Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.ArticleJOURNAL OF POULTRY SCIENCE. 51(1):87-95 (2014)journal articl

Effect of Long-Term Selection for Non-Destructive Deformation on Egg Shape in White Leghorns

Several conventional traits, including eggshell thickness, are commonly being improved genetically as a means to increase eggshell strength. At the same time, researchers have come to recognize that factors related to egg geometry, such as egg shape, are important determinants of the variability remaining in eggshell strength, after conventional traits have been considered. Therefore, given that the value of the egg shape index -the egg’s width to length ratio- depends highly on the hen strain, it is necessary to examine the relationship between eggshell strength and shape index more closely in a variety of breeds. From this perspective, by using REML methodology under a five-trait animal model, we analyzed a two-way selection experiment for non-destructive eggshell deformation in 31 generations of White Leghorns, to evaluate the effect of selection for eggshell strength on egg shape. In the strong line, which refers to the line that was selected for decreased non-destructive deformation value, the genetic correlation between eggshell breaking strength and shape index was 0.285±0.055, whereas that between non-destructive deformation and shape index was -0.021±0.063. In the weak line, these values were 0.244±0.055 and -0.093±0.060, respectively. The heritability estimates were 0.381±0.033 for non-destructive deformation, 0.349±0.029 for eggshell breaking strength, and 0.544±0.027 for shape index in the strong line, and 0.408±0.031, 0.468±0.032, and 0.484±0.028, respectively, in the weak line. The genetic correlation between eggshell breaking strength and shape index suggests that rounder eggs are somewhat more resistant to breakage than more elongated eggs. The moderately high heritability estimates for shape index indicate the potential to improve egg shape through genetic gain

Cryopreservation of Primordial Germ Cells (PGCs) from White Leghorn Embryos Using Commercial Cryoprotectants

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the large size of their yolk structure. Cryopreservation of primordial germ cells (PGCs) is an alternative way to preserve both female and male genetic material in chicken. In the present study, fertilized eggs were incubated for about 53 hours to obtain embryos at stage 14-16. The blood of the whole embryo was collected from the terminal vein or dorsal aorta using a fine glass micro pipette under a microscope. The PGCs were then purified using Nycodenz density gradient centrifugation. Four commercially available cryoprotectants (A, B, C and D) were used to preserve the PGCs, and DMSO was used as a control. The average recovery rate of PGCs after thawing was 58.2%, 52.9%, 46.5% and 39.4% with the A, B, C and D treatments, respectively. There was no significant difference between the A, B and C treatments and control (DMSO), which showed an average recovery rate of 49.9%. However, the recovery rate obtained using D cryoprotectant was significantly lower than using treatment A. The average viability of the PGCs after thawing were 86.0%, 82.3%, 81.1% and 84.8% for cryoprotectants A, B, C and D, respectively, and the control (DMSO) was 85.3%. There was no statistically significant difference between the four treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos ; however, the use of cryoprotectant D should be avoided since the recovery rate was significantly lower, although the viability was not affected

Estimation of the time of divergence between Japanese Mishima island cattle and other cattle populations using microsatellite DNA markers

We applied the theory of random genetic drift to determine the divergence history of a closed cattle population over the relatively short timescale of several hundred years. The divergence history of the closed population of Mishima Island cattle, a national natural treasure of Japan, was examined, and the results were compared with historical documents. Inbreeding depression in the isolated population was investigated for body size and fertility. Twenty-one DNA microsatellite markers in Mishima Island cattle and 3 major breeds from the mainland were genotyped. For the mainland breeds, all 21 or 20 markers were segregating. However, nearly half the number of loci (9 of 21) was fixed in the Island cattle. The average number of alleles per locus of Island cattle was markedly lower than that in the mainland breeds. These results support the theory that Island cattle have been isolated for a considerable period of time. The number of generations of isolation was estimated as 14.1-22.6, and the year of divergence was calculated as 1778-1846. In view of these findings, we propose that Island cattle diverged from the mainland population at around 1800 and were isolated for about 200 years. These estimates are in agreement with historical documents showing that divergence occurred between 1672 and 1880. The total inbreeding coefficient of the present population was predicted to be in the range of 0.51-0.60. However, historical reports dated over 100 years do not support changes in fertility, so that there is no evidence for inbreeding depression

Effects of Busulfan Sustained-release Emulsion on Depletion and Repopulation of Primordial Germ Cells in Early Chicken Embryos

The current study aimed to determine the optimal time of busulfan (1,4-butanediol dimethanesulfonate) administration for the preparation of suitable recipient embryos to generate germline chimeras in chickens. We compared two different administration times, with regards to the degree of endogenous primordial germ cell (PGC) depletion and donor PGC incorporation in recipient embryos. A dose of 75μg busulfan in 50μL sustained-release emulsion was injected into the yolk of chicken embryos incubated for 0h or 24h. Both busulfan treatments resulted in significant reduction in the number of PGCs compared with that of controls (P<0.01). The number of PGCs in the blood at stage 14 was significantly lower after busulfan treatment at 0h of incubation than after treatment at 24h of incubation (P<0.01). No significant difference was observed in the numbers of PGCs in the gonads at stage 29 between the two treatment groups. The number of donor PGCs after busulfan treatment at 0h was significantly higher than that after treatment at 24h and in controls (P<0.01). The ratios of donor to recipient PGCs in the gonads of both busulfan treated groups were higher than in controls (P<0.01). In conclusion, injecting busulfan sustained-release emulsion into the yolk of recipient embryos at 0h of incubation was a suitable method of preparing them for the generation of germline chimeras