Effect of Broussonetia papyrifera L. (paper mulberry) on growth performance, carcase traits, meat quality and immune performance in Hu ram lambs

Abstract This study was carried out in an attempt to evaluate the impact of Broussonetia papyrifera as a roughage substitute at different levels on carcase traits, growth performance, meat quality and immune performance in Hu ram lambs. Sixty Hu rams (5 months of age, 26.70 ± 2.14 kg body weight) were randomly divided into four groups. The treatments comprised B. papyrifera supplementation at levels of 0% (G0), 30% (G30), 60% (G60) and 100% (G100) of roughage feed. The results suggested that diet supplemented with B. papyrifera (G100 group) caused a higher average weight gain (AWG) and average daily gain (ADG) than those of G0 group. The highest carcase weight was observed in the G60 group. The chemical and physical properties of the longissimus dorsi muscle of Hu rams showed no significant differences (p>.05). For fatty acid, the G60 group had significantly lower content of saturated fatty acids (SFAs), higher contents of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) and higher PUFA/SFA ratio than other groups (p<.05). In terms of the immune response, G60 and G100 groups significantly showed a significant rise in the immunoglobulins (IgG, IgA and IgM) (p<.05). This study indicated multiple beneficial effects of the inclusion of B. papyrifera (60% of the roughage feed) on the growth performance, carcase traits, meat quality and immune response in Hu rams. It could be used as a high-quality unconventional feedstuff for rams. HIGHLIGHTS The addition of Broussonetia papyrifera to the diet resulted in a significant improvement in the average weight gain (AWG) and average daily gain (ADG) of Hu rams. Adding appropriate proportion of B. papyrifera in diets positively modulates fatty acid percentage in the longissimus dorsi muscle, and also increases immunoglobulins (IgG, IgA and IgM) in blood. Broussonetia papyrifera could be used as a high-quality unconventional feedstuff for rams and has a positive effect on their growth performance, meat quality and immune performance.


Introduction
With the increasing demand for animal production in the contemporary world as well as the inadequate quality of conventional forage and scarcity of crude protein-feedstuffs in many developing countries, including China, there is an increasing demand for sufficient and nutritious forage (Liu and Wang 2008;Li et al. 2017). For sustainable intensification of ruminant industries, it is imperative to explore the potential of high-quality unconventional feedstuff resources.
Broussonetia papyrifera L. (paper mulberry), an important plant native to eastern Asia, is used in many applications such as in papermaking, bark-cloth making, medicine and livestock breeding and has proved to be an ecologically, economically and medicinally important plant (Zhai et al. 2012;Sakamoto and Okada 2013;Peng et al. 2019). Chinese scientists have discovered and cultivated a new hybrid of B. papyrifera species with a high crude protein content (Shen and Peng 2017). The stems and leaves of this plant contain crude protein content up to 18-22%, which is similar to that of alfalfa (20%) (Peng et al. 2019). This species of B. papyrifera has been identified to contain more than 40 flavonoids and terpenes that result in its antioxidant, anti-inflammatory and antineoplastic properties (Ko et al. 1997;Feng et al. 2008). Moreover, these factors have proven that this hybrid B. papyrifera species is a potential source of superior quality feed that can replace protein ingredients in ruminant production, and thus, contribute to resolve the feedstuff deficiency.
The current work aims at determining the influence of B. papyrifera on growth performance, meat quality, carcase traits, blood characteristics and immune performance in Hu rams.

Experimental design and treatments
A total of 60 Hu rams of around five-month of age, with average initial body weight (IBW) of 26.70 ± 2.14 kg, were randomly divided into four treatment groups comprising 15 animals each, and 5 rams were raised in a same pen. The treatments involved B. papyrifera (hay) supplementation at 0%, 30%, 60% and 100% of roughage feed (Table 1). Broussonetia papyrifera consisted of (mean values, g/kg DM): 317.50 of dry matter, 194.7 of crude protein, 78.56 of crude fat, 116.96 of crude fibre, 136.1 of crude ash and 5.8 of phosphorus. All diets were kept at a concentrate roughage ratio of 60:40, and diet nutrients were formulated to meet the NRC requirements. Following a 14-d adaptation period, the actual duration of the experiment was 60 d. After the adaptation period, the rams were weighed and the observed weight was considered as the initial weight. The diets were fed twice daily at 08:00 and 16:00. Animals were allowed ad libitum access to feed and water throughout the experiment period.

Sampling and measurements
Feed refusals were collected and weighed daily. The dry matter intake (DMI) was obtained from the amounts of feed offered and the refusals during the experiment period. The weight of each ram was individually recorded on day 1 and day 60 to determine the IBW and final body weight (FBW). Average weight gain (AWG) and average daily gain (ADG) were estimated by subtracting IBW from FBW. At the end of the feeding trial, the feed was withheld overnight with free access to water and a total of 20 rams (five rams from each treatment group) were slaughtered. Following slaughter, the fore and hind feet were removed at the carpal and tarsal joints, respectively, whereas the head was removed at the atlanto-occipital joint. Carcase components were weighed immediately after slaughter. Loin eye area (cm 2 ) was recorded on the cut surface of the longissimus dorsi at the interface of the 12th and 13th rib. Longissimus dorsi was collected within 20 min of slaughter, trimmed of fat and stored at 4 C for further meat quality traits analysis. The pH of longissimus dorsi was measured by pH metre at 0, 24 and 48 h. Determination of cook loss percentage was made by recording weight loss after cooking of meat at 80 C for 1 h in a water bath (Babiker et al. 1990). For shear force, meat samples were placed in an unsealed bag submerged in a water bath (80 ns). When the internal temperature of the sample reached 72-75 C, samples were removed from the water bath, and then were cut into 1 cm Â 5 cm. The shear force value was determined by tenderness metre (RHN50, Nanjing Mingao Inc., Nanjing, China). Conventional components analysis of longissimus dorsi was carried out according to AOAC (1990). A colorimeter was used to measure meat colour (L Ã , a Ã and b Ã ) (CR-10 Plus, Konica Minolta).
For the analysis of fatty acids, lipids were extracted according to the method defined by Folch et al. (1957). Fatty acids composition analyses were carried out with a gas chromatograph (GC-MS; Shimadzu QP 2010 Ultra, Kyoto, Japan) equipped with a 100 m Â 0.25 mm ID Â 0.2 lm fused silica capillary column. Helium was used as the carrier gas. The temperature was set at 100 C for 5 min, then it was increased at a rate of 4 C/min to 240 C, and was maintained at 240 C for 30 min. Individual fatty acid peaks were identified by comparing the retention times with those for authentic FAME standards run under the same operating conditions. The results are expressed as the percentage of the total identified fatty acids.
For hydrolysed amino acid analysis, the sample was weighed into a glass bottle and 6 mol/L of HCl was added. After filling nitrogen, the mixture was The treatments involved B. papyrifera (hay) supplementation at 0%, 30%, 60% and 100% of roughage feed.
hydrolysed at 110 C for 22-24 h. Subsequently, the hydrolysate was transferred to a 50 mL volumetric flask and diluted to calibration tail with ultrapure water. The amino acid analyser (LA-8080 system: Hitachi Inc., Tokyo, Japan) was employed for the hydrolysed amino acid analysis. Before slaughter, samples of blood were drawn from the jugular vein of individual rams into a 5 mL heparinised collection tubes. Samples were centrifuged for 15 min at 3000 g at 4 C. For further analysis, the supernatant was separated. An automatic biochemistry analyser (IDEXX Catalyst One chemistry analyser) was used to estimate the lysozyme, catalase (CAT), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), malondialdehyde (MDA), high-density lipoprotein (HDL), total cholesterol (T-CHO), low-density lipoprotein (HDL), blood urea N (BUN), albumin (ALB), total protein (TP), globulin (GLB), glucose (GLU) and alkaline phosphatase (ALP). Immunoglobulin G (IgG), Immunoglobulin A (IgA) and immunoglobulin M (IgM) were estimated using their respective ELISA test kit (Nanjing Jiancheng Technology Co., Ltd, Nanjing, China).

Statistical analysis
Analysis of all obtained data was made using a oneway analysis of variance. Differences among means were determined using Duncan's new multiple tests for all the four treatments. All the statistical analysis was conducted using SPSS version 22.0 software (SPSS Inc., Chicago, IL). Table 2 shows growth performance, including IBW, FBW, DMI, AWG and ADG. There were no significant differences in DMI among all groups (p>.05). While IBW and FBW were found to be unaffected by B. papyrifera (p>.05), a significant increase in AWG and ADG was seen with B. papyrifera supplementation at levels of 100% (G100 group) (p<.05). There were no significant differences in ADG among the other groups (p>.05).

Growth performance and carcase traits
Diet supplemented with B. papyrifera resulted in a significant improvement in the lion-eye area of rams (p<.05). A significant increase in live body weight was seen with B. papyrifera supplementation at levels of 30% and 60% (G30 and G60 groups) (p<.05). G60 treatment group showed a significant higher carcase weight than G100 group (p<.05), while no difference was observed between the other treatment groups (p>.05). The dressing percentage was found to be unaffected by B. papyrifera (p>.05) ( Table 2). Table 3 shows the chemical-physical properties of longissimus dorsi muscle in Hu rams receiving diets supplemented with different levels of B. papyrifera (0%, 30%, 60% and 100%). No significant differences were observed between all treatment groups in terms of pH-0 h, ph-24 h, moisture, crude protein, intramuscular fat percentage, crude ash, water loss rate, shear force value and L Ã , a Ã , b Ã values of the longissimus dorsi muscle (p>.05).

Meat quality
No differences in Asp, Thr, Ser, Glu, Ala, Val, Met, Phe, Arg and Pro contents in longissimus dorsi muscle were found in this study when B. papyrifera was added (p>.05). However, there was a significant reduction of the Gly, Ile, Leu and His values in the G60 treatment group (p<.05). Except for Leu, G0 and G60 groups were not significantly different (p>.05) ( Table 4). Table 5 elaborates on the fatty acid compositions of longissimus dorsi muscle. Among the saturated fatty acids (SFAs) that were significantly influenced by the treatment G60, there were decreased levels of C14:0, C16:0 and C18:0 (p<.01). Among the monounsaturated fatty acids (MUFAs), we observed increased levels of C15:1 and C17:1 in G60 group (p<.01). The levels of C16:1 (cis-9) and C18:1 (cis-9) were significantly reduced in the G60 and G100 groups (p<.01). Among the polyunsaturated fatty acids (PUFAs), the levels of C18:2 (n-6) and C20:4 (n-6) were significantly increased in G60 group (p<.01). The content of total SFA was significantly reduced in G60 group, however, the content of MUFA and PUFA were significantly increased in G60 group. The PUFA/SFA ratio was also significantly higher in G60 group (p<.01) ( Table 5).

Blood characteristics and immune performance
No differences were observed in lysozyme, CAT, MDA, T-SOD, T-CHO, TP or ALB among four treatment groups (p>.05). From the immune response, IgG, IgA and IgM of G60 and G100 groups were found to be higher than that of G0 and G30 groups (p<.05), except for IgM, there was no significant difference between G30 and G100 (p>.05). T-AOC was significant greater for the G100 group compared with G0 and G30 groups (p<.05), besides, no difference was observed between the other three treatment groups (p>.05). G30 and G60 groups showed significantly higher HDL and LDL contents compared with G100 group (p<.05), and there was no significant difference between G0 and G100 groups (p>.05). For GLU content, the G60 and G100 groups showed significantly higher value than other two groups (p<.05). Inclusion of B. papyrifera significantly increased BUN, ALP, IL-2, IL-6, IL-1b, TNF-a and ifn-c of Hu rams (p<.05) ( Table 6).

Discussion
In this work, a diet supplemented with B. papyrifera resulted in a significant improvement in the growth performance of Hu rams. The outcome of our investigation indicated that AWG and ADG of Hu rams manifested a significant increase (p<.05) as the content of B. papyrifera supplementation reached 100% of roughage feed. This is an indication that supplementation of B. papyrifera to diet is a beneficial way to accelerate the growth performance of Hu rams. This might be attributed to the low fibre and high protein content of B. papyrifera (Peng et al. 2019). The lower fibre content and higher protein content might cause higher protein and energy digestibility of diet, which may subsequently have increased the provision of energy for the animal. Furthermore, the loin eye area measurement in B. papyrifera groups were significantly higher than the control group, indicating 5.58 ± 0.60 5.75 ± 0.42 6.13 ± 0.13 6.89 ± 0.58 NS 'NS' means no significant difference (p>.05); ' Ã ' means significant difference (p < 0.05). The treatments comprised B. papyrifera supplementation at levels of 0% (G0), 30% (G30), 60% (G60) and 100% (G100) of roughage feed. 2.52 ± 0.24 2.64 ± 0.14 NS 'NS' means no significant difference (p > 0.05); ' Ã ' means significant difference (p<.05). The treatments comprised B. papyrifera supplementation at levels of 0% (G0), 30% (G30), 60% (G60) and 100% (G100) of roughage feed. supplementation of B. papyrifera to diet is benefit to muscular development.
The fatty acid concentration and composition of meat is an important aspect of meat quality from consumer perception as well as public health (Yousefi et al. 2012), they correlated with the development and/or prevention of non-communicable chronic diseases in humans. The vast majority of fatty acids in the longissimus dorsi muscle of sheep are in general regarded as C16 and C18 (Yakan and € Unal 2010) and consist of stearic acid, palmitic acid, and oleic acid (Park and Washington 1993;Wood et al. 2008), followed by linoleic acid. Similar results were found in our present study. In our study, we found that the G60 group had lower content of SFA, higher contents of MUFA and PUFA, and higher PUFA/SFA ratio, which indicated that adding appropriate proportion of B. papyrifera in diets positively modulate fatty acid percentage. Previous studies have shown that SFA raise LDL-cholesterol levels, which could increase the risk of developing cardiovascular, however, MUFA and PUFA lowers it (Wood and Enser 2017). Many authorities have recommended that the contributions of SFA to dietary energy intake should be reduced (Wood and Enser 1997). Our dramatic effects on fatty acid concentration might be due to the different rumen microbial activities. All the changes of fatty acid content are related to the manipulation of rumen fermentation patterns (Wood and Enser 1997). Unlike monogastric animals, the meat fatty acid composition in ruminants can be altered by the dietary changes and action of rumen microorganisms. From our results, we found  that a high proportion of B. papyrifera negatively modulate fatty acid percentage. This might be due to the high tannin and lignin contents in B. papyrifera, which are not conducive to the rumen microbial fermentation. Previous studies have shown that high anti-nutrient factors (such as tannin and lignin, etc.) concentrations are always associated with poor rumen function (Mcsweeney et al. 2001). In many countries around the world, the objectionable cooking smell and the flavour of the cooked meat directly affect consumer acceptance of mutton (Wong et al. 1975). Previous studies have shown that the key flavouring agents responsible for mutton flavour are short-chain fatty acids and stearic acid (Caporaso et al. 1977). For both taste panels, the odour and flavour of mutton were found to correlate positively with the concentration of stearic acid (Sanudo et al. 2000). In this study, short-chain fatty acid was not detected in all groups and stearic acid concentration was not affected by B. papyrifera, which demonstrates that no flavour changes resulted in mutton from being fed on the B. papyrifera.
An integrated index of nutrient supply in relation to the utilisation of nutrients of animal is represented by the blood metabolites (Si et al. 2018). This study suggests that diets supplemented with B. papyrifera could potentially induce positive effects on serum biochemical parameters in Hu rams. BUN and GLU concentrations are commonly regarded as key indicators for the status of protein and carbohydrate metabolism in animals which are relevant for growth performance (Zhou et al. 2015;Liu et al. 2018), BUN is categorised as the in vivo protein degradation product (Zhou et al. 2015), whereas serum GLU level is the most commonly used indicator in animals indicating their ability to supply energy (Wang et al. 2011). Besides ALP is the enzyme contributing to the absorption of Ca and P and protein synthesis (Wang et al. 2011). In this investigation, BUN, ALP, and GLU levels were significantly improved by B. papyrifera supplementation in the diet. These positive effects in serum parameters can in part explain the improvement observed in growth performance.
The immune system is complex, controlling a group of cells with an integrated function that is essential to the maintenance of health (Si et al. 2018). IgA, IgM and IgG are key components of the humoral immunity in animals, which could protect the extravascular compartment against microorganisms and pathogenic viruses (Kong et al. 2007). In this study, rams in G60 and G100 groups had a considerably higher IgA, IgG and IgM values compared to those in the G0 group, which might due to the phenolic compounds and flavonoids in the bark and leaves of B. papyrifera, which apparently are the main components responsible for the antioxidant activity (Du et al. 2008;Kai et al. 2015). Previous studies have shown that plant flavonoids are a predominant class of plant secondary metabolites with immunity-boosting activities (Middleton 1998;Proestos et al. 2006;Zhao et al. 2011), they can extend the activity of vitamin C, act as antioxidants, and may, therefore, enhance immune functions (Acamovic and Brooker 2005). Si et al. (2018) also observed an increase in IgA, IgG and IgM in the serum of dairy cows with the addition of B. papyrifera silage (Si et al. 2018). Furthermore, the increased activity of T-AOC in the serum with B. papyrifera addition also indicated that B. papyrifera enhanced the antioxidant status of Hu rams.

Conclusions
Broussonetia papyrifera supplementation at levels of 60% to the diet resulted in a significant improvement in the concentration of MUFA and PUFA in the longissimus dorsi muscle as well as IgG, IgA and IgM in blood. It can, therefore, be concluded that B. papyrifera could be used as a high-quality unconventional feedstuff for rams and adding appropriate proportion of B. papyrifera in diets has a positive effect on their growth performance, meat quality, and immune performance. The investigation regarding the mechanism of effects of B. papyrifera upon growth and immune function of rams needs to be taken up in the future.

Ethical approval
All the experiments performed in this study were reviewed and duly passed by the Jiangxi Academy of Sciences Animal Care and Use Committee.