Equipment
Respiration boxes of about 2 m3 volume were built around commercial farrowing crates and fitted with a feeder and drinker. Air was drawn through these boxes via an inlet at the rear and an outlet above the trough at rates of approximately 240 L/min. After passing through a cold water condenser to remove water from the air, air flow was divided between a series of gas washing bottles for CO2 collection and a separate air line for gas analysis ('bypass'). The flow rate through each diversion was measured with two commercial air meters (Canadian Meter Corp., Cambridge, Canada). The CO2 collection stream consisted of five gas washing bottles (four in parallel and a final bottle) followed by a solvent trap. Part of the bypass stream of air was drawn with a small air pump (Gast Model 0531, Gast Mfg. Corp., Benton Harbour, MI) and delivered to oxygen (Taylor-Servomex, Crowborough, UK) and CO2 analysers (Beckman LB2, Beckman, Irvine, CA). Air flow to the analysers was regulated by ball-type flowmeters (Scienceware Size 2, Fisher Scientific, Mississauga, Canada). The analog output (mV) was converted to digital data by an analog-digital converter (Datagrabber, Data Electronics, Australia) and recorded by a computer. Data acquisition was set for maximum rate (four readings per second) and the average gas concentration for each minute was recorded.
Calibration of respiration systems
The equipment was pressure-tested to ensure that the system contained no air leaks. The recovery of CO2 and 14CO2 by the system was determined by releasing a known quantity of CO2 and 14CO2 in the respiration chambers. Four moles of NaHCO3, equivalent to the hourly CO2 production of a 250-kg sow being fed maintenance energy intake, were dissolved in 4 L of water and spiked with 37 kBq of NaH14CO3 (American Radiolabeled Chemicals, St. Louis, MO). The solution was placed on a magnetic stirrer inside the respiration chamber and air was collected immediately thereafter. The CO2 was released by the addition of 4.5 mol of concentrated HCl over 1 h using syringe pumps. After 2 h of air collection in 30 min collection intervals, the quantitative release of CO2 was confirmed by sampling the bicarbonate solution and measuring the remaining radioactivity. The CO2 concentration in the bypass flow was recorded at 1 min intervals throughout this 2 h period.
On each study day, the O2 and CO2 analysers were calibrated for their zero and gain readings with either pure N2 (zero) or calibration gas (21% O2, 1.5% CO2, 77.5% N2). The average readings for the zero and calibration gases were each recorded for 5 min immediately before and after each study to correct for any change in analyser readings during the study.
Collection of CO2
Each gas washing bottle was filled with 100 mL of CO2 absorber solution (monoethanolamine:2-methoxyethanol, 1:2; Caledon Laboratories, Edmonton, Canada). After each 30 min collection, the pooled volume in the first four bottles and separately, in the fifth bottle, were weighed and sampled. For the first four bottles, 0.5 g of the sample was weighed in a scintillation vial, diluted with 0.5 ml of 2-methoxyethanol, and mixed with 5 mL of a scintillation cocktail (Atomlight, Canberra Packard, Mississauga, Canada); for the fifth bottle, 1 g was weighed and mixed with 5 mL of scintillant. Absorber from the final solvent trap was also weighed, sampled and counted at the end of each study day to verify complete collection. All samples were collected in duplicate and counted in a scintillation counter (Beckman LS 3000, Beckman, Irvine, CA) for 15 min or to a 2 σ error of 2%.
CO2 recovery in animals: experimental design and diets
Six adult sows (235 kg, SD 5), non-pregnant after their fourth parity, were surgically implanted with catheters in the cephalic vein for isotope infusion. After surgery, they were left to recover for at least 7 d. Between studies, the animals were housed individually in pens 1.5 by 2.1 m fitted with rubber mats. The animal room was temperature controlled at 21°C. All procedures were approved by the Faculty Animal Policy and Welfare Committee of the faculty Agriculture, Food and Human Ecology, University of Alberta.
The semi-synthetic diet contained (g/kg diet) casein (27.5), cornstarch (200.0), corn oil (20.0), sulkaflok (150.0), sugar (527.4), L-cystine (1.67), L-threonine (1.96), L-tryptophan (0.27), L-isoleucine (0.24), L-aspartate (2.02), L-glutamate (2.02), chromic oxide (5.0), limestone (11.0), di-calcium-phosphate (23.0), magnesium sulfate (1.0), potassium bicarbonate (6.2), sodium bicarbonate (3.5), sodium chloride (2.2), thiamin (0.005), vitamin B6 (0.005), vitamin and mineral premix (15.0); the premix contained (per kg) Cu (2000 mg), I (20 mg), Fe (15 g), Mn (1200 mg), Se (30 mg), Zn (12 g), vitamin A (1,000,000 IU), vitamin D (100,000 IU), vitamin E (8000 IU), vitamin K (200 mg), biotin (20 mg), choline 100,000 mg), folacin (160 mg), niacin (4000 mg), pantothenic acid (2500 mg), riboflavin (1200 mg), and vitamin B12 (3000 μg). This diet provided twice the maintenance requirement for amino acids and at least 120% of the mineral and vitamin recommendations of NRC [5] for gestating sows. The diets were offered to exceed the maintenance energy requirement [5] by 10%. Between study days, the animals were fed twice daily. On study days, sows were offered half their daily allowance in two modes: a single meal 2 h into the infusion experiment after 12 h of food deprivation (F1), or, 2 to 4 days later, in eight hourly meals starting 2 h before the infusion experiment (F8). The hourly feeding regimen was imposed on the day of study only. Each animal was subjected to both treatments in a randomized crossover manner.
Isotope infusion
The recovery of 14CO2 was determined during primed, constant infusions of a saline solution containing NaH14CO3. The priming dose of 20% of the hourly infusion dose of NaH14CO3 was administered within 1 min. The constant dose was infused by syringe pump (Fisher Scientific, Mississauga, Canada) at an approximate rate of 95 kBq/h for either 8 h (F1) or 4 h (F8); the infusion solution was weighed and sampled immediately after each infusion to measure actual radioactivity delivered. Throughout the study, 14CO2 was collected in 30-min intervals. Because repeated measurements were conducted in each animal, the background radioactivity in breath was determined during a 30 min CO2 collection before each isotope infusion. This background collection was started as soon as the CO2 content in the air drawn from the box had reached an equilibrium as measured by the CO2 analysers. Oxygen consumption and CO2 production were monitored in 1 min average measurements throughout the infusion studies.
Calculations
The air flow in the respiration system was corrected to 0°C and an atmospheric pressure of 760 mm Hg. Oxygen consumption and CO2 production were calculated by multiplying the corrected air flow by the 1 min average gas concentration after correcting for room air. The respiratory quotient (RQ) was calculated by dividing the CO2 production by the O2 consumption. The energy expenditure was calculated using caloric factors for CO2 and O2 (Brouwer formula) [6]. Methane production and urinary N excretion were omitted from the formula because they were not measured. The CO2 production was divided into three phases: 1) the fasting phase in F1 prior to feeding, 2) the period of high CO2 production immediately after each feeding in both F1 and F8, and 3) the postprandial phase following this period of high CO2 production in both F1 and F8. A non-significant regression of CO2 production on time was used to define plateaus during these phases. The resulting time periods were used to estimate the O2 consumption, RQ and heat production during the three phases. The effect of digestion was calculated by subtracting the fasting data from the postprandial data. The effect of physical activity was estimated by subtracting the data from the postprandial phase from the data directly after feeding. To compare the CO2 production to the 14CO2 recovery, 30-min values were calculated for the CO2 production to coincide with the collection of 14CO2.
Statistics
Data are presented as means and SD. One-minute means were calculated within feeding frequencies and phases assuming 'sow' to be a random effect. Data across three phases within F1 feeding were analyzed by ANOVA using Fisher's (protected LSD) multiple comparisons between groups and were considered significant when P < 0.05 [7]. Differences between the two phases of F8 feeding as well as the effects of the feeding regimen on CO2 excretion, O2 consumption, 14CO2 recovery, and RQ were evaluated using Student's t-test. The relationship between CO2 production and 14CO2 recovery was assessed using Pearson's correlation coefficients.