Characterization of structure and protein of vitelline membranes of precocial (ring-necked pheasant, gray partridge) and superaltricial (cockatiel parrot, domestic pigeon) birds.

Of all the known oviparous taxa, female birds lay the most diverse types of eggs that differ in terms of shape, shell pigmentation, and shell structure. The pigmentation of the shell, the weight of the egg, and the composition of the yolk correlate with environmental conditions and the needs of the developing embryos. In this study, we analyzed the structure and protein composition of the vitelline membrane (VM) of ring-necked pheasant, gray partridge, cockatiel parrot, and domestic pigeon eggs. We found that the VM structure is characteristic of each species and varies depending on whether the species is precocial (ring-necked pheasant and gray partridge) or superaltrical (cockatiel parrot and domestic pigeon). We hypothesize that a multilayer structure of VM is necessary to counteract the aging process of the egg. The multilayer structure of VM is only found in species with a large number of eggs in one clutch and is characterized by a long incubation period. An interesting discovery of this study is the three-layered VM of pheasant and partridge eggs. This shows that the formation of individual layers of VM in specific sections of the hen's reproductive system is not confirmed in other species. The number of protein fractions varied between 19 and 23, with a molecular weight ranging from 15 to 250 kDa, depending on the species. The number of proteins identified in the VM of the study birds' eggs is as follows: chicken-14, ring-necked pheasant-7, gray partridge-10, cockatiel parrot-6, and domestic pigeon-23. The highest number of species-specific proteins (21) was detected in the VM of domestic pigeon. This study is the first to present the structure and protein composition in the VM of ring-necked pheasant, gray partridge, cockatiel parrot, and domestic pigeon eggs. In addition, we analyzed the relationship between the hatching specification of birds and the structure of the VM.

Effect of guinea fowl egg storage duration on embryonic and physiological parameters, and keet juvenile growth.

This study investigated the effects of guinea fowl hatching eggs storage time on embryo parameters and post-hatch juvenile growth. A total 1,800 eggs of guinea fowl were used. They were numbered, weighed, and divided into four groups of 450 eggs each according to storage time of 3, 7, 11, and 15 D before storage at a temperature of 18°C. Then, they were incubated at 37.7°C and 55% relative humidity for 28 D in a forced-draft incubator. Egg weight loss, albumen pH and weight, embryo weight, hatching events, and keet growth up to 7 D post-hatch were recorded. In addition, thyroid hormone and corticosterone levels were determined. The results indicate that during storage, relative egg weight loss increased with storage duration. However, albumen pH increased with storage time up to 11 D of storage and remained unchanged between 11 and 15 D. In addition, from 19 to 22 D of incubation, albumen weight was higher for eggs stored for 15 D compared to that of eggs stored for 3 to 11 D. But, from 16 D of incubation, embryos from eggs stored for 3 D grew faster than those from eggs stored for 7 to 15 D. Incubation durations up to internal pipping (IP), external pipping (EP), and hatching events increased with egg storage duration. At IP, corticosterone and triiodothyronine (T3) concentrations of eggs stored for 15 D had the lowest (P < 0.05) compared to those of eggs stored for 3 to 11 D. Moreover, the levels of thyroxine (T4) decreased with storage duration (P < 0.05). At hatch, corticosterone levels increased while T4 levels decreased with storage duration (P < 0.05). Also, hatchability decreased with egg storage duration. In addition, 7-day-old keets from eggs stored for 3 and 7 D had comparable weight and were heavier than those from eggs stored for 11 D. It was concluded that storage of guinea fowl hatching eggs more than 7 D negatively affects egg quality and subsequently depresses embryo and post-hatch growth.

Effect of sex ratio, storage time and temperature on hatching rate, fertility and embryonic mortality in Chukar partridge (Alectoris chukar).

Chukar partridges (Alectoris chukar) are frequently reared in captivity with the aim of producing fertile eggs and chicks. The aims of the present study were to evaluate the effect of male to female sex ratio on hatching rate and fertility and to determine the optimal temperature and time for egg storage and its effect on hatching rate and embryonic mortality in Chukar partridges. Maximum hatching rate and fertility rate were affected by male:female ratios during breeding (P < 0.05). The greatest embryonic mortality rate was with eggs that were stored at 25 °C for 15-20 d, however, the greatest hatching rate was when eggs were stored at 10 and 15 °C for 5 days. Storage time of Chukar partridge eggs can be extended for as long as 5 d, but longer storage periods can negatively affect hatching rate. In conclusion, a reduction of storage temperature and duration of time of storage in combination with reducing the sex ratio of the egg producing females and males during the breeding period might improve fertility, hatching rate and fertility of Chukar partridge eggs.

The periodic coloration in birds forms through a prepattern of somite origin.

The periodic stripes and spots that often adorn animals' coats have been largely viewed as self-organizing patterns, forming through dynamics such as Turing's reaction-diffusion within the developing skin. Whether preexisting positional information also contributes to the periodicity and orientation of these patterns has, however, remained unclear. We used natural variation in colored stripes of juvenile galliform birds to show that stripes form in a two-step process. Autonomous signaling from the somite sets stripe position by forming a composite prepattern marked by the expression profile of agouti Subsequently, agouti regulates stripe width through dose-dependent control of local pigment production. These results reveal that early developmental landmarks can shape periodic patterns upstream of late local dynamics, and thus constrain their evolution.

Developmental basis of phallus reduction during bird evolution.

BACKGROUND: One of the most puzzling events in evolution is the reduction and loss of the phallus in birds. All birds reproduce by internal fertilization, but only ∼3% of birds have retained a phallus capable of intromission. A number of hypotheses have been proposed for the evolutionary mechanisms that drove phallus reduction; however, the underlying developmental mechanisms are unknown. RESULTS: We investigated genital development in two sister clades of birds, Galliformes (land fowl), most of which lack an intromittent phallus, and Anseriformes (waterfowl), which have well developed phalluses; and in two outgroups, Paleognathae (emus) and Crocodilia (alligators). Galliform embryos undergo cryptic development of a genital tubercle, the precursor of the phallus, but this later undergoes apoptosis, leading to regression of the tubercle. At the molecular level, a derived pattern of Bmp4 expression was identified in chick (a galliform) genital tubercles. Inhibition of Bmp signaling in chick genitalia rescues cells from apoptosis and prevents phallus regression, whereas activation of Bmp signaling in duck (an anseriform) genitalia induces a galliform-like pattern of apoptosis. Thus, distal Bmp activity is necessary and sufficient to induce apoptosis in Galloanserae genital tubercles. CONCLUSIONS: Our results indicate that evolutionary reduction of the intromittent phallus in galliform birds occurred not by disruption of outgrowth signals but by de novo activation of cell death by Bmp4 in the genital tubercle. These findings, together with discoveries implicating Bmps in evolution of beak shape, feathers, and toothlessness, suggest that modulation of Bmp gene regulation played a major role in the evolution of avian morphology.

Genital evolution: cock-a-doodle-don't.

Losing the penis in species with internal fertilization may seem paradoxical, but birds have managed to do it multiple times. A new study addresses one developmental mechanism responsible for penis reduction in birds, and opens the door to further examination of this little understood evolutionary phenomenon.

Induction of cytochrome P4501A by highly purified hexachlorobenzene in primary cultures of ring-necked pheasant and Japanese quail embryo hepatocytes.

Primary cultures of ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica) embryo hepatocytes were used to compare the potencies of highly purified hexachlorobenzne (HCB-P), reagent-grade HCB (RG-HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. HCB-P, RG-HCB and TCDD all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNA. Induction was not caused by contamination of HCB with polychlorinated dibenzo-p-dioxins, dibenzofurans or biphenyls. Based upon a comparison of the EC(50) and EC(threshold) values for EROD and CYP1A4/5 concentration-response curves, the potency of HCB relative to TCDD was 0.001 in ring-necked pheasant and 0.01 in Japanese quail embryo hepatocytes. Differences in species sensitivity to HCB were found to be mainly dictated by differences in species sensitivity to TCDD rather than differences in the absolute potency of HCB. Consequently, ring-necked pheasant and Japanese quail embryo hepatocytes were found to be equally sensitive to HCB exposure. Species sensitivity comparisons were also made with chicken (Gallus gallus domesticus) and revealed that chicken embryo hepatocytes were less responsive to EROD induction (lower maximal response) by HCB compared to the embryo hepatocytes of pheasant and quail.

Effect of sight barriers in pens of breeding ring-necked pheasants (Phasianus colchicus) II. Reproductive parameters.

1. The effects of sight barriers in the pens of breeding ring-necked pheasants were investigated on a commercial game farm over a 10-week laying season. 2. Reproductive performance was recorded as egg production, numbers of eggs rejected for hatching together with measures of fertility, embryonic mortality and hatchability for 11 pens with barriers and 11 pens that were left open and acted as controls. 3. Egg production per pen and the numbers of rejected eggs were not significantly affected by the presence of the barriers. 4. Fertility was significantly higher and persisted for longer in the barrier pens, particularly towards the end of the laying season. 5. Embryonic mortality was unaffected by the presence of the barriers but hatchability was significantly lower in the open pens, which was associated with lower levels of fertility. 6. Establishing sight barriers in breeder pens for commercial pheasants would appear not only to offer improved welfare but also significant commercial advantages.

Developmental and posthatch effects of in ovo exposure to 2,3,7,8-TCDD, 2,3,4,7,8-PECDF, and 2,3,7,8-TCDF in Japanese quail (Coturnix japonica), common pheasant (Phasianus colchicus), and white leghorn chicken (Gallus gallus domesticus) embryos.

An egg injection study was conducted to confirm a proposed model of relative sensitivity of three avian species to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like chemicals. It was previously reported that the order of species sensitivity to in ovo exposure to TCDD, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) at doses ranging from 0.044 to 37 picomoles (pmol)/g egg was the chicken (Gallus gallus domesticus), common pheasant (Phasianus colchicus), and Japanese quail (Coturnix japonica) based on embryo mortality and hepatic enzyme induction. In the present study, the incidence of developmental deformities, changes in body and relative organ masses, and organ pathology of hatchlings as additional indicators of species sensitivity were assessed; in addition, embryo mortality in the three species was categorized by stage of development. Embryo mortality varied temporally with significant increases generally occurring after organogenesis and just prior to hatching. A significant increase in the percentage of developmental deformities was observed only in Japanese quail exposed to TCDF. Body and relative organ masses of quail, pheasants, and chickens dosed in ovo with TCDD, PeCDF, or TCDF were not consistently affected. Chemical-related pathology occurred only in livers of quail at the greatest doses of each compound. These results indicated that the incidence of developmental deformities, changes in body and relative organ masses and organ pathology could not be used as indicators of species sensitivity or chemical potency.

Sensitivity of Japanese quail (Coturnix japonica), Common pheasant (Phasianus colchicus), and White Leghorn chicken (Gallus gallus domesticus) embryos to in ovo exposure to TCDD, PeCDF, and TCDF.

Egg injection studies were performed to confirm a proposed model of relative sensitivity of birds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this model, species are classified as belonging to one of three categories of sensitivity based on amino acid substitutions in the ligand-binding domain of the aryl hydrocarbon receptor. Embryo lethality and relative potencies of 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) were compared with TCDD for Japanese quail (Coturnix japonica; least sensitive), Common pheasant (Phasianus colchicus; moderately sensitive), and White Leghorn chicken (Gallus gallus domesticus; most sensitive). Doses ranging from 0.044 to 37 pmol/g egg (0.015-12 ng/g egg) were injected into the air cell of eggs prior to incubation. LD(50) (95% confidence intervals) values, based on rate of hatching for TCDD, PeCDF, and TCDF, were 30 (25-36), 4.9 (2.3-9.2), and 15 (11-24) pmol/g egg for the quail, 3.5 (2.3-6.3), 0.61 (0.28-1.2), and 1.2 (0.62-2.2) pmol/g egg for pheasant, and 0.66 (0.47-0.90), 0.75 (0.64-0.87), and 0.33 (0.23-0.45) pmol/g egg for chicken, respectively. LD(50)-based relative potencies of PeCDF and TCDF were 6.1 and 2.0 for quail, 5.7 and 2.9 for pheasant, and 0.88 and 2.0 for chicken, respectively. TCDD was not the most potent compound among the species tested, with PeCDF and TCDF being more potent than TCDD in the quail and pheasant. TCDF was the most potent in chicken. Species sensitivity was as expected for TCDD and TCDF, whereas for PeCDF, the chicken and pheasant were similar in sensitivity and both were more sensitive than the quail. Results from companion in vitro studies are generally similar to those reported here with a few exceptions.

Effect of long-term storage on the hatchability of red-legged partridge (Alectoris rufa) eggs.

The effect of 0-, 7-, 14-, 21-, 28-, and 35-d storage periods on hatchability and weight loss during storage and incubation of red-legged partridge (Alectoris rufa) eggs stored at 15 degrees C and 80% RH was studied. There was a significant effect of storage period on egg weight loss during storage (P<0.001), a significant increase occurring in weight loss for each additional week the eggs were stored, and this weight loss amounted to more than 3% for the eggs stored for 35 d. Storage period affected egg weight loss during incubation (P<0.05). An increase in egg weight loss during incubation occurred, increasing from 21 d of storage and amounting to more than 10% for the eggs stored for 35 d. Storage period had a significant effect on hatchability (P<0.05). Storage of red-legged partridge eggs up to 28 d does not affect subsequent hatchability, but a significant decline in hatchability occurs when the eggs are stored for 35 d. This research confirms, as occurs in other Alectoris species, that long-term storage of red-legged partridge eggs (to 28 d) does not have a negative effect on hatchability. These findings will be useful in terms of successful hatchery management, mainly by allowing prolonged storage of eggs while gathering a sufficient-sized batch to be incubated, and shipment of long shelf life hatching eggs at game farms while losing little hatchability until further incubation.

Storage time and eggshell colour of pheasant eggs vs. the number of blastodermal cells and hatchability results.

The aim of this study was to investigate the number of the embryo blastodermal cells and hatchability of pheasant from eggs of different eggshell colours depending on the length of the storage period before hatching. On the day of collection, dark-brown and olive eggs were characterised by a similar and significantly higher (by about 48%) number of embryo BCs in comparison with light-brown and blue eggs. Dark-brown eggs stored longer than one day had the highest, while blue-shelled eggs the lowest, number of BCs. The number of BCs found in eggs with blue and light-brown coloured eggshells stored for 10 days was similar and significantly lower (by 27.7%) in comparison with dark-brown eggs. With the lengthening of the storage period, the number of blastodermal cells in eggs of all eggshell colours declined as a result of necrobiosis. In comparison with the dark-brown and olive-shelled eggs, eggs with blue eggshells had higher (by about 7.0%) weight loss during the 21 days until hatching. The dark-brown and olive eggs were found to have a 10.3% higher proportion of eggs considered as fertilised in comparison with the blue-shelled eggs. Eggs with dark-brown shells stored for 2-4 days prior to hatching, in comparison with blue-shelled eggs, had a higher proportion of fertilised eggs. The dark-brown and olive eggs stored for 7 and more days before hatching possessed a higher value of this trait in comparison with the eggs of light-brown and blue eggshells (x = 80.9 against 66.4%). The highest drop in the share of fertilised eggs, which amounted on average to 3.25% for each day of storage, was observed in the blue-shelled eggs. The dark-brown eggs stored for 7 days before being placed in an incubator had higher hatchability from fertilised eggs (by 17.8%) in comparison with the eggs with blue eggshells. In the case of eggs stored for 8 to 10 days, values for this trait were higher for the dark-brown and olive-coloured eggs than for the blue-shelled eggs. The highest mean decrease of chick hatchability from fertilised eggs was observed in the case of the blue-shelled eggs (7.93% for each day). The dark-brown eggs had significantly higher (by about 22.0%) chick hatchability from fertilised eggs than the blue-shelled eggs. Moreover, the dark-brown and olive eggs, in comparison with the blue-shelled eggs, were characterised by a significantly higher hatchability after each period of storage before incubation. The highest negative trend-cycle was observed for eggs with blue shells, while the smallest--for olive-shelled eggs. Directly after laying, pheasant eggs differed with regard to the developmental advancement of the blastodermal embryo depending on eggshell colour. Longer storage time caused the numbers of blastodermal cells in eggs to decrease. The group with blue shells had a lower proportion of fertilised eggs and lower hatching results than the dark-brown eggshell group. It was also demonstrated that the value of hatchability indices decreased significantly irrespective of eggshell colour after seven days of storage prior to hatching.

Development of a pheasant interspecies primordial germ cell transfer to chicken embryo: effect of donor cell sex on chimeric semen production.

This study was conducted to evaluate whether the sex of donor primordial germ cells (PGCs) influences production of chimeric semen from recipient hatchlings produced by interspecies transfer between pheasant (Phasianus colchicus) and chicken (Gallus gallus). Pheasant PGCs were retrieved from 7-d-old embryos and subsequently transferred into circulatory blood of 2.5-d-old (Stage 17) embryos. The sex of embryos was discerned 3 to 6 days after laying, and in preliminary study, overall rate of embryo survival after sexing was 74.6% with male-to-female ratio of 0.49 to 0.51. In Experiment 1, magnetic-activated cell sorting (MACS) using QCR1 antibody was effective for enriching the population of male and female PGCs in gonadal cells (9.2- to 12.5-fold and 10.8- to 19.5-fold increase, respectively). In Experiment 2, an increase in the number of hatchlings producing chimeric semen was detected after the homosexual transfer of male-to-male compared with that after the heterosexual transfer of female-to-male (68% to 88%). Significant increase was found in the frequency of chimeric semen production (0.96 to 1.68 times); production of pheasant progenies by artificial insemination using chimeric semen was also increased in the homosexual transfer (0 to 3 cases). In conclusion, the homosexual PGC transfer of male-to-male yielded better rate of generating pheasant progenies after test cross-reproduction than that of the heterosexual transfer of female-to-male, which could improve the efficiency of interspecies germ cell transfer system.

Reproduction of wild birds via interspecies germ cell transplantation.

The present study was conducted to apply an interspecies germ cell transfer technique to wild bird reproduction. Pheasant (Phasianus colchicus) primordial germ cells (PGCs) retrieved from the gonads of 7-day-old embryos were transferred to the bloodstream of 2.5-day-old chicken (Gallus gallus) embryos. Pheasant-to-chicken germline chimeras hatched from the recipient embryos, and 10 pheasants were derived from testcross reproduction of the male chimeras with female pheasants. Gonadal migration of the transferred PGCs, their involvement in spermatogenesis, and production of chimeric semen were confirmed. The phenotype of pheasant progenies derived from the interspecies transfer was identical to that of wild pheasants. The average efficiency of reproduction estimated from the percentage of pheasants to total progenies was 17.5%. In conclusion, interspecies germ cell transfer into a developing embryo can be used for wild bird reproduction, and this reproductive technology may be applicable in conserving endangered bird species.

Comparative embryonic development of the skeleton of the domestic turkey (Meleagris gallopavo) and other galliform birds.

Ossification sequences are poorly known for birds in general, even for common domestic and experimental species. Such sequences constitute a rich source of data on character evolution, and may even provide phylogenetic information. It is not clear, however, what factors influence ossification sequences and what the relative importance of phylogeny is to the sequences. Galliformes constitute a good group to examine these variables. These birds are osteologically conservative, have precocial young, but have a broad spectrum of body sizes and incubation periods. Here, I describe the embryonic ossification of the skeleton in the domestic turkey (Meleagris gallopavo), and compare it to the domestic chicken (Gallus gallus) and the Japanese quail (Coturnix coturnix). Ossification sequences in this group are not affected by egg size or incubation period. They also appear to be independent of both the spatial location and the embryonic tissue from which the osteogenic cells originated. Accumulation of a wider sample of ossification sequences from more morphologically variable avian taxa will be necessary in order to test functional and phylogenetic effects.

Identification of the avian B-cell-specific Bu-1 alloantigen by a novel monoclonal antibody.

A panel of monoclonal antibodies was generated against the guinea fowl's bursal cells. One of the antibodies, designated BoA1, recognized both cortical and medullary B cells of bursal follicles and B cell dependent regions of peripheral lymphoid organs, like germinal centers and splenic periellipsoidal regions. The staining pattern of this monoclonal antibody is similar to other antibodies (L22, 11G2, AV20), which also identify the Bu-1 antigens. Under reducing conditions, the molecular weight of the BoA1 antigen is 70 to 73 kDa, and after immunoprecipitation it proved to be identical with the antigen recognized by the AV20 antibody. It is unique for this novel monoclonal antibody that it shows wide range cross-reactivity with different avian species, like chicken, quail, guinea fowl, and turkey. Therefore, this Bu-1-specific monoclonal antibody could be a versatile tool for studying the B cell development in different domesticated birds.

Consequences of prenatal androgen exposure for the reproductive performance of female pheasants (Phasianus colchicus).

Maternal hormones in vertebrate eggs can mediate important forms of maternal effects. However, the function of hormone transfer to the eggs is still debated, especially because long-term fitness consequences have been little studied. We investigated the effect of prenatal exposure to physiologically elevated yolk testosterone (T) levels on reproduction of female pheasants (Phasianus colchicus) in captivity. We found that females hatching from T-injected eggs (T-females) had a lower egg-laying rate than controls, and their eggs were more frequently infertile than those laid by control females. There were no effects of prenatal maternal treatment on egg size and yolk T concentration, but eggs carrying a female embryo laid by T-females had smaller yolks than eggs with a male embryo, while there was no sex difference in yolk size among the eggs laid by control females. Progeny sex ratio was unaffected by maternal treatment. These findings suggest that the transfer of high androgen levels to the eggs by the mother is constrained by complex trade-offs between direct effects on her daughters' reproduction and by trans-generational differential consequences on male and female descendants.

Detection and characterization of primordial germ cells in pheasant (Phasianus colchicus) embryos.

The developmental similarity between the chicken and pheasant (Phasianus colchicus) allows the novel biotechnologies developed in the chicken to be applied to the production of transgenic pheasants and interspecies germline chimeras. To detect pheasant primordial germ cells (PGCs) efficiently, which is important for inducing germline transmission, the ultrastructure of PGCs and their reactivity to several antibodies (2C9, QB2, anti-SSEA-1, and QCR1) and periodic acid-Schiff's solution (PAS) were examined. To obtain PGCs, blood was taken from embryos incubated for 62-72 h or from gonads from embryos incubated for 156-216 h. The PGCs collected from both sources had the typical ultrastructure of pluripotent cells: a large nucleus with a distinct nucleolus, a high ratio of nuclear to cytoplasmic volume, and a distinct cytoplasmic membrane. In comparing the morphology of PGCs collected from different sites, more mitochondria and better-developed membrane microvilli were found in gonadal PGCs than in circulating PGCs. The nucleus of gonadal PGCs was flattened and had a large eccentrically positioned nucleolus. Of the antibodies tested, only QCR1 antibody reacted with an epitope in pheasant PGCs, and no specific signal was detected to other antibodies. The temporal change in the PGC populations in the blood and gonads of embryos was examined. In blood, the population was greater (P < 0.0001) in embryos incubated for 64 h than in embryos incubated for 62 or 66-72 h (31.4 versus 5.6-16.2 microL(-1)). In embryonic gonads, the number of PGCs increased continuously from 156 to 216 h of incubation (193-2,718 cells/embryo), although the ratio of PGCs to total gonadal cells did not change significantly (0.50-0.61%). In conclusion, pheasant PGCs have typical germ cell morphology and possess the QCR1 epitope. Circulating blood and the gonads of embryos incubated for 64 and 216 h, respectively, are good sources of PGCs.