Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone 1,2 J. M. Oatley* 3 , A. Tibary†, D. M. de Avila*, J. E. Wheaton‡, D. J. McLean*, and J. J. Reeves* *Department of Animal Sciences, †College of Veterinary Medicine, and Center for Reproductive Biology, Washington State University, Pullman 99164; and ‡Department of Animal Sciences, University of Minnesota, St. Paul 55108 ABSTRACT: Spermatogonial stem cell transplanta- tion is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient’s seminiferous tu- bules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immu- nization against LHRH (IMM), localized testicular irra- diation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotro- pin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also Key Words: Depletion, Germ Cell, Ram 2005 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2005. 83:604–612 Introduction The continual process of spermatogenesis relies on spermatogonial stem cells. In rodents and goats, trans- 1 Supported by Washington Sheep Producers grant to J. J. Reeves and an Achievement Rewards for Collegiate Scientists (ARCS) Fel- lowship to J. M. Oatley. 2 The authors gratefully acknowledge A. Schmidt, J. Stevens, and H. Adams for assistance in data collection and animal handling, and Serono Corp. for donation of Perganol. 3 Correspondence and current address: Dept. of Anim. Biol., Rm. 100E, School of Vet. Med., Univ. of Pennsylvania, Philadelphia 19104 (phone: 215-898-8805; fax: 215-898-0667; e-mail: joatley@vet. upenn.edu). Received September 22, 2004. Accepted November 29, 2004. 604 received an ultrasound-guided injection of PBS con- taining 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P < 0.05) average per- centages of seminiferous tubule cross sections with de- pleted germ cells compared with controls. Serum testos- terone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remain- der of the study compared with controls and IR rams, which were not different from each other. Serum in- hibin concentration was unchanged in all rams follow- ing treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation. plantation of these cells from the testis of a donor male into the seminiferous tubules of an azoospermic recipi- ent has been shown to result in donor-derived spermato- genesis and generation of offspring with the donor geno- type (Brinster and Avarbock, 1994; Brinster and Zimmerman, 1994; Honamarooz et al., 2003a). Sperma- togonial stem cell transplantation technology has po- tential utility in livestock as an alternative means to enhance genetic gain and generate transgenic off- spring. Two main concepts must be developed in large ani- mals for this technique to be efficiently applied. First, a means for preparing recipient animals for transplant is necessary. In goats, recipients were sexually imma- ture and only one of several transfers was successful. A system that destroys or depletes endogenous germ