Salivary Gland Adaptation to Dietary Inclusion of Hydrolysable Tannins in Boars

Abstract

The ingestion of hydrolysable tannins as a potential nutrient to reduce boar odor in entire males results in the significant enlargement of parotid glands (parotidomegaly). The objective of this study was to characterize the effects of different levels of hydrolysable tannins in the diet of fattening boars (n = 24) on salivary gland morphology and proline-rich protein (PRP) expression at the histological level. Four treatment groups of pigs (n = 6 per group) were fed either a control (T0) or experimental diet, where the T0 diet was supplemented with 1% (T1), 2% (T2), or 3% (T3) of the hydrolysable tannin-rich extract Farmatan^®. After slaughter, the parotid and mandibular glands of the experimental pigs were harvested and dissected for staining using Goldner's Trichrome method, and immunohistochemical studies with antibodies against PRPs. Morphometric analysis was performed on microtome sections of both salivary glands, to measure the acinar area, the lobular area, the area of the secretory ductal cells, and the sizes of glandular cells and their nuclei. Histological assessment revealed that significant parotidomegaly was only present in the T3 group, based on the presence of larger glandular lobules, acinar areas, and their higher nucleus to cytoplasm ratio. The immunohistochemical method, supported by color intensity measurements, indicated significant increases in basic PRPs (PRB2) in the T3 and acidic PRPs (PRH1/2) in the T1 groups. Tannin supplementation did not affect the histo-morphological properties of the mandibular gland. This study confirms that pigs can adapt to a tannin-rich diet by making structural changes in their parotid salivary gland, indicating its higher functional activity.

Keywords: dietary supplements; histology; immunohistochemistry; mandibular gland; parotid gland; pigs; proline-rich proteins (PRP); tannins.

Figure 1

Serous acini in parotid glands from fattening boars fed diets supplemented with different tannin levels. Group fed with the standard mixture (T0; control group), and the standard mixture supplemented with 1% (T1), 2% (T2), or 3% (T3) tannin-rich extract Farmatan^®. Goldner’s Trichrome method, 10× magnification.

Figure 2

Nucleus to cytoplasm ratio measured in parotid glands. Groups were fed with a standard mixture (T0; control group), or the standard mixture supplemented with 1% (T1), 2% (T2), or 3% (T3) tannin-rich extract Farmatan^®. ^a,b Different superscript letters indicate significant differences (p < 0.05).

Figure 3

Immunohistochemical staining with antibodies against basic proline-rich proteins (PRB2) in the parotid gland, 40× magnification. Groups were fed with the standard mixture (T0; control group), or the standard mixture supplemented with 1% (T1), 2% (T2), or 3% (T3) tannin-rich extract Farmatan^®.

Figure 4

Assessment of immunohistochemical staining with antibodies against basic proline-rich proteins (PRB2) in the parotid salivary gland. (a) Distance to black; (b) RGB channel (red, green, blue). Groups were fed with the standard mixture (T0; control group) or standard mixture supplemented with 1% (T1), 2% (T2), or 3% (T3) tannin-rich extract Farmatan^®. Values represent mean ± SEM for each channel. ^a,b Different superscript letters indicate significant differences (p < 0.05).

Figure 5

Immunohistochemical staining with antibodies against acid proline-rich proteins (PRH1/2) in the parotid gland, 40× magnification. Groups were fed with the standard mixture (T0; control group) or standard mixture supplemented with 1% (T1), 2% (T2), or 3% (T3) tannin-rich extract Farmatan^®.

Figure 6

Assessment of immunohistochemical staining with antibodies against acidic proline-rich proteins (PRH1/2) in the parotid salivary gland. (a) Distance to black; (b) RGB channel (red, green, and blue). Groups were fed the standard mixture (T0; control group) or standard mixture supplemented with 1% (T1), 2% (T2), or 3% (T3) tannin-rich extract Farmatan^®. Values represent mean ± SEM for each channel. ^a,b Different superscript letters within a row indicate significant differences (p < 0.05).

Figure 7

Mucous and mixed acini of the mandibular gland from fattening boars fed diets supplemented with different tannin levels. Groups were fed with the standard mixture (T0; control group) or standard mixture supplemented with 1% (T1), 2% (T2), or 3% (T3) tannin-rich extract Farmatan^®. Goldner’s Trichrome method, 10× magnification.

Figure 8

Nucleus to cytoplasm ratios were measured in (a) serous cells and (b) mixed cells within mandibular glands. Groups were fed with the standard mixture (T0; control group) or standard mixture supplemented with 1% (T1), 2% (T2), or 3% (T3) tannin-rich extract Farmatan^®. Values represent mean ± SEM.