cows in lactation showed a reduction of 600 steps/d. Daily lying time increased by 26 min and lying bout length increased by 8 min in severely lame cows compared with nonlame cows. These results indicate a high prevalence of lameness on Dutch dairy farms, with an increase in higher locomotion scores from the dry period into early lactation. Time budgets for multiparous dairy cows differed between the dry period and the lactating period, with a higher locomotion score (increased lameness) having an effect on cows' complete behavioral profile. Body condition score loss in early lactation was associated with poor locomotion postpartum, whereas lameness resulted in less eating time in the dry period and early lactation, creating a harmful cycle.

Relationships between metabolic profiles and gene expression in liver and leukocytes of dairy cows in early lactation.

Homeorhetic mechanisms assist dairy cows in the transition from pregnancy to lactation. Less successful cows develop severe negative energy balance (NEB), placing them at risk of metabolic and infectious diseases and reduced fertility. We have previously placed multiparous Holstein Friesian cows from 4 herds into metabolic clusters, using as biomarkers measurements of plasma nonesterified fatty acids, ß-hydroxybutyrate, glucose and IGF-1 collected at 14 and 35 d in milk (DIM). This study characterized the global transcriptomic profiles of liver and circulating leukocytes from the same animals to determine underlying mechanisms associated with their metabolic and immune function. Liver biopsy and whole-blood samples were collected around 14 DIM for RNA sequencing. All cows with available RNA sequencing data were placed into balanced (BAL, n = 44), intermediate (n = 44), or imbalanced (IMBAL, n = 19) metabolic cluster groups. Differential gene expression was compared between the 3 groups using ANOVA, but only the comparison between BAL and IMBAL cows is reported. Pathway analysis was undertaken using DAVID Bioinformatic Resources (https://david.ncifcrf.gov/). Milk yields did not differ between BAL and IMBAL cows but dry matter intake was less in IMBAL cows and they were in greater energy deficit at 14 DIM (-4.48 v -11.70 MJ/d for BAL and IMBAL cows). Significantly differentially expressed pathways in hepatic tissue included AMPK signaling, glucagon signaling, adipocytokine signaling, and insulin resistance. Genes involved in lipid metabolism and cholesterol transport were more highly expressed in IMBAL cows but IGF1 and IGFALS were downregulated. Leukocytes from BAL cows had greater expression of histones and genes involved in nucleosomes and cell division. Leukocyte expression of heat shock proteins increased in IMBAL cows, suggesting an unfolded protein response, and several key genes involved in immune responses to pathogens were upregulated (e.g., DEFB13, HP, OAS1Z, PTX3, and TLR4). Differentially expressed genes upregulated in IMBAL cows in both tissues included CD36, CPT1, KFL11, and PDK4, all central regulators of energy metabolism. The IMBAL cows therefore had greater difficulty maintaining glucose homeostasis and had dysregulated hepatic lipid metabolism. Their energy deficit was associated with a reduced capacity for cell division and greater evidence of stress responses in the leukocyte population, likely contributing to an increased risk of infectious disease.

Genome-wide association for metabolic clusters in early-lactation Holstein dairy cows.

The aim of this study was to detect the genomic region or regions associated with metabolic clusters in early-lactation Holstein cows. This study was carried out in 2 experiments. In experiment I, which was carried out on 105 multiparous Holstein cows, animals were classified through k-means clustering on log-transformed and standardized concentrations of blood glucose, insulin-like growth factor I, free fatty acids, and ß-hydroxybutyrate at 14 and 35 d in milk (DIM), into metabolic clusters, either balanced (BAL) or other (OTR). Forty percent of the animals were categorized in the BAL group, and the remainder were categorized as OTR. The cows were genotyped for a total of 777,962 SNP. A genome-wide association study was performed, using a case-control approach through the GEMMA software, accounting for population structure. We found 8 SNP (BTA11, BTA23, and BTAX) associated with the predicted metabolic clusters. In experiment II, carried out on 4,267 second-parity Holstein cows, milk samples collected starting from the first week until 50 DIM were used to determine Fourier-transform mid-infrared (FT-MIR) spectra and subsequently to classify the animals into the same metabolic clusters (BAL vs. OTR). Twenty-eight percent of the animals were categorized in the BAL group, and the remainder were classified in the OTR category. Although daily milk yield was lower in BAL cows, we found no difference in daily fat- and protein-corrected milk yield in cows from the BAL metabolic cluster compared with those in the OTR metabolic cluster. In the next step, a single-step genomic BLUP was used to identify the genomic region(s) associated with the predicted metabolic clusters. The results revealed that prediction of metabolic clusters is a highly polygenic trait regulated by many small-sized effects. The region of 36,258 to 36,295 kb on BTA27 was the highly associated region for the predicted metabolic clusters, with the closest genes to this region (ANK1 and miR-486) being related to hematopoiesis, erythropoiesis, and mammary gland development. The heritability for metabolic clustering was 0.17 (SD 0.03), indicating that the use of FT-MIR spectra in milk to predict metabolic clusters in early-lactation across a large number of cows has satisfactory potential to be included in genetic selection programs for modern dairy cows.

Potential of milk mid-infrared spectra to predict nitrogen use efficiency of individual dairy cows in early lactation.

Improving nitrogen use efficiency (NUE) at both the individual cow and the herd level has become a key target in dairy production systems, for both environmental and economic reasons. Cost-effective and large-scale phenotyping methods are required to improve NUE through genetic selection and by feeding and management strategies. The aim of this study was to evaluate the possibility of using mid-infrared (MIR) spectra of milk to predict individual dairy cow NUE during early lactation. Data were collected from 129 Holstein cows, from calving until 50 d in milk, in 3 research herds (Denmark, Ireland, and the UK). In 2 of the herds, diets were designed to challenge cows metabolically, whereas a diet reflecting local management practices was offered in the third herd. Nitrogen intake (kg/d) and nitrogen excreted in milk (kg/d) were calculated daily. Nitrogen use efficiency was calculated as the ratio between nitrogen in milk and nitrogen intake, and expressed as a percentage. Individual daily values for NUE ranged from 9.7 to 81.7%, with an average of 36.9% and standard deviation of 10.4%. Milk MIR spectra were recorded twice weekly and were standardized into a common format to avoid bias between apparatus or sampling periods. Regression models predicting NUE using milk MIR spectra were developed on 1,034 observations using partial least squares or support vector machines regression methods. The models were then evaluated through (1) a cross-validation using 10 subsets, (2) a cow validation excluding 25% of the cows to be used as a validation set, and (3) a diet validation excluding each of the diets one by one to be used as validation sets. The best statistical performances were obtained when using the support vector machines method. Inclusion of milk yield and lactation number as predictors, in combination with the spectra, also improved the calibration. In cross-validation, the best model predicted NUE with a coefficient of determination of cross-validation of 0.74 and a relative error of 14%, which is suitable to discriminate between low- and high-NUE cows. When performing the cow validation, the relative error remained at 14%, and during the diet validation the relative error ranged from 12 to 34%. In the diet validation, the models showed a lack of robustness, demonstrating difficulties in predicting NUE for diets and for samples that were not represented in the calibration data set. Hence, a need exists to integrate more data in the models to cover a maximum of variability regarding breeds, diets, lactation stages, management practices, seasons, MIR instruments, and geographic regions. Although the model needs to be validated and improved for use in routine conditions, these preliminary results showed that it was possible to obtain information on NUE through milk MIR spectra. This could potentially allow large-scale predictions to aid both further genetic and genomic studies, and the development of farm management tools.

Prediction of metabolic clusters in early-lactation dairy cows using models based on milk biomarkers.

The aim of this study was to describe metabolism of early-lactation dairy cows by clustering cows based on glucose, insulin-like growth factor I (IGF-I), free fatty acid, and ß-hydroxybutyrate (BHB) using the k-means method. Predictive models for metabolic clusters were created and validated using 3 sets of milk biomarkers (milk metabolites and enzymes, glycans on the immunogamma globulin fraction of milk, and Fourier-transform mid-infrared spectra of milk). Metabolic clusters are used to identify dairy cows with a balanced or imbalanced metabolic profile. Around 14 and 35 d in milk, serum or plasma concentrations of BHB, free fatty acids, glucose, and IGF-I were determined. Cows with a favorable metabolic profile were grouped together in what was referred to as the "balanced" group (n = 43) and were compared with cows in what was referred to as the "other balanced" group (n = 64). Cows with an unfavorable metabolic profile were grouped in what was referred to as the "imbalanced" group (n = 19) and compared with cows in what was referred to as the "other imbalanced" group (n = 88). Glucose and IGF-I were higher in balanced compared with other balanced cows. Free fatty acids and BHB were lower in balanced compared with other balanced cows. Glucose and IGF-I were lower in imbalanced compared with other imbalanced cows. Free fatty acids and BHB were higher in imbalanced cows. Metabolic clusters were related to production parameters. There was a trend for a higher daily increase in fat- and protein-corrected milk yield in balanced cows, whereas that of imbalanced cows was higher. Dry matter intake and the daily increase in dry matter intake were higher in balanced cows and lower in imbalanced cows. Energy balance was continuously higher in balanced cows and lower in imbalanced cows. Weekly or twice-weekly milk samples were taken and milk metabolites and enzymes (milk glucose, glucose-6-phosphate, BHB, lactate dehydrogenase, N-acetyl-ß-d-glucosaminidase, isocitrate), immunogamma globulin glycans (19 peaks), and Fourier-transform mid-infrared spectra (1,060 wavelengths reduced to 15 principal components) were determined. Milk biomarkers with or without additional cow information (days in milk, parity, milk yield features) were used to create predictive models for the metabolic clusters. Accuracy for prediction of balanced (80%) and imbalanced (88%) cows was highest using milk metabolites and enzymes combined with days in milk and parity. The results and models of the present study are part of the GplusE project and identify novel milk-based phenotypes that may be used as predictors for metabolic and performance traits in early-lactation dairy cows.

Association between metabolic diseases and the culling risk of high-yielding dairy cows in a transition management facility using survival and decision tree analysis.

The objective of this study was to assess the association between individual metabolic diseases (MD) and multiple MD (MD+) in the transition period (±3 wk relative to calving) and the culling risk in the first 120 d in milk (DIM) in Holstein-Friesian dairy cows. Health records from a transition management facility in Germany with 1,946 calvings were analyzed in a 1-yr cohort via survival analysis and a decision tree model. The recorded MD were milk fever (MF), retained placenta (RP), metritis (METR), ketosis (KET), displaced abomasum (DA), twinning (TWIN), and clinical mastitis (MAST). The overall culling within 120 DIM was 18%. The 120 DIM culling risk for healthy cows (64.8% of the total) was 13%, whereas it was 25% for MD (24.5%) and 33% for MD+ (10.7%) cows. The 120 DIM culling risk (%) for each MD and MD+, respectively, was 34.6 and 48 for MF and MF+, 15 and 31.5 for RP and RP+, 9.4 and 22.2 for METR and METR+, 30.7 and 37.3 for KET and KET+, 56.1 and 46.8 for DA and DA+, 30.3 and 34 for TWIN and TWIN+, and 36.6 and 27.8 for MAST and MAST+. Moreover, the incidence risk (%) for each MD and MD+, respectively, was 4 and 2.6 for MF and MF+, 1 and 2.8 for RP and RP+, 8.7 and 6 for METR and METR+, 4.5 and 6.1 for KET and KET+, 0.8 and 2.4 for DA and DA+, 1.7 and 2.7 for TWIN and TWIN+, and 3.6 and 1.8 for MAST and MAST+. Setting the healthy cows as the referent, the 120 DIM hazard ratios (HR) for culling were MD 2.1, MD+ 2.9, MF 3.3, MF+ 4.6, RP+ 2.7, METR+ 1.8, KET 2.6, KET+ 3.3, DA 5.5, DA+ 4.5, TWIN 2.8, TWIN+ 3.0, MAST 3.1, and MAST+ 2.3. According to both decision tree and random forest analyses, MF was the most significant disease influencing survival, followed by DA, MAST, METR, and TWIN. In conclusion, the presence of MD or MD+ during the transition period was associated with increased culling risk in the first 120 DIM. The culling hazard was greater when an MD was complicated with another MD. In this study performed in a well-managed large farm, uncomplicated cases of RP (HR = 1.2) and METR (HR = 0.7) did not have an influence on the 120 DIM culling risk. Interestingly, both decision tree and random forest analyses pointed to MF and DA as main culling reasons in the first 120 DIM in the present dairy herd.

Influence of adipocyte size and adipose depot on the number of adipose tissue macrophages and the expression of adipokines in dairy cows at the end of pregnancy.

The aim of this study was to determine the number of adipose tissue macrophages (ATM) and the mRNA expression of adipokines [adiponectin (ADIPOQ), leptin (LEP), interleukin 6 (IL6), tumor necrosis factor (TNF), and interleukin 10 (IL10)] in different adipose depots from cows with a variable body condition score (BCS) at the end of the dry period. We hypothesized that the number of ATM and the expression of these adipokines depend on adipocyte size and the anatomical location of the adipose depot. Subcutaneous, omental, mesenteric, perirenal, and intrapelvic adipose tissue samples were taken immediately after euthanasia of 10 Holstein Friesian dairy cows (upcoming parity 2 to 5, age 3.9 ± 1.4 yr; mean ± standard deviation) at the end of pregnancy (actual days of pregnancy at the moment of euthanasia: 269 ± 5 d). During the dry period, all animals received similar diets to meet but not exceed requirements. Five animals were considered to have a normal BCS (2.5-3.5) and 5 animals were considered to be over-conditioned (BCS = 3.75-5). Body weight of the animals at the moment of euthanasia was 717 ± 77 kg. Expression of the different genes was determined by reverse transcription quantitative real-time PCR. Adipocyte size was determined by measuring the area of 100 adipocytes on histological sections. Average adipocyte area was 10,475 ± 1,019, 8,500 ± 780, 10,383 ± 1,227, 11,466 ± 1,039, and 11,087 ± 1,632 µm2 for the subcutaneous, mesenteric, omental, intrapelvic, and perirenal adipose depot, respectively. Immunohistochemistry using anti-bovine CD172a antibodies was performed to determine the proportion of ATM (the number of CD172a-positive cells per 100 adipocytes, given as a percentage). Expression of LEP, IL6, and TNF was positively associated with adipocyte size, whereas no association could be detected between ADIPOQ and IL10 with the size of the adipocytes. The omental adipose depot was especially infiltrated with ATM (1.92 ± 0.55, 1.10 ± 0.33, and 8.28 ± 2.24% for the subcutaneous, mesenteric, and omental adipose depot, respectively). The proportion of ATM was positively associated with the size of the adipocytes in the omental and mesenteric adipose depot. Expression of ADIPOQ, LEP, IL6, TNF, and IL10 differed among depots, which suggests differences in inflammatory characteristics depending on the anatomical location of depots. In conclusion, the results of the present study confirm the adipose tissue as a potential source of inflammatory mediators and demonstrate ATM infiltration, especially in the omental adipose depot.

Cytological endometritis diagnosed at artificial insemination in repeat breeder dairy cows.

This study aimed to (i) assess the prevalence of cytological endometritis (CYTO) diagnosed at artificial insemination (AI); (ii) evaluate the effect of CYTO on the pregnancy outcome of the same AI sample; and (iii) determine the risk factors associated with CYTO diagnosed at AI in repeat breeder (RB) dairy cows. We analysed the productive and reproductive performances of 146 RB Holstein-Friesian cows. To obtain a CYTO sample at AI, we used the cytotape technique. Generalized mixed effect models were computed to find the risk factors associated with the pregnancy and CYTO outcome. Based on ≥1% PMN cut-off point, the CYTO prevalence at AI in RB cows was 25.3%. The overall pregnancy at AI was 44.2%. The conception rate in CYTO-positive (n = 37) RB cows was 29.7% versus 49.5% for CYTO-negative (n = 109) cows. A RB cow diagnosed CYTO positive at AI had 0.47 [odds ratio (OR)] odds to become pregnant in comparison with a CYTO-negative cow. Cows that produced more milk than their counterparts in this study had increased odds (OR = 1.01) to be CYTO positive at AI. A novel risk factor positively associated with CYTO diagnosed at AI in RB cows was the level of daily milk urea (OR = 1.11). To conclude, CYTO at the moment of AI had a significantly negative effect on the pregnancy outcome in RB dairy cows. However, as only one of fourth of RB cows is affected with CYTO at AI, it may not be considered a key element associated with the RB syndrome.

Cytological endometritis at artificial insemination in dairy cows: Prevalence and effect on pregnancy outcome.

The aims of our field study in dairy cows were (1) to consolidate cytotape (CT) as a valid technique to diagnose cytological endometritis (CYTO) during artificial insemination (AI); (2) to establish a cutoff point concerning the polymorphonuclear cells (PMN) proportion to diagnose CYTO at AI; (3) to assess the prevalence of CYTO at AI; and (4) to evaluate the effect of CYTO on the pregnancy outcome of that AI. The investigation was performed using 1,625 AI-CT samples harvested from 873 Holstein-Friesian cows from 18 dairy farms in the Flemish region of Belgium. The CT device consisted of adapting a 1.5-cm piece of paper tape on the top of a conventional AI catheter covered with a double guard sheet, allowing an endometrial cytology sample to be taken when performing an AI. A receiving operator characteristic curve was built to assess the threshold level above which the PMN proportion significantly affected the AI success. Multilevel generalized mixed-effect models were built to identify factors affecting the pregnancy outcome of the AI under investigation. Only 7 samples (0.4%) harvested in 5 cows were discarded because of low-quality parameters. The cutoff point for CYTO at AI was set at ≥1% PMN (sensitivity=33.8%, specificity=88.6%). Prevalence of CYTO at AI was 27.8%. The conception rate for CYTO-positive samples was 32.7%, whereas it was 47% for CYTO-negative samples. A CYTO-negative AI had 1.8 [odds ratio (OR)] more chances to become pregnant than a CYTO-positive one. Other factors identified as detrimental for the pregnancy outcome were body condition score ≤1.5 (OR=0.6), relative 305-d milk yield (OR=0.9), dystocia (OR=0.3), parity ≥2 (OR=0.7), and warm months of the year. In conclusion, CT is a consolidated technique to diagnose CYTO at AI, PMN 1% is the threshold level to diagnose CYTO at AI, around one-quarter of inseminated uteri suffer from CYTO, and affected uteri having a significantly lower chance to become pregnant from that insemination.

Novel approaches to assess the quality of fertility data stored in dairy herd management software.

Scientific journals and popular press magazines are littered with articles in which the authors use data from dairy herd management software. Almost none of such papers include data cleaning and data quality assessment in their study design despite this being a very critical step during data mining. This paper presents 2 novel data cleaning methods that permit identification of animals with good and bad data quality. The first method is a deterministic or rule-based data cleaning method. Reproduction and mutation or life-changing events such as birth and death were converted to a symbolic (alphabetical letter) representation and split into triplets (3-letter code). The triplets were manually labeled as physiologically correct, suspicious, or impossible. The deterministic data cleaning method was applied to assess the quality of data stored in dairy herd management from 26 farms enrolled in the herd health management program from the Faculty of Veterinary Medicine Ghent University, Belgium. In total, 150,443 triplets were created, 65.4% were labeled as correct, 17.4% as suspicious, and 17.2% as impossible. The second method, a probabilistic method, uses a machine learning algorithm (random forests) to predict the correctness of fertility and mutation events in an early stage of data cleaning. The prediction accuracy of the random forests algorithm was compared with a classical linear statistical method (penalized logistic regression), outperforming the latter substantially, with a superior receiver operating characteristic curve and a higher accuracy (89 vs. 72%). From those results, we conclude that the triplet method can be used to assess the quality of reproduction data stored in dairy herd management software and that a machine learning technique such as random forests is capable of predicting the correctness of fertility data.

Short communication: Limitations of glucose tolerance tests in the assessment of peripheral tissue insulin sensitivity during pregnancy and lactation in dairy heifers.

The aim of the present study was to point at the limitations of glucose tolerance tests (GTT) to assess peripheral tissue insulin sensitivity in dairy heifers in different physiological states (pregnancy and lactation). Intravenous GTT were performed in 5 nonpregnant, nonlactating heifers, 5 heifers at the end of pregnancy (12-7 d before calving), and 5 lactating primiparous cows (11-14 d after calving). Glucose and insulin concentrations were determined and area under the curve (AUC) and clearance rate of glucose and insulin were calculated. Additionally, data were analyzed using the minimal model to derive the insulin sensitivity parameter (Si). Basal glucose and insulin concentrations were greater in the nonpregnant, nonlactating heifers. Clearance rate of glucose and Si were lowest, whereas the AUC for glucose was greatest in the nonpregnant, nonlactating heifers. Insulin concentrations during the GTT were greater for the nonpregnant, nonlactating heifers. Results from the GTT in pregnant heifers and lactating primiparous cows are biased by the fact that a large part of the glucose disappearance during an intravenous GTT occurs independently of insulin by the pregnant uterus or the lactating mammary gland. As such, greater AUC of glucose, lower clearance rate of glucose, or lower Si derived from GTT performed in nonpregnant, nonlactating dairy heifers in the present study might indicate decreased peripheral tissue insulin sensitivity of the glucose metabolism or decreased insulin-independent glucose disappearance. Based on the results from a GTT, it is impossible to discriminate between both metabolic pathways. It can be concluded that parameters derived from GTT are not suited to compare peripheral tissue insulin sensitivity of the glucose metabolism between dairy heifers in different physiological states due to the large variation in insulin secretion and the substantial difference in insulin-independent glucose disposal associated with these physiological states.

Prevalence of cytological endometritis and effect on pregnancy outcomes at the time of insemination in nulliparous dairy heifers.

The objectives of the present study were to assess the prevalence of cytological endometritis (CYTO) at the time of artificial insemination (AI) and its effect on pregnancy outcomes in nulliparous dairy heifers. In total, 512 endometrial cytology samples were taken during AI from 351 nulliparous Holstein-Friesian heifers using cytotape (a 1.5-cm piece of paper tape rolled on the top of an AI catheter covered with a double guard sheet). After sampling, the top of the AI catheter was gently rolled onto a glass slide, air-dried, and stained using Diff-Quick (Fisher Diagnostics, Newark, DE). For each slide, 300 nucleated cells were counted, and the polymorphonuclear cell ratio (% PMN) was assessed at 400× magnification. We constructed a receiver operating characteristic curve to find the cutoff point at which sensitivity and specificity (% PMN) affected pregnancy outcomes. The receiver operating characteristic curve revealed that the threshold level for diagnosing CYTO in nulliparous dairy heifers was 1% PMN. An insemination was considered successful when pregnancy was confirmed by rectal palpation at least 45d post-AI. Heifers were considered not pregnant when they received a subsequent insemination or were diagnosed empty by rectal palpation. We built multilevel generalized mixed-effect models to test factors affecting pregnancy outcomes and the occurrence of CYTO at AI. We excluded 16 samples harvested from 12 heifers due to poor sample quality or unavailability of reproductive data. Of the 496 AI samples, the prevalence of CYTO at AI was 7.86% (n=39). The conception rate was 62.8% (n=287) in CYTO-negative samples (n=457) and 38.46% (n=15) in CYTO-positive samples. Risk factors for non-pregnancy were a previous AI (odds ratio 2.96; 95% confidence interval: 1.21-7.26) and the interaction between CYTO and previous AI. The only risk factor identified as being associated with the occurrence of CYTO was a previous AI (odds ratio 4.7; 95% confidence interval: 2.15-10.34). The performance of unsuccessful inseminations significantly affects reproductive outcomes in subsequent AI and may lead to CYTO in nulliparous dairy heifers.

Influence of adipocyte size and adipose depot on the in vitro lipolytic activity and insulin sensitivity of adipose tissue in dairy cows at the end of the dry period.

The aim of the present research was to describe characteristics of adipose tissue lipolysis in dairy cows with a variable body condition score (BCS). Ten clinically healthy Holstein Friesian cows were selected based on BCS and euthanized 10 to 13 d before the expected parturition date. Immediately after euthanasia, adipose tissue samples were collected from subcutaneous and omental fat depots. In both depots, we observed an increase in adipocyte size with increasing BCS. Using an in vitro explant culture of subcutaneous and omental adipose tissue, we aimed to determine the influence of adipocyte size and localization of adipose depot on the lipolytic activity in basal conditions and after addition of isoproterenol (nonselective ß-agonist) and insulin in different concentrations. Glycerol release in the medium was used as a measure for lipolytic activity. We observed that the basal lipolytic activity of subcutaneous and omental adipose tissue increased with adipocyte volume, meaning that larger fat cells have higher basal lipolytic activity independent of the location of the adipose depot. Dose-response curves were created between the concentration of isoproterenol or insulin and the amount of glycerol released. The shape of the dose-response curves is determined by the concentration of isoproterenol and insulin needed to elicit the half-maximal effect and the maximal amount of stimulated glycerol release or the maximal inhibitory effect of insulin. We observed that larger fat cells released more glycerol upon maximal stimulation with isoproterenol and this was more pronounced in subcutaneous adipose tissue. Additionally, larger fat cells had a higher sensitivity toward lipolytic signals. We observed a trend for larger adipocytes to be more resistant to the maximal antilipolytic effect of insulin. The insulin concentration needed to elicit the half-maximal inhibitory effect of insulin was within the physiological range of insulin and was not influenced by adipocyte size or adipose depot. We conclude that overconditioned cows have larger adipocytes and are predisposed to excessive mobilization of body fat due to a higher basal and stimulated lipolytic activity of large adipocytes while the antilipolytic effect of insulin is preserved.

Distribution of inflammation and association between active and chronic alterations within the endometrium of dairy cows.

Objectives of this study were twofold: (i) to assess the association between polymorphonuclear (PMN) counts and chronic alterations within the bovine endometrium and (ii) to determine the distribution of inflammation throughout the endometrium of clinically healthy dairy cows. Holstein-Friesian cows (n = 32) from a single dairy farm were selected for this experiment. Before slaughtering, a complete reproductive examination was performed to discard any type of clinical disease. After slaughtering, reproductive tracts were collected, and the endometrium was sampled at 8 pre-defined locations. At each location, endometrial biopsies (EBs) and cytology (CY) samples were harvested. Histopathology samples were stained with haematoxylin-eosin (EB-HE) and naphthol-AS-D-chloroacetate-esterase (EB-naphthol), while CY samples were stained with Wright-Giemsa. In the EB-HE samples, parameters assessed were epithelium height, mononuclear cells infiltration, lymphocytic aggregates, periglandular fibrosis, angiosclerosis and haemorrhage. In EB-naphthol and CY slides, PMNs counts were evaluated. Binomial logistic regression was used to assess the association between the number of PMNs present in both the EB-naphthol and CY samples and alterations identified in the EB-HE samples and to analyse the distribution of the histopathological alterations (EB-HE). A Poisson mixed-effect model was used to analyse the distribution of PMNs within the endometrium. A significant positive association was found between the PMN counts and the mononuclear cells infiltration. The presence of erythrocytes was associated with higher odds to detect PMNs in the stratum compactum. Significantly, higher infiltration of PMNs and mononuclear cells were detected in the uterine body and the right horn region. Concluding, CY is a technique that allows the evaluation of PMN counts and therefore only evaluates active inflammation. A complete assessment of endometrial health can only be obtained using EB. To optimize the sensitivity to diagnose endometrial inflammation in cows, adjacencies of the corpus uteri should be considered as the preferred region to harvest samples.

Insulin response of the glucose and fatty acid metabolism in dry dairy cows across a range of body condition scores.

The objective of the present research was to determine the insulin response of the glucose and fatty acid metabolism in dry dairy cows with a variable body condition score (BCS). Ten pregnant Holstein Friesian dairy cows (upcoming parity 2 to 5) were selected based on BCS at the beginning of the study (2mo before expected parturition date). During the study, animals were monitored weekly for BCS and backfat thickness and in the last 2wk, blood samples were taken for determination of serum nonesterified fatty acid (NEFA) concentration. Animals underwent a hyperinsulinemic euglycemic clamp test in the third week before the expected parturition date. The hyperinsulinemic euglycemic clamp test consisted of 4 consecutive insulin infusions with increasing insulin doses: 0.1, 0.5, 2, and 5mIU/kg per minute. For each insulin infusion period, a steady state was defined as a period of 30min where no or minor changes of the glucose infusion were necessary to keep the blood glucose concentration constant and near basal levels. During the steady state, the glucose infusion rate [steady state glucose infusion rate (SSGIR) in µmol/kg per minute] and NEFA concentration [steady state NEFA concentration (SSNEFA) in mmol/L] were determined and reflect the insulin response of the glucose and fatty acid metabolism. Dose response curves were created based on the insulin concentrations during the steady state and the SSGIR or SSNEFA. The shape of the dose response curves is determined by the concentration of insulin needed to elicit the half maximal effect (EC50) and the maximal SSGIR or the minimal SSNEFA for the glucose or fatty acid metabolism, respectively. The maximal SSGIR was negatively associated with variables reflecting adiposity of the cows (BCS, backfat thickness, NEFA concentration during the dry period, and absolute weight of the different adipose depots determined after euthanasia and dissection of the different depots), whereas the EC50 of the glucose metabolism was positively associated with these variables. These results reflect a decreased insulin sensitivity and a decreased insulin responsiveness of the glucose metabolism in overconditioned dry dairy cows. The minimal SSNEFA and the EC50 of the fatty acid metabolism were not associated with variables reflecting adiposity of the cows, meaning that the insulin response of the fatty acid metabolism was not associated with the level of fat accumulation in dry dairy cows. Additionally, within individual cows, the EC50 of the glucose metabolism was higher than the EC50 of the fatty acid metabolism, meaning that the response of the fatty acid metabolism occurs at lower insulin concentrations compared with the response of the glucose metabolism. It can be concluded that a negative association exists between the level of fat accumulation in pregnant dairy cows at the end of the dry period and the insulin response of the glucose metabolism.

Effect of ß-hydroxybutyric acid, parity, and body condition score on phenotype and proliferative capacity of colostral mononuclear leukocytes of high-yielding dairy cows.

In neonatal calves, the ingestion of colostrum is imperative for preventing infectious diseases. Investigations into the transfer of passive immunity of cattle have primarily focused on the importance of colostral immunoglobulins, with a recent increase in focus on understanding the role of colostral leukocytes. The main objective of the present study was to measure the influence of parity, body condition score, serum nonesterified fatty acids, and serum ß-hydroxybutyrate concentrations of periparturient cows on phenotype and mitogen- and antigen-induced proliferative capacity of bovine colostral leukocytes. Holstein-Friesian cows (n=141) were intramuscularly vaccinated at 60 and 30 d before the expected parturition date with a tetanus toxoid vaccine. Of these 141 animals, 28 primiparous and 72 multiparous cows were sampled immediately after parturition. Colostrum mononuclear cell populations were identified by flow cytometry using bovine cluster of differentiation markers, and the proliferative capacity of these cells was determined using a (3)H-thymidine proliferation assay. Under-conditioned cows had a significantly higher percentage of colostral macrophages than normal-conditioned animals, whereas over-conditioned cows had significantly more colostral B-lymphocytes. Serum ß-hydroxybutyrate was significantly associated with higher numbers of colostral T-lymphocytes and macrophages. Heifers had significantly higher mitogen- and antigen-induced proliferation of their colostral leukocytes than third parity or older cows. In conclusion, body condition score, parity, and serum ß-hydroxybutyrate concentration of periparturient high-yielding dairy cows were shown to influence the number of colostral macrophages or the mitogen- and antigen-induced proliferation of colostral leukocytes, possibly influencing the cellular immunity of the newborn calf.