Use of pomegranate peel and pomegranate peel treated with tannase- producing bacteria on the histomorphometry and histopathology of rumen, liver and kidneys in fattening lambs

Digestion and absorption are related to the growth and development of ruminal villi and intestinal villi and one of the most important factors affecting the growth of ruminal epithelium is diet and increasing the villi height and villi surface increases the absorption capacity and helps maintain ruminal pH (Wang et al. 2009). Epithelial tissue cells play an important role in maintaining stable ruminal conditions, and changes in morphology predispose to the dysfunction of these tissues (Korasani et al. 2021). Pomegranate peel contains large amounts of secondary metabolites such as tannins, saponins, and polyphenolic compounds with various properties such as antimicrobial, antioxidant, anti-inflammatory, and immune-stimulating effects (Adams et al. 2006). The main feature of tannins is their binding to proteins, which inhibits the action of enzymes. Tannins can inhibit the activity of microbes and inactivate extracellular endoglucanase in some fiber-digesting bacteria (Babadi et al. 2018). Tannins prevent the digestion of lignocellulosic substances that are dependent on extracellular enzymes, prevent microbes from attaching to food particles, reduce their digestibility, and make them more persistent in the rumen (McSweeney et al. 2001). Bacteria, yeasts, and filamentous fungi are capable of producing tannins. Bacterial tannase can effectively break down and hydrolyze natural tannins and tannic acid (Kumar et al. 2015). Tanase produced by Klebsiella pneumoniae is more stable in the acidic pH range of 4.5 to 5.5. Therefore, the tannase produced by Klebsiella pneumoniae is of high value for various industrial applications due to its acidophilic nature and thermal stability (Kumar et al. 2015). Several researchers have reported that strains of Klebsiella pneumoniae are able to degrade phenolic compounds; in fact, using bacteria could overcome tannins by altering, destroying, and inactivating tannins. These isolates are likely to increase pectinase and cellulase activity by altering the pH and reducing the toxic effects of tannin; in fact, reducing phenols and tannins increases digestibility by ruminal microorganisms. Therefore, in tropical and subtropical regions, the processing of tannin-rich trees with tannase is very important in animal nutrition systems (Mohammadabadi et al. 2020).

The present experiment was performed in the educational-research farm and laboratories of Khuzestan University of Agricultural Sciences and Natural Resources. Fifteen Arabic male lambs with nine or ten months old and initial body weight of 39.75±7.91kg were used in a completely randomized design with three treatments and five replicates. The trial period consisted of 35 days including 14 days of habituation period and 21 days of recording period. The lambs were randomly assigned to one of the three treatments: 1- control (diet without pomegranate peel) 2- control diet + 20% pomegranate peel 3- control diet + 20% pomegranate peel treated with Klebsiella pneumoniae, which produces tannase. The diets were adjusted using the small ruminant's nutrition requirements (NRC 2007). The lambs were fed a fully mixed ratio at two meals (8 and 16 hours) with free access to water. At the end of the experiment, the lambs of each treatment were slaughtered. The parts about one square centimeter were separated from the abdominal area of rumen and reticulum and one cubic centimeter from liver and kidney and each sample were fixed separately in a 10% solution of neutral formalin (khorasani et al 2021). Micrometric studies including villi height, villi thickness, villi depth, epithelial thickness, muscle layer thickness, and ruminal wall thickness were examined histomorphometrically.

The thickness of epithelial tissue and papillae thickness of reticulum in the treatment containing pomegranate peel treated with bacteria were less than other treatments (P <0.05). The thickness of epithelial tissue and papillae thickness of rumen in the treatment containing pomegranate peel treated with bacteria were less than other treatments and were significant compared with the treatment containing pomegranate peel (P <0.05). The height and depth of ruminal villi in the control treatment were significantly higher than other treatments (P <0.05) and this difference was greater than the treatment containing bacteria. The most important pathological finding in the rumen was translucent corneum hyperkeratosis which was more strongly observed in the treatment containing pomegranate peel. In the treated group with bacteria, basophilic casts (possibly minerals deposition) in the kidneys was reduced compared with the treatment of pomegranate peel. In the treatment containing pomegranate peel, due to the reduction of digestibility of nutrients including cellulosic substances and the decrease in the activity of cellulolytic bacteria in the rumen, the thickness of epithelial tissue and villi thickness increased compared with the treatment containing pomegranate peel processed with bacterial, Because bacterial tannase reduces tannin and actually increases the activity of cellulolytic bacteria and increases acetate production (Mohammadabadi et al. 2020). The reduction of translucent corneum hyperkeratosis in the treatment of pomegranate peel treated with tannase-producing bacteria can be attributed to the reduction of tannin in this treatment and its role in increasing the uptake of ruminal fatty acids compared with the treatment containing pomegranate peel (Khafipour et al. 2009). The presence of basophilic casts into the urinary tubules and glomerular hyperemia can be attributed to oxalate and phytate in pomegranate peel (Romelle et al. 2016).

Compared with control and pomegranate peel treatments, the treatment of pomegranate peel treated with Klebsiella pneumoniae can improve the status of rumen and reticulum tissue markers and relative reduction of ruminal tissue lesions and the severity of basophilic casts in the kidneys compared with pomegranate peel treatment. Therefore, considering the total of these benefits, the use of Klebsiella pneumoniae in the consumption of foods containing tannins can be recommended.