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.

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.

Methodological guidelines: Cow milk mid-infrared spectra to predict reference enteric methane data collected by an automated head-chamber system.

Various methodological protocols were tested on milk samples from cows fed diets affecting both methanogenesis and milk synthesis to identify the best approach for the prediction of GreenFeed system (GF) measured methane (CH4) emissions by milk mid-infrared (MIR) spectroscopy. The models developed were also tested on a data set from cows fed chemical inhibitors of CH4 emission [3-nitrooxypropanol (3NOP)] that just marginally affect milk composition. A total of 129 primiparous and multiparous Holstein cows fed diets with different methanogenic potential were considered. Individual milk yield (MY) and dry matter intake were recorded daily, whereas fat- and protein-corrected milk (FPCM) was recorded twice a week. The MIR spectra from 2 consecutive milkings were collected twice a week. Twenty CH4 spot measurements with GF were taken as the basic measurement unit (BMU) of CH4. The equations were built using partial least squares regression by splitting the database into calibration and validation data sets (excluding 3NOP samples). Models were developed for milk MIR spectra by milking and on day spectra obtained by averaging spectra from 2 consecutive milkings. Models based on day spectra were calibrated by using CH4 reference data for a measurement duration of 1, 2, 3, or 4 BMU. Models built from the average of the day spectra collected during the corresponding CH4 measurement periods were developed. Corrections of spectra by days in milk (DIM) and the inclusion of parity, MY, and FPCM as explanatory variables were tested as tools to improve model performance. Models built on day milk MIR spectra gave slightly better performances that those developed using spectra from a single milking. Long duration of CH4 measurement by GF performed better than short duration: the coefficient of determination of validation (R2V) for CH4 emissions expressed in grams per day were 0.60 vs. 0.52 for 4 and 1 BMU, respectively. When CH4 emissions were expressed as grams per kilogram of dry of matter intake, grams per kilogram of MY, or grams per kilogram of FPCM, performance with a long duration also improved. Coupling GF reference data with the average of milk MIR spectra collected throughout the corresponding CH4 measurement period gave better predictions than using day spectra (R2V = 0.70 vs. 0.60 for CH4 as g/d on 4 BMU). Correcting the day spectra by DIM improved R2V compared with the equivalent DIM-uncorrected models (R2V = 0.67 vs. 0.60 for CH4 as g/d on 4 BMU). Adding other phenotypic information as explanatory variables did not further improve the performance of models built on single day DIM-corrected spectra, whereas including MY (or FPCM) improved the performance of models built on the average of spectra (uncorrected by DIM) recorded during the CH4 measurement period (R2V = 0.73 vs. 0.70 for CH4 as g/d on 4 BMU). When validating the models on the 3NOP data set, predictions were poor without (R2V = 0.13 for CH4 as g/d on 1 BMU) or with (R2V = 0.31 for CH4 as g/d on 1 BMU) integration of 3NOP data in the models. Thus, specific models would be required for CH4 prediction when cows receive chemical inhibitors of CH4 emissions not affecting milk composition.

Prediction of nitrogen excretion from data on dairy cows fed a wide range of diets compiled in an intercontinental database: A meta-analysis.

Manure nitrogen (N) from cattle contributes to nitrous oxide and ammonia emissions and nitrate leaching. Measurement of manure N outputs on dairy farms is laborious, expensive, and impractical at large scales; therefore, models are needed to predict N excreted in urine and feces. Building robust prediction models requires extensive data from animals under different management systems worldwide. Thus, the study objectives were (1) to collate an international database of N excretion in feces and urine based on individual lactating dairy cow data from different continents; (2) to determine the suitability of key variables for predicting fecal, urinary, and total manure N excretion; and (3) to develop robust and reliable N excretion prediction models based on individual data from lactating dairy cows consuming various diets. A raw data set was created based on 5,483 individual cow observations, with 5,420 fecal N excretion and 3,621 urine N excretion measurements collected from 162 in vivo experiments conducted by 22 research institutes mostly located in Europe (n = 14) and North America (n = 5). A sequential approach was taken in developing models with increasing complexity by incrementally adding variables that had a significant individual effect on fecal, urinary, or total manure N excretion. Nitrogen excretion was predicted by fitting linear mixed models including experiment as a random effect. Simple models requiring dry matter intake (DMI) or N intake performed better for predicting fecal N excretion than simple models using diet nutrient composition or milk performance parameters. Simple models based on N intake performed better for urinary and total manure N excretion than those based on DMI, but simple models using milk urea N (MUN) and N intake performed even better for urinary N excretion. The full model predicting fecal N excretion had similar performance to simple models based on DMI but included several independent variables (DMI, diet crude protein content, diet neutral detergent fiber content, milk protein), depending on the location, and had root mean square prediction errors as a fraction of the observed mean values of 19.1% for intercontinental, 19.8% for European, and 17.7% for North American data sets. Complex total manure N excretion models based on N intake and MUN led to prediction errors of about 13.0% to 14.0%, which were comparable to models based on N intake alone. Intercepts and slopes of variables in optimal prediction equations developed on intercontinental, European, and North American bases differed from each other, and therefore region-specific models are preferred to predict N excretion. In conclusion, region-specific models that include information on DMI or N intake and MUN are required for good prediction of fecal, urinary, and total manure N excretion. In absence of intake data, region-specific complex equations using easily and routinely measured variables to predict fecal, urinary, or total manure N excretion may be used, but these equations have lower performance than equations based on intake.

Identification of chronic stress biomarkers in dairy cows.

Stress in dairy herds can occur from multiple sources. When stress becomes chronic because of a long duration and inability of animals to adapt, it is likely to deeply affect the emotional state, health, immunity, fertility and milk production of cows. While assessing chronic stress in herds would be beneficial, no real consensus has emerged from the literature regarding the indicators of interest. The goal of this study was to compare and evaluate potential biomarkers for chronic stress after inducing stress over a 4-week period through severe overstocking, restricted access to feed and isolated unusual events. A total of 30 cows were involved in the experiment and two similar groups were constituted. Over a 4-week period, the 15 cows of the stress group were housed in overstocked conditions, with 4.6 m2 per cow, including resting and feeding areas. In this area, only seven individual places at the feeding area were available for the 15 cows to generate competition for feed access. Twice during the trial and over a period of 2 h, an additional stress was induced by moving cows to an unfamiliar barn and diffusion of stressing noises (dog barking). Meanwhile, the 15 cows of the control group stayed in the original barn, with more than 10 m2 per cow and more individual places at the feeding area than cow number. On a weekly basis, several variables considered as potential biomarkers for chronic stress were recorded. Collected data were analysed using single trait linear repeated mixed models. No differences were observed regarding milk yield, BW of cows or body condition score but the milk loss was more pronounced in the stress group. The activity was more heterogeneous and the rumination of cows was lower in the stress group. The heart rate was lower in the stress group and showed more heterogeneity at the end of the stress period. No differences were observed regarding salivary cortisol, blood glucose, ß-endorphin, thyroxine and leucocyte profile. A higher level of hair cortisol and blood fructosamine were observed in the stress group at the end of the stress period. Regarding the practical use of the highlighted biomarkers, milk loss may be an effective and easy way to detect general problems, including stress. The blood fructosamine and the hair cortisol concentrations are promising indicators to assess chronic stress in commercial farms.

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.

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.

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.

Between- and within-herd variation in blood and milk biomarkers in Holstein cows in early lactation.

Both blood- and milk-based biomarkers have been analysed for decades in research settings, although often only in one herd, and without focus on the variation in the biomarkers that are specifically related to herd or diet. Biomarkers can be used to detect physiological imbalance and disease risk and may have a role in precision livestock farming (PLF). For use in PLF, it is important to quantify normal variation in specific biomarkers and the source of this variation. The objective of this study was to estimate the between- and within-herd variation in a number of blood metabolites (ß-hydroxybutyrate (BHB), non-esterified fatty acids, glucose and serum IGF-1), milk metabolites (free glucose, glucose-6-phosphate, urea, isocitrate, BHB and uric acid), milk enzymes (lactate dehydrogenase and N-acetyl-ß-D-glucosaminidase (NAGase)) and composite indicators for metabolic imbalances (Physiological Imbalance-index and energy balance), to help facilitate their adoption within PLF. Blood and milk were sampled from 234 Holstein dairy cows from 6 experimental herds, each in a different European country, and offered a total of 10 different diets. Blood was sampled on 2 occasions at approximately 14 days-in-milk (DIM) and 35 DIM. Milk samples were collected twice weekly (in total 2750 samples) from DIM 1 to 50. Multilevel random regression models were used to estimate the variance components and to calculate the intraclass correlations (ICCs). The ICCs for the milk metabolites, when adjusted for parity and DIM at sampling, demonstrated that between 12% (glucose-6-phosphate) and 46% (urea) of the variation in the metabolites' levels could be associated with the herd-diet combination. Intraclass Correlations related to the herd-diet combination were generally higher for blood metabolites, from 17% (cholesterol) to approximately 46% (BHB and urea). The high ICCs for urea suggest that this biomarker can be used for monitoring on herd level. The low variance within cow for NAGase indicates that few samples would be needed to describe the status and potentially a general reference value could be used. The low ICC for most of the biomarkers and larger within cow variation emphasises that multiple samples would be needed - most likely on the individual cows - for making the biomarkers useful for monitoring. The majority of biomarkers were influenced by parity and DIM which indicate that these should be accounted for if the biomarker should be used for monitoring.

Potential of milk mid-IR spectra to predict metabolic status of cows through blood components and an innovative clustering approach.

Unbalanced metabolic status in the weeks after calving predisposes dairy cows to metabolic and infectious diseases. Blood glucose, IGF-I, non-esterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB) are used as indicators of the metabolic status of cows. This work aims to (1) evaluate the potential of milk mid-IR spectra to predict these blood components individually and (2) to evaluate the possibility of predicting the metabolic status of cows based on the clustering of these blood components. Blood samples were collected from 241 Holstein cows on six experimental farms, at days 14 and 35 after calving. Blood samples were analyzed by reference analysis and metabolic status was defined by k-means clustering (k=3) based on the four blood components. Milk mid-IR analyses were undertaken on different instruments and the spectra were harmonized into a common standardized format. Quantitative models predicting blood components were developed using partial least squares regression and discriminant models aiming to differentiate the metabolic status were developed with partial least squares discriminant analysis. Cross-validations were performed for both quantitative and discriminant models using four subsets randomly constituted. Blood glucose, IGF-I, NEFA and BHB were predicted with respective R 2 of calibration of 0.55, 0.69, 0.49 and 0.77, and R 2 of cross-validation of 0.44, 0.61, 0.39 and 0.70. Although these models were not able to provide precise quantitative values, they allow for screening of individual milk samples for high or low values. The clustering methodology led to the sharing out of the data set into three groups of cows representing healthy, moderately impacted and imbalanced metabolic status. The discriminant models allow to fairly classify the three groups, with a global percentage of correct classification up to 74%. When discriminating the cows with imbalanced metabolic status from cows with healthy and moderately impacted metabolic status, the models were able to distinguish imbalanced group with a global percentage of correct classification up to 92%. The performances were satisfactory considering the variables are not present in milk, and consequently predicted indirectly. This work showed the potential of milk mid-IR analysis to provide new metabolic status indicators based on individual blood components or a combination of these variables into a global status. Models have been developed within a standardized spectral format, and although robustness should preferably be improved with additional data integrating different geographic regions, diets and breeds, they constitute rapid, cost-effective and large-scale tools for management and breeding of dairy cows.

Short communication: Development of an equation for estimating methane emissions of dairy cows from milk Fourier transform mid-infrared spectra by using reference data obtained exclusively from respiration chambers.

Evaluation and mitigation of enteric methane (CH4) emissions from ruminant livestock, in particular from dairy cows, have acquired global importance for sustainable, climate-smart cattle production. Based on CH4 reference measurements obtained with the SF6 tracer technique to determine ruminal CH4 production, a current equation permits evaluation of individual daily CH4 emissions of dairy cows based on milk Fourier transform mid-infrared (FT-MIR) spectra. However, the respiration chamber (RC) technique is considered to be more accurate than SF6 to measure CH4 production from cattle. This study aimed to develop an equation that allows estimating CH4 emissions of lactating cows recorded in an RC from corresponding milk FT-MIR spectra and to challenge its robustness and relevance through validation processes and its application on a milk spectral database. This would permit confirming the conclusions drawn with the existing equation based on SF6 reference measurements regarding the potential to estimate daily CH4 emissions of dairy cows from milk FT-MIR spectra. A total of 584 RC reference CH4 measurements (mean ± standard deviation of 400 ± 72 g of CH4/d) and corresponding standardized milk mid-infrared spectra were obtained from 148 individual lactating cows between 7 and 321 d in milk in 5 European countries (Germany, Switzerland, Denmark, France, and Northern Ireland). The developed equation based on RC measurements showed calibration and cross-validation coefficients of determination of 0.65 and 0.57, respectively, which is lower than those obtained earlier by the equation based on 532 SF6 measurements (0.74 and 0.70, respectively). This means that the RC-based model is unable to explain the variability observed in the corresponding reference data as well as the SF6-based model. The standard errors of calibration and cross-validation were lower for the RC model (43 and 47 g/d vs. 66 and 70 g/d for the SF6 version, respectively), indicating that the model based on RC data was closer to actual values. The root mean squared error (RMSE) of calibration of 42 g/d represents only 10% of the overall daily CH4 production, which is 23 g/d lower than the RMSE for the SF6-based equation. During the external validation step an RMSE of 62 g/d was observed. When the RC equation was applied to a standardized spectral database of milk recordings collected in the Walloon region of Belgium between January 2012 and December 2017 (1,515,137 spectra from 132,658 lactating cows in 1,176 different herds), an average ± standard deviation of 446 ± 51 g of CH4/d was estimated, which is consistent with the range of the values measured using both RC and SF6 techniques. This study confirmed that milk FT-MIR spectra could be used as a potential proxy to estimate daily CH4 emissions from dairy cows provided that the variability to predict is covered by the model.

Relationships between milk mid-IR predicted gastro-enteric methane production and the technical and financial performance of commercial dairy herds.

Considering economic and environmental issues is important in ensuring the sustainability of dairy farms. The objective of this study was to investigate univariate relationships between lactating dairy cow gastro-enteric methane (CH4) production predicted from milk mid-IR (MIR) spectra and technico-economic variables by the use of large scale and on-farm data. A total of 525 697 individual CH4 predictions from milk MIR spectra (MIR-CH4 (g/day)) of milk samples collected on 206 farms during the Walloon milk recording scheme were used to create a MIR-CH4 prediction for each herd and year (HYMIR-CH4). These predictions were merged with dairy herd accounting data. This allowed a simultaneous study of HYMIR-CH4 and 42 technical and economic variables for 1024 herd and year records from 2007 to 2014. Pearson correlation coefficients (r) were used to assess significant relationships (P<0.05). Low HYMIR-CH4 was significantly associated with, amongst others, lower fat and protein corrected milk (FPCM) yield (r=0.18), lower milk fat and protein content (r=0.38 and 0.33, respectively), lower quantity of milk produced from forages (r=0.12) and suboptimal reproduction and health performance (e.g. longer calving interval (r=-0.21) and higher culling rate (r=-0.15)). Concerning economic results, low HYMIR-CH4 was significantly associated with lower gross margin per cow (r=0.19) and per litre FPCM (r=0.09). To conclude, this study suggested that low lactating dairy cow gastro-enteric CH4 production tended to be associated with more extensive or suboptimal management practices, which could lead to lower profitability. The observed low correlations suggest complex interactions between variables due to the use of on-farm data with large variability in technical and management practices.

Standardization of milk mid-infrared spectrometers for the transfer and use of multiple models.

An increasing number of models are being developed to provide information from milk Fourier transform mid-infrared (FT-MIR) spectra on fine milk composition, technological properties of milk, or even cows' physiological status. In this context, and to take advantage of these existing models, the purpose of this work was to evaluate whether a spectral standardization method can enable the use of multiple equations within a network of different FT-MIR spectrometers. The piecewise direct standardization method was used, matching "slave" instruments to a common reference, the "master." The effect of standardization on network reproducibility was assessed on 66 instruments from 3 different brands by comparing the spectral variability of the slaves and the master with and without standardization. With standardization, the global Mahalanobis distance from the slave spectra to the master spectra was reduced on average from 2,655.9 to 14.3, representing a significant reduction of noninformative spectral variability. The transfer of models from instrument to instrument was tested using 3 FT-MIR models predicting (1) the quantity of daily methane emitted by dairy cows, (2) the concentration of polyunsaturated fatty acids in milk, and (3) the fresh cheese yield. The differences, in terms of root mean squared error, between master predictions and slave predictions were reduced after standardization on average from 103 to 17 g/d, from 0.0315 to 0.0045 g/100 mL of milk, and from 2.55 to 0.49 g of curd/100 g of milk, respectively. For all the models, standard deviations of predictions among all the instruments were also reduced by 5.11 times for methane, 5.01 times for polyunsaturated fatty acids, and 7.05 times for fresh cheese yield, showing an improvement of prediction reproducibility within the network. Regarding the results obtained, spectral standardization allows the transfer and use of multiple models on all instruments as well as the improvement of spectral and prediction reproducibility within the network. The method makes the models universal, thereby offering opportunities for data exchange and the creation and use of common robust models at an international level to provide more information to the dairy sector from direct analysis of milk.

Genetic parameters of mid-infrared methane predictions and their relationships with milk production traits in Holstein cattle.

Many countries have pledged to reduce greenhouse gases. In this context, the dairy sector is one of the identified sectors to adapt production circumstances to address socio-environmental constraints due to its large carbon footprint related to CH4 emission. This study aimed mainly to estimate (1) the genetic parameters of 2 milk mid-infrared-based CH4 proxies [predicted daily CH4 emission (PME, g/d), and log-transformed predicted CH4 intensity (LMI)] and (2) their genetic correlations with milk production traits [milk (MY), fat (FY), and protein (PY) yields] from first- and second-parity Holstein cows. A total of 336,126 and 231,400 mid-infrared CH4 phenotypes were collected from 56,957 and 34,992 first- and second-parity cows, respectively. The PME increased from the first to the second lactation (433 vs. 453 g/d) and the LMI decreased (2.93 vs. 2.86). We used 20 bivariate random regression test-day models to estimate the variance components. Moderate heritability values were observed for both CH4 traits, and those values decreased slightly from the first to the second lactation (0.25 ± 0.01 and 0.22 ± 0.01 for PME; 0.18 ± 0.01 and 0.17 ± 0.02 for LMI). Lactation phenotypic and genetic correlations were negative between PME and MY in both first and second lactations (-0.07 vs. -0.07 and -0.19 vs. -0.24, respectively). More close scrutiny revealed that relative increase of PME was lower with high MY levels even reverting to decrease, and therefore explaining the negative correlations, indicating that higher producing cows could be a mitigation option for CH4 emission. The PME phenotypic correlations were almost equal to 0 with FY and PY for both lactations. However, the genetic correlations between PME and FY were slightly positive (0.11 and 0.12), whereas with PY the correlations were slightly negative (-0.05 and -0.04). Both phenotypic and genetic correlations between LMI and MY or PY or FY were always relatively highly negative (from -0.21 to -0.88). As the genetic correlations between PME and LMI were strong (0.71 and 0.72 in first and second lactation), the selection of one trait would also strongly influence the other trait. However, in animal breeding context, PME, as a direct quantity CH4 proxy, would be preferred to LMI, which is a ratio trait of PME with a trait already in the index. The range of PME sire estimated breeding values were 22.1 and 29.41 kg per lactation in first and second parity, respectively. Further studies must be conducted to evaluate the effect of the introduction of PME in a selection index on the other traits already included in this index, such as, for instance, fertility or longevity.

Invited review: Large-scale indirect measurements for enteric methane emissions in dairy cattle: A review of proxies and their potential for use in management and breeding decisions.

Efforts to reduce the carbon footprint of milk production through selection and management of low-emitting cows require accurate and large-scale measurements of methane (CH4) emissions from individual cows. Several techniques have been developed to measure CH4 in a research setting but most are not suitable for large-scale recording on farm. Several groups have explored proxies (i.e., indicators or indirect traits) for CH4; ideally these should be accurate, inexpensive, and amenable to being recorded individually on a large scale. This review (1) systematically describes the biological basis of current potential CH4 proxies for dairy cattle; (2) assesses the accuracy and predictive power of single proxies and determines the added value of combining proxies; (3) provides a critical evaluation of the relative merit of the main proxies in terms of their simplicity, cost, accuracy, invasiveness, and throughput; and (4) discusses their suitability as selection traits. The proxies range from simple and low-cost measurements such as body weight and high-throughput milk mid-infrared spectroscopy (MIR) to more challenging measures such as rumen morphology, rumen metabolites, or microbiome profiling. Proxies based on rumen samples are generally poor to moderately accurate predictors of CH4, and are costly and difficult to measure routinely on-farm. Proxies related to body weight or milk yield and composition, on the other hand, are relatively simple, inexpensive, and high throughput, and are easier to implement in practice. In particular, milk MIR, along with covariates such as lactation stage, are a promising option for prediction of CH4 emission in dairy cows. No single proxy was found to accurately predict CH4, and combinations of 2 or more proxies are likely to be a better solution. Combining proxies can increase the accuracy of predictions by 15 to 35%, mainly because different proxies describe independent sources of variation in CH4 and one proxy can correct for shortcomings in the other(s). The most important applications of CH4 proxies are in dairy cattle management and breeding for lower environmental impact. When breeding for traits of lower environmental impact, single or multiple proxies can be used as indirect criteria for the breeding objective, but care should be taken to avoid unfavorable correlated responses. Finally, although combinations of proxies appear to provide the most accurate estimates of CH4, the greatest limitation today is the lack of robustness in their general applicability. Future efforts should therefore be directed toward developing combinations of proxies that are robust and applicable across diverse production systems and environments.

Assessing the effect of pregnancy stage on milk composition of dairy cows using mid-infrared spectra.

Changes in milk production traits (i.e., milk yield, fat, and protein contents) with the pregnancy stage are well documented. To our knowledge, the effect of pregnancy on the detailed milk composition has not been studied so far. The mid-infrared (MIR) spectrum reflects the detailed composition of a milk sample and is obtained by a nonexhaustive and widely used method for milk analysis. Therefore, this study aimed to investigate the effect of pregnancy on milk MIR spectrum in addition to milk production traits (milk yield, fat, and protein contents). A model including regression on the number of days pregnant was applied on milk production traits (milk yield, fat, and protein contents) and on 212 spectral points from the MIR spectra of 9,757 primiparous Holstein cows from Walloon herds. Effects of pregnancy stage were expressed on a relative scale (effect divided by the squared root of the phenotypic variance); this allowed comparisons between effects on milk traits and on 212 spectral points. Effect of pregnancy stage on production traits were in line with previous studies indicating that the model accounted well for the pregnancy effect. Trends of the relative effect of the pregnancy stage on the 212 spectral points were consistent with known and observed effect on milk traits. The highest effect of the pregnancy was observed in the MIR spectral region from 968 to 1,577 cm-1. For some specific wavenumbers, the effect was higher than for fat and protein contents in the beginning of the pregnancy (from 30 to 90 or 120 d pregnant). In conclusion, the effect of early pregnancy can be observed in the detailed milk composition through the analysis of the MIR spectrum of bovine milk. Further analyses are warranted to explore deeply the use of MIR spectra of bovine milk for breeding and management of dairy cow pregnancy.

Changes throughout lactation in phenotypic and genetic correlations between methane emissions and milk fatty acid contents predicted from milk mid-infrared spectra.

The aim of this study was to estimate phenotypic and genetic correlations between methane production (Mp) and milk fatty acid contents of first-parity Walloon Holstein cows throughout lactation. Calibration equations predicting daily Mp (g/d) and milk fatty acid contents (g/100 dL of milk) were applied on milk mid-infrared spectra related to Walloon milk recording. A total of 241,236 predictions of Mp and milk fatty acids were used. These data were collected between 5 and 305 d in milk in 33,555 first-parity Holstein cows from 626 herds. Pedigree data included 109,975 animals. Bivariate (i.e., Mp and a fatty acid trait) random regression test-day models were developed to estimate phenotypic and genetic parameters of Mp and milk fatty acids. Individual short-chain fatty acids (SCFA) and groups of saturated fatty acids, SCFA, and medium-chain fatty acids showed positive phenotypic and genetic correlations with Mp (from 0.10 to 0.16 and from 0.23 to 0.30 for phenotypic and genetic correlations, respectively), whereas individual long-chain fatty acids (LCFA), and groups of LCFA, monounsaturated fatty acids, and unsaturated fatty acids showed null to positive phenotypic and genetic correlations with Mp (from -0.03 to 0.13 and from -0.02 to 0.32 for phenotypic and genetic correlations, respectively). However, these correlations changed throughout lactation. First, de novo individual and group fatty acids (i.e., C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, SCFA group) showed low phenotypic or genetic correlations (or both) in early lactation and higher at the end of lactation. In contrast, phenotypic and genetic correlations between Mp and C16:0, which could be de novo synthetized or derived from blood lipids, were more stable during lactation. This fatty acid is the most abundant fatty acid of the saturated fatty acid and medium-chain fatty acid groups of which correlations with Mp showed the same pattern across lactation. Phenotypic and genetic correlations between Mp and C17:0 and C18:0 were low in early lactation and increased afterward. Phenotypic and genetic correlations between Mp and C18:1 cis-9 originating from the blood lipids were negative in early lactation and increased afterward to become null from 18 wk until the end of lactation. Correlations between Mp and groups of LCFA, monounsaturated fatty acids, and unsaturated fatty acids showed a similar or intermediate pattern across lactation compared with fatty acids that compose them. Finally, these results indicate that correlations between Mp and milk fatty acids vary following lactation stage of the cow, a fact still often ignored when trying to predict Mp from milk fatty acid profile.

Development of Fourier transform mid-infrared calibrations to predict acetone, ß-hydroxybutyrate, and citrate contents in bovine milk through a European dairy network.

To manage negative energy balance and ketosis in dairy farms, rapid and cost-effective detection is needed. Among the milk biomarkers that could be useful for this purpose, acetone and ß-hydroxybutyrate (BHB) have been proved as molecules of interest regarding ketosis and citrate was recently identified as an early indicator of negative energy balance. Because Fourier transform mid-infrared spectrometry can provide rapid and cost-effective predictions of milk composition, the objective of this study was to evaluate the ability of this technology to predict these biomarkers in milk. Milk samples were collected in commercial and experimental farms in Luxembourg, France, and Germany. Acetone, BHB, and citrate contents were determined by flow injection analysis. Milk mid-infrared spectra were recorded and standardized for all samples. After edits, a total of 548 samples were used in the calibration and validation data sets for acetone, 558 for BHB, and 506 for citrate. Acetone content ranged from 0.020 to 3.355mmol/L with an average of 0.103mmol/L; BHB content ranged from 0.045 to 1.596mmol/L with an average of 0.215mmol/L; and citrate content ranged from 3.88 to 16.12mmol/L with an average of 9.04mmol/L. Acetone and BHB contents were log-transformed and a part of the samples with low values was randomly excluded to approach a normal distribution. The 3 edited data sets were then randomly divided into a calibration data set (3/4 of the samples) and a validation data set (1/4 of the samples). Prediction equations were developed using partial least square regression. The coefficient of determination (R(2)) of cross-validation was 0.73 for acetone, 0.71 for BHB, and 0.90 for citrate with root mean square error of 0.248, 0.109, and 0.70mmol/L, respectively. Finally, the external validation was performed and R(2) obtained were 0.67 for acetone, 0.63 for BHB, and 0.86 for citrate, with respective root mean square error of validation of 0.196, 0.083, and 0.76mmol/L. Although the practical usefulness of the equations developed should be further verified with other field data, results from this study demonstrated the potential of Fourier transform mid-infrared spectrometry to predict citrate content with good accuracy and to supply indicative contents of BHB and acetone in milk, thereby providing rapid and cost-effective tools to manage ketosis and negative energy balance in dairy farms.

Capitalizing on fine milk composition for breeding and management of dairy cows.

The challenge of managing and breeding dairy cows is permanently adapting to changing production circumstances under socio-economic constraints. If managing and breeding address different timeframes of action, both need relevant phenotypes that allow for precise monitoring of the status of the cows, and their health, behavior, and well-being as well as their environmental impact and the quality of their products (i.e., milk and subsequently dairy products). Milk composition has been identified as an important source of information because it could reflect, at least partially, all these elements. Major conventional milk components such as fat, protein, urea, and lactose contents are routinely predicted by mid-infrared (MIR) spectrometry and have been widely used for these purposes. But, milk composition is much more complex and other nonconventional milk components, potentially predicted by MIR, might be informative. Such new milk-based phenotypes should be considered given that they are cheap, rapidly obtained, usable on a large scale, robust, and reliable. In a first approach, new phenotypes can be predicted from MIR spectra using techniques based on classical prediction equations. This method was used successfully for many novel traits (e.g., fatty acids, lactoferrin, minerals, milk technological properties, citrate) that can be then useful for management and breeding purposes. An innovation was to consider the longitudinal nature of the relationship between the trait of interest and the MIR spectra (e.g., to predict methane from MIR). By avoiding intermediate steps, prediction errors can be minimized when traits of interest (e.g., methane, energy balance, ketosis) are predicted directly from MIR spectra. In a second approach, research is ongoing to detect and exploit patterns in an innovative manner, by comparing observed with expected MIR spectra directly (e.g., pregnancy). All of these traits can then be used to define best practices, adjust feeding and health management, improve animal welfare, improve milk quality, and mitigate environmental impact. Under the condition that MIR data are available on a large scale, phenotypes for these traits will allow genetic and genomic evaluations. Introduction of novel traits into the breeding objectives will need additional research to clarify socio-economic weights and genetic correlations with other traits of interest.

Hot topic: Innovative lactation-stage-dependent prediction of methane emissions from milk mid-infrared spectra.

The main goal of this study was to develop, apply, and validate a new method to predict an indicator for CH4 eructed by dairy cows using milk mid-infrared (MIR) spectra. A novel feature of this model was the consideration of lactation stage to reflect changes in the metabolic status of the cow. A total of 446 daily CH4 measurements were obtained using the SF6 method on 142 Jersey, Holstein, and Holstein-Jersey cows. The corresponding milk samples were collected during these CH4 measurements and were analyzed using MIR spectroscopy. A first derivative was applied to the milk MIR spectra. To validate the novel calibration equation incorporating days in milk (DIM), 2 calibration processes were developed: the first was based only on CH4 measurements and milk MIR spectra (independent of lactation stage; ILS); the second included milk MIR spectra and DIM information (dependent on lactation stage; DLS) by using linear and quadratic modified Legendre polynomials. The coefficients of determination of ILS and DLS equations were 0.77 and 0.75, respectively, with standard error of calibration of 63g/d of CH4 for both calibration equations. These equations were applied to 1,674,763 milk MIR spectra from Holstein cows in the first 3 parities and between 5 and 365 DIM. The average CH4 indicators were 428, 444, and 448g/d by ILS and 444, 467, and 471g/d by DLS for cows in first, second, and third lactation, respectively. Behavior of the DLS indicator throughout the lactations was in agreement with the literature with values increasing between 0 and 100 DIM and decreasing thereafter. Conversely, the ILS indicator of CH4 emission decreased at the beginning of the lactation and increased until the end of the lactation, which differs from the literature. Therefore, the DLS indicator seems to better reflect biological processes that drive CH4 emissions than the ILS indicator. The ILS and DLS equations were applied to an independent data set, which included 59 respiration chamber measurements of CH4 obtained from animals of a different breed across a different production system. Results indicated that the DLS equation was much more robust than the ILS equation allowing development of indicators of CH4 emissions by dairy cows. Integration of DIM information into the prediction equation was found to be a good strategy to obtain biologically meaningful CH4 values from lactating cows by accounting for biological changes that occur throughout the lactation.