Article pubs.acs.org/JAFC
Nutritional and Metabolic Impacts of a Defatted Green Marine Microalgal (Desmodesmus sp.) Biomass in Diets for Weanling Pigs and Broiler Chickens Ricardo Ekmay, Stephanie Gatrell, Krystal Lum, Jonggun Kim, and Xin Gen Lei* Department of Animal Science, Cornell University, Ithaca, New York 14853, United States S Supporting Information *
ABSTRACT: Two experiments were conducted to determine the nutritional and metabolic impacts of defatted green microalgal (Desmodesmus sp.) biomass (DGM), protease, and nonstarch polysaccharide degrading enzymes (NSPase) in diets for weanling pigs and broiler chicks. Pigs fed 10% DGM for 28 days had growth performance comparable to the controls, but 23−39% lower (P < 0.05) plasma urea nitrogen concentrations. Broilers fed 15% DGM had 16% greater (P < 0.05) gain/feed efficiency than the control (0.78 vs 0.67) over the 42 day period. Supplemental protease (0.06%) decreased (P < 0.03) plasma uric acid concentrations in pigs on day 14, whereas supplemental NSPase showed negative effects in broilers. Dietary inclusions of DGM or enzymes altered (P < 0.05−0.1) hepatic and muscle protein levels of key regulators in the mTOR pathway. In conclusion, weanling pigs and broiler chicks tolerated dietary inclusions of 10 and 15% DGM, respectively, and adding protease might help digestion. KEYWORDS: microalgal biomass, poultry, production, protein metabolism, swine
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INTRODUCTION Corn and soybean acreage account for 29 and 26% of national crop land, respectively.1 Approximately 60% of corn and 47% of soy produced in the United States is used by the feed industry, mainly to raise chickens and pigs.2 Although they represent the two major meat-producing species, their massive consumption of corn and soy directly competes with the human food supply. Thus, alternative feed ingredients are needed to sustain animal agriculture and human food security. Various marine microalgae have been tested in diets for poultry3−5 and swine.6−8 Along with recent interests in using microalgae as feedstock for the third generation of biofuel production,9 our laboratory has determined the efficacy of inclusions of defatted diatom microalgae Staurospira sp. (DFA) into diets for broiler chicks5 and weanling pigs.8 In three consecutive chick experiments, we demonstrated that the inclusion of 7.5% DFA in the diets for broiler chicks to replace corn and soybean meal did not impair their growth performance or biochemical status during the full production cycle (days 1−42).5 However, including DFA at 10% replacing corn and soybean meal or at 7.5% replacing soybean meal alone resulted in negative impacts on the growth performance of chicks. Because DFA contained a lower level of crude protein than soybean meal (19.1 vs 48.5%), a practical question was if the broiler chicks could tolerate higher inclusions of other types of defatted microalgal biomass that have better profiles of protein, amino acids, ash, and other components. Whereas supplementing DFA up to 10% in the diets showed little effect on a number of plasma biochemical indicators of chicks, the effects on plasma uric acid, the end product and sensible biomarker of avian nitrogen metabolism,10,11 remained inconclusive,5,12 and effects of the supplemental DFA on plasma amino acid profile were not determined. Our previous © 2014 American Chemical Society
study tested the efficacy of a supplemental commercial protease in the 7.5% DFA-containing diets, but other microalgae may exhibit a different susceptibility to the proteolytic actions of the enzyme.5 Meanwhile, nonstarch polysaccharide (NSP) degrading enzymes are also commonly incorporated into diets for broiler chicks13−16 and have been shown to improve the nutritive values of diets based on oats,17 barley,18,19 and wheat.20,21 Similar to these plant ingredients, many types, if not all, of microalgal biomass contain relatively high levels of complex carbohydrates,22,23 including cellulose24,25 and xylose.26 In fact, the previously tested DFA contained 14% neutral detergent fiber.5 However, effects of supplementing NSPdegrading enzymes to diets containing microalgal biomass have not been well studied. Although microalgae are often explored as new or alternative protein supplements, previous research by us and others27,28 did not examine impacts of feeding microalgae on body metabolism or underlying regulatory mechanism. Specifically, the mammalian target of rapamycin (mTOR) pathway is a key regulator of cell growth, which integrates signals from nutrients, energy status, and growth factors to regulate metabolism.29 Feeding regimens,30 amino acid abundance and availability,31,32 protein abundance,30 and protein source33 are able to affect this pathway. Downstream targets of mTOR include eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4E (eIF4E) that mediate steps in translation initiation and ribosomal protein S (S6) and ribosomal protein S6 kinase (S6K1) that are thought to Received: Revised: Accepted: Published: 9783
March 7, 2014 August 5, 2014 September 12, 2014 September 12, 2014 dx.doi.org/10.1021/jf501155n | J. Agric. Food Chem. 2014, 62, 9783−9791
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regulate protein translation.34 Because broiler chicks go through intensive protein synthesis during the growth, it is not only novel but also relevant to determine effects of the dietary microalgal biomass inclusion on their functional expression of signal proteins involved in the mTOR pathway. Just as broiler chicks, weanling pigs grow at a fast rate with intensive protein metabolism.35 Likewise, our previous research13 showed that weanling pigs grew well with diets containing 7.5% DFA to replace corn and soybean meal, but not with 15% DFA replacing corn and soybean meal or 7.5% DFA replacing soybean meal. Subsequently, the same question arose as to whether weanling pigs could tolerate higher inclusion levels of the other apparently “superior” types of defatted microalgal biomass. However, there were different considerations of the pig experiment from those of the broilers. Comparatively, effects of the added microalgal biomass on plasma biochemical measures of nutritional status including tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (AKP), and alanine aminotransferase (ALT)8 were less certain than those in the broiler studies. Meanwhile, plasma urea nitrogen (PUN) and amino acid profiles are often used to estimate nutritional values of feed proteins.36 Apparently, it is more difficult and expensive to sample tissues from pigs than from chicks to study the metabolic regulation of the full production cycle that is likely very different between weanling and finishing pigs.37,38 Although various sources of NSPases are increasingly added into swine diets containing nonconventional ingredients,39,40 the general notion remains that these enzymes are more consistently effective41 in diets for poultry. Therefore, we conducted two parallel pig and broiler experiments to determine nutritional and metabolic values of a newly acquired defatted green microalgal (DGM, Desmodesmus sp., Cellana, Kailua-Kona, HI, USA) biomass from biofuel research that contained 31.2% crude protein. The objective of the pig experiment (I) was to determine impacts of including 10% DGM and (or) 0.06% protease on growth performance and plasma levels of TRAP, AKP, ALT, urea nitrogen, uric acid, and amino acids. The objective of the chick experiment (II) was to determine impacts of including 15% DGM and (or) 0.06% protease or NSP enzymes on growth performance, plasma levels of uric acid and amino acids, and production of signal proteins related to the mTOR pathway in liver and muscle.
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Table 1. Nutritional Composition of Defatted Green Marine Microalgal DGM (Desmodesmus sp.) Biomassa % “as is”
nutrient dry matter crude protein crude fat acid detergent fiber neutral detergent fiber ash calcium (Ca) phosphorus (P) sodium (Na) potassium (K) magnesium (Mg)
96.0 31.2 1.50 15.4 23.2 17.1 0.33 0.65 3.24 0.89 0.63
mineral iron (Fe) copper (Cu) manganese (Mn) zinc (Zn) molybdenum (Mo) selenium (Se)
mg/kg 1900 16.0 154 34.0 2.4 0.12
amino acid
% “as is”
alanine arginine aspartic acid cysteine glutamic acid glycine histidine hydroxylysine hydroxyproline isoleucine leucine lysine methionine ornithine phenylalanine proline serine taurine threonine tryptophan tyrosine valine
2.27 1.45 2.69 0.33 2.93 1.72 0.50 0.37 0.07 1.10 2.29 1.61 0.48 0.04 1.34 2.73 1.10 0.02 1.26 0.43 1.01 1.59
a
Proximate analysis was carried out by Dairy One Coop Inc. (Ithaca, NY, USA), and amino acids were determined by the Agricultural Experiment Station Chemical Laboratories at the University of Missouri (Columbia, MO, USA).
arrangement (n = 8 pigs per treatment). The experimental diet compositions are presented in Table 2. The inclusion rate of ProAct was based on our previous experiment.5 All diets were formulated to meet the nutrient requirements of weanling pigs.42 Pigs were housed in individual pens with a feeder and nipple water drinker. Appropriate feed was weighed out each morning to minimize the amount of spillage. Any spillage was collected and weighed the following morning prior to the addition of fresh feed. Individual pig weights were recorded biweekly for determination of body weight gain and feed conversion efficiency. Blood samples were collected biweekly from the anterior vena cava into heparinized tubes and kept on ice until analyses were completed on the same day. Plasma Biochemical Analyses. Blood samples were centrifuged at 1500g for 20 min to collect plasma. Plasma AKP activity was determined according to the method of Bowers and McComb.43 Plasma TRAP activity was determined according to the method of Lau et al.44 with a modification of pH to 5.8. Total plasma amino acid concentration was determined using a modified o-phthaldehyde (OPA) derivatization assay. Briefly, phthaldehyde complete reagent was added at equal volume to a plasma aliquot, vortexed, and read within 2 min at 340 nm on a 96-well plate reader (Biotek Instruments, Inc., Winooski, VT, USA). Plasma uric acid, urea nitrogen, and ALT were assayed using the above-described commercial kits. Broiler Experiment. Experimental Design, Growth Performance, and Sample Collection. Ross 308 broiler chicks were received at 1 day of age and assigned to one of six treatment groups in a randomized complete block design. A 2 (DGM: 0 or 15%, on an “as is” basis) × 3 (enzyme: none, ProAct, and NSPase) factorial arrangement was used within each block. Each treatment consisted of five battery cages (five birds/cage), and each cage was considered a replicate. The compositions of the control and the DGM-containing diets are shown in Table 3. Both protease and NSPase were incorporated at 0.06% on the basis of our previous experiments.5 Birds were housed within Petersime battery cages and allowed ad libitum access to feed and water. Birds were fed the starter diet from day 1 to day 21 and the grower diet from day 22 to day 42 (Table 3). All diets were formulated
MATERIALS AND METHODS
Chemicals. The nutrient composition of the DGM is presented in Table 1. Protease Ronozyme ProAct and NSPase mixture (50% Roxazyme G2, 40% Ronozyme A, and 10% Ronozyme WX) were obtained from DSM Nutritional Products Inc. (Parsippany, NJ, USA). Phthaldehyde complete solution reagent was purchased from SigmaAldrich (St. Louis, MO, USA). Kits for determining uric acid, urea nitrogen, and alanine aminotransferase (ALT) were obtained from Thermo Scientific, Inc. (Waltham, MA, USA). Rabbit-anti-β-actin (4967), rabbit-anti-mTOR (7C10), rabbit-antiphospho-mTOR (S2448), rabbit-anti-S6 (5G10), rabbit-antiphospho-S6 (S235/236), rabbit-anti-S6K1 (49D7), rabbit-antiphospho-S6K1 (T389), rabbitantiphospho-4E-BP1 (S65), and rabbit-anti-eIF4E (C46H6) monoclonal antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Pig Experiment. Experimental Design, Growth Performance, and Blood Sample Collection. All animal experimental procedures were approved by the Cornell University Institute Animal Care and Use Committee. Weanling Yorkshire × Hampshire × Landrace pigs (n = 32) were allotted into one of four treatment groups in a 2 (DGM: 0 or 10%, on an “as is” basis) × 2 (ProAct: 0 or 0.06%) factorial 9784
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Table 2. Nutritional Composition of Swine Experimental Diets ingredient
control
control + protease
DGMa
DGM + protease
corn (yellow) (%) soybean meal (48.5% CP) (%) DGM (%) corn oil (%) dicalcium phosphate (%) plasma protein (%) limestone (%) L-lysine HCl (%) DL-methionine (%) L-threonine (%) salt (%) vitamin/mineral mixb (%) choline (%) enzymec or corn starch (%) antibioticd (%) total (%)
64.0 28.1
64.0 28.1
62.3 20.0
62.3 20.0
0.00 2.00 1.50 0.00 0.70 0.25 0.01 0.10 0.18 0.20
0.00 2.00 1.50 0.00 0.70 0.25 0.01 0.10 0.18 0.20
10.0 3.00 1.50 1.20 0.70 0.35 0.01 0.10 0.00 0.20
10.0 3.00 1.50 1.20 0.70 0.35 0.01 0.10 0.00 0.20
0.10 2.36
0.10 2.36
0.10 0.06
0.10 0.06
0.50 100
0.50 100
0.50 100
0.50 100
3380 18.0 1.09 0.64 0.70
3380 18.0 1.09 0.64 0.70
3370 18.1 1.11 0.64 0.65
3370 18.1 1.11 0.64 0.65
nutritional composition ME (kcal/kg) protein (%) lysine methionine + cysteine threonine
Table 3. Nutritional Composition of Poultry Experimental Diets ingredient corn (yellow) (%) soybean meal (48.5% CP) (%) DGMa (%) corn oil (%) dicalcium phosphate (%) limestone (%) L-lysine HCl (%) DL-methionine (%) salt (%) mineralb/vitaminc premix (%) choline (%) enzymed or starch (%) total (%) nutritional composition ME (kcal/kg) protein (%) lysine (%) methionine + cysteine (%) threonine (%) Ca (%) available P (%)
control starter
DGMa starter
control grower
DGM grower
57.0 36.9
49.0 28.0
54.4 37.0
48.2 28.2
0.00 1.74 1.95 1.30 0.30 0.35 0.25 0.05
15.0 3.45 1.19 1.90 0.75 0.50 0.00 0.05
0.00 4.25 1.95 1.30 0.30 0.35 0.25 0.05
15.0 5.30 1.19 1.90 0.00 0.00 0.00 0.05
0.10 0.06 100
0.10 0.06 100
0.10 0.06 100
0.10 0.06 100
2970 21.8 1.49 1.07
3030 21.6 1.46 1.19
3100 21.6 1.50 1.10
3160 21.7 1.20 0.70
0.95 1.05 0.40
0.95 1.07 0.40
0.84 1.10 0.40
0.85 1.10 0.40
a
Defatted green microalgal (Desmodesmus sp.) biomass (Cellana, Kailua-Kona, HI, USA) that contained 31.2% crude protein. bProvided (in mg//kg of diet): CuSO4·5H2O, 31.42; KI, 0.046; FeSO4·7H2O, 224.0; MnSO4·H2O, 61.54; Na2SeO3, 0.13; ZnO, 43.56; Na2MoO4· 2H2O, 1.26. cProvided (in IU/kg of diet): vitamin A, 6500; vitamin D3, 3500; vitamin E, 25; and (in mg/kg of diet) riboflavin, 25; D-calcium pantothenate, 25; nicotinic acid, 150; cyanocobalamin, 0.011; choline chloride, 1250; biotin, 1.0; folic acid, 2.5; thiamin hydrochloride, 7.0; pyridoxine hydrochloride, 25.0; menadione sodium bisulfite, 5.0; and ethoxyquin, 66. dEach of the four experimental diets contains either starch (control) or protease (Ronozyme ProAct, DSM Nutritional Products Inc., Parsippany, NJ, USA) or NSPase (a mixture of 50% Roxazyme G2, 40% Ronozyme A, and 10% Ronozyme WX (DSM Nutritional Products Inc., Parsippany, NJ, USA). Protease activity was measured in PROT units, where 1 unit is defined as the amount of enzyme that releases 1 μmol of p-nitroaniline from 1 μM substrate (Suc-Ala-Ala-Pro-Phe-p-nitroaniline) per minute at pH 9.0 and 37 °C. Ronozyme WX activity was measured in FXU units, where the minimum activity of the endo-1,4-β-xylanase is 1000 FXU/g, where an FXU unit is the amount of enzyme that releases 7.8 μmol of reducing sugar (xylose equivalents) from azo-wheat arabinoxylan per minute at pH 6.0 and 50 °C. Roxazyme G2 contains a minimum of 8000 U/g of endo-1,3-β-glucanase, 18000 U/g of endo-1,3(4)-β-glucanase, and 26000 U/g of endo-1,4-β-xylanase, where 1 U is the amount of enzyme that liberates 0.1 μmol of glucose from carboxymethylcellulose, barley β-glucan, or oat spelt xylan per minute at pH 5.0 and 40 °C, for each enzyme, respectively. Ronozyme A (CT) consisted of 200 kilo-Novo α-amylase units and 350 fungal β-glucanase units/g of enzyme concentrate.
a
DGM, defatted green microalgal (Desmodesmus sp.) biomass (Cellana, Kailua-Kona, HI, USA) that contained 31.2% crude protein. b Vitamin and mineral premix supplied the following amounts (per kilogram of diet): vitamin A, 2200 IU; vitamin D3, 220 IU; vitamin E, 16 IU; vitamin K, 0.5 mg; biotin, 0.05 mg; choline, 0.5 g; folacin, 0.3 mg; niacin, 15 mg; pantothenic acid, 10 mg; riboflavin, 3.5 mg; thiamin, 1 mg; vitamin B6, 1.5 mg; vitamin B12, 17.5 μg; Cu, 6 mg; I, 0.14 mg; Mn, 4 mg; Zn 100 mg; Se, 0.3 mg; Mg, 0.4 mg; and Fe, 80 mg. cRonozyme ProAct (DSM Nutritional Products Inc., Parsippany, NJ, USA). Protease activity was measured in PROT units, where 1 unit is defined as the amount of enzyme that releases 1 μmol of pnitroaniline from 1 μM substrate (Suc-Ala-Ala-Pro-Phe-p-nitroaniline) per minute at pH 9.0 and 37 °C. dAntibiotic additive (Tylan 10) contained tylosin (as tylosin phosphate) at 22 g/kg. to meet the nutrient requirements for both periods45 except for no supplemental lysine or methionine in the DGM grower diets to test the maximal potential of the microalgal protein. Body weight and feed intake were recorded weekly by cage. Feed spillage was collected and weighed daily. Blood samples (∼1−3 mL, not exceeding 10% of blood volume) were collected at days 21 and 42 via right wing vein in heparinized needles. At day 42, one bird per cage was euthanized by CO2 asphyxiation and the liver and right pectoralis muscle were excised and immediately frozen for protein quantitation and Western blot analysis. Plasma Biochemical and Western Blot Analyses. Blood samples were centrifuged at 1500g for 20 min to collect plasma. Total plasma amino acid content and uric acid were assayed using the same methods described above under Pig Experiment. Frozen liver and muscle samples were ground in liquid nitrogen. Powdered tissue (10 mg) was then homogenized on ice with ice-cold buffer (80 μL) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 mM KCl, 50 mM NaF, 1 mM dithiothreitol, 0.5 mM sodium orthovanate, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM benzamidine. Homogenates were centrifuged at 1000g for 30 min at 4 °C, and
the supernatant was centrifuged at 10000g for 10 min at 4 °C. The final supernatants were aliquoted and stored at −80 °C. Tissue lysates (50 μg of protein) were subjected to sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using the appropriate antibody (Supplemental Table 1 in the Supporting Information). 9785
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Table 4. Effects of Defatted Green Microalgal Biomass (DGM) and Protease on Growth Performance of Pigsa diet:
control
enzyme:
none
body weight, kg day 0 9.49 day 14 18.0 day 28 28.6 body weight gain, g/day days 0−14 609 days 14−28 757 days 0−28 683 feed intake, g/day days 0−14 1040 days 14−28 1220 days 0−28 1130 gain/feed efficiency days 0−14 0.59 days 4−28 0.62 days 0−28 0.61
P value
DGM protease
none
protease
SEM
DGM
enzyme
interaction
9.63 17.2 26.2
9.66 17.5 26.7
9.95 18.4 28.1
0.15 0.26 0.45
0.47 0.51 0.96
0.55 0.94 0.60
0.73 0.14 0.03b
538 648 593
562 648 605
600 695 648
15.4 18.6 15.0
0.77 0.39 0.71
0.58 0.39 0.40
0.09 0.24 0.02b
1020 1190 1110
935 1100 1020
1070 1140 1110
26.7 33.7 27.1
0.58 0.25 0.32
0.22 0.90 0.51
0.17 0.66 0.36
0.54 0.55 0.54
0.61 0.60 0.60
0.56 0.61 0.59
0.02 0.02 0.01
0.44 0.52 0.45
0.10 0.34 0.15
1.00 0.17 0.45
a Data are expressed as mean (n = 7−8). Main effects of DGM, protease, and their interaction were analyzed by two-way ANOVA. bThe significant interactions indicate that the protease exerted negative effects in the control diet but positive effects in the DGM diet. However, Duncan’s multiplerange test showed no significant difference among the treatment means.
Table 5. Effects of Defatted Green Microalgal Biomass (DGM) and Protease on Plasma Biochemical Indicators of Pigsa diet: enzyme:
control none
tartrate-resistant acid phosphatase, day 0 168 day 14 202 day 28 145 alkaline phosphatase, U/L day 0 87.1 day 14 224 day 28 94.5 alanine aminotransferase, U/L day 0 16.8 day 14 14.0 day 28 16.0 uric acid, μmol/L day 0 19.2 day 14 30.8a day 28 36.0 urea nitrogen, mg/dL day 0 8.3 day 14 5.9 day 28 7.3a amino acid, μmol/mL day 0 9.7 day 14 17.8 day 28 27.4
P value
DGM protease
none
U/L 169 189 145
162 183 146
86.9 175 80.0
protease
SEM
DGM
enzyme
interaction
160 175 145
4.13 4.22 0.32
0.39 0.05b 0.51
0.99 0.92 0.34
0.86 0.47 0.53
86.9 222 122
82.3 179 111
1.45 11.6 6.88
0.44 0.95 0.03d
0.44 0.05c 0.36
0.47 0.92 0.66
14.2 17.0 16.1
13.5 13.5 16.9
15.8 16.6 15.0
0.77 0.67 0.68
0.54 0.73 0.89
0.88 0.02e 0.60
0.13 0.78 0.54
16.8 23.5ab 27.5
18.2 24.5ab 27.0
15.3 16.7b 28.3
0.67 1.74 1.01
0.40 0.07 0.38
0.06 0.03f 0.62
0.70 0.44 0.22
9.9 7.3 7.7a
8.8 5.5 6.2ab
7.5 5.2 4.6b
0.40 0.32 0.37
0.27 0.04g 0.01h
0.80 0.39 0.45
0.07 0.14 0.10
9.6 17.2 26.2
9.7 17.5 26.6
9.7 17.8 27.6
0.15 0.28 0.51
0.91 0.74 0.79
1.00 0.79 0.94
0.82 0.48 0.34
a
Data are expressed as mean (n = 7−8). Main effects of DGM, protease, and their interaction were analyzed by two-way ANOVA. Treatment means in the same row without a common letter differ, P < 0.05, according to Duncan’s multiple-range test. bDGM effect, P < 0.05: control, 196 vs DGM, 179 U/L, n = 14−16. cProtease effect, P < 0.05: none, 223 vs protease, 177 U/L, n = 14−16. dDGM effect, P < 0.03: control, 87 vs DGM, 117 U/L, n = 14−16. eProtease effect, P < 0.02: none, 13.8 vs protease, 16.8 U/L, n = 14−16. fProtease effect, P < 0.03: none, 27.7 vs protease, 20.1 μmol/L, n = 14−16. gDGM effect, P < 0.04: control, 6.60 vs DGM, 5.35 mg/dL, n = 14−16. hDGM effect, P < 0.01: control, 7.50 vs DGM, 5.40 mg/dL, n = 14−16. Statistical Analyses. Data were analyzed with the GLM procedure of PC-SAS 8.1 (SAS Institute, Cary, NC, USA). The overall main effects of DGM and the enzymes and their interactions were determined using two-way ANOVA (2 × 2 factorial for the pig experiment and 2 × 3 factorial for the chick experiment). Growth
parameters were analyzed as time-repeated measurements. Mean comparisons were conducted using Duncan’s multiple-range test. If there was an interaction between DGM and the enzyme(s), only conditional (within the same level of the other variable) treatment mean comparisons were considered. Due to the semiquantitative 9786
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Table 6. Effects of Defatted Green Microalgal Biomass (DGM) and Hydrolytic Enzymes on Growth Performance of Broilersa diet:
control
enzyme:
none
weight gain, g/week·chick days 0−21 221 days 22−42 493b days 0−42 384b feed intake, g/week·chick days 0−21 360a days 22−42 828b days 0−42 596 feed efficiency days 0−21 0.58b days 22−42 0.76a days 0−42 0.67bc
P value
DGM
protease
NSPase
none
protease
NSPase
SEM
DGM
enzyme
interaction
236 569a 436a
227 554ab 423ab
221 525ab 404ab
239 539ab 419ab
216 418b 338c
3.70 12.2 7.80
0.73 0.02 0.02
0.16 0.01 0.01
0.73 0.01b 0.01b
360a 910ab 635
356a 888ab 622
291b 1004a 647
358a 939ab 648
338ab 973ab 656
7.80 21.4 10.4
0.05c 0.03d 0.13
0.16 0.95 0.68
0.13 0.34 0.75
0.62b 0.82a 0.71ab
0.59b 0.82a 0.72ab
0.80a 0.76a 0.78a
0.68b 0.78a 0.73ab
0.65b 0.59b 0.62c
0.02 0.02 0.01
0.01 0.02 0.59
0.10 0.10 0.03
0.03b 0.03b 0.01b
a Data are expressed as mean (n = 5). Main effects of DGM, enzyme, and their interaction were analyzed by two-way ANOVA. Treatment means in the same row without a common letter differ, P < 0.05, according to Duncan’s multiple-range test. bWith significant interactions between DGM and enzyme, only conditional treatment mean comparisons should be considered: comparing the DGM effect at each of three enzyme treatments and the enzyme effect within each of the two diets (control and DGM). cDGM effect, P < 0.05: control, 359 vs DGM, 329 g/week·chick, n = 15. dDGM effect, P < 0.03: control, 875 vs DGM, 972 g/week·chick, n = 15.
Table 7. Effects of Defatted Green Microalgal Biomass (DGM) and Hydrolytic Enzymes on Plasma Biochemical Indicators of Broilersa diet: enzyme:
control none
protease
plasma amino acids, μmol/mL day 21 0.32b 0.66a day 42 3.72a 1.53b plasma uric acid, mmol/L day 21 0.25ab 0.21b day 42 0.51 0.57
P value
DGM NSPase
none
protease
NSPase
pooled SEM
DGM
enzyme
interaction
0.26b 2.12ab
0.40b 2.20ab
0.29b 2.36ab
0.38b 3.53a
0.04 0.25
0.01b 0.60
0.03 0.12
0.01c 0.01c
0.28ab 0.52
0.27ab 0.54
0.24b 0.48
0.31a 0.53
0.01 0.01
0.07 0.68
0.02 0.98
1.00 0.08
a Data are expressed as mean (n = 5). Main effects of DGM, enzyme, and their interaction were analyzed by two-way ANOVA. Treatment means in the same row without a common letter differ, P < 0.05, according to Duncan’s multiple-range test. bEnzyme effect: none, 0.26ab; protease, 0.24b; and NSPase, 0.30a mmol/L. Means without a common letter differ, P < 0.05, n = 10. cWith significant interactions between DGM and enzyme, only conditional treatment mean comparisons should be considered: comparing the DGM effect at each of three enzyme treatments and the enzyme effect within each of the two diets (control and DGM).
nature and limited sample size (n = 3) of the Western blot analyses, only selected treatment effects were directly compared with the respective controls using the t test. Data are expressed as means ± SEM, and P < 0.05 was considered statistically significant.
amino acid concentrations were not affected by either the DGM or the protease inclusion. Broiler Experiment. There were significant (P < 0.02) effects of DGM, enzyme, and their interaction on body weight gains of chicks during the grower period (days 22−42) and (or) the entire period (days 0−42) (Table 6). The gains for both periods were improved (P < 0.05) by the protease addition into the control diet, but the gain for the entire period decreased (P < 0.05) with NSPase addition to the DGM diet. Chicks fed the DGM diet had 9% lower (P < 0.05) feed intake than those fed the control diet during the starter period (days 0−21), but this difference was reversed with an 11% increase during the grower period. Thus, there was no difference in the overall feed intake between these two diets. There were significant interactions of DGM and enzyme (P < 0.03) on the gain/feed ratios for all of the assayed periods. During the starter period and the entire period, chicks fed the DGM diet without the enzyme supplementation had 38 and 16% greater (P < 0.05) gain/feed efficiency, respectively, than those fed the control diet. Supplementing the NSPase to the DGM diets decreased (P < 0.05) the gain/feed efficiencies for all three periods, whereas supplementing the protease to the DGM diets decreased (P < 0.05) the gain/feed efficiency for the starter period. The enzyme supplementations in the control diets
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RESULTS Pig Experiment. Neither the 10% DGM nor the 0.06% protease addition alone affected body weight, average daily gain, average daily feed intake, or feed efficiency at any time points of the 28 day experiment (Table 4). However, there was an interaction between DGM and protease on the day 28 body weight (P = 0.03) and overall average body weight gain for the entire 28 day experiment (P = 0.02), showing a beneficial trend of the protease to both measures in the presence of DGM, but an adverse effect in the control diet. The DGM inclusion decreased (P < 0.05) the day 14 plasma TRAP activity by 9%, increased (P < 0.03) the day 28 plasma AKP activity by 34%, decreased the day 14 plasma uric acid concentrations (P = 0.07) by 32%, and decreased the day 14 and 28 plasma urea nitrogen concentrations (P < 0.05) by 23 and 39%, respectively (Table 5). The protease addition decreased (P = 0.05) the day 14 plasma AKP activity by 26%, increased (P < 0.05) the day 14 plasma ALT activity by 22%, and decreased (P < 0.05) the day 14 plasma uric acid concentrations by 38%, respectively. Plasma 9787
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Figure 1. Effects of defatted green microalgal biomass (DGM) and hydrolytic enzymes on protein and phosphorylation levels of protein biosynthesis regulators in livers of broilers. Data are expressed as means ± SE (n = 3). Values are expressed as a ratio to β-actin and then normalized to the control. Blots are representative of three independent replicate gels. (∗) Significant effect of DGM versus control (P < 0.05), (∗∗) trending effect of DGM versus control (P < 0.10), (†) significant effect of protease versus treatment control (P < 0.05), (††) trending effect of protease versus treatment control (P < 0.10), (‡) significant effect of NSPase versus treatment control (P < 0.05), (‡‡) trending effect of NSPase versus treatment control (P < 0.10). eIF4E, eukaryotic initiation factor 4E; p70, p70 S6 kinase; S6, S6 ribosomal protein; pS6, phospho-S6 ribosomal protein; mTOR, mammalian target of rapamycin.
Figure 2. Effects of defatted green microalgal biomass (DGM) and hydrolytic enzymes on protein and phosphorylation levels of protein biosynthesis regulators in muscles of broilers. Data are expressed as means ± SE (n = 3). Values are expressed as a ratio to β-actin and then normalized to the control. Blots are representative of three independent replicate gels. (∗) Significant effect of DGM versus control (P < 0.05), (∗∗) trending effect of DGM versus control (P < 0.10), (†) significant effect of protease versus treatment control (P < 0.05), (††) trending effect of protease versus treatment control (P < 0.10), (‡) significant effect of NSPase versus treatment control (P < 0.05), (‡‡) trending effect of NSPase versus treatment control (P < 0.10). eIF4E, eukaryotic initiation factor 4E; p70, p70 S6 kinase; S6, S6 ribosomal protein; pS6, phospho-S6 ribosomal protein; mTOR, mammalian target of rapamycin.
showed no such effects on the gain/feed efficiency. Plasma amino acid concentrations were elevated (P < 0.05) and decreased (P < 0.05) by the addition of the protease into the control diet on days 21 and 42, respectively (Table 7). Plasma uric acid concentrations at day 21 appeared to be decreased by protease, but the only significant difference (P < 0.05) was seen between the two enzyme-supplemented DGM diets. The inclusion of DGM consistently decreased (P < 0.05− 0.1) all four proteins (eIF4E, mTOR, S6, and pS6) and the ratio of pS6/S6 in the liver compared with the control diet without supplemental enzymes (Figure 1). However, the same inclusion exhibited variable effects on the same four proteins, the ratio of pS6/S6, and p70 in the breast muscle (Figure 2). Supplementing NSPase into the control diet decreased hepatic mTOR (P < 0.05), S6 (P < 0.05), and pS6 (P < 0.1) and muscle eIF4E (P < 0.1), p70 (P < 0.05), and pS6 (P < 0.05), respectively. Supplementing NSPase into the DGM diet decreased (P < 0.05) both hepatic and muscle mTOR levels. Supplementing the protease into the control diet decreased (P < 0.05) hepatic pS6 level and elevated (P < 0.05) muscle eIF4E
level, whereas supplementing the same enzyme into the DGM diet decreased (P < 0.05) hepatic eIF4E and S6 levels and enhanced muscle pS6 (P < 0.1) and the ratio of pS6/S6 (P < 0.05).
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DISCUSSION The main finding of the pig experiment was that the 10% inclusion of DGM produced no appreciable adverse effects on growth performance of the pigs except for numerically a 7% decrease in the body weight at day 28 and a 13% decrease in overall daily body weight gain. Interestingly, these decreases were restored by the supplemental protease in the DGMcontaining diet. These data are in agreement with past reports that pigs tolerate moderate levels of various species of microalgal inclusion.6,8,46 As shown in our previous broiler,5 hen,12 and pig13 experiments, feeding weanling pigs with the 10% DGM diet did not alter their plasma ALT activity. Although pigs fed the DGM diet showed elevated plasma AKP activity at day 28 and decreased plasma TRAP activity at day 14, these changes were neither consistent nor sufficient to 9788
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suggest altered phosphorus nutrition status.47 Our previous studies5 also supported this notion. Apparently, digestibility and bioavailability of phosphorus in this and other sources of microalgal biomass need to be determined directly. Effects of supplemental DGM and protease on dietary protein digestion and utilization in pigs were assessed by the responses of plasma uric acid, urea nitrogen, and amino acid concentrations. Whereas the DGM diet did not alter plasma amino acid concentration, it lowered substantially plasma uric acid and urea nitrogen concentrations. These favorable decreases seem to indicate that pigs utilized their ingested N more efficiently from the DGM-containing diet than from the control diet. As plasma urea nitrogen is highly correlated with urinary nitrogen excretion rate,36 its decrease may result in attenuated nitrogen excretion to the environment from the swine production. It is worth mentioning that 1.2% plasma protein was added in the DGM-containing diet to match the protein and amino acid concentrations of the control diet. It is unknown if the superior plasma protein48,49 helped overcome any possible deficiencies of the DGM-containing diet. Meanwhile, supplementing 0.06% protease in both control and DGM-containing diet also improved plasma uric acid concentration. Along with the above-described positive effect on growth performance of pigs, these improvements suggest an enhanced proteolysis by the extrinsic protease in the DGMcontaining diet. Results of the broiler experiment showed a potential of the inclusion of 15% DGM to improve growth performance of chicks. Gain/feed efficiency of chicks fed the DGM-containing diet was indeed greater than that of chicks fed the control diet during the starter and the entire 42 day period. The decreased feed intake during the starter period followed by an elevated feed intake during the grower period in chicks fed the DGMcontaining diet, compared with those fed the control diet, might reflect an adaptation of the animals to the microalgal biomass. Notably, synthetic lysine and methionine were added into the DGM-containing diet only during the starter period. The DGM-containing grower diet was formulated to match only the protein but not the two limiting amino acids of the control diet, which was designed to determine the maximum potential of the DGM protein. Remarkably, chicks fed such grower diet displayed a growth performance similar to that of those fed the control diet. In comparison with the compromised body weight and body weight gain in the weanling pigs fed only 10% DGM, the tolerance of the broiler chicks to the inclusion of 15% DGM without exogenous lysine or methionine implied their better capacity to utilize DGM than shown by the weanling pigs. Nevertheless, thought should be given to possible confounding effects of the rather different lysine and methionine profiles between the control and DGM grower diets and (or) a slight extra methionine supplementation in the DGM starter diets. Along with the positive control diet as used in the present study, an additional negative control diet may be formulated to match the lower levels of lysine and methionine of the DGM grower diet for the future exploration of the full potential and mechanism of DGM in meeting protein and amino acid requirements for chicks and pigs. Supplementing protease and NSPase to the broiler diets produced mixed or opposite effects on their growth performance. Protease supplementation to the control diets led to an increased weight gain. However, the same supplementation to the DGM-containing diet, as in the case of 7.5% DFA supplementation in our previous research,5 showed little or
no consistent effects. Presumably, the DGM contained a high level of NSPs within the cell wall structure.50 Because NSPs possess antinutritive activity even at low levels (
