In-Life Testicular Volume as an Indicator of Histological Testicular Development Suitable for Testicular Toxicity Evaluation in Male Cynomolgus Macaques (Macaca fascicularis)

Reliable evaluation of testicular toxicity in nonclinical safety studies requires animals with sufficient histological testicular maturity. However, chronological age alone is an unreliable indicator of reproductive maturity in cynomolgus macaques because substantial inter-individual variation exists in pubertal development. Since histological maturity can only be confirmed after necropsy, a practical, non-invasive method for prospective animal selection is needed. This retrospective study evaluated whether in-life testicular volume could predict histological maturity suitable for routine histopathological evaluation. Testicular length and width were measured immediately before scheduled necropsy in 61 male cynomolgus macaques (Macaca fascicularis) using digital calipers, and testicular volume was calculated using the ellipsoid formula. Histological maturity was classified according to the six-grade system of Haruyama et al., with bilateral Grade ≥4 selected as the primary endpoint because this represents the earliest stage at which routine histopathological evaluation of spermatogenesis is considered reliable. Logistic regression and receiver operating characteristic (ROC) analyses demonstrated a strong association between mean testicular volume and bilateral Grade ≥4 status (AUC = 0.976), with an optimal ROC-derived cut-off value of approximately 8.7 cm3. Although age and body weight were significantly associated with histological maturity, testicular volume remained an independent predictor after adjustment for these variables. Testicular width also showed high diagnostic performance, whereas postmortem indices, including testicular weight and the mean relative testicular weight, cannot be applied for prospective animal selection. These findings suggest that externally measured testicular volume may facilitate prospective identification of animals likely to possess testes suitable for routine histopathological evaluation before study initiation.