Effect of dietary inclusion of dried citrus pulp on growth performance, carcass characteristics, blood metabolites and hepatic antioxidant status of rabbits

ABSTRACT Citrus pulp is a major by-product of the citrus processing industry. A total of 192 post-weaning, 35-day-old Hyla rabbits were randomly divided into four groups. Each group contained four replicates and each replicate comprised 12 rabbits. The four groups were given 0 (control group), 7%, 14% and 21% citrus pulp in the feed. Results indicated no significant difference in the performance of rabbits (P >.118). Rabbits fed with increasing citrus pulp demonstrated an increase in hot carcass weight (linear, P =.041) and liver weight (linear, P =.015). An upward trend (linear, P =.087) was found in thymus with increasing citrus pulp. The experimental groups all displayed an increase of at least 8.28%, 11.7% and 5.07% in serum albumin (ALB), ALB:GLO ratio and calcium (Ca) levels, respectively, compared with the control group (P <.05), and the experimental groups also presented high serum P (linear, P =.023). Hepatic total antioxidant capacity activities in the experimental groups were significantly higher than those in the control group (P <.05), but no difference was found in glutathione peroxidase activities (P >.05). In summary, inclusion of increased citrus pulp did not impair growth performance, could improve immunization, serum Ca and hepatic antioxidant status of growing rabbits.


Introduction
Citrus pulp is a by-product of citrus juicing industries (Grasser et al. 1995). The use of agroindustrial by-product as feed may effectively lower the cost of waste processing and management (Salvador et al. 2014), which has been a hot topic in the livestock industry. Dried citrus pulp (DCP) is one of the by-products of citrus, which is a mixture of citrus peel, pulp and seeds. The ingredients of DCP include 20-40% sugar and 22-44% neutral detergent-soluble fibre carbohydrates (Sunvold et al. 1995;Hall et al. 2010), which can be used as a source of energy (Bampidis and Robinson 2006). In addition, flavonoids and vitamin C in DCP can present antioxidant properties (Williams et al. 2004;Santos et al. 2014), antibacterial (Nordi et al. 2014) and immunestimulating activities (Lee et al. 2010;Ebrahimi et al. 2015;Pourhossein et al. 2015) and meat oxidative stability (Inserra et al. 2014). Currently, DCP is being commonly used as a common ingredient in the diet of domestic animals. 0-3% DCP had no effects on growth performance (Ebrahimi et al. 2013;Abbasi et al. 2015). DCP (Abbasi et al. 2015) or Citrus sinensis peel extract (Ebrahimi et al. 2014) can lower abdominal fat content of broiler chicken. DCP can also optimize the gastrointestinal microbiota of broiler chicken (Ebrahimi et al. 2015). However, Mourao et al. (2008) reported that 5-10% DCP in diets impaired the growth of broiler chickens. No side effects were observed on growth performance and egg quality by adding 6% DCP in the daily feed of laying quails (Florou-Paneri et al. 2001) and adding 10-12% DCP in the daily feeds of laying hens (Yang and Choung 1985;Nazok et al. 2009). In the study of Hon et al. (2009), no difference was found in growth performance after 20% dried sweet orange (Citrus sinensis) pulp meal was added in rabbit feeds. Moreover, fermentation of structural carbohydrates in citrus pulp can benefit the animal (Hernández et al. 2012). Information related to the effects of dietary citrus pulp on the performance, blood metabolites and antioxidant status of growing rabbits is limited.
In this experiment, we attempted to study the optimal fraction of citrus pulp in daily feed of rabbits and investigate how dietary citrus pulp could influence growth performance, serum biochemical indexes and immunization.

Rabbits, diets and experimental protocol
In this study, DCP was provided by Chongqing Three Gorge Fruit Industry Ltd (Huiyuan Juice Group Limited). After juice processing, fresh citrus pulps were dried by a hot breeze dryer. The chemical composition of DCP was 92.23% dry matter, 5.50% crude protein, 9.61% crude fibre, 17.70% neutral detergent fibre, 20.30% acid detergent fibre, 3.82% ash, 2.41% calcium and 1.29% phosphorus.
The experiment was conducted in accordance with the Animal Care and Use Guidelines of College of Animal Science and Technology, Southwest University, Chongqing, China. A total of 192 post-weaning 35-day-old male French Hyla rabbits, with similar heredity, generation and body weight, were randomly divided into four groups, with each group containing four replicates, and each replicate comprising 12 rabbits. The rabbits were housed in galvanized mental wire cages. Natural lighting and automatic ventilation were used in the rabbit house. Of the four groups, the control group was not administered DCP. The other three groups received 7%, 14% and 21% DCP mixed into daily feeds. The feeding experiment lasted 35 days. The first 7 days was a pre-test, and the remaining 28 days was the formal experimental study. The rabbits were fed ad libitum during the 28-day experiment, and water was constantly available. Table 1 shows the diet composition and nutrient levels that were determined following rabbit nutrient demand suggested by France, as well as considerations of local feed sources in Chongqing. The feed was made into pellets approximately 4 mm in diameter and 10 mm long in length.

Experimental measurements
On the start and end of the experiment, rabbits were weighed before feeding in the morning. The feed intake was recorded daily.
At the end of the experiment, 12 rabbits were weighed and slaughtered. The carcasses were measured and divided, following the methods recommended by the World Rabbit Science Association (Blasco and Ouhayoun 1996). After slaughtering, the skin, head, distal part of legs, spleen and visceral organs (such as lung, genitals, urinary bladder and gastrointestinal tract) were removed. Hot carcass contained only meat, heart, liver, kidney, fat and bone. Dressing yield was the percentage of hot carcass weight divided by live weight. Liver, kidney, heart, spleen and thymus gland were weighed and expressed as the percentage of live weight, in which spleen-and thymus gland-to-body weights were immunization indexes.
At the end of the experiment, 12 rabbits were chosen. About 10 mL blood was obtained by cardiac puncture from each rabbit. Subsequently, about 5 g of liver was collected, put in N 2 and stored at −80°C. The blood was centrifuged for 15 min at 3000 r/min. Blood serum was extracted and stored at −20°C for the analysis of serum metabolites. An autobiochemical analyzer (Beckman DXC800, USA) was used to analyse the concentration of aspartate aminotransferase (AST), alkaline phosphatase (AKP), total protein (TP), albumin (ALB), globulin (GLO), glucose (GLU), total cholesterol (TC), calcium (Ca), and phosphorus (P) using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
After homogenization of liver (1:10, w:v) and centrifugation at 3000 r/min for 4 min at 4°C, the supernatants were used to determine the activities of total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), and liver protein concentration using commercially available kits, according to the manufacturers' instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The T-AOC, GSH-Px activity and protein concentration were determined using procedures by Miller et al. (1993), Maral et al. (1977) and Bradford (1976), respectively.

Statistical analysis
Data were analysed using one-way ANOVA with SPSS 20.0. A completely randomized design was used for growth analysis with pen as the experimental unit. Contrast was established using linear, and quadratic polynomial methods to determine the effects of increasing citrus pulps. Multiple comparisons were made using the Turkey multiple range test. Differences were considered significant at P < .05.

Growth performance
Performance data are presented in Table 2. Although fluctuations in live weight were observed at 70 days (P < .05, linear), the concentration of dietary citrus pulp did not affect the daily feed intake, average daily gain and feed conversion ratio (P > .118).

Carcass traits and internal organs
Rabbits fed with increasing citrus pulp exhibited increased hot carcass weight (linear, P = .041, Table 3) and liver weight (linear, P = .015). An upward trend (linear, P = .087) for thymus was observed with increasing citrus pulp. No difference was found in spleen (P > .05).

Serum metabolites and hepatic antioxidant enzyme activities
No significant difference was found in serum AST, TP, GLO, GLU and TC (P > .05). The experimental groups all displayed an increase of at least 8.28%, 11.7% and 5.07% in serum ALB, ALB:GLO ratio and Ca levels, respectively, compared with the control group (P < .05; Table 4). The experimental groups also presented high serum P (linear, P = .023). Hepatic T-AOC activities in the experimental groups were significantly higher than those in control group (P < .05), but no difference was found in GSH-Px activities (P > .05).

Discussion
Feed intake slightly increased in the present study, because rabbits ate 30-40 meals a day, and their gastric volumes accounted for about 30% of their entire digestive tracts (Gidenne et al. 2009). Pascual and Carmona (1980) found that the digestibilities of dry matter, organic matter and fibre were improved with the increased addition of citrus pulp in the feed of 6-week-old New Zealand male rabbits. The inclusion of DCP in the daily feed improved the total tract apparent digestibility of ingredients such as dry matter, crude protein, ether extract and nonfibrous carbohydrates, which improved milking performance (Santos et al. 2014). The increased digestibility possibly accounted for the high live weight of rabbits in the current citrus pulp groups. In this study, utilization of DCP of up to 21% had no any adverse effects on feed intake, body weight gain and feed conversion ratio, which were similar to the results of Hon et al. (2009). The weights of slaughtered rabbits were close to average weights, so live weight and hot carcass in the 21% citrus pulp groups were higher than those in the control, and   demonstrated the same weight trend at the end of experiment. The dressing yield, an important economic index for manufacturers, showed no difference from the control group. Over 60 flavonoids have been found in citrus (Benavente-Garcia et al. 1997) and these compounds can be digested in the small intestine (Walsh et al. 2009); therefore, the level of these flavonoids can significantly increase in blood serum. Flavonoids have antioxidant, anti-inflammatory, antibacterial and immune-stimulating effects (Harborne and Williams, 2000). Pourhossein et al. (2015) found that C. sinensis peel extract significantly improves the serum levels of IgM, and IgG in broiler chickens. The immunization index, to some degree, indicates the immunization functionality of animals. In this experiment, the thymus indexes of the citrus pulp inclusion groups demonstrated an increased trend compared with those of the control group, which indicated that the dietary inclusion of citrus pulp benefitted the health of rabbits. AKP is a hydrolase that helps to improve the immunization of organisms (Ming et al. 2012). In this study, serum AKP levels in the citrus groups were higher than those in the control group. Hepatic T-AOC, the ferric reduction antioxidant power, increased in the citrus pulp groups. Zhu et al. (2002) demonstrated the relationship between the amount of total phenolic compounds and ferric reduction antioxidant power. This association was also supported by previous findings of Santos et al. (2014), who reported that the activity of ferric reduction antioxidant power in milk improves with the citrus pulp additions of 9% and 18%.
Serum AST is an indicator of liver function and health. Increased AST concentration is a reflective response of an organism (Ozardali et al. 2004). Research showed that the inclusion of citrus maxima peel power in the diet of mice with CC14-induced hepatic damage significantly reduced the AST concentration in serum and liver (Chowdhury et al. 2015). In our study, AST showed no change (P = .931), which indicated no adverse side effects on the liver. When evaluating the health status scores of cows during calving, Akbar et al. (2015) found that cows with high and medium scores showed high levels of ALB in serum. Daudu et al. (2013) reported high serum ALB levels in DCP-fed rabbits. Plasma proteins are part of the immune response, in which antibodies are made of ALB. ALB is the major protein in serum. In this study, the ALB and ALB:GLO ratio levels in the citrus groups were higher than those in the control group, which possibly indicated good overall health status of the rabbits in the citrus groups. Moreover, the dietary inclusion of DCP increased serum Ca concentration (P < 0.05), because DCP is a good source of Ca (Nazok et al. 2009). No difference in the plasma glucose was determined in lactating Holstein cows (Santos et al. 2014), demonstrating the same trend with this study. Although the concentration of phosphorus in DCP (Nazok et al. 2009) is low, phosphorus linearly improved in DCP diets in the current study. This improvement possibly contributed to the high phosphorus concentration in the experimental diet (0.7%).

Conclusion
The experimental results showed that citrus pulp could be used as an available feed resource for rabbits. Up to 21% DCP in diet of rabbits had no adverse effects on growth performance, and also helped to improve serum Ca concentration and liver antioxidant status.

Disclosure statement
No potential conflict of interest was reported by the authors.