Nutritional characteristics of meat from lambs fed diets containing mulberry hay

L.G.A. Cirne, A.G.S. Sobrinho, E.A. Oliveira, G.G.P. Carvalho, G.M.B. Moreno, R.L. Valença, F.A. Almeida, V. Endo & N.M.B.L. Zeola 1 Institute of Biodiversity and Forestry, Federal University of Western Pará. Santarém, Brazil 2 Department of Animal Science, São Paulo State University. Jaboticabal, Brazil 3 Department of Animal Science, Federal University of Bahia. Salvador, Brazil 4 Department of Animal Science, Federal University of Alagoas. Arapiraca, Brazil


Introduction
Meat is considered a 'noble' food for humankind, as it contributes to the human diet with proteins, fat, minerals and vitamins, which are essential nutrients for life.At the present time, important changes are taking place in the eating habits of the world's population, who seeks healthier and better-quality products.Meats with these nutritional characteristics have become a preference because of their functional properties (Costa et al., 2008).In sheep meat, polyunsaturated fatty acids (PUFAs) recognized as beneficial to human health such as linoleic (omega-6) and linolenic (omega-3) acids can compose up to 72% of desirable fatty acids (Banskalieva et al., 2000).
The feedlot is an alternative system for finishing lambs in which the animal reaches the degrees of conformation and fatness required for slaughter in a short period, providing high-quality standardized carcasses.One of the obstacles in this method is the cost of feed, which often raises the cost of the production system.Conventional sources of energy and protein such as soybean meal and corn grain elevate feed costs greatly.Using different protein and energy sources are thus an interesting option for the production chain, provided that they do not compromise animal performance or final product quality.
Many authors have investigated the use of mulberry (Morus sp.) in ruminant nutrition (Kandylis et al., 2009;Vu et al., 2011).However, most studies evaluate performance data without addressing qualitative aspects of the meat, although both factors are greatly influenced by the diet.
Mulberry (Morus sp.), a member of the family, Moraceae, is used as a forage ingredient in ruminant feeding by virtue of its grass-like features such as adaptation to different soils and climates, biomass yield of 25 to 30 t/ha/yr, crude protein (CP) levels of 18% to 28%, total digestible nutrients content of 76%, good acceptance by animals and a dry matter (DM) digestibility of 75% to 85% (Sanchez, 2002;Ba et al., 2005;Bamikole et al., 2005).These properties render this plant a potential alternative feedstuff in ruminant diets.
Given the importance of nutrition in production and general aspects of meat, research should be undertaken to discover alternative feedstuffs that can improve quality traits in sheep meat.The consumption of lamb meat of higher nutritional quality can deliver benefits to human health.On this basis, this study was developed to evaluate the nutritional characteristics of meat from lambs fed diets containing mulberry hay.

Materials and Methods
The study complied with the Ethical Principles of Animal Experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and was approved by the Ethics Committee in Animal Use (CEUA) of the Faculty of Agriculture and Veterinary Sciences, FCAV -UNESP, Protocol 014105/11.
The experiment was conducted at the Sheep Husbandry Section in the Department of Animal Science, Faculty of Agriculture and Veterinary Sciences, FCAV -UNESP, in Jaboticabal -SP, Brazil.Twenty-four weaned Île-de-France lambs at approximately 60 days old, with an initial body weight (BW) of 15.48 ± 0.07 kg, were housed in a shed in individual pens (1 m 2 ) on a raised wooden slatted floor, equipped with individual feeders and water troughs.At the beginning of the experiment, lambs were identified, dewormed, vaccinated against clostridial diseases, supplemented with vitamins A, D and E, and randomly allotted to the treatments.
Experimental diets (Table 1) were formulated to meet the requirements of weaned lambs with an average weight gain of 300 g/day, as suggested by the National Research Council (2007).The percentages of the main fatty acids in the concentrates and in the ingredients (Table 2) were determined according to methodologies proposed by Bligh & Dyer (1959) and ISO (1978), as detailed below in the analysis of meat.
The forage to concentrate ratio in the diets was 50 : 50.Dietary treatments were: sugarcane + concentrate without mulberry hay; sugarcane + concentrate containing 12.5% mulberry hay; and sugarcane + concentrate with 25.0% mulberry hay.Mulberry hay substituted corn grain in the experimental diets.The sugarcane was variety IAC 86-2480, which was chopped to a particle size of approximately 1.0 cm and was provided fresh.The mulberry branches, obtained from the Department of Sericulture at FCAV-UNESP, were cut after 60 days of regrowth and sundried until reaching the point of hay, below 20% moisture.Once hayed, the material was ground using a 0.8-mm sieve to improve the homogeneity of the concentrate ingredients and to prevent animal selectivity.
The feed was supplied at 7:00 and at 17:00 to ensure at least 10% as orts, and fresh feed and orts were weighed daily to determine DM intake.Animals were weighed every 14 days to determine their weight gain.On reaching 32.2 ± 0.49 kg BW, lambs underwent a water-only diet for 16 hours.The slaughtering procedures began with stunning with a 220-W electric discharge for two seconds followed by bleeding by sectioning the jugular veins and carotid arteries.After skinning, evisceration and removal of head and limbs, carcasses were moved to a cold-storage room at 6 ºC, where they remained for 24 hours, hanging by the gastrocnemius tendons on appropriate hooks spaced 17 cm apart.
The carcasses were then split lengthwise into two half-carcasses.The left half was sectioned into five anatomical regions: neck, shoulder, rib, loin and leg.Longissimus lumborum muscles were identified individually, vacuum packed and stored at −18 °C until analysis.
For the analysis of the samples, the loins were thawed inside plastic bags in a biochemical oxygen demand (BOD) incubator at 10 ºC for 12 hours and dissected with a scalpel and a knife.Longissimus lumborum samples were harvested to determine the chemical composition, cholesterol concentration and fatty acid profile.
For the chemical composition, samples were lyophilized for 72 hours and then ground through a ball mill to generate the laboratory sample.The chemical composition of the meat was determined in moisture, CP, crude fat and mineral contents, following the methodology proposed by AOAC (1995).
The meat cholesterol concentration was obtained according to the methodology of Bohac et al. (1988).For this, lipid extraction was carried out using a 2 : 1 ratio of chloroform to methanol in a 10-g raw sample of the longissimus lumborum muscle.Subsequently, 5 mL of the chloroform extract was evaporated with nitrogen gas and subjected to saponification with potassium hydroxide in 12% ethanol.The unsaponifiable fraction (cholesterol) was extracted with hexane, purified and subjected to a colour reaction using acetic and sulphuric acids and ferrous sulphate as the catalyst.Absorbance was then determined with a spectrophotometer at 490 nm.The calibration curve for cholesterol was established using 0.01 g of p.a. cholesterol diluted in 50 mL hexane, and aliquots of 40, 80, 120, 160 and 200 mg/mL were taken.The total lipids of the longissimus lumborum muscle were extracted according to the methodology described by Bligh & Dyer (1959).Afterwards, triacylglycerols were transesterified using method 5509 of ISO (1978) in n-heptane and KOH/methanol solution.The fatty acid esters were isolated and analysed in a Shimadzu 14B gas chromatograph equipped with a flame ionization detector and a fused silica capillary column (30 m length, 0.25 mm internal diameter and 0.25 μm Omega wax 250).The initial column flame temperature was 50 °C, which was held for 2 min and raised to 220 °C at 4 °C/min for 25 min.Methyl ester standards were obtained from Sigma-Aldrich, and data were expressed as percentage compositions of each fatty acid.
A completely randomized design was adopted for three treatments with eight replicates, totalling 24 experimental units.Results were assessed by analyses of variance and regression, with degrees of freedom decomposed into linear or quadratic effects, according to the percentages of mulberry hay.The significance of regressions was obtained by the F-test at the 1% or 5% probability levels using the Sisvar statistical programme (Ferreira, 2011).

Results and Discussion
There were no alterations (P >0.05) in chemical composition or cholesterol levels of the meat from mulberry hay-fed lambs in comparison with the meat from control-fed lambs (Table 3).The chemical compositions observed in this study (Table 3) were in agreement with those observed in sheep meat by Tornberg (2005), which included 75% moisture, 20% CP, 3% crude fat and 2% non-protein substances (vitamins, minerals, carbohydrates, among others).The cholesterol level in the sheep meat observed in this trial is considered low (< 90 mg/100 g), according to Madruga et al. (2008), characterizing the meat as a healthy food.
The increased levels of α-linolenic (ω3) and ɣ-linolenic (ω6) acids in the lamb meat might also have stemmed from the higher percentage of these acids in the mulberry hay that possibly escaped rumen fermentation.α-linolenic acid is a precursor of eicosapentaenoic acid, which was likewise found to increase in the meat of the animals.ɣ-linolenic acid, in turn, is a precursor of dihomo-gamma-linolenic acid, which, despite not having been studied here, is a precursor of arachidonic acid (Perini et al., 2010).Eicosapentaenoic and arachidonic acids are precursors of eicosanoids (prostaglandins, thromboxanes and leukotrienes), inflammatory mediators of lipid origin that regulate and protect the organism against effects such as platelet aggregation (by virtue of their anti-thrombotic action).They also control blood pressure and inflammation and reduce immune responses.Arachidonic acid also acts on reproductive function, foetal growth and cerebral and retinal development.Additionally, eicosapentaenoic and arachidonic acids participate in a number of other processes important for human health (Yu & Funk, 2006;Perini et al., 2010), imparting functional properties to the meat of lambs fed mulberry hay.
The increasing concentrations of polyunsaturated α-linolenic (ω-3) and ɣ-linolenic (ω-6) acids are related to their higher concentrations in mulberry hay (Table 2).The increased CLA concentration in the meat from animals fed diets containing 12.5% mulberry hay is probably due to the increase in dietary linoleic acid, since CLA derives from the partial biohydrogenation of this fatty acid in the rumen resulting from the microbial fermentation of Butyrivibrio fibrisolvents (Jenkins et al., 2008).This assertion is based on the increased contents of linoleic acid found in the lamb meat (Table 4).
An increase was observed in the percentage of omega-3 fatty acids (P <0.01) (Table 5), which was mainly the result of the higher concentration of α-linolenic acid in the meat of lambs fed diets containing mulberry hay (Table 4).Although the concentration of omega-3 fatty acids in the meat of the animals studied here was small, these acids play a role in elevating the 'good' cholesterol, represented by the high-density lipoproteins (HDL).Higher percentages of omega-3 in the diet reduce cardiovascular risks (Simopoulos, 2002), which suggests that the meat of mulberry hay-fed lambs may be a healthy human foodstuff.Omega-3 fatty acids are essential because the human body is incapable of synthesizing them and, therefore, they must be incorporated in the diet.Long-chain fatty acids such as eicosapentaenoic and docosahexaenoic acids, in turn, are synthesized by the body (Coletta et al., 2010).
Although some PUFAS were present in high levels in the diets (Table 2), as were linoleic (ω6) and α-linoleic (ω3) acids, which are the main fatty acids in ruminant feeding, their deposition in the lamb meat was low.This was probably a result of the biohydrogenation of these acids occurring during rumen fermentation, a process from which only 10% to 35% of the PUFAs escapes (Medeiros et al., 2015).
The omega-6 to omega-3 ratio in the meat of mulberry hay-fed lambs decreased (P <0.01), ranging from 20.99 (control treatment) to 8.06 (inclusion of 25.0% DM mulberry hay).Nutritionists have emphasized the importance of keeping the omega-6 to omega-3 ratio lower than 4 to reduce coronary complications, and the ratio of 4 : 1 was associated with a 70% decrease in total mortality (Simopoulos, 2004).However, most foodstuffs in the human diet have omega-6 to omega-3 ratios higher than these values.
The activities of desaturase and elongase enzymes, AI and TI indices, hypocholesterolemic to hypercholesteraemic (h:H) ratios, and concentrations of hypercholesterolemic, hypocholesterolemic and neutral fatty acids of the meat of lambs fed diets containing mulberry hay were similar (P >0.05) to those of the meat of animals receiving the control diet (Table 6).The desaturase enzyme activities in the meat of lambs fed diets containing mulberry hay were similar to those observed in the meat of those fed the control diet (Table 6).According to Malau-Aduli et al. (1997), the Δ 9 desaturase 16 and 18 enzymes are responsible for the desaturation of SFAs with 16 and 18 carbons and convert these into their monounsaturated counterparts, with a double bond in carbon 9.The mulberry hay-containing diets probably did not facilitate the formation of MUFAs (C16:1 and C18:1) from the desaturation of saturated acids (C16:0 and C18:0), since no changes were observed in these percentages (Table 4).
The AI and TI indices of 0.65% and 1.73%, respectively, were close to the 0.63% (AI) and 1.46% (TI) reported by Arruda et al. (2012) for the meat of Santa Ines feedlot lambs fed Tifton 85 hay and concentrate.These indices connect the pro-and anti-atherogenic acids and indicate the stimulus potential for platelet aggregation, i.e. the low AI and TI indices observed in this trial indicate a larger amount of anti-atherogenic fatty acids and, as a consequence, greater potential for preventing the onset of coronary diseases (Ulbright & Southgate, 1991).

Conclusions
Inclusion of mulberry hay at levels of up to 25% in the concentrate portion of lamb diets increased the concentrations of unsaturated α-linolenic, ɣ-linolenic, CLA and omega-3 acids and considerably reduced the omega-6 to omega-3 ratio in their meat; improving its nutritional quality.The results of this trial characterize this forage plant as an important source to manipulate the fatty acid profile and validate its use as an alternative ingredient for improving the nutritional quality of meat from feedlot lambs.

Table 1
Chemical composition of ingredients and experimental diets containing mulberry hay

Table 2
Percentage compositions of fatty acids of diets containing mulberry hay and of the ingredients used in the diets

Table 3
Chemical composition and cholesterol levels of the longissimus lumborum muscle from lambs fed diets containing mulberry hay

Table 4
Fatty acid profile of the longissimus lumborum muscle of lambs fed diets containing mulberry hay

Table 5
Concentrations and ratios of fatty acids in the longissimus lumborum muscle of lambs fed diets containing mulberry hay

Table 6
Enzyme activities and indices in the longissimus lumborum muscle of lambs fed diets containing mulberry hay