Phenotypic responses of chickens to long-term, bidirectional selection for juvenile body weight--historical perspective.

A long-term selection experiment for high (HWS) and low (LWS) BW at 8 wk of age (BW8) was conducted in White Plymouth Rock chickens. Over 54 generations of selection, responses to bidirectional selection were profound. Increase in BW8 in line HWS was linear, and there was a significant quadratic response in line LWS for BW at both 4 and 8 wk of age. Although there is no indication that line HWS has come close to approaching a selection limit in more than 50 generations, selection limits occurred in line LWS chickens at generation 48 for females and generation 50 for males. Evidence also exists that one or more beneficial mutations have occurred in line HWS, aiding in progressive increases in BW8 over generations. Analyses of ratios of BW at 4 wk of age with those at 8 wk of age (ratio 4/8) revealed that LWS females grew proportionately faster through 4 wk of age than LWS males or HWS chickens. Comparisons of the selected lines with contemporary lines in which selection had been relaxed (discontinued) indicated that, in line HWS, the relaxed lines generally regressed toward original (preselection) values, suggesting that the linear response to single-trait selection was at least partially due to continued genetic variance. In LWS chickens, a series of plateaus in selection response occurred, but relaxed contemporary lines still regressed toward preselection values for BW8. In spite of the length of this selection experiment (54 generations), genetic variance and beneficial mutations have allowed continued, linear response to selection for increased BW8. Response to selection for decreased BW8 has been tempered by physiological barriers that have decreased survival of young chicks or the ability of females to reproduce. These findings are discussed in a historical perspective.

Effect of route of inoculation on humoral immune response of White Leghorn chickens selected for high or low antibody response to sheep red blood cells.

Effects of route of SRBC inoculation and antigen dosage on primary and secondary antibody response of White Leghorn lines selected for high (HA) or low (LA) 5-d antibody response to a single i.v. inoculation with 0.1 mL of a 0.25% suspension of SRBC were studied in two trials. In the first trial, chicks from parents of generation S24 of each line were randomly assigned to one of four treatments. At 35 d of age, they were inoculated into the brachial vein with 0.1 mL of 0.25% suspension of SRBC or into the breast muscle with 0.1 mL of 0.25, 2.50, or 25.00% SRBC. Plasma SRBC antibody was measured 3, 6, 10, and 20 d later. In the second trial, chicks from parents of generation S25 of each line were randomly assigned to treatment groups. At 28 d of age they were inoculated with 0.1 mL of 0.25% SRBC into the brachial vein, 0.1 mL of 25.00% SRBC into the thigh (T-L) or breast muscle (B-L), or 0.5 mL of 25.00% SRBC into the thigh (T-H) or breast muscle (B-H). Twenty-one days later, chicks (except five per group) were given a booster inoculation of 0.1 mL of 25.00% SRBC into the thigh muscle. Six and 10 d after each inoculation, plasma SRBC antibody, IgG, and IgM titers were measured. The SRBC antibody titers after primary i.v. inoculation with SRBC were always higher for HA than LA chicks. When inoculations were i.m., differences between lines varied with dosage. Low dosages inoculated into the breast failed to induce line differences consistently, whereas at higher dosages, titers were greater for HA than LA chicks regardless of inoculation site. For Line LA, inoculation into the thigh elicited higher titers than inoculations into the breast. Antibody titers to the booster inoculation of SRBC were similar for the lines.

Antibody responses to different dosages of sheep red blood cells in lines of chickens selected for high and low antibody response to sheep red blood cells.

Temporal patterns of SRBC antibody response after primary and secondary inoculations were measured in White Leghorn males from lines selected 24 generations for high (HA) or low (LA) 5-d antibody titers to an i.v. inoculation with 0.1 mL of a 0.25% suspension of SRBC. Primary i.v. inoculations were administered at 50 d of age as 0.1 mL of either 0.025 or 0.25% suspension of SRBC. Antibody levels of SRBC were measured 3, 5, 7, 9, 11, 13, and 20 d after inoculation. Also, IgG levels were measured on samples obtained 3, 7, 13, and 20 d after inoculation. At 70 d of age, half of the cockerels in each line-dosage subclass were given a booster inoculation of 0.1 mL of 0.25% SRBC. The SRBC antibody was measured 3, 6, 9, and 13 d later in chicks both receiving and not receiving the second inoculation. Patterns of antibody response to SRBC dosage differed according to line, resulting in interactions of line by dosage by day. Concentrations of IgG were greater for Line HA than for LA at 7 and 11 d after inoculation with SRBC but not at 3 and 20 d. Antibody responses to the booster inoculation differed between lines with a dosage effect present for LA but not for HA chicks. The greater anamnestic response observed in LA than in HA chicks is explained in the context of the resource allocation paradigm.

Heterosis following long-term bidirectional selection for mating frequency in male Japanese quail.

Reciprocal crosses (sire line shown first and dam line second) among high (H) and low (L) selected lines and the randombred control line (C), which was the base population for the selected lines, were made after 40 generations of bidirectional selection for mating frequency of male Japanese quail. Significant heterosis for the selected trait was found only in crosses between Lines C and L, being 62 and 92% for LC and CL, respectively. Heterosis for percentage of maters was present in all crosses, ranging from 8% for HC and CH to 46% for HL. Three (HC, LH, and CL) of the six crosses had significant heterosis for both 4- and 8-wk BW. Heterosis for 4- and 8-wk BW was also significant for the HL and CH crosses, respectively. For area of the cloacal gland, heterosis was significant in five crosses. Although crosses tended to exhibit higher relative aggressiveness than their respective midparent means, heterosis for this trait was not significant. Reciprocal effects, although not important for most traits, were present for BW in crosses between Lines C and L and Lines H and C. In general, long-term selection for mating frequency of males changed the genetic basis of selected and correlated traits with considerable nonadditive genetic effects observed for most traits in specific crosses.

Kinetics of antibody responses in hens from chicken lines divergently selected for response to sheep red blood cells.

Changes in antibody titers to SRBC were monitored for 180 d after inoculation in hens from two lines divergently selected for 24 generations for high (HAS) or low (LAS) antibody response to SRBC. The HAS hens not only had a higher peak of antibody response (12.9 vs 9.4), but also showed greater persistence in maintaining antibody levels than LAS hens. As a result, HAS hens exhibited higher antibody titers for the 180-d assay period than LAS hens. Antibodies to SRBC were detected in all day-old chicks hatched from HAS eggs collected 10 to 14 d after inoculation as well as 92 to 119 d after inoculation. Only a portion (20 to 75%) of progeny for LAS hens had detectable levels of antibody during the same periods. Among responders, antibody titers were higher for HAS than for LAS progeny. There was a positive correlation among antibody titers taken at different times after the inoculation with SRBC.

Heterosis and developmental stability of body and organ weights at hatch for parental line broiler breeders and specific crosses among them.

Body, yolk sac, left and right shanks with toes, empty left and right ceca, left and right lungs, heart, and bursa of Fabricius weights were obtained at hatch for 50 chicks from each of five commercial broiler parental lines (three sire and two dam) and three F1 crosses involving them. Differences among stocks and between sexes were inconsistent among mating combinations. Although correlation coefficients between yolk-free chick weight with organ weights were generally stock specific, they were high (> 0.75) with shank weight, intermediate with heart and lung weights, and low (< 0.25) with ceca and bursa weights. Heart:lung ratios of all F1 crosses were greater than those for their respective parental lines; however, the degree of heterosis differed among populations. Developmental stability, as measured by percentage relative asymmetry, was less in two of the sire parental lines than in their respective dam lines and F1 crosses.

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.

Diet and immunological memory of lines of White Leghorn chickens divergently selected for antibody response to sheep red blood cells.

Antibody responses to a first, second, and third injection with SRBC, and growth were studied in lines of White Leghorn chickens selected for high (HA) or low (LA) 5-d antibody titers to an i.v. inoculation with 0.1 mL of a 0.25% suspension of SRBC. The experiment involved parallel studies on two groups of chicks hatched from the same matings of parental lines HA and LA at a 14-d interval. Chicks of each age-line subclass were fed either a high or low nutrient density diet from hatch onwards. When chicks of Hatches 1 and 2 were 28 and 14 d of age (doa) respectively, they were injected with 0.1 mL of 0.25% suspension of SRBC, and antibody titers measured 3 and 6 d later. A second and a third injection of the same concentration of SRBC was given to chicks of each age-line-diet subclass at 10-d intervals and antibody titers measured 3 and 6 d after each injection in different chicks randomly sampled from each age-line-diet subclass. After the first injection, antibody (primary) responses of HA chicks were higher than those of LA chicks regardless of age and diet. This difference (HA > LA) observed for the primary response was seldom evident in the responses to the second (secondary) and third (tertiary) injections. Antibody responses of LA chicks after the second and third injections were anamnestic. For HA chicks given the first injection at 28 doa, neither the secondary nor tertiary responses suggested anamnestic capacities, whereas there was apparent memory exhibited by the secondary and tertiary responses of HA chicks initially injected at 14 doa. The LA chicks were significantly heavier than HA chicks at all ages. Even though the higher nutrient density diet increased BW of chicks of both lines, its effect on memory responses was sporadic. The results of this experiment show that, even though divergent selection has been successful in the primary responses, correlated responses in immunological memory were not always observed, suggesting that the two types of responses might be under different genetic control.

Immunocompetence and resistance to marble spleen disease of broiler- and layer-type pure lines of chickens.

The intent of this study was to evaluate, under concurrent conditions, certain responses that may be important in chicken breeding and growing. Three commercial broiler pure lines (A, B, and C) and two experimental White Leghorn lines selected for high (HAS) and low (LAS) antibody response to sheep red blood cells were evaluated concurrently for humoral and cell-mediated immunocompetence, resistance to marble spleen disease virus (MSDV), relative asymmetry (RA), and comb weight. Chicks were injected with 0.1 ml of 0.25% SRBC at 21 days of age. Antibody response 6 days after injection was highest in line HAS. Titres for the commercial lines were similar to those in line LAS. The cutaneous basophil hypersensitivity test, an in vivo cell-mediated immune response, was measured as the increase in toe-web thickness 24 h after an injection with T-cell mitogen phytohaemagglutinin (PHA)-P or -M into a sample of chicks at 9 days of age and a different sample of chicks at 20 days of age. PHA-P elicited greater responses than PHA-M at both ages. The pattern among stocks, however, differed depending upon age. Responses at 9 days were greater for the Leghorn than broiler lines, while at 20 days, responses were greater in lines A and LAS than in lines B, C, and HAS. Resistance to MSDV challenge differed among stocks, with the ranking for resistance being C>(A=B=LAS)>HAS. Rankings of RA for normal thickness of the toe web between the third and fourth digits at 9 days of age were (HAS=LAS)>(A=B=C). There were no differences in RA among stocks at 20 days of age. There was a significant line by sex interaction for relative comb weight, due to differences between lines for males but not females. Data from this study suggest that competence in one arm of the immune system is not a reliable measure of general immunocompetence, nor is it a measure of resistance in general.

Forty generations of bidirectional selection for mating frequency in male Japanese quail.

A bidirectional replicated selection experiment for high (H1 and H2) or low (L1 and L2) cumulative number of complete matings (CNCM) in male Japanese quail was conducted for 40 generations. In the S32 generation, a subline was taken from each selected line and selection was relaxed. In the randombred control line (C), CNCM and unselected traits changed significantly over generations. Means of the selected lines were adjusted each generation for deviations from the control means. After 40 generations of selection, there was a 21-fold difference in CNCM (59.4 vs 2.8) between Lines H1 and L1. Whereas means increased and variation decreased in the high lines, means decreased and variation increased in the low lines. Regressions of mean CNCM on generation of Lines H1, H2, L1, and L2 were 1.15 +/- 0.08, 0.61 +/- 0.08, -0.26 +/- 0.04, and -0.34 +/- 0.03, respectively. Although responses to selection were observed throughout the 40 generations in Line H1, the low lines appeared to have reached a limit to selection after the S30 generation. Relaxed lines provided supporting evidence for this conclusion. Mean CNCM decreased in the relaxed high lines to that of the control, whereas the low relaxed lines remained at the same level as their corresponding selected lines. Line H2 went into extinction in the 37th generation as a result of reduced fitness. Realized heritabilities of CNCM were 0.09, 0.07, 0.06, and -0.15 in Lines H1, H2, L1, and L2, respectively. As correlated responses to the selection, male quail in the high lines were heavier, exhibited greater relative aggressiveness, and had larger cloacal glands than those of the control and low lines.

Hematocrit values in weight-selected and relaxed lines of White Rock chickens.

Hematocrits (PCV) were measured at 29 and 106 d of age (PCV1 and PCV2, respectively) in male and female White Plymouth Rocks. Four lines were used, two of which had undergone 40 generations of divergent selection for 8-wk BW (HWS, LWS), and two respective sublines (HWR, LWR), in which selection had been relaxed for five generations. At both ages, males and females did not differ for PCV in lines HWR, LWR, and LWS. For line HWS there was an age by sex interaction that resulted from an age effect for males but not for females, and from a sex effect at each age. At both ages, PCV was higher for the HW than the LW lines. Initially, there was no difference between the selected and their respective relaxed lines, but by 106 d, HWR chickens had a higher PCV than HWS chickens. In lines HWR and LWR, PCV increased with age. There was a negative correlation in HWS males for PCV1 with 28 and 56 d BW. The HWR males also had a negative correlation for PCV1 with BW at 28 d, but not between PCV2 and BW. The correlation for PCV1 with PCV2 was high and positive for HWR males and females.

Diet and humoral responsiveness of lines of chickens divergently selected for antibody response to sheep red blood cells.

Growth and humoral immune response were studied in lines of white leghorn chickens selected for high (HA) or low (LA) 5-day antibody titers to an i.v. inoculation with 0.1 ml of a 0.25% suspension of sheep red blood cells (SRBC) antigen(s). Chicks were fed either a high (E) or low (A) nutrient density diet from hatch onward. Chicks from each line-diet subclass were inoculated i.v. with 0.1 ml of either 0.25% or 2.50% suspension of SRBC at either 7, 14, 21, or 28 days of age. Antibody titers were measured 5, 10, and 20 days after inoculation. LA chicks were heavier than HA chicks at 7 days of age and thereafter. Chicks fed diet E were heavier than those fed diet A. Feed efficiency was influenced by diet (E > A) at 21 and 28 days of age and line (LA > HA) at 28 days of age. In all but one case, antibody titers to SRBC were higher in HA than LA chicks. Also, the frequency of nonresponders of chicks inoculated with SRBC at 7 days of age was higher for LA than HA chicks. The higher dosage elicited greater 5-day antibody responses in LA but not in HA chicks inoculated at 28 days of age. Dietary effects on SRBC antibody were generally unimportant except for occasional interactions of dosage and line.

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.

Mode of inheritance of unselected traits in lines of chickens selected for high or low antibody response to sheep red blood cells. 2. Heterophils, lymphocytes, and hematocrits.

The nuclear lines for this experiment were White Leghorns that had undergone long-term selection for high (HH) or low (LL) antibody response to sheep red blood cell antigen(s). Sixteen progeny types consisting of parental lines, reciprocal F1 and F2 crosses, and backcrosses were produced in a single hatch from age-contemporary parents. At 30 d of age, blood was obtained from a random sample of 10 males per progeny type (n = 160) and slides prepared for subsequent determination of number of heterophils and lymphocytes. Twelve days later, blood was collected from random samples of 10 males and 10 females per progeny type (n = 320) for measuring hematocrits. There were no differences between parental lines for heterophils, lymphocytes, or the heterophil:lymphocyte ratio. Reciprocal effects were evident in the F1 crosses and directional heterosis was present in one cross but not the other. Neither maternal heterosis nor recombination effects were significant for either heterophils or lymphocytes. Although hematocrits were similar for males and females and parental lines, sex-linked and recombination effects appeared to be important.

Developmental stability in different genetic stocks of white rock chickens.

Asymmetries were determined for several bilateral traits in females from a line of chickens selected for 39 generations for low 56 day body weight (LWS) and in a subline of LWS where selection had been relaxed for four generations (LWR). Because of reduced food intake under ad libitum feeding, some LWS females do not commence egg production, a condition that can be overcome by relaxing selection for a generation or two. Bilateral traits, measured at 240 days of age in LWS non-layers, LWS layers, and LWR layers, were shank length and diameter, distance between the auditory canal and the posterior junction of the upper and lower mandible, and weight and length of the first primary wing feather. Other traits measured were body weights at 56, 168, and 240 days of age and age at first egg. Fluctuating asymmetry, a good overall measure of developmental stability, was lower in the relaxed than selected line. Means of relative asymmetries were also lower for LWR females than LWS layers and nonlayers which were similar.

Comparison of sex-linked dwarf genes in chickens from two sources when introgressed into unrelated genetic backgrounds.

1. Sex-linked dwarfing genes from 2 broiler stock origins (EU and US) were each introgressed into 2 White Leghorn populations that had been divergently selected for antibody response to sheep erythrocytes. 2. When the resulting backcrossed populations were 87.5% of their respective. White Leghorn line, non-dwarf pullets were assessed for body weights, shank lengths, immunoresponsiveness, age and body weight at sexual maturity, egg production, average egg weight, and duration of fertility. For measurements where there were no differences between non-dwarf pullets from the 2 origins of the dwarfing genes, then the dwarf pullets (which were full sisters to the non-dwarfs) were compared. 3. Shank length at 8 weeks of age and mature (24-week) body weights were higher for dwarf pullets from EU than US dwarf origin. Immune response and several egg production traits were higher for dwarf pullets from the high antibody backcross than from those of the low antibody backcross. 4. There were few differences in expression of the dwarfing genes from 2 origins in the unrelated backcross populations used in this study. Also each of the dwarfing genes, when introgressed into different genomic backgrounds, was not discernibly different in its expression in terms of antibody response or egg production characteristics.

Effects of season and diet on tensile strength and mineral content of the equine hoof wall.

Studies evaluating nutritional and seasonal influences on hoof strength and composition in horses, as well as the scientific justification for feeding supplements to improve hoof quality, are lacking. The horseman and veterinarian need controlled studies in this area to make informed decisions. This project quantified, in 2 trials, relative elasticity, tensile strength, % moisture, and mineral composition of hooves of 48 mature Thoroughbred mares maintained on different nutritional/management regimens, sampled quarterly over 12 month periods. Tensile strength was positively associated with sulphur content. Per cent moisture was not significantly associated with tensile strength, relative elasticity, or mineral content measures. Results indicated that both seasonal trends and nutritional regimes greatly affected hoof wall strength and mineral composition.

Responses of meat-type chickens to choice feeding of diets differing in protein and energy from hatch to market weight.

Chickens from three genetic stocks known to differ in growth potential consumed ad libitum either a single (control) diet or a choice of two diets that differed in protein and energy. Formulation of the choice diets was such that when mixed in specific proportions they provided single diets that decreased in protein and increased in energy over the experimental period. When comparisons of feeding regimens were made at a common age, body weights and feed efficiencies for all stocks were enhanced in chicks fed a single diet. When comparisons were made at a common body weight for controls, chickens fed the single diet were about 15% heavier than those given a dietary choice. For feed efficiency, however, the pattern remained for the faster growing stock whereas there was no difference between feeding regimens for the slower growing stock. Chicks provided a choice of diets had heavier abdominal fat pads and lighter breasts relative to body weight than those fed a single diet. With choice feeding, there were stock by diet interactions for dietary preferences through the first 9 d after hatch. Early on, the interactions resulted from the faster growing stocks exhibiting a greater preference for the diet higher in protein and lower in energy than the slower growing stock. By Day 5, however, the interaction occurred because stocks exhibited either no dietary preference or preferred the diet that was lower in protein and higher in energy. Regardless of genetic stock, at 9 d of age and thereafter there was a clear preference for the diet lower in protein and higher in energy than the diet higher in protein and lower in energy. These data for feed intake were consistent with behavioral observations that showed a preponderance of chicks eating from the feeder containing the diet lower in protein and higher in energy. Compared to a single diet, under choice feeding, energy utilization was negatively influenced more in the faster than slower growing stocks. Protein and sulfur amino acid utilization was not affected by feeding regimen in faster growing stocks, but was enhanced under choice feeding in the slower growing stock. Although lysine utilization was enhanced by choice feeding in all stocks, the effect was greater in the slower than in the faster growing ones. These data demonstrate that although broiler diets are formulated to enhance growth and overall feed efficiency, chicks that are provided a dietary choice of protein and energy do not eat to maximize growth or feed efficiency.

Genetic selection strategies--population genetics.

This paper provides an overview of the association between population genetics and selection strategies in poultry. Relationships between artificial and natural selection and among causes contributing to limits to artificial selection are discussed. Homeostasis and resource allocations at the individual and at the population level are reviewed. Examples from poultry demonstrate where human intervention has circumvented biological limits. Lastly, this paper considers the role of population genetics in future breeding strategies for poultry.

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.