Endocrine disrupting chemicals (EDCs) are compounds that alter the production, secretion, action, and elimination of endogenous hormones. In the environment, organisms are exposed to many different types of EDCs at any one time, each with different mechanisms of action, many of which are not fully understood at present. Most research done with EDCs has focused on the effects of these chemicals on the estrogen and thyroid systems, however, many of these same chemicals also exert strong effects on the androgen system. Also, many studies assessing the effects of EDCs on wildlife have focused on reproductive measures of exposure, often overlooking potential effects on the immune system. We have demonstrated that embryonic exposure to androgen-active EDCs, anti-androgenic p,p'-DDE and androgenic trenbolone acetate, impairs the development and function of both the immune and reproductive systems in Japanese quail. The most sensitive measured immunological target of androgen-active EDCs was the bursa of Fabricius, the primary immune organ responsible for the development of the humoral part of the immune system. Specifically, embryonic development the bursa is extremely sensitive to androgen-active EDC exposure, providing a possible mechanism for EDC-induced immunosuppression. We have also demonstrated that the bursa in Japanese quail can be resilient to embryonic exposure to EDCs, if post-hatch exposure to these chemicals is prevented. Rates to reproductive maturation and copulatory behavior were the most sensitive reproductive measures of exposure. Male and female rates to sexual maturity were altered by the one-time in ovo exposure to DDE and trenbolone. Male reproductive behavior, as measured by attempts to mount and successful cloacal contacts, was suppressed by both chemicals. The myriad of endpoints used in this study has been compiled to provide toxicologists with a list of sensitive and persistent measures that can be used as reliable biomarkers of exposure to androgen-active EDCs in birds.

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In this experiment, 728 blood samples were collected from 22 breeds (28 populations) of native Japanese chickens that have been designated as Natural Monuments of Japan [Chabo, Gifu-Jidori (2 populations), Hinai-dori, Jitokko, Kinpa, Koeyoshi, Kuro-Kashiwa, Ko-Shamo (2 populations), Kawachi-Yakko, Minohiki-dori, Mie-Jidori, Ohiki, Onaga-dori, Oh-Shamo (2 populations), Satsuma-dori, Shoukoku (2 populations), Tosa-Jidori, Toumaru, Toutenkou, Ukokkei (2 populations), Uzurao and Yakido (2 populations)] and two foreign breeds (White Leghorn and Rhode Island Red) to examine the genetic variability and relationships by using microsatellite DNA polymorphisms. Genetic variabilities in 20 microsatellites examined varied depending on the populations. The mean number of alleles per locus, the proportion of the polymorphic loci, and the expected average heterozygosity ranged from 1.75 to 4.70, from 0.55 to 1.00, and from 0.21 to 0.67, respectively. Microsatellite alleles, being unique to a particular population, were detected in some breeds/populations. A phylogenetic tree was constructed based on the D_A genetic distance and using the neighbour-joining method. Using the tree, genetic relationships were examined between breeds/populations. Some were in accordance with earlier hypotheses, and some did not support earlier hypotheses.

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Considering the importance of fluoroquinolones in animal health and the fact that the enrofloxacin patent has expired, a bioequivalence study of seventeen preparations of enrofloxacin vs. the reference preparation (Baytril®) in broilers was carried out. Oral pharmacokinetics of seventeen commercial enrofloxacin preparations and Baytril as reference preparation were assessed in five trials in a total of 1760 broilers. Ten mg/kg of each preparation was administered to a total of 80 clinically healthy 3 weeks old broilers. After drug administration, blood was collected 8 times from ten birds within 24h. Using a microbiological agar diffusion analysis, serum concentration of enrofloxacin and metabolites were determined by correlating the composite antibacterial activity of enrofloxacin and metabolites with actual concentration. Bioequivalence limits were set between 80 to 125% reference values for maximum serum concentration (Cs_max) and area under the curve (AUC). Results display mean Baytril® values of Cs_max=1.662μg/ml±0.034 ; AUC=4.073±0.814μg/ml/hr. Only three preparations out of seventeen could be regarded as bioequivalent while the rest gave AUC and/or Cs_max values smaller than the accepted lower limit of the mean values obtained for the reference enrofloxacin (P<0.05). The use of non-bioequivalent preparations of enrofloxacin will consequently undermine clinical outcomes and could play a significant role in accelerating the emergence of bacterial resistance in the poultry industry.

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One thousand two hundred and fifty sexed 21 day-old commercial broilers (Ross-308) were randomly divided into five dietary treatment groups of 250 broilers each. Each treatment group was further sub-divided into five replicates of 50 broilers (25 male and 25 female) per replicate. The treatments groups were control ; low phosphorus ; low phosphorus plus 500FTU phytase/kg diet ; low phosphorus, energy, protein ; and low phosphorus, energy, protein plus 500FTU phytase/kg diet. There were significant effects of dietary treatments on body weight, body weight gain, feed intake and feed conversion ratio at 21 to 42 days. The body weight and the body weight gain of the broilers fed the control and low phosphorus plus phytase diet were heavier than other treatment. Feed intake of broilers was not affected by the supplementation of phytase to the low phosphorus diet at 21 to 42 days. Feed conversion ratio of broiler fed on low phosphorus, energy and protein diet plus phytase was significantly better than that of broilers fed on low phosphorus, energy and protein diet. Neither phytase supplementation nor diet nutrient density and dietary phosphorus level had a significant effect on broiler mortality. The percentage of tibia ash and phosphorus was significantly increased by the addition of microbial phytase to low phosphorus, energy and protein diet. This study demonstrates that microbial phytase can compensate for reduced available phosphorus levels, but could not compensate for reduced dietary protein and energy.

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Growth hormone secretagogues (GHS), including ghrelin, are known to stimulate feeding behavior in mammals. In contrast, intracerebroventricular (ICV) injection of rat or chicken ghrelin, strongly inhibits food intake in chicks. The difference prompted us to compare the effect of growth hormone releasing peptide-6 (GHRP-6), one of synthetic GHSs on feeding behavior of chicks with that of ghrelin. Gastric motility stimulating action of GHRP-6 was also examined in vivo measuring retention of food in digestive tracts. ICV injection of GHRP-6 (0.38nmol-1.5nmol) dose-dependently inhibited food intake of neonatal chicks under both ad libitum and fasting conditions. The anorexic effect of GHRP-6 was attenuated by D-Lys³-GHRP-6 (GHS receptor antagonist) in a dose dependent manner. Central injection of GHS agonist and/or antagonist did not affect food retention in crop, proventriculus and gizzard. These results suggest that central injection of synthetic GHS, GHRP-6 inhibits feeding behavior as dose ghrelin without affecting gastrointestinal motility in layer chicks.

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Effect of feeding astaxanthin (Ax) provided as Phaffia rhodozyma on T cell population and responsiveness to mitogen, concanavalin A (Con A) or phytohemaggulutinin (PHA), of splenic mononuclear cells (MNC) was determined in male broiler chicks. Chicks at1 week of age, were fed ad libitum on diets containing 0, 20 or 100ppm Ax for 7 days. Feeding the diet containing Ax decreased percentage of CD3-positive cells, an indicator of the T cell population in splenic lymphocytes. Feeding the diet containing 20ppm Ax decreased percentage of CD4-positive cells estimated as the indicator of helper T cells, but no modulation was observed by feeding the diet containing100ppm Ax. Percentage of CD8-positive cells, an indicator of cytotoxic T cell, was not affected by feeding diets containing either 20 or 100ppm Ax. T cell proliferation stimulated with Con A was enhanced by feeding diet containing 20ppm Ax, but not affected by 100ppm Ax. Feeding diets containing 20 and 100ppm Ax enhanced the proliferation of splenocytes against PHA. Feeding diets containing Ax showed no effect on interleukin(IL)-2mRNA expression in splenocytes stimulated by Con A estimated as the T cell proliferation promoting factor. IL-2 like activity was slightly, but significantly, decreased by feeding diet containing 20ppm Ax. IFN-γmRNA expression, estimated as a T cell activator under condition of Con A stimulation, was higher in chicks fed the diet containing 20ppm Ax. These results suggest that proliferation and cytokine expressions of splenic T cells in response to feeding Phaffia rhodozyma containing Ax were independent of the dietary Ax concentration or total Ax intake. The enhancement of T cell proliferation by feeding 20ppm Ax might be caused by changes in IFN-γ rather than IL-2 production in the chick splenocytes.

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The effect of quail semen dilution with three extenders : Lake's, Ringer's and Tyrod's on morphology and fertility trials of unstored spermatozoa was evaluated. Semen was collected twice a week by male stimulation by female method, from 60 quail males randomly divided into four groups (I-IV). Forty-eight females divided into four respective groups (A-D) were inseminated intravaginally with 10μl of fresh or 20μl of 2-folds diluted semen. Semen dilution did not affect the number of live spermatozoa in total, but the number of live, morphologically normal spermatozoa depended on the diluent. The highest number of morphologically normal spermatozoa -65.3% vs. 67.5% in fresh semen was stated in semen diluted with Lake's extender. It was significantly higher (P≤0.01) than in semen diluted with Ringer's diluent (56.2%). Egg fertility (99.2%), hatchability from set (91.2%) and fertile eggs (93.9%) in naturally mated group were significantly higher (P≤0.01) when comparing with females inseminated artificially both, with fresh and diluted semen. The highest fertility (60.1%) was obtained for spermatozoa diluted with Lake's extender, the lowest for spermatozoa treated with Ringer's solution (30.2%).

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The effect of proctodeal gland foam addition to fresh net or diluted semen supplemented with (DMA) dimethylacetamide, on morphology and fertilising potency of quail spermatozoa was evaluated. Every stage of quail semen preparation caused an increase in sperm deformations. The addition of foam, diluent and DMA decreased the number of morphologically intact spermatozoa by 28.3 percent points, in relation to the fresh semen. Foam addition to the fresh semen allowed to obtain 91.4% fertility, which was similar to results obtained during natural mating (93.0%), but in diluted and DMA supplemented semen, equilibrated for 5 minutes at 4°C, the foam had a detrimental effect on fertilising ability of spermatozoa.

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This is the first successful report on producing the endangered domestic fowl, Kureko Dori (KD), which is a Kumamoto prefectural natural treasure, using germline chimeras transplanted from primordial germ cells (PGCs) in practice. The attempt was made to produce germline chimeras between KD fowl, as a model of rare and/or endangered poultry, and common White Leghorn (WL) for the proliferation of KD individuals. The PGCs of KD were collected from embryonic blood at stages 13 to 16 (H & H) and preserved in liquid nitrogen until injection. A certain number of frozen-thawed PGCs were picked up under a phase contrast microscope, and injected into the peripheral vein of WL embryos (common poultry) at stages 12 to 14. We obtained one male and 6 females as the putative germline chimera, they were raised until sexual maturity, and the progeny test was performed by mating them with KD of the opposite sex to analyze the efficiency of germline chimera. The average rate of offspring originating from transplanted PGCs of KD was only 1.4%, and the maximum was 5.3% from all 577. These KD offspring derived from the KD-WL germline chimeras also achieved fertility as normal. These results show that rare and/or endangered birds can proliferate through producing germline chimeras between the same species of bird.

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The aim of this study was to examine the effects of diethylstilbestrol (DES) administration on sperm penetration into the inner perivitelline layer (IPL) of ovum, and the interaction between sperm and ovum in Japanese quail (Coturnix japonica). DES was used as a model of endocrine disrupters in this experiment. We employed the in vitro penetration test, and the in vitro competition assay which was used to detect the interaction between sperm and ovum. Male and female quail were intramuscularly injected with 1mg of DES for 3 days. When sperm and IPL derived from DES-treated quail were incubated with untreated IPL and untreated sperm, respectively, the number of holes formed in the IPL resulting from sperm penetration were 42.5±5.3 and 48.3±6.2 per 1mm². The values were significantly lower than that of control group without DES treatment (119.1±2.7). In a series of assay from pre-incubation of untreated chicken sperm (cS) with DES-treated solubilized qIPL to co-incubation with a sheet of untreated cIPL, the number of holes in the untreated cIPL was 107.3±46, whereas the number of holes in the untreated cIPL was significantly reduced to 25.5±2.3 when cS was pre-incubated with untreated solubilized qIPL. These results suggest that administration of DES into male and female quail caused an impairment of penetrability of sperm and receptivity of ovum to sperm, and thereby resulted in the disorder of the fertilization system in quail.

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Incubating, laying, and nonlaying Gifujidori hens were restrained on a board, and their plasma corticosterone concentration was measured by radioimmunoassay. The corticosterone concentration was observed to increase within 5min after the onset of restraint. The degree of the increase was greater in the incubating hens than in the laying and nonlaying hens. These results suggest that stress condition of incubating hens are thought to be high than laying and nonlaying hens before giving stress and after having given stress.

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The aim of the study was to evaluate the growth rate and post mortem values of two groups of White Koluda® goslings. Each group consisted of 5 males and 5 females. The first group originated from geese inseminated with fresh semen, the second group of goslings obtained from geese of the same breed, inseminated with frozen-thawed semen. Semen was collected from 10 White Koluda® ganders. Part of semen was used once a week for fresh semen insemination, the remained ones was frozen and used after thawing for insemination twice a week. Body weight at the Day 1, 21, 35, 42, 56, 70, 84 and 105 (day of slaughter) and growth rates were examined. At post-mortem analysis there were evaluated (in g and % in relation to live body weight) : eviscerated carcass with neck, heart, liver, gizzard, edible giblets, breast muscles with skin, legs with skin, wings with skin and remainder of carcass with skin. Character of semen (fresh or frozen-thawed) do not affect the quality of progeny resulted from insemination. There were no significant differences in goslings obtained after fresh or frozen-thawed semen insemination with regard to the growth rate, final body weight (5140g- for fresh and 4941g- for frozen-thawed semen) and slaughter values (slaughter yeld : 69 vs. 72% ; edible giblets : 373.9 vs. 371.5g ; breast muscles : 762.0 vs. 682.3g).

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Wrong:Han Y and Baker DH. Lysine requirements of fast and slow-growing broiler chicks. Poultry Science, 73 : 1739-1745. 1991.
Right:Poultry Science, 70 : 2108-2114. 1991.

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Wrong:Mather CM and Laughlin KF. Storage of hatching eggs : The effect on early embryonic deve-lopment. British Poultry Science, 17 : 471-479. 1977.
Right:British Poultry Science, 18 : 597-603. 1977.

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