# Oxidative Stress and Ultrastructural Changes in Laminar Tissue of Dairy Cows with Acute Laminitis Induced by Oligofructose Overload

## TL;DR

This study shows that oxidative stress and structural damage in the hoof tissue of dairy cows are linked to laminitis caused by excessive oligofructose intake.

## Contribution

The study identifies dysregulation of the Keap1-Nrf2 pathway and ultrastructural damage in laminar tissue as key factors in OF-induced laminitis in dairy cows.

## Key findings

- Oligofructose overload increased Keap1 and decreased Nrf2, Ho1, and Nqo1 gene and protein expression in laminar tissue.
- Ultrastructural changes included loss of hemidesmosomes and ruptured anchoring fibers in OF-treated cows.
- Oxidative stress was confirmed as a contributor to epidermal detachment in laminar tissue.

## Abstract

Bovine laminitis is the leading cause of lameness, resulting in significant financial losses and animal welfare issues in the worldwide dairy sector. Indeed, its pathogenesis remains poorly understood. Oxidative stress may play an important role in the pathophysiology of dairy cow laminitis. This study evaluated the oxidative stress status in the laminar tissue of dairy cows with oligofructose (OF)-induced laminitis at the gene and protein levels. Decreased gene and protein expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (Ho1), and NAD(P)H: quinone oxidoreductase 1 (Nqo1) were observed in the OF group’s laminar tissue of dairy cows. However, the gene and protein expression levels of Kelch-like ECH-Associated protein 1 (Keap1) were enhanced. The distribution of Keap1 expression increased, while that of the Nrf2 decreased in the OF group relative to the control group. In addition, ultrastructural analysis of the laminar tissue showed that the OF led to shrinkage, loss of hemidesmosomes (HDs), and rupture of anchoring fibers. These findings suggest that the unbalanced expression of oxidative stress-associated genes and proteins may contribute to epidermal detachment, confirming that oxidative stress was enhanced in the laminar tissue of OF-treated cows. Therefore, targeting the Keap1-Nrf2 pathway may lead to improved prevention and treatment strategies for bovine laminitis. However, further large-scale gene- and protein-based research is needed to fully understand the pathophysiology of dairy cows’ laminitis.

This study explored ultrastructural changes and the expression of oxidative stress-related genes and proteins in the laminar tissue of dairy cows with acute laminitis induced by oligofructose (OF) overload. Twelve clinically healthy, non-pregnant Chinese Holstein cows were randomly allocated into two groups: the OF-overload group (n = 6) and the control group (n = 6). 17 g/kg BW of oligofructose (OF) dissolved in 20 mL/kg BW of deionized water was provided to the OF-treated group, while the control group received 20 mL/kg BW of deionized water via a stomach tube. Laminar tissue samples were collected at 72 h post-OF administration. RT-qPCR revealed significantly increased Keap1 mRNA expression (p = 0.0097) and significantly decreased Nrf2 (p < 0.0001), Ho1 (p < 0.0001), and Nqo1 (p = 0.0101) mRNA expression in the OF group compared to the control group. Western blot analysis confirmed corresponding protein-level changes, with significantly increased Keap1 (p = 0.0062) and significantly decreased Nrf2 (p = 0.0008), Ho1 (p = 0.0297), and Nqo1 (p = 0.0004) in the OF group compared with the control group. Immunohistochemical analysis revealed significantly increased cytoplasmic Keap1 distribution (p = 0.0200) and significantly decreased nuclear Nrf2 localization (p = 0.0032) in the OF group than the control group. Ultrastructural examination revealed significant pathological changes in the OF group, including a reduced number of hemidesmosomes (p < 0.01), an increased distance from epidermal basal cells to the lamina densa (p < 0.01), thickened and damaged lamina densa with disorganized collagen fibers, and deformed basal cell nuclei with reduced chromatin relative to the control group. In conclusion, these findings demonstrate that OF-induced acute laminitis is associated with significant dysregulation of the Keap1-Nrf2 antioxidant pathway and severe ultrastructural damage to the dermal–epidermal interface, suggesting that oxidative stress contributes to laminar tissue injury in dairy cows.