We have established an efficient method of testicular busulfan injection that required lower dose of busulfan to prepare recipients for GCT while achieving the objective of maximum Gc depletion without any cytotoxic effect in other organs. Our method is very efficient in terms of offspring generated (56 %) from SSC-transplanted males; rapid in terms of time required to evacuate testis using lowest effective dose of busulfan (75 μg) without any systemic ill effect. The success rate of our method for obtaining donor-derived offspring is very high (71 %) as compared to such methods reported recently by other group (5.5 %) as only 1 out of 18 Gc-transplanted mice could generate offspring from transplanted SSC in their study .
For this study, we have used F1B6SJL strain of mice for initial standardization of busulfan dosage for direct administration into the testes to prepare recipient mice prior to SSCT. We tested several doses of testicular busulfan (25 μg, 50 μg, 75 μg, and 100 μg) and found that the 75 μg dose provided desirable results as compared to a recently reported method  where 6 mg/kg-1dose (approximately 180 μg busulfan per testis) was selected as an appropriate dose for recipient preparation. This is 2.4 times more than ours (75 μg/testis).
There have been several reports which showed that i.p. injections of busulfan can cause systemic toxicity including severe bone marrow depression and/or death of animals [4, 12, 31]. In our study, no mortality was observed in testicular BST mice while i.p. injection of busulfan caused 60 % mortality. This confirmed that direct administration of busulfan in testis at a lower dose did not induce any cytotoxic effect on the hematopoietic system.
The body weight of T-BST and control group mice increased with age unlike the IP-BST group where the body weight remained low as compared to age-matched untreated animals.
Testicular size and weight proportionally decreased with increase in dose of busulfan injection. Lowest testicular weight was found in T-BST-75 and T-BST-100 at day 15 post BST. This was followed by a significant decrease in the tubular diameter. The prominent decrease in the diameter of the seminiferous tubule might have occurred due to depletion of the luminal contents. This was supported by histological observations like depletion of Gc, presence of multiple vacuoles, absence of sperm and presence of hollow cavities in the seminiferous tubules of T-BST. Maximum depletion of Gc was observed in T-BST-75 and T-BST-100 group mice 15 days post BST but T-BST-100 mice showed severe tubular disorganization which was not discernible in T-BST-75 mice. The depletion of Gc with our method was 7 days earlier than the one reported recently using 6 mg/ kg-1 testicular busulfan where maximum depletion was noticed at day 21 after BST [20, 21]. The major difference between our procedures is the use of two diagonally opposite sites for testicular injection of busulfan by us and from a single site through the tail end of testis by them [20, 21]. We injected busulfan from two sites (10 μl at each site) through both ends (the upper as well as lower end of the testis) covering the whole testicular area, thereby the effect of busulfan was more pronounced. In a previously reported study, injection was given from the testicular tail side along the long axis of the testis. In the process of travelling from one end to another, busulfan may get diluted by testicular lymph nodes thereby requiring higher doses and duration for the desirable effect.
None of the T-BST-75 mice required bone marrow transplantations as hematopoietic suppression was not observed in any of the mice, as opposed to conventional way of busulfan administration (i.p.) where whole body gets exposed to this cytotoxic drug causing depletion of various stem cell populations in other body organs too.
Restoration of endogenous spermatogenesis without SSCT was observed in few seminiferous tubules at 2 months after busulfan injection and gradually increased with time showing maximum restoration by 4 months. Since non-spermatogenic state of the BST-T-75 mice was attained at 15 days post BST, transplantation experiments may be performed 25 days earlier in contrast to the conventional method (i.p.). In the conventional method, Gc depletion and removal of sloughed off Gc occur by 6 weeks after busulfan injection, after which the transplantation experiments are performed . However, by our method, GCT can be performed 25 days earlier. Also with our method, the depleted state of the testis is sustained for a longer period (>1 month) thereby increasing the window for undertaking transplantation studies.
Donor-derived germ cell colonies in testes of T-BST-75 recipients showing GFP expression were observed within 2 months post transplantation, indicating successful GCT. In all, 24 out of 34 such males could successfully impregnate females within 10 days of cohabitation. However, males treated with busulfan but not transplanted with SSC (n = 3) could impregnate females only at 4.5 months after BST. Based on this, it may be assumed that out of 34 T-BST-75 mice, attempted for GCT, 24 were transplanted successfully and efficiently because of which they (71 % success rate) sired pups at about 2.5–3 months after BST. Out of 69 offspring analyzed, 39 were found to be transgene positive (56 % transgenic efficiency). This transgenic efficiency rate with our method of BST is very high in comparison to 1.2 % shown previously as only 4 out of 335 pups were found to be transgene positive originated from donor SSC which were transplanted exogenously  and all these offspring were born from only 1 of the 18 recipient mice, with success rate of 5.5 % only . The reason for their low efficiency might reside in remarkably reduced cytoplasm of Sertoli cells observed in the cross section of testes due to busulfan treatment, at doses higher than ours (120–180 μg as compared to 75 μg used by us). They also have transplanted SSC at a time when the testis was not sufficiently depleted of Gc, reducing their efficiency. Our method is hence, superior to the previously suggested method.
Unilateral testicular busulfan treatment for depleting Gc in single testis was quite effective by our method without any nonspecific cytotoxic effect of busulfan on any other organ including contralateral testis. While in the conventional i.p. route of busulfan administration, whole body get exposed to this cytotoxic drug, which causes the depletion of other stem cell population in the body, this unilateral testicular treatment may be advantageous for many studies where one testis can be used for experimental purpose and contralateral testis may be used as an internal control.
Our method of recipient preparation using the F1B6SJL strain of mice, when checked in other strains of mice was found to be equally effective and successful in evacuating the seminiferous tubules in 15 days after busulfan injection. We also suggest that species-wise doses of busulfan could be selected for better effect at the lowest dose, causing damage to SSC but sparing other cell types of the testes like Sertoli cells or Leydig cells, which help restoration of spermatogenesis. It has also been reported in the past that higher doses of busulfan also affect the Sertoli cells and Leydig cells .
Here, we have established an efficient method of Gc depletion by testicular busulfan injection using a lower dose of busulfan to prepare GCD recipients while achieving the objective of maximum Gc depletion without any cytotoxic effect to other organs. The success rate by our method is 56 % which is severalfold more as compared to 1.2 % shown earlier . Our method is fast, efficient, and ethically superior as it will utilize less animals to generate more information.