Genome-wide association analysis of haematological and biochemical traits in blood and wool under basal and environmental stress conditions in Rasa Aragonesa sheep

Extensive sheep production systems in semi-arid regions are becoming more vulnerable to the effects of climate change, such as water scarcity and heat stress, which can compromise animal health, welfare, and productivity. Local breeds such as Rasa Aragonesa are considered well adapted to these challenging environmental conditions; although, the genetic basis of their physiological responses to environmental stress remains poorly understood. This study investigated the genetic basis of haematological, biochemical, and endocrine traits associated with responses to combined water and heat stress in Rasa Aragonesa ewes. A total of 202 ewes were subjected to five days of complete water restriction during a naturally occurring heat wave, resulting in concurrent exposure to heat-stress conditions (temperature-humidity index ≥ 22.2) for approximately 55% of the experimental period and providing a realistic model of combined water and heat stress under semi-arid Mediterranean production conditions. Blood samples were collected before (day 0) and after the challenge (day 5), while wool samples were collected on day 0 and 28. Phenotypes included haematological profiles, blood metabolites (glucose, non-esterified fatty acids, and total proteins), and stress-related hormones (cortisol, dehydroepiandrosterone ( DHEA ), and DHEA sulphate ( DHEA-S )), measured in blood and wool. Animals were genotyped using the Illumina AgResearchSheep HD 600K SNP chip. Genetic parameters were estimated using genomic animal models, and genome-wide association studies ( GWAS ) were performed using a multi-locus linear mixed model (FarmCPU), followed by pathway enrichment analyses with Multi-marker Analysis of GenoMic Annotation ( MAGMA ). Moderate-to-high heritabilities and generally positive genetic correlations between basal and stress conditions were observed for several blood-cell traits, suggesting relatively stable genetic control across environments. In contrast, metabolic and endocrine traits appeared more environmentally sensitive. GWAS identified candidate genomic regions associated with stress-response traits, highlighting a missense variant in HBB as the strongest positional and functional candidate, consistent with its role in haemoglobin function and adaptation to dehydration and heat exposure. MAGMA analyses highlighted pathways related to oxidoreductase activity, cell-cycle regulation, cellular homeostasis, and amino-acid metabolism, including mechanisms involving betaine and ornithine. Although exploratory, these results provide new insights into the biological mechanisms underlying resilience to combined water and heat stress and identify candidate genes and pathways that warrant further validation for future resilience-oriented breeding strategies.