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research-article |
Agassiz Research Centre, Agriculture and Agri-Food Canada, Agassiz, British Columbia, Canada V0M 1A0
ABSTRACT
Transplantation of testicular tissue onto the back of immunodeficient nude mice provides a tool to examine testicular development and preserve fertility in mammals. There is no immunodeficient model in birds, but we recently transplanted ovarian tissue between newly hatched chicks from two lines of chickens and produced donor-derived offspring, showing that experimental transplantation is possible in newly hatched chicks. In the present study testicular tissue from newly hatched Barred Plymouth Rock (BPR) chicks was transplanted under the skin of the back, under the skin of the abdomen, or in the abdomen of White Leghorn chicks that had been surgically castrated and immunocompromised. Recipient birds were killed at 10 mo of age. Transplanted tissue was observed in one of five hosts receiving tissue under the skin of the back, two of five hosts receiving tissue under the skin of the abdomen, and three of five chicks with grafts inside the abdominal cavity. In recipients with no regeneration of host testes, testicular transplants grew to the size of normal testes, and histologic analysis showed active spermatogenesis. Subsequent collection of sperm from two successful transplants and surgical insemination of the sperm into the magna of the oviducts of BPR hens resulted in the production of 24 donor-derived chicks. These results demonstrate that the combination of testicular tissue transplantation with intramagnal insemination can produce viable, normal chicks, which could provide a simple approach for the recuperation of live offspring in avian species.
assisted reproductive technology, sperm, spermatogenesis, testis
Advances in transplantation of testicular germ cells and tissue may provide uniquely valuable approaches for the preservation of the male germline [1]. Transplantation of spermatogonial cells was first developed in mice [2, 3] and further extended to farm animals [4, 5] and chickens [6, 7]. Transplantation of testicular tissue onto the back of immunodeficient mice has been developed for interspecific production of mammalian sperm [8]. Spermatogenesis has been maintained by grafting of testicular tissue from the mouse, pig, goat, hamster, marmoset, cow, horse, and human [8–15] into an immunodeficient mouse host. Live progeny were generated from sperm obtained from mouse-mouse grafts and subsequent intracytoplasmic injection of sperm into mouse oocytes [16], suggesting that sperm produced by transplantation of testicular tissue within the same species maintain their full fertilizing potential.
We recently developed a surgical technique for orthotopic transplantation of chicken ovarian tissue between newly hatched chicks [17] and produced donor-derived offspring from transplantation of Barred Plymouth Rock (BPR) ovarian tissue into White Leghorn (WL) recipients [18], thus demonstrating the feasibility of transplantation of ovarian tissue between two lines of chickens. This transplantation technique should allow cryopreservation of female avian genetic material, because it allows recuperation of stored material in living birds. In this report we describe the transplantation of testicular tissue between newly hatched chicks and the production of donor-derived offspring by surgical insemination using a fluid suspension from the transplanted testis tissue.
BPR and WL chicks from pure lines maintained at the Agassiz Research Centre [19] were used as donors and recipients, respectively. The WL chickens are white feathered and homozygous for the dominant white gene (II) [20], and the BPR chickens are black feathered and homozygous for the wild-type allele at this locus (ii). A cross of WL and BPR chickens produces offspring that are white with a few randomly distributed black spots (Ii). Feather color was used as a genetic marker to distinguish donor-derived (black, ii) from host-derived (white, Ii) offspring when sperms were used to inseminate BPR hens. All methods used were approved by the Animal Care Committee of the Agassiz Research Centre and followed principles described by the Canadian Council of Animal Care (1993).
Donor testes were isolated from newly hatched BPR chicks that had been freshly killed by cervical dislocation. Each testicle was cut into four to five pieces (from 1.0 to 1.5 mm3 in size) after removal of the tunica albuginea and tunica vaginalis membranes and was kept on ice in Dulbecco modified Eagle medium until transplantation within 4 h.
A surgical technique developed to prepare recipient chicks for orthotopic transplantation of chicken ovaries [17] was used to castrate the WL recipients. Newly hatched (less than 24 h old) chicks were anesthetized by intramuscular injection of 0.5 mg ketamine and 0.1 mg xylazine. Each chick was placed on its back on a heated surgical table. The abdominal skin was washed with 70% ethanol, and the down on the abdomen was removed with small scissors. A transverse incision (2.5–3 cm) was made 1 cm from the last rib, and the peritoneal cavity was opened. The yolk sac and intestine were carefully displaced, and after the yolk stalk was tied with surgical suture the yolk sac was removed and discarded. Yolk removal has been used extensively in nutrition research and results in only slightly delayed growth without influencing absorption dietary energy and lipids [21]. In the newly hatched chick, the paired testicles are located in the extreme craniodorsal part of the abdomen, parallel to the celiac artery, on either side of the median plane. To prevent hemorrhage, host testicles were removed in pieces with fine forceps (Dumont Medical no. 5–45; Fine Science Tools).
Testicular tissue was transplanted into three different locations. In one series of transplantations (n = 5), five to eight pieces of donor tissue were placed on the cranial mesenteric membrane inside the abdominal cavity, and the abdominal incision was closed by two layers of continuous suturing (absorbable polyglycolic acid suture, size 4–0). In another set of transplantations, five to eight pieces of testicular tissue were placed under the abdominal skin (n = 5) before the surgical opening was sutured. In a third series of transplantations (n = 5), the abdomen was closed with sutures, and five to eight pieces of testicular tissue were inserted under the skin of the back through a small incision that was subsequently closed by a single suture. Immediately after surgery chicks were given an intramuscular injection of 5 mg EXCENEL (Pharmacia Animal Health) to prevent infection.
Surgically manipulated birds were kept in a battery brooder for 2 wk with an initial temperature of 33°C and were subsequently raised in a floor pen with a temperature of 25°C. An oral dose of an immunosuppressant, mycophenolate mofetil (CellCept; Hoffmann-LaRoche), was administered at 100 mg/kg per day for 2 weeks, and then once a week until the birds were 2 mo of age to prevent immunologic rejection of the transplanted tissue. This treatment was shown to increase the number of donor-derived offspring from recipients of grafted ovarian tissue [18].
The transplanted birds were raised for 10 mo for analysis of their grafts, except that recipients of grafts under the skin were killed early if no lumps were identified under the skin in the region of the grafts. At approximately 10 mo of age, the roosters were killed by cervical dislocation, and the donor testicular tissue was removed. Pieces of tissue approximately 0.25 cm3 were removed for histologic examination. Samples from the grafts and from the testicles of day-old and adult BPRs (used as controls) were fixed in Bouin solution overnight, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin. Samples were evaluated under a light microscope. Seminiferous tubules, lumens, epithelium, and interstitial gaps were measured in all the tubules in three microscopic fields of sections from testicular grafts and the control testes, using a slide micrometer as a reference. The seminiferous tubules and their lumens were measured as the shortest diameter of the exterior and interior of the tubules, respectively, and the epithelium was calculated as half of the difference between the two. The interstitial gaps were calculated as the average of the largest and smallest values. The area of the seminiferous epithelium in the tubule was calculated as the area of the tubule minus the area of the lumen of the same tubule. The microimages were captured with Qimaging Retiga 1300R digital camera (Qimaging Corp., Burnaby, BC, Canada) and Olympus BX51 microscopy.
Unlike mammalian spermatozoa, which require a period of capacitation within the female reproductive tract, sperm isolated directly from the chicken testicle are capable of fertilizing eggs [22]. The transplanted testicular tissue was cut into small pieces, and the fluid that exuded from the surface was collected and used for intramagnal insemination without dilution and within 1.5 h. Surgical insemination was performed according to the procedure of Engel et al. [23], with some modification. The BPR hens were anesthetized by intramuscular injection of 0.4 ml xylazine (20 mg/ml). The left leg of the hen was drawn up while the hen was lying on its right side. Feathers overlying the left abdominal wall were removed, and the area around the site of the surgery was wetted with 70% ethanol. An incision of approximately 2.5 cm was made in the skin between the thigh and the breast close to the last rib, and an incision of approximately 1 cm was made in the underlying muscle where it is thinnest. A retractor was used to expose the magnum, which lies alongside the body wall in this area of the abdomen. A section of the magnum was held with large forceps while 0.25 to 0.75 ml testicular fluid suspension was injected using a 1-ml syringe equipped with a 20-gauge needle. The exposed loop of the magnum was then returned to the peritoneal cavity, and the skin of incision was closed by continuous sutures. Testicular sperm remaining after insemination were used for microscopic analysis.
The Proc GLM procedure of SAS was used to compare the average sizes of seminiferous tubules, seminiferous epithelium, lumen, and interstitium. Contingency chi-square was used to determine the fertility of eggs laid from 2 to 10 days after intramagnal insemination with testicular sperm. Statistical significance was set at P < 0.05.
Growth of Testicular Transplants
Testicular tissue from newly hatched BPR chicks (Fig. 1a) was transplanted to the abdominal cavity, under the abdominal skin, or under the skin of the back of WL chicks of the same age. When recipients were 10 mo of age, growth of transplanted testicular tissue was observed in 60%, 40%, and 20% of birds, respectively, receiving tissue in the three locations (Fig. 1, c and e; Table 1). Host testes were removed before transplantation, but complete castration was observed in only two birds. Testicular transplants in the completely castrated hosts grew to the size of a normal testis (Fig. 1, b, d, and f), but in hosts with regenerated tissue, transplants grew to only 20% to 30% of the size of normal testis.
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Production of Donor-Derived Offspring by Intramagnal Insemination
Fluid was easily collected from the transplanted tissue in completely castrated hosts, but not from transplanted tissue from hosts that were incompletely castrated. Fluid could be collected from the regenerated testes of incompletely castrated hosts. A volume of 2.75 ml was collected from the transplant (12.5 g in weight) under the skin of the back (Fig. 2d), and 1.5 ml was collected from the transplant (23.2 g in weight) in the abdominal cavity (Fig. 2f). Testicular fluid from the transplanted tissue contained motile sperm, and surgical insemination of this fluid into the magnum of hens' oviducts produced fertile eggs resulting in only black, donor-derived chicks from tissue transplanted under the skin of the back (Fig. 1g) and in the abdominal cavity (Fig. 1h). Heterotopic transplantation eliminated the possibility of contribution by the host testes. In total, 24 black (BPR) chicks were produced from testicular tissue transplanted into WL chickens (Table 2).
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Testicular sperm from the transplanted tissue showed the same fertilizing ability as normal BPR testicular sperm when 0.75 ml of the fluid suspension was surgically inseminated into each hen (chi-square = 2.04, P > 0.05; Table 2). Insemination with 0.25 ml of testicular fluid suspension resulted in 20% fertility, which is significantly lower (chi-square = 9.72, P < 0.05) than that obtained using 0.75 ml of fluid suspension from the same transplants.
Histology of Testicular Transplants
In the testis of newly hatched chicks, germ cells were surrounded by somatic cells without seminiferous tubules (Fig. 2a). Normal adult testes contained seminiferous tubules with active spermatogenesis (Fig. 2b). In completely castrated hosts, the transplanted testes contained seminiferous tubules and lumen with diameters that were significantly larger than those of the control (Fig. 2, c and e; Table 3), with active spermatogenesis in all of the sections from the testicular transplants under the skin of the back (Fig. 2d) or in the abdominal cavity (Fig. 2e). All of the seminiferous tubules contained spermatogonia, spermatocytes, round spermatids, and sperm. The average height of the seminiferous epithelium was significantly lower in the transplants than in the control, but the average area of seminiferous epithelium within the tubules was not significantly smaller in the transplants than in the control (Table 3), indicating that the decrease in the height of seminiferous epithelium was due to the enlargement of the tubules by the accumulation of fluid inside the tubule rather than reduced spermatogenesis. The epithelial gap was greater than the control in the transplant in the abdomen but not under the skin of the back. The morphology of individual spermatozoa from transplanted tissue appeared to be normal. In hosts with regenerated testes, seminiferous tubules in the transplanted tissue were at varied stages of development (Fig. 3, a–d). About 40% of seminiferous tubules contained round spermatids as the most advanced germ cells (Fig. 3, a and b), 50% of seminiferous tubules developed lumen with a few sperm inside (Fig. 3c), and only 10% of tubules underwent active spermatogenesis in part of the seminiferous tubules (Fig. 3d).
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In the present report we demonstrate that testes of newly hatched chicks transplanted under the skin of the back or inside the abdominal cavity produce sperm that when inseminated intramagnally can give rise to healthy offspring. Approximately 150 years ago chicken testes were transplanted to castrated hosts, leading to the discovery of the endocrine effect of the testes [24, 25]. However, to the best of our knowledge, the extent of spermatogenesis in chicken testicular transplants has not been documented in the literature. We report here not only spermatogenesis in transplanted chicken testes, but also the production of offspring from the sperm produced in heterotopic testicular transplants inseminated intramagnally. Others [16] generated progeny in mice by intracytoplasmic injection of sperm retrieved from grafted mouse testes. We previously transplanted ovaries between newly hatched chicks and produced donor-derived offspring from the transplanted tissue [18]. This report confirms our previous finding that there is a window just after hatch that allows grafting in chickens and that offspring can be produced from grafted gonadal tissue. Successful production of donor-derived offspring from the transplantation of chicken testes and ovaries is a significant step toward the development of a simple universal protocol for the conservation of avian germplasm. These techniques can also be used to rescue valuable breeders or transgenic birds with developmental defects after hatching.
Spermatogenesis is a complex cellular interaction between germ and somatic cells (mainly Sertoli cells) that is regulated by growth factors and endocrine, autocrine, and paracrine hormones. Heterotopic transplantation of testes provides the organ structure and cell components for normal spermatogenesis, but the grafted tissue lacks the original vascularization. The organization of testes in birds is less complex than that of mammals, and the tubular structure is not obvious in the testis of newly hatched chicks, suggesting a primitive stage of testicular development. In the present study, transplantation of testicular tissue from newly hatched chicks into castrated hosts resulted in active spermatogenesis and produced enough sperm to fertilize eggs when deposited into the magnum of the oviduct of hens, suggesting that spermatogenesis reached quantitatively normal levels in the transplanted tissue. Histologic analysis showed that the seminiferous tubules undergo active spermatogenesis in the transplants to completely castrated hosts, with an enlargement of the luminal space presumably due to the accumulation of fluid. There are fewer mitotic divisions during spermatogonial proliferation in birds than in mammals. The process of stem cell renewal and spermotogonial proliferation in birds also is less complex, resulting in much more rapid spermatogenesis and the production of more spermatozoa per gram of testis than in mammals [26]. Chickens normally reach sexual maturity before 6 mo of age, and the transplant hosts were killed at 10 mo. Results from the present study suggest that chicken sperm in the testes maintain their fertilizing ability for very long periods of time. In addition, extensive sperm production in the absence of efferent ducts did not to lead to increased defects in spermatogenesis, which has been observed in mouse grafts [16].
As in mammals, complete castration of the recipient is essential for the development of normal spermatogenesis in the testicular transplants [8, 27]. In the present study, recipient chicks were castrated before transplantation, but regeneration of endogenous testicular tissue occurred in most manipulated birds, resulting in abnormal spermatogenesis in the transplants. Active spermatogenesis was only observed in transplants with complete castration. In newly hatched chicks, complete removal of the recipient testes appears to be difficult, and a small portion of remaining testis can regenerate and result in the failure. A second castration before sexual maturity may be needed to improve this transplantation technique.
The testes of birds undergo spermatogenesis at a body temperature that causes apoptosis of germ cells in mammals [28]. Two heat shock proteins, HSP70 and ubiquitin, may play important roles in preventing heat-induced damage to the testis during chicken spermatogenesis [29]. In the present study, normal spermatogenesis was observed in the testicular transplants growing in the abdominal cavity and under the skin of the back. This suggests that a high body temperature is not required for spermatogenesis in birds, because the temperature under the skin of the back is lower than the temperature in the abdominal cavity. Transplantation of testicular tissue under the skin provides an easy method for monitoring growth of the transplanted tissue and may allow retrieval of sperm from the transplants using a needle without sacrificing the entire transplant.
Germline chimeras have been produced by transfer of primordial germ cells [30] and by transplantation of testicular germ cells [6, 7]. These techniques have been developed primarily for the production of transgenic poultry and could be used for the preservation of avian germplasm, but successful colonization of donor germ cells in the recipients is dependent on the depletion of endogenous germ cells [7, 31]. Using germline chimeras for the preservation of poultry lines requires the development of screening protocols to distinguish the donor line from the recipient line, because not all endangered lines are pigmented [32]. In this study, we combined testicular tissue transplantation with intramagnal insemination to demonstrate a simple approach for the recuperation of live birds from tissue in which depletion of endogenous germ cell and identification of the donor offspring are not needed.
ACKNOWLEDGMENTS
The authors would like to thank Beth McCannel, Lee Struthers, Harold Hanson, Cathy Ingram, Wendy Clark, and Karly Ryde for the care of the experimental birds. Appreciation is also expressed to Dr. Tom Forge, in whose lab microphotography was performed.
FOOTNOTES
1Supported by Agriculture and Agri-Food Canada contribution number: 751 and the Canadian Poultry Industry Council. 
Correspondence: 2F.G. Silversides, Agassiz Research Centre, P.O. Box 1000, Agassiz, BC, Canada V0M 1A0. FAX: 604 796 0359; e-mail: silversidesf{at}agr.gc.ca
Received: 10 October 2006.
First decision: 27 October 2006.
Accepted: 12 December 2006.
REFERENCES
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  Y. Song and F. G. Silversides Production of Offspring from Cryopreserved Chicken Testicular Tissue Poult. Sci., July 1, 2007; 86(7): 1390 - 1396. [Abstract] [Full Text] [PDF]
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