Epigenetic modifiers identified as regulators of food intake in a unique hypophagic chicken model.

DNA methylation is an epigenetic modification that influences gene transcription; however, the effects of methylation-influencing chemicals on appetite are unknown. We evaluated the effects of single administration of a methyl donor, S-Adenosylmethionine (SAM), or methylation inhibitor, 5-Azacytidine (AZA), on immediate and later-age food intake in an anorexic chick model. The doses of intracerebroventricularly-injected SAM were 0 (vehicle), 0.1, 1, and 10 µg, and of AZA were 0 (vehicle), 1, 5, and 25 µg. When injected on day 5 posthatch, there was no effect of SAM on food intake in either fed or fasted chicks, whereas AZA increased food consumption in the fasted state but decreased it in fed chicks. We then performed a single injection (same doses) at hatch and measured food intake on day 5 in response to neuropeptide Y (NPY; 0.2 µg) injection. Irrespective of NPY, chicks injected with 1 µg of SAM ate more than others on day 5. In contrast, chicks injected with AZA (5 and 25 µg doses) consumed less on day 5. In conclusion, we identified DNA methylation-regulating chemicals as regulators of food intake. AZA but not SAM affected food intake in the short-term, feeding state dependently. Later, both chemicals injected on the day of hatch were associated with food intake changes at a later age, suggesting that feeding pathways might be altered through changes in methylation.

Immunological responses to Clostridium perfringens alpha-toxin in two genetically divergent lines of chickens as influenced by major histocompatibility complex genotype.

Chickens genetically selected for low (LA) or high (HA) antibody response to SRBC displayed a correlated change in MHC, so that LA chickens were 96% B13 and HA chickens were 96% B21. The LA line appears to be less susceptible to invasion by extracellular pathogens, whereas HA chickens are more resistant to infection by intracellular organisms. Resistance to Clostridium perfringens is one instance in which the lines do not follow their established trend of pathogen susceptibility, where during a clinical outbreak of necrotic enteritis, B21B21 genotypes experienced significantly less mortality than B13B13 genotypes. A study was carried out to assess immunological differences between LA and HA lines during exposure to C. perfringens α-toxin. Peripheral blood mononuclear cells were isolated from each genetic line, cultured with or without lipopolysaccharide (4 h), and exposed to varying concentrations of α-toxin (1; 10; 100; and 1,000 U/L) for 2 and 4 h. Evaluation of cellular proliferation, percentage of cytotoxicity, and immunological gene expression was carried out in a series of experiments. Cells isolated from HA chickens had significantly increased proliferation than those from LA chickens at low toxin levels (1 and 10 U/L) and significantly decreased proliferation at high toxin levels (100 and 1,000 U/L). Following exposure to lipopolysaccharide, the percentage of cytotoxicity was higher for LA than HA cells. In both assays, HA cells displayed superior performance following lipopolysaccharide-stimulation. Gene expression analysis of immune transcripts by quantitative real-time PCR revealed significantly upregulated expression of interferon (IFN)-γ, interleukin (IL)-8, IL-13 (2 h), IL-15, and CXCLi1 (4 h) in HA than LA chickens. Cells isolated from the LA line displayed significantly elevated expression of IL-2, IL-10, IL-13 (4 h), IL-16, IL-18, inducible nitric oxide synthase (iNOS), CXCLi1 (2 h), and lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF) compared with the HA line. Clearly, these 2 genetic lines display highly divergent immune responses in regards to C. perfringens toxin exposure.

Differential appetite-related responses to central neuropeptide S in lines of chickens divergently selected for low or high body weight.

The anorexigenic 20 amino acid neuropeptide S (NPS) has not been studied in an animal model of hypo- or hyperphagia. The present study aimed to elucidate whether central NPS appetite-related effects are different in lines of chickens that had undergone long-term divergent selection for low (LWS) or high (HWS) body weight and that were hypo- and hyperphagic, respectively. It took a longer time for food intake to be reduced in LWS than HWS chicks administered the lowest dose of NPS tested (0.14 nmol) and, at the highest dose tested (0.56 nmol), they had a greater reduction in food intake than did HWS chicks. HWS chicks responded with a similar magnitude of food intake reduction that was independent of NPS dose. Although water intake was reduced concurrently with food intake after central NPS in both lines, blood glucose concentrations were not affected. Hypothalamic signalling was different between the lines. Although both lines respond to central NPS with decreased c-Fos immunoreactivity in the lateral hypothalamus, the periventricular nucleus had increased c-Fos immunoreactivity in LWS but not HWS chicks. After central NPS treatment, there was increased c-Fos immunoreactivity in the paraventricular nucleus in HWS but not LWS chicks. These data support the notion of differences in the central NPS system between the LWS and HWS lines and infer that central NPS may differentially affect appetite-related processes in other species that contain hypo- and hyperphagic individuals.

Mode of inheritance of unselected traits in lines of chickens selected for high or low antibody response to sheep red blood cells. 1. Resistance to marble spleen disease virus and juvenile body weight.

Two lines of White Leghorns that had undergone long-term selection for high (HH) or low (LL) antibody response to sheep red blood cell antigen(s) formed the nuclear lines for this experiment. Matings were made in a full diallel cross to produce in a single hatch from age-contemporary breeders the parental lines, reciprocal F1 and F2 crosses, and backcrosses for 16 progeny types. For males and females, there were parental line differences in BW to 42 d of age, after which there was decline between lines for males. Differences in BW between reciprocal F1 crosses and maternal heterosis declined with age, primarily reflecting dissipation of effects of egg weight. Heterosis of BW was dependent on the particular F1 cross and recombination effects were not important. At 50 d of age chicks were inoculated with either a 1 or 10% suspension of spleen extract from chickens infected with marble spleen disease virus (MSDV). A third group served as uninjected controls. Response to MSDV was evaluated by spleen weight 6 d after inoculation. Spleen weights relative to BW of control chicks were heavier for the HH than LL line with evidence from the crosses of sexlinkage and negative heterosis. Line LL chicks were more resistant to MSDV than Line HH chicks was F1 crosses intermediate to and different from either parental line with no evidence of heterosis.

Effect of tumor necrosis factor-alpha on progesterone production by granulosa cells in laying hens of different genetic lines.

In vitro progesterone production by granulosa cells in the presence or absence of human recombinant tumor necrosis factor-alpha (hrTNF-alpha) was measured at 10, 20, and 30 wk of egg production in White Leghorn hens selected for high (HA)- or low-antibody (LA) response to sheep red blood cell challenge. Isolated granulosa cells from the three largest preovulatory follicles (F1-F3) were incubated with 5 or 250 ng/ml hrTNF-alpha, and progesterone production was determined by the use of a validated radioimmunoassay. F1, F2 and F3 granulosa cells from HA hens produced more (P < or = 0.05) progesterone (140.8, 107.2, and 49.7 ng/ml) than LA hens (109.4, 78.9, and 26.9 ng/ml). The treatment of granulosa cells with hrTNF-alpha consistently inhibited (P < or = 0.05) progesterone secretion by all follicles among HA and LA hens, but not always at both doses. Generally, 5 ng/ml hrTNF-alpha was the maximum inhibitory dose. In the laying hen, a decrease in steroid production in response to cytokines may upset the steroid balance created by follicular hierarchy and inhibit or delay ovulation.

Intubation of weight-selected chicks with soybean oil or residual yolk: effect on early growth and development.

Experiments were conducted to study the role of residual yolk on growth and development of chicks during the 1st wk after hatch. Surgical removal of the yolk sac at hatch reduced early body weight gains. Intubating chicks with a mixture of soybean meal and soybean oil or with residual yolk obtained from chicks at hatch increased body weights and enhanced development of the liver and pancreas. Whereas intubation with residual yolk reduced absorption of yolk sac contents, absorption was similar for chicks that consumed mash ad libitum or were intubated with the soybean meal-soybean oil diet. After cessation of yolk intubation, absorption of residual yolk resumed. Removal of the yolk sac decreased amylase and lipase activity in the pancreas. In the intestinal chyme, activity of amylase, trypsin, and chymotrypsin was reduced by yolk sac removal. Intubation with yolk increased enzyme activity (amylase excepted) in the pancreas or intestinal chyme only in chicks that had their yolk sacs removed. There was evidence that contents of the yolk sac did not enter the intestine via the yolk stalk, but were absorbed directly via the blood.

Removal of residual yolk at hatch influences food choice and feeding activity in lines of chickens selected for high or low juvenile body weight.

The effect of residual yolk removal at hatch on growth and feeding behavior was assessed in lines of chickens divergently selected for low (LW) or high (HW) 56-day body weight. At hatch chicks were assigned to 1 treatment: removal of the residual yolk (R) or no surgery (I). Twenty-four chicks per line (12 I and 12 R) were assigned to an all-mash regimen (M) and 48 per line (24 I and 24 R) to a choice (C) between mash and dietary residual yolk. HW were heavier than LW chicks regardless of treatment. On day 5 and thereafter, chicks given a choice were heavier than those eating mash. HW chicks ate more than LW ones. The percentage of yolk eaten increased during the 2-3 d after hatch, remained constant for 2 d, then decreased. Results are discussed in term of yolk need and development of the gastrointestinal tract in the divergent lines.

Effect of yolk sac removal at hatch on growth and feeding behavior in lines of chickens differing in body weight.

Influence of residual yolk on growth, feeding behavior, and development of certain internal organs during the first week after hatch were measured in lines of chickens that had undergone long-term selection for high (HW) or low (LW) 56-day body weight. At hatch chicks were assigned to one of the following treatments: removal of residual yolk (Y), sham operated (S), or no surgery (I). Body weight gains of HW chicks began on day 1 while LW chicks did not begin to gain weight until several days after hatch. Food intake increased linearly in both lines, but at a higher rate for HW than LW chicks. Seven days after hatch, relative to body weight, shanks were longer and pancreases and small intestines heavier for HW than LW chicks. Among treatments, body weight gains were greater for I and S than for Y chicks in the HW line, but not the LW line, demonstrating a differential response by fast and slow growing chicks to removal of the yolk sac. Feed intake differed on day 3, with Y chicks eating less than S chicks; I chicks were intermediate. Results indicate that presence of residual yolk sac during the first three days after hatch are critical to growth and development of chicks.

Performance of early and late feathering broiler breeder females with different feeding regimens.

1. Growth, reproductive characteristics and immunocompetence were evaluated in a line of broiler breeder females segregating at the sex-linked feathering locus when maintained on three feeding regimens with two forms of food in a factorial design. 2. Long-term obesity but not short-term weight gain had deleterious consequences for reproductive characteristics, response to sheep red blood cell antigen, resistance to Escherichia coli and livability. 3. Reproductive performance of early feathering females was superior to that of late feathering ones. Differences were attributed to an association between the allele for late feathering and an endogenous viral locus ev21, which encodes for avian leucosis virus. 4. Reproductive performance of breeders fed crumbles was superior to that of breeders fed a combination of crumbles and fines.

Genetic and environmental effects on the response of chickens to avian adenovirus group II infection.

The effects of husbandry procedures on an intravenous challenge with avian adenovirus group II were studied in lines of White Leghorns selected for high (HH) and low (LL) antibody response to sheep erythrocytes and their reciprocal crosses (HL and LH). Husbandry procedures were deprivation or no deprivation of water during the first week after hatching (ES), habituation or no habituation to humans throughout life, and flock stability or instability (LS) 24 hours prior to an intravenous challenge with avian adenovirus group II. Spleens of chickens were weighed 6 days after challenge. HL chicks were most susceptible and LH chicks were most resistant to avian adenovirus group II. Chicks subjected only to ES or no stress responded similarly and were less affected than those subjected to LS or to both ES and LS. There were major genetic-environment interactions which affected spleen size.

Cellular defense of the avian respiratory tract: paucity of free-residing macrophages in the normal chicken.

Avian respiratory macrophages (ARM) were obtained from lungs and air sacs of 122 White Plymouth Rock chickens, ranging from 376 to 3800 g in weight. Procedures involved lavaging through the surgically prepared trachea with either a 15-g cannula or French #8 pediatric urinary catheters. Factors, in different combinations, investigated for their effects on the ARM yield, were: lavage fluids (0.85% physiologic saline, 0.1 M phosphate-buffered saline, Ca-Mg-free Hanks' solution, Eagle's minimum essential medium); additives (10 U heparin/ml, 0.1% EDTA, 12 mM lidocaine); lavage repetitions (from 3 to 10); fluid temperature (room and 41 C); and lavage time (fluid retention up to 35 min). None of the lavage methods emerged clearly as the best, with phosphate-buffered saline and 0.85% physiologic saline alone as good as when combined with additives. Although 10 lavages yielded more ARM, it appeared that the majority of ARM washed off into the early lavages. Chickens from a line selected for large body size had more ARM than those from a line selected for small body weight. Regardless of genetic line, however, the chickens yielded a very low number of ARM compared with mammalian species of the same or smaller weight. Most of the birds yielded only 200,000 to 300,000 ARM, with minimum yields being less than 10,000, the maximum being 2 million ARM. Either these results point to a deficiency in the defense system of the chicken's respiratory tract against bacteria, mycoplasma, fungi, and viruses, or mechanisms other than macrophages are primary in resistance to pathogens.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection for body weight at eight weeks of age. 17. Overfeeding.

Female chicks from lines selected for high and low body weight and their reciprocal F1 crosses were overfed via force feeding to crop capacity from hatching to 21 days of age. Low-weight line chicks could be overfed at an earlier age to a greater extent than those from the high-weight line and F1 progeny. The degree of overfeeding appeared to be associated with the relative size of certain gastrointestinal components. Differences among lines in their capacity to be overfed were reflected in concomitant changes in body weight and carcass fat. Relationships among feed consumption and growth are discussed within the context of selection for body weight and heterosis under various feeding regimens.

In vivo aromatization of [3H]testosterone in high and low mating lines of Japanese quail.

Japanese quail selected bidirectionally for adult mating frequency were utilized to study in vivo aromatization of testosterone (T) in relation to masculine copulatory behavior. Functionally castrated high (HM) and low mating (LM) line quail were injected with 75 microCi of [3H]T. One hour after the injection, all radioactivity recovered in telencephalic-diencephalic brain tissue was in the form of T, dihydrotestosterone (DHT), or estradiol (E2). Neither the total 3H nor the [3H]T metabolite radioactivity differed between the two genetic lines. Of all [3H]T metabolic radioactivity, [3H]E2 represented 45 +/- 6 % in the HM line and 46 +/- 6% in the LM line, indicating that the line difference in mating frequency was not due to a corresponding difference in aromatase activity. Inasmuch as both the HM and LM line birds actively converted T to E2, these results implicate a neural mechanism involving E2-receptor interactions as the cause of the behavioral differences between the HM and LM lines.

Testosterone aromatization in high and low mating lines of gallinaceous birds.

Capons from high (HML) and low (LML) mating lines of Japanese quail and chickens received testosterone propionate, 5 alpha-dihydrotestosterone (5 alpha-DHT) or estradiol benzoate injections after which copulatory behavior was observed during exposure to live females and to a female model. With live females, the testosterone-treated HML capons mated significantly more than the other HML groups. All LML capons mated infrequently and at comparable levels. When tested with the model, estrogen-treated LML capons mated significantly more frequently than testosterone-treated LML capons, suggesting testosterone aromatization rate was affecting mating activity. This possibility was tested with HLM and LML intact cocks and capons receiving silastic implants of either testosterone, 5 alpha-DHT or a combination of estradiol and 5 alpha-DHT (E + DHT). Mating activity of intact HML cocks, T-treated, and E + DHT-treated HML capons were similar, and all groups mated significantly more than the control or 5 alpha-DHT-treated capons. There were no significant differences between any of the LML groups. The data suggested that limited testosterone aromatization was not the cause of relatively reduced sexual activity in the LML males.

The effects of exogenous steroidal hormones on the testes of quail from selected high and low mating lines.

Several steroid hormones were tested for their effects on the testes of lines of Japanese quail selected for high and low mating frequency. Estradiol benzoate and testosterone propionate caused a complete and partial regression of the testes, respectively, while dihydrotestosterone did not affect testicular weight. Spermatogenesis was adversely affected by both estradiol benzoate and testosterone propionate but not by dihydrotestosterone. Dihydrotestosterone caused a significant increase in the diameter of the seminiferous tubules while estradiol benzoate and testosterone propionate caused a significant decrease in tubular diameter. Significant line differences were found; the high mating line exceeded the low mating line for each of the treatments except extradiol benzoate. Extradiol benzoate caused testicular regression in both lines.

Cell-mediated immunity in Marek's disease virus-infected chickens genetically selected for high and low concentrations of plasma corticosterone.

Lines of chickens genetically selected for high (HPC)-and low-plasma corticosterone (LPC) titers were assessed for cell-mediated immunity during Marek's disease tumorigenesis. Lymphocyte transformation (using phytohemagglutinin and 2,4-dinitrofluorobenzene-bovine serum complex) and mitogen-induced cellular cytotoxicity were evaluated with peripheral blood leukocytes. Chickens from the LPC line showed a greater lymphocyte transformation response than those from the HPC line. All tumor-bearing chickens demonstrated a markedly suppressed blastogenic response. Although the blastogenic response was suppressed in those chickens infected with Marek's disease virus but not having gross tumors when compared with the steroid level, this response was greater than that of chickens with tumors and less than that of normal controls. In comparison with LPC-line chickens, HPC-line chickens demonstrated a decreased mitogen-induced cellular cytotoxicity. A decreased mitogen-induced cellular cytotoxicity was observed in both steroid lines concomitant with the onset and severity of tumors. The impaired cell-mediated response observed in the HPC line was associated with an increased tumor incidence and a greater mortality than that observed in the LPC line. Metyrapone, a chemical which blocks adrenal corticosterone synthesis, transiently enhanced mitogen-induced cellular cytotoxicity and initiated tumor regression in chickens debilitated with Marek's disease tumors.

Ultrastructural observations of the adenohypophyseal gonadotrophs from growth selected lines of chickens.

The ultrastructure of adenohypophyseal gonadotrophs (GT-cells) from pullets of lines selected for high (HWS) and low (LWS) body weight was studied at various physiological ages. Increasing the photoperiod beyond 14 hours in a 24-hour cycle did not have a significant effect on the ultrastructure of GT-cells in either line. A qualitative analysis, based on the extent and complexity of the protein synthetic apparatus, suggested that the synthetic activity of gonadotrophs from HWS pullets was greater than that of LWS pullets at each of the ages studied. Although no significant differences were found in the frequency of pullets showing evidence of intracellular digestion of secretory products, the condition was more severe in the HWS than in the LWS females. Crinophagy tended to be positively correlated with cell activity in both lines. These observations suggest that selection for growth rate had a correlated effect on hypophyseal proteinaceous secretions. They also indicate that interactions between fluctuations in gonadotrophic cell activity, secreting mechanisms and neural thresholds for hormone action may be implicated in the manifestation of the erratic ovulation patterns.