Sensitivity of spermatogonia to irradiation varies with age in pre-pubertal ram lambs

Although germ cells from donor rams transplanted into irradiated recipient testes have produced donor derived offspring, efficiency is low. Further optimization of recipient irradiation protocols will add precision to the depletion of recipient spermatogonia prior to germ cell transplant. Three irradiation doses (9,12,15 Gy) were administered to ram lambs aged 14 weeks (Group 1) and 20 weeks (Group 2), then testicular biopsies were collected 1, 2 and 3 months after irradiation. At 1 month after irradiation of Group 1, only the largest dose (15 Gy) reduced spermatogonia numbers below 10% of non-irradiated controls, whereas in Group 2 lambs, each irradiation dose reduced spermatogonia below 10% of controls. In both Groups, fewer differentiated germ cells were present in seminiferous tubules compared to controls. At 2 months after irradiation, spermatogonia numbers in both Groups increased more than sixfold to be similar to controls, whereas fewer differentiated germ cells were present in the tubules of both Groups. At 3 months in Group 1, each irradiation dose reduced spermatogonia numbers t

A New and Fast Technique to Generate Offspring after Germ Cells Transplantation in Adult Fish: The Nile Tilapia (Oreochromis niloticus) Model

Background: Germ cell transplantation results in fertile recipients and is the only available approach to functionally investigate the spermatogonial stem cell biology in mammals and probably in other vertebrates. In the current study, we describe a novel non-surgical methodology for efficient spermatogonial transplantation into the testes of adult tilapia (O. niloticus), in which endogenous spermatogenesis had been depleted with the cytostatic drug busulfan. Methodology/Principal Findings: Using two different tilapia strains, the production of fertile spermatozoa with donor characteristics was demonstrated in adult recipient, which also sired progeny with the donor genotype. Also, after cryopreservation tilapia spermatogonial cells were able to differentiate to spermatozoa in the testes of recipient fishes. These findings indicate that injecting germ cells directly into adult testis facilitates and enable fast generation of donor spermatogenesis and offspring compared to previously described methods. Conclusion: Therefore, a new suitable methodology for biotechnological investigations in aquaculture was established, with a high potential to improve the production of commercially valuable fish, generate transgenic animals and preserv

Current status and future direction of cryopreservation of camelid embryos

Over the past 3 decades, and similar to the horse industry, fresh embryo transfer has been widely practiced on large commercial scales in different camelid species, especially the dromedary camel and alpaca. However, the inability to cryopreserve embryos significantly reduces its broader application, and as such limits the capacity to utilize elite genetic resources internationally. In addition, cryopreservation of the semen of camelids is also difficult, suggesting an extreme sensitivity of the germplasm to cooling and freezing. As a result, genetic resources of camelids must continue to be maintained as living collections of animals. Due to concerns over disease outbreaks such as that of the highly pathogenic Middle East Respiratory Syndrome in the Middle East and Asia, there is an urgent need to establish an effective gene banking system for camelid species, especially the camel. The current review compares and summarizes recent progress in the field of camelid embryo cryopreservation, identifying four possible reasons for the slow development of an effective protocol and describing eight future directions to improve the current protocols. At the same time, the results of a recent dromedary camel embryo transfer study which produced a high morphologic integrity and survival rate of Open Pulled Straw-vitrified embryos are also discussed. © 2016 Elsevier Inc

Leptin has concentration and stage-dependent effects on embryonic development in vitro

There is accumulating evidence that leptin may be directly involved in pre-implantation embryonic development, however, it is unclear whether there is a concentration and stage-dependent regulatory pattern. In this study, the addition of 10 ng/ml human recombinant leptin to the culture medium significantly increased the percentage of two-cell mouse embryos that developed into blastocysts and hatched blastocysts, whereas in the presence of 100 ng/ml leptin, the development rate was significantly inhibited. The total cell numbers in the hatched blastocysts were significantly higher in the presence of 10 ng/ml leptin compared with controls and higher concentrations. The differential sensitivity to leptin was found to vary among embryos at different stages of development. Supplementation of leptin (10 ng/ml) to culture medium at two- to eight-cell stages resulted in a consistent stimulatory effect on embryo development. Most interestingly, the inhibitory effect of high leptin concentration (100 ng/ml) on embryo development was diminished when it was added to the culture medium at the eight-cell stage of development. The concentration-dependent regulation pattern was confirmed using sheep embryos, under similar conditions although sheep embryos appeared to be more sensitive in responding to leptin. Having established the effect of exogenous leptin on embryo development, the expression pattern of leptin and its receptors were also investigated. Leptin mRNA was not detected in mouse two-, four-, eight-cell and blastocyst stage embryos, whereas three isoforms of leptin receptor (Ob-Ra, Ob-Rb and Ob-Re) were identified in these cells, indicating that leptin is likely to modulate embryo development via a paracrine signalling system

Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel ('Camelus dromedarius')

The objective of this study was to modify and optimize a vitrification protocol (open pulled straw) that was originally designed for human oocytes and embryos, to make it suitable for the cryopreservation of camel hatched blastocysts. The original open pulled straw protocol was a complex process with 15-minute exposure of oocytes/embryos in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (Me₂SO) for equilibration, and cooling in 16% EG + 16% Me₂SO + 1 M sucrose. Recognizing a need to better control the cryoprotectant (CPA) concentrations, while avoiding toxicity to the embryos, the effects on the survival rate and developmental potential of camel embryos in vitro were investigated using two different methods of loading the CPAs into the embryos (stepwise and semicontinuous increase in concentration), two different loading temperature/time (room temperature ~°C/15 min and body 37°C/3 min), and the replacement of Me₂SO with EG alone or in combination with glycerol (Gly). A total of 145 in vivo-derived embryos were subjected to these processes, and after warming their morphological quality and integrity, and reexpansion was assessed after 0, 2, 24, 48, 72, and 96 hours of culture. Exposure of embryos in a stepwise method was more beneficial to the survival of embryos than was the semicontinuous process, and loading of CPAs at 37°C with a short exposure time (3 minutes) resulted in an outcome comparable to the original processing at room temperature with a longer exposure time (15 minutes). The replacement of the Me₂SO + EG mixture with EG only or a combination of EG + Gly in the vitrification medium significantly improved the outcome of all these evaluation criteria (

Current status and future direction of cryopreservation of camelid embryos

Over the past 3 decades, and similar to the horse industry, fresh embryo transfer has been widely practiced on large commercial scales in different camelid species, especially the dromedary camel and alpaca. However, the inability to cryopreserve embryos significantly reduces its broader application, and as such limits the capacity to utilize elite genetic resources internationally. In addition, cryopreservation of the semen of camelids is also difficult, suggesting an extreme sensitivity of the germplasm to cooling and freezing. As a result, genetic resources of camelids must continue to be maintained as living collections of animals. Due to concerns over disease outbreaks such as that of the highly pathogenic Middle East Respiratory Syndrome in the Middle East and Asia, there is an urgent need to establish an effective gene banking system for camelid species, especially the camel. The current review compares and summarizes recent progress in the field of camelid embryo cryopreservation, identifying four possible reasons for the slow development of an effective protocol and describing eight future directions to improve the current protocols. At the same time, the results of a recent dromedary camel embryo transfer study which produced a high morphologic integrity and survival rate of Open Pulled Straw-vitrified embryos are also discussed. © 2016 Elsevier Inc

Transplanted germ cells persist long-term in irradiated ram testes

Testicular germ cell transplantation provides a tool to study transgenesis, spermatogenesis and to increase production efficiency in livestock industries. Isolated testicular germ cells can be transplanted into testes of livestock breeds to generate sperm of donor origin. In sheep, methods have been developed previously to isolate cell populations from ram testes and transplant these into irradiated testes of recipient rams. This has resulted in rams producing sperm derived from the donor cells and a number of the recipient animals have produced donor-derived offspring from the introduced spermatogonial cells. Microsatellite genotyping data presented here demonstrates that these rams continue to produce sperm of donor origin for at least 5 years post-transplantation. This research provides new evidence of the stability of transplanted germ cells in a commercially important species, and with further refinements to cell isolation, transplantation and recipient preparation, this technology should find use in breeding systems to increase livestock production efficiency