Ethinyl estradiol–levonorgestrel contraceptive bait impairs testicular function and induces germ-cell apoptosis in male rats

Hormonal baiting is being studied as an innovative fertility control method in rodents, with most studies focusing on quinestrol + levonorgestrel (EP1). However, no study has investigated ethinylestradiol-levonorgestrel (EE-LNG) bait formulation for male rats, and no studies have reported investigating multi-organ tissue integrity using AO/PI, DAPI, and H&E staining for histopathological analysis. The objective of the present study is to investigate the toxicological and antifertility effects of an EE-LNG bait in male rats. Eighteen adult male rats were divided into three groups, namely control, low dose, and high dose. They were fed the EE-LNG bait for 14 days. Serum testosterone levels were quantified using ELISA. Furthermore, histopathology and the DNA and nuclear integrity of the testis, liver, and kidney were carried out using H&E, DAPI, and AO/PI staining methods. The results revealed that both EE-LNG doses result in the reduced weight of the testes and epididymis relative to the control and significantly suppressed serum testosterone levels in a dose-dependent manner (P<0.00000001). Testicular, hepatic, and renal tissues showed degenerative histopathology, with fragmented DNA and nuclear condensation confirmed by the H&E, AO/PI, and DAPI staining. This study represents the first research on an EE-LNG contraceptive bait in male rats, revealing that even 14 days of bait feeding is sufficient to suppress testosterone levels and impair multi-organ integrity. The innovative application of AO/PI and DAPI provided evidence of tissue-level toxicity, supporting the potential of EE-LNG bait as a candidate for fertility control.