Validation of milk mid-infrared spectroscopy for predicting the metabolic status of lactating dairy cows in Australia.

Increased concentrations of some serum biomarkers are known to be associated with impaired health of dairy cows. Therefore, being able to predict these biomarkers, especially in the early stage of lactation, would enable preventive management decision. Some health biomarkers may also be used as phenotypes for genetic improvement for improved animal health. In this study, we validated the accuracy and robustness of models for predicting serum concentrations of ß-hydroxybutyrate (BHB), fatty acids, and urea nitrogen, using milk mid-infrared (MIR) spectroscopy. The data included 3,262 blood samples of 3,027 lactating Holstein-Friesian cows from 19 dairy herds in Southeastern Australia, collected in the period from July 2017 to April 2020. The models were developed using partial least squares regression and were validated using 10-fold random cross-validation, herd-year by herd-year external validation, and year by year validation. The coefficients of determination (R2) for prediction of serum BHB, fatty acids, and urea obtained through random cross-validation were 0.60, 0.42, and 0.87, respectively. For the herd-year by herd-year external validation, the prediction accuracies held up comparatively well, with R2 values of 0.49, 0.33, and 0.67 for of serum BHB, fatty acids, and urea, respectively. When the models were developed using data from a single year to predict data collected in future years, the R2 remained comparable, however, the root mean squared errors increased substantially (4-10 times larger than compared with that of herd-year by herd-year external validation) which could be due to machine differences in spectral response, the change in spectral response of individual machines over time, or other differences associated with farm management between seasons. In conclusion, the mid-infrared equations for predicting serum BHB, fatty acids, and urea have been validated. The prediction equations could be used to help farmers detect cows with metabolic disorders in early lactation in addition to generating novel phenotypes for genetic improvement purposes.

The use of milk mid-infrared spectroscopy to improve genomic prediction accuracy of serum biomarkers.

Breeding objectives in the dairy industry have shifted from being solely focused on production to including fertility, animal health, and environmental impact. Increased serum concentrations of candidate biomarkers of health and fertility, such as ß-hydroxybutyric acid (BHB), fatty acids, and urea are difficult and costly to measure, and thus limit the number of records. Accurate genomic prediction requires a large reference population. The inclusion of milk mid-infrared (MIR) spectroscopic predictions of biomarkers may increase genomic prediction accuracy of these traits. Our objectives were to (1) estimate the heritability of, and genetic correlations between, selected serum biomarkers and their respective MIR predictions, and (2) evaluate genomic prediction accuracies of either only measured serum traits, or serum traits plus MIR-predicted traits. The MIR-predicted traits were either fitted in a single trait model, assuming the measured trait and predicted trait were the same trait, or in a multitrait model, where measured and predicted trait were assumed to be correlated traits. We performed all analyses using relationship matrices constructed from pedigree (A matrix), genotypes (G matrix), or both pedigree and genotypes (H matrix). Our data set comprised up to 2,198 and 9,657 Holstein cows with records for serum biomarkers and MIR-predicted traits, respectively. Heritabilities of measured serum traits ranged from 0.04 to 0.07 for BHB, from 0.13 to 0.21 for fatty acids, and from 0.10 to 0.12 for urea. Heritabilities for MIR-predicted traits were not significantly different from those for the measured traits. Genetic correlations between measured traits and MIR-predicted traits were close to 1 for urea. For BHB and fatty acids, genetic correlations were lower and had large standard errors. The inclusion of MIR predicted urea substantially increased prediction accuracy for urea. For BHB, including MIR-predicted BHB reduced the genomic prediction accuracy, whereas for fatty acids, prediction accuracies were similar with either measured fatty acids, MIR-predicted fatty acids, or both. The high genetic correlation between urea and MIR-predicted urea, in combination with the increased prediction accuracy, demonstrated the potential of using MIR-predicted urea for genomic prediction of urea. For BHB and fatty acids, further studies with larger data sets are required to obtain more accurate estimates of genetic correlations.

Genetic parameters for mid-infrared spectroscopy-predicted fertility.

Female fertility is a challenging trait to improve genetically because of its low heritability, its unfavorable genetic correlation with milk yield, and its relatively small number of records. The MFERT trait is the probability of conception to first insemination predicted using mid-infrared (MIR) spectroscopy of a milk sample collected during lactation as part of routine milk recording, age at calving, days in milk, and milk production. As such, MFERT could become available on many more cows than traditional fertility traits. Our objectives were (1) to estimate the heritability of MFERT; (2) to estimate genetic correlations between MFERT, traditional fertility traits, and milk production traits; and (3) to assess the potential of MFERT to be used as an indicator trait for fertility in a selection index. The MFERT trait had a heritability of 0.16, which was higher than that (0.05) obtained for traditional fertility traits. Genetic correlations between MFERT and traditional fertility traits were low to moderate. The weakest and strongest correlations (mean ± standard error) were with pregnancy at the end of the mating season (0.13 ± 0.05) and calving to first service (-0.61 ± 0.03), respectively. Based on our estimates, including MFERT in a fertility index will only substantially increase the accuracy of the index when there are many more records available for MFERT than for the traditional fertility traits. This is likely to be the case because the number of milk samples from commercial machines belonging to milk recording companies in Australia that are capable of generating MIR spectra is growing. Hence, the number of records for MFERT is expected to increase substantially in the near future.

Predicting the likelihood of conception to first insemination of dairy cows using milk mid-infrared spectroscopy.

The objective of this study was to examine the ability of milk mid-infrared (MIR) spectroscopy and other on-farm data, such as milk yield, milk composition, stage of lactation, calving age, days in milk at insemination, and somatic cell count, to identify cows that were most or least likely to conceive to first insemination. A total of 16,628 spectral and milk production records of 7,040 cows from 29 commercial dairy herds across 3 Australian states were used. Three models, comprising different explanatory variables, were tested. Model 1 included features that are readily available on farms participating in milk recording, such as milk yield, milk composition, somatic cell count, days from calving to insemination, and calving season. Days in milk and age at calving were incorporated into model 1 to form model 2. In model 3, MIR was added to model 2, but to avoid double counting, milk composition traits of model 2 were removed. The models were first trained on extreme data [i.e., including cows that (1) conceived to first insemination and (2) cows with no conception event recorded and with only 1 insemination]. Then, the models were validated in a fresh data set with all cows regardless of conception outcomes present to test for their ability to identify cows that conceived or did not conceive to first insemination. To do this, we ranked the predicted probability of all cows in the validation set and then selected the top and bottom records in varying proportions from 5 to 40% (i.e., where the model predicted the highest versus lowest likelihood of conception to first insemination, respectively) and compared with the actual values. The model's performance was evaluated through herd-year by herd-year external validation and measured as the proportion of selected records being correct. The results show that when more cows are selected (i.e., descending confidence), the accuracy of the models was reduced, and selecting the 10% of cows with the highest confidence of predictions produces optimal accuracy. Irrespective of the proportions, none of the models could predict cows that conceived to first insemination, with an accuracy around 0.48. When attempting to predict the bottom 10% of cows, which had the least likelihood of conception to first insemination, model 1 had prediction accuracy around 0.64. Compared with model 1, the addition of days in milk and calving age (model 2) resulted in a negligible improvement in prediction accuracy (0.01 to 0.03). Model 3 had the highest prediction accuracy (0.76), which implies that in the models tested, MIR is of primary importance in the prediction of fertility of dairy cows. In conclusion, this study indicates that MIR and other milk recording data could be used to identify cows with potential difficulty in getting pregnant to first insemination with promising accuracy.

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.

Usefulness of milk mid-infrared spectroscopy for predicting lameness score in dairy cows.

The objective of this study was to evaluate the ability of milk infrared spectra to predict cow lameness score (LMS) for use as an indicator of cow health on Australian dairy farms, or as an indicator trait for genetic evaluation purposes. The study involved 3,771 cows from 10 farms in Australia. Milk infrared spectra collected during the monthly herd testing were available in all the farms involved in the study. Lameness score was measured once in each herd, within 72 h from a test day, and merged to the closest spectra records. Lameness score was expressed on a scale from 0 to 3, where 0 is assigned to sound cows and scores 1 to 3 are assigned to cows with increased lameness severity. Partial least squares discriminant analysis was used to develop prediction models for classifying sound (score 0) and not-sound cows (i.e., cows walking unevenly, score greater than 0). Discriminant models were tested in a 10-fold random cross-validation process. Milk infrared spectra correctly classified only 57% of the cows walking unevenly and only 59% of the sound cows. When additional predictors (parity, age at calving, days in milk, and milk yield) were included in the prediction model, the model correctly classified 57% of the cows walking unevenly and 62% of the sound cows. The same model applied only to the cows in the first third of lactation correctly classified 66% of the cows walking unevenly and 57% of the sound cows. When the prediction model was used to identify lame cows (scores 2 and 3), only 49% of them were classified as such. These results are considered to be too poor to envisage a practical application of these models in the near future as on-farm tools to provide an indication of LMS. To investigate whether, at this stage, predictions of the LMS could be useful as large-scale phenotypes for animal breeding purposes, we estimated (co)variance components for actual and predicted LMS using 2,670 and 24,560 records, respectively. As the genetic correlation between actual and predicted LMS was not significantly different from zero, predictions of lameness from milk spectra and additional on-farm variables cannot be used, at this stage, as an indicator trait for actual LMS. More research is needed to find better strategies to predict lameness.

Classifying the fertility of dairy cows using milk mid-infrared spectroscopy.

The objective of this study was to investigate the potential of milk mid-infrared (MIR) spectroscopy, MIR-derived traits including milk composition, milk fatty acids, and blood metabolic profiles (fatty acids, ß-hydroxybutyrate, and urea), and other on-farm data for discriminating cows of good versus poor likelihood of conception to first insemination (i.e., pregnant vs. open). A total of 6,488 spectral and milk production records of 2,987 cows from 19 commercial dairy herds across 3 Australian states were used. Seven models, comprising different explanatory variables, were examined. Model 1 included milk production; concentrations of fat, protein, and lactose; somatic cell count; age at calving; days in milk at herd test; and days from calving to insemination. Model 2 included, in addition to the variables in model 1, milk fatty acids and blood metabolic profiles. The MIR spectrum collected before first insemination was added to model 2 to form model 3. Fat, protein, and lactose percentages, milk fatty acids, and blood metabolic profiles were removed from model 3 to create model 4. Model 5 and model 6 comprised model 4 and either fertility genomic estimated breeding value or principal components obtained from a genomic relationship matrix derived using animal genotypes, respectively. In model 7, all previously described sources of information, but not MIR-derived traits, were used. The models were developed using partial least squares discriminant analysis. The performance of each model was evaluated in 2 ways: 10-fold random cross-validation and herd-by-herd external validation. The accuracy measures were sensitivity (i.e., the proportion of pregnant cows that were correctly classified), specificity (i.e., the proportion of open cows that were correctly classified), and area under the curve (AUC) for the receiver operating curve. The results showed that in all models, prediction accuracy obtained through 10-fold random cross-validation was higher than that of herd-by-herd external validation, with the difference in AUC ranging between 0.01 and 0.09. In the herd-by-herd external validation, using basic on-farm information (model 1) was not sufficient to classify good- and poor-fertility cows; the sensitivity, specificity, and AUC were around 0.66. Compared with model 1, adding milk fatty acids and blood metabolic profiles (model 2) increased the sensitivity, specificity, and AUC by 0.01, 0.02, and 0.02 unit, respectively (i.e., 0.65, 0.63, and 0.678). Incorporating MIR spectra into model 2 resulted in sensitivity, specificity, and AUC values of 0.73, 0.63, and 0.72, respectively (model 3). The comparable prediction accuracies observed for models 3 and 4 mean that useful information from MIR-derived traits is already included in the spectra. Adding the fertility genomic estimated breeding value and animal genotypes (model 7) produced the highest prediction accuracy, with sensitivity, specificity, and AUC values of 0.75, 0.66, and 0.75, respectively. However, removing either the fertility estimated breeding value or animal genotype from model 7 resulted in a reduction of the prediction accuracy of only 0.01 and 0.02, respectively. In conclusion, this study indicates that MIR and other on-farm data could be used to classify cows of good and poor likelihood of conception with promising accuracy.

The use of mid-infrared spectra to map genes affecting milk composition.

The aim of this study was to investigate the feasibility of using mid-infrared (MIR) spectroscopy analysis of milk samples to increase the power and precision of genome-wide association studies (GWAS) for milk composition and to better distinguish linked quantitative trait loci (QTL). To achieve this goal, we analyzed phenotypic data of milk composition traits, related MIR spectra, and genotypic data comprising 626,777 SNP on 5,202 Holstein, Jersey, and crossbred cows. We performed a conventional GWAS on protein, lactose, fat, and fatty acid concentrations in milk, a GWAS on individual MIR wavenumbers, and a partial least squares regression (PLS), which is equivalent to a multi-trait GWAS, exploiting MIR data simultaneously to predict SNP genotypes. The PLS detected most of the QTL identified using single-trait GWAS, usually with a higher significance value, as well as previously undetected QTL for milk composition. Each QTL tends to have a different pattern of effects across the MIR spectrum and this explains the increased power. Because SNP tracking different QTL tend to have different patterns of effect, it was possible to distinguish closely linked QTL. Overall, the results of this study suggest that using MIR data through either GWAS or PLS analysis applied to genomic data can provide a powerful tool to distinguish milk composition QTL.

Prediction of blood ß-hydroxybutyrate content and occurrence of hyperketonemia in early-lactation, pasture-grazed dairy cows using milk infrared spectra.

The objective of this study was to evaluate the ability of milk infrared spectra to predict blood ß-hydroxybutyrate (BHB) concentration for use as a management tool for cow metabolic health on pasture-grazed dairy farms and for large-scale phenotyping for genetic evaluation purposes. The study involved 542 cows (Holstein-Friesian and Holstein-Friesian × Jersey crossbreds), from 2 farms located in the Waikato and Taranaki regions of New Zealand that operated under a seasonal-calving, pasture-based dairy system. Milk infrared spectra were collected once a week during the first 5 wk of lactation. A blood "prick" sample was taken from the ventral labial vein of each cow 3 times a week for the first 5 wk of lactation. The content of BHB in blood was measured immediately using a handheld device. After outlier elimination, 1,910 spectra records and corresponding BHB measures were used for prediction model development. Partial least square regression and partial least squares discriminant analysis were used to develop prediction models for quantitative determination of blood BHB content and for identifying cows with hyperketonemia (HYK). Both quantitative and discriminant predictions were developed using the phenotypes and infrared spectra from two-thirds of the cows (randomly assigned to the calibration set) and tested using the remaining one-third (validation set). A moderate accuracy was obtained for prediction of blood BHB. The coefficient of determination (R2) of the prediction model in calibration was 0.56, with a root mean squared error of prediction of 0.28 mmol/L and a ratio of performance to deviation, calculated as the ratio of the standard deviation of the partial least squares model calibration set to the standard error of prediction, of 1.50. In the validation set, the R2 was 0.50, with root mean squared error of prediction values of 0.32 mmol/L, which resulted in a ratio of performance to deviation of 1.39. When the reference test for HYK was defined as blood concentration of BHB ≥1.2 mmol/L, discriminant models indicated that milk infrared spectra correctly classified 76% of the HYK-positive cows and 82% of the HYK-negative cows. The quantitative models were not able to provide accurate estimates, but they could differentiate between high and low BHB concentrations. Furthermore, the discriminant models allowed the classification of cows with reasonable accuracy. This study indicates that the prediction of blood BHB content or occurrence of HYK from milk spectra is possible with moderate accuracy in pasture-grazed cows and could be used during routine milk testing. Applicability of infrared spectroscopy is not likely suited for obtaining accurate BHB measurements at an individual cow level, but discriminant models might be used in the future as herd-level management tools for classification of cows that are at risk of HYK, whereas quantitative models might provide large-scale phenotypes to be used as an indicator trait for breeding cows with improved metabolic health.