# Comparative Evaluation of High-Throughput In Vitro Digestion Methods for Predicting In Vivo Digestibility and Fecal Odor Emissions in Pigs

## TL;DR

This study compares lab methods to predict how pigs digest feed and produce manure odors, finding that dialysis-based methods best mimic real digestion and reduce the need for animal testing.

## Contribution

The study introduces a combined shaking plus dialysis method that balances accuracy and efficiency in predicting pig digestion and odor emissions.

## Key findings

- Dialysis-based methods (D and SD) better represent real digestion by preventing enzyme inhibition from metabolite accumulation.
- The SD method offers a practical balance between accuracy and efficiency for predicting digestibility and odor compounds.
- The D method achieved the highest agreement with in vivo digestibility and fecal profiles of odor-related compounds.

## Abstract

Predicting how pigs digest feed and produce manure odors usually requires animal trials, which are costly, time-consuming, and raise animal welfare concerns. This study tested three laboratory-based (in vitro) digestion methods—shaking (S), dialysis (D), and a combined shaking plus dialysis (SD)—to see how well they could mimic what actually happens in pigs at different growth stages. After simulating stomach and small-intestinal digestion, we let pig fecal microbes ferment the remaining feed. We found that methods that removed digested products by dialysis (D and SD) better represented real digestion because they prevented build-up of metabolites that can block enzymes. The D method gave the most accurate predictions of whole-tract digestibility, while the SD method offered a practical balance between accuracy and efficiency. Both D and SD closely matched real pig manure in terms of odor-producing compounds and microbial enzyme activities. Overall, the SD method provides a reliable, high-throughput laboratory tool to evaluate pig feeds and predict manure odors, reducing the need for animal testing and helping design diets that improve nutrient use and lessen environmental impacts.

Accurate and physiologically relevant in vitro models are needed to predict nutrient digestibility and hindgut fermentation in pigs, as conventional in vivo trials are resource-intensive and raise animal welfare concerns. This study evaluated and compared the predictive performance of three in vitro digestion approaches—shaking (S), dialysis (D), and a combined shaking plus dialysis (SD) method—for estimating in vivo apparent total tract digestibility (ATTD) and fermentation characteristics across weaning, growing, and finishing pigs. Commercial diets were subjected to simulated gastric and small-intestinal digestion using S, D, or SD, followed by fecal inoculation to model hindgut fermentation for 12 and 48 h. During the gastrointestinal phase, crude protein digestibility was highest with D (>75%), intermediate with SD, and lowest with S (50–60%), indicating that product removal by dialysis mitigated enzyme inhibition from metabolite accumulation. After 48 h of fermentation, all methods showed strong linear correlations with in vivo ATTD (r > 0.93), but only D achieved high absolute agreement (Lin’s CCC > 0.95 for dry matter and crude protein). Moreover, D and SD at 48 h closely reflected in vivo fecal profiles of skatole, indole, and microbial enzyme activities, with D at 12 h showing an especially strong correlation for protease (r = 0.98). While D provided the most precise predictions of absolute values, the SD method offered an optimal balance between physiological relevance and operational efficiency, supporting its use as a robust, high-throughput platform for porcine feed evaluation and fecal nitrogenous odorant prediction.