Study of ex-ovo embryonic development of Gallus gallus domesticus / Estudio inicial del desarrollo embrionario ex-ovo de Gallus gallus domesticus

SUMMARY: The domestic chicken is a species of bird that has been extensively studied in regard to its biology and as a model organism for science. The reproduction of the species is by the laying of fertilized eggs, which in a period of 21 days will develop a chick inside. Several methods have been described to develop embryos ex-ovo, allowing the observation and manipulation of the organism. This work has the propose to standardize a method that allows the development of the embryos inside the artificial incubation system, which has a low cost and is easy to make. In this work, 100 chicken eggs were used to study the effects of humidity, mineral supplementation, and the preincubation time of the egg on the incubation ex-ovo of the embryos. Embryo development was documented through the different days. Pulverized eggshell was selected as an optimal source to provide calcium, magnesium, phosphorus, and other minerals to the developing embryo. By providing 900-1200 mg of pulverized eggshell, 40 mL of the 0.001 % solution of benzalkonium chloride, and a preincubation time of approximately 56 h, the embryos were able to develop until 19 days, and even though they did not reach hatching, the incubation conditions that allowed the survival and development of embryos until late stages were achieved. Thus, due to the conditions established for calcium, humidity and preincubation time, in the present work, the chicks reached 19 days of development.

El pollo doméstico es una especie de ave que ha sido ampliamente estudiada en cuanto a su biología y como organismo modelo para la ciencia. La reproducción de la especie es por la puesta de huevos fecundados, que en un período de 21 días desarrollarán un polluelo en su interior. Se han descrito varios métodos para desarrollar embriones ex-ovo, permitiendo la observación y manipulación del organismo. Este trabajo tuvo como objetivo estandarizar un método que permita el desarrollo de los embriones dentro del sistema de incubación artificial, el cual tiene un bajo costo y es fácil de realizar. En este trabajo se utilizaron 100 huevos de gallina para estudiar los efectos de la humedad, la suplementación mineral y el tiempo de preincubación del huevo sobre la incubación ex-ovo de los embriones. El desarrollo embrionario se documentó a través de los diferentes días. Se seleccionó la cáscara de huevo pulverizada como una fuente óptima para proporcionar calcio, magnesio, fósforo y otros minerales al embrión en desarrollo. Al suministrar 900-1200 mg de cáscara de huevo pulverizada, 40 mL de la solución de cloruro de benzalconio al 0.001 % y un tiempo de preincubación de aproximadamente 56 h, los embriones lograron desarrollarse hasta los 19 días, y aunque no llegaron a eclosionar, los embriones lograron desarrollarse hasta los 19 días. Se lograron condiciones de incubación que permitieron la supervivencia y desarrollo de los embriones hasta etapas tardías. Así, debido a las condiciones establecidas de calcio, humedad y tiempo de preincubación, en el presente trabajo los pollitos alcanzaron los 19 días de desarrollo.

Shielding the Negative Impact of Cadmium in Avian Embryos by Supplementing with Beetroot Juice.

The increased correlation between maternal cadmium (Cd) exposure and restricted fetal growth has become a matter of global concern. Because dietary nitrate has been demonstrated to offer a range of beneficial vascular effects, we attempt to unveil the effect of one such nitrate-rich food, beetroot, on recovering Cd-induced fetal growth retardation. Using chick embryos as a model, our results confirm that retarded growth is a result of exposure to Cd at an early stage of development and that Cd hinders vasculogenesis and angiogenesis. We find that beetroot juice (BRJ) recovers fetal growth retardation effects of Cd via its vasodilatory effect and promotes embryonic angiogenesis. In conclusion, this study confirms that BRJ reduces the rate of congenital malformations with Cd exposure in utero and suggests the importance of dietary supplements for pregnant women.

Embryo malposition as a potential mechanism for mercury-induced hatching failure in bird eggs.

We examined the prevalence of embryo malpositions and deformities in relation to total mercury (THg) and selenium (Se) concentrations in American avocet (Recurvirostra americana), black-necked stilt (Himantopus mexicanus), and Forster's tern (Sterna forsteri) eggs in San Francisco Bay (CA, USA) during 2005 to 2007. Overall, 11% of embryos were malpositioned in eggs > or =18 d of age (n = 282) and 2% of embryos were deformed in eggs > or =13 d of age (n = 470). Considering only those eggs that failed to hatch (n = 62), malpositions occurred in 24% of eggs > or =18 d of age and deformities occurred in 7% of eggs > or =13 d of age. The probability of an embryo being malpositioned increased with egg THg concentrations in Forster's terns, but not in avocets or stilts. The probability of embryo deformity was not related to egg THg concentrations in any species. Using a reduced dataset with both Se and THg concentrations measured in eggs (n = 87), we found no interaction between Se and THg on the probability of an embryo being malpositioned or deformed. Results of the present study indicate that embryo malpositions were prevalent in waterbird eggs that failed to hatch and the likelihood of an embryo being malpositioned increased with egg THg concentrations in Forster's terns. We hypothesize that malpositioning of avian embryos may be one reason for mercury-related hatching failure that occurs late in incubation, but further research is needed to elucidate this potential mechanism.

Selenium distribution in eggs of avian species.

We studied the effect of egg mass of eight different avian species on Se distribution between egg components and the effect of incubation on Se accumulation by chicken eggshell and shell membrane. Eight groups of birds received a diet without Se supplementation. Unfertile eggs were collected after 35 days of feeding; yolk, albumen, shell and shell membrane were assayed separately for Se. All avian species studied showed identical Se concentration in yolk-albumen complex equal to 38.7 microg Se/100 g, reflecting a linear correlation between yolk-albumen mass and Se content. Shells and shell membrane Se accumulation showed quadratic correlation with the appropriate mass thus explaining unusually high Se concentration in ostrich shell and shell membrane, that reached values 1785 and 1904 microg Se/kg respectively. Incubation of fertile eggs decreased eggshell Se content, the effect being more expressed in eggs from hens fed sodium selenite compared to organic Se utilization (Sel-Plex). It was concluded that shell might be an additional Se source for an embryo.

Transgenic birds for the production of recombinant proteins.

Transgenic birds were expected to be an excellent transgenic bioreactor for the production of recombinant pharmaceutical proteins. However, the only successful transgenic bioreactors have been based on mammals. We have developed two key techniques for obtaining transgenic birds. For bird embryo culture, we identified that the low rate of hatchability of cultured embryos is caused by limited oxygen and calcium availability. In quail embryo culture using a chicken eggshell as a culture vessel, hatchability increased to 80% by the supplement of calcium lactate in addition to oxygen aeration. A fully artificial vessel for quail embryo culture using a gas-permeable Teflon membrane was also designed. Although the hatchability was lower than that of cultures using a surrogate eggshell, we succeeded in hatching of bird embryos using a fully artificial vessel. For transgene introduction, a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G) was injected to laid embryos at the blastodermal stage, and the embryos were hatched in vitro to generate G0 birds. The viral vector sequence was detected in the tissues of all G0 birds. The germ-line transmission efficiency was more than 80%. Plural copies of the transgene were inserted into the genome of G1 transgenic progeny.

Effects of size and temperature on developmental time.

Body size and temperature are the two most important variables affecting nearly all biological rates and times. The relationship of size and temperature to development is of particular interest, because during ontogeny size changes and temperature often varies. Here we derive a general model, based on first principles of allometry and biochemical kinetics, that predicts the time of ontogenetic development as a function of body mass and temperature. The model fits embryonic development times spanning a wide range of egg sizes and incubation temperatures for birds and aquatic ectotherms (fish, amphibians, aquatic insects and zooplankton). The model also describes nearly 75% of the variation in post-embryonic development among a diverse sample of zooplankton. The remaining variation is partially explained by stoichiometry, specifically the whole-body carbon to phosphorus ratio. Development in other animals at other life stages is also described by this model. These results suggest a general definition of biological time that is approximately invariant and common to all organisms.

Selenium in a Wyoming grassland community receiving wastewater from an in situ uranium mine.

Water, soil, vegetation, grasshoppers, bird eggs, and bird livers collected at a 23.5-ha (58 acres) grassland irrigated with wastewater from an in situ uranium mine (study area) and a reference site in 1998 were analyzed for selenium and other trace elements. Selenium concentrations in the uranium mine wastewater applied onto the grassland ranged from 340 to 450 microg/L. Selenium in the upper 15 cm (6 in) of soil from the irrigated grassland at the mine ranged from 2.6 to 4.2 microg/g dry weight (DW). Mean selenium concentrations in soil and water were 5 and 15 times higher at the study area than at the reference site. Selenium concentrations in grasses and grasshoppers ranged from 6.8 to 24 microg/g and 11 to 20 microg/g DW, respectively. Selenium in red-winged blackbird eggs and livers collected from the study area ranged from 13.2 to 22 microg/g and 33 to 53 microg/g DW, respectively, and concentrations were well in excess of toxic thresholds. Mean selenium concentrations in grasses, grasshoppers, and bird eggs and livers were 5.8 to 30 times higher at the study area than at the reference site. Elevated selenium concentrations in water, soil, grasshoppers, and red-winged blackbird eggs and livers collected from the study area demonstrate that selenium is being mobilized and is bioaccumulating in the food chain.

Sublethal effects in avocet and stilt hatchlings from selenium-contaminated sites.

Excess selenium (Se) in the aquatic food chain is embryotoxic and teratogenic to avocets, stilts, and other waterbirds. American avocet (Recurvirostra americana) and black-necked stilt (Himantopus mexicanus) eggs were collected from three sites in the Tulare Lake Basin of California, USA, and hatched in the laboratory. These sites included the Tulare Lake Drainage District-north (TLDD-N, water 2.5 ppb Se), TLDD-south (TLDD-S, water 8.6 ppb Se), and Westfarmers (WF, water 190 ppb Se). Highest egg Se concentrations occurred at WF (geometric mean 31.4 ppm dry wt for avocets and 20.5 ppm dry wt for stilts). Mean egg Se concentrations were 6.7 ppm for avocets and 8.4 ppm for stilts at TLDD-S, and 3.3 ppm for avocets and 2.3 ppm for stilts at TLDD-N. Hatching success and incidence of malformations did not differ among sites, but yolk sac-free hatching weights and bone lengths were less for avocets at the WF site, whereas liver weights and liver to body weight ratios were greater at that site. With increasing Se concentration, oxidative stress was most apparent in avocet hatchlings from WF: hepatic glutathione (GSH) peroxidase activity increased, glucose-6-phosphate dehydrogenase activity decreased, and oxidized glutathione (GSSG) concentration as well as the ratio of GSSG to reduced GSH concentration increased. In stilts, hepatic GSH concentration was lower in WF hatchlings. In conclusion, our findings of Se-impaired embryo growth and hepatotoxicity in avocet hatchlings suggest that oxidative stress observed in hatchlings may be related to these biological effects and may serve as a potential bioindicator of subsequent impaired functions.

Transfer of n-3 and n-6 polyunsaturated fatty acids from yolk to embryo during development of the king penguin.

This study examines the transfer of lipids from the yolk to the embryo of the king penguin, a seabird with a high dietary intake of n-3 fatty acids. The concentrations of total lipid, triacylglycerol (TAG), and phospholipid (PL) in the yolk decreased by ~80% between days 33 and 55 of development, indicating intensive lipid transfer, whereas the concentration of cholesteryl ester (CE) increased threefold, possibly due to recycling. Total lipid concentration in plasma and liver of the embryo increased by twofold from day 40 to hatching due to the accumulation of CE. Yolk lipids contained high amounts of C(20-22) n-3 fatty acids with 22:6(n-3) forming 4 and 10% of the fatty acid mass in TAG and PL, respectively. Both TAG and PL of plasma and liver contained high proportions of 22:6(n-3) ( approximately 15% in plasma and >20% in liver at day 33); liver PL also contained a high proportion of 20:4(n-6) (14%). Thus both 22:6(n-3) and 20:4(n-6), which are, respectively, abundant and deficient in the yolk, undergo biomagnification during transfer to the embryo.

Contaminants in eggs of aquatic birds from the grasslands of central California.

Eggs were collected from the Grasslands of western Merced County, California, during 1986 and 1987, and at the Mendota Wildlife Area in Fresno County in 1987, as part of a reproductive study of nesting ducks and shorebirds. The eggs were analyzed to evaluate the effects of contaminants in agricultural drainwater on avian reproduction. Agricultural drainwater was the major source of water for irrigation and fall flooding in the Grasslands until the fall of 1985 when drainwater was mostly replaced by uncontaminated water. Drainwater had not been used at Mendota. Concentrations of arsenic and boron in eggs from all sites were lower than those known to harm avian reproduction. However, mean concentrations of selenium, a trace element known to impair avian reproduction at high dietary levels, were higher in eggs from the Grasslands than in eggs from the uncontaminated site. Although some selenium concentrations were as high as those found to have caused death or deformities at other sites, reproductive impairment was not observed in any ducks or shorebirds nesting in the Grasslands in 1986 or 1987.

Selenium toxicosis in wild aquatic birds.

Severe gross and microscopic lesions and other changes were found in adult aquatic birds and in embryos from Kesterson Reservoir (a portion of Kesterson National Wildlife Refuge), Merced County, Calif., during 1984. Adult birds from that area were emaciated, had subacute to extensive chronic hepatic lesions, and had excess fluid and fibrin in the peritoneal cavity. Biochemical changes in their livers included elevated glycogen and non-protein-bound sulfhydryl concentrations and glutathione peroxidase activity but lowered protein, total sulfhydryl, and protein-bound sulfhydryl concentrations. Congenital malformations observed grossly in embryos were often multiple and included anophthalmia, microphthalmia, abnormal beaks, amelia, micromelia, ectrodactyly, and hydrocephaly. Mean concentrations of selenium in livers (94.4 ppm, dry weight) and kidneys (96.6 ppm) of birds collected at the Kesterson ponds were about 10 times those found at a nearby control area (8.3 and 12.2 ppm). We conclude that selenium present in the agricultural drainage water supplied to the Kesterson ponds accumulated in the food chain of aquatic birds to toxic concentrations and caused the lesion and other changes observed.

Toxicity of petroleum crude oils and their effect on xenobiotic metabolizing enzyme activities in the chicken embryo in ovo.

Microliter quantities of a Prudhoe Bay crude oil (PBCO) applied to the shell of fertile chick eggs during various stages of development induced cytochrome P-450 levels and mixed-function oxidase activities within the liver of the embryo. PBCO (5 microliter) applied on Day 11 of incubation was found to maximally induce within 24 hr embryo hepatic cytochrome P-450 levels (fourfold), naphthalene hydroxylase (sixfold), benzo[a]pyrene 3-hydroxylase (14-fold), and 7-ethoxyresorufin O-deethylase (24-fold). Glutathione S-transferase was not induced. Crude oils are known to be highly toxic to avian embryos, especially during the early stages of development. The LD50 of PBCO and Hibernia crude oil applied to the egg shell on Day 8 of incubation was found to be 1.3 and 2.2 microliter, respectively. Mixed-function oxidase-dependent metabolism of crude oil components may be required for toxicity since administration of 20 micrograms of disulfiram in dioxane 1 hr prior to application of 1.3 microliter of PBCO reduced embryo mortality from 60 to 20%.

Comparative teratological study of insecticide Wofatox 50 EC (50% methyl-parathion) on chicken and pheasant fetuses.

An experimental insecticide Wofatox 50 EC, injected into the embryonated eggs from hens and pheasants at 12th day of incubation, caused a significantly diminished body mass, a high incidence of developmental malformations and embryonic mortalities at higher dose-levels. The lower, in plant protection practice used concentration was no teratogenic or lethal on embryos.

Review of in vitro systems with potential for use in teratogenicity screening.

In this review an in vitro system is considered to be any developing tissue, organ, or organism other than mammalian embryo in situ. Before the various test systems that have been used or proposed are discussed, attention is given to the questions of whether all chemicals in the environment are in need of biological testing and what criteria should be used in making this judgment. Consideration is also given to selection of the appropriate level of rigorousness to be used in tests of different categories of chemicals. To aid in this, the characteristics of an optimal test are assembled and used as a standard for estimating the potential usefulness of the various in vitro systems. The systems discussed include bacteria and other unicellular organisms, somatic cells in culture, tissue culture, organ culture, intact invertebrate embryos (e.g., drosophila, sea urchins, sand dollars), intact lower vertebrate embryos (e.g., frogs, other amphibians, fish), cultured mammalian embryos, and incubating chick embryos. None of these are regarded as sufficiently validated in terms of comparisons with known teratogenic responses in pregnant mammals to warrant adoption as a reliable test at this time. Intact embryos of drosophila, sea urchins, amphibians, and fish are regarded as promising, but much research is needed to ascertain their predictive validity for mammals. The incubating chick embryo, however, possesses more of the essential features of the optimal system than any of the others. A tentative proposal using the chick is outlined, but it will require considerable further comparison with currently used procedures in pregnant mammals before its reliability can be fully evaluated.