We examined the energetic costs of immune activation by measuring both basal (BMR) and exercise-induced maximal metabolic rates (MMR) in house sparrows before and after being injected with either saline (sham) or two novel antigens (keyhole limpet haemocyanin and sheep red blood cells; KLH and SRBC, respectively) after the primary and two subsequent vaccinations. We also examined the effect of experimentally-induced breeding levels of testosterone (T) on immune responses and their metabolic costs in both males and females. 

Experimental animals

We captured free-living house sparrows (Passer domesticus) residing in the Illawarra region of NSW. The 31 males and 31 females were distributed among 4 outdoor flight cages (4.5 x 3.6 x 2.5 m) exposed to ambient conditions. Birds had free access to commercial finch seed mix (Golden Cob, Mars Birdcare Australia), mineralised grit, and water.

Experimental protocols

All male birds were bilaterally castrated, under anaesthetic, one or two days after initial capture and returned to the flight aviaries.  Birds were allowed two weeks to adjust to captivity before measuring their basal and exercise-induced maximal metabolic rates (BMR and MMR, respectively). Blood was then sampled to characterise hormonal and immune status, and each bird implanted with either an empty or testosterone-filled silastic tubule (see below). Metabolic evaluations and blood sampling were undertaken 2 weeks later to determine T levels and immune response parameters (below). Based on these MMR rankings, male and female birds were distributed randomly into 4 experimental groups: antigenic injection/testosterone implant (IC(immune challenge)/T), antigenic injection/control implant (IC/C), sham injection/testosterone implant (S/T), sham injection/control implant (S/C).

Testosterone implant procedure

Birds were implanted subcutaneously with either an empty silastic capsule (control birds) or a silastic tube filled with crystalline testosterone (Sigma Chemical Co.; T-1500). The capsules were prepared from 6 mm lengths of medical-grade silastic tubing (1.47 mm inner diameter; 1.96 mm outer diameter; Dow Corning), which were sealed at each end with silastic glue (Dow Corning). Birds were given anaesthetic (methoxyflurane) and had a small incision made to permit subcutaneous insertion of implants on one side of their thorax. The resultant wounds were sealed with medical-grade cyanoacrylate adhesive (Vetbond).

Immune challenge through hypervaccination

Two novel antigens, keyhole limpet haemocyanin (KLH; Sigma-Aldrich) and sheep red blood cells (SRBC; Institute of Medical and Veterinary Science, Adelaide, South Australia) were used to elicit adaptive immune responses in the immune-challenge groups. Inoclation using KLH followed Hasselquist et al. (1999 Behavioral Ecology and Sociobiology), with 1 mg KLH/ml sterile water emulsified 1:1 with Freund’s incomplete adjuvant (Sigma-Aldrich) for the immune challenge groups (IC/T and IC/C) and sterile water emulsified 1:1 with Freund’s incomplete adjuvant for the sham challenge groups (S/C and S/C).  Birds were injected with 100 ml of either mixture into their pectoral muscle. Immune-challenged birds were also injected intra-abdominally with 100 ml of SRBC (10% by volume in phosphate-buffered saline (PBS)), whereas sham-challenged birds were injected with 100 ml PBS. These injections were administered three times, 2-3 weeks apart, to achieve hypervaccination. Metabolic measurements and blood samples were collected 12 days after the first injection and 6 days after the second and third injections.

Quantifying adaptive immunity

Following venepuncture, ~200 μl of blood was collected from an alar vein in heparinised microhaematocrit tubes. Blood samples were immediately centrifuged at 6000 rpm for 5 min and plasma stored at -20 ˚C for hormonal (see Supplementary material) and immune analyses. Plasma concentrations of anti-KLH antibodies were determined using enzyme-linked immunosorbent assay (ELISA) described by Hasselquist et al. (1999 Behavioral Ecology and Sociobiology) and Martinez et al. (2003 Functional Ecology). Constitutive innate immunity and specific immunity to SRBC were determined by quantifying agglutination and lysis activity of plasma before and after exposure to SRBC, respectively, following Matson et al. (2005 Developmental and Comparative Immunology), modified to use 1% suspension of SRBC instead of rabbit RBC.

Basal metabolic rate

Measurement of BMR was conducted when birds were post-absorptive, during the birds’ rest-phase, and at temperatures within the thermoneutral zone for this species (29-31˚C). At least 3 h after they had last fed, birds were placed individually in 2-litre metal chambers fitted with a perch and supplied with room air at 500 ml min^-1 (Tylan mass flow controllers FC-280S). Oxygen content of inlet and outlet air for each chamber was measured using a Sable Systems Oxzilla II oxygen analyser in combination with an electronic stream selector (Sable Systems Respirometer Multiplexer V 2.0). Basal rates of oxygen consumption (BMR) were defined as the mean of the two lowest 5-minute periods of oxygen uptake recorded over the 12-hour measurement period.

Maximum metabolic rate

Oxygen consumption rates ( O₂) were measured during intense exercise within an enclosed 5-litre drum with clear sides and carpet lining the inner rim (18). A mass-flow controller (Tylan Corp.) supplied air to the chamber at 5 l min^-1 and the oxygen content of inlet and outlet ports was measured with an oxygen analyser (Sable Systems FC-1).  A single bird was placed in the drum and, once settled, the cover was removed and the drum rotated. The Ping-pong balls within the drum encouraged birds to maintain a series of rapid takeoffs and short-term flights interspersed with vigorous hopping. The  O₂ data were adjusted with ‘instantaneous’ conversion procedures to account for gas mixing characteristics of the wheel and accurately resolve short-term oxygen content variation. The highest continuous 60-sec instantaneous oxygen consumption rate was designated as MMR.

Statistical analysis

Immune parameters after each of the three injection stages were quantified as individual’s change, positive or negative, in a given parameter compared to their ‘baseline’ for that parameter. Changes were expressed as a difference above or below an individual’s baseline value (e.g. KLH_{change@stage1} = KLH_baseline – KLH_stage1). Metabolic parameters were likewise expressed as a departure from baseline (e.g. BMR_{proportion@stage1} = BMR_{stage1 -} BMR_baseline).

Description of the data variables and any missing values can be found in the README file. 

Please read the README file and contact the authors if you would like to reuse this dataset.