Methionine supplementing effects on intestine, liver and uterus morphology, and on positivity and expression of Calbindin-D28k and TRPV6 epithelial calcium carriers in laying quail in thermoneutral conditions and under thermal stress

Abstract

This study aimed to provide the performance, localization and expression of the epithelial calcium transporter channels Calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quail under heat stress (HS), and with methionine supplementation (MS). This study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels in the kidneys, intestine and uterus of 504 laying quails under different MS (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under HS (32°C) had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb, and increased positivity in proximal CTs. Deleterious effects of HS were minimized with MS for: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney (P≤0.05). Epithelial calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the MS, increasing egg production in HS and quality in termoneutrality (P≤0.05). MS further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity of Calb, and increase Calb and TRPV6 expression in the kidney (P≤0.001) under thermoneutrality. It was concluded that the use of MS (120%) is justifiable in order to partially reverse the deleterious effects of HS on the production, in the epithelial calcium carriers, and in the digestory and reproductive morphology of laying quail.

Fig 1. Photomicrographs of anti-Calbindin-D28k immunohistochemistry in laying quail intestine at different magnifications.

Positive (brown stain) anti-calbindin-D28k intestinal epithelium (arrows) and non-positive goblet cells (arrowheads) (A and B) are observed. Non-antibody-positive crypts (asterisk) are also observed (C). Lower epithelial positivity (brown stain) is observed under 28°C (D) when compared to other temperature treatments. Chromogen staining diaminobenzidine+hematoxylin.

Fig 2. Photomicrographs of anti-calbindin-D28k immunohistochemistry on the kidney of quail laying at different temperatures.

A) 20°C: Positivity (brown stain) occurs mainly in distal contorted tubules near large vessels (asterisks). B and C) 24 and 28°C: Lower antibody positivity (brown stain). D) 32°C: There are more positive distal contorted tubules and slightly positive proximal contorted tubules as well. More positivity (brown stain) is observed at temperatures 20 and 32°C. Chromogen staining diaminobenzidine + hematoxylin. Magnification 100x.

Fig 3. Immunohistochemistry photomicrographs of anti-Calbindin-D28k in the kidney of laying quail at different temperatures.

A) 24°C: Note poor positivity (brown stain) in distal contorted tubules (arrowheads) and no positivity in proximal contorted tubules (asterisks). B) 32°C: In thermal stress there is intense positivity (brown stain) of the distorted contorted tubules (arrowheads) and low positivity (brown stain) in the proximal contorted tubules (asterisks). Chromogen staining diaminobenzidine + hematoxylin. Magnification 400x.

Fig 4. Photomicrographs of anti-Calbindin-D28k immunohistochemistry in the uterus of laying quail at different temperatures (20, 24, 28 and 32°C) and supplemented with 120% methionine at 32°C.

A) 20°C: observe lower positivity (brown stain) in the uterine glands (asterisk). B) 24°C: observe greater positivity (brown stain) in the uterine glands (asterisk). C) 28°C: observe greater positivity (brown stain) in the uterine glands (asterisk). D) 32°C: observe lower positivity (brown stain) in the uterine glands (asterisk). E) 32°C: supplemented with 120% methionine. More positive than treatment without methionine supplementation. Arrowheads (uterine epithelium), asterisks (uterine glands). Chromogen staning diaminobenzidine + hematoxylin staining. 400x magnification.

Fig 5

Graphs of the effects of methionine supplementation (100% and 120%) at different temperatures on the Calb 28 (A) and TRPV6 (B) genes expressions in the kidneys; Calb 28 (C) and TRPV6 (D) in the intestine; and Calb 28 (E) and TRPV6 (F) in the uterus of Japanese quail (Coturnix japonica) in production phase. ^a,b,A,B Averages followed by the same lower case letter for 100% supplementation and upper case for 120% supplementation do not differ from each other (p<0,05); ^ns not significant, *p<0.05, **p<001.