The influence of dietary taurine and reduced housing density on hepatic functions in laying hens Zili Ma,*†1 Jinqiu Zhang,‡1 Haitian Ma,* Bin Dai,* Liuhai Zheng,* Jinfeng Miao,*2 and Yuanshu Zhang* *College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China; †Animal Husbandry and Veterinary Bureau of Dongyang City in Zhejiang Province, Dongyang 322100, China; and ‡National Research Center for Veterinary Vaccine Engineering and Technology of China, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China decreased by taurine (P < 0.05). Taurine reduced the expression of tumor necrosis factor-α mRNA in all 3 rearing patterns, IL-4 mRNA expression in the highdensity group, and IL-10 in the low-density group (P < 0.05). Malondialdehyde levels decreased in serum and liver from T groups and serum total antioxidation capability levels increased significantly (P < 0.05) in the low-density group. Dietary taurine supplementation decreased acetyl-CoA and sterol regulatory elementbinding protein-1c mRNA expression in the high-density groups (P < 0.05). Taurine significantly increased lipoprotein lipase mRNA expression in the high-density group and peroxisome proliferator receptor mRNA expression both in the low- and high-density groups (P < 0.05). Taurine supplementation reduced total cholesterol levels in the low- and high-density groups, decreased triglyceride and low-density lipoprotein cholesterol levels in high-density groups, and increased high-density lipoprotein cholesterol levels in all 3 rearing patterns (P < 0.05). Our data demonstrate that dietary taurine and reduced housing density offer significant protection from hepatic damage in laying hens.
Key words: rearing pattern, welfare, taurine, laying hen, liver injury 2014 Poultry Science 93:1724–1736 http://dx.doi.org/10.3382/ps.2013-03654
INTRODUCTION Agricultural animals are sentient beings. Increasing concern about animal welfare has been observed in food production systems in modern society. When animal welfare is being discussed, the general public pays attention to movement restriction, rapid growth,
©2014 Poultry Science Association Inc. Received September 28, 2013. Accepted March 17, 2014. 1 These authors contributed equally to this work. 2 Corresponding author: [email protected]
high production rates, and cruel practices (Robins and Phillips, 2011; Shimmura et al., 2011; Ingenbleek et al., 2012). Good health is an absolute necessity in animal rearing and is one of the core values of animal welfare, as poor health leads to poor welfare. The domestic chicken provides a major protein source, via meat and eggs, for human populations throughout the world (Wicker et al., 2005). A highdensity caged environment is one of the most common and economical systems employed by the commercial layer industry (Davis et al., 2000). Associated with the resultant economic success of this system are several health problems, including hepatic lipidosis, renomeg-
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Downloaded from http://ps.oxfordjournals.org/ at Universite Laval on June 23, 2014
ABSTRACT To investigate the influence of dietary taurine and reduced housing density on hepatic functions in laying hens, green-shell laying hens were randomly assigned to 3 groups: a free-range group, a caged group with low-density, and a caged group with highdensity. Each group was further divided into the control (C) and taurine-treatment (T) groups. All the test birds were fed the same basic diet, except that the T groups were supplemented with 0.1% taurine. After 15 d, sera and liver were aseptically collected. The results show that dietary taurine supplementation and reduced housing density significantly attenuated physiopathological changes in the liver. When compared with the free-range group, serum alanine aminotransterase and aspartate aminotransterase in the caged hens were significantly higher and were deceased by taurine (P < 0.05). Serum inducible nitric oxide synthase activity in caged hens was higher than that in free-range hens, and taurine reduced serum inducible nitric oxide synthase activities in the low-density group (P < 0.05). Nuclear factor-κB DNA-binding activity increased significantly in the high-density housing group when compared with the other 2 housing patterns and was
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DIETARY TAURINE AND REDUCED HOUSING DENSITY
MATERIALS AND METHODS Birds Fifteen thousand green-shell laying hens (local cross strain) were reared in the Nanjing Jinshuiwan Ecological Park (Nanjing, China). The chicks were reared in a shed. At 12 wk of age they were randomly assigned to 1 of 3 groups: a free-range group and caged groups with low- or high-density housing. Each group was further divided into control (C) and taurine-treatment (T) groups (2,500 hens per group). Thus, a total of 6 groups were used: free-range (with and without taurine), lowdensity (with and without taurine), and high-density housing (with and without taurine). For the free-range group, 2 pens, C and T, were used. Five thousand hens were allocated to 1,666 cages with 3 hens per larger cage (the remaining 2 hens were allocated to 2 smaller cages), resulting in 351 cm2/hen (high-density group).
Table 1. Ingredient composition and nutrient content of diets Item Ingredient (%) Corn Wheat bran Soybean meal Limestone Salt Calcium phosphate dl-Met Vitamin-mineral premix1 Calculated composition (%) ME (kcal/kg) CP Ca Lys Available P
Measurement 63.2 2.5 23.4 6.0 0.4 1.7 0.1 2.7 2,600 16.7 3.8 0.9 0.46
1Vitamin-mineral premix supplied the following per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU; vitamin K3, 0.5 mg; vitamin B12, 0.014 mg; thiamine, 1.8 mg; riboflavin, 5.2 mg; d-pantothenic acid, 11 mg; folic acid, 0.70 mg; pyridoxine, 2.5 mg; niacin, 38 mg; biotin, 0.15 mg; microelement, 0.1 mg; betaine, 0.03 mg; choline chloride, 0.5 mg; allicin, 0.05 mg; Fe, 80 mg; Cu, 11 mg; Mn, 60 mg; Zn, 30 mg; I, 0.35 mg; Se, 0.1 mg.
Each group (C and T) had 833 larger cages and 1 smaller cage. The remaining 5,000 birds were allocated to 2,500 cages with 2 hens per cage, resulting in 526 cm2/hen (low-density group). Each group (C and T) had 1,250 cages. The cages or pens are the experimental units. Caged layers were maintained under artificial lighting at constant temperature (20 ± 3°C) and humidity (50 ± 3%). The free-range group was housed in pastured woods during daylight and confined to indoor pens at night. All birds had free access to drinking water. Dietary nutrient levels are listed in Table 1.. All experiments followed the guidelines of the regional Animal Ethics Committee.
Treatment The birds in each group were fed the same basic diet. After 9 wk (21 wk of age) the treatment groups were fed a diet supplemented with 0.1% taurine. Fifteen days later, 10 layers selected randomly in each group were euthanized, and then sera and liver tissues were aseptically collected, immersed in Bouin’s fixative, or stored at −80°C until analyzed. All procedures were preapproved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University.
Preparation of Liver and Serum Liver samples were weighed and homogenized (Kinematica AG, Luzern, Switzerland) with sterile physiological saline (1:4 wt/vol) on ice and then centrifuged at 2,000 × g for 40 min at 4°C. Fat was removed and the supernatant was collected and stored at −20°C for later analysis. Protein concentration was determined using the Bradford method. Serum was centrifuged at 2,000 × g for 15 min at room temperature and the
Downloaded from http://ps.oxfordjournals.org/ at Universite Laval on June 23, 2014
aly, osteoporosis, cage layer fatigue, ascites, lameness, and inflammation (Burt, 2002; Robins and Phillips, 2011; Buijs et al., 2012). Metabolic lipid disorders leading to liver injury are common and most often occur during the most productive egg-laying cycles (Chou et al., 2012). The mechanism of hepatic lipidosis is similar to nonalcoholic fatty liver disease in humans, which is defined as pathological fat deposition in hepatocytes (Di Minno et al., 2012). It has been reported that an adjusted dietary composition and an increase in physical activity represent the most common and effective therapeutics (Hickman et al., 2004; Federico et al., 2008). Whether these methods can be applied to laying hens is the subject of the current study. Taurine (2-aminoethane sulfonic acid) is present in most animal tissues and plays an important role in several essential biological processes (Grimble, 2006). It has been demonstrated that taurine is an antioxidant and regulates the release of proinflammatory cytokines (Das et al., 2008, 2012; Roy and Sil, 2012). Thus, in animals and humans, taurine and its derivatives are used in the prevention and treatment of various topical infections and chronic inflammatory diseases, in the amelioration of metabolic lipid disorders (Chen et al., 2009), and in protection of the liver from injury (Chen et al., 2009; Das et al., 2012). The potential prophylactic and therapeutic functions of dietary supplementation with taurine have been reported (Gentile, et al., 2011). Although taurine can be synthesized from other dietary sulfur-containing amino acids (i.e., Met, Cys), endogenous production is generally inadequate in hens. Therefore, supplementary taurine is predicted to be beneficial (Das et al., 2008, 2012; Roy and Sil, 2012). Herein, we report the influence of dietary taurine and reduced housing density (thus increasing physical activity) on hepatic functions, thus enhancing the health and welfare of laying hens.
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supernatant was collected and stored at −20°C until analyzed.
Histologic Examination Tissue specimens were fixed in Bouin’s solution for 24 h. Standard dehydration and paraffin wax-embedding procedures were used to produce tissue blocks, which were cut as 5-μm thick serial sections. After deparaffinization and dehydration, hematoxylin- and eosinstained slides were prepared using standard methods. Groups were compared using a point score for the number of hepatocytes demonstrating lipid accumulation as 0 =
Key words: rearing pattern, welfare, taurine, laying hen, liver injury 2014 Poultry Science 93:1724–1736 http://dx.doi.org/10.3382/ps.2013-03654
INTRODUCTION Agricultural animals are sentient beings. Increasing concern about animal welfare has been observed in food production systems in modern society. When animal welfare is being discussed, the general public pays attention to movement restriction, rapid growth,
©2014 Poultry Science Association Inc. Received September 28, 2013. Accepted March 17, 2014. 1 These authors contributed equally to this work. 2 Corresponding author: [email protected]
high production rates, and cruel practices (Robins and Phillips, 2011; Shimmura et al., 2011; Ingenbleek et al., 2012). Good health is an absolute necessity in animal rearing and is one of the core values of animal welfare, as poor health leads to poor welfare. The domestic chicken provides a major protein source, via meat and eggs, for human populations throughout the world (Wicker et al., 2005). A highdensity caged environment is one of the most common and economical systems employed by the commercial layer industry (Davis et al., 2000). Associated with the resultant economic success of this system are several health problems, including hepatic lipidosis, renomeg-
1724
Downloaded from http://ps.oxfordjournals.org/ at Universite Laval on June 23, 2014
ABSTRACT To investigate the influence of dietary taurine and reduced housing density on hepatic functions in laying hens, green-shell laying hens were randomly assigned to 3 groups: a free-range group, a caged group with low-density, and a caged group with highdensity. Each group was further divided into the control (C) and taurine-treatment (T) groups. All the test birds were fed the same basic diet, except that the T groups were supplemented with 0.1% taurine. After 15 d, sera and liver were aseptically collected. The results show that dietary taurine supplementation and reduced housing density significantly attenuated physiopathological changes in the liver. When compared with the free-range group, serum alanine aminotransterase and aspartate aminotransterase in the caged hens were significantly higher and were deceased by taurine (P < 0.05). Serum inducible nitric oxide synthase activity in caged hens was higher than that in free-range hens, and taurine reduced serum inducible nitric oxide synthase activities in the low-density group (P < 0.05). Nuclear factor-κB DNA-binding activity increased significantly in the high-density housing group when compared with the other 2 housing patterns and was
1725
DIETARY TAURINE AND REDUCED HOUSING DENSITY
MATERIALS AND METHODS Birds Fifteen thousand green-shell laying hens (local cross strain) were reared in the Nanjing Jinshuiwan Ecological Park (Nanjing, China). The chicks were reared in a shed. At 12 wk of age they were randomly assigned to 1 of 3 groups: a free-range group and caged groups with low- or high-density housing. Each group was further divided into control (C) and taurine-treatment (T) groups (2,500 hens per group). Thus, a total of 6 groups were used: free-range (with and without taurine), lowdensity (with and without taurine), and high-density housing (with and without taurine). For the free-range group, 2 pens, C and T, were used. Five thousand hens were allocated to 1,666 cages with 3 hens per larger cage (the remaining 2 hens were allocated to 2 smaller cages), resulting in 351 cm2/hen (high-density group).
Table 1. Ingredient composition and nutrient content of diets Item Ingredient (%) Corn Wheat bran Soybean meal Limestone Salt Calcium phosphate dl-Met Vitamin-mineral premix1 Calculated composition (%) ME (kcal/kg) CP Ca Lys Available P
Measurement 63.2 2.5 23.4 6.0 0.4 1.7 0.1 2.7 2,600 16.7 3.8 0.9 0.46
1Vitamin-mineral premix supplied the following per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU; vitamin K3, 0.5 mg; vitamin B12, 0.014 mg; thiamine, 1.8 mg; riboflavin, 5.2 mg; d-pantothenic acid, 11 mg; folic acid, 0.70 mg; pyridoxine, 2.5 mg; niacin, 38 mg; biotin, 0.15 mg; microelement, 0.1 mg; betaine, 0.03 mg; choline chloride, 0.5 mg; allicin, 0.05 mg; Fe, 80 mg; Cu, 11 mg; Mn, 60 mg; Zn, 30 mg; I, 0.35 mg; Se, 0.1 mg.
Each group (C and T) had 833 larger cages and 1 smaller cage. The remaining 5,000 birds were allocated to 2,500 cages with 2 hens per cage, resulting in 526 cm2/hen (low-density group). Each group (C and T) had 1,250 cages. The cages or pens are the experimental units. Caged layers were maintained under artificial lighting at constant temperature (20 ± 3°C) and humidity (50 ± 3%). The free-range group was housed in pastured woods during daylight and confined to indoor pens at night. All birds had free access to drinking water. Dietary nutrient levels are listed in Table 1.. All experiments followed the guidelines of the regional Animal Ethics Committee.
Treatment The birds in each group were fed the same basic diet. After 9 wk (21 wk of age) the treatment groups were fed a diet supplemented with 0.1% taurine. Fifteen days later, 10 layers selected randomly in each group were euthanized, and then sera and liver tissues were aseptically collected, immersed in Bouin’s fixative, or stored at −80°C until analyzed. All procedures were preapproved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University.
Preparation of Liver and Serum Liver samples were weighed and homogenized (Kinematica AG, Luzern, Switzerland) with sterile physiological saline (1:4 wt/vol) on ice and then centrifuged at 2,000 × g for 40 min at 4°C. Fat was removed and the supernatant was collected and stored at −20°C for later analysis. Protein concentration was determined using the Bradford method. Serum was centrifuged at 2,000 × g for 15 min at room temperature and the
Downloaded from http://ps.oxfordjournals.org/ at Universite Laval on June 23, 2014
aly, osteoporosis, cage layer fatigue, ascites, lameness, and inflammation (Burt, 2002; Robins and Phillips, 2011; Buijs et al., 2012). Metabolic lipid disorders leading to liver injury are common and most often occur during the most productive egg-laying cycles (Chou et al., 2012). The mechanism of hepatic lipidosis is similar to nonalcoholic fatty liver disease in humans, which is defined as pathological fat deposition in hepatocytes (Di Minno et al., 2012). It has been reported that an adjusted dietary composition and an increase in physical activity represent the most common and effective therapeutics (Hickman et al., 2004; Federico et al., 2008). Whether these methods can be applied to laying hens is the subject of the current study. Taurine (2-aminoethane sulfonic acid) is present in most animal tissues and plays an important role in several essential biological processes (Grimble, 2006). It has been demonstrated that taurine is an antioxidant and regulates the release of proinflammatory cytokines (Das et al., 2008, 2012; Roy and Sil, 2012). Thus, in animals and humans, taurine and its derivatives are used in the prevention and treatment of various topical infections and chronic inflammatory diseases, in the amelioration of metabolic lipid disorders (Chen et al., 2009), and in protection of the liver from injury (Chen et al., 2009; Das et al., 2012). The potential prophylactic and therapeutic functions of dietary supplementation with taurine have been reported (Gentile, et al., 2011). Although taurine can be synthesized from other dietary sulfur-containing amino acids (i.e., Met, Cys), endogenous production is generally inadequate in hens. Therefore, supplementary taurine is predicted to be beneficial (Das et al., 2008, 2012; Roy and Sil, 2012). Herein, we report the influence of dietary taurine and reduced housing density (thus increasing physical activity) on hepatic functions, thus enhancing the health and welfare of laying hens.
1726
Ma et al.
supernatant was collected and stored at −20°C until analyzed.
Histologic Examination Tissue specimens were fixed in Bouin’s solution for 24 h. Standard dehydration and paraffin wax-embedding procedures were used to produce tissue blocks, which were cut as 5-μm thick serial sections. After deparaffinization and dehydration, hematoxylin- and eosinstained slides were prepared using standard methods. Groups were compared using a point score for the number of hepatocytes demonstrating lipid accumulation as 0 =
