The potent hepatotoxin and carcinogen aflatoxin B1 (AFB1) is a common mycotoxin contaminant of grains used in animal feeds. Aflatoxin M1 (AFM1) is the major metabolite of AFB1 in mammals, being partially excreted into milk, and is a possible human carcinogen. The maximum permitted concentration of AFM1 in cows’ milk is 0.05 μg/kg in Israel and the European Union. Since milk yield and the carry-over of AFB1 in the feed to AFM1 in the milk are highly correlated, it was considered important to determine the AFM1 carry-over in Israeli-Holstein dairy cows, distinguished by world record high milk production. Twelve such cows were used to determine AFM1 carry-over following daily oral administration of feed containing ~86 μg AFB1 for 7 days. The mean carry-over rate at steady-state (Days 3–7) was 5.8% and 2.5% in mid-lactation and late-lactation groups, respectively. The carry-over appears to increase exponentially with milk yield and could be described by the equation: carry-over% = 0.5154 e^{0.0521 × milk yield}, with r² = 0.6224. If these data truly reflect the carry-over in the national Israeli dairy herd, the maximum level of AFB1 in feed should not exceed 1.4 μg/kg, a value 3.6 times lower than the maximum residue level currently applied in Israel.

The main pregnancy test method for dairy cows is rectal palpation performed around 40–45 days from last insemination. This is an invasive examination that disrupts the cow’s routine, potentially affecting its well-being. We examined the effectiveness of a commercial kit for performing pregnancy tests on milk samples compared to rectal palpation at 28- and 42-days post insemination. Accordingly, the purpose of the current study was to check if performing early pregnancy test instead of rectal palpation would result in better welfare and improvement of the reproductive performance, at least in part, of the cows. At 28 days, we examined the effect of early pregnancy testing on reproductive performance, especially number of days open. At 42 days, we determined the pregnancy test methods’ effects on cow welfare measures and milk production. For the day 28 experiment, cows on one farm were divided into two groups: one milk-tested for pregnancy on 28 to 30 days after insemination, and the other tested by routine veterinary examination (42 to 45 days after insemination). The milk test resulted in a 16-day reduction in days open for cows identified as non-pregnant on day 28 compared to day 42 (rectal examination), potentially improving reproductive performance on the dairy farm. For the day 42 experiment, test effects on cow welfare as seen in pedometer data and milk production were examined on two large dairy farms. Test-day milk production did not differ significantly between milk-tested and rectally palpated groups, but well-being indices of milk-tested cows improved, especially in winter months. The reason for this can be because, in the summer, cows are interrupted due to colling activities. In addition to fewer days open and economic improvement, early milk tests for pregnancy can free up the practitioner for other tasks on the dairy farm.

Introduction: Freedom from pain is a key component of animal welfare. However, unlike in humans, objective methods for assessing and managing pain in farm animals remain limited. Pain assessment is particularly challenging in cattle, due to their inherently stoic nature. Objective: We investigated biotinidase, an enzyme with amino-exopeptidase activity on opioid neuropeptides, as a novel, short-term pain-biomarker in dairy cattle, in comparison with cortisol and substance P. Methods: Plasma levels of biotinidase, substance P, and cortisol were quantified by ELISA in Holstein cows subjected to 3 distinct pain stimuli: freeze branding (routine husbandry), topical application of capsaicin cream (nonclinical pain model), and spontaneous clinical mastitis. Results: Biotinidase levels increased significantly within 30 minutes after freeze branding compared to controls (1.36 difference of normalized values; P = 0.021), and after capsaicin application (from 18.5 ± 2.5 to 21.5 ± 2.7 ng/mL; P = 0.002). Neither cortisol nor substance P exhibited significant changes after freeze branding or capsaicin exposure. Biotinidase levels elevated significantly in mastitic cows (28.1 ± 8.2 ng/mL) compared to healthy controls (13.63 ± 5.62 ng/mL; U = 74.0, P = 0.038), whereas substance P and cortisol showed no significant differences. Receiver operating characteristic analysis suggests that biotinidase (AUC: 0.84, 0.52, 0.81) outperformed substance P and cortisol in distinguishing painful from painless states. Conclusion: These findings provide compelling new evidence that plasma biotinidase responds robustly to diverse pain stimuli in nonhuman mammals. Further studies should investigate its diagnostic performance individually, and alongside other biochemical, physiological, and behavioral indicators, to enhance pain assessment accuracy and of noninvasive pain detection protocols for farm animals.