251. Production of donor-derived live lambs following testis germ cell transplantation

Testes germ cell transplantation in livestock has the potential for amplification of transgenic genotypes and for use as an alternative to artificial insemination. This study investigated a workable protocol for testis germ cell transplantation in sheep between animals of the same breed and different breeds. Testes of two groups of recipients at the stage of pre-pubertal (transition from gonocytes to spermatogonia, n = 2) or peri-pubertal (spermatogenesis initiated, n = 2) were treated with a single dose of 9 grey (Gy) or 15 Gy with a 6MV photon beam irradiation, respectively. In the first experiment, using pre-pubertal irradiated animals, testis germ cell transplantation between the same breed was performed at 16 weeks post irradiation. The left testes of recipient rams were injected with donor cells labelled with fluorescent dye PHK26, while the right testes were given unlabelled cells. The left testes of recipients were removed by castration after 2 weeks following transplantation to evaluate the location of the transferred cells, while the right testes were kept in place for long-term assessment of sperm output. In cryosections of the left testes, PKH26 positive cells were found both on the basement membrane as single cells or in the interstitial area. In the second experiment, animals irradiated at the peri-pubertal stage, received donor cells at 5 weeks post irradiation and animals were kept intact for semen production. For a period of two years after transplantation, semen samples were collected routinely from two groups of rams and analysed using microsatellite markers. Two recipients (50%) demonstrated the presence of donor DNA in their ejaculates. In order to investigate the fertility of the donor-origin sperm in recipient ejaculates, 99 ewes were artificially inseminated with semen from two positive rams. Four lambs (8%) have been identified as being sired by donor-derived sperm produced in the recipient ram that received a Merino to Merino transplantation, while no donor-derived offspring was obtained from the recipient with Border Leicester to Merino transplantation. This study represents the first report of the production of live progeny following testis germ cell transplantation in sheep.

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MORPHOLOGICAL CHARACTERISTIC OF SPERMATOGONIA AND TESTES DISSOCIATION : A Preliminary Study for the Germ Cell Transplantation in Giant Gouramy (Osphronemus gouramy)

Indonesian Aquaculture Journal ◽

10.15578/iaj.5.2.2010.163-172 ◽

2010 ◽

Vol 5 (2) ◽

pp. 163

Author(s):

Irma Andriani ◽

Ita Djuwita ◽

Komar Sumantadinata ◽

Muhammad Zairin Junior ◽

Harton Arfah ◽

...

Keyword(s):

Germ Cell ◽

Cell Transplantation ◽

Incubation Time ◽

Trypan Blue Exclusion ◽

Endangered Fish ◽

Germ Cell Transplantation ◽

Donor Cells ◽

Viable Population ◽

Preliminary Study ◽

Optimal Incubation

The recent study were attempting to develop spermatogonial germ cell transplantation as a tool to preserve and propagate male germ-plasm from endangered fish species, as well as to produce surrogate broodstock of commercially valuable fish. Spermatogonia identification and testes dissociation were the first necessary steps to obtain highly amount and viable population of spermatogonia as donor cells for transplantation. Using giant gouramy testes as a model, spermatogonia was histological characterized and two methods of testes dissociations were compared (i.e. medium A contained 0.5% trypsin in PBS and medium B contained 0.5% trypsin and DNase 10 IU/μL in PBS complemented with CaCl2, Hepes and FCS). Optimal incubation times (1, 2, 3, 4 and 5 hours) in dissociation medium were also determined. Freshly isolated testes of immature giant gouramy were minced in dissociation medium and then incubated to get monodisperce cell suspension. Parameters observed were number and viability of spermatogonia (ø > 10 μm). The viability was analyzed using trypan blue exclusion dye. The results showed that the average number of spermatogonia observed in medium B was higher than in medium A (P<0.05), meanwhile the viability of spermatogonia between medium A and B were not significantly different (P>0.05). The viability of spermatogonia decreased by the increasing duration time of dissociation. The viability of spermatogonia started to decrease significantly in 2 hours incubation time in medium A and 4 hours incubation time in medium B (P<0.05). In conclusion, application of dissociation medium B yielded higher number of viable spermatogonia than dissociation medium A.

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269. Optimal testicular size of donor and recipient for testicular germ cell transplantation in the bovine

Reproduction Fertility and Development ◽

10.1071/srb05abs269 ◽

2005 ◽

Vol 17 (9) ◽

pp. 110

Author(s):

M. Herrid ◽

R. Davey ◽

S. Vignarajan ◽

K. Hutton ◽

J. Hill

Keyword(s):

Germ Cell ◽

Cell Transplantation ◽

Cell Number ◽

Rete Testis ◽

Colonization Rate ◽

Cell Transplant ◽

Testicular Germ Cell ◽

Scrotal Circumference ◽

Germ Cell Transplantation ◽

Donor Cells

The maturity status of donor and recipient testis appears to be important in the efficiency of testicular germ cell transplantation. When neonatal mice were used as recipients, they show 9.4 times greater colonization and 4 times larger colony size than in the adult.1 The objective of this study in cattle was to investigate the effect of testicular maturity of donor and recipient calves on success of the testis cell transplant procedure. Testicular maturity was measured indirectly by scrotal circumference and donor testicular cells were enzymatically isolated from 8 Angus calves aged 5–7 months (scrotal circumference, SC of 18–22cm) and injected into both testes of 12 recipient calves aged 4–6 months (SC 15–21cm. Donor cells were labeled with the red fluorescent dye PKH26 then transferred into the rete testis under ultrasonographic guidance. Castration of recipients was performed 2–6 months following injection and then frozen sections were used to localize the PKH26 positive donor cells. Five sections from different 5 areas in each testis were prepared and 100 tubules were counted. In 15 of the 24 (63%) testes, PKH positive donor cells were identified. There was no correlation between colonization rate and maturity of donor animal testis for the range of testis sizes studied. Testis cells from donors of SC 18-20 cm or of SC 21-22 did not result in different number of recipient testis with positive cells (7/10 (70%) v. 8/14 (57%)) or the number of positive cells per testis (1.92 ± 0.67% v. 2.5 ± 1.01%). Recipient maturity (SC of 15–18 cm v. SC of 19–21 cm) had no effect on the colonization rate (7/11 (64%) v. 8/13 (62%)); however, there were significantly more positive cells per testis in less mature (SC of 15–18) recipients (3.18±1.21% v. 1.52 ± 0.64% P < 0.05)). In summary we have demonstrated successful testicular germ cell transplantation between calves and while donor testis cell age appeared to have little effect on the efficiency of colonization, less mature testis provided more suitable conditions for colonization. (1)Shinohara T, Orwig KE, Avarbock MR and Brinster RL. (2001) Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. PNAS 98(11), 6186–6191.

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Induced sterility in fish and its potential and challenges for aquaculture and germ cell transplantation technology: a review

Biologia ◽

10.1515/biolog-2016-0118 ◽

2016 ◽

Vol 71 (8) ◽

Cited By ~ 9

Author(s):

Amin Golpour ◽

Mohammad Abdul Momin Siddique ◽

Diógenes Henrique Siqueira-Silva ◽

Martin Pšenička

Keyword(s):

Germ Cell ◽

Cell Transplantation ◽

Large Scale ◽

Hormonal Treatment ◽

Important Species ◽

Genetic Ablation ◽

Germ Cell Transplantation ◽

Commercially Important ◽

Alternative Approaches ◽

Hormone Signalling

AbstractInterest in reproductively sterile fish in aquaculture has prompted research into their production. Several methods are available for inducing sterility and optimizing its application in the global fishery industry. Sterilization can potentially be accomplished through irradiation, surgery, or chemical and hormonal treatment. Alternative approaches include triploidization, hybridization, and generation of new lines via advanced biotechnological techniques. Triploids of many commercially important species have been studied extensively and have been produced on a large scale for many years. Novel approaches, including disruption of gonadotropin releasing hormone signalling and genetic ablation of germ cells, have been developed that are effective in producing infertile fish but have the disadvantage of not being 100% reliable or are impractical for large-scale aquaculture. We review currently used technologies and recent advances in induction of sterility in fish, especially those intended for use in germ cell transplantation. Knowledge of the implications of these approaches remains incomplete, imposing considerable limitations.

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Testicular stem cells for fertility preservation: Preclinical studies on male germ cell transplantation and testicular grafting

Pediatric Blood & Cancer ◽

10.1002/pbc.22002 ◽

2009 ◽

Vol 53 (2) ◽

pp. 274-280 ◽

Cited By ~ 78

Author(s):

Stefan Schlatt ◽

Jens Ehmcke ◽

Kirsi Jahnukainen

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Fertility Preservation ◽

Cell Transplantation ◽

Male Germ Cell ◽

Preclinical Studies ◽

Germ Cell Transplantation

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Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation1

Biology of Reproduction ◽

10.1095/biolreprod.103.016519 ◽

2003 ◽

Vol 69 (2) ◽

pp. 412-420 ◽

Cited By ~ 104

Author(s):

Clayton J. Brinster ◽

Buom-Yong Ryu ◽

Mary R. Avarbock ◽

Levent Karagenc ◽

Ralph L. Brinster ◽

...

Keyword(s):

Germ Cell ◽

Cell Transplantation ◽

Germ Cell Transplantation ◽

Restoration Of Fertility

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Male germ cell transplantation in livestock

Reproduction Fertility and Development ◽

10.1071/rd05123 ◽

2006 ◽

Vol 18 (2) ◽

pp. 13 ◽

Cited By ~ 52

Author(s):

J. R. Hill ◽

I. Dobrinski

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Cell Transplantation ◽

Large Scale ◽

Developmental State ◽

Male Germ Cell ◽

Seminiferous Tubules ◽

Cell Transfer ◽

Donor Sperm ◽

Germ Cell Transplantation

Male germ cell transplantation is a powerful approach to study the control of spermatogenesis with the ultimate goal to enhance or suppress male fertility. In livestock animals, applications can be expanded to provide an alternative method of transgenesis and an alternative means of artificial insemination (AI). The transplantation technique uses testis stem cells, harvested from the donor animal. These donor stem cells are injected into seminiferous tubules, migrate from the lumen to relocate to the basement membrane and, amazingly, they can retain the capability to produce donor sperm in their new host. Adaptation of the mouse technique for livestock is progressing, with gradual gains in efficiency. Germ cell transfer in goats has produced offspring, but not yet in cattle and pigs. In goats and pigs, the applications of germ cell transplantation are mainly in facilitating transgenic animal production. In cattle, successful male germ cell transfer could create an alternative to AI in areas where it is impractical. Large-scale culture of testis stem cells would enhance the use of elite bulls by providing a renewable source of stem cells for transfer. Although still in a developmental state, germ cell transplantation is an emerging technology with the potential to create new opportunities in livestock production.

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