Production of Offspring from Cryopreserved Chicken Testicular Tissue 1

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1 Production of Offspring from Cryopreserved Chicken Testicular Tissue 1 Y. Song and F. G. Silversides 2 Agassiz Research Centre, British Columbia, Canada, V0M 1A0 ABSTRACT Cryopreservation of avian germplasm provides In the present study, a simple freezing protocol was used a means of genetic banking for future needs in biological research and animal production. The sperm of birds can be cryopreserved and used to fertilize eggs. However, the fertility of frozen-thawed avian semen is generally much lower than that of mammalian semen and varies among species or among lines, reducing the value of semen for the preservation of genetic resources. to cryopreserve testicular tissue of day-old chicks, and after subsequent transplantation, the frozen-thawed testicular tissue developed functional seminiferous tubules that produced sufficient sperm to fertilize eggs, resulting in donor-derived offspring. This study provides an alternative to semen cryopreservation for storage of the male germline in birds. Key words: chicken, testicular tissue, transplantation, cryopreservation 2007 Poultry Science 86: INTRODUCTION Poultry genetic resources are presently maintained as live animals, which is costly and leaves the populations vulnerable to disease outbreaks or environmental disasters. Another strategy for conserving genetic diversity in birds is cryopreservation of germplasm, providing genetic banking for future needs in biological research and poultry production. Freezing avian embryos is difficult or impossible because the embryo is attached to a large yolk. Germline chimeras can be produced by transfer of frozen-thawed blastodermal cells (Kino et al., 1997) or primordial germ cells (Naito et al., 1994a; Tajima et al., 1998), allowing cryopreservation of avian germplasm. Unfortunately, the procedures to produce germline chimeras are complex, the efficiency of germline constitution is low, and depletion of the host germ cells is difficult (Naito et al., 1994b; Song et al., 2005). Cryopreservation of poultry semen has been investigated extensively since the discovery of the properties of glycerol as a cryoprotectant (Polge, 1951). However, the fertility of frozen-thawed poultry semen has been much lower than that of mammalian semen and the techniques may not be sufficiently reliable for the cryopreservation of avian genetic resources (Long, 2006). In addition, there is significant variation in fertility of frozen-thawed poultry semen among species, among lines (Blanco et al., 2000; Fulton, 2006), and even within lines (Donoghue et al., 2003). Although cryopreservation of 2007 Poultry Science Association Inc. Received January 26, Accepted March 9, Agriculture and Agri-Food Canada Contribution Number Corresponding author: silversidesf@agr.gc.ca semen is still an important technique for conserving the male germline, poultry genetic stocks should not been stored solely as cryopreserved semen because reconstitution of a line may require a redundancy of techniques to ensure recovery when needed. Transplantation and cryopreservation of testicular tissue have been studied as means of preserving fertility in mammals (Woods et al., 2004; Pukazhenthi et al., 2006), but these techniques have not been explored in birds. Surgical techniques have recently been developed to transplant chicken ovarian (Song and Silversides, 2006) and testicular tissue between newly hatched chicks, with subsequent production of donor-derived offspring (Song and Silversides, 2007a,b). These transplantation techniques provide an opportunity for cryopreservation of avian genetic material because living birds can be produced from stored material. The research described here reports the production of offspring from cryopreserved testicular tissue. MATERIALS AND METHODS Birds Barred Plymouth Rock (BPR) and White Leghorn (WL) chicks from pure lines maintained at the Agassiz Research Centre (Silversides et al., 2007) were used as donors and recipients of testicular tissue, respectively. All methods used were approved by the Animal Care Committee of the Agassiz Research Centre and followed principles described by the Canadian Council on Animal Care (1993). Cryopreservation of Testicular Tissue Donor testes were isolated from newly hatched BPR chicks that had been freshly euthanized by cervical dislo- 1390

2 CRYOPRESERVATION OF CHICKEN TESTIS 1391 Table 1. Transplantation of frozen-thawed testicular tissue from Barred Plymouth Rock to White Leghorn chicks Without comb development With comb at 3 mo development at Item Total of age 1 3moofage 2 Number of chicks receiving transplants Number of recipients containing transplanted testicular tissue Number of recipients with complete castration Birds were killed at 3 mo of age. 2 Birds were killed at 11 mo of age. cation. Each testicle was cut into 4 to 5 pieces (from 1.0 to 1.5 mm 3 in size) after removal of the tunica albuginea and tunica vaginalis membranes. Testicular tissue was transferred into 1.2-mL cryovials (2 birds per vial) and equilibrated for 25 min at 0 C in Dulbecco s modified Eagle s medium (DMEM) containing 10% (vol/vol) dimethylsulfoxide and 10% fetal bovine serum. The cryovials were placed in a Nalgene Cryo1 C freezing container (cat. no , Sigma Chemical Co., St. Louis, MO) and the container was placed in a freezer at 80 C for 4 h. The container was designed to achieve a 1 C/ min rate of cooling at 80 C. The cryovials were then plunged into liquid nitrogen and stored for 4 to 5 mo. Transplantation of Frozen-Thawed Testicular Tissue The cryovials were removed from the liquid nitrogen and thawed in a 37 C water bath. The contents were immediately placed in a petri dish and the testicular tissue was washed with 3 changes of DMEM containing 10% fetal bovine serum. Eight pieces of frozen-thawed testicular tissue were transplanted into the abdominal cavity of the WL chicks using previously described methods (Song and Silversides, 2007a). Surgically manipulated birds were kept for 2 wk with an initial temperature of 33 C and subsequently reared in a floor pen with a temperature of 25 C. An oral dose of an immunosuppressant, mycophenolate mofetil (CellCept, Hoffmann-LaRoche Ltd., Mississauga, Ontario, Canada), was administered at 100 mg/kg per day for 2 wk after surgery, and then once a week until the birds were 2 mo of age to prevent immunologic rejection of the transplanted tissue. Transplanted birds with no male comb develop- ment were killed at 3 mo of age, and those with normal male comb development were kept for further study. Histology At approximately 11 mo of age, the roosters were euthanized by cervical dislocation. Both donor testicles and any regenerated host testicles were removed and weighed. Pieces of tissue of approximately 0.25 cm 3 were removed for histological examination. Tissue samples from surgically manipulated birds and adult BPR (used as controls) were fixed in Bouin s solution overnight, embedded in paraffin, sectioned at 5 m, and stained with hematoxylin. Images were captured with a Qimaging Retiga 1300R digital camera (Qimaging Corp., Burnaby, British Columbia, Canada) and an Olympus BX51 microscope (Olympus Corp., Tokyo, Japan). Intramagnal Insemination Testicular sperm were collected from the donor testicles and used for intramagnal insemination of BPR hens to test whether the transplants from the frozen-thawed tissue could produce enough sperm to fertilize eggs. Testicular sperm were first collected as a fluid suspension that was exuded from the tissue when cut into small pieces. Subsequently, the tissue was washed with the same volume of DMEM and collected as a washed suspension. A dose of 0.4 ml of fluid suspension or 0.5 ml of washed suspension was surgically inseminated into BPR hens using previously described procedures (Engel et al., 1991; Song and Silversides, 2007a). The eggs were collected for 2 wk and incubated to evaluate the fertility of sperm from cryopreserved testes. Table 2. Recovery of testes after transplantation of frozen-thawed testicular tissue Fluid Number Weight suspension Weight of of donor of pooled collected Wingband regenerated testes donor from donor of recipient host testes (g) identified testes (g) testes (ml) 62, , , ,

3 1392 SONG AND SILVERSIDES Table 3. Production of offspring from sperm obtained from the transplantation of frozen-thawed testicular tissue Collection Day after intramagnal insemination method for testicular sperm d 2 to 8 d 9 d 10 d 11 d 12 d 13 Total Fluid suspension 1 Eggs collected Fertile eggs Chicks hatched Washed suspension 2 Eggs collected Fertile eggs Chicks hatched Two hens were surgically inseminated with a dose of 0.4 ml of fluid suspension from the transplanted testicle. 2 Two hens were surgically inseminated with a dose of 0.5 ml of washed suspension from the same transplanted testicle. RESULTS Growth of Testicular Transplants from Frozen-Thawed Tissue Frozen-thawed testicular tissue from day-old BPR chicks was transplanted into the abdominal cavity of 9 castrated WL chicks of the same age. Four recipient birds showed the characteristic male comb development by 3 mo of age, but 5 did not (Table 1). Those recipients not showing comb development by 3 mo of age were killed. Of these 5 birds, transplanted testicular tissue (Figure 1, panel A) and complete castration were observed in 4 of them. The 4 recipients with male comb development were kept and killed at 11 mo of age. Of these 4 birds, transplanted testicular tissue was observed in all of them (Figure 1, panels B to E) but only 1 was completely castrated (Table 1 and Figure 1, panel E). Among the 3 hosts with regenerated testes, 1 of the transplanted testis clearly predominated over the regenerated testis (Table 2 and Figure 1, panel D). Production of Offspring from Sperm Collected from Testicular Transplants Testicular fluid was easily collected from the transplanted testes when the host was completely castrated or the transplanted testes outgrew the regenerated host testes (Table 2), but not when the regenerated host testes predominated over the transplanted testes (Figure 1, panels B and C). Surgical insemination of sperm collected from 1 transplant (3.1 g in weight; Figure 1, panel D) produced a total of 23 donor-derived offspring from the fluid and washed suspensions (Table 3 and Figure 1, panel F). Both fluid and washed suspensions resulted in fertility for up to 13 d after insemination. Hens surgically inseminated with testicular suspensions from another transplant failed to produce any eggs within 2 wk of surgery. Histology of Testicular Transplants from Frozen-Thawed Tissue All the seminiferous tubules from the normal adult testis contained evidence of active spermatogenesis (Figure 2, panel A). In hosts with predominant regenerated testes, seminiferous tubules in the transplanted tissue were at various stages of development and only a small proportion of tubules underwent spermatogenesis (Figure 2, panel B). In the host with a predominant transplanted testis, active spermatogenesis was observed in seminiferous tubules from the donor testis (Figure 2, panel C), but spermatogenesis in the regenerated testis was restricted to a small part of seminiferous tubules (Figure 2, panel D). In the center of this transplanted testis, some tubules contained no seminiferous epithelium (Figure 2, panel E) but were full of sperm (Figure 2, panel F). DISCUSSION In this study, a simple freezing protocol was used to preserve chicken testicular tissue, and after thawing and subsequent transplantation, the frozen-thawed testicular tissue developed functional seminiferous tubules that produced sufficient sperm to fertilize eggs. This study extended our previous observations on heterotopic transplantation of fresh chicken testes between newly hatched chicks (Song and Silversides, 2007a) and provides an alternative method to semen cryopreservation for storage of the male germline in birds. Cryopreservation of avian semen has been studied for over 50 years, with the results being described in more than 200 scientific publications. However, a simple universal freezing protocol has not been established for any species of bird because the susceptibility of avian sperm to freezing damage varies significantly among lines and species (Fulton, 2006; Long, 2006). Our successful production of offspring from cryopreserved chicken testicular tissue demonstrated that germ cells in the tissue from newly hatched chicks can easily be frozen, and that spermatogenesis in the frozen-thawed transplanted tissue is maintained. Given that the morphology of the testes and the development of spermatogenesis are essentially similar in most avian species (Johnson, 1986), we can expect that newly hatched testicular tissue of most or all avian species can be preserved in liquid nitrogen and subsequently used to generate mature sperm when transplanted into appropriate hosts. Chicken ovaries can be transplanted between newly hatched chicks with subsequent production of donor-derived offspring (Song and Silversides, 2007b). Chicken ovaries that were frozen using the same simple protocol were also transplanted and underwent development in host chicks (Song and Silversides, unpublished data). Therefore, cryopreservation and transplantation of testes and ovaries could provide a simple universal protocol for the conservation of avian germplasm of all species and lines.

4 CRYOPRESERVATION OF CHICKEN TESTIS 1393 Figure 1. Morphology of testes that developed from frozen-thawed testicular pieces and offspring produced. Donor testicles from hosts in which the male characteristics were not seen at 3 mo of age (A). The regenerated host testes predominated over the transplanted testes (B, C). The transplanted testis outgrew the regenerated host testis (D). In a completely castrated host, 5 pieces of donor testes were identified (E) but no eggs were obtained after surgical insemination of sperm collected. Seven of the 23 Barred Plymouth Rock chicks (F) produced by intramagnal insemination of sperm collected from the donor testicle shown in D. Bar = 1.27 cm (0.5 in.). Castration of recipient birds appears to be a critical factor that affects spermatogenesis in the testicular transplants. In our previous study on transplantation of fresh chicken testes, sperm could be collected for the production of offspring only in the completely castrated host (Song and Silversides, 2007a). The present study demon-

5 1394 SONG AND SILVERSIDES Figure 2. Spermatogenesis in transplanted donor and regenerated host testes. Seminiferous tubules with active spermatogenesis in a control Barred Plymouth Rock testis (A). In the host with a predominant regenerated testis, seminiferous tubules and spermatogenesis in the transplanted donor testis were not fully developed (B). In the host with a predominant transplanted testis, active spermatogenesis was observed in the donor testis (C) but that in the regenerated testis (D) was restricted, and some tubules in the center of the transplanted testis of this individual lacked seminiferous epithelium (E) but contained sperm (F). Bar = 100 m. strated that sperm could be collected from the donor testis if the host was completely castrated or if the transplanted testis was predominant over the regenerated host testis. It is generally believed that in avian species, as in mammals, the development of the testes and the maintenance of spermatogenesis are dependent on hor-

6 CRYOPRESERVATION OF CHICKEN TESTIS 1395 mone-controlled interactions in the hypothalamic-pituitary-testis axis (Johnson, 1986). Heterotopic testicular transplants lack the original vascularization, and transplanted tissue likely needs several days to revascularize in the host. If the host is completely castrated, serum levels of luteinizing hormone and follicle-stimulating hormone rise significantly (Knight et al., 1981), which provides a positive stimulus for the development of the transplanted testes (Schlatt et al., 2003; Honaramooz et al., 2004). After revascularization, the transplanted testis releases testosterone to establish feedback on gonadotropin release in the recipient chick. The coordinated hormonal interactions between the host and the transplant induce and maintain active spermatogenesis in the transplanted testis. However, if the host is not completely castrated, the feedback system in the hypothalamic-pituitary-testis axis is controlled by the regenerated host testis and the development of the transplanted testis is restricted. In the present study, active spermatogenesis was observed in the seminiferous tubules from the frozenthawed testicular tissue and the morphology of most seminiferous tubules appears similar to that of the control testis, which suggests that most germ cells survived the cryogenic process. In our previous study on transplantation of fresh testicular tissue (Song and Silversides, 2007a), the seminiferous tubules of the transplants were enlarged by continued production of sperm with no release via the efferent duct. In the present study, the diameter of the seminiferous tubules in transplanted frozen-thawed tissue was not enlarged, but the amount of fluid collected from the transplants was similar to that obtained from the fresh transplants. Histological analysis suggested that a small number of tubules, in which the germ cells may have failed to survive the cryogenic process, became the storage tubules for sperm. Successful production of 23 offspring from a relatively small testicle developed from frozen-thawed tissue demonstrated that, although germ cells may have been lost during the freezing process, the surviving testicular tissue could maintain spermatogenesis and produce enough sperm for fertilization. The main purpose of our present research was to demonstrate the feasibility of recuperation of live offspring from frozen-thawed testes. However, several aspects of this technique could be refined. First, the efficiency of castration needs to be improved. In this experiment, 5 out of 9 birds operated on were completely castrated, and we previously reported (Song and Silversides 2007a) that only 2 out of 15 chicks were completely castrated, suggesting that the technique of neonatal castration could be improved. Second, more than half the chicks receiving transplants failed to develop male combs by 3 mo of age, indicating a low level of testosterone in the castrated host. It may be useful to characterize hormonal changes in recipient birds and administer appropriate hormones to stimulate development of the transplanted testes. Finally, the techniques of surgical insemination sometimes interrupt the egg production cycle, and development of a nonsurgical technique for artificial insemination with testicular sperm would be helpful. ACKNOWLEDGMENTS The authors would like to thank Beth McCannel, Lee Struthers, Harold Hanson, Cathy Ingram, Wendy Clark, and Karli Ryde for care of the experimental birds. Appreciation is also expressed to Tom Forge, in whose laboratory the microphotography was performed. This research was funded by the Canadian Poultry Industry Council, the Canadian Poultry Research Council, and Agriculture and Agri-Food Canada. REFERENCES Blanco, J. M., G. Gee, D. E. Wildt, and A. M. 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