Recipient Chickens Research Articles

PiggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens

Transgenic birds embody one of the most potent and exciting research tools in biotechnology for agriculture, medicine, and model animals. To date, retrovirus- or lentivirus-mediated transgenesis has been established in chickens and quail. However, despite having a valid technique for viral transduction to achieve transgenic birds, many obstacles exist for practical applications because of relatively low and variable rates of germ-line transmission and transgenic offspring showing transgene silencing, as well as safety issues related to viral vector use. Thus, the generation of transgenic poultry by nonviral integration is a prerequisite for the introduction of biotechnology to practical applications. Herein, we show that a germ-line-competent chicken primordial germ-cell (PGC) line was established with high efficiency of transmission to offspring and that piggyBac transposition into PGCs improved the efficiency of transgenic chicken production and led to high-level transgene expression. GFP transgene-expressing donor PGC-transferred recipient chickens produced donor-derived progenies, and the germ-line transmission efficiency of donor PGCs was 95.2% on average. Subsequently, half of the donor-derived offspring (52.2%) were transgenic chicks because GFP-expressing donor PGCs, in which a transgene was inserted into one chromosome 20, were heterozygous. In all of the transgenic chickens, GFP expression was constant and strong, regardless of age. Our results demonstrate that piggyBac transposition into the chicken PGC line could be the surest way to generate transgenic chickens safely for practical applications.

Establishing Germline Chimeric Chickens Using Primordial Germ Cells

Chicken primordial germ cells (PGCs) are used to restore and conserve genetic resources of some endangered birds or flocks of domestic chickens. When PGCs are transferred from a donor to a recipient, they can survive and gain sexual maturity in the recipient chicken. This indicates that PGCs retaining exogenous genes can proliferate and develop in recipient gonads, and proves that transferred PGCs are effective sources for establishing transgenic and germline chimeric chickens. The PGC transfer method has been used to produce germline chimeric chickens and may become popular in the field of reproductive science. Germline chimeric chickens have the potential to reconstruct and reproduce endangered birds, including both domestic and wild species.

Interspecific Duck–Chicken Chimeras

The production of quail–Peking duck, Peking duck–quail and Maya duck–chicken chimeras by transferring donor (quail, Peking duck and Maya duck) blastoderm cells into the subgerminal cavity of the recipient (Peking duck, quail and chicken) eggs is described. Production of chimeras by transferring blastoderm cells into the recipient embryo should provide a basis for the investigation of fertilisation barriers in interspecies germline chimeras. It might help to protect endangered birds, contribute to further understanding of physiology and embryonic development in poultry, and provide a technical method for poultry transgenics.

Identification of the Rate of Chimerism of Different Tissues with Microsatellite Markers in Chicken Chimeras

The goal of our study was to evaluate whether private alleles can be defined in microsatellite markers for the breeds under investigation; to evaluate if these private alleles distinguish chicken chimera when using different tissues; to trace them back to the donor: Green-Legged Partridgelike and recipient: White Leghorn chicken breeds, and further on, to estimate the level of chimerism in each tissue. Private and common alleles were defined for donor and recipient chicken breeds in 3 loci. The rate of chimerism was defined based on private alleles present in liver, heart, breast muscle, femoral muscle and gonads. The highest rate of chimerism was observed in liver. A lower rate of chimersim was observed in gonads, and femoral muscle, and finally the lowest rate of chimerism was observed in breast muscle and heart.

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.

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract.

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12-15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.

Establishment of an in vitro culture system for chicken preblastodermal cells

To develop an alternative source for chicken pluripotent cells, we examined (1) whether undifferentiated preblastodermal cells could be subcultured in vitro for an extended period and (2) how subculturing affected the physiological properties of preblastodermal cells. The average number of preblastodermal cells was 2,397 in stage V embryos and 36,345 in stage VII embryos; stage X embryos had an average of 53,857 blastodermal cells. The average cell size decreased significantly (70.63-18.83 microm in diameter; P < 0.0001) as the embryo grew; this was closely related to a reduction in the size and number of lipid vesicles in the cell cytoplasm. The culture conditions were optimized for the stage V preblastodermal cells and the control stage X blastodermal cells. On STO feeder cells, the preblastodermal cells achieved stable growth in vitro only in HES medium or a mixed medium of the Knockout DMEM and HES media. However, more than 10 passages of preblastodermal cells at intervals of 3-4 days was possible only by using the Knockout/HES mixed medium and BRL cell-conditioned HES medium for the primary cultures and subcultures, respectively. Colony-forming preblastodermal cells had well-delineated cytoplasm, which was positively stained for stem cell-specific markers by anti-stage-specific embryo antigen-1 antibody, periodic acid-Schiff's solution, and alkaline phosphatase. When preblastodermal cells with or without culturing were transferred into the blastodermal cavity of stage X embryos, only in vitro-cultured preblastodermal cells at stage V (4/5 = 80%) and stage VII (2/8 = 25%) induced somatic chimerism in recipient chickens. In conclusion, undifferentiated preblastodermal cells could be subcultured, and only the colony-forming preblastodermal cells that stained positively for stem cell markers could induce somatic chimerism.

Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos.

This study reports for the first time the production of chicken germline chimeras by transfer of embryonic germ (EG) cells into recipient embryos of different strain. EG cells were established by the subculture of gonadal tissue cells retrieved from stage 28 White Leghorn (WL) embryos with I/I gene. During primary culture (P(0)), gonadal primordial germ cells (gPGCs) in the stromal cells began to form colonies after 7 days in culture with significant (P < 0.0001) increase in cell population. Colonized gPGCs were then subcultured with chicken embryonic fibroblast monolayer for EG cell preparation. Prepared EG cells or gPGCs at P(0) were transferred to stage 17 Korean Ogol chicken (KOC) embryos with i/i gene. The recipient chickens were raised for 6 months to sexual maturity, then a testcross analysis by artificial insemination was conducted for evaluating germline chimerism. As results, transfer of EG cells and gPGCs yielded total 17 germline chimeras; 2 out of 15 (13.3%) and 15 of 176 sexually matured chickens (8.5%), respectively. The efficiency of germline transmission in the chimeras was 1.5-14.6% in EG cells, while 1.3-27.6% in gPGCs. In conclusion, chicken germline chimeras could be produced by the transfer of EG cells, as well as gPGCs, which might enormously contribute to establishing various innovative technologies in the field of avian transgenic research for bioreactor production.

Experimental Transfer of Adult Ascaridia galli to Recipient Chickens via Cloacal Intubation

A transplantation technique for transferring adult worms of Ascaridia galli to parasite-naive recipient chickens is described. The aim was to find a less traumatic transplantation technique than surgical transplantation, as well as a technique that would avoid worm injuries due to worm passage through the chicken proventriculus and gizzard. Thirty experimental growers were divided into 3 groups. Group 1 animals were fasted for 24 hr before transplantation, and group 2 received an antiperistaltic treatment before and after the transplantation. Animals in group 3 received no special treatment. All animals were fasted for 2 hr before anesthesia and pretreated with atropine. The chickens were anesthetized, and each received 2 male and 2 female worms of Ascarid. galli by cloacal intubation. The tube was inserted and the worms flushed out of the tube as anteriorly as possible in the rectum. Fecal samples were examined during the experimental period to monitor the eventual parasite egg excretion. Two weeks after transplantation the birds were slaughtered and the intestines examined for adult Ascarid. galli. Infection was established in 20% of the animals belonging to groups 1 and 2, and in 60% of the animals in group 3. This transplantation technique enables direct establishment of infections of adult Ascarid. galli in chickens, although the exact worm sex composition of the infection is still unpredictable because of expulsion of worms after transplantation. The method is less traumatic to the experimental animal than are surgical techniques.

Transmissibility of adenovirus-induced adiposity in a chicken model.

We previously reported that human adenovirus Ad-36 induces adiposity and paradoxically lower levels of serum cholesterol (CHOL) and triglycerides (TG) in animals. To evaluate the transmissibility of Ad-36 and Ad-36 induced adiposity using a chicken model. Experiment 1--four chickens were housed (two per cage) and one from each cage was inoculated with Ad-36. Duration of presence of Ad-36 DNA in the blood of all chickens was monitored. Experiment 2--two groups of chickens were intranasally inoculated with Ad-36 (infected donors, I-D) or media (control donors, C-D). Blood drawn 36 h later from I-D and C-D groups was inoculated into wing veins of recipient chickens (infected receivers, I-R, and control receivers, C-R, respectively). On sacrifice, 5 weeks post-inoculation, blood was drawn, body weight noted and visceral fat was separated and weighed. Experiment 1--Ad-36 DNA appeared in the blood of the inoculated chickens and that of uninoculated chickens (cage mates) within 12 h of inoculation and the viral DNA persisted up to 25 days in the blood. Experiment 2--compared with C-D, visceral and total body fat were significantly greater and CHOL significantly lower for the I-D and I-R. TG were significantly lower for the I-D. Ad-36 was isolated from 12 out of 16 blood samples of the I-D that were used for inoculating I-R chickens. Ad-36 DNA was present in the blood and the adipose tissue of the I-D and I-R but not in the skeletal muscles of animals selected randomly for testing. As seen in experiment 1, Ad-36 infection can be transmitted horizontally from an infected chicken to another chicken sharing the cage. Additionally, experiment 2 demonstrated blood-borne transmission of Ad-36-induced adiposity in chickens. Transmissibility of Ad-36-induced adiposity in chicken model raises serious concerns about such a possibility in humans that needs further investigation.

Introgression of the avian naked neck gene assisted by DNA fingerprints

SummaryTheoretical predictions suggest that DNA markers can be useful tools for genomic selection in gene introgression programmes. An experiment was carried out to evaluate the efficiency of using multi‐locus DNA markers in an introgression programme designed to transfer the naked neck gene from a donor to a recipient chicken line. The donor line was a commercial egg layer chicken stock heterozygous at the naked neck locus (Na/na+), while the recipients were from a Cornish broiler line. These two lines differ markedly in their average body weight, a quantitative trait that can also represent the comprehensive differences between the genomes of the two lines involved. Three groups of naked neck BC1 individuals were selected according to the following criteria: (i) low band‐sharing with their donor grandsires evaluated by multi‐locus DNA markers, (ii) high body weight at six weeks of age, and (iii) selection at random as a control group. Birds from each of these groups were mated at random to individuals from the heavier Cornish line to produce three groups of BC2 individuals whose body weights were recorded weekly from three to seven weeks of age. Results indicated that BC2 birds obtained from BC1 parents selected for band‐sharing levels and those selected for body weight, performed equally well at 4–7 weeks of age; both were 3.1–3.9% heavier than birds from the randomly selected group. The additional genome recovery of the heavier broiler line, obtained by DNA markers, was found to be in agreement with theoretically predicted values.

Enrichment of Cecal Species of Avian Eimeria in a Mixed Culture by Transfer of Sporozoites from Foreign Host to Natural Host Birds

An effective method for the enrichment of two cecal species of the avian Eimeria in mixed cultures has been developed. The method involves the transfer of sporozoites of Eimeria tenella and Eimeria adenoeides from their specific site of invasion in a foreign host bird to the natural host, and markedly increases the numbers of the cecal species in relation to the other species in the mixture. Donor turkeys and chickens were inoculated with mixed cultures containing equal numbers of oocysts of the chicken coccidia, E. tenella + Eimeria acervulina, or the turkey coccidia, E. adenoeides + Eimeria meleagrimitis, respectively. After 8 to 24 h, the cecal tissues of the donor birds were scraped into phosphate-buffered saline and transferred, per os, to the natural host. On Day 6 to 7 posttransfer, numerous oocysts of E. tenella and E. adenoeides were found in the ceca of the recipient chickens and turkeys, respectively. In contrast, only occasional oocysts were found in a few birds in other areas of the intestine.

Eimeria tenella and E. acervulina: Differences in Ability to Elicit Cross-Species Protection as Compared with the Turkey Coccidium, E. adenoeides

Repeated oral inoculation of turkey poults with large doses (1 x 10(6) oocysts) of the chicken coccidia, Eimeria tenella or E. acervulina, failed to prevent weight loss, poor feed conversion, and intestinal pathology in turkeys challenged with the turkey coccidium, E. adenoeides. Invasion by E. tenella in turkeys was significantly greater than invasion by E. adenoeides in chickens; by 24 hr postinoculation (PI), the numbers of E. tenella and E. adenoeides sporozoites in the ceca had decreased markedly as compared with the numbers that initially invaded, and they did not differ significantly from each other. At 24 hr PI, however, transfer of cecal scrapings from chickens or turkeys inoculated with E. adenoeides produced infection in 53% of the recipient turkeys, but transfer of scrapings from either chickens or turkeys inoculated with E. tenella failed to produce infection in 20 attempts with recipient chickens. Cultured chicken peripheral blood monocytes (PBMs) that were inoculated with E. adenoeides sporozoites contained numerous vesicles that were recognized by the refractile body-specific monoclonal antibody 1209; the number of vesicles was markedly decreased in PBM cultures inoculated with gamma-irradiated E. adenoeides sporozoites. Very few vesicles were detected in the cytoplasm of turkey PBMs that contained E. tenella sporozoites, and none were detected in turkey PBMs containing E. adenoeides sporozoites. The survival of infective sporozoites, along with the secretion of refractile body antigen, may be more critical to the development of cross-species immunity than the number of sporozoites that initially invade the foreign host.

Production of germ line chimera by transfer of primordial germ cells in the domestic chicken ( [formula omitted][formula omitted

Blood was collected from Stage 13 to 14 (1) chick embryos. Primordial germ cells (PGCs) were separated from blood cells by Ficoll density gradient centrifugation. One hundred Rhode Island Red PGCs per embryo were transferred to the blood stream of Stage 14 to 15 White Leghorn embryos. Also, one hundred White Leghorn PGCs per embryo were transferred to the blood stream of Stage 14 to 15 Rhode Island Red embryos. Hatched male and female chicks were raised until sexual maturity, and progeny tests were performed by mating these PGC recipients with Rhode Island Red chickens of the opposite sex. Chicks apparently derived from the transferred PGCs, based on the feather color of the chicks, were produced from all 4 possible mating combinations. The present results indicate that the germ line of PGC recipient chickens consists of 2 distinct populations of germ cells.

Pattern of electroencephalographic activity during light induced seizures in genetic epileptic chicken and brain chimeras.

Genetic epilepsy was studied in Fayoumi epileptic (F.Epi) chickens and in neural chimeras obtained by selective substitution of embryonic brain vesicles of F.Epi donors in normal recipient chickens. Typical motor seizures accompanied by convulsions were evoked by intermittent light stimulation in F.Epi and in chimeras having embryonic substitution of the prosencephalon and the mesencephalon. The motor seizure was less severe in chimeras receiving only the prosencephalon. In the F.Epi, as well as in all the chimeras, the EEG during seizures was characterized by a desynchronized (or a flattening) pattern of activity. F.Epi and chimeras had a lower threshold to Metrazol induced seizures than control chickens. The experimental animals show that, in this model, large prosencephalic and mesencephalic areas are involved in the epileptic disease. The epileptic character of this genetic dysfunction is discussed.

Transfer of genetic epilepsy by embryonic brain grafts in the chicken.

In the Fayoumi chicken, a spontaneous recessive autosomal mutation (F.Epi) is responsible for high susceptibility to seizures that are especially inducible by intermittent light stimulation. Substitution of defined areas of the encephalic neuroepithelium in normal chicken embryos at 2 days of incubation by their counterparts from homozygous F.Epi embryos generates the epileptic phenotype in the chimeras. It was found that grafting primordia of both prosencephalon and mesencephalon of homozygous F.Epi birds is necessary and sufficient for transfer of the full disease. When grafted alone, the homozygous F.Epi prosencephalon, although showing the typical epileptic interictal electroencephalogram, does not allow the complete epileptic seizures to occur in the hosts. Grafts of mesencephalon and/or rhombencephalon modify neither the behavior nor the electroencephalographic pattern of the recipient chickens. Cooperation of forebrain and midbrain activities is therefore required to yield epileptic seizures in this model.

The extraintestinal stages of Eimeria tenella and E. maxima in the chicken

Donor chickens given feed medicated with one or two levels of decoquinate or given non-medicated feed were infected with oocysts of Eimeria tenella or E. maxima per os. Twelve hours after inoculation with oocysts liver, mid-intestine or ceca homogenates were fed to previously uninfected recipient chickens. The results showed that continuous medication with decoquinate was effective in preventing the transfer of sporozoites from the intestine to the liver. Oocysts were detected in the feces of all recipients of tissue from non-medicated donors, showing that some sporozoites of E. maxima and E. tenella are normally transferred to liver. Young broiler chickens were immunized by oral inoculation of E. maxima oocysts. The immune status of similar chickens inoculated with sporozoites of the same species directly into the liver or spleen were assessed. During the experimental period half of the chicks were provided with non-medicated food and the remainder were given feed supplemented with decoquinate; decoquinate was effective in arresting the development of the sporozoites. Two weeks after initial infection the birds were challenged with oocysts of E. maxima per os. Injection of sporozoites into the spleen did not protect against challenge. Birds inoculated with sporozoites into the liver were unable to develop a significant level of immunity. When the drug pressure was removed from these birds, parasitism of the intestine occurred and immunity developed.

Transfer of Blood Lymphocytes and Macrophages Between Histocompatible Progressor and Regressor Chickens Infected With Rous Sarcoma Virus&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

The development of histocompatible White Leghorn (progressor) and Arkansas Regression (regressor) chicken lines was described. When challenged with Rous sarcoma virus, progressor chickens developed fatal tumors while the regressor chickens eliminated the sarcoma. When sensitized histocompatible peritoneal macrophages and blood lymphocytes were transferred from regressor donors to progressor recipients, they both eradicated growing tumors. Histoincompatible cells were ineffective in inducing tumor remission. Within the two age groups tested, the sensitized blood lymphocytes and macrophages were only effective when transferred between age-matched donor and recipient chickens.

Some Evidence of Mechanical Transmission of Reticuloendotheliosis Virus by Mosquitoes

The possible role of mosquitoes in the mechanical transmission of reticuloendotheliosis virus (REV) in chickens was examined. A carbon-dioxide-baited trap was used to capture mosquitoes in poultry pens for one year to determine the seasonal occurrence and the species of mosquitoes prevalent in the area. Homogenates of captured mosquitoes were used for REV isolation in chicken embryo fibroblast cultures. Sera from sentinel chickens were also examined for REV antibodies. Population peaks of mosquitoes were seen in February, March, and April, and the dominant species, Culex quinqufasciatus, was present throughout the year. Of the 39 batches of mosquitoes examined, REV was isolated on seven occasions: once in summer and six times in autumn. Isolation of REV twice from unengorged mosquitoes from a pen with persistently viremic chickens and once from engorged mosquitoes in a pen with sentinel chickens implied that feeding on the viremic birds had been interrupted. Mechanical transmission of REV could have occurred when unengorged mosquitoes contaminated with REV completed their feeding on sentinel chickens. The birds developed REV antibodies following REV isolation from sentinel pens. REV was experimentally transmitted from persistently viremic donor chickens to a recipient chicken using Culex annulirostris.

Effect of histoincompatibility on migration of labeled bursa cells in the chicken.

A comparison was made of the localization of i.v. injected 3H-adenosine-labeled bursa cells in gamma-irradiated B-identical and B-different recipient chickens. There was no difference in the localization in nonlymphoid organs or in bursa and thymus, but the spleen showed more radioactivity at 24 hr after injection in B-identical than in B-different recipients. This difference was more reproducible in 3 to 4 week or older than in 7- to 10-day-old recipients. Histologically, the greatest difference in the older recipients was seen in the number of labeled cells localizing in germinal centers of spleen. Thymectomy or bursectomy of recipients did not appear to affect the bursa cell localization. syngeneity at the major histocompatibility B locus between donor and recipients appeared sufficient to obtain maximal spleen localization of donor bursa cells.