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Abstract Chronic exposure to elevated temperatures affects livestock production and welfare resulting in decreased performance and testicular function during breeding season. Nevertheless, there is a lack of research on the effects of heat stress during the peripubertal growth period. The objective of this study was to identify the effects of heat stress exposure during peripubertal growth on testicular physiology and evaluate if N-carbamylglutamate (NCG) and melatonin (MEL) supplementation could mitigate the adverse effects. We hypothesized that supplementation of NCG and MEL will increase testicular artery blood flow mitigating the effects of heat stress. This study utilized peripubertal Dorper rams (n = 35) placed into 1 of 3 groups balanced by sire, testicular circumference and body weight. These groups consisted of a cooled environment with shade without supplementation (CL-CON; n = 11), a heat stressed environment without supplementation (HS-CON; n = 12), and a heat stressed environment supplemented with 1.4g/h/d of NCG and 8mg/h/d of melatonin (HS-NCGMEL; n = 12). Temperature humidity index was recorded during the trial with an average of 77.23. On day 49, testicular artery blood flow, testicular circumference, and testicular temperature were assessed. Testicular blood flow was evaluated using a Doppler ultrasound, whereas a thermal imaging camera was used to collect testicular temperature. On day 54, rams were slaughtered, and testicular weights were collected. Data were evaluated through MIXED measures using SAS v 9.4. There was a significant difference in testicular blood flow between treatment groups (P = 0.033), where HS-CON had a decreased testicular blood flow (21.90±2.30 mL/min) compared with CL-CON (29.14±2.39 mL/min) and HS-NCGMEL (28.60±2.27 mL/min). There was no significant difference in testicular blood flow between CL-CON and HS-NCGMEL (P > 0.05). Final testicular circumference was decreased (P = 0.037) in HS-CON (27.42±0.7074 cm) compared with CL-CON (30.00±0.7389 cm) and HS-NCGMEL (29.75±0.7074 cm), with no significant difference in testicular circumference between CL-CON and HS-NCGMEL (P > 0.05). In addition, HS-CON had a decreased testicular weight (414.77±33.18 g) compared with CL-CON (544.45±34.66 g; P = 0.015). However, the testicular weight of HS-NCGMEL rams was not different from any of the aforementioned groups (P > 0.05). Lastly, testicular temperature was not different between treatments (P > 0.05), although a negative correlation between testicular temperature and testicular artery blood flow was observed (r = -0.46; P = 0.007). An increase in testicular artery blood flow and final testicular circumference in HS-NCGMEL suggests that supplementation of N-carbamylglutamate (NCG) and melatonin (MEL) to heat stressed peripubertal rams enhance testicular parameters.