Quantification of stress sensitive markers in single fecal samples do not accurately predict excretion of these in the pig

Abstract

All feces produced during 24 h were collected from five pigs and cortisol and immunoreactive cortisol metabolites (CICM), and IgA were quantified. Within pigs, the concentrations of CICM and IgA varied extensively between random samples obtained from a single fecal dropping, and deviated in most cases significantly from the true concentration measured in total fecal output (CV 6.7–130%). The CICM and IgA contents varied considerably (CV 8.1–114%) within and between individual fecal droppings from the same pig compared to the total fecal excretion. In conclusion, single random samples could not be used to reliably quantify the total fecal concentration or excretion of CICM or IgA in pigs. Analyses of all feces collected during shorter periods than 24 h did not provide an accurate estimate of the daily excretion of CICM. Thus, the concentration of stress sensitive molecules in random single fecal samples as an indicator of animal welfare should be interpreted with prudence.

Introduction

There is considerable public concern about the welfare of farm animals as well as laboratory animals. Unfortunately, there are few methods available to reliably assess animal welfare non-invasively. A method that would make it possible to objectively compare the impact on animal welfare of different housing and husbandry conditions as well as of different modes of transportation with the aim to improve the well-being of domestic and laboratory animals is highly desirable.

Non-invasive sampling methods, based on quantification of stress sensitive molecules, are important in objective assessment of animal welfare as an alternative to quantification of such molecules in blood. Blood sampling usually involves restraint of the animal and may often be perceived as painful, which is likely to stress the animal. Analyses of the concentration of corticosteroids and their metabolites as well as the concentration of immunoglobulin A (IgA) in feces are increasingly used with the aim to monitor stress of short and long duration, respectively (Miller et al., 1991, Monfort et al., 1998, Whitten et al., 1998, Möstle and Palme, 2002, Eriksson et al., 2004, Royo et al., 2004). Corticosteroids are predominantly excreted via the urine in most species (Palme et al., 1996). However, collection of urine requires either restraint or other manipulation of the animal or the use of metabolic cages, procedures which may confound the results (Hopster et al., 1999, Cook et al., 2000). Although, urine is simpler to analyze, feces is preferred for non-invasive sampling especially in a field situation. Measuring molecules in excretions like feces must be assumed to be complicated by multiple sources of error. Firstly the excretion rate and volume of feces are influenced by the psycho-physiological state of the animal as well as the intake of water and food. Secondly feces may not constitute a homogenous biological compartment like blood or to a certain extent even urine does. Thirdly, feces contain a plethora of bacteria and enzymes that may rapidly degrade and transform large molecules rendering uniform sampling, storing, sample processing and assaying conditions crucial for meaningful analyses of fecal molecules. Possible coprophagy may, under normal housing and husbandry conditions, further complicate the estimation of excreted molecules.

When measuring fecal corticosteroids many scientists have chosen to simply measure concentrations in fecal samples. In rats, however, variation in concentration of fecal markers between individual neighboring fecal pellets is substantial (Hau et al., 2001, Pihl and Hau, 2003). The time of day when the sample was collected is not always stated in the reports, although it is well known that the concentration of corticosteroids vary with the diurnal rhythm (Barnett et al., 1981, De Jong et al., 2000, Pihl and Hau, 2003).

The aims of the present study were, using the pig as a model, to investigate (i) stability of immunoreactive CICM and IgA in feces, (ii) the variation of water contents in fecal droppings and its implications for quantification of the two markers, (iii) the spatial distribution or variation of CICM and IgA in fecal droppings, and (iv) to devise a method to estimate the daily excretion of CICM and IgA in pig feces.