Genetic Divergence in Domestic Japanese Quail Inferred from Mitochondrial DNA D-Loop and Microsatellite Markers.

To assess the genetic diversity of domestic Japanese quail (Coturnix japonica) populations, and their genetic relationships, we examined mitochondrial DNA (mtDNA) D-loop sequences and microsatellite markers for 19 Japanese quail populations. The populations included nine laboratory lines established in Japan (LWC, Quv, RWN, WE, AWE, AMRP, rb-TKP, NIES-L, and W), six meat-type quail lines reimported from Western countries (JD, JW, Estonia, NIES-Br, NIES-Fr, and NIES-Hn), one commercial population in Japan, and three wild quail populations collected from three Asian areas. The phylogenetic tree of mtDNA D-loop sequences revealed two distinct haplotype groups, Dloop-Group1 and Dloop-Group2. Dloop-Group1 included a dominant haplotype representing most of the quail populations, including wild quail. Dloop-Group2 was composed of minor haplotypes found in several laboratory lines, two meat-type lines, and a few individuals in commercial and wild quail populations. Taking the breeding histories of domestic populations into consideration, these results suggest that domestic quail populations may have derived from two sources, i.e., domestic populations established before and after World War II in Japan. A discriminant analysis of principal components and a Bayesian clustering analysis with microsatellite markers indicated that the domestic populations are clustered into four genetic groups. The two major groups were Microsat-Group1, which contained WE, and four WE-derived laboratory lines (LWC, Quv, RWN, and AWE), and Microsat-Group2 consisting of NIES-L, JD, JW, Estonia, NIES-Br, NIES-Fr, NIES-Hn, W, and commercial and wild populations. The remaining two lines (AMRP and rb-TKP) were each clustered into a separate clade. This hierarchical genetic difference between domestic quail populations is attributed to the genetic background derived from two different genetic sources-the pre-war and post-war populations-which is well supported by their breeding histories.

Long-term selection using a single trait criterion, non-destructive deformation, in White Leghorns: Effect over time on genetic parameters for traits related to egg production.

Although non-destructive deformation is relevant for assessing eggshell strength, few long-term selection experiments are documented which use non-destructive deformation as a selection criterion. This study used restricted maximum likelihood-based methods with a four-trait animal model to analyze the effect of non-destructive deformation on egg production, egg weight and sexual maturity in a two-way selection experiment involving 17 generations of White Leghorns. In the strong shell line, corresponding to the line selected for low non-destructive deformation values, the heritability estimates were 0.496 for non-destructive deformation, 0.253 for egg production, 0.660 for egg weight and 0.446 for sexual maturity. In the weak shell line, corresponding to the line selected for high non-destructive deformation values, the heritabilities were 0.372, 0.162, 0.703 and 0.404, respectively. An asymmetric response to selection was observed for non-destructive deformation, egg production and sexual maturity, whereas egg weight decreased for both lines. Using non-destructive deformation to select for stronger eggshell had a small negative effect on egg production and sexual maturity, suggesting the need for breeding programs to balance selection between eggshell traits and egg production traits. However, the analysis of the genetic correlation between non-destructive deformation and egg weight revealed that large eggs are not associated with poor eggshell quality.

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.

Production of functional gametes from cryopreserved primordial germ cells of the Japanese quail.

The Japanese quail (Coturnix japonica) is a valuable bird as both an experimental animal, for a wide range of scientific disciplines, and an agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs would be a feasible strategy for the conservation of both male and female fertility cells in Japanese quail. However, the effects of freeze-thaw treatment on viability, migration ability and germline transmission ability of quail PGCs still remain unclear. In the present study, male and female PGCs were isolated from the blood of 2-day-old embryos, which were cooled by slow freezing and then cryopreserved at -196 C for 77-185 days, respectively. The average recovery rate of PGCs after freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P<0.05) (85.5% vs. 95.1%). Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into recipient embryos migrated toward and were incorporated into recipient gonads, although the number of PGCs settled in the gonads was 48.5% lower in the frozen group than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that one female and two male recipients produced live progeny derived from the frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than that of unfrozen controls, but the difference was not significant (4.0 vs. 14.0%). These results revealed that freeze-thaw treatment causes a decrease in viability, migration ability and germline transmission ability of PGCs in quail.

Characterization of the intronic VNTR polymorphisms found in a paralog of chicken serotonin transporter gene.

Polymorphisms in the neurotransmitter-related genes can be a major source of behavioral variations. We searched for polymorphic sites in chicken neurotransmitter-related genes and identified two variable number of tandem repeat (VNTR) loci encompassing the paralog of chicken serotonin transporter gene (5-HTT). Both intronic VNTR were highly polymorphic across chicken breeds and the other Galliformes species, even though predominant alleles were considerably different among breeds. One VNTR locus contained sequences complementary to a conserved motif of CCCTC-binding factor (CTCF) within each repetitive unit, indicating that transcription of chicken 5-HTT paralog may be regulated by the CTCF protein. It is of great interest to contrast these results with previous knowledge on the human 5-HTT that also has CTCF binding sites in the repetitive units of intronic VNTR. Additionally, we measured the degree of impulsiveness in domestic chicks for their preference of immediate/small to large/delayed rewards. A significant difference in the impulsiveness score was detected between two chicken breeds (White Leghorn vs. Boris Brown; P < 0.01), as well as between White Leghorn chicks with different 5-HTT genotypes. These findings imply the possibility that 5-HTT VNTR genotypes may have some impact on chicks' impulsive choice by modifying the serotonergic neurotransmission.

Comparison of production systems for efficient use of indigenous pig breeds in developing countries.

Conserving pig genetic resources and improving their productivity is important to increase returns over investment in developing countries. The purebred, first-cross, rotational cross and backcross matings representing production systems based on pig breeds indigenous to the country and exotic pig breeds were investigated. The number of pigs in the nucleus and commercial herds necessary to produce a defined quantity of pork was considered. The amount of heterosis between the indigenous and exotic breeds, superiority in meat production, and degree of inferiority in reproductive performance of the exotic breed compared with that of the indigenous breed were investigated. The number of breeding pigs in the whole system was in the following order: pure breeding (PB) > first-cross (F1) > rotational cross (RC) > backcross (BC) systems. The number of breeding pigs in the nucleus herds of the RC and BC systems was smaller than that in the nucleus herds of the PB and F1 systems. The degree of inferiority in reproductive performance of the exotic breed compared with that of the indigenous breed affected the efficiency of the production system.

X-irradiation removes endogenous primordial germ cells (PGCs) and increases germline transmission of donor PGCs in chimeric chickens.

Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6- and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gy-irradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens.

Influence of intergeneric/interspecies mitochondrial injection; parthenogenetic development of bovine oocytes after injection of mitochondria derived from somatic cells.

Interspecies/intergeneric mitochondrial heteroplasmy can occur in interspecies/intergeneric hybrid embryos or following nuclear transfer. In the present study, intergeneric buffalo (Bubalus bubalis) mitochondria (WB-mt) or interspecies murine (Mus spretus) mitochondria (M-mt) were injected into bovine (Bos taurus) oocytes, and the subsequent embryonic development was characterized. Fibroblast mitochondria (WB-mt or M-mt) were microinjected into in vitro matured bovine oocytes followed by oocyte activation by a combination of electrical stimulation and 6-dimethylaminopurine treatment. After seven days of culture, embryo development was evaluated. The copy number of specific mtDNA populations (introduced and native mtDNA) from heteroplasmic oocytes was estimated using real-time PCR. The results illustrated that oocytes injected with either WB-mt or M-mt can develop to the blastocyst stage (20.6% and 19.6%). Cleavage division rates and development to the morula stage in oocytes injected with WB-mt were lower (76.2% and 45.9%, respectively) in comparison with uninjected oocytes (89.2% and 59.1%, respectively) (P<0.05). However, no differences were found in comparing M-mt injected oocytes and controls (P>0.05). An increase in bovine mtDNA copy number was observed at the expanded blastocyst stage of injected embryos (P<0.01), while the number of injected mtDNA was stable throughout development. This study demonstrates that interspecies/intergeneric mitochondrial injected bovine oocytes have the ability to develop to the blastocyst stage after parthenogenetic activation and that injected mtDNA was neither selectively destroyed nor enhanced through development. Moreover, injected intergeneric mitochondria had a demonstrated influence on bovine parthenogenetic development and mtDNA replication.

Comparative proteomic analysis of liver mitochondrial proteins derived from cloned adult pigs reconstructed with Meishan pig fibroblast cells and European pig enucleated oocytes.

Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P<0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

Constant transmission of mitochondrial DNA in intergeneric cloned embryos reconstructed from swamp buffalo fibroblasts and bovine ooplasm.

Although interspecies/intergeneric somatic cell nuclear transfer (iSCNT) has been proposed as a tool to produce offspring of endangered species, conflict between donor nucleus and recipient cytoplasm in iSCNT embryos has been identified as an impediment to implementation for agricultural production. To investigate the nuclear-mitochondrial interactions on the developmental potential of iSCNT embryos, we analyzed the mtDNA copy numbers in iSCNT embryos reconstructed with water buffalo (swamp type) fibroblasts and bovine enucleated oocytes (buffalo iSCNT). As controls, SCNT embryos were derived from bovine fibroblasts (bovine SCNT). Buffalo iSCNT and bovine SCNT embryos showed similar rates of cleavage and development to the 8-cell stage (P>0.05). However, buffalo iSCNT embryos did not develop beyond the 16-cell stage. Both bovine and buffalo mtDNA content in buffalo iSCNT embryos was stable throughout the nuclear transfer process, and arrested at the 8- to 16-cell stage (P>0.05). In bovine SCNT embryos that developed to the blastocyst stage, mtDNA copy number was increased (P<0.05). In conclusion, both the donor cell and recipient cytoplast mtDNAs of buffalo iSCNT embryos were identified and maintained through the iSCNT process until the 8-16-cell stage. In addition, the copy number of mtDNA per embryo was a useful monitor to investigate nuclear-mitochondrial interactions.

Comparison of liver mitochondrial proteins derived from newborn cloned calves and from cloned adult cattle by two-dimensional differential gel electrophoresis.

Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT-derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two-dimensional (2-D) fluorescence differential gel electrophoresis (DIGE) using CyDye™ dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P < 0.05). 2D-DIGE analysis revealed differential expression of three proteins for SCNT cattle (n = 4) and seven proteins for SCNT calves (n = 6) compared to controls (P < 0.05). Different protein patterning was observed among SCNT animals even if animals were generated from the same donor cell source. No differences were detected in two of the SCNT cattle. Moreover, there was no novel protein identified in any of the SCNT cattle or calves. In conclusion, variation in mitochondrial protein expression concentrations was observed in non-viable, neonatal SCNT calves and among SCNT individuals. This result implicates mitochondrial-related gene expression in early developmental loss of SCNT embryos. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

The habitat disruption induces immune-suppression and oxidative stress in honey bees.

The honey bee is a major insect used for pollination of many commercial crops worldwide. Although the use of honey bees for pollination can disrupt the habitat, the effects on their physiology have never been determined. Recently, honey bee colonies have often collapsed when introduced in greenhouses for pollination in Japan. Thus, suppressing colony collapses and maintaining the number of worker bees in the colonies is essential for successful long-term pollination in greenhouses and recycling of honey bee colonies. To understand the physiological states of honey bees used for long-term pollination in greenhouses, we characterized their gene expression profiles by microarray. We found that the greenhouse environment changes the gene expression profiles and induces immune-suppression and oxidative stress in honey bees. In fact, the increase of the number of Nosema microsporidia and protein carbonyl content was observed in honey bees during pollination in greenhouses. Thus, honey bee colonies are likely to collapse during pollination in greenhouses when heavily infested with pathogens. Degradation of honey bee habitat by changing the outside environment of the colony, during pollination services for example, imposes negative impacts on honey bees. Thus, worldwide use of honey bees for crop pollination in general could be one of reasons for the decline of managed honey bee colonies.

Efficient system for preservation and regeneration of genetic resources in chicken: concurrent storage of primordial germ cells and live animals from early embryos of a rare indigenous fowl (Gifujidori).

The unique accessibility of chicken primordial germ cells (PGCs) during early development provides the opportunity to combine the reproduction of live animals with genetic conservation. Male and female Gifujidori fowl (GJ) PGCs were collected from the blood of early embryos, and cryopreserved in liquid nitrogen for >6 months until transfer. Manipulated GJ embryos were cultured until hatching; fertility tests indicated that they had normal reproductive abilities. Embryos from two lines of White Leghorn (24HS, ST) were used as recipients for chimera production following blood removal. The concentration of PGCs in the early embryonic blood of 24HS was significantly higher than in ST (P < 0.05). Frozen-thawed GJ PGCs were microinjected into the bloodstream of same-sex recipients. Offspring originating from GJ PGCs in ST recipients were obtained with a higher efficiency than those originating from GJ PGCs in 24HS recipients (23.3% v. 3.1%). Additionally, GJ progeny were successfully regenerated by crossing germline chimeras of the ST group. In conclusion, the cryogenic preservation of PGCs from early chicken embryos was combined with the conservation of live animals.

Germline replacement by transfer of primordial germ cells into partially sterilized embryos in the chicken.

We report a novel technique for almost complete replacement of the recipient germline with donor germ cells in the chicken. Busulfan solubilized in a sustained-release emulsion was injected into the yolk of fertile eggs before incubation. A dose of 100 microg was found to provide the best outcome in terms of reducing the number of endogenous primordial germ cells (PGCs) in embryonic gonads (0.6% of control numbers) and hatchability (36.4%). This was applied for preparing partially sterilized embryos to serve as recipients for the transfer of exogenous PGCs. Immunohistochemical analysis showed that the proportion of donor PGCs in busulfan-treated embryos was significantly higher than in controls (98.6% vs. 6.4%). Genetic cross-test analysis revealed that the germline transmission rate in busulfan-treated chickens was significantly higher than in controls (99.5% vs. 6.0%). Of 11 chimeras, 7 produced only donor-derived progenies, suggesting that these produced only donor-derived gametes in the recipient's gonads. This novel germline replacement technique provides a powerful tool for studying germline differentiation, for generating transgenic individuals, and for conserving genetic resources in birds.

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes.

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P<0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P>0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P<0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P>0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.

Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens.

The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca(2+)- and Mg(2+)-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 microg per 50 microL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.

Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm.

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.

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.

Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages.

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.

A novel method to isolate primordial germ cells and its use for the generation of germline chimeras in chicken.

A novel method was developed to isolate chick primordial germ cells (PGCs) from circulating embryonic blood. This is a very simple and rapid method for the isolation of circulating PGCs (cPGCs) using an ammonium chloride-potassium (ACK) buffer for lysis of the red blood cells. The PGCs were purified as in vitro culture proceeded. Most of the initial red blood cells were removed in the first step using the ACK lysis buffer. The purity of the cPGCs after ACK treatment was 57.1%, and the recovery rate of cPGCs from whole blood was 90.3%. The ACK process removed only red blood cells and it did not affect cPGC morphology. In the second step, the red blood cells disappeared as the culture progressed. At 7 days of in vitro culture, the purity of the PGCs was 92.9%. Most of these cells expressed germline-specific antibodies, such as those against chicken vasa homolog (CVH). The cultured PGCs expressed the Cvh and Dazl genes. Chimeric chickens were produced from these cultured PGCs, and the donor cells were detected in the gonads, suggesting that the PGCs had biological function. In conclusion, this novel isolation system for PGCs should be easier to use than previous methods. The results of the present study suggest that this novel method will become a powerful tool for germline manipulation in the chicken.