Acquired Pigmentation of Porcine Lymph Nodes: Dietary Polyphenolic Compounds as Biological Markers?

Abstract

The investigation aimed at exploring whether 1) high contents of natural polyphenols from the diet can induce pigment accumulation in lymph nodes (LNs); 2) if so, whether polyphenolic compounds and derivates can be used as biological markers; 3) and whether a lymph node from a specific anatomical region can be univocally identified, so as to be considered as a sentinel for the identification of the dietary origin of pigments. A paired match approach was used to switch 20 pigs (range of initial body weight, BW: 113 -121 kg) to two experimental diets, for four weeks: ten pigs (pair housed) were fed with an experimental acorn based diet (acorns: 50% in the diet, as fed; total polyphenols, 78.1 g TP/Kg DM in the diet; tannic acid equivalent, 25.8 g TAE/kg DM); the remainder ten, received a pelleted complete diet for finishers (0% acorns in the diet). Daily feed intake in the last two weeks of the experimental feeding was recorded per pair of pigs in both groups of animals, showing an average intake of 610 mg TAE/kg BW/d. At an average final BW of between 127 to 137 kg, all pigs were slaughtered and LNs from different anatomical regions of the carcass were removed and analysed. At gross inspection, LNs from both groups displayed different grades of intensity and diffusion of pigmentation: a partial and incidental pigmentation was randomly detected in renal or sub-iliac LNs in the control group; a constant and uniform pigmentation of LNs was observed in acorns fed pigs: a dark brown staining diffused to the whole LN associated with a brownish colour of the muscles was found systematically. At light microscope intracytoplasmic granules were found in macrophages and dendritic cells from both groups, but, at confocal laser analysis, an intense auto-fluorescence was observed in medial-iliac LNs from the carcasses of acorn-fed pigs (green emission). However, intracellular sources of blue and green fluorescence at different wavelengths, likely due to tryptophan, indoleamine and derivates were also found in medial-iliac and inguinal LNs from the control group. A dietary origin was attributed to the different discoloration of LNs between the carcasses of the two groups: such acquired pigmentation is relevant in the left sub-iliac LN, but the confocal laser microscopic test to elicit auto-fluorescence of polyphenolic compounds (biological markers) displayed a 76.9% specificity, despite a 100% of sensitivity for the univocal identification of the carcass from acorn-fed pigs. Cranial sternal LNs resulted to suit the sentinel role in the distinction of carcass from acorns fed pigs at confocal laser microscopic analysis.