James A. Marsh

Is avian humoral immunocompetence suppressed by testosterone

Abstract A key issue in sexual selection theory is how a correlation between male secondary sexual characters and male genetic quality can be maintained. The immunocompetence-handicap hypothesis proposes that testosterone-dependent male characters remain honest signals because of the immunosuppressive effect of elevated steroid hormone levels. The hypothesis requires that physiological levels of testosterone depress immune system function. We quantified testosterone titers and humoral immunocompetence of captive male and female red-winged blackbirds (Agelaius phoeniceus) at three points in the annual cycle (autumn, prebreeding, and breeding). We also conducted an implant experiment on the males to assess the effects of prolonged, above-normal testosterone titers on humoral immune responses. Humoral immunocompetence was measured as secondary antibody production to a non-pathogenic protein antigen, keyhole limpet hemocyanin, using an enzyme-linked immunosorbent assay we developed for A. phoeniceus. Secondary antibody responses of individuals were highly repeatable between sampling periods. Neither physiological nor above-normal levels of plasma testosterone suppressed secondary antibody production. In paired tests of the same individuals between prebreeding and breeding, and between breeding and implant, plasma testosterone increased significantly but secondary antibody responses were unaffected. We are confident in these results because with 80% power, an 11–14% difference in antibody titers would have been detected. There was no relationship between plasma testosterone levels and humoral immunocompetence in free-ranging males tested at the peak of breeding. These results cast doubt on a key assumption of the immunocompetence-handicap hypothesis.

Influence of dietary selenium and vitamin E on the humoral immune response of the chick.

Abstract Nutritional deficiencies in vitamin E and/or selenium (Se) caused impaired immune function, as measured by the humoral response to ovine erythrocytes by young chicks, but only at low antigen doses. In the 2-week-old chick, both vitamin E and Se were required for optimum immune function; however, at 3 weeks of age, either vitamin E or Se was sufficient for optimum immune function. At this developmental stage, Se appeared to be capable of replacing vitamin E with regard to the immune system. Titrations of dietary vitamin E in the presence of adequate Se and the reciprocal experimental regime did not result in immune enhancement. However, the range of concentrations of each factor employed were within nutritional and nonpharmacological levels. In contrast, high dietary Se produced significant immune suppression in male but not female chicks. These data suggest that Se may be an important component of immune function, and its effect is influenced by antigen concentration, sex, and the ontogenic state.

Effect of selenium and vitamin E dietary deficiencies on chick lymphoid organ development

Abstract Diets specifically deficient in selenium (Se) and/or vitamin E or adequate in both nutrients were fed to chicks from the time of hatching. Lymphoid organs (bursa, thymus, and in some instances, spleen) were collected from chicks 7-35 days of age. Growth of the chicks fed these diets was monitored over the experimental period as was lymphoid organ growth. The development of the primary lymphoid organs was further assessed by histological techniques and the organ contents of vitamin E (α-tocopherol) and Se were determined. Specific deficiencies of either Se or vitamin E were found to significantly impair bursal growth as did a combined deficiency. Thymic growth was impaired only by the combined deficiency diet. Severe histopathological changes in the bursa resulted from the combined deficiency and these were detectable by 10-14 days after hatching. These changes were characterized by a gradual degeneration of the epithelium and an accompanying depletion of lymphocytes. Similar changes, although slower to develop and less severe, were observed in the thymus as a result of the combined deficiency. When both serum and tissue levels of vitamin E and Se were monitored, it was observed that these were rapidly and independently depleted by the specific deficiency diets. These data suggest that the primary lymphoid organs are major targets of Se and vitamin E dietary deficiencies and provide a possible mechanism by which immune function may be impaired.

Diminished Hepatic Growth Hormone Receptor Binding in Sex-Linked Dwarf Broiler and Leghorn Chickens:

Abstract Hepatic growth hormone (GH) receptor binding was compared in normal and sex-linked dwarfs (SLD) from both Hubbard and Cornell strain chickens. At 6, 8, and 20 weeks of age, hepatic GH receptor binding in the Hubbard SLD chickens was significantly lower than that of normal fast-growing birds. At 20 weeks of age, only 2 of 22 SLD chickens in the Hubbard broiler strain showed positive binding at a high enough level to allow for Scatchard analysis. The affinity constants and binding capacities of these two SLD chickens were numerically (but not significantly) lower than those of the normal fast-growing birds. We further examined hepatic GH receptor binding in two closely related White Leghorn strains of chickens that have been maintained as closed breeding populations for many years. We observed no detectable hepatic GH binding in the Cornell SLD chickens (N = 20), as compared to the normal-growing control strain (K strain). In both SLD strains, pretreatment with 4 M MgCl2 did not enhance GH binding, suggesting that there was no endogenous GH binding to the receptor. Based on these data, we suggest that the lack, or greatly reduced number, of GH receptors may be a major contributing factor to the dwarfism observed in these strains.

Enhanced Growth and Immune Development in Dwarf Chickens Treated with Mammalian Growth Hormone and Thyroxine

Abstract The effects of growth hormone and/or thyroxine treatments on antibody production, primary lymphoid organ development, and general body growth were examined in two dwarf strains (sex-linked dwarf—SLD, and autosomal dwarf—ADW) and in a normal-growing strain (K) of White Leghorn chickens. One-day-old male chicks were assigned to experimental groups and were treated either with thyroxine (T4) feed supplements or daily mammalian growth hormone (GH) injections or a combination of these treatments (T4/GH). Within the SLD strain, GH treatments resulted in a significant enhancement (P < 0.005) of humoral immune responsiveness and bursal growth while T4 treatments significantly (P < 0.05) stimulated thymic growth. Overall growth in the SLD was also stimulated by T4 treatments. GH and/or T4 treatments had no specific effects on primary lymphoid organ growth in the ADW strain but either treatment separately resulted in a significant (P < 0.05) increase in overall body size. None of the treatments significantly (P > 0.05) affected any of these parameters in the K-strain controls. Supplementation with T4 significantly elevated serum T4 levels within all strains. There were, however, no significant differences in the serum levels of T4 between strains within any of the treatment groups. Serum triiodothyronine (T3) levels were significantly lower in all treatment groups of the SLD as compared to the K-strain control. ADW serum T3 levels were significantly lower than the K-strain only in the T4-treated group. No differences (P > 0.05) were found between the dwarf and control strains in endogeneous GH levels, although mammalian GH treatments did produce significant changes in serum T3 and T4 levels within the K-strain. These results provide evidence that hormonal manipulations can significantly affect body growth, primary lymphoid organ development, and immune function in the dwarf strains studied and suggest these strains may be useful models for future studies on hormonal interactions and immune function.

Effect of dietary vitamin E and selenium deficiency on chicken splenocyte proliferation and cell surface marker expression.

Beginning at hatching, chicks were fed a Basal diet, without vitamin E or selenium (Se) or the same diet supplemented with vitamin E (100 IU/kg) and Se (0.2 ppm). The effect of these treatments on the expression of cell surface markers (CT-1a, CD3, CD4, CD8, sIgs, and Ia) defining specific thymocyte and peripheral blood leukocyte (PBL) subpopulations were examined using flow cytometric analyses. In parallel studies the effect of the dietary deficiencies on splenocyte proliferative responses to ConA or PHA stimulation was examined. The mean expression of CD3 and CT-1a per cell was increased while CD8 and CD4 expression was decreased on thymocytes from chicks fed the Basal diet. The proportion of double negative (CD4-, CD8-) thymocytes and single positive CD8+ thymocytes was significantly decreased while single positive CD4+ and double positive (CD4+, CD8+) thymocytes were significantly increased by the dietary vitamin E and Se deficiencies. The dietary deficiencies resulted in a decreased proportion of peripheral T cells and specifically decreased the number of CD4+ PBL. The proliferative response to both ConA and PHA was impaired by the vitamin E and Se dietary deficiencies. The proliferative response could be fully reconstituted but only after vitamin E and Se supplementation for periods longer than 1 week. Plasma SeGSHpx and alpha-tocopherol levels paralleled the mitogen responsiveness observed. These results support the conclusion that vitamin E and Se deficiencies may affect both the maturation of specific lymphocyte subpopulations and the functional and proliferative capabilities of the peripheral lymphocytes.

Effects of Triiodothyronine Treatments on Body and Organ Growth and the Development of Immune Function in Dwarf Chickens

Abstract The effects of triiodothyronine (T3) treatments on general body growth, long bone growth, primary lymphoid organ development, antibody production, and serum growth hormone (GH) and thyroid hormone levels were examined in two dwarf strains (sex-linked dwarf—SLD, and autosomal dwarf—ADW) and in a normal-growing control strain (K) of White Leghorn chickens. One-day-old male chicks from each of these strains were assigned to either an untreated control group or to one of the groups receiving a T3 supplement ranging from 0.01 to 1.0 ppm. General body growth and long bone growth were significantly (P < 0.05) stimulated only within the SLD strain by the intermediate T3 dosages. The 1.0-ppm T3 dosage level resulted in depressed body weights within both the K and ADW strains but produced no significant changes within the SLD strain. Thymic growth was significantly stimulated due to treatments of 0.1 ppm T3 in the SLD strain (P < 0.05) and 1.0 ppm T3 in both the SLD and ADW strains (P < 0.001 and P < 0.05, respectively). Bursal growth was significantly depressed (P < 0.05) at all T3 dosage levels within the SLD strain while 0.01 and 0.1-ppm T3 treatments resulted in significant bursal growth stimulation in the K and ADW strains, respectively. Concomitant with the depressed bursal growth, antibody production was significantly depressed (P < 0.05) within the SLD strain at the 1.0-ppm T3 dosage level. Antibody production was not significantly affected by any of the T3 treatments within the control K or ADW strains. Serum T3 levels were significantly increased in all strains by the T3 supplementation but thyroxine (T4) serum levels were affected only within the SLD strain. The 0.01-ppm T3 treatment resulted in a significant increase (P < 0.05) in serum T4 levels within this strain and treatment group.

Effect of hypophysectomy and growth hormone on immune development in the domestic fowl

The effect of hypophysectomy and recombinant growth hormone (rcGH) treatment on the growth and development of the immune system was investigated in young chickens. Flow cytometric analysis of cell surface markers revealed no changes in the proportion of thymocytes expressing CT-1a, CD4, and/or CD8 among any of the treatment groups. In contrast, the proportion of both single positive CD4 and CD8 peripheral blood lymphocytes (PBL) was altered in hypox birds treated with rcGH compared to the vehicle-treated group. Specifically, rcGH treatment produced a decrease in the proportion of CD8+ cells and an increase in the percentage of CD4+ PBL. There was little change in the labeling intensity of PBL or thymocytes associated with any treatment; however, double positive (CD4+CD8+) thymocytes from hypophysectomized chicks that were not given rcGH had increased fluorescence relative to rcGH supplemented hypox chicks. As expected, hypophysectomy reduced body, skeletal, and thymic growth. Treatment of hypox chicks with rcGH enhanced body weight while thymic weights were somewhat increased. Skeletal growth was not significantly altered by rcGH. Bursal growth appeared refractory to either treatment. These studies support the conclusion that growth hormone influences thymic growth and the maturation of thymus-derived lymphocytes. These results also demonstrate a biological activity for chicken growth hormone derived through recombinant technology.

Effect of triiodothyronine on the expression of T cell markers and immune function in thyroidectomized white leghorn chickens

Abstract Hypothyroid K-strain chickens were produced by neonatal thyroidectomy and treatment with 6-propyl-2-thiouracil. Thyroidectomized birds were given 0, 1.5, 4.5, 15, or 45 μg/kg body wt of triiodothyronine (T3) by daily injection. At 5 weeks of age, thymocytes were prepared for flow cytometric analysis of CT-1a, CD3, CD4, and CD8 expression. Sham-operated birds had the smallest proportion of CT-1a+ cells and the brightest CT-1a+ cells. Unsupplemented thyroidectomized birds presented an inverse picture, while T3-Vtreated thyroidectomized birds were intermediate. Fewer and less brightly labeled CD3+, CD4+, and CD8+ cells were associated with sham-operated birds or with higher levels of T3 replacement. Low levels (1.5 μg/kg body wt) or no T3 treatment produced a greater proportion of positive, highly fluorescent cells. The ratios of CD4+ to CD8+ thymocytes were increased (P ≤ 0.05) by T3 supplementation. Functionally, thyroidectomy produced a decrease in mitogen-stimulated proliferation of peripheral blood lymphocytes. This effect was ameliorated by T3 supplementation. Further, thyroidectomy produced an elevation of plasma growth hormone concentrations. These results suggest that thyroid factors and alterations of thymic status significantly affect the generation of specific thymus-derived lymphocyte populations and their functional capabilities, perhaps due to changes in the thymic microenvironment. These alterations may have important consequences for the development of immunocompetence and disease resistance in chickens.

Stimulation of growth hormone secretion in dwarf chickens by thyrotrophin-releasing hormone (TRH) or human pancreatic growth-hormone-releasing factor (hpGRF)

The basal plasma growth hormone (GH) level in adult sex-linked dwarf hens was elevated in comparison with autosomal dwarf hens and with control (Cornell K strain) laying hens. The iv administration of thyrotrophin-releasing hormone (TRH) (10 micrograms/kg) had no effect on GH secretion in control hens but slightly (1.2-fold) and transiently (for 10 min) increased the GH level in the autosomal dwarfs and greatly (8.7-fold) increased the GH level in the sex-linked dwarfs, in which it remained elevated for at least 30 min after injection. The iv administration of human pancreatic GH-releasing factor (hpGRF) (10 micrograms/kg) stimulated GH release in each strain. The response in the sex-linked dwarfs was greater than that in the autosomal dwarfs and the control hens but less than that elicited by TRH. These results suggest that the increased basal GH level in the sex-linked dwarfs results from an increased responsiveness to provocative stimulation.