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Introduction The integrity of the gastro-intestinal tract and the intestinal absorption of nutrients may contribute to inter-individual variation in feed efficiency in beef cattle (Kenny et al., 2018). The aim of this study was to compare the intestinal epithelium integrity and the jejunum and ileum electrogenic transport in growing heifers from three different crossbreeds. Material and Methods Ethics committee of Switzerland approved all the procedures. Thirty growing heifers from three crossbreeding (Brown Swiss as dam and Limousin, Angus or Simmental as sire, n = 10 of each) were used. After weaning at the age of 2.5 months (mo), animals were fed according to two type of grass-based diets and were slaughtered at different ages leading to five experimental treatments (n = 6 for each): Grass silage-based diet continuously, slaughtered at 200 ± 10 days old (GS-7mo); 350 ± 10 days (GS-11mo) or 470 ± 10 days (GS-15mo); or grass silage-based diet until 7 mo and pasture only thereafter, slaughtered at 290 ± 10 days (PA-9mo) or 350 ± 10 days (PA-11mo). Intestinal segments from jejunum and ileum (210 cm and 5 cm cranium from ileo-cecal valve, respectively) were removed within 50 min after slaughter. To evaluate L-Glutamate (L-Glut), L-Arginine (L-Arg), L-Methionine (L-Meth) and D-Glucose (D-Glu) uptakes across intestinal tissues, jejunum and ileum segments were mounted on Ussing chambers. The trans-epithelial potential difference (TEER) and short-circuit current (Isc) were continuously monitored. Mucosal addition of 5 mM L-Glut was followed by the addition 15 min apart of L-Arg, L-Meth and D-Gluc at the same concentration. The results were analyzed by ANOVA with the R software (v 4.0.5). Linear mixed-effects regression (lmer) models were used as mixed models. Model contained the crossbreed, tissue (jejunum vs ileum) and treatment as fixed effects and the animal as a random effect. The interactions crossbreed x tissue and tissue x treatment were evaluated, while the interactions crossbreed x treatment and crossbreed x tissue x treatment were not addressed due to the reduced number of observations. For pairwise comparisons, Sidak function was performed for a modified Tukey test for multiple comparisons of means. Means and SEM were reported from the lsmeans function from the package emmeans. Residuals of lmer models were checked for normality and homoscedasticity. For L-Glut and L-Meth, the values were logarithmic transformed. Results and Discussion Whatever the slaughter age and dietary treatment, the jejunum TEER of the Limousin crossbreed was higher (P < 0.01) compared to the jejunum and ileum of the Angus and Simmental crossbreeds. Conversely, none differences were observed according to crossbreed or treatment in the amino acids and D-Glu uptakes (Table 1). The L-Arg uptake was lower (P = 0.01) in GS-7mo compared to GS-11mo, whatever the tissue segment (Figure 1). Conclusion and Implications The TEER measurements constitute a method to monitor the barrier integrity of epithelial layers (Chen et al., 2015) and it mostly depends by tight-junctions integrity. The barrier function of tight-junctions regulates host nutrition, maintenance of homeostasis and protection of the host against pathogen invasion that can cause severe intestinal infection (Gareau et al., 2010). Therefore, based on these results, we speculate that the Limousin crossbred heifers could be less susceptible to gastrointestinal disorders and further analyses are planned to further investigate the differences between the three crossbreeds in the tight-junctions protein expression. The reasons and the implications of the increased L-Arg uptake for GS-11mo treatment and subsequent decrease after 4 mo on the same diet will be also the subject of future studies.