Canadian Journal of Animal Science

Effects of flavonoids from Allium mongolicum regel as a dietary additive on meat quality and composition of fatty acids related to flavor in lambs

Journal:For Canadian Review Journal of Animal ScienceOnly Manuscript ID CJAS-2018-0008.R1

Manuscript Type: Article

Date Submitted by the Author: 15-Mar-2018

Complete List of Authors: Liu, Wangjing Ding, He; College of Animal Science Khas-Erdene, Khas-Erdene; Inner Agricultural University, college of animal science Chen, renwei; College of Animal Science Mu, qier; College of Animal Science Ao, ChangJin; Agricultural University, College of Animal Science

Keywords: Allium Mongolicum regel; flavonoids; lambs; meat quality; fatty acids.

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Effects of flavonoids from Allium mongolicum regel as a dietary additive on meat quality and composition of fatty acids related to flavor in lambs Wangjing Liua, He Dinga, Khas-Erdenea, Renwei Chen, Muqier, Changjin AO* College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, a Co-first Author names and affiliations These authors contributed equally to this work Wangjing Liua College of Animal Science, Inner Mongolia AgriculturalFor ReviewUniversity, Hohhot 010018,Only China Tel.: +8618248112856; Email: [email protected] He Dinga College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China Tel.: +8615204717075; Email: [email protected] KhasErdenea College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China Tel.: +8618647138393; Email: hesd[email protected] *Corresponding author: Prof. Changjin AO College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China Tel.: +8613947119088; Email: [email protected]

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ABSTRACT Sixty male Smalltailed Han Sheep (initial body weight: 42.5 ± 4.1 kg) were assigned randomly and averagely into four groups to evaluate effects of flavonoids extracted from Allium mongolicum Regel on meat quality and composition of fatty acids related to flavor in longissimus dorsi muscle of lambs. Lambs of four groups were fed a basal control diet (C), and basal diet + three different amounts of flavonoids extracted from the Allium mongolicum Regel (AMR) those were11 mg/kg

(Flav 11), 22 mg/kg (Flav 22), or 33 mg/kg (Flav 33), respectively. Dressing percentage, loin eye area, and cooking loss, pressing loss were affected by treatments (P < 0.05). PercentageFor of intramuscular Review fat, and the Only concentrations of eicosapentaenoic acid, C18:0, total saturated fatty acids, monounsaturated fatty acid and 4methyloctanoic acid in longissimus dorsi muscle were changed by treatments (P < 0.05). Addition of AMR to diet improved the meat quality and had favorable effects on fatty acid composition related to meat flavor in longissimus dorsi muscle.

Keywords: Allium Mongolicum regel; flavonoids; lambs; meat quality; flavor; fatty acids.

Introduction Allium mongolicum Regel is a typical of the Liliaceous family, which grows in the desertified grassland in Mongolia, Siberia, and the north of China (Schmitt, Schulz, Storsberg, & Keusgen, 2005). Allium mongolicum Regel has been shown to have a variety of nutrients for human, in addition to having a pleasant taste. According to the Mongolian Pharmacopoeia, it has many important physiological effects, including antihypertensive, hypolipidemic and antiinflammatory (Obmann et al., 2010) . The herdsmen living in the Inner Mongolia China find that sheep fed with Allium mongolicum Regel had a special flavor of mutton which was well accepted by most of people, at the same time the sheep had few diseases and grew faster after eating the Allium mongolicum Regel (Bao et al., 2015). We have previously reported that Allium Mongolicum Regel and its extracts decreased saturated fatty acids content 2

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of subcutaneous fat, Longissimus dorsi muscle and perinephric fat and increased content of monounsaturated fatty acids and polyunsaturated fatty acids, improved mutton quality and decreased odor of mutton (Zhang et al., 2008). Allium are rich in flavonoids which are important active ingredients (Maisashvili, Gvazava, & Kuchukhidze, 2009), and Allium mongolicum Regel produces many flavonoids compounds as well. Flavonoids containing diphenylchromone are widely present in plants (Bodas, Prieto, LópezCampos, Giráldez, & Andrés, 2011). All flavonoids produce polyphenolic compounds (RichEvans, Miller, & Paganga, 1996), which play important roles on growth development, antibiosis,For and Reviewdisease prevention (Block, Only 1992). Flavonoids extracted from leaves of Allium plants had positive effects on texture, odor, color, flavor and nutritive value in meat (Iwashina, 2003; Devatkal, Narsaiah, & Borah, 2010; Terevinto et al., 2010). Young growing ruminants may have evolutionarily depended on flavonoids (Heinrichs, 2005). Yaghoubi et al. found that young Holstein calves provided with high dose (3.6×103 g/Kg body weight) of flavonoids from bee propolis had been improved growth and body weight (Yaghoubi, Ghorbani, Rahmani, & Nikkhah, 2008). Flavonoids from soybean significantly increased dressing percentage, loin eye area, and cooking loss of pigs (Wenk, Fernandez, & Dupuis, 2000). Because of a consequence of these benefits, there are a growing number of studies in benefits of flavonoids (Andrés et al., 2013; Simitzis, IliasDimopoulus, Charismiadou, Biniari, & Deligeorgis, 2013). However, there were few works about flavonoids from Allium mongolicum Regel as feed additives whether having effects on meat quality. The cooked flavor was closely associated with branched chain fatty acids which are related to mutton flavor (Wong, Nixon, & Johnson, 1975). Higher acceptance of the final cooked meat product was found with lower concentrations of 4methyloctanoic acid (MOA) (Watkins et al., 2014). Obviously, ameliorating impacts of MOA and 4ethyloctanoic acid (EOA) on the sensory components of meat would result in higher acceptance of the final product by consumers. Based on the abovementioned facts, we especially studied whether the inclusion of the flavonoids extracted from Allium mongolicum Regel in a concentrate mix ration could decrease composition of fatty 3

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acids related to flavor (MOA, MNA, and EOA). The aim of this study was to evaluate the effects of inclusion of the flavonoids from Allium mongolicum Regel in a concentrate mix ration on lambs meat quality, and composition of fatty acids related to flavor in longissimus dorsi muscle of lambs. Materials and methods Extraction process of Allium Mongolicum regel flavonoids Allium Mongolicum regel was collected from natural pasture in Alxa League of Inner Mongolia area, and was identified by the Institute of Ecology and Environment of Inner Mongolia Agricultural University (Zhang et al, 2014). Fresh Allium Mongolicum regelFor was thoroughly Review cleaned by tap Onlywater, and dried in 65ºC , then pulverized and screened by 80 mesh sieve. The powder was then defatted and decolored by petroleum ether, where the ratio of powder to petroleum ether was 1:10. After evaporation, crude extracts were extracted by ultrasonic assisted extraction method (Maryam et al, 2016). Briefly, Allium Mongolicum regel powder was mixed with ethanol (50%) at a ratio of 1:30 (w/v), and extracted at 40ºC for 15 min. After centrifugation at 2000 r/min for 10 min, the supernatant was collected and then concentrated using a rotary evaporator (model: R1002B, Yamato Scientific, China). Finally the extracts was lyophilized in a freeze dryer (model: CA301, Yamato Scientific, China). The extracts were mainly flavonoids reported by our previous studies (Zhao, 2008) and then the structure of flavonoids were identified by liquid chromatography ESItandem mass spectrometry technology (LCESIMSMS). The results showed that the extracts contain seven kinds of flavonoids (Luteolin 5'Oglucose4hydroxy phenylpropionate, Quercetin7O3Orutaceae glucoside, Kaempferol4'O3,7OO2glucosidase, 7O5,4'dimethoxy3'hydroxyflavonol, 3',4'epoxy7O5methoxyflavonol, Quercetin7O3Oglucoside and Kaempferol7O3Oglucoside, respectively). Experimental animals and rearing The feeding trial was conducted with Smalltailed Han Sheep. All procedures were undertaken following the guidelines of the China Agricultural University Animal Care and Use Committee on animal ethics. The experiment was carried out at Fu Chuan 4

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breeding Technology Co. Ltd in Bayannuoer city, Inner Mongolia, China (40ο 79′ N, 107ο 42′ E, 1038 m above sea level). The ambient temperature and relative humidity values during the study were in the range of 23 28ºC and 28 33%, respectively. The trial adopted onefactor completely random design. Sixty growing Smalltailed Han Sheep (initial bodyweight, 42.5 ± 4.1 kg) around 6 monthold were randomly distributed into four groups of fifteen animals for a period of 75 d. Lambs were allowed 15 d to acclimate to the diets and experimental measurements were taken in 60 d. All lambs were dewormed and vaccinated against common sheep diseases before the experiment. BasedFor on our Review previous studies, the Only supplementation amounts were primarily determined by the amounts of fresh Allium Mongolicum regel or its dried powder addition to the lamb diets that had favorable effects on growing performance, meat quality and meat flavor, and the final amounts were calculated according to the extractive rate of crude flavonoids from Allium Mongolicum regel. Respective rations were offered to the animals twice daily, and water is available ad libitum. Four groups of experimental animals were fed the composition of diets as follows, feeding basal diet (C), treatment groups were supplemented with, 11 mg/kg (Flav 11), 22 mg/kg (Flav 22) or 33 mg/kg (Flav 33) flavonoids on a dry matter basis. Based on our previous studies, the supplementation amounts were primarily determined by the amounts of fresh Allium Mongolicum regel or its dried powder addition to the lamb diets that had favorable effects on meat quality and meat flavor, and the final amounts were calculated according to the extractive rate of crude flavonoids from Allium Mongolicum regel. Experimental basal diets The experimental basal diets referred to mutton sheep fattening standard of NRC (2012). Ingredient compositions are presented in Table 1. The experimental basal diets samples were analyzed for nitrogen using ‘Terbotherm’ and ‘Vapodest’ (Gerhard, Germany) analyzer based on the microKjeldhal method (Braghieri et al., 2011). The neutral detergent fiber (NDF) was estimated using sodium sulfite and acid detergent fibre (ADF) described by Van Soest ( Soest et al, 1991). The NDF and ADF fractions 5

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included residual ash. Calcium was estimated as per the method described by Talapatra (Talapatra et al, 1940). Phosphorus was determined colorimetrically as per the method of Ward and Johnston (Ward, & Johnston et al, 1960). Carcass parameters At the end of experiment, three lambs per group were randomly selected for slaughter, twelve lambs totally from 4 groups and those were fasted for 18 h on the day prior to transportation to the slaughterhouse. After slaughter, carcass were transferred to a cooler at 4°C and chilled for 24 h. At 24 h postmortem the longissimus dorsi muscle was excised from the carcass, and was vacuum packed and stored at 2°C to ageFor for 5 days. Review Subcutaneous fat andOnly silver skin were removed, and frozen at 20°C for subsequent chemical analysis. The longissimus dorsi muscle was collected, for subsequent measurement of compositions and contents of fatty acids. Indicators of meat production ability The live weights before slaughter were recorded. Lambs were slaughtered in the local abattoir using Halal methods.The bodies were skinned and organs removed such as head, feet and viscera except kidney and the adipose tissue surrounding of kidney in order to measure the carcass weight. Dressing carcass percentage for individual lamb was calculated using the weight of carcass divided by the live weights before slaughter. Loin eye muscle area is positively associated with the amount of meat production, reflecting the ability of meat production of lamb. Loin eye muscle area (cm2) of each lamb was recorded on the cutsurface of the longissimus dorsi muscle at the interface of 12th and 13th rib on both sides of the carcass. Mutton quality The moisture, fat, protein and ash contents were determined according to the official methods 950.46, 991.36, 981.10 and 920.153, respectively (AOAC, 1990). The pH was measured by a portable pH meter (testo 250, TestoStraße 179853 Lenzkirch, Germany) equipped with a glass electrode at 45 minutes and 24 hours after slaughter.The IF content was determined using the Soxhlet petroleumether extraction method according to a previous report (Zhang et al., 2014). The shear force of the samples was determined using a texture analyzer (TAXT plus, Stable Micro 6

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System,Surrey, U.K.). The pressing (2g) and cooking loss (1 cm x 1cm x long sample after cooking) were determined using the following equations: Cooked meat ratio = (weight after steaming) / (weight before steaming) × 100% Pressing loss = (the total water loss of water) / the total water × 100% (in mutton sample) Determination of fatty acids composition Determination of the fatty acid composition of longissimus dorsi muscle was performed on approximately 1 g of sample (Wonnacott, Kwong & Hughes, 2010). The fatty acidTMS esters were separated by injection (1 L) onto a DB5MS fused silica capillary columnFor (HP88, Review 100 m×0.25 mm i.d.×0.25Only m film thickness) in a Varian 3400 gas chromatograph(GC) and detected by a Saturn 2000 ion trap mass spectrometer operating in full scan mode. The septumless programmable injector (SPI) was programmed starting at 45 °C and increased to 300 °C at 180 °C min−1. The GC oven was held at 75 °C for 2 min then increased to 300 °C at 10 °C min1 and held at this temperature for 8 min. Helium was used as the carrier gas at a constant pressure of 190 kPa. The mass spectrometer transfer line was at 280 °C. Mass spectra were acquired using an ion source temperature of 220 °C and an electron multiplier voltage of 2300 V. Determination of fatty acids related to flavor The fat extracts of raw muscle tissue of longissimus dorsi muscle of the same chop (without bones) was analyzed. The preparation of the samples was undertaken according to the method of Kaffarnik (Kaffarnik, Preuß, & Vetter, 2014). The fat of muscle tissue samples (dried homogenized muscle tissue, subcutaneous fat removed) was extracted by means of a Soxtherm apparatus (Kaffarnik et al., 2014). Measurement of branched chain fatty acids was described by Schiller (Schiller et al., 2015). Statistical analysis Data were analyzed using PROC MIXED of SAS (2002) (version. 9.2; SAS Institute, Cary, NC, USA). The fixed effects were addition of AMR, amounts of the addition, and addition of AMR by the amounts, and the random effect was 7

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experimental sheep. Polynomial contrasts were constructed to partition the addition of AMR by the amounts into single degree of freedom comparisons. Least square means were calculated and are presented with their standard errors throughout. Statistic significant was declared at P ≤ 0.05, and tendencies at P ≤ 0.15. Results Meat production ability and quality Effects of flavonoids extracted from Allium mongolicum Regel on meat production ability and quality are presented in Table 2. The results showed that, there were no significant differences in pH at 1 h (pH1) and 24 h (pH24) after the slaughter (P > 0.05). Dietary supplementationFor Review of different added Onlyconcentrations flavonoids did significantly increase the dressing percentage, loin eye area and cooking loss (P < 0.05) as the pressing loss decreased (P < 0.05) in the experimental of lambs. Although there was no significant difference, the shear force was numerically lower in the supplemented group than C (P > 0.05). Dressing percentage, loin eye area and cooking loss of AMR treatments increased linearly with different added concentrations of flavonoids , however, the pressing loss was decreased linearly with different added concentrations of flavonoids .The moisture, protein and ash percentage were not significantly different (P > 0.05) in table 3, while the percentage of intramuscular fat increased linearly with different added concentrations of flavonoids (P < 0.05). Fatty acid composition of the longissimus dorsi muscle Effects of dietary flavonoids on the fatty acids compositions of the longissimus dorsi muscle of lambs are shown in Table 4. The concentrations of saturated fatty acid and C18:0 were decreased linearly with different concentrations of flavonoids (P < 0.05) without affecting the sum of total free fatty acids. The concentrations of eicosapentaenoic acid and monounsaturated fatty acid increased linearly with different added concentration flavonoids (P < 0.05). The concentrations of fatty acids related to flavor Effects of feeding diets with different ratios of flavonoids on fatty acids related to flavor in longissimus dorsi muscle are presented in Table 5. Dietary supplementation 8

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with the treatments tended to reduce the concentration of 4methylnonanoic acid, while significantly reduce the amount of 4methyloctanoic acid (P < 0.05), except Flav 33, without affecting the sum of 4ethyloctanoic acid. The concentration of 4methyloctanoic acid decreased quadratically with different concentrations of flavonoids. Discussion Meat production ability and quality According to Sen (Sen, Sirin, Ulutas, & Kuran, 2010) argued that “ the pH has a significant role in the quality assessment of meat ”. In the present study, pH1 and pH24 after the slaughter,For pH Reviewvalues of all groups wereOnly found to be insignificant (P >

0.05) in the Table 2. The results of pH values were similar to the Martı́nez (Martı́nez et al., 2005). pH24 values decreased in this study with increasing the concentrations of flavonoids varied between 5.72 5.78. Moreover, Yakan (Yakan & Ünal, 2010b; Majdoub et al., 2013) acknowledge that “ it can be said that the final pH ranges were both appropriate and inside normal range (5.6 6.4) ”. Supplementation with different added concentrations flavonoids significantly increased the dressing percentage and loin eye area of the lambs. This was in agreement with Hagos and Melaku who reported that supplementation improved carcass parameters of Afar rams. There was also significant difference (P < 0.05) among the supplemented groups for the dressing percentage and loin eye area (Hagos, & Melaku, 2009). Furthermore, Wenk (Wenk, Fernandez, & Dupuis, 2010) illustrated that “ pressing loss is a significant index reflecting the meat quality, which has an important meaning on the physics form flavor and flesh color of muscles ”. Then, Savage (Savage, Warriss, & Jolley, 1990) implied that “ per milliliter of pressing loss water contains 112 mg of protein ”. Luciano (Luciano et al., 2009) defined that “decreasing heme pigments with pressing loss water, which was positively related to sensory appreciation of meat colour ”. Pressing loss was significantly (P < 0.05) reduced linearly with different concentrations of flavonoids in treatments. The importance is that the pressing loss showed decreasing trend as flavonoids added to lambs diets increased. Zhang (Zhang et al., 2013) pointed out that “ it may be because flavonoids have antioxidant abilities, 9

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and dietary antioxidant can reduce Pressing loss of meat ”. Based on the assumption that the different ratios of flavonoids can improve water holding capacities of mutton. The cooking loss values increased as pressing loss decreased (Table 3). In agreement with GÖKALP (GÖKALP etal,1998) showed that “ lower cooking loss values for high pressing loss meats ”. Similar results were observed in our present study. According to (Hopkins et al., 2010) speculated that “ tenderness can be evaluated by objectively measuring the shear force ”. As a whole, meat quality of flavonoids treatment groups were superior to C. While the differences among groups were not found significant with regard to moisture, protein andFor Ash ( PReview > 0.05). (Table 3). TheOnly longissimus dorsi muscle moisture contents of smalltailed han sheeps of the present study (71 73%) was similar to values reported by other researchers for the some muscle (Ekiz et al., 2009). In addition, (Hopkins & Mortimer, 2014) denied that “ intramuscular fat has important effects on organoleptic traits ”. In recent studies, Pannier (Pannier et al., 2013) commented that “ the intramuscular fat range from 2.5 to 7.0%, the sensory scores for the loins increased by 10.7, 10.0, 9.1 and 5.9 units for juiciness, overall liking, flavor and tenderness respectively on a 0∼100 scale ”. Intramuscular fat in the treatments range (6.55 6.66%) was higher than C (6.51%) (P < 0.05). What is more, Warner (Warner, Greenwood, Pethick, & Ferguson, 2010) claimed that “ IF breaks up the structure of intramuscular connective tissue and it separates and dilutes the perimysial collagen fibers of the muscles ”. So, greater amounts of intramuscular fat improves meat tendness. The composition of fatty acids An increasingly important aspect of meat quality is its nutritive value, especially fatty acids compositions. Indeed, Yakan (Yakan & Ünal, 2010) echoed that “ almost all of the fats are localized as triglycerides in adipose of ruminants, and fatty acids are regarded as C16 and C18 ”. In our present study, we have determined the contents and ratios of the key fatty acids related to human health (Table 4). As a matter of fact, Sañudo (Sañudo et al., 2000) affirmed that “ for both taste panels, odor and flavor intensity were positively correlated with the amounts and percentages of C18:0 and 10

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negatively correlated with those of C18:2 ”. In this study, dietary supplementation with flavonoids reduced concentrations of C18:0. It was indicated that flavonoids reduced a certain extent the smell of mutton odor. Moreover, Cao (Cao et al., 2012) revealed that “ substituting dietary saturated fatty acids with monounsaturated fatty acids and polyunsaturated fatty acids has been reported to be associated with reducing risk of diseases about heart by lowering the lowdensity lipoprotein cholesterol ”. Laderia (Laderia et al., 2014) labeled that saturated fatty acid is of paramount importance in the production of healthier meat for consumers. On the one hand, Sierra (Sierra et al., 2008) mentioned that “ scientist has mainly considered a simultaneous reduction of saturatedFor fatty acidsReview and increase polyunsaturated Only fatty acids to prevent health problems ”. In our present experiment, meat from lambs fed the different added concentrations flavonoids diet tended to increase concentrations of polyunsaturated fatty acid and decreased saturated fatty acids (P < 0.05). Eicosapentaenoic acid is one of the beneficial fatty acids. On the other hand, Cottin (Cottin, Alsaleh, Sanders, & Hall, 2016; Ramprasath, Eyal, Zchut, Shafat, & Jones, 2014) surmised that “ the contents of eicosapentaenoic acids in a normal serving of meat are below the recommended levels to obtain significant human health benefit, which is why research into increasing the level of these fatty acids in the diet is an important goal ”. Eicosapentaenoic acid in treatments was higher then that in C (P < 0.05). We could concluded that the content of eicosapentaenoic acids increased linearly with different added concentration of flavonoids. The concentrations of fatty acids related to flavor According to Prescott (Prescott, Young, & O’Neill, 2001) emphasized that “ Typical sheep odor and flavor are often associated with an unpleasant smell and therefore lower consumer acceptance of sheep products such as lamb ”. For the consumer, it would be rather simple and practicable opportunity to achieve better consumer acceptance if the mutton reduced speciesspecific odor and flavor. Moreover, Wong (Wong et al., 1975) recommended that “ the branched chain fatty acids (BCFAs) 4methyloctanoic acid, 4methylnonanoic acid and 4ethyloctanoic acid were thought to be mainly responsible for speciesrelated 11

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flavor ”, especially, 4methyloctanoic acid (Prescott et al., 2001). Each BCFA, along with octanoic acid, have mutton and goatlike odors (Brennand et al., 1989) and so, when present in sufficient concentration, each of these compounds will contribute to the‘mutton’odor produced during the cooking process. The further study also showed that the meat products with higher acceptance contained lower concentrations of 4methyloctanoic acid (Watkins et al., 2014). Combined the result with those above studies’ conclusions, it demonstrated that the diets added into flavonoids can reduced the concentration of 4methyloctanoic acid, which improved the mutton speciesspecific odor and flavor. But the concentrations of 4methylnonanoic acid in treatments were insignificantFor Review with C. A lack of significant Only results concerning correlations could be due to other substances besides the two BCFAs tested being involved in lamb flavor. Another possibility would be a different fatty acids compositions in subcutaneous as opposed to intramuscular fat as observed for some fatty acids and reviewed by Wood (Wood et al., 2008). Differences in the fatty acid compositions of intramuscular fat with regard to BCFAs remain unclear but might be an interesting objective for further studies. Meanwhile, the presence of these BCFAs is not the only factor which will affect consumer acceptance; tenderness and juiciness are also important considerations (Thompson et al., 2005). Additionally, the overall odor and flavor of mutton will be affected by other factors as well; ultimate pH, for example (Braggins, 1996). Thus, while strategies to reduce 4methyloctanoic acid and 4methylnonanoic acid in meat would assist product acceptance by consumers, other factors need to be considered since these would also impact on the overall sheep meat quality. Conclusions In summary, dietary supplementation of diets with different added concentrations flavoniods can improve dressing percentage, loin eye area, and the content of intramuscular fat, which improved the meat tendness, with lower pressing loss. Dietary supplementation of complete diets with different added concentration flavoniods could decrease C18:0 and increase the EPA, PUFA and MUFA. All are recognized as being for human health through helping prevent cardiovascular and

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inflammatory diseases. The same time, appropriate additive amounts of flavonoids in diets could decrease the concentration of MOA related to flavor, which improved mutton speciesspecific odor and flavor. Acknowledgment Great thanks are due to Chen Bai and Ni Dan (Inner Mongolia Agricultural University) for him and her comments and corrections on this manuscript. We would like to express our sincere gratitude and appreciation to the projects of The National Natural Science Foundation of China (31260558, 31160474, 31460611) for providing the financial supportFor for this Reviewstudy. Only

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Table 1. Percent Compositions and nutrient levels of basal diets (g/Kg on dry matter basis)

Ingredient,%

Chinese wildrye gress hay 32.00

Alfalfa 17.80

Corn 23.00

Wheatbran 2.87 Sunflower meal expellerFor Review Only 16.92 The pea plant 2.45

Pomace 2.45

Dicalcium phosphate 0.72

Nacl 0.79

Premixa 1.00

Total 100.00

Nutrient levels

Digestible energy (MJ/Kg)b 16.88

Crude fat 16.33

Neutral detergent fiber 37.92

Acid detergent fiber 29.57

Calcium 1.39

Phosphorus 0.51

a The premix provided the following per kg of diets: 25 mg Fe(as ferrous sulfate), 29 mg Zn (as zinc sulfate), 8mg Cu(as copper sulfate), 30 mg Mn(as manganese sulfate), 0.04 mg I(as potassium iodide),0.1 mg Co(as cobalt sulfate), 3200 IU Vitamin A, 1200 IU Vitamin D, 20 IU Vitamin E. b Digestible energy was a calculated value, while the others were measured values.

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Table 2. Effects of feeding diets with different added concentrations flavonoids on meat quality traits and production performance in lambs

Groups1 Pvalue Parameters SEM2 C Flav 11 Flav 22 Flav 33 AMR×C Linear Quadratic Cubic pH1 6.07 For6.03 Review6.07 6.08 0.013Only 0.782 0.187 0.075 0.068 pH24 5.77 5.78 5.74 5.72 0.021 0.465 0.101 0.415 0.526

DP, % 45.6 47.8 47.2 50.9 0.050 0.0001 0.0001 0.0001 0.0001

LEA (cm2) 19.6 20.6 20.7 20.9 0.048 0.0001 0.0001 0.0001 0.006

PL, % 5.23 5.15 5.13 5.12 0.012 0.0001 0.0002 0.031 0.276

CL, % 58.4 58.5 58.6 58.6 0.016 0.0001 0.0001 0.029 0.435

SF (N) 46.1 46.5 46.2 46.3 0.010 0.145 0.078 0.120 0.004 1 C = complete diets alone, Flav 11 = complete diets+11 mg/Kg flavonoids, Flav 22 complete diets+22 mg/Kg flavonoids, Flav 33 = complete diets+33 mg/Kg flavonoids, DP = Dressing percentage, LEA = Loin eye area, PL = Pressing loss, CL = Cooking loss, SF = Shear force 2 SEM: standard error of the mean

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Table 3. Effects of feeding diets with different added concentrations flavonoids on general chemical composition of Longissimus dorsi muscle on fresh basis (%) in lambs

Groups1 Pvalue Parameters SEM2 C Flav 11 Flav 22 Flav 33 AMR×C Linear Quadratic Cubic

Moisture, % 72.3 72.4 72.2 72.4 0.014 0.073 0.090 0.070 0.0001

Protein,% 19.7 19.7 19.8 19.7 0.023 0.599 0.100 0.530 0.082

IF, % 6.51 6.55 6.63 6.66 0.008 0.0001 0.0001 0.549 0.0152

Ash, % 1.08 For1.08 Review1.08 1.10 0.008Only 0.433 0.092 0.191 0.541 1 C = complete diets alone, Flav 11 = complete diets+11 mg/Kg flavonoids, Flav 22 = complete diets+22 mg/Kg flavonoids, Flav 33 = complete diets+33 mg/Kg flavonoids, IF = intramuscular fat 2 SEM: standard error of the mean

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Table 4. Effects of feeding diets with different added concentrations flavonoids on fatty acids of Longissimus dorsi muscle on fresh basis (mg/g) in lambs

Groups1 Pvalue Fatty acids SEM2 C Flav 11 Flav 22 Flav 33 AMR×C Linear Quadratic Cubic

C10:0 0.051 0.053 0.051 0.051 0.0006 0.491 0.546 0.274 0.047

C12:0 0.061 0.062 0.063 0.061 0.0008 0.414 0.926 0.174 0.782

C14:0 0.058 0.056 0.059 0.056 0.0005 0.105 0.195 0.545 0.003

C14:1 0.032 0.032 0.034 0.032 0.0007 0.798 0.555 0.659 0.102

C15:0 0.078 For0.076 Review0.078 0.076 0.0007 Only 0.101 0.189 0.659 0.102

C16:0 4.580 4.571 4.593 4.588 0.005 0.422 0.132 0.863 0.212

C16:1 0.464 0.466 0.463 0.463 0.001 0.946 0.504 0.546 0.336

C17:0 0.152 0.154 0.153 0.153 0.0006 0.063 0.494 0.005 0.189

C17:1 0.146 0.147 0.146 0.147 0.0007 0.332 0.347 1.000 0.347

C18:0 2.807 2.794 2.795 2.791 0.003 0.005 0.010 0.167 0.163

C18:1n9t 0.397 0.396 0.398 0.399 0.002 0.877 0..594 0.690 0.905

C18:1n9c 6.435 6.424 6.403 6.418 0.0009 0.090 0.110 0.184 0.274

C18:2n6c 1.136 1.147 1.146 1.121 0.018 0.926 0.591 0.357 0.898

C20:0 0.028 0.025 0.025 0.026 0.002 0.215 0.321 0.388 0.965

C18:3n6 0.021 0.022 0.025 0.025 0.002 0.236 0.121 0.868 0.527

C18:3n3 0.089 0.100 0.101 0.384 0.146 0.546 0.212 0.381 0.667

C21:0 0.068 0.070 0.067 0.067 0.001 0.920 0.166 0.244 0.062

C20:4 0.316 0.320 0.324 0.329 0.001 0.0002 0.001 0.591 0.093

C20:5 0.052 0.056 0.057 0.059 0.002 0.047 0.138 0.110 0.902

C22:6 0.020 0.022 0.021 0.021 0.001 0.388 0.587 0.545 0.683

EPA 0.057 0.069 0.079 0.085 0.0009 0.0001 0.0001 0.045 0.658

TFFA 17.464 16.836 17.006 17.311 0.152 0.052 0.681 0.515 0.358

SFA 8.013 7.708 7.818 7.743 0.007 0.005 0.0001 0.0001 0.048

PUFA 1.734 1.735 1.753 2.022 0.147 0.581 0.679 0.149 0.959

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MUFA 7.517 7.393 7.435 7.546 0.009 0.0005 0.014 0.0001 0.054 1 C = complete diets alone, Flav 11 = complete diets+11 mg/Kg flavonoids, Flav 22 = complete diets+22 mg/Kg flavonoids, Flav 33 = complete diets+33 mg/Kg flavonoids. EPA = eicosapentaenoic acid, TFFA = total free fatty acid, SFA= saturated fatty acid, PUFA = polyunsaturated fatty acid, MUFA = monounsaturated fatty acid 2 SEM: standard error of the mean

For Review Only

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Table 5. Effects of feeding diets with different added concentrations flavonoids to on branched chain fatty acids in Longissimus dorsi muscle in lambs (mg/Kg)

Groups1 Pvalue BCFAS SEM2 C Flav 11 Flav 22 Flav 33 AMR×C Linear Quadratic Cubic

MOA 1.830 0.290 0.437 1.543 0.358 0.031 0.668 0.006 0.661

MNA 1.703 1.103 1.050 1.640 0.448 0.421 0.904 0.221 0.963

EOA 0 0 0 0 0 − − − − 1 C = complete diets alone, Flav 11 = complete diets + 11 mg/Kg flavonoids, Flav 22 = complete diets+22 mg/Kg flavonoids, Flav 33 = complete diets+33 mg/Kg flavonoids, MOA = 4methyloctanoic acid, MNA = 4methylnonanoic acid, EOA = 4ethyloctanoic acidFor Review Only 2 SEM: standard error of the mean

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