Animal Reproduction (AR)
Animal Reproduction (AR)
Original Article

Spermatogonial stem cell survival in ram lambs following busulfan treatment

Mohammad Hadi Rasouli; Mohammad Zandi; Ali Asghar Sadeghi; Naser Emamjomeh-Kashan

Downloads: 1
Views: 181


Abstract: To clarify the effect of busulfan on the depletion of spermatogonial stem cells (SSCs) from shal rams testis, in the first experiment, lambs were treated by intraperitoneal injection with 4 mg/kg busulfan. In the second experiment, different concentrations of busulfan (1, 2 and 4 mg/kg) were injected directly into both sides of the left testis. The testes of 8 lambs were collected by standard castration procedure for histological analysis five weeks after the treatments and the left testis of remaining lambs were collected after eight weeks and a two-time enzymatic digestion process was used to isolate SSCs. The results showed that all rams that had received intraperitoneal injections of busulfan died. But by testicular injecting of same dose of the drug, 40% of the animals died. The testicular injection of rams with 1, 2 and 4 mg/kg of busulfan resulted in a dose dependent decrease in testis size and also spermatocytes population after 5 weeks of treatments. From the results of colony formation 8 weeks after treatment with busulfan, it can be concluded that only in 1 and 2 mg/kg of busulfan, recovery of endogenous germ cells was performed. In conclusion, the results demonstrated that intra-testicular injections of busulfan (2 mg/kg) reduced spermatocytes population in ram testis within 5 weeks of treatments, and this effect was reversible within 8 weeks of injection. However, it was not recommended to inject 4 mg/kg busulfan into the peritoneal cavity or testis of lambs based on its side effects.


spermatogonial stem cells, busulfan, recipients, sheep


Ganguli N, Wadhwa N, Usmani A, Kunj N, Ganguli N, Sarkar RK, Ghorai SM, Majumdar SS. An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation. Stem Cell Res Ther. 2016;7(1):142. PMid:27659063.

Goharbakhsh L, Mohazzab A, Salehkhou S, Heidari M, Zarnani AH, Parivar K, Akhondi MM. Isolation and culture of human spermatogonial stem cells derived from testis biopsy. Avicenna J Med Biotechnol. 2013;5(1):54-61. PMid:23626877.

Hermann BP, Sukhwani M, Lin CC, Sheng Y, Tomko J, Rodriguez M, Shuttleworth JJ, McFarland D, Hobbs RM, Pandolfi PP, Schatten GP, Orwig KE. Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques. Stem Cells. 2007;25(9):2330-8. PMid:17585169.

Herrid M, Olejnik J, Jackson M, Suchowerska N, Stockwell S, Davey R, Hutton K, Hope S, Hill JR. Irradiation enhances the efficiency of testicular germ cell transplantation in sheep. Biol Reprod. 2009;81(5):898-905. PMid:19571259.

Honaramooz A, Behboodi E, Hausler CL, Blash S, Ayres S, Azuma C, Echelard Y, Dobrinski I. Depletion of endogenous germ cells in male pigs and goats in preparation for germ cell transplantation. J Androl. 2005;26(6):698-705. PMid:16291964.

Honaramooz A, Behboodi E, Megee SO, Overton SA, Galantino-Homer H, Echelard Y, Dobrinski I. Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats. Biol Reprod. 2003;69(4):1260-4. PMid:12801978.

Izadyar F, Den Ouden K, Stout TA, Stout J, Coret J, Lankveld DP, Spoormakers TJ, Colenbrander B, Oldenbroek JK, Van der Ploeg KD, Woelders H, Kal HB, De Rooij DG. Autologous and homologous transplantation of bovine spermatogonial stem cells. Reproduction. 2003;126(6):765-74. PMid:14748695.

Jafarnejad A, Aminafshar M, Zandi M, Sanjabi MR, Emamjomeh Kashan N. Optimization of in vitro culture and transfection condition of bovine primary spermatogonial stem cells. S Afr J Anim Sci. 2018;48(1):108-16.

Kubota H, Brinster RL. Germ Cell Transplantation. Encyclopedia of Reproduction, 2nd edition, Volume 1. 2018.

Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM. Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol Pathol. 2002;30(4):524-33. PMid:12187944.

Lin Z, Bao J, Kong Q, Bai Y, Luo F, Songyang Z, Wu Y, Huang J. Effective production of recipient male pigs for spermatogonial stem cell transplantation by intratesticular injection with busulfan. Theriogenology. 2017;89:365-373.e2. PMid:27919445.

Ma W, An L, Wu Z, Wang X, Guo M, Miao K, Ma W, Tian J. Efficient and safe recipient preparation for transplantation of mouse spermatogonial stem cells: pretreating testes with heat shock. Biol Reprod. 2011;85(4):670-7. PMid:21593478.

Ogawa T, Ohmura M, Yumura Y, Sawada H, Kubota Y. Expansion of murine spermatogonial stem cells through serial transplantation. Biol Reprod. 2003;68(1):316-22. PMid:12493728.

Olejnik J, Suchowerska N, Herrid M, Jackson M, Hinch G, Hill J. Spermatogonia survival in young ram lambs following irradiation, busulfan or thermal treatment. Small Rumin Res. 2018;166:22-7.

Olejnik JA, Herrid M, Davey R, Hutton K, Hinch G, Hill J. The successful use of busulfan to deplete endogenous spermatogonia in ram testes. Reprod Fertil Dev. 2005;17(9):107.

Qin Y, Liu L, He Y, Ma W, Zhu H, Liang M, Hao H, Qin T, Zhao X, Wang D. Testicular injection of busulfan for recipient preparation in transplantation of spermatogonial stem cells in mice. Reprod Fertil Dev. 2016a;28(12):1916-25. PMid:26111862.

Qin Y, Liu L, He Y, Wang C, Liang M, Chen X, Hao H, Qin T, Zhao X, Wang D. Testicular busulfan injection in mice to prepare recipients for spermatogonial stem cell transplantation is safe and non-toxic. PLoS One. 2016b;11(2):e0148388. PMid:26871566.

Rodriguez-Sosa JR, Dobson H, Hahnel A. Isolation and transplantation of spermatogonia in sheep. Theriogenology. 2006;66(9):2091-103. PMid:16870245.

Stockwell S, Hill JR, Davey R, Herrid M, Lehnert SA. Transplanted germ cells persist long-term irradiated in ram testes. Anim Reprod Sci. 2013;142(3-4):137-40. PMid:24139695.

Takashima S, Shinohara T. Culture and transplantation of spermatogonial stem cells. Stem Cell Res. 2018;29:46-55. PMid:29587218.

Trefil P, Micáková A, Mucksová J, Hejnar J, Poplstein M, Bakst MR, Kalina J, Brillard JP. Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken. Biol Reprod. 2006;75(4):575-81. PMid:16807385.

Zeng W, Tang L, Bondareva A, Honaramooz A, Tanco V, Dores C, Megee S, Modelski M, Rodriguez-Sosa JR, Paczkowski M, Silva E, Wheeler M, Krisher RL, Dobrinski I. Viral transduction ofmale germline stem cells results in transgene transmission after germcell transplantation in pigs. Biol Reprod. 2013;88(1):27. PMid:23221397.

Zhang Z, Renfree MB, Short RV. Successful intra- and inter specific male germ cell transplantation in the rat. Biol Reprod. 2003;68(3):961-7. PMid:12604649.

5f0774e90e8825b54db160c9 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections