Elsevier

Livestock Science

Volume 123, Issues 2–3, August 2009, Pages 215-220
Livestock Science

A novel cytometric approach to study intestinal mucosa rebuilding in weaned pigs fed with dietary nucleotides

https://doi.org/10.1016/j.livsci.2008.11.012Get rights and content

Abstract

In the weaning period, transition from sow's milk to the post-weaning diet causes the withdrawal of important nutrients as milk nucleotides, which are known to be determinant for the development of the gastrointestinal tract and immune function. The role of dietary nucleotides in the rebuilding of gut epithelium remains unclear. The aim of the study was to evaluate using appropriate markers the rate of mitosis (Ki67) in the crypt epithelial cell, and the rate of apoptosis (active caspase 3), autophagy (MAP I LC3) and DNA damage (p53) in the crypt and villi epithelial cells of the small intestine of weaned pigs fed diets supplemented without/with nucleotides. An in-tissue cytometry method, based on confocal imaging and automated quantitative analysis was implemented. The studies allowed us to understand molecular bases of animal performance which could not be accessed by a routine histometric approach. Namely, the dietary nucleotides provided more uniform small intestine epithelium with considerably less animal-to-animal variation in respect to mitosis (SEM = 1.02 in control vs. 0.62 in supplemented group), autophagy (SEM = 1.22 vs. 0.99) and expression of p53 protein (SEM = 0.72 vs. 0.28). A significant increase in ratio of apoptosis and autophagy, and significantly lower p53 expression was found in the nucleotide-supplemented pigs as compared to control. The mitosis/apoptosis index was lower in the nucleotide-supplemented group suggesting slower rebuilding of gut epithelium in these pigs as compared to the control, respectively 0.454 to 0.191. Finally, in the supplemented group a drop in p53 expression was observed, however, it remains uncertain whether the reduction in DNA damage index is due to the supplementation with nucleotides utilized as a source for repair processes or due to the reduced DNA alterations.

Introduction

Intestinal tissue poses a challenge for quantitative evaluation of growth and remodeling. Its complex architecture consists of a variety of cell and tissue types from different germ layers that tend to react in different way to the stimuli. In the present study we propose in-tissue cytometry, the quantitative automated method of evaluation of intestine mucosa remodeling based on the molecular markers of mitosis, programmed cell death (PCD) and DNA damage indexes. Microphotographs of immuno-labeled tissue sections were acquired by means of confocal microscopy and later quantitatively evaluated using automated image analysis software (Godlewski et al., 2007). At present, the most commonly accepted mitosis marker in the normal tissues is the expression of Ki67 protein which is required for cell progression through the division cycle as it organizes chromatin structure. It is expressed in every phase of cell-division cycle except G0 phase thus allowing quantification of whole population of dividing cells in the intestinal mucosa (Godlewski et al., 2006), but not the mature functional enterocytes. Among PCD, the apoptosis and autophagy are the most relevant for the rebuilding of small intestinal mucosa (Godlewski et al., 2005). The rate of apoptosis in the intestinal epithelium was analyzed using active caspase 3 expression as a marker of executive (irreversible) step of apoptosis. The rate of autophagy on the other hand was measured using MAP I LC3 expression as an only reliable marker of autophagosome formation (Yoshimori, 2004). In previous studies we demonstrated that the extent of mucosa rebuilding can be assessed by calculating the mitosis/apoptosis ratio (Godlewski et al., 2005, Godlewski et al., 2007). During intensive mitosis DNA replication errors are the common fact. Analysis of the expression of p53 protein (a so called genome guard) in the intestinal epithelium was used to measure the rate of DNA damage (Potten et al., 1994).

Weaning young piglets contributes to a number of gastrointestinal disorders and deaths in the intensive pig production. The problem is caused by the conflict between the physiological timing of gastrointestinal tract development and the trend to intensify animal production by reducing the number of days spent by the newborn pig with the sow. Among many strategies developed, the dietary nucleotides have been suggested to improve the development of the gastrointestinal tract and immune function after weaning. Martinez-Puig et al. (2007) showed that supplementation of weaned pig diet with a product containing the composition of nucleotides of sow's milk led to a lesser reduction in the villous height/crypt depth ratio as well as to a significant reduction of diarrhea and mortality as compared to control pigs. Their results are important for product commercialization but do not clarify the biological mechanisms involved in, for instance the rebuilding of intestinal mucosa which is crucial in understanding the digestive processes in fast growing pigs.

The aim of present study was to evaluate the intestinal remodeling in weaned pigs fed diets without/with nucleotide supplementation using a novel automated quantitative cytometry method based on confocal microscopy coupled with quantitative analysis by the image analysis software and the set of appropriate markers of mitosis in the crypts and apoptosis, autophagy and DNA damage in the crypt and villi epithelial cells.

Section snippets

Animal studies

The experiment was performed at the Experimental Unit of the Universitat Autònoma de Barcelona and received prior approval from the Local Ethical Committee for Animal Experimentation of the institution. The treatment, housing, husbandry and slaughtering conditions conformed to the European Union Guidelines (The Council of the European Communities, 1986). Twelve piglets (Landrace × Large White × Duroc) from six litters were selected and randomly divided in 2 groups (n = 6) according to body weight,

Results

Animal performance, health/mortality and gastrointestinal histometry data were published earlier by Martinez-Puig et al. (2007). Concerning the in-tissue cytometry results, no significant differences in the range of mitosis (Fig. 2) in the intestinal crypts epithelium between the control and supplemented pigs were found (Table 1). In contrast to control, pigs fed with the diet supplemented with nucleotides showed little animal-to-animal variability of measured intestinal mucosa parameters. The

Discussion

Until recently the most common method used to characterize the gut mucosa was its thickness and the villous height/crypt depth ratio (Pluske et al., 1996, Martinez-Puig et al., 2007). Unfortunately it does not reflect solely the maturation changes. The crypt size depends on the intensity of water and electrolyte secretion by the crypt enterocytes, and little if any, depends on the intensity of cell mitoses since there are less then 10% dividing cells in the crypts (Godlewski et al., 2005,

Conclusion

The proposed automated in-tissue-cytometry quantitative approach proved valuable in accurate description and better understanding of the molecular mechanisms involved in the dynamic process of small intestine epithelium remodeling. Presented cytometry results when read together with morphological and functional data (Martinez-Puig et al., 2007) indicate a coincidence between the low mitosis/apoptosis and DNA damage indexes and the improved animal health. It also helps to understand the way by

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