Locally elevated cortisol in lymphoid organs of the developing zebra finch but not Japanese quail or chicken.

Glucocorticoids are important for production of functional lymphocytes and immunity. In altricial neonates, adrenal glands are unresponsive and local glucocorticoid synthesis in lymphoid organs may be necessary to support lymphocyte development. Precocial neonates, in contrast, have fully responsive adrenal glucocorticoid production, and lymphoid glucocorticoid synthesis may not be necessary. Here, we found that in altricial zebra finch hatchlings, lymphoid organs had dramatically elevated endogenous glucocorticoid (and precursor) levels compared to levels in circulating blood. Furthermore, while avian adrenals produce corticosterone, finch lymphoid organs had much higher levels of cortisol, an unexpected glucocorticoid in birds. In contrast, precocial Japanese quail and chicken offspring did not have locally elevated lymphoid glucocorticoid levels, nor did their lymphoid organs contain high proportions of cortisol. These results show that lymphoid glucocorticoids differ in identity, concentration, and possibly source, in hatchlings of three different bird species. Locally-regulated glucocorticoids might have species-specific roles in immune development.

Recovery of fertility from adult ovarian tissue transplanted into week-old Japanese quail chicks.

Fertility of cryopreserved ovarian tissue from immature chickens and Japanese quail has been recovered by transplantation. This is of special importance for non-mammalian vertebrates in which cryopreservation and in vitro maturation of oocytes are challenging because their oogenesis is characterised by vitellogenesis. This study tested whether fertility of adult quail ovarian tissue could be recovered by transplantation. Ovaries were isolated from mature Japanese quail hens, trimmed, cut into 3- to 4-mm2 pieces and transplanted into ovariectomised, week-old chicks. Recipients were administered an immunosuppressant for two weeks. Ten of 12 recipients survived until sexual maturity and seven laid eggs, but all stopped laying by 17 weeks of age. The age at first egg of recipients laying eggs (75.7±4.2 days) was greater than that of untreated hens (51.8±1.7 days) and egg production of recipients during the laying period (21.7±5.7) was less than that of untreated hens (60.8±3.5). Recipients were paired with males from the WB line for test mating. Only two hens laid eggs during the test period but both produced 100% donor-derived offspring. This research demonstrated that the reproductive potential of ovarian tissue from adult quail hens can be restored by transplantation.

A model for cryobanking female germplasm in Japanese quail (Coturnix japonica).

Cryopreservation and transplantation of ovarian tissue can be used for cryobanking female germplasm of avian species. Here we report a model of cryobanking ovarian tissue of Japanese quail. Ovaries of 1-wk-old quail chicks from the recessive white-breasted (WB) line were cryopreserved using a vitrification method. The cryopreserved tissue was warmed and transplanted orthotopically into ovariectomized, 1-wk-old female quail chicks from the homozygous wild-type QO line. At sexual maturation, recipients were mated with WB males and the plumage coloration was used as a marker to determine the origin of their ovaries. Seven of the 15 recipients laid eggs and produced donor-derived offspring, and 5 produced 100% donor-derived offspring. No difference was seen in egg production between recipients and untreated birds. The high efficiency of the vitrification and transplantation procedures in preserving and recovering quail ovarian tissue shows that this model can be used for maintaining commercial and experimental quail strains and may benefit other poultry species and endangered avian species.

Production of live offspring from testicular tissue cryopreserved by vitrification procedures in Japanese quail (Coturnix japonica).

Cryopreservation of testicular tissue can be used for ex situ conservation of male germplasm of avian species. The possibility of using vitrification and transplantation of testicular tissue for fertility preservation and recovery was tested in Japanese quail. Testes were removed from 1-wk-old Japanese quail; transfixed on acupuncture needles; equilibrated with dimethyl sulphoxide, ethylene glycol, and sucrose; plunged into liquid nitrogen; and stored in 2-ml straws. Cryopreserved tissue was warmed in sucrose solution at room temperature or at 40°C. Fresh and cryopreserved tissue were transplanted subcutaneously into castrated, 1-wk-old recipients. Twenty of 21 recipients survived the surgery, and 18 had viable transplants at maturity, with no difference in transplantation success between fresh and cryopreserved tissue. Fluid extrusion from 11 of the transplants was collected and inseminated surgically into the magnum of 22 quail hens, and 10 inseminations included foam from the proctodeal gland of the same recipients. Egg production in the 2 wk after insemination was reduced, and none of the hens inseminated with foam produced fertile eggs. Five hens inseminated without foam produced a total of eight live offspring; four of these hens had been inseminated with fluid extrusion from cryopreserved tissue. Histological examination showed spermatogenesis in the transplants, and the tubules, lumens, and epithelium of the seminiferous tubules were of comparable size to those of testicular tissue from intact males. These results demonstrate that testicular tissue of Japanese quail can be preserved using vitrification procedures and recovered through transplantation.

Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica).

Cryopreservation of ovarian tissue has been the only effective way of ex situ conservation of female germplasm in avian species. A novel needle-in-straw (NIS) vitrification method was developed to store tissue in straws instead of cryovials. Fragments of ovarian tissue from one-week old Japanese quail were transfixed on an acupuncture needle. They were immersed in equilibration and vitrification solutions containing dimethyl sulphoxide, ethylene glycol and sucrose. A layer of tin foil was rolled over the tissue fragments and the tin foil package was plunged into liquid nitrogen and inserted into a pre-cooled, 0.5-ml straw which was stored in liquid nitrogen. Tissue was also preserved using a needle immersed vitrification (NIV) method, in which tissue fragments transfixed on needles without tin foil and were stored in cryovials filled with liquid nitrogen. Cryopreserved tissue was warmed at room temperature (RT) or 37°C and the ratio of normal follicles to total visible follicles was determined by histological methods. In addition, cryopreserved and warmed tissue was cultured on the chorioallantoic membranes of fertilized chicken eggs for 5-6 days. The viability and vascularization of the grafts were evaluated. The tissue cryopreserved by NIS and warmed at RT showed comparable follicle morphology to fresh tissue and to that preserved by NIV and warmed at RT. No significant impairment on the viability or vascularization of the grafted tissue was observed. The NIS method allows tissue to be stored and transported safely and efficiently and can be used instead of cryovials in tissue cryobanking.

Production of donor-derived offspring from cryopreserved ovarian tissue in Japanese quail (Coturnix japonica).

Cryopreservation of avian ova and embryos is challenging because of the yolky structure of the egg. As an alternative, with the development of effective cryopreservation protocols, ovarian tissue cryopreservation could be used for cryobanking for birds. Pieces of ovarian tissue of week-old Japanese quail (Coturnix japonica) were frozen at 0.5 degrees C/min in a programmable freezer or vitrified by immersion in liquid nitrogen. Straws containing slow-frozen samples were thawed in ice water, and vitrified samples were removed from the vials and transferred into sucrose, with the concentration lowered in sequence at room temperature. Cell viability of tissue was estimated by trypan blue assay, and tissue histology was examined by light microscopy. Frozen-thawed or vitrified-warmed tissue from WB (recessive plumage color) chicks was transplanted into week-old ovariectomized QO (wild-type plumage) chicks, with some chicks receiving fresh tissue as a control group. At sexual maturity, QO recipients were mated to WB males, and the production of WB offspring demonstrated successful cryopreservation and transplantation. Donor-derived offspring were obtained from the ovarian tissue that had been cryopreserved by either slow-freezing or vitrification. The vitrification protocol used in this study showed better outcomes at each level of evaluation. This study demonstrated that the function of ovarian tissue in avian species can be successfully preserved at subzero temperatures and recovered by transplantation. The vitrification protocol is recommended because of high efficiency and overall simplicity.

Inbreeding in Japanese quail estimated by pedigree and microsatellite analyses.

Accurately estimating inbreeding is important because inbreeding reduces fitness and production traits in populations. We analyzed information from pedigrees and from microsatellite markers to estimate inbreeding in a line of Japanese quail derived from a randombred line (QO) and maintained for 17 generations by pedigreed matings of brothers to groups of sisters. Pedigree data were used to calculate the inbreeding coefficient (F(IT)), which is the level of inbreeding based on a reference ancestor. Data from analysis of 14 microsatellite markers in the inbred and QO lines were used to calculate the population differentiation (F(ST)) of the lines caused by inbreeding. The F(IT) was then calculated as F(IT) = F(IS) + (1 - F(IS)) x F(ST), where F(IS) is the level of inbreeding in the inbred line. Observed heterozygosity from analysis of the microsatellite markers of the QO and inbred lines was 0.43 and 0.21, respectively, and the number of alleles was 3.29 and 1.93, demonstrating a reduction of genetic diversity in the inbred line. The F(IT) of the inbred line calculated from the pedigree and microsatellite marker analyses was 0.69 +/- 0.07 and 0.57 +/- 0.33, respectively. These data suggest that pedigree analysis was more accurate than microsatellite marker analyses for estimating inbreeding in this line of Japanese quail.