Selection lines originated from an Institut Séléction Animal (ISA) Warren cross, selected for 15 nonoverlapping generations on high or low primary antibody response at day 5 after primary intramuscular immunization with sheep erythrocytes at 37 days of age. A random-bred control line was also maintained. Details regarding immunological methods and the selection process are described elsewhere (20, 31). Briefly, in each generation approximately 300 chicks were reared in each line. In each selection line, the 25–40 males and 50–70 females with most extreme antibody titers were selected to produce the next generation, and the control line was random bred. Realized selection differentials were in the order of 1.7–2.1 antibody titers per generation (31). Birds were individually housed in battery cages (floor 46 × 35 cm, height 40 cm) and were approximately 1 yr old when measured.
Blood samples (1 ml) were collected from the wing web within 3 min of removal from the cage. Plasma was stored at −20°C for 3 mos before analysis. Concentrations of testosterone were estimated by a solid-phase 125I RIA method (Coat-A-Count TKTT; Diagnostic Products, Los Angeles) according to the manufacturer by using duplicate aliquots of 50 μl plasma. Calculation of results was performed by applying a spline approximation using riasmart (Packard). The main crossreactivities were 3.3 and 0.5% for dihydrotestosterone and androstenedione, respectively, and
After the blood sample was taken, birds were weighed and morphometric data were collected with vernier callipers (to nearest 0.1 mm) by observers unaware of the selection regime from which a bird stemmed. Comb length (maximum length) and height (above the eye perpendicular to longitudinal axis of head) were measured and multiplied to give an index of comb size in cm2. Combs were clipped at hatch, and combs that were clipped unsuccessfully were not measured (birds with serrated edge of the comb were not measured; n = 5; three high line, one control line, one low line). Combs would have been larger in the absence of clipping, but because the immune system is not yet active at this age (32), and all lines were treated equally, there is no reason to expect that such an effect would differ between selection lines. Furthermore, independent support for the immunocompetence effect on comb size comes from the testosterone data (see Fig. 2, below). Tarsus and head lengths were measured as indicators of structural body size. Tarsus length was measured according to Svensson (33), and head length was measured from bill tip to back of head in a straight line over the eye. Tarsus and head length were correlated (r = 0.44, n = 87, P
Sample sizes in the present study were 28–30 males for each of the three lines. Data were analyzed by using analysis of variance and analysis of covariance. To enhance statistical power, the results of these analyses were combined with the ordered heterogeneity test (34), which yields a new test statistic (rsPc). This test is appropriate because the selection lines are ordered with respect to immunocompetence. Before analysis, testosterone titers were natural logarithm transformed to normalize the distribution.