Effects of dietary energy level on growth performance, blood parameters and meat quality in fattening male Hu lambs

2.1 Ethics statement

All animal experimental procedures were performed according to protocols approved by the Animal Care Advisory Committee of Hunan Normal University, Changsha, Hunan, China.

2.2 Diets, animals and experimental procedures

This experiment was performed at the Hubei Zhiqinghe Agriculture and Animal Husbandry. Sixty 4-month-old male Hu lambs with initial body weight (IBW) of 20.16 ± 0.38 kg were used in this study. All lambs were randomly assigned to five possible treatments based on IBW. Each treatment was fed in individual pens (length × width × height = 5.0 × 2.5 × 1.0 m) with individual feeding and automatic water. The groups (n = 12) were then randomly assigned one of the following five diets with different metabolizable energy levels: 9.17, 9.59, 10.00, 10.41 and 10.82 MJ/kg. Diet formula design references by NRC 2007 according to lamb weight of 20 kg and daily gain of 200 g, except for energy, and other nutrients met the requirements for all test lambs. Feed was formulated as a mixed diet. Ingredients and nutrient levels are shown in Table 1. Lambs were fed twice daily at 07:00 and 17:00 hr during the trial period. Feed supply was adjusted for each group in the morning according to prior day dry matter intake (DMI) to assure that 5% remained.

2.3 Animal performance and serum biochemical indices

During the experiment, all the lambs were weighed individually in the morning on an empty stomach at the beginning and each 30-day intervals of the trial. The ADG was calculated on the basis of the average of three measurements for individual ADG. The FCR was calculated as DMI per ADG (Yin, Baidoo, Schulze, & Simmins, 2001).

At the end of the trial, blood samples were collected in a 5-ml vacuum tube without anticoagulant (Changsha Yiqun Medical Equipment) from the jugular vein of the five lambs from each group that was nearest to the average weight of the group before the morning feeding. Blood samples were centrifuged at 1006.2g for 10 min after standing for 2–3 hr; then, the supernatant was stored at −20°C. A TBA-120FR Automatic Biochemistry Radiometer (Hitachi) was used to measure the concentrations of serum biochemical indices (Chen et al., 2019; Yin et al., 2018).

2.4 Slaughtering and carcass traits

According to the results of growth performance, five lambs with mostly closing to the average weight of each group were selected for slaughter at the end of experiment from dietary energy levels of 9.17 (low energy, LE), 10.00 (medium energy, ME) and 10.82 MJ/kg (high energy, HE). Total of fifteen lambs were exsanguinated via the jugular vein in compliance with veterinary police rules after fasting for 12 hr and recording live body weight. At slaughter, the non-carcass parts of the head, feet, rumen, reticulum, omasum, abomasum, heart, liver, spleen, double kidneys, double kidney fats and mesenteric fats were removed and weighed. Carcasses were subsequently weighed and spilt longitudinally. Viscera indexes and fat ratios were calculated as a percentage of live weight before slaughter (LBWS). Dressing percentage = (carcass weight/LBWS) × 100%.

2.5 Meat quality physical analyses

A cross section of the area between the 12th and 13th spinal ribs was drawn on sulphur paper. A planimeter (KP-21C; Koizumi) was used to determine the area, this procedure was repeated twice, and the mean of the two measurements is reported as the loin muscle area. The longissimus dorsi (LD) muscle of the left half carcass was used to measure the pH, meat colour, amino acids (AA), fatty acids and muscle fibre characteristics. Amino acids, fatty acids and muscle fibre characteristics were also measured from the left biceps femoris (BF). LD muscle pH values were measured at slaughter plus 45 min (pH_45min) and after 24 hr (pH₂₄) of carcass refrigeration at 4°C (Russell CD700; Russell pH Limited). The average of the three measurements is the pH value of each sample. Meat colour imetric characteristics (L^*, a^*, b^*) were measured in three different places using a colorimeter (Minolta CR-300; Minolta Camera), and the average was reported as the meat colour value (Yin et al., 2017).

2.6 Muscle nutritional profile analysis

Amino acids contents of muscle were determined by Liu et al. (2019). Approximately 0.50 g of a freeze-dried sample was hydrolysed in 10 ml HCl of 6 mol/L at 110°C for 22 hr. The hydrolysate was diluted with deionized water to a volume of 100 ml. One ml of the settled solution was diluted 200 times, and then the diluent was filtered by a 0.22-μm membrane. One ml filtrate was used for analysis by using an ion-exchange AA analyser (Hitachi).

Intramuscular fatty acid profiles were measured. Lipids were extracted from the freeze-dried mutton samples via a benzene-petroleum ether (1:1, v/v) procedure. Fatty acid methyl esters were prepared for GC determination using KOH/methanol. A fatty acid methyl ester analysis was performed using an Agilent 7890A gas chromatographer fitted with a special capillary column SP-2560 (100m × 0.25 mm × 0.2 μm). It was equipped with a flame ionization detector (Agilent Technologies). An injection volume of 1 μl was used. The injector and detector temperatures were set at 280°C. The initial column temperature was set at 140°C for 5 min. It was then raised to 220°C at 3°C/min and kept at 220°C for 40 min. Hydrogen carrier gas of hydrogen flow rate was 30 ml/min. Individual fatty acid methyl esters were identified by retention times using an authenticated standard. Individual fatty acid concentrations were quantified according to the peak area and expressed as a percentage (wt/wt) of total methylated fatty acids (Yin et al., 2000).

2.7 Muscle fibre characteristics and myosin heavy chain (MyHC) gene expression

Within 2 hr post-slaughter, each one piece of muscle tissue (approximately 3.0 × 1.5 × 0.5 cm) was gently removed from the left LD and BF muscles, which was saved in a the neutral formalin solution immediately at 4°C, and then replaced by a fresh fixed liquid after 24 hr. Samples from the fixative solution were treated by washed, transparent, dipped in wax and embedded, and then were cut into 4-μm sections with a slicer at room temperature. The obtained slices were stained with haematoxylin–eosin and sealed for later microscopic examination. Thirty typical representative visual fields from each sample were selected from muscle tissue sections for each sample, and Image-Pro Plus software was used to measure the fibre diameter, area and density (Wang et al., 2019).

Approximately 100 mg of meat samples from the LD and BF muscles, respectively, was taken immediately post-slaughter. The samples were placed, put into an aseptic cryopreservation tube without RNA enzyme, then rapidly frozen in liquid nitrogen and refrigerated at stored in −80°C refrigerator for later total RNA extraction (Zong et al., 2019). Using the method described by Yang et al. (2013), total RNA was separated via a TRIzol reagent (Invitrogen). The RNA quantity and quality were examined via an ultraviolet spectrophotometer (NanoDrop ND-1000; Thermo Fisher Scientific). Real-time quantitative PCR analyses were performed (ABI 7900HT Fast Real-Time PCR System Applied Biosystems) with a total volume of 10 μl. The selected gene primer sequences are shown in Table 2. The mRNA expression abundance (A) of target genes was calculated as A = 2^−ΔΔCt [Ct(GAPDH)-Ct(target)]. The relative expression of MyHC genes in LD and BF muscles was examined for this study.

2.8 Statistical analysis

excel (Microsoft) was used for preliminary processing of test data processing. spss 18.0 software (SPSS) was used for data variance analysis of data, and Duncan's tests were used for multiple comparisons among different groups. The final test results were presented with mean ± SEM. A p-value of <.05 was significant, and p < .01 was extremely significant.

3.1 Body weight and daily weight gain

The growth performance is shown in Table 3 and Figure 1. Increasing energy levels from 9.17 to 10.82 MJ/kg, there were no significant differences for IBM and FBW (p > .05), but the FBW showed an increasing trend. The DMI and ADG had extremely increased (p < .001) by increasing the dietary metabolizable energy level. Feed conversion ratio (FCR) resulted in a significantly decrease (p < .01).

3.2 Blood biochemical indexes

The blood parameters were presented in Table 4. Our results were similar to those of previous studies (Abdel-Ghani, Solouma, Abd Elmoty, Kassab, & Soliman, 2011; El-Barody, Abdalla, & El-Hakeam, 2002). Dietary treatment did not significantly affect the concentrations of TP, ALB, ALT, AST, LDH, BUN, GLU, TG, CHOL, HDL, LDLC, LACT and NH₃ in lamb serum. ALB and GLU concentrations had a tendency to increase (p < .10) by increasing dietary energy levels. HDL concentration was greater (p < .10) in dietary energy of 9.17 MJ/kg than that 9.59 and 10.00 MJ/kg of dietary energy levels, and there were no significant differences with the increasing of dietary metabolizable energy levels from 9.17 to 10.82 MJ/kg.

3.3 Slaughter performance, organ index and meat quality

The slaughter performance for live weight before slaughter (LWBS) (p < .05), carcass weight (p < .05) and dressing percentage (p < .10) showed a significantly positive correlation by increasing the dietary energy level, and non-carcass fat, mesenteric fat and perirenal fat ratios had not obviously differences (Table 5). Compared with LE and ME levels, the HE level lambs had a higher non-carcass fat and perirenal fat ratios. There were no apparent effects on organ indexes and meat quality indicators of loin muscle area, pH_45min, pH_24h, L^*, a^* and b^* by increasing dietary energy levels (Table 5).

3.4 Muscle hydrolysate amino acid profiles

The amino acid profiles in muscle were less affected by dietary energy levels (Table 6). In LD muscle, except for enhanced glycine (Gly) concentration (p < .05) in group LE compared with the groups of ME and HE, no other amino acid concentrations were influenced by increasing dietary energy levels. In BF muscle, all of the amino acid concentration had no significant difference. However, the concentrations of total amino acid (TAA), essential amino acid (EAA) and most of the flavoured amino acid (FAA) concentrations except for Gly, cysteine (Cys) and proline (Pro) tended to increase by increasing the dietary energy levels. Overall, EAA, FAA and TAA concentrations in both LD and BF muscles were not notably different. The concentrations of all measured amino acids, EAA, FAA and TAA in BF muscle were numerically, greater than those in the LD muscle.

3.5 Intramuscular fatty acid profiles

In LD muscle, most of the fatty acid concentrations were unaffected by a high energy diet (Table 7). The concentrations of C17:0 (p < .01), C18:3n-3 (p < .01), C18:1n-9t (p < .05) and n-3 PUFA (p < .01) were decreased with increasing the dietary energy levels, while the ∑n-6/∑n-3 ratio was higher (p < .01) in the HE group than that in the LE and ME groups. Most of fatty acid concentrations were also not significantly affected by dietary energy levels in BF muscle. The concentrations of C17:0, C18:3n-3, (p < .01) and n-3 PUFA (p < .05) were notably influenced by energy, which were negatively correlated with the energy level of the diet. C20:1 concentration and the ∑n-6/∑n-3 ratio were significantly higher (p < .01) in the group HE than that in the LE and ME groups. Overall, the fatty acid profiles both in LD and BF muscles were not much affected by dietary energy levels. SFA and UFA concentration tendencies were similar in LD and BF muscles. The concentrations of SFA and UFA were not significantly affected by the dietary energy levels, but the SFA concentration turned to a downward trend, and the UFA concentration showed a rising tendency by increasing dietary energy levels.

3.6 Muscle fibre characteristics and MyHC gene expression

The muscle fibre diameter, area and density are summarized in Table 8 and Figure 2. At least ten areas were randomly sampled. The number of muscle fibres was counted within each area for density statistics. Diameters and areas for approximately 20 fibres were measured from the same ten areas. The results showed that muscle fibre diameter, area and density for LD and BF muscles were not significantly affected by different dietary energy levels.

The relative expressions of MYHC-IIa and MYHC-IIx in BF muscle decreased significantly (p < .05) as dietary energy levels increased (Table 9). In LD muscle, MYHC-IIa relative expression notably decreased (p < .10) as dietary energy levels increased. MYHC-IIb relative expression in the HE group was higher than in the LE and ME groups.

4.1 Body weight and daily weight gain

Digestible nutrients are the material basis of animal growth and development. Energy intake levels directly determine animal growth rates. Studies show that animals can adjust their feed intake according to the energy level in the diet within a certain range, and increasing energy levels of the diet can improve weight gain rates and feed rewards in mutton sheep (Yerradoddi et al., 2015; Kabir et al., 2014; Sayed, 2011). In this experiment, metabolizable energy and DMI differences may be the main reasons for a group's different growth performances. ADG was significantly increased by increasing dietary energy level for lambs aged 120–180 days. This is consistent with Kabir et al. (2014) and Yerradoddi et al. (2015). When metabolizable energy reached a certain level (10.41 MJ/kg), the growth rate of male Hu lambs was slowed, and there was no difference between 10.41 and 10.82 MJ/kg. Previous study shows that DMI reduces with an increase in dietary energy (Sayed, 2011; Kabir et al., 2014), and Kabir et al. (2014) also find that too low or too high energy levels affect DMI. However, our study was inconsistent with their findings, and this may be related to the proportion of roughage and concentrate. We regulated dietary energy level mainly by adjusting the concentrations of silage corn and corn, and the concentrations of NDF and ADF reduced by the increase in dietary metabolizable energy level in this study, which may improve the palatability and eventually lead to the increase in DMI. Meanwhile, the roughage surplus in the low-energy group was higher than that in the high energy group in the actual feeding process. Additionally, the differences may relate to sheep breeds, physiological stages or different test seasons (Arsenos et al., 2002; Pereira et al., 2017). FCR significantly decreased by increasing dietary energy levels from 9.17 to 10.41 MJ/kg, but there were no significant differences between 10.41 and 10.82 MJ/kg of dietary energy levels, which suggested that too high dietary energy levels inhibited the DMI of male Hu lambs. From the standpoint of improvement economic efficiency, when the dietary metabolizable energy level was 10.41 MJ/kg, the highest comprehensive economic benefit and the highest feed reward were obtained by evaluating the weight gain income and removing the feed cost.

4.2 Blood biochemical indexes

Dietary nutrients are digested and absorbed, and then, blood circulation transports them to the tissue, organs and cells. Blood biochemical indexes are important indicators that reflect nutritional levels. Research shows that changing nutrition levels of feed affect energy metabolism and changes in serum biochemical indexes reflect the metabolism in such things as the GLU serum content which can reflect the energy metabolism of the body (Graugnard et al., 2012). Insufficient animal dietary energy intake can lead to low GLU blood levels. Chelikani, Ambrose, and Kennelly (2003) found that GLU in serum increased in step with dietary energy level increases. The results of this experiment are similar to these research results. The GLU concentration increased as the dietary energy level increasing except for the group of LE level. Serum BUN content can be estimated and is an indicator measure of protein and amino acid metabolism in vivo, and low BUN content suggests that nitrogen metabolism efficiency is high (Song et al., 2018). This study indicated that there were no significant differences in serum BUN concentration among different dietary energy levels. This suggests that dietary energy levels may have little effect on protein and amino acid metabolism in male Hu lambs.

4.3 Slaughter performance, organ index and meat quality

Slaughter traits are an important index for evaluating ruminant production performances. They can directly reflect the body composition proportions of the edible parts. Studies have reported that slaughter rates trend upward as LWBS increases (Gökdal, Atay, Eren, & Demircioğlu, 2012; Hawkins et al., 1985; Sen, Sirin, Ulutas, & Kuran, 2011). This experiment had results consistent with those found above. A proper increase in energy level improved growth performance significantly. LWBS differences were significant, and dressing percentage slaughter rates also rose notably. There were no significant increases in non-carcass fat ratio. Under a condition of increasing dietary energy levels, muscle growth and fat deposition rates accelerate, resulting in a decrease in viscera index (Fluharty & Mcclure, 1997). Although there were no significant differences between internal organs indices for each group, this result was consistent with previous studies (Khliji, Ven, Lamb, Lanza, & Hopkins, 2010). Livers and spleens were more developed in HE than those for either ME or LE, and this suggests that HE had a higher digestive metabolism and protein synthesis efficiency causing it to have better weight gain performance in production trait. Meat colour is the preferred apparent indicator for consumer judgements of meat quality. It is an extremely important factor influencing consumer choice (WHO, 1985). The results of this study suggest that properly increasing dietary energy levels did not significantly affect meat colour.

4.4 Muscle hydrolysate amino acid profiles

In this study, amino acid composition was not affected by energy levels. EAA was abundant, approximately 42%–44%, in meat samples. This exceeds the FAO/WHO recommended ratio of 40% which appears (Wood et al., 2008). The EAA-to-non-essential amino acid (NEAA) ratio in each group was more than 70%. This is significantly higher than the FAO/HWO high-quality protein standard of 60% (Fluharty & Mcclure, 1997; Webb and O'Neill, 2008). Hu lamb meat appears to be an excellent consumer choice which meets human requirements for high-quality protein food.

4.5 Intramuscular fatty acid profiles

Dietary composition affects muscle total fatty acid composition (Luciano et al., 2013; Wood et al., 2008). Fatty acids are either bio-hydrogenated or transformed by rumen micro-organisms. Diets with high forage proportion stimulate rumen activity (Beam et al., 2000). Song et al. (2017) found that energy-restricted feeding alters meat fatty acid composition. The results of this study are similar to Song et al. (2017). LD and BF muscle fatty acid compositions were reflected in dietary fatty acid composition. Medium and long-chain fatty acid content was not significantly different between the treatments. It was dominated by MUFA, followed by SFA and PUFA. This is consistent with other studies (Maleki, Kafilzadeh, Meng, Rajion, & Ebrahimi, 2015; Lestingi, Facciolongo, Marzo, Nicastro, & Toteda, 2015). The primary UFA in LD and BF muscles was cis-9-octadecanoic acid (C18:1n-9c) and linoleic acid (18:2n-6c). The main SFA was palmitic (C16:0) and stearic (C18:0) acid. There were considerable differences attributable to dietary energy levels. Odd chain fatty acid (C17:0) concentration decreased significantly with increased dietary energy levels in LD and BF muscles. Trans-9-octadecanoic acid (C18:1n-9t) lessened in LD for high energy fed lambs. This may have been due to altered stearoyl-CoA desaturase activity. That can catalyse stearic acid desaturation at carbon 9 to produce elaidic acid (C18:1) (Zhang, Zhang, Shao, Wang, & Gong, 2013). The primary n-3 PUFA was C18:3n-3. The concentrations of C18:3n-3 and n-3 PUFA were significantly decreased in LD and BF muscles as energy increased in the present study, which may be related to the concentration of C18:3n-3 in the feed. Red meat contains n-3PUFA which is beneficial to human health (Daley, Abbott, Doyle, Nader, & Larson, 2010). The minimum ∑n-6/∑n-3 ratio value was 11.80. It was much higher than some nutritional advice recommendations (<4.0) (Scollan et al., 2006). Di Luccia et al. (2003) reported that dietary energy levels influence UFA composition. MUFA concentrations increased, and PUFA percentages decreased by increasing dietary energy levels in this study. Lopes et al. (2014) reported that UFA and MUFA concentrations were greater for ad libitum fed goats, and n-3 PUFA concentrations were lower compared with feed restricted goats. Our findings were in agreement with these results.

4.6 Muscle fibre characteristic and MyHC gene expression

Muscle fibre is the main component of muscle tissue. The LD and BF muscles are the primary constituents of sheep trunk and posterior drive muscle development. The number of muscle fibres is fixed soon after birth, and lamb weight increase depends primarily on muscle fibre growth including the gradual increases in muscle fibre diameter, area and length (Joubert, 1956). There is a close relationship between the muscle fibre morphological characteristics and meat quality (de Araújo et al., 2017; Arsenos et al., 2002). Several studies have confirmed that muscle fibre diameter and density closely relate to muscle tenderness and water power (Mandell et al., 1997; Wood et al., 2008). The smaller the diameter and the greater the density of muscle fibre suggest that the more intramuscular fat is accumulated; the meat is more tender. Conversely, the meat becomes harder or tougher (Eddinger, Moss, & Cassens, 1985; Fahey, Brameld, Parr, & Buttery, 2005). This study found no significant differences in LD or BF muscle fibre diameters, areas or densities as increasing dietary energy levels. These results suggested that dietary energy within the 9.17–10.82 MJ/kg ranges had little effect on meat quality or tenderness.

Muscle fibre types greatly influence meat quality (Joo et al., 2013). Dietary nutrient levels affect muscle fibre type composition and MyHC expression. Solomon and Lynch (1988) found that dietary energy densities affect sheep muscle fibre composition. LD muscles have more type I and fewer type IIB fibres when fed a low-energy diet. Four types of MyHC: I, IIa, IIx and IIb genes express in most mature mammalian muscles. MyHC is also classifiable as slow (type I) and fast (types IIa, IIx and IIb) according to their ATPase activity and contraction speed (Anne et al., 2016). In this study, MyHC relative expression showed there are no significant differences in LD muscle with increasing dietary energy level. MyHC-IIa and MyHC-IIx relative expression in BF muscle significantly decreased by increasing dietary metabolizable energy level. These results suggested that increasing energy levels reduced the relative expression in fast muscle fibre, and different dietary energy levels had a certain effect on MyHC expression, especially in BF muscle.