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.

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, β-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 10fold 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-byherd 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 ...

While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre­(2–59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC­(60–343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.