Effects of Casein Phosphopeptide-Selenium Complex on the Immune Functions in Beagle Dogs

Abstract

The health of pets is becoming a growing concern for the pet industry and its owners. Immunity is one of the foundational supports for health, thus developing a functional bioactive substance that can boost pets' immunity is essential. Many studies have shown that casein phosphopeptide (CPP) and selenium (Se) can individually regulate immunity in many species, but there has been no reported research on the immunomodulatory function of casein phosphopeptide-selenium complex (CPP-Se). The objective of this study was to investigate the function of CPP-Se on immunomodulation in dogs. Twenty Beagle dogs were equally divided into two groups and fed either a control snack or a test snack supplemented with 0.03% CPP-Se for 30 days. Anticoagulated blood, serum and peripheral blood lymphocytes (PBL) were collected from dogs at 0 d, 10 d, 20 d and 30 d to detect the change in the number of immune cells and the expression of cytokine-related mRNAs and proteins. PBL isolated from blood were exposed to CPP-Se in vitro to measure the proliferative responses and cytokine-related mRNAs expression. During the time the test snack was fed, the number of lymphocytes increased significantly, whereas neutrophils and monocytes remained unaltered. The expression of interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), CD4 molecule (CD4) and CD8α molecule (CD8α) was up-regulated, while interleukin-1β (IL-1β) was down-regulated, and interleukin-10 (IL-10) declined initially and subsequently increased. ELISA detection revealed a significant increment in serum IL-4, IL-6, Immunoglobulin M (IgM) and IFN-γ, except for IgG. Furthermore, CPP-Se treatment increased the proliferation and the expression of cytokine-related mRNAs in PBL cultured in vitro. This is the first study to demonstrate that CPP-Se can improve immunity in the dog.

Keywords: casein phosphopeptide–selenium complex (CPP-Se); cytokines; dogs; immunity; lymphocytes.

Figure 1

The schematic diagram of experimental design.

Figure 2

Changes in the number of lymphocytes (A), monocytes (B) and neutrophils (C) during the feeding period. Values are means with standard error bars, means with different letters are different (p < 0.01).

Figure 3

Changes in the expression level of cytokine-related genes in PBL of dogs fed 0 d (A), 10 d (B), 20 d (C) and 30 d (D). “*” indicates statistical significance at p < 0.05, “**” indicates statistical significance at p < 0.01.

Figure 4

Changes in the content of IgM (A), IL-4 (B), IFN-γ (C), IL-6 (D) and IgG (E) during the feeding period. Different lowercase letters represent the level of significance at p < 0.05; different uppercase letters represent the level of significance at p < 0.01.

Figure 5

Morphology and viability identification of PBL. (A) Morphological characteristics of Wright-Giemsa stained PBL. Under the microscope, a small number of granulocytes (rod-shaped or lobulated nuclei, indicated by green arrows), and a large number of lymphocytes (round nuclei with little cytoplasm and light dark color, indicated by red arrows) were observed. (B) Viability detection of PBL using trypan blue staining. Live cells refuse to stain (black arrows) while dead cells are stained blue (white arrows). All pictures were screened at 400×, scale bar = 50 μm.

Figure 6

Changes in cell proliferation (A) and cytokine-related mRNAs expression (B) after PBL treated with 5 μg/mL CPP-Se in vitro. “*” indicates statistical significance at p < 0.05, “**” indicates statistical significance at p < 0.01.