Single-step genome-wide association analyses for milk urea concentration in Walloon Holstein cows.

The aims of this study were to conduct a single-step genome-wide association to identify genomic regions associated with milk urea (MU) and to estimate genetic correlations between MU and milk yield (MY), milk composition (calcium content [CC], fat percentage [FP], protein percentage [PP], and casein percentage [CNP]), and cheese-making properties (CMP; coagulation time [CT], curd firmness after 30 min from rennet addition [a30], and titratable acidity [TA]). The used data have been collected from 2015 to 2020 on 78,073 first-parity (485,218 test-day records) and 48,766 second-parity (284,942 test-day records) Holstein cows distributed in 671 herds in the Walloon Region of Belgium. Data of 565,533 SNP located on 29 BTA of 6,617 animals (1,712 males) were used. Random regression test-day models were used to estimate genetic parameters through the Bayesian Gibbs sampling method. The SNP solutions were estimated using a single-step genomic BLUP approach. The proportion of the total additive genetic variance explained by windows of 50 consecutive SNPs (with an average size of ∼216 kb) was calculated, and the top-3 genomic regions explaining the largest rate of the genetic variance were considered promising regions and used to identify potential candidate genes. Mean (SD) MU was 25.38 (8.02) mg/dL and 25.03 (8.06) mg/dL in the first and second lactation, respectively. Mean heritability estimates for daily MU were 0.21 and 0.23 for the first and second lactation, respectively. The genetic correlations estimated between MU and MY, milk composition, and CMP were quite low (ranged from -0.10 [CC] to 0.10 [TA] and -0.05 [CT] to 0.13 [TA] for the first and second lactations, respectively). The top-3 regions associated with MU were located from 80.61 to 80.74 Mb on BTA6, 103.26 to 103.41 Mb on BTA11, and 1.59 to 2.15 Mb on BTA14. Genes including KCNT1, MROH1, SHARPIN, TSSK5, CPSF1, HSF1, TONSL, DGAT1, SCX, and MAF1 were identified as positional candidate genes for MU. The findings of this study can be used for a better understanding of the genomic architecture underlying MU in Holstein cattle.

Prediction of key milk biomarkers in dairy cows through milk mid-infrared spectra and international collaborations.

At the individual cow level, suboptimum fertility, mastitis, negative energy balance, and ketosis are major issues in dairy farming. These problems are widespread on dairy farms and have an important economic impact. The objectives of this study were (1) to assess the potential of milk mid-infrared (MIR) spectra to predict key biomarkers of energy deficit (citrate, isocitrate, glucose-6 phosphate [glucose-6P], free glucose), ketosis (ß-hydroxybutyrate [BHB] and acetone), mastitis (N-acetyl-ß-d-glucosaminidase activity [NAGase] and lactate dehydrogenase), and fertility (progesterone); (2) to test alternative methodologies to partial least squares (PLS) regression to better account for the specific asymmetric distribution of the biomarkers; and (3) to create robust models by merging large datasets from 5 international or national projects. Benefiting from this international collaboration, the dataset comprised a total of 9,143 milk samples from 3,758 cows located in 589 herds across 10 countries and represented 7 breeds. The samples were analyzed by reference chemistry for biomarker contents, whereas the MIR analyses were performed on 30 instruments from different models and brands, with spectra harmonized into a common format. Four quantitative methodologies were evaluated to address the strongly skewed distribution of some biomarkers. Partial least squares regression was used as the reference basis, and compared with a random modification of distribution associated with PLS (random-downsampling-PLS), an optimized modification of distribution associated with PLS (KennardStone-downsampling-PLS), and support vector machine (SVM). When the ability of MIR to predict biomarkers was too low for quantification, different qualitative methodologies were tested to discriminate low versus high values of biomarkers. For each biomarker, 20% of the herds were randomly removed within all countries to be used as the validation dataset. The remaining 80% of herds were used as the calibration dataset. In calibration, the 3 alternative methodologies outperform the PLS performances for the majority of biomarkers. However, in the external herd validation, PLS provided the best results for isocitrate, glucose-6P, free glucose, and lactate dehydrogenase (coefficient of determination in external herd validation [R2v] = 0.48, 0.58, 0.28, and 0.24, respectively). For other molecules, PLS-random-downsampling and PLS-KennardStone-downsampling outperformed PLS in the majority of cases, but the best results were provided by SVM for citrate, BHB, acetone, NAGase, and progesterone (R2v = 0.94, 0.58, 0.76, 0.68, and 0.15, respectively). Hence, PLS and SVM based on the entire dataset provided the best results for normal and skewed distributions, respectively. Complementary to the quantitative methods, the qualitative discriminant models enabled the discrimination of high and low values for BHB, acetone, and NAGase with a global accuracy around 90%, and glucose-6P with an accuracy of 83%. In conclusion, MIR spectra of milk can enable quantitative screening of citrate as a biomarker of energy deficit and discrimination of low and high values of BHB, acetone, and NAGase, as biomarkers of ketosis and mastitis. Finally, progesterone could not be predicted with sufficient accuracy from milk MIR spectra to be further considered. Consequently, MIR spectrometry can bring valuable information regarding the occurrence of energy deficit, ketosis, and mastitis in dairy cows, which in turn have major influences on their fertility and survival.

Invited review: From heat stress to disease-Immune response and candidate genes involved in cattle thermotolerance.

Heat stress implies unfavorable effects on primary and functional traits in dairy cattle and, in consequence, on the profitability of the whole production system. The increasing number of days with extreme hot temperatures suggests that it is imperative to detect the heat stress status of animals based on adequate measures. However, confirming the heat stress status of an individual is still challenging, and, in consequence, the identification of novel heat stress biomarkers, including molecular biomarkers, remains a very relevant issue. Currently, it is known that heat stress seems to have unfavorable effects on immune system mechanisms, but this information is of limited use in the context of heat stress phenotyping. In addition, there is a lack of knowledge addressing the molecular mechanisms linking the relevant genes to the observed phenotype. In this review, we explored the potential molecular mechanisms explaining how heat stress affects the immune system and, therefore, increases the occurrence of immune-related diseases in cattle. In this regard, 2 relatively opposite hypotheses are under focus: the immunosuppressive action of cortisol, and the proinflammatory effect of heat stress. In both hypotheses, the modulation of the immune response during heat stress is highlighted. Moreover, it is possible to link candidate genes to these potential mechanisms. In this context, immune markers are very valuable indicators for the detection of heat stress in dairy cattle, broadening the portfolio of potential biomarkers for heat stress.

Single-step genome-wide association for selected milk fatty acids in Dual-Purpose Belgian Blue cows.

The aim of this study was to estimate genetic parameters and identify genomic regions associated with selected individual and groups of milk fatty acids (FA) predicted by milk mid-infrared spectrometry in Dual-Purpose Belgian Blue cows. The used data were 69,349 test-day records of milk yield, fat percentage, and protein percentage along with selected individual and groups FA of milk (g/dL milk) collected from 2007 to 2020 on 7,392 first-parity (40,903 test-day records), and 5,185 second-parity (28,446 test-day records) cows distributed in 104 herds in the Walloon Region of Belgium. Data of 28,466 SNPs, located on 29 Bos taurus autosomes (BTA), of 1,699 animals (639 males and 1,060 females) were used. Random regression test-day models were used to estimate genetic parameters through the Bayesian Gibbs sampling method. The SNP solutions were estimated using a single-step genomic best linear unbiased prediction approach. The proportion of genetic variance explained by each 25-SNP sliding window (with an average size of ~2 Mb) was calculated, and regions accounting for at least 1.0% of the total additive genetic variance were used to search for candidate genes. Average daily heritability estimated for the included milk FA traits ranged from 0.01 (C4:0) to 0.48 (C12:0) and 0.01 (C4:0) to 0.42 (C12:0) in the first and second parities, respectively. Genetic correlations found between milk yield and the studied individual milk FA, except for C18:0, C18:1 trans, C18:1 cis-9, were positive. The results showed that fat percentage and protein percentage were positively genetically correlated with all studied individual milk FA. Genome-wide association analyses identified 11 genomic regions distributed over 8 chromosomes [BTA1, BTA4, BTA10, BTA14 (4 regions), BTA19, BTA22, BTA24, and BTA26] associated with the studied FA traits, though those found on BTA14 partly overlapped. The genomic regions identified differed between parities and lactation stages. Although these differences in genomic regions detected may be due to the power of quantitative trait locus detection, it also suggests that candidate genes underlie the phenotypic expression of the studied traits may vary between parities and lactation stages. These findings increase our understanding about the genetic background of milk FA and can be used for the future implementation of genomic evaluation to improve milk FA profile in Dual-Purpose Belgian Blue cows.

Single-step genome-wide association analyses for selected infrared-predicted cheese-making traits in Walloon Holstein cows.

This study aimed to perform genome-wide association study to identify genomic regions associated with milk production and cheese-making properties (CMP) in Walloon Holstein cows. The studied traits were milk yield, fat percentage, protein percentage, casein percentage (CNP), calcium content, somatic cell score (SCS), coagulation time, curd firmness after 30 min from rennet addition, and titratable acidity. The used data have been collected from 2014 to 2020 on 78,073 first-parity (485,218 test-day records), 48,766 second-parity (284,942 test-day records), and 21,948 third-parity (105,112 test-day records) Holstein cows distributed in 671 herds in the Walloon Region of Belgium. Data of 565,533 single nucleotide polymorphisms (SNP), located on 29 Bos taurus autosomes (BTA) of 6,617 animals (1,712 males), were used. Random regression test-day models were used to estimate genetic parameters through the Bayesian Gibbs sampling method. The SNP solutions were estimated using a single-step genomic BLUP approach. The proportion of the total additive genetic variance explained by windows of 50 consecutive SNPs (with an average size of ∼216 KB) was calculated, and regions accounting for at least 1.0% of the total additive genetic variance were used to search for positional candidate genes. Heritability estimates for the studied traits ranged from 0.10 (SCS) to 0.53 (CNP), 0.10 (SCS) to 0.50 (CNP), and 0.12 (SCS) to 0.49 (CNP) in the first, second, and third parity, respectively. Genome-wide association analyses identified 6 genomic regions (BTA1, BTA14 [4 regions], and BTA20) associated with the considered traits. Genes including the SLC37A1 (BTA1), SHARPIN, MROH1, DGAT1, FAM83H, TIGD5, MROH6, NAPRT, ADGRB1, GML, LYPD2, JRK (BTA14), and TRIO (BTA20) were identified as positional candidate genes for the studied CMP. The findings of this study help to unravel the genomic background of a cow's ability for cheese production and can be used for the future implementation and use of genomic evaluation to improve the cheese-making traits in Walloon Holstein cows.

Genome-wide association study and functional annotation analyses for nitrogen efficiency index and its composition traits in dairy cattle.

The aims of this study were (1) to identify genomic regions associated with a N efficiency index (NEI) and its composition traits and (2) to analyze the functional annotation of identified genomic regions. The NEI included N intake (NINT1), milk true protein N (MTPN1), milk urea N yield (MUNY1) in primiparous cattle, and N intake (NINT2+), milk true protein N (MTPN2+), and milk urea N yield (MUNY2+) in multiparous cattle (2 to 5 parities). The edited data included 1,043,171 records on 342,847 cows distributed in 1,931 herds. The pedigree consisted of 505,125 animals (17,797 males). Data of 565,049 SNPs were available for 6,998 animals included in the pedigree (5,251 females and 1,747 males). The SNP effects were estimated using a single-step genomic BLUP approach. The proportion of the total additive genetic variance explained by windows of 50 consecutive SNPs (with an average size of about 240 kb) was calculated. The top 3 genomic regions explaining the largest rate of the total additive genetic variance of the NEI and its composition traits were selected for candidate gene identification and quantitative trait loci (QTL) annotation. The selected genomic regions explained from 0.17% (MTPN2+) to 0.58% (NEI) of the total additive genetic variance. The largest explanatory genomic regions of NEI, NINT1, NINT2+, MTPN1, MTPN2+, MUNY1, and MUNY2+ were Bos taurus autosome 14 (1.52-2.09 Mb), 26 (9.24-9.66 Mb), 16 (75.41-75.51 Mb), 6 (8.73-88.92 Mb), 6 (8.73-88.92 Mb), 11 (103.26-103.41 Mb), 11 (103.26-103.41 Mb). Based on the literature, gene ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction, 16 key candidate genes were identified for NEI and its composition traits, which are mainly expressed in the milk cell, mammary, and liver tissues. The number of enriched QTL related to NEI, NINT1, NINT2+, MTPN1, and MTPN2+ were 41, 6, 4, 11, 36, 32, and 32, respectively, and most of them were related to the milk, health, and production classes. In conclusion, this study identified genomic regions associated with NEI and its composition traits, and identified key candidate genes describing the genetic mechanisms of N use efficiency-related traits. Furthermore, the NEI reflects not only its composition traits but also the interactions among them.

Imputation of missing milk Fourier transform mid-infrared spectra using existing milk spectral databases: A strategy to improve the reliability of breeding values and predictive models.

The use of milk Fourier transform mid-infrared (FT-MIR) spectrometry to develop management and breeding tools for dairy farmers and industry is growing and supported by the availability of numerous new predicted phenotypes to assess the nutritional quality of milk and its technological properties, but also the animal health and welfare status and its environmental fingerprint. For genetic evaluations, having a long-term and representative spectral dairy herd improvement (DHI) database improves the reliabilities of estimated breeding values (EBV) from these phenotypes. Unfortunately, most of the time, the raw spectral data used to generate these estimations are not stored. Moreover, many reference measurements of those phenotypes, needed during the FT-MIR calibration step, are available from past research activities but lack spectra records. So, it is impossible to use them to improve the FT-MIR models. Consequently, there is a strong interest in imputing those missing spectra. The innovative objective of this study was to use the existing large spectral DHI database to estimate missing spectra by selecting probable spectra using, as the match criteria, common dairy traits recorded for a long time by DHI organizations. We tested 4 match criteria combinations. Combination 1 required to have equal fat and protein contents between the sample for which a spectrum was to be estimated and the reference samples in the DHI database. Combination 2 also required an equal urea content. Combination 3 requested equal fat, protein, and lactose contents. Finally, combination 4 included all criteria. When more than one spectrum was found during the search, their average was the estimated spectrum for the query sample. Concretely, this study estimated missing spectra for 1,700 samples using 2,000,000 spectral DHI records. For assessing the effect of this spectral estimation on the prediction quality, FT-MIR equations were used to predict 11 phenotypes, selected as their quantification used different FT-MIR regions. They were related to the milk fat and mineral composition, lactoferrin content, quantity of eructed methane, body weight (BW), and dry matter intake. The accuracy between predictions obtained from actual and estimated spectra was evaluated by calculating the mean absolute error (MAE). The criteria in the fourth and second combinations were too strict to estimate a spectrum for most samples. Indeed, for many samples, no spectra with the same values for those matching criteria was found. The third match criteria combination had a poorer prediction performance for all studied traits and spectral absorptions than the first combination due to fewer matched samples available to compute the missing spectrum. By allowing a range for matching lactose content (±0.1 g/dL milk), we showed that this new combination increased the number of selected samples to compute missing spectra and predict better the infrared absorption at different wavenumbers, especially those related to the lactose quantification. The prediction performance was further improved by performing queries on the entire Walloon DHI spectral database (6,625,570 spectra), and it varied among the studied phenotypes. Without considering the traits used for the matching, the best predictions were obtained for the content of saturated fatty acids (MAE = 0.15 g/dL milk) and BW (MAE = 12.80 kg). Yet, the predictions for the unsaturated fatty acids were less accurate (MAE = 0.13 and 0.018 g/dL milk for monounsaturated and polyunsaturated fatty acids), likely because of the poorer predictions of spectral regions related to long-chain fatty acids. Similarly, poorer predictions were observed for the amount of methane eructed by dairy cows (MAE = 47.02 g/d), likely because it is not directly related to fat content or composition. Prediction accuracies for the remaining traits were also low. In conclusion, we observed that increasing the number of relevant matching criteria helps improve the quality of FT-MIR predicted phenotypes and the number of spectra used during the search. So, it would be of great interest to test in the future the suitability of the developed methodology with large-scale international spectral databases to improve the reliability of EBV from these FT-MIR-based phenotypes and the robustness of FT-MIR predictive models.

Validating genomic prediction for nitrogen efficiency index and its composition traits of Holstein cows in early lactation.

Nitrogen (N) use efficiency (NUE) is an economically important trait for dairy cows. Recently, we proposed a new N efficiency index (NEI), that simultaneously considers both NUE and N pollution. This study aimed to validate the genomic prediction for NEI and its composition traits and investigate the relationship between SNP effects estimated directly from NEI and indirectly from its composition traits. The NEI composition included genomic estimated breeding value of N intake (NINT), milk true protein N (MTPN) and milk urea N yield. The edited data were 132,899 records on 52,064 cows distributed in 773 herds. The pedigree contained 122,368 animals. Genotypic data of 566,294 SNP was available for 4514 individuals. A total of 4413 cows (including 181 genotyped) and 56 bulls (including 32 genotyped) were selected as the validation populations. The linear regression method was used to validate the genomic prediction of NEI and its composition traits using best linear unbiased prediction (BLUP) and single-step genomic BLUP (ssGBLUP). The mean theoretical accuracies of validation populations obtained from ssGBLUP were higher than those obtained from BLUP for both NEI and its composition traits, ranging from 0.57 (MTPN) to 0.72 (NINT). The highest mean prediction accuracies for NEI and its composition traits were observed for the genotyped cows estimated under ssGBLUP, ranging from 0.48 (MTPN) to 0.66 (NINT). Furthermore, the SNP effects estimated from NEI composition traits, multiplied by the relative weight were the same as those estimated directly from NEI. This study preliminary showed that genomic prediction can be used for NEI, however, we acknowledge the need for further validation of this result in a larger dataset. Moreover, the SNP effects of NEI can be indirectly calculated using the SNP effects estimated from its composition traits. This study provided a basis for adding genomic information to establish NEI as part of future routine genomic evaluation programs.

Large-scale phenotyping in dairy sector using milk MIR spectra: Key factors affecting the quality of predictions.

Methods and technologies enabling the estimation at large scale of important traits for the dairy sector are of great interest. Those phenotypes are necessary to improve herd management, animal genetic evaluation, and milk quality control. In the recent years, the research was very active to predict new phenotypes from the mid-infrared (MIR) analysis of milk. Models were developed to predict phenotypes such as fine milk composition, milk technological properties or traits related to cow health, fertility and environmental impact. Most of models were developed within research contexts and often not designed for routine use. The implementation of models at a large scale to predict new traits of interest brings new challenges as the factors influencing the robustness of models are poorly documented. The first objective of this work is to highlight the impact on prediction accuracy of factors such as the variability of the spectral and reference data, the spectral regions used and the complexity of models. The second objective is to emphasize methods and indicators to evaluate the quality of models and the quality of predictions generated under routine conditions. The last objective is to outline the issues and the solutions linked with the use and transfer of models on large number of instruments. Based on partial least square regression and 10 datasets including milk MIR spectra and reference quantitative values for 57 traits of interest, the impact of the different factors is illustrated by evaluating the influence on the validation root mean square error of prediction (RMSEP). In the displayed examples, all factors, when well set up, increase the quality of predictions, with an improvement of the RMSEP ranging from 12% to 43%. This work also aims to underline the need for and the complementarity between different validation procedures, statistical parameters and quality assurance methods. Finally, when using and transferring models, the impact of the spectral standardization on the prediction reproducibility is highlighted with an improvement up to 86% with the tested models, and the monitoring of individual spectrometer stability over time appears essential. This list inspired from our experience is of course not exhaustive. The displayed results are only examples and not general rules and other aspects play a role in the quality of final predictions. However, this work highlights good practices, methods and indicators to increase and evaluate quality of phenotypes predicted at a large scale. The results obtained argue for the development of guidelines at international levels, as well as international collaborations in order to constitute large and robust datasets and enable the use of models in routine conditions.

Can unsupervised learning methods applied to milk recording big data provide new insights into dairy cow health?

Among the dairy sector's current concerns, the assessment of global animal health status is a complex challenge. Its multidimensionality means that global monitoring tools are rarely considered. Instead, specific disease detection is often studied separately and, due to financial and ethical issues, uses small-scale data sets focusing on few biomarkers. Several studies have already been conducted using milk Fourier transform mid-infrared (FT-MIR) spectroscopy to detect mastitis and lameness or to quantify health-related biomarkers in milk or blood. Those studies are relevant but they focus mainly on one biomarker or disease. To solve this issue and the small-scale data set, in this study, we proposed a holistic approach using big data obtained from milk recording, including milk yield, somatic cell count, and 27 FT-MIR-based predictors related to milk composition and animal health status. Using 740,454 records collected from 114,536 first-parity Holstein cows in southern Belgium, we performed repeated unsupervised learning algorithms based on Ward's agglomerative hierarchical clustering method to find potential interesting patterns. A divide-and-conquer approach was used to overcome the limitation of computational resources in clustering a relatively large data set. Five groups of records were identified. Differences observed in the fourth group suggested a relationship to metabolic disorders. The fifth group seemed to be related to mastitis. In a second step, we performed a partial least squares discriminant analysis (PLS-DA) to predict the probability of belonging to those specific groups for the entire data set. The obtained global accuracy was 0.77 and the balanced accuracy (i.e., the mean between sensitivity and specificity) of discriminating the fourth and fifth groups was 0.88 and 0.96, respectively. Then, a validation of the interpretation of those groups was performed using 204 milk and blood reference records. The predicted probability associated with the metabolic disorders issue had significant correlations of 0.54 with blood ß-hydroxybutyrate, 0.44 with blood nonesterified fatty acids, -0.32 with blood glucose, -0.23 with milk glucose-6-phosphate, and 0.38 with milk isocitrate. In contrast, the predicted probability of belonging to the mastitis group had correlations of 0.69 with milk lactate dehydrogenase, 0.46 with milk N-acetyl-ß-d-glucosaminidase, -0.18 with milk free glucose, and 0.16 with milk glucose-6-phosphate. Consequently, these results suggest that the obtained quantitative traits indirectly reflect some of the main health disorders in dairy farming and could be used to monitor dairy cows on a large scale. By using unsupervised learning on large-scale milk recording data and then validating the pattern using reference laboratory measures, we propose a new approach to quickly assess dairy cow health status.

Genome-wide associations for heat stress response suggest potential candidate genes underlying milk fatty acid composition in dairy cattle.

Contents of milk fatty acids (FA) display remarkable alterations along climatic gradients. Detecting candidate genes underlying such alterations might be beneficial for the exploration of climate sensitivity in dairy cattle. Consequently, we aimed on the definition of FA heat stress indicators, considering FA breeding values in response to temperature-humidity index (THI) alterations. Indicators were used in GWAS, in ongoing gene annotations and for the estimation of chromosome-wide variance components. The phenotypic data set consisted of 39,600 test-day milk FA records from 5,757 first-lactation Holstein dairy cows kept in 16 large-scale German cooperator herds. The FA traits were C18:0, polyunsaturated fatty acids (PUFA), saturated fatty acids (SFA), and unsaturated fatty acids (UFA). After genotype quality control, 40,523 SNP markers from 3,266 cows and 930 sires were considered. Meteorological data from the weather station in closest herd distance were used for the calculation of maximum hourly daily THI, which were allocated to 10 different THI classes. The same FA from 3 stages of lactation were considered as different, but genetically correlated traits. Consequently, a 3-trait reaction norm model was used to estimate genetic parameters and breeding values for FA along THI classes, considering either pedigree (A) or genomic (G) relationship matrices. De-regressed proofs and genomic estimated breeding values at the intermediate THI class 5 and at the extreme THI class 10 were used as pseudophenotypes in ongoing genomic analyses for thermoneutral (TNC) and heat stress conditions (HSC), respectively. The differences in de-regressed proofs and in genomic estimated breeding values from both THI classes were pseudophenotypes for heat stress response (HSR). Genetic correlations between the same FA under TNC and HSC were smallest in the first lactation stage and ranged from 0.20 for PUFA to 0.87 for SFA when modeling with the A matrix, and from 0.35 for UFA to 0.86 for SFA when modeling with the G matrix. In the first lactation stage, larger additive genetic variances under HSC compared with TNC indicate climate sensitivity for C18:0, PUFA, and UFA. Climate sensitivity was also reflected by pronounced chromosome-wide genetic variances for HSR of PUFA and UFA in the first stage of lactation. For all FA under TNC, HSC, and HSR, quite large genetic variance proportions were explained by BTA14. In GWAS, 30 SNP (within or close to 38 potential candidate genes) overlapped for HSR of the different FA. One unique potential candidate gene (AMFR) was detected for HSR of PUFA, 15 for HSR of SFA (ADGRB1, DENND3, DUSP16, EFR3A, EMP1, ENSBTAG00000003838, EPS8, MGP, PIK3C2G, STYK1, TMEM71, GSG1, SMARCE1, CCDC57, and FASN) and 3 for HSR of UFA (ENSBTAG00000048091, PAEP, and EPPK1). The identified unique genes play key roles in milk FA synthesis and are associated with disease resistance in dairy cattle. The results suggest consideration of FA in combination with climatic responses when inferring genetic mechanisms of heat stress in dairy cows.

Defining a nitrogen efficiency index in Holstein cows and assessing its potential effect on the breeding program of bulls.

The purposes of this study were (1) to explore the relationship between 3 milk mid-infrared predicted features including nitrogen intake (NINT), milk true protein N (MTPN), and milk urea-N yield (MUNY); (2) to integrate these 3 features into an N efficiency index (NEI) and analyses approximate genetic correlations between the NEI and 37 traits (indices) of interest; and (3) to assess the potential effect of including the NEI into breeding programs of bulls. The edited data were 1,043,171 test-day records on 342,847 cows in 1,931 herds and 143,595 test-day records on 53,660 cows in 766 herds used for estimating breeding values (EBV) and variance components, respectively. The used records were within 5 to 50 d in milk. The records were grouped into primiparous and multiparous. The genetic parameters for the included mid-infrared features and EBV of the animals included in the pedigree were estimated using a multiple-trait repeatability animal model. Then, the EBV of the NINT, MTPN, MUNY were integrated into the NEI using a selection index assuming weights based on the N partitioning. The approximate genetic correlations between the NEI and 37 traits of interest were estimated using the EBV of the selected bulls. The bulls born from 2011 to 2014 with NEI were selected and the NEI distribution of these bulls having EBV for the 8 selected traits (indices) was checked. The heritability and repeatability estimates for NINT, MTPN, and MUNY ranged from 0.09 to 0.13, and 0.37 to 0.65, respectively. The genetic and phenotypic correlations between NINT, MTPN, and MUNY ranged from -0.31 to 0.87, and -0.02 to 0.42, respectively. The NEI ranged from -13.13 to 12.55 kg/d. In total, 736 bulls with reliability ≥0.50 for all included traits (NEI and 37 traits) and at least 10 daughters distributed in at least 10 herds were selected to investigate genetic aspects of the NEI. The NEI had positive genetic correlations with production yield traits (0.08-0.46), and negative genetic correlations with the investigated functional traits and indices (-0.71 to -0.07), except for the production economic index and functional type economic index. The daughters of bulls with higher NEI had lower NINT and MUNY, and higher MTPN. Furthermore, 26% of the bulls (n = 50) with NEI born between 2011 to 2014 had higher NEI and global economic index than the average in the selected bulls. Finally, the developed NEI has the advantage of large-scale prediction and therefore has the potential for routine application in dairy cattle breeding in the future.

Genome-wide association study for selected cheese-making properties in Dual-Purpose Belgian Blue cows.

This study aimed to estimate genetic parameters and identify genomic region(s) associated with selected cheese-making properties (CMP) in Dual-Purpose Belgian Blue (DPBB) cows. Edited data were 46,301 test-day records of milk yield, fat percentage, protein percentage, casein percentage, milk calcium content (CC), coagulation time (CT), curd firmness after 30 min from rennet addition (a30), and milk titratable acidity (MTA) collected from 2014 to 2020 on 4,077 first-parity (26,027 test-day records), and 3,258 second-parity DPBB cows (20,274 test-day records) distributed in 124 herds in the Walloon Region of Belgium. Data of 28,266 SNP, located on 29 Bos taurus autosomes (BTA) of 1,699 animals were used. Random regression test-day models were used to estimate genetic parameters through the Bayesian Gibbs sampling method. The SNP solutions were estimated using a single-step genomic BLUP approach. The proportion of the total additive genetic variance explained by windows of 25 consecutive SNPs (with an average size of ∼2 Mb) was calculated, and regions accounting for at least 1.0% of the total additive genetic variance were used to search for candidate genes. Heritability estimates for the included CMP ranged from 0.19 (CC) to 0.50 (MTA), and 0.24 (CC) to 0.41 (MTA) in the first and second parity, respectively. The genetic correlation estimated between CT and a30 varied from -0.61 to -0.41 and from -0.55 to -0.38 in the first and second lactations, respectively. Negative genetic correlations were found between CT and milk yield and composition, while those estimated between curd firmness and milk composition were positive. Genome-wide association analyses results identified 4 genomic regions (BTA1, BTA3, BTA7, and BTA11) associated with the considered CMP. The identified genomic regions showed contrasting results between parities and among the different stages of each parity. It suggests that different sets of candidate genes underlie the phenotypic expression of the considered CMP between parities and lactation stages of each parity. The findings of this study can be used for future implementation and use of genomic evaluation to improve the cheese-making traits in DPBB cows.

Estimation of genetic parameters for predicted nitrogen use efficiency and losses in early lactation of Holstein cows.

The objective of this study was to estimate genetic parameters of predicted N use efficiency (PNUE) and N losses (PNL) as proxies of N use and loss for Holstein cows. Furthermore, we have assessed approximate genetic correlations between PNUE, PNL, and dairy production, health, longevity, and conformation traits. These traits are considered important in many countries and are currently evaluated by the International Bull Evaluation Service (Interbull). The values of PNUE and PNL were obtained by using the combined milk mid-infrared (MIR) spectrum, parity, and milk yield-based prediction equations on test-day MIR records with days in milk (DIM) between 5 and 50 d. After editing, the final data set comprised 46,163 records of 21,462 cows from 154 farms in 5 countries. Each trait was divided into primiparous and multiparous (including second to fifth parity) groups. Genetic parameters and breeding values were estimated by using a multitrait (2-trait, 2-parity classes) repeatability model. Herd-year-season of calving, DIM, age of calving, and parity were used as fixed effects. Random effects were defined as parity (within-parity permanent environment), nongenetic cow (across-parity permanent environment), additive genetic animal, and residual effects. The estimated heritability of PNUE and PNL in the first and later parity were 0.13, 0.12, 0.14, and 0.13, and the repeatability values were 0.49, 0.40, 0.55, and 0.43, respectively. The estimated approximate genetic correlations between PNUE and PNL were negative and high (from -0.89 to -0.53), whereas the phenotypic correlations were also negative but relatively low (from -0.45 to -0.11). At a level of reliability of more than 0.30 for all novel traits, a total of 504 bulls born after 1995 had also publishable Interbull multiple-trait across-country estimated breeding values (EBV). The approximate genetic correlations between PNUE and the other 30 traits of interest, estimated as corrected correlations between EBV of bulls, ranged from -0.46 (udder depth) to 0.47 (milk yield). Obtained results showed the complex genetic relationship between efficiency, production, and other traits: for instance, more efficient cows seem to give more milk, which is linked to deeper udders, but seem to have lower health, fertility, and longevity. Additionally, the approximate genetic correlations between PNL, lower values representing less loss of N, and the 30 other traits, were from -0.32 (angularity) to 0.57 (direct calving ease). Even if further research is needed, our results provided preliminary evidence that the PNUE and PNL traits used as proxies could be included in genetic improvement programs in Holstein cows and could help their management.

Climate sensitivity of milk production traits and milk fatty acids in genotyped Holstein dairy cows.

The aim of this study was the evaluation of climate sensitivity via genomic reaction norm models [i.e., to infer cow milk production and milk fatty acid (FA) responses on temperature-humidity index (THI) alterations]. Test-day milk traits were recorded between 2010 and 2016 from 5,257 first-lactation genotyped Holstein dairy cows. The cows were kept in 16 large-scale cooperator herds, being daughters of 344 genotyped sires. The longitudinal data consisted of 47,789 test-day records for the production traits milk yield (MY), fat yield (FY), and protein yield (PY), and of 20,742 test-day records for 6 FA including C16:0, C18:0, saturated fatty acids (SFA), unsaturated fatty acids (UFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). After quality control of the genotypic data, 41,057 SNP markers remained for genomic analyses. Meteorological data from the weather station in closest herd distance were used for the calculation of maximum hourly daily THI. Genomic reaction norm models were applied to estimate genetic parameters in a single-step approach for production traits and FA in dependency of THI at different lactation stages, and to evaluate the model stability. In a first evaluation strategy (New_sire), all phenotypic records from daughters of genotyped sires born after 2010 were masked, to mimic a validation population. In the second strategy (New_env), only daughter records of the new sires recorded in the most extreme THI classes were masked, aiming at predicting sire genomic estimated breeding values (GEBV) under heat stress conditions. Model stability was the correlation between GEBV of the new sires in the reduced data set with respective GEBV estimated from all phenotypic data. Among all test-day production traits, PY responded as the most sensitive to heat stress. As observed for the remaining production traits, genetic variances were quite stable across THI, but genetic correlations between PY from temperate climates with PY from extreme THI classes dropped to 0.68. Genetic variances in dependency of THI were very similar for C16:0 and SFA, indicating marginal climatic sensitivity. In the early lactation stage, genetic variances for C18:0, MUFA, PUFA, and UFA were significantly larger in the extreme THI classes compared with the estimates under thermoneutral conditions. For C18:0 and MUFA, PUFA, and UFA in the middle THI classes, genetic correlations in same traits from the early and the later lactation stages were lower than 0.50, indicating strong days in milk influence. Interestingly, within lactation stages, genetic correlations for C18:0 and UFA recorded at low and high THI were quite large, indicating similar genetic mechanisms under stress conditions. The model stability was improved when applying the New_env instead of New_sire strategy, especially for FA in the first stage of lactation. Results indicate moderately accurate genomic predictions for milk traits in extreme THI classes when considering phenotypic data from a broad range of remaining THI. Phenotypically, thermal stress conditions contributed to an increase of UFA, suggesting value as a heat stress biomarker. Furthermore, the quite large genetic variances for UFA at high THI suggest the consideration of UFA in selection strategies for improved heat stress resistance.

Genetic analysis of milk urea concentration and its genetic relationship with selected traits of interest in dairy cows.

The aim of this study was to estimate genetic parameters of milk urea concentration (MU) and its genetic correlations with milk production traits, longevity, and functional traits in the first 3 parities in dairy cows. The edited data set consisted in 9,107,349 MU test-day records from the first 3 parities of 560,739 cows in 2,356 herds collected during the years 1994 to 2020. To estimate the genetic parameters of MU, data of 109 randomly selected herds, with a total of 770,016 MU test-day records, were used. Genetic parameters and estimated breeding values were estimated using a multiple-trait (parity) random regression model. Herd-test-day, age-year-season of calving, and days in milk classes (every 5 d as a class) were used as fixed effects, whereas effects of herd-year of calving, permanent environment, and animal were modeled using random regressions and Legendre polynomials of order 2. The average daily heritability and repeatability of MU during days in milk 5 to 365 in the first 3 parities were 0.19, 0.22, 0.20, and 0.48, 0.48, 0.47, respectively. The mean genetic correlation estimated among MU in the first 3 parities ranged from 0.96 to 0.97. The average daily estimated breeding values for MU of the selected bulls (n = 1,900) ranged from -9.09 to 7.37 mg/dL. In the last 10 yr, the genetic trend of MU has gradually increased. The genetic correlation between MU and 11 traits of interest ranged from -0.28 (milk yield) to 0.28 (somatic cell score). The findings of this study can be used as the first step for development of a routine genetic evaluation for MU and its inclusion into the genetic selection program in the Walloon Region of Belgium.

Development of a subacute ruminal acidosis risk score and its prediction using milk mid-infrared spectra in early-lactation cows.

A routine monitoring for subacute ruminal acidosis (SARA) on the individual level could support the minimization of economic losses and the ensuring of animal welfare in dairy cows. The objectives of this study were (1) to develop a SARA risk score (SRS) by combining information from different data acquisition systems to generate an integrative indicator trait, (2) the investigation of associations of the SRS with feed analysis data, blood characteristics, performance data, and milk composition, including the fatty acid (FA) profile, (3) the development of a milk mid-infrared (MIR) spectra-based prediction equation for this novel reference trait SRS, and (4) its application to an external data set consisting of MIR data of test day records to investigate the association between the MIR-based predictions of the SRS and the milk FA profile. The primary data set, which was used for the objectives (1) to (3), consisted of data collected from 10 commercial farms with a total of 100 Holstein cows in early lactation. The data comprised barn climate parameters, pH and temperature logging from intrareticular measurement boluses, as well as jaw movement and locomotion behavior recordings of noseband-sensor halters and pedometers. Further sampling and data collection included feed samples, blood samples, milk performance, and milk samples, whereof the latter were used to get the milk MIR spectra and to estimate the main milk components, the milk FA profile, and the lactoferrin content. Because all measurements were characterized by different temporal resolutions, the data preparation consisted of an aggregation into values on a daily basis and merging it into one data set. For the development of the SRS, a total of 7 traits were selected, which were derived from measurements of pH and temperature in the reticulum, chewing behavior, and milk yield. After adjustment for fixed effects and standardization, these 7 traits were combined into the SRS using a linear combination and directional weights based on current knowledge derived from literature studies. The secondary data set was used for objective (4) and consisted of test day records of the entire herds, including performance data, milk MIR spectra and MIR-predicted FA. At farm level, it could be shown that diets with higher proportions of concentrated feed resulted in both lower daily mean pH and higher SRS values. On the individual level, an increased SRS could be associated with a modified FA profile (e.g., lower levels of short- and medium-chain FA, higher levels of C17:0, odd- and branched-chain FA). Furthermore, a milk MIR-based partial least squares regression model with a moderate predictability was established for the SRS. This work provides the basis for the development of routine SARA monitoring and demonstrates the high potential of milk composition-based assessment of the health status of lactating cows.

Predicting milk mid-infrared spectra from first-parity Holstein cows using a test-day mixed model with the perspective of herd management.

The use of test-day models to model milk mid-infrared (MIR) spectra for genetic purposes has already been explored; however, little attention has been given to their use to predict milk MIR spectra for management purposes. The aim of this paper was to study the ability of a test-day mixed model to predict milk MIR spectra for management purposes. A data set containing 467,496 test-day observations from 53,781 Holstein dairy cows in first lactation was used for model building. Principal component analysis was implemented on the selected 311 MIR spectral wavenumbers to reduce the number of traits for modeling; 12 principal components (PC) were retained, explaining approximately 96% of the total spectral variation. Each of the retained PC was modeled using a single trait test-day mixed model. The model solutions were used to compute the predicted scores of each PC, followed by a back-transformation to obtain the 311 predicted MIR spectral wavenumbers. Four new data sets, containing altogether 122,032 records, were used to test the ability of the model to predict milk MIR spectra in 4 distinct scenarios with different levels of information about the cows. The average correlation between observed and predicted values of each spectral wavenumber was 0.85 for the modeling data set and ranged from 0.36 to 0.62 for the scenarios. Correlations between milk fat, protein, and lactose contents predicted from the observed spectra and from the modeled spectra ranged from 0.83 to 0.89 for the modeling set and from 0.32 to 0.73 for the scenarios. Our results demonstrated a moderate but promising ability to predict milk MIR spectra using a test-day mixed model. Current and future MIR traits prediction equations could be applied on the modeled spectra to predict all MIR traits in different situations instead of developing one test-day model separately for each trait. Modeling MIR spectra would benefit farmers for cow and herd management, for instance through prediction of future records or comparison between observed and expected wavenumbers or MIR traits for the detection of health and management problems. Potential resulting tools could be incorporated into milk recording systems.

Diagnosing the pregnancy status of dairy cows: How useful is milk mid-infrared spectroscopy?

Pregnancy diagnosis is an essential part of successful breeding programs on dairy farms. Milk composition alters with pregnancy, and this is well documented. Fourier-transform mid-infrared (MIR) spectroscopy is a rapid and cost-effective method for providing milk spectra that reflect the detailed composition of milk samples. Therefore, the aim of this study was to assess the ability of MIR spectroscopy to predict the pregnancy status of dairy cows. The MIR spectra and insemination records were available from 8,064 Holstein cows of 19 commercial dairy farms in Australia. Three strategies were studied to classify cows as open or pregnant using partial least squares discriminant analysis models with random cow-independent 10-fold cross-validation and external validation on a cow-independent test set. The first strategy considered 6,754 MIR spectra after insemination used as independent variables in the model. The results showed little ability to detect the pregnancy status as the area under the receiver operating characteristic curve was 0.63 and 0.65 for cross-validation and testing, respectively. The second strategy, involving 1,664 records, aimed to reduce noise in the MIR spectra used as predictors by subtracting a spectrum before insemination (i.e., open spectrum) from the spectrum after insemination. The accuracy was comparable with the first approach, showing no superiority of the method. Given the limited results for these models when using combined data from all stages after insemination, the third strategy explored separate models at 7 stages after insemination comprising 348 to 1,566 records each (i.e., progressively greater gestation) with single MIR spectra after insemination as predictors. The models developed using data recorded after 150 d of pregnancy showed promising prediction accuracy with the average value of area under the receiver operating characteristic curve of 0.78 and 0.76 obtained through cross-validation and testing, respectively. If this can be confirmed on a larger data set and extended to somewhat earlier stages after insemination, the model could be used as a complementary tool to detect fetal abortion.

A comparison of 4 different machine learning algorithms to predict lactoferrin content in bovine milk from mid-infrared spectra.

Lactoferrin (LF) is a glycoprotein naturally present in milk. Its content varies throughout lactation, but also with mastitis; therefore it is a potential additional indicator of udder health beyond somatic cell count. Condequently, there is an interest in quantifying this biomolecule routinely. First prediction equations proposed in the literature to predict the content in milk using milk mid-infrared spectrometry were built using partial least square regression (PLSR) due to the limited size of the data set. Thanks to a large data set, the current study aimed to test 4 different machine learning algorithms using a large data set comprising 6,619 records collected across different herds, breeds, and countries. The first algorithm was a PLSR, as used in past investigations. The second and third algorithms used partial least square (PLS) factors combined with a linear and polynomial support vector regression (PLS + SVR). The fourth algorithm also used PLS factors, but included in an artificial neural network with 1 hidden layer (PLS + ANN). The training and validation sets comprised 5,541 and 836 records, respectively. Even if the calibration prediction performances were the best for PLS + polynomial SVR, their validation prediction performances were the worst. The 3 other algorithms had similar validation performances. Indeed, the validation root mean squared error (RMSE) ranged between 162.17 and 166.75 mg/L of milk. However, the lower standard deviation of cross-validation RMSE and the better normality of the residual distribution observed for PLS + ANN suggest that this modeling was more suitable to predict the LF content in milk from milk mid-infrared spectra (R2v = 0.60 and validation RMSE = 162.17 mg/L of milk). This PLS +ANN model was then applied to almost 6 million spectral records. The predicted LF showed the expected relationships with milk yield, somatic cell score, somatic cell count, and stage of lactation. The model tended to underestimate high LF values (higher than 600 mg/L of milk). However, if the prediction threshold was set to 500 mg/L, 82% of samples from the validation having a content of LF higher than 600 mg/L were detected. Future research should aim to increase the number of those extremely high LF records in the calibration set.