This review describes the oestrus-to-ovulation interval, the possibility of predicting the time of ovulation, and the optimum time for insemination relative to oestrus in dairy cows. The duration of oestrus in dairy cows is approximately 8–20 h, with differences possibly related to the methods of oestrus detection and the frequency of observations. Most cows ovulate approximately 24–33 h after the onset of oestrus and 15–22 h after the end of oestrus. The interval from the preovulatory luteinising hormone (LH) surge to ovulation is approximately 4–30 h. Ovulation occurs when follicle diameter averages 18–20 mm. When it is possible to correctly determine the beginning of oestrus, artificial insemination can be performed utilizing the “a.m.–p.m. rule”, and only one insemination may be applied. In cows with too long or too short oestrus-to-ovulation intervals, fertility can be compromised. One important factor that can alter the oestrus-to-ovulation interval is acute or chronic heat stress during the warm season. When there is a risk that insemination may occur too early or too late with respect to the time of ovulation, GnRH administration can be considered.

Melatonin, an endogenous indolamine derived from tryptophan, is primarily synthesized by the pineal gland in mammals and regulated by a complex neural system. Its release follows a circadian rhythm, which is crucial for regulating physiological processes in response to light–dark cycles in both humans and animals. In this review, we report that the presence of this hormone in bovine milk, with significant differences in concentration between daytime and nighttime milking, has increased interest in milk as a natural source of bioactive molecules. Melatonin lowers cortisol levels at night, reduces body temperature and blood pressure, coinciding with decreased alertness and performance, acts as an antioxidant and anti-inflammatory agent, modulates the immune system, offers neuroprotective benefits, and supports gastrointestinal health by scavenging free radicals and reducing oxidative stress in dairy cows. Many factors influence the release of melatonin, such as the intensity of artificial lighting during nighttime milking, the frequency of milkings, milk yield, and genetic differences between animals. Nocturnal milking under low-intensity light boosts melatonin, potentially reducing oxidative damage and mastitis risk. Additionally, ultra-high temperature (UHT) treatment does not significantly affect the melatonin content in milk. However, further research on its stability during milk processing and storage is crucial for ensuring product efficacy. In some countries, nighttime milk with naturally elevated melatonin content is already commercialized as a natural aid for sleep. Thus, naturally melatonin-rich milk may be a promising alternative to synthetic supplements for promoting better sleep and overall well-being.

Mastitis remains a prevalent and economically detrimental disease within the dairy industry, profoundly affecting animal welfare, milk quality, and overall production output. Nowadays, Somatic Cell Count (SCC) is widely recognized as the gold-standard indicator for the detection of mastitis; however, its limitations in pathogens discrimination and the lack of early-stage characterization of mastitis highlight the need for complementary diagnostic approaches. This review synthesizes recent research into the development and validation of novel biomarkers for the early and accurate identification of mastitis in dairy cows. The investigation encompasses a range of biological molecules for improving mastitis diagnosis. Biomarkers such as lactoferrin (LTF), β-defensin 4 (DEFB4), vitronectin, paraoxonase 1 (PON1), and N-acetyl-β-D-glucosaminidase (NAGase) show promise in distinguishing between cows not susceptible and cows susceptible to mastitis. Concurrently, nucleic acid-based biomarkers are emerging as a particularly promising frontier. While mitochondrial DNA (mtDNA) has demonstrated insufficient specificity, microRNAs (miRNAs) are gaining attention as highly stable and sensitive indicators of intramammary inflammation, potentially enabling the detection of subclinical infections before they become clinically apparent. Despite these advances, significant challenges related to specificity, reliability, and cost-effectiveness currently hinder the widespread practical application of any single biomarker. Therefore, future research should be directed towards the validation of a synergistic panel of multiple biomarkers to improve mastitis management in dairy cow farms.

Mastitis is the leading cause of economic losses in dairy farming, significantly impairing animal welfare and the quality and quantity of milk production. MicroRNAs are increasingly gaining attention, in both human and veterinary medicine, as biomarkers for various diseases. This study evaluated the diagnostic potential of four circulating microRNAs (miR-26-5p, miR-142-5p, miR-146a, and miR-223-3p) by examining changes in their expression in milk samples from dairy cows at different immune-cell subpopulations correlated to different stage of mastitis with a validated method. Additionally, this study has analyzed the possible source of these circulating microRNAs by the measurement of their secretion from activated immune cells (lymphocytes, monocytes, and neutrophils). miR-223-3p has been significantly expressed in an acute stage of mastitis (p < 0.01) but not in the chronic or susceptible stages. Conversely, mir-26-5p has been significantly reduced in acute, chronic, and susceptible groups of animals. In immune-cell cultures, miR-26 has been shown to be down-regulated in lipopolysaccharide (LPS)-stimulated neutrophils, while miR-223 has been shown to be up-regulated in phytohemagglutinin (PHA)-stimulated lymphocytes. The differential expression of miR-223-3p and miR-26-5p, combined with differential and total somatic cell count, could serve as a useful tool for identifying the evolutionary stage of mastitis-related inflammatory pathology.

Mastitis is the disease that causes the greatest economic losses in dairy farming, significantly im-pairing animal welfare and the quality and quantity of milk produced. MicroRNAs are increasingly gaining attention, both in human and veterinary medicine, as biomarkers for various diseases. This study evaluated the diagnostic potential of four circulating microRNAs (miR-26-5p, miR-142-5p, miR-146a, and miR-223-3p) by examining changes in their expression in milk samples from dairy cows at different immune cells subpopulations correlated to different stage of mastitis in validated method. Additionally, the project has analysed the possible source of these circulating microRNA by the measurement of their secretion from activated immune cells (lymphocytes, monocytes, and neutrophils). miR-223-3p has been significantly expressed in acute stage of mastitis (p<0.01) but not in chronic or susceptible stages. mir-26-5p has been significantly reduced in acute, chronic and susceptible groups of animals. In immune cell cultures miR-26 has been shown to downregulate in LPS-stimulated neutrophils while miR-223 has been shown to be upregulated in PHA-stimulated lymphocytes. The differential expression of miR-223-3p and miR-26-5p together with differential and classic somatic cell count could be utilized for identifying the evolutionary stage of masti-tis-related inflammatory pathology.