1. Introduction
With the rapid development of animal husbandry in recent years, the contradiction between the supply and demand of corn has become increasingly prominent [
1]. According to data from the Chinese National Statistics Bureauso, in 2022, China’s corn production reached 277 million tons, with a demand of 298 million tons and an import volume of 20.62 million tons. Rice is the most important grain crop, and the annual output has reached more than 200 million tons in China [
2]. The inventory of rice in China has remained stable at over 100 million tons in recent years, with an estimation of over 14 million tons of long-term stored rice. On the condition that food rations are absolutely safe, the overstocked grain can be considered as an alternative energy source. Therefore, research on the nutritional characteristics and utilization values of stored rice in animal feeding has great theoretical and practical significance.
Rice is a variety of grain that is not resistant to long-term storage, as aging and deterioration can occur after the second year, and the warranty storage life is about three years. During grain storage, the composition and structure of the main nutrients (protein, starch, and fat) will change due to their own respiration, oxidation, and the action of microorganisms, causing a decrease in nutritional values [
3]. Higher disulfide bond content and surface hydrophobicity and lower free sulfhydryl content were produced during storage, which led to the deterioration of the rice’s protein value [
4]. Compared to fresh rice, the solubility of total starch and amylose decreased, resulting in a lower gelatinization temperature, greater hardness, and lower viscosity of the rice [
5]. Moreover, the lipid oxidation increased the contents of free fatty acids and volatile carbonyl compounds, such as glutaraldehyde and hexanal, which reduced the palatability of the stored grain [
6].
Several studies have reported changes in the energy values and nutrient digestibility of stored corn and wheat in animal diets. Zhang et al. reported that the nutrient availability of corn, including the digestible energy (DE) and metabolizable energy (ME) values, decreased after being stored at room temperature for 10 months [
7]. Although there were no significant changes in the ME, the digestibility of crude protein, histidine, arginine, and starch decreased quadratically with corn stored for 4 years at room temperature [
8]. Some studies have reported that the nutritional value of stored corn and wheat had little change under standard storage conditions [
9,
10,
11]. The major nutrients of brown rice obtained from rice hulling were higher than or equal to corn and can be effectively used as an energy ingredient [
12,
13]. However, few studies have been completed on stored brown rice regarding the available energy values, amino acid digestibility, and its application in animal diets.
The objective of the present study was to evaluate the available energy value and amino acid (AA) digestibility of brown rice stored for 1 or 6 years and to investigate the effects of stored brown rice on growth performance, nutrient digestibility, serum biochemical parameters, intestinal enzyme activities in weaned piglets, carcass characteristics, and meat quality in fully grown pigs. The hypothesis was that if brown rice was stored under proper conditions, its available energy value and amino acid digestibility would not change significantly, and the growth performance and meat quality of the pigs would not be influenced by the inclusion of stored brown rice in the diets.
2. Materials and Methods
All experiments were conducted in accordance with the Chinese Guidelines for Animal Welfare and Experimental Protocol, and prior approval was obtained from the Animal Care and Use Committee of Academy of National Food and Strategic Reserves Administration (ethical approval code: 20230316006).
The paddy rice used in this study was stored in brick concrete barns (36 × 24 × 6 m, storage capacity 5000 tons) at National Grain Reserve Barn of Heilongjiang Province, as it has the greatest rice yield and inventory in China. Rice stored up to 6 years has been selected as a representative of long-term storage rice, and rice stored for 1 year has also been collected as fresh rice. The temperature and relative humidity in all grain barns was controlled within 20 °C and 70% all year around. Before the start of the experiment, the paddy rice was hulled into brown rice and crushed for subsequent experiments. All the diet, feces, and digesta samples collected in animal experiments were ground to pass through a 1 mm sieve for chemical analysis (
Table A1). Dry matter (DM, method 934.01), crude protein (CP, method 990.03), ash (method 942.05), ether extract (EE, method 920.39), calcium (method 985.01), and total phosphorus (method 985.01) contents were determined according to the procedures of Association of Official Analytical Chemists (AOAC) International (2006) [
14]. The contents of 18 AAs were measured according to the methods in AOAC. Tryptophan was hydrolyzed with LiOH at 110 °C for 22 h and then analyzed using High-Performance Liquid Chromatography (Agilent 1200 Series, Santa Clara, CA, USA). Cysteine and methionine were firstly oxidized with performic acid and hydrolyzed with 7.5 mol/L HCl at 110 °C for 24 h and then analyzed using AA analyzer. Another 15 AAs were hydrolyzed with 6 mol/L HCl at 110 °C for 24 h firstly and then analyzed using AA analyzer (L-8900, Hitachi; Tokyo, Japan). Neutral detergent fiber (NDF) was determined using α-amylase treated method without correction for insoluble ash, and acid detergent fiber (ADF) was expressed as inclusive of residual ash according to the procedures of the ANKOM200 Fibre Analyzer (Ankom Technology, Macedon, NY, USA). The gross energy (GE) was analyzed using automatic isoperibol oxygen bomb calorimeter (IKA C6000; IKA, GER). The fatty acid value was analyzed according to the procedures of GB/T 20569-2006 (Guidelines for evaluation of paddy storage character) [
15].
2.1. Exp. 1: Evaluating the DE and ME Values of Stored Brown Rice
Eighteen Landrace × Yorkshire (L × Y) barrows with an initial body weight (IBW) of 25.48 ± 3.21 kg were randomly allotted to 3 treatment groups with 6 replicated pigs per treatment. The diet was formulated to contain 96.9% corn (Corn) or brown rice stored for 1 (BR1) or 6 years (BR6) and 3.1% minerals and vitamins to meet the nutrient requirements for growing pigs recommended by the National Research Council (NRC, 2012) (
Table A2) [
16]. Each pig was individually raised in metabolism crates (1.4 × 0.45 × 0.6 m), and the room temperature was controlled at 23 ± 2 °C. Pigs were provided the feed equivalent of 4% of their IBW daily and fed twice at 08:30 and 16:30, respectively.
Pigs were first fed commercial diet for 7 d to adapt to the metabolic chamber. Animal experiments lasted for 12 d, of which 7 d were used to adapt to the experimental diet and 5 d to collect feces and urine samples [
17]. Feces sample of each pig was collected separately and stored at −20 °C. Urine of each pig was collected in barrels containing 50 mL 6 mol/L HCl, and 10% of the total urine collected daily was stored at −20 °C. At the end of the experiment, the fecal and urine samples were thawed and merged to obtain the sub-sample by pig. Fecal sub-sample was dried at 65 °C for 3 d using a drying oven while 4 mL of urine sample was added onto a quantitative filter paper in crucibles and then dried at 65 °C for 8 h using a drying oven for analysis of GE.
2.2. Exp. 2: Evaluating the AA Digestibility of Stored Brown Rice
Eighteen barrows (L × Y) with T-cannula at the terminal ileum (IBW: 22.16 ± 2.42 kg) were allocated to 3 experiment diets in a completely randomized design with 6 replicated pigs per treatment. The diets were formulated to contain 96.6% of corn (Corn) or brown rice stored for 1 (BR1) or 6 years (BR6) and 3.1% minerals and vitamins to meet the nutritional requirements for growing pigs recommended by the NRC (2012) [
16]. N-free diet containing 73.35% corn starch and 15% sucrose was used to evaluate the losses of basal ileal endogenous N and AAs. In addition, 0.3% chromic oxide (Cr
2O
3, ≥99.0%; 10,006,918, SINOPHARM, Beijing, China) was included as exogenous indicator (
Table A2). Feeding management was the same as above.
After a 15 d recovery postoperative period, pigs were fed commercial diet for 7 d to adapt to the environment. The animal experiment lasted for 7 d, of which 5 d were used to adapt to the experimental diets and 2 d to collection of digesta, which lasted for 9 h daily beginning at 08:30 [
17]. The sample bag was fixed to the cannula to collect the digesta and then stored at −20 °C. After the collection period, the digesta samples were thawed and merged to obtain the sub-sample by pig, and then lyophilized by vacuum freeze drier. The contents of chromium in the diets and digesta samples were measured using an Atomic Absorption Spectrophotometer (model Z-5000, Hitachi Corp., Tokyo, Japan) according to the method of García-Rico et al. [
18]
2.3. Exp. 3: Growth Trail on Weaned Piglets
One hundred and eight weaned piglets (L × Y, male; IBW: 9.16 ± 0.89 kg) were selected from a commercial herd, and randomly allocated into 3 diet treatments with 6 replicate pens per treatment and 6 pigs per pen (2.1 × 1.8 × 0.6 m, 0.5 m above the ground). The treatment included 1 control diet (Control) and 2 experimental diets formulated by completely replacing corn with brown rice stored for 1 (BR1) or 6 years (BR6), respectively (
Table 1). The diets were formulated based on the ME value in Exp. 1 and the SID AA value in Exp. 2, and meet the nutritional requirements for weaned piglets recommended by NRC (2012) [
16]. The ME and SID lysine, methionine, threonine, and tryptophan in 3 diets were kept the same. In the last 2 weeks of the experiment, 0.3% chromic oxide was added to each diet as an exogenous indicator.
Pigs were housed in pens with drinkers, feeders, and slatted floors, and were provided water and feed freely. The environment temperature was controlled at 22 ± 2 °C. The experiment lasted for 28 days. Pigs and feed were weighed at the beginning (d 1) and the end of the experiment (d 28) to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR).
From d 25 to d 27, approximately 100 g of fresh feces were collected daily from each pen and immediately stored at −20 °C. All samples were pooled by pen and then dried at 65 °C in a drying oven for 72 h. After fasting for 16 h, blood samples were collected through intravenous puncture on the morning (07:00) of d 28 and then injected into a 10 mL vacuum tube. After centrifugation at 3000× g for 15 min (4 °C), serum samples were collected and stored at −20 °C for the further determination of biochemical parameters. At the end of the experiment, 18 pigs with nearly average BW were selected from each pen. After overnight fasting, the pigs were slaughtered, and the gastrointestinal tract was ligated; then, the mucosa of duodenum, jejunal, and ileum were scraped with a glass slide and stored in liquid nitrogen.
After fasting overnight, the pigs are slaughtered, and the gastrointestinal tract of each pig is ligated. Then, the mucosa of the duodenum, jejunum, and ileum is scraped with a slide and stored in liquid nitrogen.
2.4. Exp. 4: Growth Trail on Fully Grown Pigs
Ninety growing pigs (L × Y; IBW: 48.28 ± 3.51 kg) were randomly allotted into 3 dietary treatments with 6 replicate pens per treatment and 5 pigs per pen (4.0 × 2.8 × 1 m). The treatment diets included 1 control diet (Control) and 2 experimental diets formulated by completely replacing corn with brown rice stored for 1 (BR1) or 6 years (BR1), respectively (
Table 2). The diets were formulated based on the ME value in Exp. 1 and the SID AAs value in Exp. 2 and meet the nutritional requirements for pigs in different phases recommended by NRC (2012) [
16]. The ME and SID lysine, methionine, threonine, and tryptophan in 3 diets were kept the same.
Pigs were kept in pens with drinkers, feeders, and slatted floors and were provided water and feed freely. The environment temperature was controlled at 18 ± 2 °C. The experiment lasted for 8 weeks and included 2 phases: I (d 1 to d 24); II (d 25 to d 56). At the beginning (d 1) and end of each phase (d 24 and d 56), pigs and feed were weighed to determine ADG, ADFI, and FCR.
At the end of the experiment, 15 pigs with near-average BW were selected from each pen. After overnight fasting, pigs were subjected to electric shock (250 V, 0.5 A, for 5–6 s), bleeding, and evisceration using standard commercial procedure. Approximately 10 g of longissimus dorsi muscle (LDM) were sampled from the left half of each carcass and stored at −20 °C.
2.5. Determination of Serum Biochemical Indices and Intestinal Enzyme Activities
Serum albumin (ALB), globulin (GLB), total protein (TP), triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urea nitrogen (UN) were measured by automatic biochemical analyzer (7020 series; Hitachi, Japan) and following the protocol of assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The content of serum immunoglobulin (Ig) A, IgG, and IgM were determined using enzyme-linked immunosorbent assay (ELISA) using assay kits purchased from Takara Biomedical Technology Institute (Beijing, China). Serum catalase (CAT), total antioxidant capacity (T-AOC), glutathione (GSH), glutathione peroxidase (GSH-PX), malondialdehyde (MDA), superoxide dismutase (SOD) were measured using assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
The duodenum, jejunal, and ileum mucosa samples were homogenized in cold maleic acid buffer (0.1 mol/L, pH = 6.8, 1: 10, w/v) and then centrifuged at 3000× g for 10 min. Supernatants were collected to evaluate the activities of amylase, lipase, chymotrypsin, trypsin, lactase, maltase, and sucrase, following the protocol of assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2.6. Determination of Carcass Characteristics and Meat Quality
After slaughter and scraping, the head, hooves, tail, and internal organs of pigs were removed, while the suet and kidneys were preserved to record carcass weight, and dressing percentage was calculated by dividing carcass weight by live body weight. Carcass straight length was measured from the first rib to the end of the public bone. Backfat thickness was recorded at the first rib, last rib, and last lumbar vertebra, and Loin eye height and width were measured at the 10th rib following the equation loin–eye area (cm
2) = 0.7 × loin eye height (cm) × loin eye width (cm), according to the NY/T 825-2004 (Technical Regulation for Testing of Carcass Traits in Lean-Type Pig) [
19].
The LDM on the left half of carcass between the 10th and 12th ribs were sampled for analysis of meat quality, including drip loss, shear force, pH, and muscle color, according to the NY/T 821-2019 (Technical Code of Practice for Pork Quality Assessment) and NY/T 1180-2006 (Determination of Meat Tenderness Shear Force Method) [
20,
21]. About 30 g of meat was hung in a plastic bag at 4 °C for 24 h and kept out of contact with the bag. Drip loss was calculated as a percentage of the droplet amount compared to the initial meat weight. Meat was cooked in a water bath at 70 °C for 20 min, and then ten cylindrical samples were obtained by cutting the meat parallel to the fiber direction, and shear force was determined by cutting the cylindrical sample vertically to the myofiber axis using a digital-display-muscle tenderness meter. At 45 min postmortem, initial pH of LDM was recorded using a glass penetration pH electrode, and pH of LDM was detected again at 24 h postmortem. The meat color was measured as L* (lightness), a* (redness), and b* (yellowness) using a tristimulus colorimeter three times at 24 h postmortem. About 20 g meat sample was lyophilized to determine the fatty acids profile using classical gas chromatography (6890 series; Agilent Technologies, Wilmington, DE, USA) [
22].
2.7. Statistical Analysis
PROC UNIVERSATE program (SAS Inst. Inc., Carry, NC, USA) of SAS 9.2 was used to check the normal and abnormal values of growth performance, nutrient digestibility, serum biochemical index, enzyme activity, carcass traits, and meat quality data. Cook’s distance and abandonment method was used to identify outliers. Then, the PROC GLM program of SAS was used to analyze the data. The diet was the only fixed effect, while each pig was considered an experimental unit (for growth performance data, each pen was considered an experimental unit). The LSMEANS statement was used to separate treatment means, and the Tukey test was used to adjust the data. Significant difference was declared at p < 0.05.