Background: The aim of this study was to compare consequences in single and triple testicular biopsy by biopty gun in pubertal rats using histological and immunohistochemical analysis. Methods: Thirty-two Sprague-Dawley male rats were used as the experimental model. The rats were randomly divided into three study groups. The rats from the first group (n = 12) received a single-biopsy of upper pole of the left testis, while the rats from the second group (n = 10) received triple-biopsy of upper and lower poles and lateral surface of left testis. The third group (n = 10) was a control group. On the eightieth day after the biopsy in all rats bilateral orchiectomy and funiculectomy were performed to obtain testicular tissue and sperm for analysis. The consequences of the puncture were observed by pathohistology, immunohistochemistry and semen analysis. Results: The results of the study showed lower percentage of sperm count (14.5 mill/mL vs. 16 mill/mL, p = 0.130), sperm motility (24.6% vs. 32.7%, p > 0.05), abnormal sperm (30% vs. 27%, p > 0.05), atrophic tubules (21% vs. 6%, p < 0.001), volume (1.7 mL vs. 2.28 mL, p < 0.01) and apoptotic index (1.56 vs. 1.19, p = 0.650) in the testes with a triple-biopsy compared to the testes with a single-biopsy. Semen analysis showed a borderline significant difference between the group with triple-biopsy where sperm count was lower than it in the control group (14.5 mill/mL vs. 17.5 mill/mL, p = 0.05). A single-biopsy has little effect on the testis, especially on overall fertility. A triple-biopsy showed higher degree of the testicular damage but without a significant impact on overall fertility. Semen analysis showed that single- and triple-biopsies did not have a significant effect on sperm count, motility and morphology. Conclusion: Biopty gun procedure is a cheap, simple and reliable method for testicular biopsy in rats without a significant effect on sperm count, motility and morphology.
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