Heat stress affects production and health in cows severely. Since it is difficult to define heat-tolerant animals, studies of response to heat stress are important for understanding dairy cows’ health and production. However, information on the impact of heat stress on various indicators in heat-tolerant and heat-sensitive cows is sparse. This study aimed to investigate the effects of heat stress (HS) on blood, production, and physiological indicators in heat-tolerant and heat-sensitive cows. A total of 43 dairy cows were used from 9 May to 7 August 2021, under Temperature–Humidity Index (THI) measurements that ranged from 65.9 to 86.7. We identified cows that were tolerant or sensitive to HS based on the slope of the response of physiological and production traits against THI during the HS period by using a clustering method. After HS, serum glucose (Glu), cortisol (COR), 5-hydroxytryptamine (5-HT), and interleukin-6 (IL-6) levels of cows in the heat-tolerant group were lower than in the heat-sensitive group (p < 0.05). With THI as the predictor, the R² for predicting respiration rate (RR) and body surface temperature (BT) in heat-tolerant cows was 0.15 and 0.16, respectively, whereas the R² for predicting RR and BT in heat-sensitive cows was 0.19 and 0.18, respectively. There were low to moderate, positive correlations between RR, BT, and MY with THI, with Pearson correlation coefficients ranging from r = 0.11 to 0.4 in the heat-tolerant group, and from r = 0.24 to 0.43 in the heat-sensitive group. There was a low positive correlation between VT and THI, with a Spearman correlation coefficient of r = 0.07 in the heat-sensitive group. The heat-tolerant dairy cows had lower MY losses and had lower MY (p = 0.0007) in mixed models. Heat-tolerant cows with low-stress levels, through upregulating RR rapidly, increased their adaptability to thermal environments. They have better thermoregulation capability; the hypothalamic–pituitary–adrenal (HPA) axis regulated the thermoregulatory in animals by releasing a variety of neurotransmitters and hormones.

Stereotypic behaviors are common in farm animals and often signal poor welfare. Tongue-rolling is the most prevalent stereotypic behavior in cows. In this study, we compared the plasma and milk composition of 16 high-frequency tongue-rolling cows (HTR group) and 16 non-stereotypic cows (CON group). All cows were primiparous cows. Biochemical tests, plasma metabolomics, and milk proteomics revealed higher plasma triiodothyronine levels in HTR cows, and lower levels of αs1-casein, β-casein, κ-casein, and lactoferrin in their milk. Multi-omics analyses identified 103 differential metabolites and 73 differential proteins, including various GTP-binding proteins, with the Ras signaling pathway being significantly upregulated in the HTR cows. GO enrichment analysis highlighted significant changes in molecular function, particularly related to GTP/GDP-binding proteins. Additionally, HTR cows exhibited elevated cellular metabolic activity. These findings suggest that high-frequency tongue-rolling is associated with altered endocrine and metabolic profiles, disrupted milk protein synthesis, and impaired immune function potential. The reduction in key milk proteins and lactoferrin may negatively impact milk quality and immune defense. Further research is needed to clarify the causal relationship between these physiological changes and tongue-rolling, providing insights into the underlying mechanisms of stereotypic behaviors in dairy cows and their implications for animal welfare and milk production.

Tongue-rolling behavior (TR) is commonly observed in dairy cows and is considered a stereotypic behavior indicative of compromised welfare. Chronic stress can impair lactation and immune function, yet the interaction between behavior (TR vs. normal behavior (NB)) and chronic stress (high vs. low) remains unclear. In this study, hair cortisol concentration (HCC) was used to assess stress levels in cows. The cows were first classified into high- and low-stress cows using K-means clustering. Subsequently, cows exhibiting high levels of TR and those exhibiting NB (i.e., no stereotypic behaviors) were selected from both stress categories to establish four groups (n = 8 per group): high-stress TR (HT), high-stress NB (HN), low-stress TR (LT), and low-stress NB (LN). We analyzed milk protein composition, milk proteome, and plasma immune-inflammatory indicators. Behavior (TR vs. NB) and chronic stress (high vs. low) showed significant interaction effects on plasma tumor necrosis factor-α (p = 0.046), interleukin-6 (p = 0.002), and proteomic profiles, involving multiple guanosine triphosphate-binding proteins (p < 0.05), transferrin (p = 0.001), and complement factors (p < 0.05). In addition, TR cows had significantly lower levels of αs1-casein (p = 0.019), β-casein (p < 0.001), κ-casein (p = 0.016), lactoferrin (p = 0.003), and plasma immunoglobulin A (p = 0.002). These results indicate that, under different chronic stress levels, TR cows differ markedly from NB cows in milk protein expression, immune function, and inflammatory responses. Moreover, milk from TR cows showed reduced quality, and immune dysfunction and inflammation were exacerbated under high stress. Overall, this study provides new insights into the physiological consequences of stereotypic behavior in dairy cows. These findings may help dairy farmers identify cows exhibiting TR as at risk of reduced milk quality and immune dysfunction, allowing for early management interventions to improve animal welfare and productivity.

The placenta plays a key role in fetal growth and functions to transport nutrients that are vital to the fetus. Heat stress (HS) impairs placental function in cows, affecting fetal development. There is limited information on the effects of HS on the placenta of heat-tolerant and -sensitive dry cows. The objective of this study was to assess differences in placental efficiency, morphology, apoptosis, and placental proteomics in heat-tolerant and -sensitive dry cows, and to investigate the underlying mechanisms of the effect. Sixty-six dry cows were measured for respiratory rate, body surface temperature, and microenvironmental temperature-humidity index, by 56.94 ± 16.04 bpm, 36.11 ± 0.38°C, and 80.65 ± 3.31 accordingly, indicating that the cows were exposed to HS. A mixed-effects model was applied to analyze the measured indicators, and clustering was used to classify them as heat-tolerant dry cows (n = 19) and heat-sensitive dry cows (n = 47). Placentals were collected from heat-tolerant (n = 8) and -sensitive (n = 13) dry cows. The placental weight, number of placental cotyledons, and fetal birth weight were recorded. Placental efficiency (calf birth weight: placental weight ratio), morphology, apoptosis, and proteomic differences were assessed in heat-tolerant and -sensitive dry cows. Five placental cotyledon samples from each group underwent hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and label-free proteomic analysis to assess microstructure, apoptosis, and differential protein expression, respectively. Results indicate that placental efficiency was significantly higher in heat-tolerant dry cows compared with heat-sensitive dry cows. Heat-tolerant cows exhibited a significantly lower apoptosis index (TUNEL+ cells: the total number of cells ratio) compared with heat-sensitive cows. A total of 3,691 proteins were identified, with 160 showing differential expression. The endocytosis pathway was significantly enriched in both Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Heat tolerance of heat-stressed dry cows improves placental efficiency, exhibits lower placental apoptosis, and maintains nutrient transport of placenta through the endocytosis pathway, which are likely due to the coordinated action of multiple pathways, contributing to better overall placental function in heat-tolerant cows.