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Use of Hermetia illucens larvae as a dietary protein source: Effects on growth performance, carcass traits, and meat quality in finishing pigs
Meat Science 158 (2019) 107837
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Use of Hermetia illucens larvae as a dietary protein source: Effects on growth performance, carcass traits, and meat quality in finishing pigs
T
Miao Yua,b,c,d,e, Zhenming Lia,b,c,d,e, Weidong Chena,b,c,d,e, Ting Ronga,b,c,d,e, ⁎ Gang Wanga,b,c,d,e, Jianhao Lia,b,c,d,e, Xianyong Maa,b,c,d,e, a
Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640, China State Key Laboratory of Livestock and Poultry Breeding, Guangzhou, Guangdong 510640, China c Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture, Guangzhou, Guangdong 510640, China d Guangdong Public Laboratory of Animal Breeding and Nutrition, Guangzhou, Guangdong 510640, China e Guangdong Engineering Technology Research Center of Animal Meat Quality and Safety Control and Evaluation, Guangzhou, Guangdong 510640, China b
ARTICLE INFO
ABSTRACT
Keywords: Meat quality Intramuscular fat Finishing pigs Hermetia illucens larvae Lipid metabolism
This study investigated the effects of feeding Hermetia illucens larvae (0, 4, and 8%; HI0, HI4, and HI8 groups, respectively) on growth performance, carcass traits, and meat quality of finishing pigs. Results showed that the HI4 diet increased (P < .05) final body weight and average daily gain and decreased (P < .05) feed to gain ratio compared with HI0 and HI8 group. HI4 and HI8 diets increased (P < .05) loin-eye area, marbling scores, and inosine monophosphate content of longissimus thoracis (LT) compared with HI0 diet. The intramuscular fat content was greater (P < .05) in HI4 group than in the HI0 group. Furthermore, HI4 diet up-regulated (P < .05) lipogenic genes and MyHC-IIa mRNA levels in LT compared with HI0 diet. Our results indicated that dietary inclusion of H. illucens larvae has a beneficial impact on growth performance and meat quality, and the underlying mechanism may be due to the altered lipogenic potential induced by H. illucens larvae.
1. Introduction Along with growth in the global human population, the consumption and demand for livestock products drastically increases. In parallel, this affects the demand for livestock feeds, and places heavy pressure on already overexploited resources, especially protein feeds. Soybean meal is the conventional feed resource of protein in the Chinese livestock industry, and the supply has declined in recent years while the demand has continued to rise, resulting in soaring prices for purchasing feed in livestock production. Thus, it is imperative to find new alternative, and sustainable protein sources for livestock feed to reduce the use of soybean meal. In this case, several insect species, such as Hermetia illucens, have attracted increased interest as a sustainable alternative to meet demand and either partly or completely replace conventional protein feed sources (Makkar, Tran, Heuzé, & Ankers, 2014; Van Huis, 2013). H. illucens larvae are a high-value feedstuff source, containing about 40 to 44% crude protein with a similar or better amino acid profile compared with that of soybean meal (Barragan-Fonseca, Dicke, & Loon, 2017; Veldkamp, 2015). Additionally, H. illucens larvae are also rich in fat, with the content ranging from 15 to 49% on a dry matter basis (Makkar
⁎
et al., 2014; Spranghers et al., 2017), which can be defatted and used for the preparation of biodiesel, while the remainder of the defatted meal could be used as an alternative sustainable protein rich ingredient for the feed industry (Dabbou et al., 2018). Thus, there is an urgent need to investigate their viability as alternative sustainable protein feed for animal production. Until now, the administration of H. illucens larvae as an alternative for soybean meal in diets was shown to modulate the growth performance, nutrient digestibility, and blood profiles of chickens (Cutrignelli et al., 2018; Maurer et al., 2015), weaning or growing pigs (Dabbou et al., 2018; Hale, 1973; Spranghers et al., 2017), and the carcass and meat traits of chickens (Cullere et al., 2016), without negative effects. These results indicate that H. illucens larvae can be used in the diet of monogastric animals as a potentially valuable feed ingredient. However, little information is available on the growth performance, carcass and meat traits of H. illucens larvae in finishing pigs. It is well known that intramuscular fat (IMF) is regarded as an important factor that affects pork quality. High contents of IMF could enhance meat quality as it contributes to pork tenderness and flavor (Wood et al., 2013). In laying hens, H. illucens larvae completely replacing soybean meal in the diet decreased serum and egg cholesterol
Corresponding author at: Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640, China. E-mail address: [email protected] (X. Ma).
https://doi.org/10.1016/j.meatsci.2019.05.008 Received 21 December 2018; Received in revised form 5 May 2019; Accepted 7 May 2019 Available online 08 May 2019 0309-1740/ © 2019 Published by Elsevier Ltd.
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and triglycerides levels (Marono et al., 2017). In growing pigs, dietary H. illucens larvae inclusion increased serum high density lipoprotein concentration in growing pigs (Zhang et al., 2018), suggesting an impact of H. illucens larvae on lipid metabolism. However, whether and how the lipid metabolism can be regulated by H. illucens larvae feeding to finishing pigs, remains relatively unknown and requires further investigation. Additionally, muscle fiber type composition can influence several meat quality traits, e.g. tenderness and water-holding capacity (Joo, Kim, Hwang, & Ryu, 2013). To our knowledge, no research has been conducted to test whether H. illucens larvae treatment can change meat quality and, if so, whether lipid metabolism or muscle fiber characteristics can be changed by feeding H. illucens larvae. Therefore, in the present study, we hypothesized that H. illucens larvae as a dietary protein source may alter the meat quality traits of finishing pigs by influencing lipid metabolism and muscle fiber characteristics of the longissimus thoracis muscle (LT).
Table 1 Feed ingredient and nutrient composition of experimental diets (as-fed basis). Items
Treatment HI0
Ingredient, % Corn 71.00 Soybean meal 16.98 Wheat bran 6.00 Hermetia illucens L. – Soybean oil 2.50 L-Lysine-HCl(98%) 0.38 DL-Methionine 0.06 L-Threonine 0.06 L-Tryptophan 0.02 Dicalcium phosphate 0.95 Limestone 0.75 Salt 0.30 1 Vitamin-mineral premix 1.00 Total 100.00 Calculated content2 ME3, MJ/kg 14.37 Standard ileal digestible amino acid, % Lysine, % 0.88 Methionine + Cysteine 0.47 Threonine 0.49 Tryptophan 0.15 Analyzed nutrient composition4 Dry matter, % 89.22 Crude protein, % 14.53 Crude ash, % 4.36 Crude fat, % 5.10
2. Material and methods The experimental proposal and procedures for the care and treatment of the pigs were approved by the Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences (authorization number GAASIAS-2016-017). 2.1. Preparation of H. illucens larvae meal H. illucens larvae were purchased from Guangzhou AnRuiJie Environmental Protection Technology Co., Ltd. (Guangzhou, Guangdong, China). The prepupae were dried at 80 °C for 30 min and subsequently air dried. Finally, the dried prepupae were crushed to powder through a 1.0-mm, and then kept in a well-closed and lightresistant place. The chemical composition, energy content, and amino acid composition of the H. illucens larvae are shown in Supplementary Table S1.
HI4
HI8
71.20 13.86 6.00 4.00 1.70 0.40 0.07 0.11 0.01 0.95 0.40 0.30 1.00 100.00
71.76 10.75 6.00 8.00 0.50 0.42 0.09 0.15 – 1.00 0.03 0.30 1.00 100.00
14.38
14.38
0.88 0.47 0.50 0.15
0.88 0.47 0.50 0.14
89.16 14.53 4.39 5.10
89.23 14.54 4.50 5.10
1
Provided per kilogram of complete diet: vitamin A, 15,000 IU; vitamin D3, 3000 IU; vitamin E, 150 mg; vitamin K3, 3 mg; vitamin B1, 3 mg; vitamin B2, 6 mg; vitamin B6, 5 mg; vitamin B12, 0.03 mg; niacin, 45 mg; vitamin C, 250 mg; calcium pantothenate, 9 mg; folic acid, 1 mg; biotin, 0.3 mg; choline chloride, 500 mg; Fe (FeSO4·H2O), 170 mg; Cu (CuSO4·5H2O), 150 mg; I (KI), 0.90 mg; Se (Na2SeO3),0.2 mg; Zn (ZnSO4·H2O), 150 mg; Mg (MgO), 68 mg; Mn (MnSO4·H2O), 80 mg; Co (CoCl2), 0.3 mg. 2 Values were estimated based on the chemical analysis. 3 ME = metabolizable energy. 4 Analytical results obtained according to AOAC(AOAC, 2007).
2.2. Animals, experimental design, and diets The present study was conducted to evaluate the effects of dietary inclusion of H. illucens larvae meal in the diet of finishing pig. A total of 72 crossbred (Duroc × Landrace × Large White) female pigs with a similar initial body weight (BW, 76.0 ± 0.52 kg) were randomly allocated to three dietary treatment groups in a completely randomized design. Pigs in the three treatments received increasing levels of H. illucens larvae meal (0, 4% and 8%; HI0, HI4, and HI8, respectively). Each treatment consisted of eight pens (replicates), with three pigs per pen. The contents of metabolizable energy (ME) and crude protein (CP) in each diet were equalized by adjusting the proportion of soybean meal, corn, and soybean oil. Meanwhile, the experimental diets were formulated to meet or exceed the nutrient recommendations of the National Research Council (NRC) (NRC, 2012) (Table 1). During the 46day experimental period, pigs were housed in environmentally controlled confinement pens (2.5 × 3.0 m) that had partial concrete slatted floors. Water and feed were provided ad libitum for pigs throughout the trial period. The feed consumption per pen was recorded every day to calculate the average daily feed intake (ADFI). The BW of all pigs was recorded at the beginning and end of the experimental period just before the morning feed to determine the average daily gain (ADG). The feed to gain ratios (F:G) were calculated from the feed intake and the increased BW during the whole experimental period.
immediately excised from the right side of the carcass and flash frozen in liquid nitrogen for later gene expression analysis. Within 30 min after slaughter, the hot carcass weight (HCW) was measured to calculate the dressing percentage. About 100 g of LT between the 9th and 10th ribs of the left side were sampled and stored at −80 °C to determine the IMF, inosine monophosphate (IMP), and fatty acid profile. 24 h after slaughter, the LT on the left half of each carcass between the 10th and 13th ribs were sampled for analysis of meat quality. 2.4. Measurement of carcass characteristics and meat quality The average backfat thickness was measured at the first rib, lumbar, and the last rib of the left side. The loin-eye area (LEA) was measured by tracing the outline of the LT area at the 10th rib and then measuring the area using a planimeter and the average of three measurements was used as LEA for each carcass. The subjective marbling score of the LT was determined on the 10th-rib using the guidelines of the National Pork Producers Council (NPPC) (NPPC, 1991). The fat-free lean index (FFLI) was calculated according to the guidelines of NPPC, FFLI = (8.588 + (0.465 × 2.2046 × HCW, kg) + (3.005 × 0.155 × 10th rib loin-eye area, cm2) – (21.896 × 0.0394 × 10th rib fat depth, mm)) / (2.2046 × HCW, kg). Post-slaughter, the pH at 45 min (pH 45 min) and the pH at 24 h (pH 24 h) were measured by a using a portable pH meter (HI 9024C; HANNA Instruments, Woonsocket, RI, USA), which was calibrated at the beginning of each measuring day using pH 4.6 and 7.0 buffers. The
2.3. Sample collection and preparation At the end of the feeding period (on day 46), one pig with medium weight from each pen (eight pigs/treatment) was randomly selected. After fasting for approximately 12 h, pigs were euthanized via electrical stunning and exsanguination. Samples (about 5 g) of LT were 2
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loin meat color, including L* (lightness), a* (redness), and b* (yellowness), was measured at 45 min and 24 h postmortem as a mean of three random readings, allowing about 10–15 min for the color to develop after cutting, and using a Minolta CR-400 Chroma meter (Konica Minolta Sensing Ins., Osaka, Japan) calibrated against a standard white plate (8 mm diameter aperture, d/0 illumination system). The drip loss was determined as previously described (Honikel, 1998). For the determination of shear force, at 24 h postmortem, samples of LT muscle (about 150 g) were placed in individual polyethylene vacuum bags and cooked to an internal temperature of 70 °C in a water bath at 75 °C. Then, cooked samples were cooled in running water to room temperature. Thereafter, the cooked samples were cut into shaped strips with a diameter of 1.27 cm and length of approximately 20 mm, and sheared parallel to the muscle fiber direction as described by a previous study (Li, Li, Zhang, Gao, & Zhou, 2017). The shear force (N) of the LT was measured using a digital-display-muscle tenderness meter (C-LT3B, Tenovo, Harbin, China) with a load cell of 15 kg and a 200 mm/min crosshead speed. Ten replicates of each sample were measured.
2.7. Statistical analysis All experimental data was analyzed with the SPSS software package (SPSS v. 20.0, SPSS Inc., Chicago IL, USA). Normal distribution of the data was confirmed by Shapiro-Wilk test before assessing differences between groups. All normally distributed data were analyzed by oneway ANOVA. Diet treatment was the fixed effect and pen was the random effect in the statistical model. Data were expressed as the mean ± SEM. Differences among treatments were compared using Tukey post hoc test. Differences were identified as significant at P ≤ .05. 3. Results 3.1. Growth performance and carcass traits The growth performance and carcass traits of pigs fed diets with varying H. illucens larvae concentrations are presented in Table 2. The HI4 diet significantly increased (P < .05) the final BW and ADG of pigs, and decreased (P < .05) the F:G compared with HI0 and HI8 diets, while there was no significant difference (P = .466) on ADFI among the HI0, HI4, and HI8 groups. Furthermore, the HI4 and HI8 diet significantly increased (P < .05) the loin-eye area compared with HI0 diet. However, the diets with H. illucens larvae had no effect (P > .05) on hot carcass weight, carcass yield, average backfat, and FFLI.
2.5. Diet and muscle chemical analysis Chemical analysis of the H. illucens larvae and experimental diets were analyzed by the methods of the Association of Official Analytical Chemists (AOAC) (AOAC, 2007) for dry matter (DM), ash, crude fat, and crude protein (CP). The LT samples were analyzed for DM and IMF, according to the procedures of AOAC (AOAC, 2007). To determine the amino acid composition of H. illucens larvae meal (except methionine and tryptophan), approximately 1.0 g of each sample was digested in 10 mL of 6 N HCl at 110 °C for 24 h; the methionine concentration was measured after oxidation with performic acid, and tryptophan concentrations were determined after alkaline hydrolysis according to AOAC (AOAC, 2007). The concentration of IMP in the LT was measured using High Performance Liquid Chromatography (HPLC) as described in a previous study (Li, Duan, et al., 2015). Lipids were extracted from the LT tissue samples by the chloroform-methanol (1:1, v/v) procedure. Fatty acid methyl esters of the H. illucens larvae and experimental diets were prepared by using HCl/methanol as described by a previous study (Luo et al., 2009). For LT, the samples freeze-dried and then fatty acid methyl esters were separated using sodium methoxide in method followed by the addition of HCl/ methanol as basic and acid catalysts, respectively. Fatty acid methyl esters were separated and determined with an Agilent 7890B GC equipped with a flame ionization detector, and with a method according to previous study (Li, He, et al., 2015; Vahmani et al., 2017). The individual fatty acid methyl esters were identified by comparing the retention times of the peak area with those of known standards, and expressed as a percentage of total fatty acids. The fatty acid profiles of H. illucens larvae and experimental diets are shown in Supplementary Tables S2.
3.2. Meat quality The meat quality of pigs is shown in Table 3. The HI4 and HI8 diets significantly increased (P < .05) marbling scores compared with HI0 diet. However, dietary H. illucens larvae inclusion did not affect (P > .05) the 45 min and 24 h pH values, the 45 min and 24 h L* values, a* value, and b* value, drip loss, and shear force. 3.3. Muscle chemical composition As shown in Table 4, HI4 diet significantly increased (P < .05) the IMF contents in the LT of pigs compared with HI0 diet. In addition, the HI4 and HI8 diets increased (P < .05) the IMP concentration compared with HI0 diet. However, dietary H. illucens larvae meal did not affect the DM content in the LT of pigs. Table 2 Effects of the dietary inclusion of Hermetia illucens larvae on the growth performance and carcass traits of finishing pigs.
2.6. RNA extraction and quantitative real-time PCR
Items
Total RNA was extracted from the LT using the TRIzol reagent (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. The RNA quality of every sample was quantified by Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Wiington, DE, USA), and the ratio (OD260:OD280) ranged from 1.8 to 2.0. Thereafter, 1 μg total RNA was reverse-transcribed to cDNA using a Synthesis Kit (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. Primers used for selected genes were presented in Table S3 in the supplemental material. Real-time PCR of the target genes and β-actin was performed on CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with TB Green™ Premix Ex Taq™ (Takara Biotechnology, Dalian, China). The reaction mixtures and real-time PCR conditions were set according to a previous study (Yu et al., 2017). The housekeeping gene β-actin was used to normalize the expression levels of each target gene, and according to the following formula 2−(∆∆Ct), where ∆∆Ct = (Cttarget – Ctβ-actin)treatment – (Cttarget – Ctβ-actin)control.
Initial BW (kg) Final BW (kg) ADG(kg/d) ADFI(kg/d) F:G Hot carcass weight, kg Carcass yield, % Back fat, cm Loin eye area, cm FFLI, % a,b
Treatment
P value
HI0
HI4
HI8
76.34 ± 0.57 115.68 ± 0.62b 0.89 ± 0.03b 2.83 ± 0.05 3.21 ± 0.06a 87.95 ± 1.24 74.89 ± 0.79 25.05 ± 1.24 45.35 ± 1.64b 50.87 ± 0.60
76.46 ± 0.57 119.60 ± 0.95a 0.98 ± 0.03a 2.77 ± 0.03 2.85 ± 0.12b 87.93 ± 1.57 73.89 ± 0.58 25.08 ± 2.86 54.68 ± 3.62a 53.96 ± 1.50
76.46 ± 0.58 114.57 ± 0.96b 0.86 ± 0.02b 2.77 ± 0.04 3.24 ± 0.05a 85.40 ± 1.21 74.82 ± 0.48 25.67 ± 1.06 49.29 ± 1.02a 52.32 ± 1.60
0.980 0.009 0.013 0.466 0.028 0.329 0.574 0.967 0.009 0.484
Means in the same row with different superscripts differ (P < .05). Abbreviations: BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; F:G, feed to gain ratio; FFLI, fat-free lean index.
3
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Table 3 Effects of the dietary inclusion of Hermetia illucens larvae on the meat quality of finishing pigs. Items
Treatment
pH45 min pH24 h L* (lightness) 45 min a* (redness) 45 min b* (yellowness) 45 min L* (lightness) 24 h a* (redness) 24 h b* (yellowness) 24 h Marbling scores Drip loss, % Shear force, N a,b
P value
HI0
HI4
HI8
6.39 ± 0.06 5.45 ± 0.03 44.82 ± 0.66 16.47 ± 0.26 1.58 ± 0.32 55.67 ± 0.90 16.26 ± 0.34 2.26 ± 0.25 2.69 ± 0.10b 2.63 ± 0.10 33.23 ± 2.33
6.42 ± 0.08 5.48 ± 0.05 45.27 ± 0.38 16.70 ± 0.12 1.62 ± 0.28 54.70 ± 0.98 16.32 ± 0.22 2.38 ± 0.22 3.13 ± 0.10a 2.64 ± 0.09 35.75 ± 4.07
6.38 ± 0.10 5.50 ± 0.04 45.22 ± 0.55 16.19 ± 0.12 1.46 ± 0.22 54.25 ± 0.70 16.24 ± 0.14 1.86 ± 0.35 3.15 ± 0.20a 2.72 ± 0.10 40.65 ± 4.07
0.930 0.712 0.821 0.222 0.911 0.514 0.972 0.350 0.049 0.771 0.282
Means in the same row with different superscripts differ (P < .05).
Table 4 Effects of the dietary inclusion of Hermetia illucens larvae on the longissimus thoracis muscle chemical composition of finishing pigs (as-fresh basis). Items
DM, % IMF,3% IMP,4mg/g
Treatment
Table 5 Effects of the dietary inclusion of Hermetia illucens larvae on the fatty acid profile of the longissimus thoracis muscle of pigs (% total fatty acids).
P value
HI0
HI4
HI8
27.30 ± 0.19 2.63 ± 0.17b 1.87 ± 0.04b
27.86 ± 0.32 3.26 ± 0.14a 2.01 ± 0.04a
27.74 ± 0.13 2.79 ± 0.20ab 2.03 ± 0.03a
Items
0.219 0.049 0.008
C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 n-7 C17:0 C18:0 C18:1 n-9 C18:2 n-6 C18:3 n-3 Cγ18:3 n-6 C20:0 C20:1 C20:2 C20:3 n-6 Cγ20:3 n-6 C20:4 n-6 C20:5 n-3(EPA) C22:0 C22:1 C22:5 n-3 (DPA) C22:6 n-3 (DHA) C24:0 SFA MUFA PUFA PUFA n-6 PUFA n-3 PUFA n-6/PUFA n3
a,b
Means in the same row with different superscripts differ (P < .05). Abbreviations: DM, dry matter; IMF, intramuscular fat; IMP, inosine monophosphate.
3.4. Fatty acid profiles in longissimus thoracis muscle The fatty acid profiles of the LT are shown in Table 5. The HI4 diet also increased (P < .05) the concentrations of C18:3, C20:4, C20:5, C22:6, and the sum of n-3 polyunsaturated fatty acid (PUFA) compared with the HI0 diet, and increased (P < .05) the concentration of C18:3 compared with the HI8 diet. Furthermore, the HI8 diet significantly increased (P < .05) the concentrations of C12:0, C14:0, C16:1, C20:4, C20:5, and C22:6 compared with the HI0 diet, but decreased (P < .05) the concentration of C18:3. Although a number of the fatty acid concentrations of the LT in finishing pigs were altered, the sum of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), PUFA, and the ratio of PUFA n-6/PUFA n-3 did not significantly differ among all three treatments. 3.5. Expression of genes related to lipid metabolism and myosin heavy-chain in longissimus thoracis muscle To gain further insight into the molecular mechanism underlying the effect of H. illucens larvae administration induced regulation of IMF content, the mRNA expression levels of critical lipid metabolism factors were measured in the LT. As shown in Fig. 1, the HI4 and HI8 diets significantly up-regulated (P < .05) the mRNA expression level of fatty acid synthase (FAS) compared with the HI0 diet. Additionally, the HI4 diet also up-regulated (P < .05) the mRNA expression level of acetyl CoA carboxylase α (ACCα) and lipoprotein lipase (LPL) compared with HI0 diet. However, dietary H. illucens larvae inclusion did not affect (P > .05) the mRNA expression level of peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element binding protein-1c (SREBP-1C), carnitine palmitoyl transferase 1B (CPT1B), fatty acid transport protein 1 (FATP1), and hormone-sensitive lipase (HSL). As shown in Fig. 2, the HI4 diet significantly up-regulated (P < .05) the mRNA expression level of myosin heavy chain (MyHC)IIa compared with the HI0 diet. However, the mRNA expression levels
Treatment
P value
HI0
HI4
HI8
0.13 ± 0.01 0.11 ± 0.00b 1.50 ± 0.02b 0.03 ± 0.00 0.03 ± 0.00 24.96 ± 0.21 3.15 ± 0.21b 0.15 ± 0.00 12.93 ± 0.51 42.58 ± 0.64 10.41 ± 0.54 0.61 ± 0.04a 0.06 ± 0.00 0.24 ± 0.01 0.81 ± 0.05 0.47 ± 0.02 0.18 ± 0.02 0.11 ± 0.01 1.01 ± 0.08b 0.04 ± 0.01b 0.03 ± 0.00 0.03 ± 0.00 ND 0.12 ± 0.01b 0.18 ± 0.01 40.27 ± 0.67 46.79 ± 0.80 12.95 ± 0.66 11.77 ± 0.60 0.71 ± 0.02b 16.85 ± 0.79
0.14 ± 0.00 0.13 ± 0.00b 1.54 ± 0.02ab 0.02 ± 0.00 0.03 ± 0.00 24.31 ± 0.36 3.33 ± 0.14ab 0.15 ± 0.01 13.02 ± 0.34 42.84 ± 0.88 10.55 ± 0.93 0.60 ± 0.04a 0.07 ± 0.01 0.23 ± 0.00 0.75 ± 0.03 0.46 ± 0.30 0.20 ± 0.02 0.11 ± 0.00 1.44 ± 0.06a 0.08 ± 0.02a 0.05 ± 0.01 0.04 ± 0.00 ND 0.16 ± 0.01a 0.24 ± 0.03 39.61 ± 0.64 46.76 ± 0.94 13.62 ± 1.28 13.00 ± 0.45 0.84 ± 0.01a 15.67 ± 1.59
0.15 ± 0.01 0.15 ± 0.01a 1.58 ± 0.03a 0.04 ± 0.00 0.03 ± 0.00 24.75 ± 0.49 3.88 ± 0.00a 0.15 ± 0.01 12.09 ± 0.40 41.93 ± 0.75 10.90 ± 0.95 0.49 ± 0.04b 0.07 ± 0.01 0.24 ± 0.01 0.78 ± 0.02 0.42 ± 0.03 0.23 ± 0.03 0.09 ± 0.00 1.64 ± 0.02a 0.10 ± 0.01a ND 0.06 ± 0.02 ND 0.18 ± 0.02a 0.24 ± 0.03 39.25 ± 0.77 46.65 ± 0.87 14.10 ± 1.32 12.92 ± 0.56 0.76 ± 0.02ab 17.17 ± 0.43
0.618 < 0.001 0.020 0.087 0.236 0.466 < 0.001 0.956 0.253 0.750 0.627 0.049 0.402 0.898 0.413 0.416 0.322 0.083 < 0.001 0.047 0.121 0.567 0.022 0.303 0.586 0.865 0.570 0.136 0.049 0.460
a,b
Means in the same row with different superscripts differ (P < .05). SFA- sum of saturated fatty acids; MUFA- sum of monounsaturated fatty acids; PUFA- sum of polyunsaturated fatty acids. ND means not detected.
of MyHC-I, MyHC-IIb, and MyHC-IIx were not affected (P > .05) by different dietary treatments. Taken together, these results indicate that dietary inclusion of H. illucens larvae affected the mRNA expression levels related to lipid metabolic and MyHC genes in the LT of finishing pigs. 4. Discussion In recent years, H. illucens larvae as an alternative dietary protein source have been widely used in diets of domestic livestock, such as 4
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Fig. 1. Effects of the dietary inclusion of Hermetia illucens larvae on relative mRNA expression of genes related to lipid metabolism in longissimus thoracis muscle of finishing pigs. The relative mRNA expression levels of peroxisome proliferator-activated receptor γ (PPARγ), fatty acid synthase (FAS), acetyl CoA carboxylase α (ACCα), Sterol regulatory element binding protein-1c (SREBP-1C), lipoprotein lipase (LPL), carnitine paLTitoyl transferase 1B (CPT1B), fatty acid transport protein 1 (FATP1), and hormone-sensitive lipase (HSL) were normalized using β-actin as an internal control. The values are means ± SEM (n = 8). a,bMeans in the different letters were significantly different (one-way ANOVA with a Tukey post hoc test, P < .05).
4.2. H. illucens larvae effects on the meat quality, fatty acid composition, and intramuscular fat in the longissimus thoracis muscle
weaning piglets (Spranghers et al., 2017), poultry species (Cullere et al., 2016; Schiavone et al., 2017), and rabbits (Dalle Zotte et al., 2018). In the current study, we analyzed the impact of dietary H. illucens larvae inclusion on growth performance, carcass traits, and meat quality of finishing pigs. Our results showed that dietary supplementation with 4% H. illucens larvae dramatically increased the ADG, partly improved the carcass traits and muscle chemical composition of finishing pigs, while decreasing the F:G. The underlying mechanism may be associated with the up-regulated expression of genes related to the lipogenic potential and muscle fiber composition.
For the pork production industry, meat quality has become an important factor for consumer acceptance, as it contributes to tenderness and flavor of pork (Nitin, Ahlawat, Sharma, & Dabur, 2015). Feeding strategies are a major factor affecting meat quantity, mediated by the effect on the ratio of muscle to fat and composition (Jinquan et al., 2011). In the current study, we provided the first evidence that dietary inclusion of H. illucens larvae increased marbling scores of the LT with no negative influence on other tested physical parameters of meat quality, which indicates a significant role of H. illucens larvae in improving meat quality of pork. IMF content in the LT is positively correlated with marbling scores (Faucitano, Rivest, Daigle, Levesque, & Gariepy, 2004). The results of the present study showed that dietary inclusion 4% H. illucens larvae also increased the IMF content in LT compared with the HI0 group. It is well known that the IMF content is one of the key indices of meat quality and is closely related to the juiciness as well as the flavor of cooked meat (Wood et al., 2008), and the human perception of juiciness is increased when the IMF content is increased in LT (Jeremiah, Gibson, Aalhus, & Dugan, 2003). Therefore, the higher IMF content in LT indicates that inclusion of 4% H. illucens larvae as a partial replacement of soybean meal may increase the meat juiciness and flavor. On the other hand, the present study also showed that dietary H. illucens larvae inclusion increased the IMP content compared with the control group. As umami flavor enhancer, IMP is considered as a very important indicator for the umami flavor of meat, and the content of IMP can directly determine the freshness of the meat after cooking (Jung et al., 2013). To date, has been no published research about the role of H. illucens larvae in regulating meat quality, and it is difficult to explain the difference among different dietary treatments. Chito-oligosaccharide, industrially produced by chitin and chitosan, positively affected the meat quality of finishing pigs evidenced by decreasing drip loss and improved pork color (Han et al., 2007). Therefore, the H. illucens larvae regulation of meat quality may be attributed to chitin in the diet, but further studies will be required to elucidate the underlying mechanisms. The fatty acid composition of IMF also plays an important role in
4.1. Impact of dietary H. illucens larvae meal on growth performance The growth performance can provide vital information for the assessment of the effect of dietary H. illucens larvae inclusion on animals. In the current study, the ADFI was not affected by the dietary treatment, indicating that 4% and 8% H. illucens larvae inclusion were both acceptable for finishing pigs. However, inclusion of 4% H. illucens larvae in the diet of finishing pigs significantly increased the ADG compared with HI0 and HI8 groups, while decreasing the F:G. This phenomenon seems to indicate that inclusion of 4% H. illucens larvae as partial replacement of soybean meal is suitable for the diets of finishing pigs, as a feed ingredient, and may positively affect growth performance of pigs. To the best of our knowledge, the current study is the first to test H. illucens larvae inclusion in the diets of finishing pigs. In broiler chickens, the ADG showed a linear and quadratic response to H. illucens larvae (0%, 5%, 10%, and 15%) with a maximum observed for the 10% group during starter and growing periods (Dabbou et al., 2018). In laying hens, diets supplemented with 1% or 5% of H. illucens larvae also improved the growth performance and productivity (AlQazzaz, Ismail, Akit, & Idris, 2016.). H. illucens larvae contain chitin (about 4.65% in the present study), which is not digestible by monogastric animals, and may have a negative affects on nutrient digestibility, such as protein (Longvah, Mangthya, & Ramulu, 2011; SánchezMuros, Barroso, & Manzano-Agugliaro, 2014). Thus, the lack of positive effects in the finishing pigs fed 8% with H. illucens larvae diet in the current trial may be due to the higher level of chitin contained in the H. illucens larvae diet. 5
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Fig. 2. Effects of the dietary inclusion of Hermetia illucens larvae on relative mRNA expression of genes related to myosin heavy-chain (MyHC) in longissimus thoracis muscle of finishing pigs. The values are means ± SEM (n = 8). a,bMeans in the different letters were significantly different (with a Tukey post hoc test P < .05).
meat quality and in past determines the nutritional value of the muscle. The current study showed that dietary supplementation with 4% H. illucens larvae could change the fatty acid composition and increase the contents of n-3 PUFA in the LT compared with the HI0 group. So far, this is the first trial that has investigated the fatty acid composition of LT from pigs fed with a diet supplemented with H. illucens larvae. Thus, the exact mechanism that H. illucens larvae increased n-3 PUFA needs further study. In general, our results indicated that dietary inclusion of 4% H. illucens larvae may have a positive effect on meat quality by increasing IMP and IMF contents of finishing pigs.
Kwon, Im, Seo, & Baik, 2012). ACCα and FAS are key regulatory enzymes in muscle fat storage (Etherton, Louveau, Sørensen, & Chaudhuri, 1993). ACCα is the rate-limiting enzyme in long-chain fatty acid de novo synthesis (Tan et al., 2011). FAS is mainly involved in elongation during fatty acid biosynthesis in muscle (Chen, Yang, Tong, & Zhao, 2004). Therefore, in the present study, the IMF content of LT was increased in response to dietary inclusion of H. illucens larvae, which may be due to an up-regulation of the expression of lipogenic genes (LPL, ACCα, and FAS). There are four different single MyHC isoforms have been identified in ATPase based fiber types in the muscle of pigs, such as slow-oxidative type I, fast-oxidative type IIa, fast-oxidative glycolytic type IIx, and fastglycolytic IIb, respectively (Klont, Brocks, & Eikelenboom, 1998; Lefaucheur, Milan, Ecolan, & Callennec, 2004). Fiber type composition is also closely related to meat quality traits (Eggert, Depreux, Schinckel, Grant, & Gerrard, 2002; Ryu & Kim, 2005). Previous studies have indicated that the IMF content of the LT negatively correlates with glycolytic muscle fiber types, while it positively correlates with type I and IIa muscle fibers (Kim et al., 2008; Ryu & Kim, 2005). An increase in the relative amount of type I and IIa fibers has been suggested to increase the water holding capacity and tenderness (Ryu & Kim, 2005). The present study provides the first evidence that inclusion of H. illucens larvae in the diet of finishing pigs may up-regulate the mRNA expression level of MyHC-IIa in LT. This finding suggests that dietary inclusion of H. illucens larvae may induce the muscle fiber transition toward more oxidative muscle fibers, and partly explains the phenomenon of how H. illucens larvae may improve meat quality.
4.3. Effects of H. illucens larvae on lipid metabolic and MyHC genes expression levels in the longissimus thoracis muscle It is well known that marbling of LT depends on the balance between fat deposition and removal, which is regulated by numerous intramuscular lipid metabolic genes (Li et al., 2018). Thus, to further investigate the molecular mechanism of dietary inclusion of H. illucens larvae implicated in regulating meat quality, we measured gene expression and found that the relative mRNA abundance of the lipid uptakerelated gene LPL, and lipogenic-related factors FAS and ACCα are higher in LT in response to dietary inclusion H. illucens larvae. It has been reported that the fatty acid availability directly influences IMF deposition. LPL is one of the genes that determines fatty acid uptake, and can participate in the process of fatty acid flux into adipocytes clustered in the muscle, and then provide the essential substrate for IMF synthesis (Jeong, 6
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5. Conclusions
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The results of the present study demonstrate that inclusion of 4% H. illucens larvae in the diet increases the ADG and partly improves carcass trait and muscle chemical composition of finishing pigs, while reducing F:G. Furthermore, this study also provides the first evidence that dietary inclusion of 4% H. illucens larvae affected the meat quality via upregulating the expression of genes related to the lipogenic potential and muscle fiber composition in the LT of finishing pigs. These findings provide a new perspective for H. illucens larvae meal as a suitable and nutrient rich alternative protein source for swine feed. Acknowledgement The present study was supported by the Presidential Foundation of the Guangdong Academy of Agricultural Sciences (201802B, 201621), Guangdong Modern Agro-industry Technology Research System (2016LT1080, 2017LT1080), and key project of Guangzhou Science and Technology Plan (201707020007). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.meatsci.2019.05.008. References Al-Qazzaz, M. F. A., Ismail, D., Akit, H., & Idris, L. H. (2016). Effect of using insect larvae meal as a complete protein source on quality and productivity characteristics of laying hens. Revista Brasileira de Zootecnia, 45, 518–523. AOAC (2007). Official methods of analysis. 18th ed.Gaithersburg, MD: Association of Official Analytical Chemists. Barragan-Fonseca, K. B., Dicke, M., & Loon, J. J. A. V. (2017). Nutritional value of the black soldier fly (Hermetia illucens L.) and its suitability as animal feed. Vol. 3, 105–120. Chen, J., Yang, X. J., Tong, H., & Zhao, R. Q. (2004). Expressions of FAS and HSL mRNA in longissimus dorsi muscle and their relation to intramuscular fat contents in pig. Journal of Agricultural Biotechnology, 12, 422–426. Cullere, M., Tasoniero, G., Giaccone, V., Miotti-Scapin, R., Claeys, E., De, S. S., & Dalle, Z. A. (2016). Black soldier fly as dietary protein source for broiler quails: Apparent digestibility, excreta microbial load, feed choice, performance, carcass and meat traits. Animal, 10, 1923–1930. Cutrignelli, M. I., Messina, M., Tulli, F., Randazzo, B., Olivotto, I., Gasco, L., ... Bovera, F. (2018). Evaluation of an insect meal of the black soldier Fly (Hermetia illucens) as soybean substitute: Intestinal morphometry, enzymatic and microbial activity in laying hens. Research in Veterinary Science, 117, 209–215. Dabbou, S., Gai, F., Biasato, I., Capucchio, M. T., Biasibetti, E., Dezzutto, D., ... Schiavone, A. (2018). Black soldier fly defatted meal as a dietary protein source for broiler chickens: Effects on growth performance, blood traits, gut morphology and histological features. Journal of Animal Science and Biotechnology, 9, 49. Dalle Zotte, A., Cullere, M., Martins, C., Alves, S. P., Freire, J. P., Falcão-e-Cunha, L., & Bessa, R. J. (2018). Incorporation of black soldier fly (Hermetia illucens L.) larvae fat or extruded linseed in diets of growing rabbits and their effects on meat quality traits including detailed fatty acid composition. Meat Science, 146, 50–58. Eggert, J. M., Depreux, F. F. S., Schinckel, A. P., Grant, A. L., & Gerrard, D. E. (2002). Myosin heavy chain isoforms account for variation in pork quality. Meat Science, 61, 117–126. Etherton, T. D., Louveau, I., Sørensen, M. T., & Chaudhuri, S. (1993). Mechanisms by which somatotropin decreases adipose tissue growth. American Journal of Clinical Nutrition, 58(2), 287S–295S Suppl. Faucitano, L., Rivest, J., Daigle, J. P., Levesque, J., & Gariepy, C. (2004). Distribution of intramuscular fat content and marbling within the longissimus muscle of pigs. Canadian Journal of Animal Science, 84, 57–61. Hale, O. M. (1973). Dried Hermetia illucens larvae (Diptera: Stratiomyidae) as a feed additive for poultry. Journal of Georgia Entomological Society, 8, 16–20. Han, K. N., Yang, Y. X., Hahn, T. W., Kwon, I. K., Lohakare, J. D., Lee, J. K., & Chae, B. J. (2007). Effects of chito-oligosaccharides supplementation on performance, nutrient digestibility, pork quality and immune response in growing-finishing pigs. Journal of Animal and Feed Sciences, 16, 607–620. Honikel, K. O. (1998). Reference methods for the assessment of physical characteristics of meat. Meat Science, 49, 447–457. Jeong, J., Kwon, E. G., Im, S. K., Seo, K. S., & Baik, M. (2012). Expression of fat deposition and fat removal genes is associated with intramuscular fat content in longissimus dorsi muscle of Korean cattle steers. Journal of Animal Science, 90, 2044–2053. Jeremiah, L. E., Gibson, L. L., Aalhus, J. L., & Dugan, M. E. R. (2003). Assessment of palatability attributes of the major beef muscles. Meat Science, 65, 949–958. Joo, S. T., Kim, G. D., Hwang, Y. H., & Ryu, Y. C. (2013). Control of fresh meat quality through manipulation of muscle fiber characteristics. Meat Science, 95, 828–836.
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Use of Hermetia illucens larvae as a dietary protein source: Effects on growth performance, carcass traits, and meat quality in finishing pigs
T
Miao Yua,b,c,d,e, Zhenming Lia,b,c,d,e, Weidong Chena,b,c,d,e, Ting Ronga,b,c,d,e, ⁎ Gang Wanga,b,c,d,e, Jianhao Lia,b,c,d,e, Xianyong Maa,b,c,d,e, a
Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640, China State Key Laboratory of Livestock and Poultry Breeding, Guangzhou, Guangdong 510640, China c Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture, Guangzhou, Guangdong 510640, China d Guangdong Public Laboratory of Animal Breeding and Nutrition, Guangzhou, Guangdong 510640, China e Guangdong Engineering Technology Research Center of Animal Meat Quality and Safety Control and Evaluation, Guangzhou, Guangdong 510640, China b
ARTICLE INFO
ABSTRACT
Keywords: Meat quality Intramuscular fat Finishing pigs Hermetia illucens larvae Lipid metabolism
This study investigated the effects of feeding Hermetia illucens larvae (0, 4, and 8%; HI0, HI4, and HI8 groups, respectively) on growth performance, carcass traits, and meat quality of finishing pigs. Results showed that the HI4 diet increased (P < .05) final body weight and average daily gain and decreased (P < .05) feed to gain ratio compared with HI0 and HI8 group. HI4 and HI8 diets increased (P < .05) loin-eye area, marbling scores, and inosine monophosphate content of longissimus thoracis (LT) compared with HI0 diet. The intramuscular fat content was greater (P < .05) in HI4 group than in the HI0 group. Furthermore, HI4 diet up-regulated (P < .05) lipogenic genes and MyHC-IIa mRNA levels in LT compared with HI0 diet. Our results indicated that dietary inclusion of H. illucens larvae has a beneficial impact on growth performance and meat quality, and the underlying mechanism may be due to the altered lipogenic potential induced by H. illucens larvae.
1. Introduction Along with growth in the global human population, the consumption and demand for livestock products drastically increases. In parallel, this affects the demand for livestock feeds, and places heavy pressure on already overexploited resources, especially protein feeds. Soybean meal is the conventional feed resource of protein in the Chinese livestock industry, and the supply has declined in recent years while the demand has continued to rise, resulting in soaring prices for purchasing feed in livestock production. Thus, it is imperative to find new alternative, and sustainable protein sources for livestock feed to reduce the use of soybean meal. In this case, several insect species, such as Hermetia illucens, have attracted increased interest as a sustainable alternative to meet demand and either partly or completely replace conventional protein feed sources (Makkar, Tran, Heuzé, & Ankers, 2014; Van Huis, 2013). H. illucens larvae are a high-value feedstuff source, containing about 40 to 44% crude protein with a similar or better amino acid profile compared with that of soybean meal (Barragan-Fonseca, Dicke, & Loon, 2017; Veldkamp, 2015). Additionally, H. illucens larvae are also rich in fat, with the content ranging from 15 to 49% on a dry matter basis (Makkar
⁎
et al., 2014; Spranghers et al., 2017), which can be defatted and used for the preparation of biodiesel, while the remainder of the defatted meal could be used as an alternative sustainable protein rich ingredient for the feed industry (Dabbou et al., 2018). Thus, there is an urgent need to investigate their viability as alternative sustainable protein feed for animal production. Until now, the administration of H. illucens larvae as an alternative for soybean meal in diets was shown to modulate the growth performance, nutrient digestibility, and blood profiles of chickens (Cutrignelli et al., 2018; Maurer et al., 2015), weaning or growing pigs (Dabbou et al., 2018; Hale, 1973; Spranghers et al., 2017), and the carcass and meat traits of chickens (Cullere et al., 2016), without negative effects. These results indicate that H. illucens larvae can be used in the diet of monogastric animals as a potentially valuable feed ingredient. However, little information is available on the growth performance, carcass and meat traits of H. illucens larvae in finishing pigs. It is well known that intramuscular fat (IMF) is regarded as an important factor that affects pork quality. High contents of IMF could enhance meat quality as it contributes to pork tenderness and flavor (Wood et al., 2013). In laying hens, H. illucens larvae completely replacing soybean meal in the diet decreased serum and egg cholesterol
Corresponding author at: Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640, China. E-mail address: [email protected] (X. Ma).
https://doi.org/10.1016/j.meatsci.2019.05.008 Received 21 December 2018; Received in revised form 5 May 2019; Accepted 7 May 2019 Available online 08 May 2019 0309-1740/ © 2019 Published by Elsevier Ltd.
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and triglycerides levels (Marono et al., 2017). In growing pigs, dietary H. illucens larvae inclusion increased serum high density lipoprotein concentration in growing pigs (Zhang et al., 2018), suggesting an impact of H. illucens larvae on lipid metabolism. However, whether and how the lipid metabolism can be regulated by H. illucens larvae feeding to finishing pigs, remains relatively unknown and requires further investigation. Additionally, muscle fiber type composition can influence several meat quality traits, e.g. tenderness and water-holding capacity (Joo, Kim, Hwang, & Ryu, 2013). To our knowledge, no research has been conducted to test whether H. illucens larvae treatment can change meat quality and, if so, whether lipid metabolism or muscle fiber characteristics can be changed by feeding H. illucens larvae. Therefore, in the present study, we hypothesized that H. illucens larvae as a dietary protein source may alter the meat quality traits of finishing pigs by influencing lipid metabolism and muscle fiber characteristics of the longissimus thoracis muscle (LT).
Table 1 Feed ingredient and nutrient composition of experimental diets (as-fed basis). Items
Treatment HI0
Ingredient, % Corn 71.00 Soybean meal 16.98 Wheat bran 6.00 Hermetia illucens L. – Soybean oil 2.50 L-Lysine-HCl(98%) 0.38 DL-Methionine 0.06 L-Threonine 0.06 L-Tryptophan 0.02 Dicalcium phosphate 0.95 Limestone 0.75 Salt 0.30 1 Vitamin-mineral premix 1.00 Total 100.00 Calculated content2 ME3, MJ/kg 14.37 Standard ileal digestible amino acid, % Lysine, % 0.88 Methionine + Cysteine 0.47 Threonine 0.49 Tryptophan 0.15 Analyzed nutrient composition4 Dry matter, % 89.22 Crude protein, % 14.53 Crude ash, % 4.36 Crude fat, % 5.10
2. Material and methods The experimental proposal and procedures for the care and treatment of the pigs were approved by the Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences (authorization number GAASIAS-2016-017). 2.1. Preparation of H. illucens larvae meal H. illucens larvae were purchased from Guangzhou AnRuiJie Environmental Protection Technology Co., Ltd. (Guangzhou, Guangdong, China). The prepupae were dried at 80 °C for 30 min and subsequently air dried. Finally, the dried prepupae were crushed to powder through a 1.0-mm, and then kept in a well-closed and lightresistant place. The chemical composition, energy content, and amino acid composition of the H. illucens larvae are shown in Supplementary Table S1.
HI4
HI8
71.20 13.86 6.00 4.00 1.70 0.40 0.07 0.11 0.01 0.95 0.40 0.30 1.00 100.00
71.76 10.75 6.00 8.00 0.50 0.42 0.09 0.15 – 1.00 0.03 0.30 1.00 100.00
14.38
14.38
0.88 0.47 0.50 0.15
0.88 0.47 0.50 0.14
89.16 14.53 4.39 5.10
89.23 14.54 4.50 5.10
1
Provided per kilogram of complete diet: vitamin A, 15,000 IU; vitamin D3, 3000 IU; vitamin E, 150 mg; vitamin K3, 3 mg; vitamin B1, 3 mg; vitamin B2, 6 mg; vitamin B6, 5 mg; vitamin B12, 0.03 mg; niacin, 45 mg; vitamin C, 250 mg; calcium pantothenate, 9 mg; folic acid, 1 mg; biotin, 0.3 mg; choline chloride, 500 mg; Fe (FeSO4·H2O), 170 mg; Cu (CuSO4·5H2O), 150 mg; I (KI), 0.90 mg; Se (Na2SeO3),0.2 mg; Zn (ZnSO4·H2O), 150 mg; Mg (MgO), 68 mg; Mn (MnSO4·H2O), 80 mg; Co (CoCl2), 0.3 mg. 2 Values were estimated based on the chemical analysis. 3 ME = metabolizable energy. 4 Analytical results obtained according to AOAC(AOAC, 2007).
2.2. Animals, experimental design, and diets The present study was conducted to evaluate the effects of dietary inclusion of H. illucens larvae meal in the diet of finishing pig. A total of 72 crossbred (Duroc × Landrace × Large White) female pigs with a similar initial body weight (BW, 76.0 ± 0.52 kg) were randomly allocated to three dietary treatment groups in a completely randomized design. Pigs in the three treatments received increasing levels of H. illucens larvae meal (0, 4% and 8%; HI0, HI4, and HI8, respectively). Each treatment consisted of eight pens (replicates), with three pigs per pen. The contents of metabolizable energy (ME) and crude protein (CP) in each diet were equalized by adjusting the proportion of soybean meal, corn, and soybean oil. Meanwhile, the experimental diets were formulated to meet or exceed the nutrient recommendations of the National Research Council (NRC) (NRC, 2012) (Table 1). During the 46day experimental period, pigs were housed in environmentally controlled confinement pens (2.5 × 3.0 m) that had partial concrete slatted floors. Water and feed were provided ad libitum for pigs throughout the trial period. The feed consumption per pen was recorded every day to calculate the average daily feed intake (ADFI). The BW of all pigs was recorded at the beginning and end of the experimental period just before the morning feed to determine the average daily gain (ADG). The feed to gain ratios (F:G) were calculated from the feed intake and the increased BW during the whole experimental period.
immediately excised from the right side of the carcass and flash frozen in liquid nitrogen for later gene expression analysis. Within 30 min after slaughter, the hot carcass weight (HCW) was measured to calculate the dressing percentage. About 100 g of LT between the 9th and 10th ribs of the left side were sampled and stored at −80 °C to determine the IMF, inosine monophosphate (IMP), and fatty acid profile. 24 h after slaughter, the LT on the left half of each carcass between the 10th and 13th ribs were sampled for analysis of meat quality. 2.4. Measurement of carcass characteristics and meat quality The average backfat thickness was measured at the first rib, lumbar, and the last rib of the left side. The loin-eye area (LEA) was measured by tracing the outline of the LT area at the 10th rib and then measuring the area using a planimeter and the average of three measurements was used as LEA for each carcass. The subjective marbling score of the LT was determined on the 10th-rib using the guidelines of the National Pork Producers Council (NPPC) (NPPC, 1991). The fat-free lean index (FFLI) was calculated according to the guidelines of NPPC, FFLI = (8.588 + (0.465 × 2.2046 × HCW, kg) + (3.005 × 0.155 × 10th rib loin-eye area, cm2) – (21.896 × 0.0394 × 10th rib fat depth, mm)) / (2.2046 × HCW, kg). Post-slaughter, the pH at 45 min (pH 45 min) and the pH at 24 h (pH 24 h) were measured by a using a portable pH meter (HI 9024C; HANNA Instruments, Woonsocket, RI, USA), which was calibrated at the beginning of each measuring day using pH 4.6 and 7.0 buffers. The
2.3. Sample collection and preparation At the end of the feeding period (on day 46), one pig with medium weight from each pen (eight pigs/treatment) was randomly selected. After fasting for approximately 12 h, pigs were euthanized via electrical stunning and exsanguination. Samples (about 5 g) of LT were 2
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loin meat color, including L* (lightness), a* (redness), and b* (yellowness), was measured at 45 min and 24 h postmortem as a mean of three random readings, allowing about 10–15 min for the color to develop after cutting, and using a Minolta CR-400 Chroma meter (Konica Minolta Sensing Ins., Osaka, Japan) calibrated against a standard white plate (8 mm diameter aperture, d/0 illumination system). The drip loss was determined as previously described (Honikel, 1998). For the determination of shear force, at 24 h postmortem, samples of LT muscle (about 150 g) were placed in individual polyethylene vacuum bags and cooked to an internal temperature of 70 °C in a water bath at 75 °C. Then, cooked samples were cooled in running water to room temperature. Thereafter, the cooked samples were cut into shaped strips with a diameter of 1.27 cm and length of approximately 20 mm, and sheared parallel to the muscle fiber direction as described by a previous study (Li, Li, Zhang, Gao, & Zhou, 2017). The shear force (N) of the LT was measured using a digital-display-muscle tenderness meter (C-LT3B, Tenovo, Harbin, China) with a load cell of 15 kg and a 200 mm/min crosshead speed. Ten replicates of each sample were measured.
2.7. Statistical analysis All experimental data was analyzed with the SPSS software package (SPSS v. 20.0, SPSS Inc., Chicago IL, USA). Normal distribution of the data was confirmed by Shapiro-Wilk test before assessing differences between groups. All normally distributed data were analyzed by oneway ANOVA. Diet treatment was the fixed effect and pen was the random effect in the statistical model. Data were expressed as the mean ± SEM. Differences among treatments were compared using Tukey post hoc test. Differences were identified as significant at P ≤ .05. 3. Results 3.1. Growth performance and carcass traits The growth performance and carcass traits of pigs fed diets with varying H. illucens larvae concentrations are presented in Table 2. The HI4 diet significantly increased (P < .05) the final BW and ADG of pigs, and decreased (P < .05) the F:G compared with HI0 and HI8 diets, while there was no significant difference (P = .466) on ADFI among the HI0, HI4, and HI8 groups. Furthermore, the HI4 and HI8 diet significantly increased (P < .05) the loin-eye area compared with HI0 diet. However, the diets with H. illucens larvae had no effect (P > .05) on hot carcass weight, carcass yield, average backfat, and FFLI.
2.5. Diet and muscle chemical analysis Chemical analysis of the H. illucens larvae and experimental diets were analyzed by the methods of the Association of Official Analytical Chemists (AOAC) (AOAC, 2007) for dry matter (DM), ash, crude fat, and crude protein (CP). The LT samples were analyzed for DM and IMF, according to the procedures of AOAC (AOAC, 2007). To determine the amino acid composition of H. illucens larvae meal (except methionine and tryptophan), approximately 1.0 g of each sample was digested in 10 mL of 6 N HCl at 110 °C for 24 h; the methionine concentration was measured after oxidation with performic acid, and tryptophan concentrations were determined after alkaline hydrolysis according to AOAC (AOAC, 2007). The concentration of IMP in the LT was measured using High Performance Liquid Chromatography (HPLC) as described in a previous study (Li, Duan, et al., 2015). Lipids were extracted from the LT tissue samples by the chloroform-methanol (1:1, v/v) procedure. Fatty acid methyl esters of the H. illucens larvae and experimental diets were prepared by using HCl/methanol as described by a previous study (Luo et al., 2009). For LT, the samples freeze-dried and then fatty acid methyl esters were separated using sodium methoxide in method followed by the addition of HCl/ methanol as basic and acid catalysts, respectively. Fatty acid methyl esters were separated and determined with an Agilent 7890B GC equipped with a flame ionization detector, and with a method according to previous study (Li, He, et al., 2015; Vahmani et al., 2017). The individual fatty acid methyl esters were identified by comparing the retention times of the peak area with those of known standards, and expressed as a percentage of total fatty acids. The fatty acid profiles of H. illucens larvae and experimental diets are shown in Supplementary Tables S2.
3.2. Meat quality The meat quality of pigs is shown in Table 3. The HI4 and HI8 diets significantly increased (P < .05) marbling scores compared with HI0 diet. However, dietary H. illucens larvae inclusion did not affect (P > .05) the 45 min and 24 h pH values, the 45 min and 24 h L* values, a* value, and b* value, drip loss, and shear force. 3.3. Muscle chemical composition As shown in Table 4, HI4 diet significantly increased (P < .05) the IMF contents in the LT of pigs compared with HI0 diet. In addition, the HI4 and HI8 diets increased (P < .05) the IMP concentration compared with HI0 diet. However, dietary H. illucens larvae meal did not affect the DM content in the LT of pigs. Table 2 Effects of the dietary inclusion of Hermetia illucens larvae on the growth performance and carcass traits of finishing pigs.
2.6. RNA extraction and quantitative real-time PCR
Items
Total RNA was extracted from the LT using the TRIzol reagent (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. The RNA quality of every sample was quantified by Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Wiington, DE, USA), and the ratio (OD260:OD280) ranged from 1.8 to 2.0. Thereafter, 1 μg total RNA was reverse-transcribed to cDNA using a Synthesis Kit (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. Primers used for selected genes were presented in Table S3 in the supplemental material. Real-time PCR of the target genes and β-actin was performed on CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with TB Green™ Premix Ex Taq™ (Takara Biotechnology, Dalian, China). The reaction mixtures and real-time PCR conditions were set according to a previous study (Yu et al., 2017). The housekeeping gene β-actin was used to normalize the expression levels of each target gene, and according to the following formula 2−(∆∆Ct), where ∆∆Ct = (Cttarget – Ctβ-actin)treatment – (Cttarget – Ctβ-actin)control.
Initial BW (kg) Final BW (kg) ADG(kg/d) ADFI(kg/d) F:G Hot carcass weight, kg Carcass yield, % Back fat, cm Loin eye area, cm FFLI, % a,b
Treatment
P value
HI0
HI4
HI8
76.34 ± 0.57 115.68 ± 0.62b 0.89 ± 0.03b 2.83 ± 0.05 3.21 ± 0.06a 87.95 ± 1.24 74.89 ± 0.79 25.05 ± 1.24 45.35 ± 1.64b 50.87 ± 0.60
76.46 ± 0.57 119.60 ± 0.95a 0.98 ± 0.03a 2.77 ± 0.03 2.85 ± 0.12b 87.93 ± 1.57 73.89 ± 0.58 25.08 ± 2.86 54.68 ± 3.62a 53.96 ± 1.50
76.46 ± 0.58 114.57 ± 0.96b 0.86 ± 0.02b 2.77 ± 0.04 3.24 ± 0.05a 85.40 ± 1.21 74.82 ± 0.48 25.67 ± 1.06 49.29 ± 1.02a 52.32 ± 1.60
0.980 0.009 0.013 0.466 0.028 0.329 0.574 0.967 0.009 0.484
Means in the same row with different superscripts differ (P < .05). Abbreviations: BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; F:G, feed to gain ratio; FFLI, fat-free lean index.
3
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Table 3 Effects of the dietary inclusion of Hermetia illucens larvae on the meat quality of finishing pigs. Items
Treatment
pH45 min pH24 h L* (lightness) 45 min a* (redness) 45 min b* (yellowness) 45 min L* (lightness) 24 h a* (redness) 24 h b* (yellowness) 24 h Marbling scores Drip loss, % Shear force, N a,b
P value
HI0
HI4
HI8
6.39 ± 0.06 5.45 ± 0.03 44.82 ± 0.66 16.47 ± 0.26 1.58 ± 0.32 55.67 ± 0.90 16.26 ± 0.34 2.26 ± 0.25 2.69 ± 0.10b 2.63 ± 0.10 33.23 ± 2.33
6.42 ± 0.08 5.48 ± 0.05 45.27 ± 0.38 16.70 ± 0.12 1.62 ± 0.28 54.70 ± 0.98 16.32 ± 0.22 2.38 ± 0.22 3.13 ± 0.10a 2.64 ± 0.09 35.75 ± 4.07
6.38 ± 0.10 5.50 ± 0.04 45.22 ± 0.55 16.19 ± 0.12 1.46 ± 0.22 54.25 ± 0.70 16.24 ± 0.14 1.86 ± 0.35 3.15 ± 0.20a 2.72 ± 0.10 40.65 ± 4.07
0.930 0.712 0.821 0.222 0.911 0.514 0.972 0.350 0.049 0.771 0.282
Means in the same row with different superscripts differ (P < .05).
Table 4 Effects of the dietary inclusion of Hermetia illucens larvae on the longissimus thoracis muscle chemical composition of finishing pigs (as-fresh basis). Items
DM, % IMF,3% IMP,4mg/g
Treatment
Table 5 Effects of the dietary inclusion of Hermetia illucens larvae on the fatty acid profile of the longissimus thoracis muscle of pigs (% total fatty acids).
P value
HI0
HI4
HI8
27.30 ± 0.19 2.63 ± 0.17b 1.87 ± 0.04b
27.86 ± 0.32 3.26 ± 0.14a 2.01 ± 0.04a
27.74 ± 0.13 2.79 ± 0.20ab 2.03 ± 0.03a
Items
0.219 0.049 0.008
C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 n-7 C17:0 C18:0 C18:1 n-9 C18:2 n-6 C18:3 n-3 Cγ18:3 n-6 C20:0 C20:1 C20:2 C20:3 n-6 Cγ20:3 n-6 C20:4 n-6 C20:5 n-3(EPA) C22:0 C22:1 C22:5 n-3 (DPA) C22:6 n-3 (DHA) C24:0 SFA MUFA PUFA PUFA n-6 PUFA n-3 PUFA n-6/PUFA n3
a,b
Means in the same row with different superscripts differ (P < .05). Abbreviations: DM, dry matter; IMF, intramuscular fat; IMP, inosine monophosphate.
3.4. Fatty acid profiles in longissimus thoracis muscle The fatty acid profiles of the LT are shown in Table 5. The HI4 diet also increased (P < .05) the concentrations of C18:3, C20:4, C20:5, C22:6, and the sum of n-3 polyunsaturated fatty acid (PUFA) compared with the HI0 diet, and increased (P < .05) the concentration of C18:3 compared with the HI8 diet. Furthermore, the HI8 diet significantly increased (P < .05) the concentrations of C12:0, C14:0, C16:1, C20:4, C20:5, and C22:6 compared with the HI0 diet, but decreased (P < .05) the concentration of C18:3. Although a number of the fatty acid concentrations of the LT in finishing pigs were altered, the sum of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), PUFA, and the ratio of PUFA n-6/PUFA n-3 did not significantly differ among all three treatments. 3.5. Expression of genes related to lipid metabolism and myosin heavy-chain in longissimus thoracis muscle To gain further insight into the molecular mechanism underlying the effect of H. illucens larvae administration induced regulation of IMF content, the mRNA expression levels of critical lipid metabolism factors were measured in the LT. As shown in Fig. 1, the HI4 and HI8 diets significantly up-regulated (P < .05) the mRNA expression level of fatty acid synthase (FAS) compared with the HI0 diet. Additionally, the HI4 diet also up-regulated (P < .05) the mRNA expression level of acetyl CoA carboxylase α (ACCα) and lipoprotein lipase (LPL) compared with HI0 diet. However, dietary H. illucens larvae inclusion did not affect (P > .05) the mRNA expression level of peroxisome proliferator-activated receptor γ (PPARγ), sterol regulatory element binding protein-1c (SREBP-1C), carnitine palmitoyl transferase 1B (CPT1B), fatty acid transport protein 1 (FATP1), and hormone-sensitive lipase (HSL). As shown in Fig. 2, the HI4 diet significantly up-regulated (P < .05) the mRNA expression level of myosin heavy chain (MyHC)IIa compared with the HI0 diet. However, the mRNA expression levels
Treatment
P value
HI0
HI4
HI8
0.13 ± 0.01 0.11 ± 0.00b 1.50 ± 0.02b 0.03 ± 0.00 0.03 ± 0.00 24.96 ± 0.21 3.15 ± 0.21b 0.15 ± 0.00 12.93 ± 0.51 42.58 ± 0.64 10.41 ± 0.54 0.61 ± 0.04a 0.06 ± 0.00 0.24 ± 0.01 0.81 ± 0.05 0.47 ± 0.02 0.18 ± 0.02 0.11 ± 0.01 1.01 ± 0.08b 0.04 ± 0.01b 0.03 ± 0.00 0.03 ± 0.00 ND 0.12 ± 0.01b 0.18 ± 0.01 40.27 ± 0.67 46.79 ± 0.80 12.95 ± 0.66 11.77 ± 0.60 0.71 ± 0.02b 16.85 ± 0.79
0.14 ± 0.00 0.13 ± 0.00b 1.54 ± 0.02ab 0.02 ± 0.00 0.03 ± 0.00 24.31 ± 0.36 3.33 ± 0.14ab 0.15 ± 0.01 13.02 ± 0.34 42.84 ± 0.88 10.55 ± 0.93 0.60 ± 0.04a 0.07 ± 0.01 0.23 ± 0.00 0.75 ± 0.03 0.46 ± 0.30 0.20 ± 0.02 0.11 ± 0.00 1.44 ± 0.06a 0.08 ± 0.02a 0.05 ± 0.01 0.04 ± 0.00 ND 0.16 ± 0.01a 0.24 ± 0.03 39.61 ± 0.64 46.76 ± 0.94 13.62 ± 1.28 13.00 ± 0.45 0.84 ± 0.01a 15.67 ± 1.59
0.15 ± 0.01 0.15 ± 0.01a 1.58 ± 0.03a 0.04 ± 0.00 0.03 ± 0.00 24.75 ± 0.49 3.88 ± 0.00a 0.15 ± 0.01 12.09 ± 0.40 41.93 ± 0.75 10.90 ± 0.95 0.49 ± 0.04b 0.07 ± 0.01 0.24 ± 0.01 0.78 ± 0.02 0.42 ± 0.03 0.23 ± 0.03 0.09 ± 0.00 1.64 ± 0.02a 0.10 ± 0.01a ND 0.06 ± 0.02 ND 0.18 ± 0.02a 0.24 ± 0.03 39.25 ± 0.77 46.65 ± 0.87 14.10 ± 1.32 12.92 ± 0.56 0.76 ± 0.02ab 17.17 ± 0.43
0.618 < 0.001 0.020 0.087 0.236 0.466 < 0.001 0.956 0.253 0.750 0.627 0.049 0.402 0.898 0.413 0.416 0.322 0.083 < 0.001 0.047 0.121 0.567 0.022 0.303 0.586 0.865 0.570 0.136 0.049 0.460
a,b
Means in the same row with different superscripts differ (P < .05). SFA- sum of saturated fatty acids; MUFA- sum of monounsaturated fatty acids; PUFA- sum of polyunsaturated fatty acids. ND means not detected.
of MyHC-I, MyHC-IIb, and MyHC-IIx were not affected (P > .05) by different dietary treatments. Taken together, these results indicate that dietary inclusion of H. illucens larvae affected the mRNA expression levels related to lipid metabolic and MyHC genes in the LT of finishing pigs. 4. Discussion In recent years, H. illucens larvae as an alternative dietary protein source have been widely used in diets of domestic livestock, such as 4
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Fig. 1. Effects of the dietary inclusion of Hermetia illucens larvae on relative mRNA expression of genes related to lipid metabolism in longissimus thoracis muscle of finishing pigs. The relative mRNA expression levels of peroxisome proliferator-activated receptor γ (PPARγ), fatty acid synthase (FAS), acetyl CoA carboxylase α (ACCα), Sterol regulatory element binding protein-1c (SREBP-1C), lipoprotein lipase (LPL), carnitine paLTitoyl transferase 1B (CPT1B), fatty acid transport protein 1 (FATP1), and hormone-sensitive lipase (HSL) were normalized using β-actin as an internal control. The values are means ± SEM (n = 8). a,bMeans in the different letters were significantly different (one-way ANOVA with a Tukey post hoc test, P < .05).
4.2. H. illucens larvae effects on the meat quality, fatty acid composition, and intramuscular fat in the longissimus thoracis muscle
weaning piglets (Spranghers et al., 2017), poultry species (Cullere et al., 2016; Schiavone et al., 2017), and rabbits (Dalle Zotte et al., 2018). In the current study, we analyzed the impact of dietary H. illucens larvae inclusion on growth performance, carcass traits, and meat quality of finishing pigs. Our results showed that dietary supplementation with 4% H. illucens larvae dramatically increased the ADG, partly improved the carcass traits and muscle chemical composition of finishing pigs, while decreasing the F:G. The underlying mechanism may be associated with the up-regulated expression of genes related to the lipogenic potential and muscle fiber composition.
For the pork production industry, meat quality has become an important factor for consumer acceptance, as it contributes to tenderness and flavor of pork (Nitin, Ahlawat, Sharma, & Dabur, 2015). Feeding strategies are a major factor affecting meat quantity, mediated by the effect on the ratio of muscle to fat and composition (Jinquan et al., 2011). In the current study, we provided the first evidence that dietary inclusion of H. illucens larvae increased marbling scores of the LT with no negative influence on other tested physical parameters of meat quality, which indicates a significant role of H. illucens larvae in improving meat quality of pork. IMF content in the LT is positively correlated with marbling scores (Faucitano, Rivest, Daigle, Levesque, & Gariepy, 2004). The results of the present study showed that dietary inclusion 4% H. illucens larvae also increased the IMF content in LT compared with the HI0 group. It is well known that the IMF content is one of the key indices of meat quality and is closely related to the juiciness as well as the flavor of cooked meat (Wood et al., 2008), and the human perception of juiciness is increased when the IMF content is increased in LT (Jeremiah, Gibson, Aalhus, & Dugan, 2003). Therefore, the higher IMF content in LT indicates that inclusion of 4% H. illucens larvae as a partial replacement of soybean meal may increase the meat juiciness and flavor. On the other hand, the present study also showed that dietary H. illucens larvae inclusion increased the IMP content compared with the control group. As umami flavor enhancer, IMP is considered as a very important indicator for the umami flavor of meat, and the content of IMP can directly determine the freshness of the meat after cooking (Jung et al., 2013). To date, has been no published research about the role of H. illucens larvae in regulating meat quality, and it is difficult to explain the difference among different dietary treatments. Chito-oligosaccharide, industrially produced by chitin and chitosan, positively affected the meat quality of finishing pigs evidenced by decreasing drip loss and improved pork color (Han et al., 2007). Therefore, the H. illucens larvae regulation of meat quality may be attributed to chitin in the diet, but further studies will be required to elucidate the underlying mechanisms. The fatty acid composition of IMF also plays an important role in
4.1. Impact of dietary H. illucens larvae meal on growth performance The growth performance can provide vital information for the assessment of the effect of dietary H. illucens larvae inclusion on animals. In the current study, the ADFI was not affected by the dietary treatment, indicating that 4% and 8% H. illucens larvae inclusion were both acceptable for finishing pigs. However, inclusion of 4% H. illucens larvae in the diet of finishing pigs significantly increased the ADG compared with HI0 and HI8 groups, while decreasing the F:G. This phenomenon seems to indicate that inclusion of 4% H. illucens larvae as partial replacement of soybean meal is suitable for the diets of finishing pigs, as a feed ingredient, and may positively affect growth performance of pigs. To the best of our knowledge, the current study is the first to test H. illucens larvae inclusion in the diets of finishing pigs. In broiler chickens, the ADG showed a linear and quadratic response to H. illucens larvae (0%, 5%, 10%, and 15%) with a maximum observed for the 10% group during starter and growing periods (Dabbou et al., 2018). In laying hens, diets supplemented with 1% or 5% of H. illucens larvae also improved the growth performance and productivity (AlQazzaz, Ismail, Akit, & Idris, 2016.). H. illucens larvae contain chitin (about 4.65% in the present study), which is not digestible by monogastric animals, and may have a negative affects on nutrient digestibility, such as protein (Longvah, Mangthya, & Ramulu, 2011; SánchezMuros, Barroso, & Manzano-Agugliaro, 2014). Thus, the lack of positive effects in the finishing pigs fed 8% with H. illucens larvae diet in the current trial may be due to the higher level of chitin contained in the H. illucens larvae diet. 5
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Fig. 2. Effects of the dietary inclusion of Hermetia illucens larvae on relative mRNA expression of genes related to myosin heavy-chain (MyHC) in longissimus thoracis muscle of finishing pigs. The values are means ± SEM (n = 8). a,bMeans in the different letters were significantly different (with a Tukey post hoc test P < .05).
meat quality and in past determines the nutritional value of the muscle. The current study showed that dietary supplementation with 4% H. illucens larvae could change the fatty acid composition and increase the contents of n-3 PUFA in the LT compared with the HI0 group. So far, this is the first trial that has investigated the fatty acid composition of LT from pigs fed with a diet supplemented with H. illucens larvae. Thus, the exact mechanism that H. illucens larvae increased n-3 PUFA needs further study. In general, our results indicated that dietary inclusion of 4% H. illucens larvae may have a positive effect on meat quality by increasing IMP and IMF contents of finishing pigs.
Kwon, Im, Seo, & Baik, 2012). ACCα and FAS are key regulatory enzymes in muscle fat storage (Etherton, Louveau, Sørensen, & Chaudhuri, 1993). ACCα is the rate-limiting enzyme in long-chain fatty acid de novo synthesis (Tan et al., 2011). FAS is mainly involved in elongation during fatty acid biosynthesis in muscle (Chen, Yang, Tong, & Zhao, 2004). Therefore, in the present study, the IMF content of LT was increased in response to dietary inclusion of H. illucens larvae, which may be due to an up-regulation of the expression of lipogenic genes (LPL, ACCα, and FAS). There are four different single MyHC isoforms have been identified in ATPase based fiber types in the muscle of pigs, such as slow-oxidative type I, fast-oxidative type IIa, fast-oxidative glycolytic type IIx, and fastglycolytic IIb, respectively (Klont, Brocks, & Eikelenboom, 1998; Lefaucheur, Milan, Ecolan, & Callennec, 2004). Fiber type composition is also closely related to meat quality traits (Eggert, Depreux, Schinckel, Grant, & Gerrard, 2002; Ryu & Kim, 2005). Previous studies have indicated that the IMF content of the LT negatively correlates with glycolytic muscle fiber types, while it positively correlates with type I and IIa muscle fibers (Kim et al., 2008; Ryu & Kim, 2005). An increase in the relative amount of type I and IIa fibers has been suggested to increase the water holding capacity and tenderness (Ryu & Kim, 2005). The present study provides the first evidence that inclusion of H. illucens larvae in the diet of finishing pigs may up-regulate the mRNA expression level of MyHC-IIa in LT. This finding suggests that dietary inclusion of H. illucens larvae may induce the muscle fiber transition toward more oxidative muscle fibers, and partly explains the phenomenon of how H. illucens larvae may improve meat quality.
4.3. Effects of H. illucens larvae on lipid metabolic and MyHC genes expression levels in the longissimus thoracis muscle It is well known that marbling of LT depends on the balance between fat deposition and removal, which is regulated by numerous intramuscular lipid metabolic genes (Li et al., 2018). Thus, to further investigate the molecular mechanism of dietary inclusion of H. illucens larvae implicated in regulating meat quality, we measured gene expression and found that the relative mRNA abundance of the lipid uptakerelated gene LPL, and lipogenic-related factors FAS and ACCα are higher in LT in response to dietary inclusion H. illucens larvae. It has been reported that the fatty acid availability directly influences IMF deposition. LPL is one of the genes that determines fatty acid uptake, and can participate in the process of fatty acid flux into adipocytes clustered in the muscle, and then provide the essential substrate for IMF synthesis (Jeong, 6
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5. Conclusions
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The results of the present study demonstrate that inclusion of 4% H. illucens larvae in the diet increases the ADG and partly improves carcass trait and muscle chemical composition of finishing pigs, while reducing F:G. Furthermore, this study also provides the first evidence that dietary inclusion of 4% H. illucens larvae affected the meat quality via upregulating the expression of genes related to the lipogenic potential and muscle fiber composition in the LT of finishing pigs. These findings provide a new perspective for H. illucens larvae meal as a suitable and nutrient rich alternative protein source for swine feed. Acknowledgement The present study was supported by the Presidential Foundation of the Guangdong Academy of Agricultural Sciences (201802B, 201621), Guangdong Modern Agro-industry Technology Research System (2016LT1080, 2017LT1080), and key project of Guangzhou Science and Technology Plan (201707020007). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.meatsci.2019.05.008. References Al-Qazzaz, M. F. A., Ismail, D., Akit, H., & Idris, L. H. (2016). Effect of using insect larvae meal as a complete protein source on quality and productivity characteristics of laying hens. Revista Brasileira de Zootecnia, 45, 518–523. AOAC (2007). Official methods of analysis. 18th ed.Gaithersburg, MD: Association of Official Analytical Chemists. Barragan-Fonseca, K. B., Dicke, M., & Loon, J. J. A. V. (2017). Nutritional value of the black soldier fly (Hermetia illucens L.) and its suitability as animal feed. Vol. 3, 105–120. Chen, J., Yang, X. J., Tong, H., & Zhao, R. Q. (2004). Expressions of FAS and HSL mRNA in longissimus dorsi muscle and their relation to intramuscular fat contents in pig. Journal of Agricultural Biotechnology, 12, 422–426. Cullere, M., Tasoniero, G., Giaccone, V., Miotti-Scapin, R., Claeys, E., De, S. S., & Dalle, Z. A. (2016). Black soldier fly as dietary protein source for broiler quails: Apparent digestibility, excreta microbial load, feed choice, performance, carcass and meat traits. Animal, 10, 1923–1930. Cutrignelli, M. I., Messina, M., Tulli, F., Randazzo, B., Olivotto, I., Gasco, L., ... Bovera, F. (2018). Evaluation of an insect meal of the black soldier Fly (Hermetia illucens) as soybean substitute: Intestinal morphometry, enzymatic and microbial activity in laying hens. Research in Veterinary Science, 117, 209–215. Dabbou, S., Gai, F., Biasato, I., Capucchio, M. T., Biasibetti, E., Dezzutto, D., ... Schiavone, A. (2018). Black soldier fly defatted meal as a dietary protein source for broiler chickens: Effects on growth performance, blood traits, gut morphology and histological features. Journal of Animal Science and Biotechnology, 9, 49. Dalle Zotte, A., Cullere, M., Martins, C., Alves, S. P., Freire, J. P., Falcão-e-Cunha, L., & Bessa, R. J. (2018). Incorporation of black soldier fly (Hermetia illucens L.) larvae fat or extruded linseed in diets of growing rabbits and their effects on meat quality traits including detailed fatty acid composition. Meat Science, 146, 50–58. Eggert, J. M., Depreux, F. F. S., Schinckel, A. P., Grant, A. L., & Gerrard, D. E. (2002). Myosin heavy chain isoforms account for variation in pork quality. Meat Science, 61, 117–126. Etherton, T. D., Louveau, I., Sørensen, M. T., & Chaudhuri, S. (1993). Mechanisms by which somatotropin decreases adipose tissue growth. American Journal of Clinical Nutrition, 58(2), 287S–295S Suppl. Faucitano, L., Rivest, J., Daigle, J. P., Levesque, J., & Gariepy, C. (2004). Distribution of intramuscular fat content and marbling within the longissimus muscle of pigs. Canadian Journal of Animal Science, 84, 57–61. Hale, O. M. (1973). Dried Hermetia illucens larvae (Diptera: Stratiomyidae) as a feed additive for poultry. Journal of Georgia Entomological Society, 8, 16–20. Han, K. N., Yang, Y. X., Hahn, T. W., Kwon, I. K., Lohakare, J. D., Lee, J. K., & Chae, B. J. (2007). Effects of chito-oligosaccharides supplementation on performance, nutrient digestibility, pork quality and immune response in growing-finishing pigs. Journal of Animal and Feed Sciences, 16, 607–620. Honikel, K. O. (1998). Reference methods for the assessment of physical characteristics of meat. Meat Science, 49, 447–457. Jeong, J., Kwon, E. G., Im, S. K., Seo, K. S., & Baik, M. (2012). Expression of fat deposition and fat removal genes is associated with intramuscular fat content in longissimus dorsi muscle of Korean cattle steers. Journal of Animal Science, 90, 2044–2053. Jeremiah, L. E., Gibson, L. L., Aalhus, J. L., & Dugan, M. E. R. (2003). Assessment of palatability attributes of the major beef muscles. Meat Science, 65, 949–958. Joo, S. T., Kim, G. D., Hwang, Y. H., & Ryu, Y. C. (2013). Control of fresh meat quality through manipulation of muscle fiber characteristics. Meat Science, 95, 828–836.
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