High vitamin levels ameliorate negative effect of rapeseed meal in meat ducks by improving antioxidant activity

High vitamin levels ameliorate negative effect of rapeseed meal in meat ducks by improving antioxidant activity

∗

Institute of Animal Nutrition, Key Laboratory for Animal Disease-Resistance Nutrition of China, Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; and † Hope College, Biology Department, Holland, MI 49423

ABSTRACT The purpose of this study was to test the hypothesis that a high level of a vitamin premix would prevent the deleterious effects of rapeseed meal (RSM) when added to the diet of Pekin meat ducks. A total of 674 fifteen-day-old ducks were randomly allocated to 6 treatments with 7 cages of 16 ducks each. Three diets were formulated that contained 5, 10, or 20% RSM to compensate for reducing levels of soybean meal. Each RSM level diet was then supplemented with either a low level (low) or a high level (high) of a vitamin premix providing a total of 6 experimental diets. Ducks were fed one of the 6 experimental diets (N = 7 pens per diet) from days 15 to 35 at which time they were euthanized. Ducks were analyzed for antioxidant activity, liver biochemistry, thyroid hormone levels, and liver and thyroid histopathology. Addition of the high

vitamin premixes to the 5 or 10% RSM diets improved BW (P < 0.05), BW gain (BWG; P < 0.05), and feed to gain ratio (F/G; P < 0.05) compared to the low vitamin premix; however, neither vitamin premix level had effects on production variables of ducks fed the 20% RSM diet. The high vitamin premix level also improved antioxidant capacity as evidenced by increased (P < 0.05) serum and liver superoxide dismutase activities over that of the low vitamin premix diets. Furthermore, the high level of vitamin premix prevented liver and thyroid pathologies in diets that contain RSM compared to diets with the low vitamin premix. These results suggested that high vitamin premix could prevent the negative effects of a 5 or 10% RSM diet in ducks by improving antioxidative capacities and alleviating liver and thyroid damage.

Key words: antioxidative ability, performance, liver histopathology, meat duck, rapeseed meal, thyroid indexes, vitamin premix 2019 Poultry Science 0:1–10 http://dx.doi.org/10.3382/ps/pez160

INTRODUCTION

compounds that adversely affect animals (Fu et al., 2016). Although water extraction, heat, CuSO4 , enzyme, and fermentation treatments were able to reduce GLS levels in RSM (Tripathi and Mishra, 2007), the remaining GLS levels still exert deleterious effects and limit animal diets that include > 10% RSM. Ingestion of GLS can lead to reduced feed intake and growth rate, increased mortality, liver and thyroid damage, and other diseases in poultry (Mandiki et al., 1999; McNeill et al., 2004). Glucosinolates themselves are biologically inactive molecules, but GLS degradation products are biologically active and known for their diversified biological effects. Major metabolites of GLS are thiocyanates (SCN), isothiocyanates (ITC), nitriles, 5-vinyl-2oxazolidinethione (OZT), and 5-vinyl-1,3-oxyzolodine2-thione (5-OZT) (Tripathi and Mishra, 2007). High intake of GLS may induce an accumulation of 5-OZT in serum, milk, muscular tissues, and other organs such as liver, lung, kidney, and thyroid. In thyroid tissue, 5-OZT inhibits the synthesis of thyroxine (T4 ) (Mabon et al., 2000) and is possibly responsible for the

China is the largest producer of duck for the human food market in the world. Protein sources such as soy are not readily available for poultry feed; thus, many feed formulas have turned to the use of rapeseed meal (RSM). Rapeseed meal is the by-product of rapeseed processing after the removal of its oil. Rapeseed meal is a potential source of protein for poultry production that can partially substitute soybean meal in diets (Khajali and Slominski, 2012), and is a major protein source for duck feed in China. However, the use of RSM has been limited in duck diets due to the presence of antinutritional factors, primarily glucosinolates (GLS). A desired amount of RSM can be used for animal feed formulation using suitable technologies that minimize or remove GLS-related  C 2019 Poultry Science Association Inc. Received July 8, 2018. Accepted March 12, 2019. 1 Corresponding author: [email protected]

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L. Hang,∗ K. Y. Zhang,∗ G. S. Fraley,† X. M. Ding,∗ S. P. Bai,∗ J. P. Wang,∗ H. W. Peng,∗ and Q. F. Zeng∗,1

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MATERIALS AND METHODS Birds and Management A total of 674 one-day-old meat ducks (Cherry Valley duck) were fed a standard diet containing 12.12 MJ/kg ME, 19.5% CP, 0.96% digestible Lys (D-Lys), 0.45% DMet, 0.63% D-Thr, 0.22% D-Trp, 0.90% calcium, and 0.42% non-phytate phosphorus from 1 to 14 D of age. At day 15, ducks were randomly allotted to 42 cages of 16 birds each so that ducklings had a similar initial BW in each cage. The experimental design includes a 2 × 3 factorial arrangement with 2 levels of vitamin premixes (low and high) and 3 levels of RSM (5, 10, and 20%). The experimental diets were fed from day 15 to 35. All ducks were housed in an environmentally controlled facility. Feed and water were provided ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University.

Vitamin Supplement Preparation A low-level vitamin premix supplement (low) was formulated to 70% of the National Research Council (NRC, 1994) recommendations with no VB1 , VB12 , folic acid, biotin, and VC supplementation. A high-level vitamin premix supplement (high) was formulated according to the recommendations of DSM Vitamin Supplementation Guidelines (DSM Nutritional Products

Table 1. Composition of the low and high vitamin premixes for meat ducks.1 Item A (IU) D3 (IU) E (mg) K3 (mg) B1 (mg) B2 (mg) B6 (mg) B12 (mg) Pantothenic acid (mg) Folic acid (mg) Biotin (mg) Nicotinic acid (mg) C (mg) 1

Low

High

1,925 308 7.7 0.39 0 3.08 1.93 0 8.47 0 0 42.35 0

13,585 4,462 71.94 4.477 2.64 6.78 6.55 0.04 14.89 1.76 0.22 78.87 88

Supplied in per kilogram of diet.

Ltd., 2011) but adjusted to the 90% DSM levels. Overall, the high premix supplement had higher levels of all vitamins compared to the low premix supplement (Table 1).

Diet Formulation Three isonitrogenous and isoenergetic experimental diets were formulated on a digestible amino acid basis with decreasing levels of soybean meal. Table 2 illustrates the diet formulation with RSM. RSM used in this study contained 38.01% CP, 1.95% EE, 8.21% ash, 466 mg/kg ITC, and 590 mg/kg OZT on analyzed basis. Antinutritional factors, including ITC and OZT, were analyzed using thiourea colorimetry method and spectrometric method, respectively, by a commercial laboratory (Laboratory of Quality & Safety Risk Assessment for Oilseed Products (Wuhan), MOA, PRC). Each of the 3 diets was then combined with either the low or the high vitamin premix supplement providing a total of 6 experimental diets. All 6 diets were fed in pellet form.

Data Collection and Sampling At day 35, ducks were fasted for 6 h prior to euthanasia at which time body weight (BW) and daily feed consumption were recorded. Feed wastage was recorded daily, and the data were used in the calculations of feed consumption. BW, BW gain (BWG), feed intake (FI), and feed-to-gain ratio (F/G) were calculated. Mortality was recorded and weighed to adjust F/G. For liver enzyme and hormone analyses, 1 duck per cage was randomly selected and blood was collected from the jugular vein, centrifuged at 3,000 × g for 10 min at 4◦ C, and serum was collected and stored at –20◦ C for later analyses. Serum analyses included aspartate aminotransferase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) enzyme activities and hormone analyses of triiodothyronine (T3 ), and thyroxine (T4 ). Liver and thyroid gland were dissected and immediately weighed (% of BW). A sample

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development of hypothyroidism and goiter. Recently, Qin et al. (2017) found that ducks that are fed a diet that contains ≥ 11.32% RSM showed poor growth performance, reduced liver enzyme activity, and a higher thyroid index compared to controls. These results suggest that RSM in meat duck diets may adversely alter liver and thyroid function. However, it is still not clear if the damage in these organs is due to oxidative stress or to compromised antioxidant capacity in ducks caused by the GLS toxins. Some success has been reported to reverse the deleterious effects of dietary toxins in other species. Yuan et al. (2015) found that glutathione supplements could ameliorate liver damage caused by RSM and GLS in aquaculture diets. In another study, 150 mg/kg vitamin C supplements in the diet could totally or partially ameliorate the deleterious effect of the mycotoxin, zearalenone, in piglets (Su et al., 2018). Further, Apaydin et al. (2017) reported that vitamins C and E could protect rat liver against bendiocarb-induced toxicity, and that co-treatment of vitamin C with vitamin E may be more effective than either vitamin alone. These studies suggest that dietary supplements may be useful to counteract the effects of dietary toxins. Therefore, the objective of this study was to investigate whether the deleterious effects of GLS in a diet that contains RSM can be ameliorated by the inclusion of a high vitamin premix supplement in meat ducks.

VITAMIN PREMIX AND RAPESEED MEAL ON MEAT DUCK Table 2. Feed ingredients and chemical composition of the experimental diets on an as-feed basis.

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ported previously by Rabie et al. (2015) and Pa´sko et al. (2018).

Dietary rapeseed meal levels, % Item

10

20

61.03 17.63 11.55 0.76 5.00 0.201 0.169 1.05 1.42 0.021 0.062 0.35 0.15 0.10 0.51 100.0

60.75 14.01 9.73 1.48 10.0 0.260 0.16 1.03 1.38 0.02 0.070 0.35 0.15 0.10 0.51 100.0

60.18 6.78 6.08 2.94 20.0 0.385 0.154 0.976 1.305 0.019 0.076 0.35 0.15 0.10 0.51 100.0

12.12 16.50 0.85 0.40 0.812 0.39 0.536 0.219

12.12 16.50 0.85 0.40 0.812 0.39 0.536 0.219

12.12 16.50 0.85 0.40 0.812 0.39 0.536 0.219

23 30

47 60

93 118

1 In high vitamin diets, vitamin premix provided per kg diet: VA 13,585U/kg; VD3 4,462 U/kg; VE 71.94 mg/kg; VK3 4.477 mg/kg; VB1 2.64 mg/kg;VB2 6.776 mg/kg; VB6 6.545 mg/kg; VB12 0.035 mg/kg; calcium pantothenate 14.894 mg/kg; niacin 78.87 mg/kg; folic acid 1.76 mg/kg; biotin 0.22 mg/kg; VC 88 mg/kg; In low vitamin diets, vitamin premix provided per kg diet: VA 1925 U/kg; VD3 308 U/kg; VE 7.7 mg/kg; VK3 0.385 mg/kg; VB2 3.08 mg/kg; VB6 1.93 mg/kg; calcium pantothenate 8.47 mg/kg; niacin 42.35 mg/kg. 2 Mineral premix provided the following per kg of the diet: Cu 8 mg, Fe 80 mg, Zn 90mg, Mn 70mg, Se 0.3mg, I 0.4 mg.

of thyroid gland (4 ducks/treatment) was collected for histopathological analyses. Liver samples (right lobes, 7 ducks/treatment) were collected for antioxidant determination, and the left lobes (4 ducks/treatment) of the liver were collected and placed into 10% neutralbuffered formalin for histopathological analyses according to Zeng et al. (2014, 2015).

Serum Biochemical Parameters Enzyme activities of serum ALP, ALT, and AST were determined spectrophotometrically using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute; Nanjing, Jiangsu, People’s Republic of China) in accordance with the manufacturer’s recommendations and as reported by our lab previously (Qin et al., 2017). Serum concentrations of T4 and T3 were determined by radioimmunoassay technique using a commercial radioimmunoassay kit (Nanjing, Jiangsu Province) as re-

Analysis of Antioxidant Activity Levels of malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant activity (TAOC) were assayed in serum and liver samples using specific detection kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu Province) with a Mullikan Spectrum Reader (Model 1500; Thermo Scientific, Nyon, Switzerland) (Ren et al., 2016a).

Liver and Thyroid Histopathology Aldehyde-fixed tissues were embedded in paraffin and 5 μm sections were collected, mounted on slides, and then stained with hematoxylin and eosin for histopathological examination by a pathologist. Histopathological lesions within liver tissue were assessed following methods published previously by our lab (Zeng et al., 2014, 2015). Thyroid tissue was analyzed for follicle number, colloid deposition, papillary formation, and epithelial hypertrophy. Thyroid lesions were scored subjectively as follows: 0 = normal histological structure (100×); 1 = approximately 25% papillary formation and epithelial hypertrophy were recognizable (100×); 2 = approximately 50% papillary formation and epithelial hypertrophy were observed (100×); 3 = 80 to 100% papillary formation and epithelial hypertrophy were found (100×) (Pa´sko et al., 2018). The pathologist was unaware of the treatment groups while evaluating slides.

Statistical Analysis All data except for histopathological score were analyzed by 2-way ANOVA using the SAS Proc GLM procedure; and the data of histopathological score were analyzed by the Proc GLIMMIX procedure (SAS Institute, 2006). The model included the main effects of dietary vitamin premix, dietary RSM levels, and their interaction. The cage was the experimental unit with a final N = 7 cages per diet. The means showing significant treatment differences at P ≤ 0.05 in ANOVA were then compared using Fisher’s protected least significant difference procedure, and an alpha level of 0.05 was considered significant. All data were tested for normality using the UNIVARIATE procedure and common variance using the GLM procedure.

RESULTS Growth Performance On day 35, an interaction (P < 0.05) between dietary RSM levels with vitamin premix on BW (Figure 1A), BWG (Figure 1B), and F/G (Figure 1D) was observed. Ducks fed high vitamin premix

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Ingredient (%) Corn Soybean meal Wheat bran Soybean oil Rapeseed meal L-Lysine-HCL DL-Methionine Calcium carbonate Dicalcium phosphate L-Threonine L-Tryptophan Sodium chloride Choline chloride Vitamin premix1 Mineral premix2 Total Calculated composition (%) ME (Kcal/kg) Crude protein Calcium Non-phytate phosphorus Digestible Lys Digestible Met Digestible Thr Digestible Trp Analyzed value Isothiocyanate(mg/kg) Oxazolidinethione(mg/kg)

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supplement showed an increase (P < 0.05) in BW and BWG, and a decrease in F/G of ducks fed diets containing either 5 or 10% RSM compared to ducks fed the low vitamin supplement. However, ducks fed 20% RSM diets showed no differences (P > 0.05) in growth performance regardless of vitamin level. On the other hand, ducks fed the 20% RSM diet showed decreased (P < 0.05) FI (Figure 1C) compared to ducks fed the 5 or 10% RSM diet regardless of vitamin levels.

Liver and Serum Antioxidant Activity An interaction (P < 0.05) between dietary RSM levels with vitamin premix on serum SOD activity (Figure 2A) was observed. Ducks fed low vitamin premix supplement showed decreased (P < 0.05) serum SOD activity with increased dietary RSM. Ducks supplemented with high vitamin premix diets showed increased (P < 0.05) serum SOD activity when fed diets that contained 10 and 20% RSM. Ducks fed diets that contained 10 and 20% RSM showed reduced (P < 0.05) serum SOD compared to ducks fed diets that contained 5% RSM. However, neither RSM levels nor vitamin premix showed an effect (P > 0.05) on serum TAOC (Figure 2B) and MDA (Figure 2C) in ducks. High vitamin premix increased hepatic T-AOC and SOD activities, but not MDA concentration, compared to low vitamin premix in all 3 RSM diets (Figure 3). Although no interaction between vitamin and RSM levels

was observed, increasing RSM levels did show decreased (P < 0.05) hepatic SOD activity.

Liver Biochemistry As shown in Table 3, ducks fed diets containing 20% RSM showed higher (P < 0.05) serum ALP activity but lower (P < 0.05) AST and ALT activities than ducks fed diets containing 5 or 10% RSM. Neither vitamin premix elicited an effect on RSM-induced changes in serum liver enzyme levels. No effects on circulating levels of serum T3 or T4 were observed at any dietary RSM or vitamin levels.

Liver and Thyroid Indexes Dietary RSM levels had a significant effect (P < 0.05) on liver and thyroid indexes of ducks. Ducks fed 20% RSM had the highest (P < 0.05) liver and thyroid indexes among the 3 RSM levels. Moreover, thyroid index was higher (P < 0.05) in ducks fed 10% RSM than ducks fed 5% RSM. There was also no interactive effect (P > 0.05) on liver and thyroid indexes between vitamin premixes and RSM levels. However, the high vitamin premix level markedly increased (P < 0.05) liver indexes in diets that contain RSM compared to diets with the low vitamin premix (Table 4).

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Figure 1. Effects of vitamin premixes and rapeseed meal levels on growth performance of ducks.

VITAMIN PREMIX AND RAPESEED MEAL ON MEAT DUCK

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Figure 2. Effects of vitamin premixes and rapeseed meal levels on serum antioxidant status of ducks.

Liver and Thyroid Histopathology The pathologist’s scores for liver and thyroid histopathology are shown in Table 5. Representative photomicrographs of livers from different experimental groups are presented in Figure 4. Analyses of liver tissue showed some congested areas, hypertrophy of hepatocytes, some necrotic areas, and pockets of edema in all samples. The severity of these pathologies appears to increase with higher percentages of dietary RSM. The high vitamin premix appears to improve histological structure of livers compared to the low vitamin premix as indicated by histological scores (P = 0.012; Table 5). Representative photomicrographs of thyroid histology are presented in Figure 5. Increased indications of hypothyroidism such as an increase in the number of thyroid follicles, increased colloid within the follicular lumen, papillary formation, and epithelial hypertrophy are observed in thyroid glands of all treatment groups. Thyroid histopathology score increased as the dietary

RSM increased. The high vitamin premix decreased the total score of thyroid histopathology in ducks fed diet containing 10 and 20% RSM compared to the low vitamin premix. However, there was no significant difference on thyroid histopathological score in main effect and interactive effect (Table 5).

DISCUSSION We set out to determine if the detrimental effects of GLS found in meat duck diets that contain RSM could be ameliorated by a high vitamin premix supplement. Our data indicated that supplementing diets that contain 5 to 10% RSM with a high vitamin premix can improve antioxidant activity. We further showed improvement by high vitamin premix in liver enzyme levels, and in liver and thyroid histology. Overall, we demonstrate that an RSM diet supplemented with high levels of the vitamin premix improves growth performance of meat ducks. Many of these findings may be due to the high levels of vitamins to augment the activity of antioxidants, such as SOD.

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Figure 3. Effects of vitamin premixes and rapeseed meal levels on hepatic antioxidant status of ducks.

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HANG ET AL. Table 3. Effects of vitamin premixes and rapeseed meal levels on serum biochemical parameters of ducks. RSM

Vitamin

ALP U/L

AST U/L

ALT U/L

T3 mg/L

T4 mg/L

1 2 3 4 5 6

5% 10% 20% 5% 10% 20% SEM 5% 10% 20% Low High

Low Low Low High High High

375.01 430.1 538.1 395.4 428.1 488.9 11.05 385.2b 429.1b 513.5a 447.7 438.9

19.55 18.45 14.42 22.53 20.04 16.92 1.37 21.04a 19.25a 15.67b 17.14 19.83

32.60 31.65 18.18 25.94 25.69 19.77 1.19 29.27a 28.67a 18.97b 27.47 23.80

2.07 1.93 1.79 1.54 1.84 1.97 0.07 1.81 1.88 1.88 1.93 1.78

9.70 9.36 7.99 8.32 8.80 8.68 0.60 8.42 8.75 9.02 8.60 8.12

0.048 0.952 0.819

0.003 0.120 0.295

0.341 0.296 0.166

0.527 0.226 0.514

RSM

Vitamin P-value

RSM Vitamin RSM∗Vitamin

0.001 0.273 0.423

Means in the same low with no common superscript are significantly different (P < 0.05). Means represent 7 pens of 1 ducks per pen.

a,b 1

Table 4. Effects of vitamin premixes and rapeseed meal levels on liver and thyroid indexes of ducks.

Table 5. Effects of vitamin premixes and rapeseed meal levels on histopathology of livers and thyroid in ducks.

Group

RSM

Vitamin

Liver index

Thyroid index

Item

1 2 3 4 5 6

5% 10% 20% 5% 10% 20% SEM 5% 10% 20% Low High

Low Low Low High High High

2.021 2.09 2.22 2.18 2.23 2.38 0.04 2.10b 2.16a,b 2.30a 2.11b 2.26a

0.015 0.020 0.022 0.016 0.021 0.025 0.001 0.016c 0.021b 0.025a 0.019 0.021

0.042 0.015 0.986

0.001 0.225 0.750

RSM

Vitamin P-value

RSM Vitamin RSM∗Vitamin

a–c Means in the same low with no common superscript are significantly different (P < 0.05). 1 Means represent 7 pens of 1 ducks per pen.

In our control diets, we found that SOD activities in serum and liver of ducks decreased with the increase of dietary RSM levels. We further showed that the highlevel vitamin supplement increased SOD activity in the serum and liver of ducks fed RSM, which suggests improved antioxidant activity. Others have also reported that vitamin supplements can ameliorate the effects of toxins in feed by improving antioxidant activity. He et al. (2013) showed that the addition of vitamin E (50 IU/kg) and selenium (0.2 mg/kg) could partially counteract adverse effects of corn contaminated with aflatoxin B1 in meat ducks. Similarly, a study by Su et al. (2018) demonstrated that a vitamin C supplement could totally or partly ameliorate the deleterious effect of zearalenone in piglets. Others reported that antioxidant (SOD, TAOC) activity is associated with oxidative stress (Yu et al., 2008; Apaydın et al., 2016). Reduced levels of antioxidant enzymes in our study could be due to increased free radical formation

Liver 5%+Low 10%+Low 20%+Low 5%+High 10%+High 20%+High

Score 01

Score 11

Score 21

Score 31

Total score2

33 0 0 2 1 0

1 2 1 1 2 2

0 2 3 1 1 2

0 0 0 0 0 0

0.25 1.50 1.75 0.75 1.00 1.50

P-value RSM Vitamin RSM∗Vitamin Thyroid 5%+Low4 10%+Low 20%+Low 5%+High 10%+High 20%+High P-value RSM Vitamin RSM∗Vitamin

0.999 0.012 0.112 1 1 1 1 1 1

3 2 1 3 3 2

0 1 0 0 0 1

0 0 2 0 0 0

0.75 1.00 1.75 0.75 0.75 1.00 0.282 0.262 0.539

1 Score 0 = normal histological structure; score 1 =approximately 25% histopathological change; score 2 =approximately 50% histopathological change; score 3 = almost 100% histopathological change. 2 Total score = (score 0×number of ducks + score 1× number of ducks + score 2×number of ducks +score 3×number of ducks)/total number of ducks, for example, total score of 5%+Low =(0 × 3)+(1 × 1)+(2 × 0)+(3 × 0)]/4 =0.25. 3 Means represent 4 ducks/treatment with 3 slices of 1 duck.

and indicative of oxidative stress. This opinion is supported by Ren et al. (2016a, b), who found that increased growth performance and antioxidative ability were observed in ducks fed a diet with high vitamin premix. Further, others have shown that developing eggs that are supplemented with vitamins improved their antioxidant activity and ultimately, bone metabolism of the developing embryo (Surai, 1996; Atencio et al., 2005). Thus, in ovo vitamin supplements influenced

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Group

VITAMIN PREMIX AND RAPESEED MEAL ON MEAT DUCK

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growth, development, and health status of the hatchling (Lin et al., 2004; Atencio et al., 2005). The high level of vitamin supplements in our study improved overall growth rates, but only at the lower concentrations of RSM. The high level of vitamin premix did not improve the growth performance of duck fed 20% RSM, although the high-level vitamin premix did have some affects to alleviate liver damage and improve antioxidant activity. This apparent contradiction my simply be due to the fact that the GLS levels in the 20% RSM diet may have

partially overwhelmed the detoxification ability of the high levels of vitamins. Levels of ITC and OZT in our 20% RSM were 93.2 and 118 mg/kg, respectively. GLS levels of 200 g/kg in a diet fed to laying hens resulted in liver hemorrhage, liver enlargement, reticulolysis (e.g., lysis of the endoplasmatic reticulum of hepatocytes), and lymphoproliferation (Martland et al., 1984). These results indicate that high levels of vitamin premix could protect liver and thyroid against GLS-induced toxicity, but not completely. Therefore, determining vitamin levels that could fully protect ducks from GLS toxicity

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Figure 4. Morphology of duck livers from all treatments on day 35 (bar = 100 μ m).

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needs to be further explored. However, in our study, the high level of vitamin supplements was able to ameliorate liver damage. The liver is one of the principal target organs for many harmful effects of toxins and their metabolites. When their cell membranes are damaged, hepatocytes secrete enzymes, such as ALP, AST, and ALT, into the blood (Ncibi et al., 2008). In fact, toxin damage to hepatocytes can be detected via analyses of liver enzymes including AST, ALT, and ALP, assays of lipid profiles, and by histology. Thus, in toxicolog-

ical studies, histopathological lesions and biochemical analyses have been largely used as biomarkers (Demir et al., 2011; El-Neweshy and El-Sayed, 2011) of organ damage. Our data support the idea that increasing levels of RSM in the diet led to a concurrent alteration in liver enzyme activity. We further demonstrated both liver and thyroid pathology in the 10 and 20% RSM-exposed ducks histologically. McKinnon and Bowland (1977) also showed epithelial hypertrophy of pig thyroids caused by dietary GLS or a commercially available RSM. Bell et al. (1972) also demonstrated

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Figure 5. Morphology of duck thyroid from all treatments on day 35 (bar = 100 μ m).

VITAMIN PREMIX AND RAPESEED MEAL ON MEAT DUCK

ACKNOWLEDGMENTS Our research was supported by grants from China Agriculture Research System (CARS-42–10), the National Scientific and Technical Supporting Program (2014BAD13B02), the 111 project of Foreign Experts Affairs and Sichuan Agricultural University 211 Foundation of China.

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in rat a significant hypertrophy of thyroids when fed rapeseeds rich in sulfur compounds. In the present study, high levels of the vitamin premix increased liver indexes and improved the total score of liver and thyroid histopathology in ducks fed RSM diets as compared to those supplemented with low levels of vitamin premix. Other studies have suggested that antioxidant compounds may prevent liver damage (Uzun and Kalender, 2013; Kalender et al., 2015). These earlier findings could indirectly explain why high levels of vitamin premix in our study improved the growth performance of ducks fed 5 or 10% RSM. Although our histological analyses suggest signs of hypothyroidism, we did not observe differences in circulating levels of T3 or T4 . Others have also had similar findings. Pa´sko et al., (2018) found rutabaga sprouts that contain harmful GLS compounds do not reduce thyroid hormone levels when fed to rats; however, the authors stressed that rutabaga sprouts could worsen thyroid gland pathology. In conclusion, we have presented promising results in which high levels of vitamins in a premix supplement nearly prevented the deleterious effects known to be induced by diets that contain 5 or 10% RSM. The high levels of vitamins appeared to partially ameliorate liver and thyroid damage by increasing antioxidant activity.

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