Animals and housing

Ethics approval for the animal trial was obtained from the Animal Ethics Committee, Massey University, Palmerston North, New Zealand (application number 17/05). Entire male pigs (n 36, PIC Camborough 46 × PIC boar 356L, mean 22·3 (se 0·32) kg bodyweight) were obtained from a commercial farm. Pigs were housed individually in metabolism crates in a room maintained on average at 22°C with a 12 h/12 h light/dark cycle. Cages and toys were washed daily. Toys were rotated daily. During the feeding and cleaning, pigs were monitored for general health, alertness, dietary intake and scouring using a scoring system to determine whether pigs remained in the study. The endpoints to exclude pigs from the study were food intake, lethargy and diarrhoea.

Diets and experimental design

Pigs were randomly allocated to each of the post-feeding time points, and some researchers and technical support staff were aware of this allocation. Pigs received a semi-synthetic diet for 8 d to ensure that they were adapted to the environment and to the diet. The adaptation diet contained wheat starch (521·2 g/kg DM diet), soyabean oil (140 g/kg DM), sucrose (100 g/kg DM), cellulose (40 g/kg DM), dicalcium phosphate (20·5 g/kg DM), vitamin and mineral premix (3 g/kg DM), salt (3 g/kg DM), the antioxidant Endox ® (0·3 g/kg DM) and whey protein isolate (172 g/kg DM; NZMP^TM Whey Protein Isolate 8855, Fonterra Co-Operative Group Ltd) as the sole protein source. To improve palatability, the diet was mixed with 400 ml of tomato soup (20 g Maggi-rich tomato soup mix (14 g carbohydrates, 0·5 g protein, 0·3 g fat), Nestlé New Zealand Limited). The diet fulfilled the nutrient requirements of the growing pig⁽⁹⁾. During the feeding period, pigs received two equal meals (2 % bodyweight/meal) at 08:00 and 17:00 h. However, on the final day of the feeding period, the last meal for each pig was provided at different times to ensure a 12 h fasting period on the following test day.

On day eight, pigs were deprived of water for 2 h before receiving their final morning test meal. The test meal did not include the vitamin and mineral supplements or the antioxidant contained in the adaptation diet^{(Reference Montoya, Maier and Banic10)}, as the test meal was also consumed by humans in a parallel but separate study. The indigestible marker TiO₂ (1 g) was included in the test meal to allow the determination of its concentration in each GIT location. The test meal was given to the pigs after a 12 h fasting period, apart from a group of pigs (n 6) that were euthanised directly. The latter group of pigs received TiO₂ in the last meal on day seven, and for the calculations, they were considered as being 12 h post-feeding of the last meal. The remaining pigs were anaesthetised 15 min before being euthanised at 1, 2, 3, 4 or 6 h post-feeding (n 6 pigs/post-feeding time). Considering the sampling time required for each pig (∼25 min), pigs received their last meal at 30 min intervals to ensure that they were euthanised at the assigned post-feeding time. In addition, the same number of animals per post-feeding time was euthanised in a day. The anaesthetic cocktail (0·12 ml/kg bodyweight of Zoletil 100 (50 mg/ml), Ketamine (50 mg/ml) and Xylazine (50 mg/ml); Provet) was administered by an intramuscular injection. Pigs were euthanised by an intracardiac injection of sodium pentobarbitone (0·3 ml/kg bodyweight of Pentobarb 300; Provet).

The body cavity was opened, and the stomach (at the oesophageal and pyloric sphincters), terminal ileum (at the ileocaecal valve) and the terminal rectum were isolated with clamps and the whole GIT was dissected out as described previously^{(Reference Montoya, Maier and Banic10)}. The stomach and the terminal ileum (last 30 cm before the ileocaecal junction) were then removed, and the remaining small intestine was uncoiled and divided into two equal lengths (proximal and distal small intestine, PSI and DSI). To ensure that all chyme and digesta were collected, the stomach and each small intestinal region were gently flushed several times with a saline solution (0·9 g NaCl/l). The caecum was removed, and the colon (including the rectum) uncoiled. Faeces excreted post-feeding were collected. The full caecum and colon were weighed, opened longitudinally, digesta were collected and caecal and colonic tissues were then washed, dried with paper towels and weighed again to determine total digesta on a wet basis. This was considered to be the most accurate way to determine total digesta in these GIT locations, as digesta adhere to these tissues. Caecal and colonic digesta, in contrast to small intestinal digesta, are less easily collected with flushing. Caecal and colonic (including faeces) digesta were thoroughly mixed before taking a representative weighed aliquot. Chyme and digesta (PSI, DSI, terminal ileal, caecal and colonic) were immediately frozen in dry ice, stored at –20°C, freeze-dried and ground. Chyme and small intestinal digesta samples (without terminal ileum) for each animal were pooled. The amounts of DM for the representative aliquots of caecal and colonic digesta were then used to calculate the total DM contents in the caecal and colonic digesta.

Chemical analysis

The test meal, stomach chyme, digesta (PSI, DSI, terminal ileal, caecal and colonic) and food refusals were analysed for DM and TiO₂ ^{(Reference Short, Gorton and Wiseman11)}. The test meal, food refusals, pooled digesta and terminal ileal digesta were analysed for standard AA (using HCl hydrolysis, o-phthalaldehyde pre-column derivatisation followed by reversed-phase HPLC)^{(Reference Rutherfurd, Bains and Moughan12)} and tryptophan (alkali hydrolysis)^{(Reference Rutherfurd, Richardson and Moughan13)}. The test meal was also analysed for starch (Kit AA/AMG, Megazyme), crude protein (nitrogen × 6·25; using an elemental analyser LECO), total fat (using a Soxhlet apparatus and petroleum ether extraction) and total dietary fibre^{(Reference Prosky, Asp and Schweizer14)}.

Calculations

The amount of each AA (AA_i) in the diet, food refusals, pooled digesta and terminal ileal digesta at each post-feeding time were calculated as shown below with terminal ileal digesta as an example. The same calculations were used to determine the TiO₂ content (g DM). The TiO₂ content of the stomach, PSI and DSI were summed to determine the TiO₂ content of the pooled sample.

$$\matrix {\rm AA_i\,content _{Terminal \,ileal \,digesta} (mg \,in \,DM \,basis)\cr=\rm AA_i \,concentration _{Terminal\, ileal\, digesta} (\%)\cr \qquad\quad\times \rm Total\,content _{Terminal\, ileal\, digesta} (g \,DM)/100}$$

The relative amounts of DM and TiO₂ exiting the stomach over time (time_i as 1, 2, 3, 4, 6 and 12 h post-feeding) were determined as follows (DM as an example):

$$\matrix {\rm Relative\, DM\, exiting\, the\, stomach\, (g \,in\, DM\, basis)\cr \rm = (DM _{Intake}-DM _{Time \,i})/DM _{Intake} \times 100}$$

To ascertain the importance of AA escaping the small intestine into the large intestine, it was necessary to determine the amounts of AA released into the large intestine. Considering that the AA in large intestinal digesta do not represent the AA escaping the small intestine, as many are expected to be of microbial origin, the AA released into the large intestine at each post-feeding time were determined considering the sum of TiO₂ content in the caecal and colonic digesta and the ratio of AA content/TiO₂ content at the terminal ileal digesta (Direct method). Alternatively, the AA released into the large intestine can be calculated based on the TiO₂ ingested in the meal and the TiO₂ measured in the upper GIT (stomach to terminal ileum) (Indirect method). Both values were then used to determine the corrected apparent AA absorption in the GIT lumen as follows:

Direct method: AA_i released into the large intestine (mg in DM basis) = (TiO₂ content_{Caecal digesta} + TiO₂ content_{Colonic digesta}) × AA_i content_{Terminal ileal digesta}/TiO₂ content_{Terminal ileal digesta}

Indirect method: AA_i released into the large intestine (mg in DM basis) = (TiO₂ content_Diet – (TiO₂ content_{Pooled digesta} + TiO₂ content_{Terminal ileal digesta})) × AA_i content_{Terminal ileal digesta}/TiO₂ content_{Terminal ileal digesta}

Apparent AA_i unabsorbed (mg in DM basis) = AA_{i pooled digesta} + AA_{i terminal ileal digesta} + AA_{i released into the large intestine}

Apparent AA_i absorbed (mg in DM basis) = AA_i content_Diet − (Apparent AA_i unabsorbed + AA content_Refusal)

Apparent AA_i absorption (%) = (AA_icontent_Diet − (Apparent AA_i unabsorbed + AA content_refusal)) / (AA_icontent_Diet− AA content_refusal) × 100

The mean food DM intake and DM in pooled digesta (stomach + PSI + DSI), terminal ileum and the large intestine as well as the lysine and TiO₂ concentrations are shown in the Supplemental Materials and Methods to demonstrate the calculations described above. Data related to the study are available upon request.

One of the pigs at 4 h post-feeding did not have enough ileal digesta for both the AA and TiO₂ analyses but enough contents in the remaining GIT locations. The TiO₂ content of the remaining GIT locations was determined and 4 % of TiO₂ reached the large intestine. To estimate the amount of AA released into the large intestine for this pig, a ratio between the average amount of AA released and the average amount of TiO₂ in the large intestine for the same post-feeding time calculated over all the relevant pigs was calculated and multiplied by the amount of TiO₂ in the large intestine for the specific animal.

Statistical analysis

For the study, a sample size of six replicates per time point was deemed satisfactory (>80 % at a two-tailed 5 % significance level), based on an effect size of 1·86 obtained from data reported for the small intestinal AA digestibility^{(Reference Montoya, Cabrera and Zou15)}.

Statistical analyses were performed using SAS (SAS/STAT version 9.4; SAS Institute Inc.). A paired-t-test was performed to compare the determined and predicted amounts of TiO₂ released into the large intestine. A polynomial analysis was conducted for the amounts of AA released into the large intestine. The best polynomial model (up to third order) for each response variable was selected after comparing higher- v. reduced-order models using the log-likelihood ratio test. Probability values of P ≤ 0·05 were considered of statistical difference, and 0·05 < P < 0·10 were considered a trend.

Non-linear models were fitted to the data, and the PROC NLIN of SAS was used to estimate the parameters of different models. To determine the transit time of the diet in each GIT location (stomach, PSI, DSI, terminal ileum, caecum and colon), the power exponential model (TiO₂ remaining_Time = α ₀ exp – [κ × Time] ^β ) was first used. α ₀ is the amount of TiO₂ consumed (0·94 g), β is the index of the curve and κ is the slope of the curve. β and κ were then used to determine the 10 % cumulative transit time (CTT₁₀ = [1/κ] × [log[1/0·9]]^[1/β]). The time difference between cumulative transit times of different GIT locations was used to calculate the transit time of specific GIT locations (e.g. TT_10PSI, h = CTT₁₀ stomach and PSI – CTT₁₀ stomach).

Based on the sigmoidal shape observed over time for the apparent relative AA absorption, different non-linear models (modified Weibull equation, Chapman–Richard equation, Logistic function and Gompertz function)^{(Reference Sit, Poulin-Costello, Bergerud and Sit16)}, commonly used for sigmoidal parameters, were firstly fitted for Asp, Glu and Leu. For these AA, the Gompertz function better fitted the data (online Supplementary Table 1). However, with the Gompertz model the point of inflection is not symmetric (i.e. the half time, T₅₀ of apparent AA absorption cannot be determined). Thus, the Logistic function (relative absorption_Time = α/[1 + exp ^{[β – (γTime)]}]), which was the model with the second best fit (online Supplementary Table 1 and Supplementary Fig. 1), was fitted for all AA. The asymptote α represents the extent of apparent AA absorption or the apparent amount AA absorbed. The slope γ, or apparent absorption rate coefficient at α/2 (or inflection point), controls the shape of the curve and β shifts the curve along the X-axis. β and γ were then used to determine the point of inflection for α (T_α/2 AA_i h = β/γ) and the time of 50 % for absorption of each AA (T₅₀ AA_i h = β/γ).

The model diagnostics for each response variable were tested after combining the PROC UNIVARIATE and the ODS GRAPHICS procedures of SAS. A natural log or square root transformation of the AA released into the large intestine was required to fulfil the model assumptions of normality and homoscedasticity. The mean values reported are both transformed and back-transformed.