# Interaction Effects Between Tongue-Rolling Behavior and Chronic Stress on Plasma Immune–Inflammatory Indicators, Milk Protein Composition, and Milk Proteome in Dairy Cows

## TL;DR

Tongue-rolling behavior in cows, combined with chronic stress, leads to poorer milk quality and weakened immune function, suggesting a link between behavior, stress, and health.

## Contribution

This study reveals interaction effects between tongue-rolling behavior and chronic stress on immune function and milk protein composition in dairy cows.

## Key findings

- Tongue-rolling cows had lower levels of protective milk proteins like αs1-casein and β-casein.
- High-stress tongue-rolling cows showed stronger inflammation and weaker immune indicators in blood.
- Milk from tongue-rolling cows showed reduced quality and exacerbated immune dysfunction under high stress.

## Abstract

Some dairy cows develop a repetitive tongue-rolling behavior due to frustrated feeding or rumination motivation, which is often seen as a sign of poor welfare. At the same time, cows that live under long-term stress may have weaker immune systems and produce milk of lower quality. However, it is still unclear how this stereotypic behavior and stress influence each other and affect the cow’s health. In this study, we measured the level of cortisol in the hair of cows to reflect their chronic stress and compared tongue-rolling cows and normal behavior cows. We examined their blood and milk to look for signs of inflammation, immune function, and changes in milk proteins. The cows with tongue-rolling had lower levels of protective proteins in their milk and weaker immune indicators in their blood. They also showed stronger signs of inflammation, especially under high stress. These results suggest that cows with tongue-rolling behavior may suffer from both physical and emotional stress, leading to poorer milk quality. Understanding this link can help farmers and researchers improve the living conditions and welfare of dairy cows, leading to healthier animals and better-quality dairy products.

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.