Pulse-wave Doppler (PWD) ultrasonography of the testicular artery was recently used to compare parameters in fertile and infertile males, or males with normal and abnormal semen quality, while no studies verified the concomitant histological feature of the testis. The experimental hypothesis of the present study was that the blood flow parameters measured by PWD at different levels of the testicular artery reflect sperm attributes evaluated on spermatozoa collected from the tail of the epididymis and testicular histological characteristics. Pulse-wave Doppler parameters were recorded on both testes from 16 dogs of unknown fertility. After routine castration, epididymal spermatozoa were collected and evaluated for sperm total and progressive motility, membrane integrity, and sperm morphology. Testes were submitted to histological and immunohistochemical evaluations to quantify microscopic attributes and connective tissue and vascular area extent, respectively. The findings showed that the increased PWD indexes were suggestive of testicular histological alteration since negative correlations were found between pulsatility index and resistive index, measured in the marginal testicular artery, and histological attributes such as the seminiferous epithelial thickness (R = -0.528, P = 0.016 and R = -0.554, P = 0.005, respectively) and the tubular area/total area ratio (R = -0.494, P = 0.024 and R = -0.563, P = 0.004, respectively). Some histological characteristics, such as tubular diameter, seminiferous epithelium thickness, and the tubular area/total area ratio of the testis are correlated with the percentage of primary abnormalities (R = -0.874, P = 0.001, and R = -0.847, P = 0.001, and R = – 0.865, P = 0.001, respectively). In testes with increased sperm primary abnormalities, higher pulsatility index was found compared with testes with normal spermatogenesis in supratesticular artery (1.07 ± 0.06 vs 0.82 ± 0.23, P < 0.05), marginal artery (0.73 ± 0.04 vs 0.61 ± 0.07, P < 0.05), and intratesticular artery (0.67 ± 0.03 vs 0.43 ± 0.1, P < 0.05). Similarly, the resistive index was found significantly higher in the same vessel tracts in testes with abnormal and normal spermatogenesis (0.62 ± 0.02, 0.53 ± 0.02, and 0.46 ± 0.02 vs 0.51 ± 0.06, 0.44 ± 0.04, and 0.32 ± 0.06, respectively; P < 0.05). Our data demonstrated for the first time the relationship between PWD indexes and the contextual testicular architecture, suggesting that the pulsatility and resistive indexes could be an effective tool in andrology. In light of different histological alterations in the testis, caution is warranted in the definition of thresholds for normal and abnormal testicular blood flow indexes.Copyright © 2020. Published by Elsevier Inc.