Dietary live yeast supplementation alleviates transport-stress-impaired meat quality of broilers through maintaining muscle energy metabolism and antioxidant status.

BACKGROUND: This experiment was to investigate the effect of dietary live yeast (LY, 1 × 1010 CFU g-1 ) supplementation on serum metabolic parameters, meat quality as well as antioxidant enzyme activity of transported broilers. A total of 192 one-day-old broilers were randomly assigned to four treatments with six replicates and eight chicks per replicate: a basal diet without transportation (CON), a basal diet containing 0 (T), 500 (T + LY500 ) and 1000 mg kg-1 (T + LY1000 ) LY with 3 h of transportation after feeding for 42 days, respectively. The serum and muscle samples of broilers were collected immediately after 3 h of transportation. RESULTS: A higher (P < 0.05) final body weight and average daily weight gain were observed in T + LY1000 group compared with CON and T groups. The T + LY1000 group reduced (P < 0.05) the serum lactate contents and improved (P < 0.05) the pH24h and decreased (P < 0.05) the drip loss in muscles of transported-broilers. Also, the T + LY1000 group enhanced (P < 0.05) the total-antioxidant capacity and reduced (P < 0.05) the malondialdehyde in serum and muscles. Besides, the messenger RNA (mRNA) expression of avian uncoupling protein (avUCP) in muscles was down-regulated (P < 0.05) of T + LY1000 group compared with T group. CONCLUSION: Dietary LY supplementation alleviates transport-stress-impaired meat quality of broilers through maintaining muscle energy metabolism and antioxidant status. Therefore, LY may serve as a potential protector for broilers under transport stress in the future. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Dietary 25-hydroxycholecalciferol modulates gut microbiota and improves the growth, meat quality, and antioxidant status of growing-finishing pigs.

Introduction: 25-Hydroxycholecalciferol (25OHD3) is the active metabolite of regular vitamin D3 in vivo, which has a stronger biological activity and is more easily absorbed by the intestine than regular vitamin D3. Our study aimed to detect the potential influences of 25OHD3 on pork quality, antioxidant status, and intestinal microbiota of growing-finishing pigs receiving low-phosphorus (P) diet. Methods and results: Forty pigs [initial body weight (BW): 49.42 ± 4.01 kg] were allocated into two groups including low-P diet (CON group) and low-P diet supplemented with 50 µg/kg 25OHD3 (25OHD3 group). The whole experiment lasted for 88 days, including phase 1 (day 1-28), phase 2 (day 29-60), and phase 3 (day 61-88). The results showed that 25OHD3 supplementation tended to decrease feed conversion ratio in phase 3 and overall phase in comparison with the CON group. 25OHD3 increased (p < 0.05) serum contents of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and decreased (p < 0.05) serum bone-specific alkaline phosphatase level. 25OHD3 increased (p < 0.05) mucosal GSH-Px activity in the duodenum and ileum, and tended to increase redness value and the activities of total antioxidant capacity and SOD in longissimus dorsi. 25OHD3 significantly upregulated the mRNA level of copper/zinc superoxide dismutase, and tended to change the mRNA levels of nuclear factor E2-related factor 2 and kelch-like ECH-associated protein 1 in longissimus dorsi. Moreover, 25OHD3 supplementation decreased (p < 0.05) n-6/n-3 and iodine value in longissimus dorsi. For bone quality, 25OHD3 supplementation increased (p < 0.05) calcium content, bone mineral content, and breaking strength in the metacarpal bones. Moreover, the colonic abundance of Lactobacillus was significantly higher in pigs fed with 25OHD3, and exhibited a positive association with serum antioxidant status, pork quality, and bone characteristics. Conclusion: Overall, the inclusion of 25OHD3 in low P diet partly improved production performance, meat quality, antioxidant capacity, bone properties, and gut microbiota composition of growing-finishing pigs.

Supplementation of Mixed Organic Acids Improves Growth Performance, Meat Quality, Gut Morphology and Volatile Fatty Acids of Broiler Chicken.

Background: Organic acid as a green feed additive is increasingly favoured by enterprises and scholars, but little emphasis has been placed on the effect of organic acids on broiler meat quality. Methods: A total of 192 male chicks (one-day-old, weighted 48.40 ± 0.64 g) were selected to investigate the effect of mixed organic acids (MOA) on growth performance, meat quality as well as fatty acids profile. Chicks were randomly allocated to three treatments with eight replicates and eight chicks per replicate, including a corn-soybean basal diet with 0 (CON), 3000 mg/kg (low MOA; LMOA), and 6000 mg/kg (high MOA; HMOA) MOA. The experiment was divided into starter (d 1-d 21) and grower (d 22-d 42) phases. Results: Broilers supplemented with LMOA and HMOA enhanced (p < 0.05) the final body weight and average daily gain in the grower and overall phases. An improved (p < 0.05) feed conversion ratio in the grower and overall phases was observed in broilers supplemented with LMOA. The breast and thigh muscles pH24h were higher (p < 0.05) in broilers fed with HMOA and the redness in thigh meat was also improved (p < 0.05). Additionally, supplementing LMOA increased (p < 0.05) the saturated fatty acids, unsaturated fatty acids and the ratio of polyunsaturated fatty acids to saturated fatty acids in breast meat. A positive effect occurred (p < 0.05) on jejunal villus height and ileal crypt depth in 21 d broilers supplemented with HMOA. Conclusion: Our findings indicated that dietary supplementation of MOA could improve the growth performance, meat quality, and fatty acids profile, as well as intestinal morphology. Furthermore, diets supplemented with mixed organic acids at 3000 mg/kg may be more desirable, considering the overall experimental results in broilers.

Live Yeast or Live Yeast Combined with Zinc Oxide Enhanced Growth Performance, Antioxidative Capacity, Immunoglobulins and Gut Health in Nursery Pigs.

This study aimed to investigate the effects of dietary LY or LY combined with ZnO supplementation on performance and gut health in nursery pigs. 192 Duroc × Landrace × Yorkshire piglets (weaned on d 32 of the age with 9.2 ± 1.7 kg BW) were allocated into four treatments with eight replicate pens, six piglets per pen. The treatments included a basal diet as control (CTR), an antibiotic plus ZnO diet (CTC-ZnO, basal diet + 75 mg/kg of chlortetracycline + ZnO (2000 mg/kg from d 1 to 14, 160 mg/kg from d 15 to 28)), a LY diet (LY, basal diet + 2 g/kg LY), and a LY plus ZnO diet (LY-ZnO, basal diet + 1 g/kg LY + ZnO). The results showed that pigs fed LY or LY-ZnO had increased (p < 0.05) average daily gain, serum IgA, IgG, superoxide dismutase, fecal butyric acid, and total volatile fatty acid concentrations, as well as decreased (p < 0.05) feed conversion ratio and diarrhea rate compared with CTR. In conclusion, pigs fed diets with LY or LY combined with ZnO had similar improvement to the use of antibiotics and ZnO in performance, antioxidant status, immunoglobulins, and gut health in nursery pigs.

Effects of dietary Forsythia suspensa extract supplementation to lactating sows and nursery pigs on post-weaning performance, antioxidant capacity, nutrient digestibility, immunoglobulins, and intestinal health.

The aim of this study was to determine the effect of dietary Forsythia suspensa extract (FSE) supplementation to lactating sows and nursery pigs on post-weaning performance, antioxidant capacity, immunoglobulins, and intestinal health. Based on backfat, body weight (BW), and parity, 24 gestating sows (Landrace × Yorkshire) with average parity of 3.38 ± 0.61 and BW of 234 ± 6.81 kg were allotted into two dietary treatments (control vs. 100 mg/kg FSE) with 12 sows per treatment from day 107 of gestation to day 21 of lactation. After weaning, based on the initial BW and source litter, 192 nursery pigs (Duroc × [Landrace × Yorkshire], average BW of 6.98 ± 0.32 kg, weaned at day 21) were allotted into four dietary treatments with eight replicate pens per treatment, six pigs per pen for a 4-wk study. The treatments included the following: 1) CC (sows and their piglets both fed control diet); 2) CF (sows fed control diet and their piglets fed FSE diet [containing 100 mg/kg FSE]); 3) FC (sows fed FSE diet and their piglets fed control diet); and 4) FF (sows and their piglets both fed FSE diet). The MIXED procedures of SAS for a split-plot arrangement with sow diet as the whole plot and nursery diet as split plot were used to analyze the data. After weaning, piglets from FSE-fed sows had improved (P < 0.05) average daily gain and feed efficiency, and lower (P < 0.05) diarrhea rate in overall (day 1 to 28) compared with those from sows fed control diet. Piglets from FSE-fed sows also had higher (P < 0.05) contents of immunoglobulin G (IgG), growth hormone, superoxide dismutase (SOD), total antioxidant capacity in serum, villus height in ileum, and villus height to crypt depth ratio in jejunum, as well as lower (P < 0.05) content of malondialdehyde (MDA) in serum and crypt depth in ileum compared with those from sows fed control diet. Piglets fed FSE during nursery had increased (P < 0.05) concentrations of IgG, SOD, and catalase, and decreased (P < 0.05) MDA and tumor nuclear factor-α levels in serum compared with those fed control diet during nursery. Piglets from FC group had increased (P < 0.05) protein expression of occludin in jejunal mucosa and relative abundance of Lactobacillus on genus level in colon compared with those from CC group. In conclusion, for the performance and intestinal health, diets supplemented with FSE during lactation phase seemed more efficient to alleviate weaning stress than the nursery phase. In terms of the antioxidant status and immunoglobulins, FSE supplemented in both phases were efficient for nursery pigs.

Effects of dietary supplementation of compound enzymes on performance, nutrient digestibility, serum antioxidant status, immunoglobulins, intestinal morphology and microbiota community in weaned pigs.

The objective of this study was to evaluate the effects of compound enzymes (CE) (containing per g 375 U amylase, 2500 U protease, 4000 U xylanase and 150 U ß-glucanase) on performance, nutrient digestibility, serum antioxidant status, immunoglobulins, intestinal morphology, volatile fatty acids contents and microbiota community in weaned pigs. Seventy-two pigs (Duroc × Landrace × Yorkshire, weaned at d 28) with an average body weight of 8.49 ± 0.87 kg were allotted into two treatments with six replicate pens per treatment (three barrows and three gilts per pen) according to sex and body weight in a randomised complete block design. The treatments contained a corn-soybean meal-barley basal diet (CON) or a basal diet supplemented with 1000 mg CE/kg (CE). The study was divided into phase 1 (d 1 to 14) and 2 (d 15 to 35). The average daily gain was increased (p < 0.05) in pigs fed CE in phase 2 and overall (d 1 to 35) compared with CON. These pigs had greater (p ≤ 0.05) serum IgA, IgG, superoxide dismutase and catalase contents, as well as tended to increase serum IgM content and apparent total tract digestibility (ATTD) of organic matter in phase 1 compared with CON. In phase 2, pigs supplemented with CE showed greater (p < 0.01) ATTD of dry matter, organic matter, crude protein and gross energy compared with CON. These pigs also had increased (p < 0.05) IgA, IgG, IgM, superoxide dismutase contents, and decreased (p < 0.05) malondialdehyde content in serum compared with CON. Moreover, pigs fed CE had higher (p < 0.05) villus height and villus height to crypt depth ratio in ileum, and tended to increased acetic acid content in colon compared with CON. Furthermore, pigs fed CE had increased (p < 0.05) relative abundance of Firmicutes at phylum level, Lactobacillales at order level, Lactobacillaceae at family level, Bacilli at class level, Lactobacillus at genus level in caecum and colon, as well as lower (p < 0.05) relative abundance of Bacteroidetes at phylum level, Bacteroidales at the order level, Bacteroidia at class level, Clostridium_sensu_stricto_6 at genus level in colon compared with CON. In conclusion, dietary inclusion of compound enzymes could effectively improve nutrient digestibility, serum antioxidant status, immunoglobulin, gut morphology, microbiota community, and therefore improve performance in weaned pigs.

Effects of Dietary Fatty Acids from Different Sources on Growth Performance, Meat Quality, Muscle Fatty Acid Deposition, and Antioxidant Capacity in Broilers.

This study aimed to investigate the efficiency of dietary fatty acids from various sources on growth performance, meat quality, muscle fatty acid deposition and antioxidant capacity in broilers. 126 Arbor Acres broilers (1 d-old, initial body weight of 45.5 ± 0.72 g) were randomly assigned to three treatments with seven cages per treatment and six broilers per cage. The dietary treatments included: (1) corn-soybean meal basal diet containing 3% soybean oil (control diet, CTL); (2) basal diet + 1% microalgae + 1% linseed oil + 1% soybean oil (ML); (3) basal diet + 2% fish oil + 1% soybean oil (FS). The trial consisted of phase 1 (day 1 to 21) and 2 (day 22 to 42). Compared with CTL, broilers fed ML or FS diet showed improved (p < 0.05) average daily gain in phase 1, 2, and overall (day 1 to 42), as well as a decreased (p < 0.05) feed conversion ratio in phase 1 and overall. On day 42, broilers supplemented with FS diet showed increased (p ≤ 0.05) the relative weights of pancreas and liver, as well as higher (p < 0.05) redness value in breast and thigh muscle compared with CTL. Broilers offered ML or FS diet had lower (p < 0.05) the relative weight of abdominal fat and total serum cholesterol content in phase 1, and increased (p < 0.05) contents of serum glucose, n-3 polyunsaturated fatty acids (PUFA), eicosacagetaenoic acid, docosahexaenoic acid, glutathione peroxidase, superoxide dismutase and total antioxidant capacity, as well as lower (p < 0.05) concentrations of malondialdehyde, n-6 PUFA, and n-6/n-3 PUFA ratio in breast and thigh muscle compared with CTL. This research indicates that diets supplemented with fish oil or a combination of microalgae and linseed oil experience improved performance, antioxidant capacities and n-3 PUFA profile in muscle of broilers compared with traditional soybean oil supplemented diets.

Effects of maternal 25-hydroxycholecalciferol on nutrient digestibility, milk composition and fatty-acid profile of lactating sows and gut bacterial metabolites in the hindgut of suckling piglets.

To investigate the effects of maternal 25-hydroxycholecalciferol (25OHD3) supplementation during lactation on nutrient digestibility and milk composition of sows and gut bacterial metabolites and their metabolites in the hindgut of suckling piglets, 24 Large White × Landrace sows were assigned randomly to one of two dietary treatments (Diet ND: 2000 IU vitamin D3/kg feed; Diet 25-D: 50 µg 25OHD3/kg feed). The experiment began on d 107 of gestation and continued until weaning on d 21 of lactation. Maternal 25OHD3 supplementation increased (p < 0.05) total litter weight gain during lactation. Milk fat content, immunoglobulin G level on d 21 of lactation and 25OHD3 concentration on d 7, 14, and 21 of lactation were higher (p < 0.05) in sows fed with 25OHD3. Apparent total tract digestibility of dietary calcium was higher (p < 0.05) in 25-D sows than ND sows. With respect to fatty-acid profile, C16:0 and saturated fatty acids in milk were higher (p < 0.05), but C20:4n-6, the ratios of monounsaturated fatty acids to saturated fatty acids and polyunsaturated fatty acids to saturated fatty acids were lower (p < 0.05) in 25-D sows than ND sows. 25OHD3 supplementation increased the mRNA expressions of acetyl-CoA carboxylase α and fatty-acid synthase in the mammary gland of lactating sows. For gut bacterial metabolites, concentration of butyrate in the caecal digesta was higher (p < 0.05) in piglets suckling 25-D sows than piglets suckling ND sows. In conclusion, 25OHD3 supplementation in maternal diets changed dietary calcium digestibility, milk composition and milk fatty-acid profile of lactating sows and altered gut bacterial metabolites in the hindgut of suckling piglets.

Determination and prediction of the apparent and standardized ileal amino acid digestibility in cottonseed meals fed to growing pigs.

This experiment was conducted to evaluate the chemical composition and apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of amino acids (AA) in cottonseed meals (CSM) and to develop prediction equations for estimating AA digestibility for growing pigs based on the chemical composition and AA content of CSM. 10 CSM were collected from 10 provinces in China. 11 barrows (initial body weight: 34.4 ± 1.9 kg), fitted with an ileal T-cannula, were allotted to 6 × 11 Youden square with 6 periods and 11 diets. The diets included one N-free diet and 10 CSM test diets containing 40% CSM as the sole source of AA. The nitrogen-free diet was used to measure the basal endogenous losses of CP and AA. The AID and SID for crude protein (CP), Lys, Met, Trp, and Thr among the 10 CSM differed (p < 0.05). With R2 values exceeding 0.30, significant linear regression equations for CP, Lys, and Thr were obtained. The best fit equations for predicting the AID and SID of Lys were: AID of Lys = 82.94-0.65 neutral detergent fiber, with R2  = 0.44, residual standard deviation (RSD) = 4.03 and p = 0.04; and SID of Lys = 135.34-1.01 neutral detergent fiber + 34.27 Thr - 58.06 Trp - 84.93 Met, with R2  = 0.90, RSD = 1.42 and p = 0.01. In conclusion, the results of AID, SID and prediction equations could be used to evaluate the digestibility of CSM in growing pigs.

Mixed organic acids improve nutrients digestibility, volatile fatty acids composition and intestinal microbiota in growing-finishing pigs fed high-fiber diet.

OBJECTIVE: The objective of this study was to investigate effects of mixed organic acids (MOA) on nutrient digestibility, volatile fatty acids composition and intestinal microbiota in growing-finishing pigs fed high wheat bran diet. METHODS: Six crossbred barrows (Duroc×Landrace×Yorkshire), with an average body weight 78.8±4.21 kg, fitted with T-cannulas at the distal ileum, were allotted to a double 3×3 Latin square design with 3 periods and 3 diets. Each period consisted of a 5-d adjustment period followed by a 2-d total collection of feces and then a 2-d collection of ileal digesta. The dietary treatments included a corn-soybean-wheat bran basal diet (CTR), mixed organic acid 1 diet (MOA1; CTR+3,000 mg/kg OA1), mixed organic acid 2 diet (MOA2; CTR+2,000 mg/kg OA2). RESULTS: Pigs fed MOA (MOA1 or MOA2) showed improved (p<0.05) apparent total tract digestibility (ATTD) of gross energy, dry matter and organic matter, and pigs fed MOA2 had increased (p<0.05) ATTD of neutral detergent fiber compared to CTR. Dietary MOA supplementation decreased (p<0.05) pH value, and improved (p<0.01) concentrations of lactic acid and total volatile fatty acids (TVFA) in ileum compared to CTR. Pigs fed MOA showed higher (p<0.05) concentration of acetic acid, and lower (p<0.05) content of formic acid in feces compared to CTR. Pigs fed MOA1 had increased (p<0.05) concentration of TVFA and butyric acid in feces. Pigs fed MOA1 showed higher concentration of Lactobacillus and lower concentration of Escherichia in feces compared to CTR. CONCLUSION: Dietary supplementation of MOA 1 or 2 could improve nutrients digestibility, TVFA concentration and intestinal flora in growing-finishing pigs fed high fiber diet.

Effects of dietary supplementation with a combination of plant oils on performance, meat quality and fatty acid deposition of broilers.

OBJECTIVE: This study was to evaluate effects of mixed plant oils (identified as mixed oil 1 [MO1] and mixed oil 2 [MO2]) on performance, serum composition, viscera percentages, meat quality, and fatty acid deposition of broilers. METHODS: A total of 126 one-day-old Arbor Acres male broiler chicks (weighing 44.91±0.92 g) were randomly allocated to 1 of 3 treatments with 7 replicate pens per treatment (6 broilers per pen). Dietary treatments included a corn-soybean basal diet supplemented with 3% soybean oil (CTR), basal diet with 3% MO1 (a mixture of 15% corn oil, 10% coconut oil, 15% linseed oil, 20% palm oil, 15% peanut oil and 25% soybean oil; MO1), or basal diet with 3% MO2 (a combination of 50% MO1 and 50% extruded corn; MO2). The trial consisted of phase 1 (d 1 to 21) and phase 2 (d 22 to 42). RESULTS: Compared to CTR, broilers fed MO (MO1 or MO2) had greater (p<0.05) average daily gain in phase 1, 2, and overall (d 1 to 42), redness in thigh muscle, concentrations of serum glucose, serum albumin, saturated fatty acids (SFA) and n-6/n-3 polyunsaturated fatty acids (PUFA) ratio in breast muscle, while these broilers also showed lower (p≤0.05) drip loss and concentrations of C18:3n-3 and PUFA/SFA ratio in breast muscle. Broilers fed MO2 had higher (p<0.05) liver percentage, while broilers fed MO1 had lower (p≤0.05) feed conversion ratio in phase 1 and increased (p<0.05) contents of C18:2n-6, C20:5n-3, C22:6n-3, and n-3 PUFA in breast muscle compared to CTR. CONCLUSION: Mixed plant oils had positive effects on performance, serum parameters, meat quality, liver percentage and fatty acid deposition in broilers, which indicates they can be used as better dietary energy feedstocks than soybean oil alone.

Effects of Chromium Methionine Supplementation with Different Sources of Zinc on Growth Performance, Carcass Traits, Meat Quality, Serum Metabolites, Endocrine Parameters, and the Antioxidant Status in Growing-Finishing Pigs.

This experiment was conducted to evaluate the effects of chromium methionine with/without zinc sulfate or zinc amino acid complex on the growth performance, carcass traits, meat quality, serum parameters, endocrine parameters, and antioxidant status of growing-finishing pigs. A total of 180 (32.0 ± 1.7 kg body weight, BW) crossbred pigs (Duroc × Landrace × Yorkshire) were used in a completely randomized design with three dietary treatments and 10 replicates per treatment (five pens of barrows and five pens of gilts with six pigs per replicate). Three treatments were corn-soybean meal-based diets supplemented with 100 mg Zn/kg from zinc sulfate (ZnSO4), 100 mg Zn/kg from ZnSO4 + 0.2 mg Cr/kg from chromium methionine complex (CrMet), or 50 mg Zn/kg from ZnSO4 + 50 mg Zn/kg from zinc amino acid complex (ZnAA) + 0.2 mg Cr/kg from CrMet, respectively. The experiment lasted 105 days, of which was divided into three stages including phase 1 (30 to 50 kg BW), phase 2 (50 to 80 kg BW), and phase 3 (80 to 110 kg BW). Results showed that supplementation with CrMet and ZnAA improved (P < 0.05) the feed conversion of the pigs in phase 2, phase 3, and the overall experiment. Hot carcass weight, dressing percentage, and a longissimus dorsi muscle area were increased (P < 0.05) in pigs fed with diets supplemented with both CrMet and ZnAA compared with pigs fed with diets containing only ZnSO4 (P < 0.05). There was also an increase (P < 0.01) pH24 h in the longissimus dorsi muscle in pigs fed with diets supplemented with CrMet and ZnAA. The concentration of serum glucose in pigs fed with diets containing CrMet and ZnAA was decreased (P < 0.05) compared with that in pigs fed with the diet containing ZnSO4. Supplementation with CrMet and ZnAA increased (P < 0.05) the circulating levels of insulin and decreased (P < 0.05) cortisol. There was an increase (P < 0.05) in total serum antioxidant capacity and Cu/Zn superoxide dismutase activity as well as a decrease (P < 0.05) in serum malondialdehyde concentrations in pigs fed with diets supplemented with CrMet and ZnAA compared with pigs fed with the diet supplemented only with ZnSO4. In conclusion, supplementation of CrMet only or CrMet together with ZnAA improved feed conversion, carcass traits, and meat quality in the growing-finishing pigs.

Effects of the standardized ileal digestible valine : lysine ratio on performance, milk composition and plasma indices of lactating sows.

To determine the effects of standardized ileal digestible (SID) valine : lysine ratio on the performance, milk composition and plasma indices of lactating sows, 32 Large White × Landrace sows (219.78 ± 7.15 kg body weight; parity 1.82 ± 0.62) were allotted to one of four dietary treatments with eight sows per treatment based on parity, back fat thickness and body weight. The sows were fed corn-soybean meal-based diets containing 63, 83, 103 or 123% SID valine : lysine from day 107 of gestation until day 28 of lactation. The average daily feed intake of sows and daily weight gain of piglets increased linearly (P < 0.05) while back fat loss decreased linearly (P < 0.05) as the SID valine : lysine ratio increased. All of the analyzed amino acids in sow colostrum and valine concentrations of sow and piglet plasma increased linearly (P < 0.05) with the increasing SID valine : lysine ratio. In conclusion, 88 and 113% dietary SID valine : lysine ratios were optimal to achieve minimum back fat loss and maximum piglet growth rate using a linear-break point model which exceeds the requirement of 85% that is estimated by the National Research Council (2012).