Intratesticular versus intraperitoneal Busulfan administration: a comparative study on spermatogenesis suppression in quails and chickens

Generation of transgenic birds can be achieved by temporal suppression of endogenous spermatogenesis in males prior to primordial germ cell implantation. One of many established methods to induce male sterility is the intraperitoneal injection of busulfan, an alkylating agent. Nevertheless, the use of busulfan injections, which may also affect hematopoietic stem cells, carries the risk of potential lethality in animals. Given their safety and non-toxic nature, it has been demonstrated that intratesticular busulfan injections in mammals are less effective than intraperitoneal injections. This study aimed to compare, for the first time, the sterility and toxicity effects of intraperitoneal vs. intratesticular busulfan injections in quail and chickens. Our experimental design involved a previously established single intraperitoneal busulfan injection of 40 mg/kg of body weight (BW). In quail, busulfan was then administered intratesticularly at 3 different concentrations (6, 12, and 20 mg/kg BW), while in chickens, the working concentration was 20 mg/kg BW. We found that a single intraperitoneal busulfan injection of 40 mg/kg of BW resulted in 100% mortality in the treated roosters. In quails, however, this concentration only caused a temporary suppression of fertility for a 15-d period. Moreover, we found that a higher dose of intratesticular injection of busulfan is required to suppress spermatogenesis in quail (20 mg/kg BW) compared to mammals (4 mg/kg BW). Following these findings, we further confirmed that intratesticular injection of 20 mg/kg BW busulfan into roosters did not affect their overall viability. However, it induced a temporary state of male sterility, consistent with the effects observed with intraperitoneal injections. Hence, our data demonstrate that quail and chicken respond differently to busulfan administration. Furthermore, the present study provides evidence that direct injection into the rooster testes causes less physiological stress than intraperitoneal injection.