Modulatory Effects of Arctostaphylos uva-urs Extract In Ovo Injected into Broiler Embryos Contaminated by Aflatoxin B1

Abstract

In ovo injection of nutrients can modulate the embryo’s physiological responses against aflatoxin B1 (AFB1) embryotoxicity. This hypothesis was tested using in ovo injection of Arctostaphylos uva-ursi (Ar. uu.) methanolic extract. The total polyphenols, total flavonoids, total antioxidant capacity, and GC-MS analysis were all assessed in the Ar. uu. methanolic extract. A total of 180 ten-day-old embryonated eggs were distributed into six groups of 30 replicates each. The first group was used as a control (non-injected), and the second, third, fourth, fifth, and sixth groups were injected with 10 µ double-distilled water (DDW), 500 µL methanol, 0.01 g Ar. uu./500 µL methanol, 50 ng AFB1/10 µL DDW, and 50 ng AFB1 in 10 µ DDW + 0.01 g Ar. uu./500 µL methanol, respectively. The relative embryo weight, residual yolk sac weight, tibia length and weight, and survival were recorded. Total and differential leukocytes, oxidative stress, and humoral immune responses were observed. The residual yolk sac was lower (p < 0.05) in the Ar. uu. group than other groups. The embryonic growth (tibia weight and length) was enhanced in AFB1 + Ar. uu.-injected embryos compared with those injected with AFB1 alone. In conclusion, in ovo injection of Arctostaphylos uva-ursi could modulate AFB1-induced toxicity in chicken embryos.

Keywords: Arctostaphylos uva-ursi; aflatoxin B1; broiler; embryotoxicity.

Figure 1

Experimental design where 1 = the 1st group served as a control (non-injected eggs), 2 = the 2nd group, only injected with 10 μL DDW, 3 = the 3rd group, injected with 500 µL Methanol, 4 = the 4th group, injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., 5 = the 5th group, injected with 10 μL Methanol 20%, containing 50 ng AFB1 (AFB1 group), and 6 = the 6th group, injected with 50 ng AFB1 + 500 µL of 0.01 g Ar. uu.

Figure 2

GC-MS Chromatographic profile of methanol extract of bearberry (Arctostaphylos uva-ursi).

Figure 3

Effect of Arctostaphylos uva-ursi fruit methanolic extract and/or AFB1 in ovo injection on the survival rate of broiler embryos. Control = non-injected eggs, DDW = eggs injected with 10 μL DDW, Methanol = eggs injected with 500 µL Methanol, Ar. uu. = eggs injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., AFB1 = eggs injected with 10 μL DDW containing 50 ng AFB1, and AFB1 + Ar. uu. = eggs injected with 10 μL DDW containing 50 ng AFB1 + 500 µL containing 0.01 g Ar. uu.

Figure 4

(A,B). Effect of Arctostaphylos uva-ursi fruit methanolic extract and/or AFB1 in ovo injection on checks relative weight and residual yolk relative weight of broiler chicks on 21 days of incubation (n = 15). Control = non-injected eggs, DDW = Eggs injected with 10 μL DDW, Methanol = eggs injected with 500 µL Methanol, Ar. uu. = Eggs injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., AFB1 = Eggs injected with 10 μL DDW containing 50 ng AFB1, and AFB1 + Ar. uu. = Eggs injected with 10 μL DDW containing 50 ng AFB1 + 500 µL containing 0.01 g Ar. uu. ^a–d Values within columns with different letters significantly (p < 0.05) differ.

Figure 5

(A,B). Effect of Arctostaphylos uva-ursi fruit methanolic extract and/or AFB1 in ovo injection on checks relative tibia weight and tibia length of broiler chicks on 21 days of incubation (n = 15). Control = non-injected eggs, DDW = Eggs injected with 10 μL DDW, Methanol = eggs injected with 500 µL Methanol, Ar. uu. = Eggs injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., AFB1 = eggs injected with 10 μL DDW containing 50 ng AFB1, and AFB1 + Ar. uu. = Eggs injected with 10 μL DDW containing 50 ng AFB1 + 500 µL containing 0.01 g Ar. uu. ^a–d Values within columns with different letters significantly (p < 0.05) differ.

Figure 6

(A–E). Effect of Arctostaphylos uva-ursi fruit methanolic extract or/and AFB1 in ovo injection on lipid profiles of broiler chicks (n = 10). Control = non-injected eggs, DDW = Eggs injected with 10 μL DDW, Methanol = eggs injected with 500 µL Methanol, Ar. uu. = Eggs injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., AFB1 = eggs injected with 10 μL DDW containing 50 ng AFB1, and AFB1 + Ar. uu. = Eggs injected with 10 μL DDW containing 50 ng AFB1 + 500 µL containing 0.01 g Ar. uu. ^a–d Values within columns with different letters significantly (p < 0.05) differ.

Figure 7

(A–C). Effect of Arctostaphylos uva-ursi fruit methanolic extract or/and AFB1 in ovo injection on thyroid function of broiler chicks (n = 10). Control = non-injected eggs, DDW = Eggs injected with 10 μL DDW, Methanol = eggs injected with 500 µL Methanol, Ar. uu. = Eggs injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., AFB1 = eggs injected with 10 μL DDW containing 50 ng AFB1, and AFB1 + Ar. uu. = Eggs injected with 10 μL DDW containing 50 ng AFB1 + 500 µL containing 0.01 g Ar. uu. ^a–c Values within columns with different letters significantly (p < 0.05) differ.

Figure 8

(A–D). Effect of Arctostaphylos uva-ursi fruit methanolic extract or/and AFB1 in ovo injection on serum total immunoglobulins, and immunoglobulins fractions of broiler chicks (n = 10). Control = non-injected eggs, DDW = Eggs injected with 10 μL DDW, Methanol = eggs injected with 500 µL Methanol, Ar. uu. = Eggs injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., AFB1 = eggs injected with 10 μL DDW containing 50 ng AFB1, and AFB1 + Ar. uu. = Eggs injected with 10 μL DDW containing 50 ng AFB1 + 500 µL containing 0.01 g Ar. uu. ^a–c Values within columns with different letters significantly (p < 0.05) differ.

Figure 9

(H1–A6). Effect of Arctostaphylos uva-ursi fruit methanolic extract and/or AFB1 in ovo injection on the spleen histology (H1–H6) and immunohistochemistry (I1–I6) and spleen apoptosis examination (A1–A6). Control = non-injected eggs, DDW = Eggs injected with 10 μL DDW, Methanol = eggs injected with 500 µL Methanol, Ar. uu. = Eggs injected with 500 µL of a freshly prepared solution of 0.01 g of Ar. uu., AFB1 = eggs injected with 10 μL DDW containing 50 ng AFB1, and AFB1 + Ar. uu. = Eggs injected with 10 μL DDW containing 50 ng AFB1 + 500 µL containing 0.01 g Ar. uu.