Epitopes for chicken monoclonal antibodies in spleens of selected Japanese quail lines.

A line of Japanese quail selected for high plasma cholesterol is highly susceptible to diet-induced atherosclerosis. Lymphocyte epitopes recognized by mouse anti-chicken monoclonal antibodies (c-mAb), TCR-1, TCR-2, TCR-3. CD-3, CD-4, CD-8, and BU-1a/b were reacted with spleens from quail selected for high (HL) and low (LL) plasma total cholesterol and their nonselected controls (CL). Cross reactivity to c-mAb and effect of line and gender were immunohistochemically evaluated. Chicken spleens were positive controls. Quail were immunologically stimulated with either sheep red blood cells (SRBC) or Brucella abortus 2 weeks before spleens were removed. Quail spleen epitopes of all lines recognized TCR-3 and CD-8 c-mAb, but no other c-mAb. Number of reacting cells and staining intensity to the TCR-3 c-mAb were greater in the HL than in the LL regardless of the stimulating Ag or dose used. For the CD-8 c-mAb, there were no differences among lines in birds receiving SRBC. In B. abortus-immunized birds, sex x line interactions indicated that males of the HL and CL had lower responses than females but LL males were not different than females. TCR-3 and CD8 c-mAb may be useful in studying immunological mechanisms for atherosclerosis in Japanese quail.

Effect of genetic selection and MHC haplotypes on lymphocyte proliferation and interleukin-2 like activity in chicken lines selected for high and low antibody production against sheep red blood cells.

Chickens from third generation matings of lines of chickens selected for high (HA) and low (LA) antibody production to sheep red blood cells (SRBC) and typed for MHC genotypes B13/13, B13/21, and B21/21 were used in this study. Chickens from both lines carried all the three genotypes B13/13, B13/21, and B21/21. To study T- and B-lymphocytes mitogenic activity, 12-week-old female chickens were injected intravenously with 0.2 ml of 9% SRBC and spleens were collected at 0, 6 h, and 6 day post-antigen injection (pAg). Isolated lymphocytes were incubated with either Concanavalin-A (Con-A) for T-cell activity, or Pokeweed mitogen (PWM) for B-cell activity and thymidine 3H uptakes were measured. To study the Interleukin-2 (IL-2)-like activity in the same lines and genotypes, splenic lymphocytes from 12-week-old chickens were passed through nylon wool columns to enrich the T-cell population. After a 24 h incubation with Con-A, the conditioned media (CM) were collected. The CM were tested for IL-2 like activity by determining whether they altered the proliferation of Con-A stimulated T cells. This proliferation effect was then compared to that of a reference conditioned media (RCM) prepared from K-strain birds and that were used as the standard for the assay. There was no significant difference (p > 0.05) in IL-2 like activity between HA and LA lines, however, the LA was significantly higher than HA (p < 0.05) in T- and B-cell mitogenic activity. The genotype B13/13 had significantly higher (p < 0.05) IL-2 like activity than the B21/21. The genotype B13/13 was also significantly higher (p < 0.05) in T- and B-cell mitogenic activity than the B21/21. At 0 h, pAg T- and B-mitogenic activity was significantly higher (p < 0.05) than 6 h. In summary, our results indicate that although the birds were selected for high antibody production to SRBC, their lymphocyte mitogenic activity was lower than those selected for low antibody production. Hence, humoral and cell-mediated immune responses appear to be under different genetic controls, and that selection for greater humoral response may be at the expense of cellular responses. Our results also suggest differences in IL-2 like activity production between chickens carrying different MHC B-haplotypes, and that genetic control of such activity is possibly linked to the MHC genes.

Impact of cage density on pullet performance and blood parameters of stress.

The effects of cage density on pullet live performance and blood indices of stress were evaluated in two commercial White Leghorn strains housed at 38, 32, 26, and 20 birds per cage from Day 1 to 6 wk of age, and 19, 16, 13, and 10 birds per cage from 6 to 18 wk. Cage densities of 26 and 13 birds per cage represent a U.S. standard of 142 and 284 cm2 per bird that is often applied in commercial pullet rearing. Cage density treatments include confounding cage, feeder, and drinker spaces per bird as might be encountered in commercial practice when growing more pullets per cage. Body weight was significantly reduced at greater bird densities in both strains; however, one strain was affected as early as 6 wk of age, whereas in the other strain, body weight was not reduced significantly until 18 wk. Feed intake was increased by more than 13% in both strains at the lowest density treatment (20 birds per cage) from Day 1 to 2 wk but reduced by more than 9% by higher cage densities during the remainder of the study. Feed conversion (FC) ratio was similarly improved (i.e., reduced) when more pullets were housed per cage, and increased when fewer birds were placed per cage compared to the standard. Despite a significant reduction in feed intake and a corresponding loss of body weight, cage density treatments had no significant affect on hemagglutinin titers to sheep red blood cell antigen, percentage heterophils (H), lymphocytes (L), or the H:L ratio. However, pullet age and strain differences were observed for all blood parameters. Overall, treatments allowing more cage, feeder, and drinker spaces per bird than the U.S. commercial standard provided no body weight advantage, and allowed for greater feed intake and poorer FC at several ages.

Total cholesterol, total triglycerides, and cholesterol distribution among lipoproteins as predictors of atherosclerosis in selected lines of Japanese quail.

The proportions of plasma high and low density lipoprotein cholesterol have been linked to inherited tendency for atherosclerosis in humans. Studies were conducted with Japanese quail males from lines genetically selected for high and low TC and a randombred (unselected) control line that were fed 0.0 or 0.5% cholesterol for 12 weeks. Atherosclerotic plaques were more severe in the high than in the low line quail and in those fed cholesterol compared to non-cholesterol-fed quail. Serum TG, TC, VLDLC, LDLC, and HDLC were also higher in the high than in the low line quail and in cholesterol-fed vs. non-cholesterol-fed quail. Significant interactions indicated that TC and LDLC concentrations were more affected by dietary cholesterol in the high line than in the low line. The low line quail maintained higher HDLC and lower LDLC than the high line. Regression and correlation analyses revealed that although VLDLC, LDLC, and TC were significant predictors of atherosclerosis in the high line birds, the TC/HDLC ratio was a better predictor in the low line. The Japanese quail lines used herein represent useful experimental models for studies of genetic differences in atherosclerosis in humans.

Dietary cholesterol metabolism in Japanese quail lines selected for plasma cholesterol levels.

Dietary cholesterol metabolism was studied, using a single dose of emulsion, per os (test meal), in lines of Japanese quail that were divergently selected for high (HL) and low (LL) plasma total cholesterol. The meal contained [3H] cholesterol, [14C] beta-sitosterol, unlabeled cholesterol, triolein, and bile salt. Recovery of the nonabsorbable beta-sitosterol in the excreta permitted determination of the percentage of cholesterol absorbed. The amounts of [3H] in the plasma, egg yolks, and the excreta neutral and acid sterols were determined. A line-x-time interaction for [3H] in plasma indicated that the level of plasma cholesterol derived from the test meal declined more rapidly in the LL than in the HL. The higher [3H] detected in the excreta acidic sterols of the LL 12 hr after the test meal indicated that bile acid excretion of cholesterol was greater in the LL than in the HL. There were no differences in cholesterol absorption between lines or sexes. Cumulative [3H] radioactivity in the eggs over 18 days following the test meal was higher in the HL yolks; however, this line effect was due to the greater number of eggs produced by the HL. Thus, one of the mechanisms by which the LL maintains low plasma cholesterol levels is by an enhanced excretion of bile acid compared with the HL. The data also suggest that the more severe atherogenic effect of dietary cholesterol observed in the HL could be, in part, due to the longer residence time of cholesterol in circulation.

Changes in blood and spleen lymphocyte populations following antigen challenge in immature male chickens.

1. The effects of antigen (Ag) injection on the distribution of lymphocyte populations of Cornell K-strain male chickens were studied. 2. Two experiments were conducted. In the first, chickens were injected with Brucella abortus (BA), a purported T-independent antigen. In the second, chickens were injected with sheep red blood cells (SRBC), a T-dependent antigen. Peripheral blood lymphocytes (PBL) and spleen lymphocytes isolated at 0, 3, 6, 9, 12 and 24 h following Ag injection were stained with monoclonal antibodies (mAb) detecting B-lymphocytes, CD4+ and CD8+ cells. 3. B-lymphocytes in the blood or spleen showed no significant changes following either BA or SRBC injection. In contrast, CD4+ cells were decreased in the blood and increased in the spleen following BA and SRBC injections. CD8+ cells were decreased in both blood and spleen following BA injection but were unchanged in either blood or the spleen following SRBC injection. 4. These results indicate that there is a change in both spleen and circulating lymphocyte populations, especially T-helper cells, following Ag injection. T-helper cells are apparently the primary population involved in the initiation of humoral immunity.

Dietary cholesterol effects on plasma and yolk cholesterol fractions in selected lines of Japanese quail.

Japanese quail from lines that had been divergently selected for high (HL) or low (LL) plasma total cholesterol and their unselected control line (CL) were fed an all vegetable diet to which 0 or 0.5% crystalline cholesterol were added. Relationships between plasma and yolk cholesterol fractions were examined at 10, 14, and 18 wk of age, which followed 2, 6, and 10 wk consumption of the cholesterol-enriched diet, respectively. Unesterified cholesterol (UC) and cholesteryl esters (CE) in plasma and yolk were analyzed using HPLC. There were no consistent correlations between yolk and plasma for UC, individual CE, total esterified cholesterol (EC), or total cholesterol in the selected lines at ages tested, whether or not 0.5% cholesterol was added to the diet. Cholesterol concentrations in milligrams per gram of yolk and in milligrams per yolk were higher in the HL than the LL at 10 and 14, but not at 18 wk of age. Yolk weights of the HL females increased from 10 to 18 wk of age, whereas those of the LL did not. Cholesterol concentrations in the LL yolks continued to increase over time, however the increases in yolk weight in the HL were not accompanied by proportional increases in cholesterol deposition in the yolk, leading to a dilution of concentration of cholesterol fractions in the HL yolk. Dietary cholesterol increased egg production rate in the selected lines but did not increase the cholesterol content of the yolk.

Atherosclerosis in Japanese Quail males selected for high or low plasma cholesterol.

Three lines of Japanese quail males, unselected controls (CL), high response (HL), and low response (LL) lines, selected for plasma total cholesterol for 18 generations, were fed all-plant source, nonatherogenic diets to which 0 or .5% cholesterol were added from 6 to 18 wk of age. Atherosclerotic scores (AS) of aorta of HL birds fed cholesterol were significantly higher than those of LL birds fed cholesterol. Scores of LL fed cholesterol were not higher than LL not fed cholesterol. Fatty infiltration of muscularis and foam cell disruption of elastic fibers were observed in HL males fed cholesterol. In a second experiment, males of the three lines were fed from 6 to 14 wk of age four plant source diets to which were added: 1) 10% glucose monohydrate (cerelose); 2) 10% cerelose + .1% cholesterol; 3) 4% corn oil; or 4) 4% coconut oil. All diets were calculated to be isocaloric and isonitrogenous. Overall, AS of HL and CL males were significantly higher than LL males, but there were no effects of diet for the 56-d feeding period.

Effects of corticosterone in vivo and in vitro on adrenocorticotropic hormone production by corticotropin releasing factor-stimulated leukocytes.

Corticotropin releasing factor (CRF) and corticosterone have been shown to affect immune cell function. Previously, we have shown that CRF stimulates immunoreactive adrenocorticotropic hormone (ACTH) production by leukocytes. In this study, splenic leukocytes from corticosterone-injected chickens failed to show a CRF-induced increase in ACTH production. In addition, corticosterone in vitro inhibited the production of leukocyte ACTH as well as the stimulatory effect of CRF on splenic leukocyte ACTH production. These findings show that, as with anterior pituitary ACTH production, CRF-stimulated leukocyte ACTH production is inhibited by glucocorticoids.

Dietary cholesterol and fat saturation effects on plasma esterified and unesterified cholesterol in selected lines of Japanese quail females.

Three lines of Japanese quail females, randombred controls (CL), high response (HL), and low response (LL) lines, selected for plasma total cholesterol for 18 generations, were fed all-plant-source, nonatherogenic diets to which 0 or .5% cholesterol were added from 6 to 18 wk of age. In all three lines, plasma cholesterol increased when cholesterol was fed; however, responses were greater in the HL than in the LL line, with CL intermediate. In a second experiment, females of the three lines were fed, from 6 to 14 wk of age, four isocaloric, isonitrogenous plant-source diets to which were added: 1) 10% glucose monohydrate (cerelose); 2) 10% cerelose + .1% cholesterol; 3) 4% corn oil; or 4) 4% coconut oil. Baseline data obtained before feeding experimental diets indicated that the HL had significantly higher plasma total, esterified (EC) and unesterified (UEC) cholesterol than LL and that nonovulating females had higher concentrations of esterified cholesterol than ovulating females. Diets used did not affect cholesterol fractions in the ovulating females, although there were significant differences among lines. Dietary cholesterol significantly increased the ratio of EC to UEC. Sclerotic lesion scores were higher in the HL than the LL birds and in birds fed the coconut oil diet.

Gordon Memorial Lecture. Stress, strains and resistance.

1. Stress describes the bird's defence mechanisms and a stressor is the situation that elicits the defence response. 2. As the environment can be viewed as a composite of interacting stressors, the bird's success in coping with its environment depends on the severity of the stressors and the physiological ability to respond properly and thus maintain homeostasis. 3. The neural, endocrine and more recently immune systems are considered to be integrators of the stress response. Although stress responses may be necessary for survival in wild bird populations, they are often detrimental to efficient growth, skeletal integrity and disease resistance in domesticated fowl. 4. Stress responses are modified by the genetic components.

Validation of an assay to measure adrenocorticotropin in plasma and from chicken leukocytes.

A RIA for mammalian adrenocorticotropic hormone (ACTH) was modified and validated to measure chicken ACTH. The assay was capable of detecting an increase in chicken plasma ACTH following treatments known to increase plasma ACTH. Both splenic and peripheral blood leukocytes stimulated with corticotropin-releasing factor (CRF) showed a significant increase in ACTH production compared with unstimulated leukocytes. This finding supports the conclusion that the substance produced by leukocytes previously shown in our laboratory to stimulate adrenal cells to secrete corticosterone is immunoreactive ACTH.

Inhibition of adrenal steroidogenesis, neonatal feed restriction, and pituitary-adrenal axis response to subsequent fasting in chickens.

White Plymouth Rock chickens placed under 60% feed restriction or ad libitum feeding, with or without metyrapone (adrenal blocking agent) treatment, from 4 to 6 days of age were subjected to either 8 or 24 hr feed deprivation at 36 days of age. Chicks subjected to the neonatal 60% feed restriction (60R) but not those provided metyrapone during the procedure (60M) had elevated heterophil/lymphocyte (H/L) ratios. However, there was no difference in plasma corticosterone and ACTH responses between 60R and 60M chicks. Except for increases in H/L and plasma corticosterone concentrations among ad libitum fed (AL) and 60M chickens, respectively, there was no indication of stress response attributable to the subsequent 8 hr fast. Feed withdrawal for 24 h did not cause rises in H/L ratios and plasma levels of corticosterone of chicks that had been subjected to early 60% feed restriction with nonmetyrapone-treated feed. In contrast, chicks of other regimens had elevated H/L and plasma corticosterone responses when exposed to a similar procedure. Except for those fed ad libitum during the neonatal stage, circulating levels of ACTH declined following the 24-hr fast. These results demonstrate that stress early in life without concurrent rises in circulating corticosteroid levels may not help the biological system in coping with subsequent stressors.

Growth, feed conversion, and carcass composition in females of four broiler crosses fed starter diets with different energy levels and energy to protein ratios.

Body weight, feed conversion, and carcass composition were studied in females of four commercial broiler crosses that differed in weight at 7 wk of age. The birds were fed starter diets for the first 3 wk containing different energy levels but the same protein level (Experiment 1) or varying energy or protein to equilibrate energy:protein ratios (Experiment 2). Experiment 1 was conducted in floor pens; Experiment 2 in batteries. All birds received a common grower diet from 4 to 7 wk of age. No significant cross by starter diet interactions were found for body weight, feed conversion, carcass weight, or protein percentages. In the floor experiment, 3- and 7-wk body weights were lower in birds receiving the highest energy starter than in those receiving lower energy starters; in the battery experiment, body weights were unaffected by energy of the starter. Feed conversion was lower in the birds receiving the high-energy starter in both experiments. Carcass weights essentially paralleled live weights in both experiments; however, percentage of protein in carcasses of 7-wk-old broilers was unaffected by starter diets. Feeding a high-energy starter for the first 3 wk posthatch resulted in higher carcass fat percentages at 7 wk of age but lower absolute and relative abdominal fat pad weights. The significant cross by starter diet interactions indicated that the fastest growing broilers responded to the high-energy starter diet by a significant reduction in abdominal fat without an overall reduction in carcass fat.

Ovine corticotropin-releasing factor increases endocrine and immunologic activity of avian leukocytes in vitro.

Treatment of splenic leukocytes from Cornell K strain male chickens (homozygous at the B15 locus of the major histocompatibility complex) with ovine corticotropin-releasing factor (oCRF), before their co-incubation with naive chicken adrenal cells, resulted in an increase in corticosterone production. Supernatants from the oCRF-treated splenic leukocytes caused a time-dependent increase in corticosterone production when incubated with chicken adrenal cells. Adding oCRF directly to chicken adrenal cells did not increase corticosterone production. Pretreatment of peripheral leukocytes with oCRF increased their activity in a concanavalin A mitogen assay. Thus, chicken leukocytes stimulated with corticotropin releasing factor appear to increase the production of an "adrenocorticotropin-like" substance (adrenocorticotropin-like because it increases corticosterone production by adrenal cells), and increased their cell-mediated immune activity.

Ethics in research.

Unethical conduct in research can be divided into five categories: 1) falsification of data, in which the researcher manipulates results, provides data without experimentation, or biases the results to give a false impression of their value; 2) failure to credit others (former colleagues, students, associates) for research results or ideas; 3) plagiarism, use of other's published material (ideas, graphs, or tabular data) without permission or credit; 4) conflicts of commitment or interest in which work or ownership in a private firm in some way conflicts or detracts from the duties to the institution they represent or allows private gain through the individual's employment at the institution; 5) biased experimental design or interpretation of data to support public or private groups that have provided financial support for research. Although none of these should be condoned or tolerated by the scientific community, the latter two are probably more serious, because they are insidious, hard to prove, and, in the long run, result in the loss of public confidence in science.

Plasma cholesterol responses to the adrenocorticotropic hormone in Japanese quail following eighteen generations of divergent selection.

Japanese quail (Coturnix coturnix japonica) lines were divergently selected for plasma cholesterol response to adrenocorticotropin (ACTH). Data for Generations 9 to 18 indicated a greater response in the low (L) line than in the high (H) line, which was contrary to selection responses for the first eight generations. During Generations 9 to 18, selection differentials were greater in the H line; however, heritability estimates were greater in the L line. These data suggest caution in the interpretation of early selection responses for plasma cholesterol. Selection for plasma cholesterol response following ACTH injection also resulted in divergence in plasma cholesterol levels in the H and L lines in the absence of ACTH. Body weights at 31 days were greater in the H line than in the L line, suggesting a possible relationship with the plasma cholesterol level.

Changes in heterophils following antigen injection in immature male chickens.

Eight-week-old Single Comb White Leghorn male chickens were injected intravenously with either .1 mL Brucella abortus (BA) or saline. Blood samples were drawn at 0 and 12 h postinjection and were examined for heterophil numbers and morphology. Differences between numbers of circulating heterophils/mm3 blood were not significant for saline and BA-treated birds at time zero. However, 12 h following injection, heterophil numbers in BA-treated birds were significantly (P less than .05) greater than those in saline-treated birds. Heterophil appearance took two forms: in the first form, granules were small, round, and strongly eosinophilic; in the second form, the granules were large, rod-shaped and more basophilic in staining. These changes indicate the possible involvement of heterophils in the initiation of a humoral immune response through enhancement of some secretory function.

Social organization in caged layers: the peck order revisited.

The dominance hierarchy that exists among free ranging chickens is a peck order. Several researchers have attempted to correlate various production parameters of caged layers with dominance rank, with mixed results. Animal welfare groups have expressed increasing concern over the effects of battery cage housing on the behavior of layers, even though several researchers have shown that the incidence of aggressive pecks decreases in these cages. The studies presented here demonstrate that agonistic interactions occur among most pairs of hens housed in pens but do not occur among most pairs of hens housed in cages. Therefore, peck orders could be constructed for hens housed in pens but not for hens housed in cages. Incidence of agonistic interactions is highest in cage-housed hens immediately following housing of the hens; this peak is matched when group membership is mixed. These results suggest that a social system does form among caged hens even if a peck order does not. A single dominant hen in each cage was involved in and won a large majority of the agonistic encounters in the cages. The lack of evidence of dominance relationships between pairs of hens in cages, other than those between a single dominant hen and its cage mates, supports the hypothesis that despotism, not a peck order, was the prevailing social organization among hens housed in high-density cages.

Changes in the profiles of circulating white blood cells, corticosterone, T3 and T4 during the initiation of humoral immunity in immature male chickens.

Eight-week-old male White Leghorn chickens were injected intravenously with either 0.1 ml of Brucella abortus (BA) or saline. Blood samples were collected before injection, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, and then daily for the following 6 days after the injection. Total white blood cells (WBC), differential counts and corticosterone (CS), T3 and T4 levels were measured in all blood samples. These data represented the results obtained from the primary response. To obtain data for the secondary response, the same birds were injected 4 weeks following the first challenge and the same procedure was followed. During primary and secondary responses, circulating lymphocytes and monocytes decreased significantly 3 hr following BA injection and then increased steadily and reached a peak approximately 5 days post-BA injection, however, circulating heterophils increased significantly 6-12 hr and returned to normal levels 2 days post-BA injection. Serum corticosterone increased significantly 3 hr following BA injection. Serum T3 decreased significantly 3 to 12 hr then increased significantly 2 days following BA injection. There were no significant changes in any of the measurements following saline injection. Correlations between circulating hormones and white blood cells were calculated and are reported. These results indicate that an early step in the initiation of humoral immunity is probably an increase in serum CS. This increase in CS is followed by a decrease in circulating lymphocyte and monocyte numbers. These decreases may be the result of lymphocyte trapping in secondary lymphoid organs. This series of events may occur to modulate and activate the immune response.