The objective of this study was to compare the thermotolerance of second-cross (SC; Poll Dorset × Merino × Border Leicester) and Dorper lambs. Dorper and SC lambs (4–5 months of age) were subjected to cyclic heat stress (HS) (28–40 °C). The temperature was increased to 38–40 °C between 800 and 1700 h daily and maintained at 28 °C for the remainder of the day (30–60% relative humidity (RH)) in climatic chambers for 2 weeks (
n = 12/group), with controls maintained in a thermoneutral (TN) (18–21 °C, 40–50% RH) environment (
n = 12/group). Basal respiration rate (RR), rectal temperature (RT) and skin temperature (ST) were higher (
p < 0.01) in SC lambs than in Dorpers. HS increased RR, RT and ST (
p < 0.01) in both genotypes, but the levels reached during HS were lower (
p < 0.01) in Dorpers. HS increased (
p < 0.01) water intake to a greater extent in SC lambs, while feed intake was reduced (
p < 0.05) by HS in SC lambs but not in Dorpers. HS increased (
p < 0.01) blood urea nitrogen and creatinine in SC lambs only. Plasma non-esterified fatty acid concentrations were reduced (
p < 0.05) by HS in SC lambs but increased (
p < 0.05) in Dorpers. There was no effect of HS on pO
2, cHCO
3− and cSO
2, but higher (
p < 0.01) blood pH and lower (
p < 0.01) pCO
2 were recorded under HS in both genotypes. Blood electrolytes and base excess were reduced (
p < 0.01) under HS, while a genotype difference (
p < 0.05) was only observed in blood K
+ and hemoglobin concentrations. Basal plasma prolactin concentrations were lower (
p < 0.01) in Dorpers but were elevated at a similar level during HS (
p < 0.01) in both genotypes. Dorper lambs are more resilient to HS than SC lambs. Future research should focus on confirming whether the better heat tolerance of Dorpers is translated to better returns in terms of growth performance and carcass traits over the summer months.
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