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Effects of dietary recombinant chlorella supplementation on growth performance, meat quality, blood characteristics, excreta microflora, and nutrient digestibility in broilers
Effects of dietary recombinant chlorella supplementation on growth performance, meat quality, blood characteristics, excreta microflora, and nutrient digestibility in broilers H. Choi, S. K. Jung, J. S. Kim, K-W. Kim, K. B. Oh, P-y. Lee, and S. J. Byun1
the control NC treatment group. Moreover, the blood contents of blood urea nitrogen (BUN), creatinine, and IgA in the broilers of the T2 treatment group were significantly increased by 28.12, 23.07, and 29.72%, respectively —more than those found in the broilers of the NC treatment group (P < 0.01). In contrast, the LDL/C in the blood from the animals in the T2 treatment group was significantly decreased by 23.23% — more than that in the blood from the NC broilers (P < 0.05). Based on these results, we suggest that the dietary supplementation of broilers with recombinant chlorella could improve their growth performance, increase the concentration of IgA and apparently metabolizable nitrogen in the blood, and decrease ammonia emissions. Therefore, our findings have important implications for the effect of recombinant chlorella supplementation through increasing the concentration of IgA and the level of metabolizable nitrogen.
ABSTRACT The use of chlorella as an immune stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported. Thus, the aim of the present study was to clarify the effect of recombinant chlorella supplementation on growth performance, meat quality, and the blood profile, excreta microflora, and nutrient digestibility in broilers. A total of 375 one-day-old ROSS 308 broilers (male and female) were allotted to 5 dietary treatments using 5 cages with 15 chicks per cage. Treatments were: 1) NC, basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) PC1, 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) T1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). The broilers in the T2 treatment groups showed higher body weight gain (BGW) by 2.55% (P < 0.01) and lower feed conversion ratio (FCR) by 2.75% (P < 0.05) compared with those fed
Key words: recombinant chlorella, growth performance, blood profiles, broilers 2016 Poultry Science 0:1–7 http://dx.doi.org/10.3382/ps/pew345
INTRODUCTION
of broiler chicken leukocytes in response to feed enrichment with chlorella (Kotrbacek et al., 1994). Moreover, a study was recently conducted to determine the effect of dietary supplementation with chlorella on the growth performance of broiler chickens (Kang et al., 2013). However, the mechanisms through which recombinant chlorella positively affects broilers have not yet been investigated in detail. The 3D8 single-chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can bind to and hydrolyze nucleic acids without sequence specificity (Kim et al., 2006). Production and local delivery of genetically engineered antibody fragments by bacteria in the gastrointestinal tract could provide efficient therapy at a low cost (Pant et al., 2006). In this study, we investigated recombinant chlorella as a delivery system for 3D8 scFv as an immune stimulant. Thus, the aim of the present study was to clarify the effect of recombinant chlorella supplementation on growth performance, meat quality, blood profile, excreta microflora, and nutrient digestibility in broilers.
The beneficial effects of prebiotics are established, and prebiotics are widely used as an alternative to antibiotics in swine and poultry (Zhao et al., 2013; Zhao et al., 2016). Researchers have demonstrated that the dietary supplementation of broiler chickens with prebiotics leads to improved performance through enhancing growth performance and stimulation of the immune system (Vicente et al., 2008; Patel et al., 2015). The use of chlorella as an immune stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported (Yan et al., 2012). In addition, it has previously been shown that there is a significant increase in the phagocytic activity
C 2016 Poultry Science Association Inc. Received June 2, 2016. Accepted August 11, 2016. 1 Corresponding author: [email protected]
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Animal Biotechnology Division, National Institute of Animal Science, RDA, 1500, Kongjwipatjwi-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea
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CHOI ET AL. Table 1. Composition of basal broiler chicken diets (as-fedbasis).1 Items
Finisher
56.95 29.25 4.44 3.61 0.91 2.07 0.32 0.33 1.68 0.18 0.10 0.06 0.10
60.44 25.33 3.83 5.00 1.02 1.93 0.37 0.37 1.28 0.18 0.10 0.05 0.10
3,050 21.00 1.40 0.90 0.71
3,200 19.00 1.20 0.90 0.66
1 Provided starter diets from wk 1 to 3 and finisher diets from w 4 to 5. 2 Provided per kg of complete diet: vitamin A, 11,025 IU; vitamin D3, 1,103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, 4 mg; pantothenic acid, 29 mg; choline, 166 mg; and vitamin B12, 33 μ g. 3 Provided per kg of complete diet: Cu, 12 mg; Zn, 85 mg; Mn, 8 mg; I, 0.28 mg; and Se, 0.15 mg.
MATERIALS AND METHODS Animals, Diets, and Design This test was performed on one-day-old ROSS 308 broilers (male and female). In the 5-week trial, a total of 375 broiler chicks with an average body weight of 45 ± 0.39 g were allotted to one of 5 groups and given one of the experimental diets according to their initial body weight. The test design was as follows: 1) negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). The use of chlorella as an antiviral agent constitutes the production and local delivery of genetically engineered 3D8 fragments by bacteria in the gastrointestinal tract. Five replicates were performed per treatment, with 5 and 15 per iteration, in a completely random arrangement. The test was conducted on the Dankook University research farm, with the tested feed corn being formulated according to National Research Council (NRC) nutrient requirements, and soybean meal diets were provided (Table 1). All birds were housed in stainless-steel cages (1.75 m × 1.55 m) with concrete floors covered with clean rice bran, and continuous light was provided. The temperature of the room was maintained at 33 ± 1o C for the first 3 d and then decreased to 24o C throughout the rest of the experiment. The diets were fed in 2 phases: a starter phase, which was administered for the first 3
Feeding, Sampling, and Measurements Weight gain from the beginning of the experiment was determined at 3 wk and at the end of the experiment (5 wk). The feed intake was calculated by measuring the weight of the feed, and the feed conversion ratio was calculated by dividing this value by the weight gain. To determine when the experiment should be terminated (5 wk), 10 animals per treatment were randomly selected for measurements, and the tibia, breast, liver, F sac, abdominal fat, and proximal splenic weights were then measured at slaughter, after which the percentage of the live weight was calculated. The pH was measured with a pH meter (Testo 205, Testo AG, Lenzkirch, Germany), and a meat color colorimeter (CR-410, Minolta Co., Osaka, Japan) was used to measure each breast sample in the second iteration. At this time, the standard was L ∗ = 89.2, a ∗ = 0.921, b ∗ = 0.783. Storage loss (drip loss) was measured after holding the samples in polyethylene bags in a 4◦ C cold room for 7 d, with each sample being shaped to a constant shape with a thickness of 2 cm at one d, 3 d, and 5 d; finally, the loss generated after 7 d was measured. Blood was collected through a random selection process of 10 animals from each treatment at the end of the experiment (5 wk). Intravenous (jugular) blood was collected in a 2 mL K3EDTA tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and was then processed using an automatic blood analyzer (ADVIA 120, Bayer, NY, USA) to determine WBC, RBC, lymphocyte, heterophil, basophil, and platelet counts. For serum biochemical tests, blood (5 mL) was collected in vacuum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and then centrifuged at 4◦ C at 4,000 rpm for 15 min to obtain serum for analysis. A separate sample for the determination of serum total cholesterol was obtained, which was then analyzed using an HDL/C, LDL/C, triglyceride, AST, ALT, BUN, creatinine, total protein, albumin, globulin, IgG, IgM, and IgA content nephelometer (Behring, Germany). After harvesting for one min at the termination of the experimental treatment (at 5 wk), the obtained sample was suspended in sterile saline and homogenized. The viable cell count was then determined by dilution in steps from 10−3 to 10−6 . The experimental treatment of the broilers was performed in a matter of min on the broilers’ Lactobacillus and E. coli bacteria. The number of bacteria was measured using Lactobacillus MRS agar and E. coli MacConkey agar (Difco Laboratories, Detroit, MI) at 38 to 37◦ C. The Hennessey gas composition of the broilers was analyzed by obtaining a minute sample of excretory ammonia, hydrogen sulfide, and total mercaptan at the
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Ingredients (%) Corn Soybean meal, 45% CP Corn gluten meal, 60% CP Tallow Limestone Dicalcium phosphate Salt DL-Methionine L-Lysine-HCI L-Threonine Choline chloride Vitamin premix2 Trace mineral premix3 Calculated composition Metabolizable energy, kcal/kg Crude protein, % Lysine, % % Calcium, % Total Phosphorus, %
Starter
wk, and a finisher phase, which was administered during wk 4 and 5. The chicks were given free access to water and mash feed throughout the entire experiment.
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EFFECT OF RECOMBINANT CHLORELLA IN CHICKEN Table 2. Effect of dietary supplementation with sea plants on the growth performance of broilers.1,2 P-value
Items
NC
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
728 1045 1.435
745 1020 1.369
734 1032 1.405
741 1034 1.396
14 8 0.033
0.2252 0.1179 0.1280
0.3141 0.6465 0.3148
0.4091 0.0533 0.1838
0.7660 0.8954 0.6505
0.7509 0.3161 0.7235
0.8271 0.2532 0.5782
1011a,b 1502 1.486
1026a 1490 1.452
1016a,b 1495 1.471
1023a,b 1495 1.462
8 21 0.022
0.0286 0.6199 0.0908
0.0528 0.7320 0.1569
0.1929 0.7119 0.2907
0.5848 0.9839 0.7429
0.6322 0.8140 0.6623
0.7578 0.8771 0.7575
1739b,c 2546 1.464a,b
1771a 2510 1.417c
1751a,b 2527 1.443a,b,c
1763a,b 2529 1.434b,c
9 20 0.013
0.0010 0.2269 0.0032
0.0032 0.5736 0.0218
0.0248 0.2097 0.0208
0.3469 0.9380 0.6078
0.3620 0.4917 0.2824
0.5771 0.5049 0.3677
1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean from 5 replicates with 10 chicks/replicate (total of 250 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b,c Means in the same row with different superscripts differ (P < 0.05).
Table 3. Effect of dietary supplementation with sea plants on the carcass quality of broilers.1,2 P-value
Items
NC
pH value 5.47 Breast muscle color Lightness (L∗ ) 52.78 10.52 Redness (a∗ ) ∗ Yellowness (b ) 8.55 WHC, % 56.87 Drip loss, % d1 5.53 d3 9.04 d5 11.32 d7 13.23a Relative organ weight, % Breast muscle 19.13b Liver 3.02 Bursa of Fabricius 0.15 Abdominal fat 1.74 Spleen 0.11 Gizzard 1.24
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
5.45
5.42
5.37
5.41
0.04
0.3869
0.2632
0.6441
0.4721
0.1547
0.7957
52.76 10.69 8.51 56.82
52.46 10.46 8.43 56.64
52.91 10.28 8.58 56.91
52.77 10.34 8.27 56.83
0.68 0.18 0.21 0.67
0.7367 0.8195 0.6913 0.8113
0.9901 0.5044 0.3525 0.9733
0.7547 0.3923 0.7715 0.8480
0.8819 0.7930 0.2919 0.9399
0.8729 0.1241 0.8152 0.9290
0.7460 0.6594 0.5915 0.8373
5.56 8.93 11.16 13.08a,b
5.51 8.71 11.20 13.03a,b
5.47 8.81 11.16 13.00a,b
5.48 8.82 11.05 12.93b
0.40 0.14 0.14 0.09
0.9735 0.1157 0.5415 0.1531
0.9316 0.3029 0.1854 0.0304
0.9275 0.2853 0.8536 0.7215
0.9832 0.9259 0.5871 0.5881
0.8693 0.5448 1.0000 0.5481
0.9581 0.5743 0.4678 0.4327
19.50a,b 3.04 0.14 1.70 0.11 1.20
20.07a 2.94 0.15 1.71 0.12 1.27
19.69a,b 2.97 0.14 1.68 0.11 1.22
19.72a,b 3.02 0.13 1.72 0.12 1.25
0.68 0.11 0.01 0.12 0.01 0.04
0.0052 0.6135 0.5484 0.8569 0.3300 0.4978
0.0666 0.9896 0.1976 0.8758 0.2739 0.8542
0.0813 0.5219 0.4245 0.9186 0.5410 0.1574
0.9095 0.7361 0.4245 0.8381 0.7133 0.5821
0.5558 0.6456 0.7637 0.9377 0.7133 0.6998
0.2855 0.6045 0.0630 0.9808 0.9024 0.6203
1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean from 5 replicates with 10 chicks/replicate (total of 250 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b Means in the same row with different superscripts differ (P < 0.05).
same time at the end of the test (5 wk) for each treatment group. The ammonia, hydrogen sulfide, and total mercaptan contents were measured while holding the samples at room temperature for 5 d; 300 g of the sample was placed in a sealed plastic container with a 2,600 mL volume for 24 h of fermentation in a Gastec (Model GV - 100, Gastec Corp, Kanagawa, Japan) with ammonia and hydrogen sulfide, and measurements were
performed using a total detecting tube (No. 3 L, No. 4LT and No. 70 L, Gastec, Japan). Nutrient digestibility was assessed at the end of the experiment (5 wk), after 6 d in 0.2%, by adding fodder chromium oxide (Cr2 O3 ) as an indicator of water. The sample was collected after 72 h, and after drying in a dryer at 60◦ C, it was subjected to analysis via pulverization in a Willey mill. Cr was then mixed with
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d 1-21 BWG, g 720 FI, g 1040 FCR 1.444 d 21 to 35 BWG, g 999b FI, g 1506 FCR 1.508 Overall BWG, g 1719c FI, g 2545 FCR 1.481a
PC1
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CHOI ET AL. Table 4. Effect of dietary supplementation with sea plants on the blood profile of broilers.1,2 P-value
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
WBC, 103 /μ RBC, 106 /μ Lymphocyte, % Heterophil, % Basophil, % Platelet, 109 / Total Cholesterol, mg/dL HDL/C, mg/dL LDL/C, mg/dL Triglyceride, mg/dL AST, U/L ALT, U/L BUN, mg/dL Creatinine, mg/dL Total Protein, g/dL Albumin, g/dL Globulin, g/dL IgG, g/dL IgM, g/dL IgA, g/dL
22.9 2.81 61.8 31.3 2.87 21.78 111b 82 26.9a 69 187.2 14.2a 3.2b 0.26b 2.7 1.2 1.5 234 154 37b
23.1 2.91 63.3 31.9 2.93 21.97 115b 83 24.7a,b 67 183.7 12.8a,b 3.6a,b 0.28a,b 3.1 1.3 1.9 239 155 40a,b
23.9 2.94 64.1 32.4 2.98 22.02 120a,b 87 22.7a,b 66 183.0 12.3a,b 3.6a,b 0.29a,b 3.2 1.4 1.8 239 158 44a,b
24.0 3.10 65.8 33.1 3.04 22.08 121a,b 90 22.2a,b 66 181.7 11.5b 4.0a 0.31a 3.1 1.4 1.7 244 164 45a,b
24.5 3.22 65.9 33.8 3.10 22.14 126a 95 21.8b 66 180.1 10.9b 4.1a 0.32a 3.3 1.3 2.0 248 163 48a
1.4 0.2 2.8 1.30 0.22 0.54 4 6 2 7 5.6 0.6 0.2 0.01 0.2 0.1 0.2 13 4 3
0.6117 0.6027 0.5468 0.5738 0.7060 0.7597 0.0910 0.5256 0.0681 0.7560 0.5950 0.0406 0.1584 0.1542 0.0890 0.2426 0.2955 0.7865 0.4958 0.0771
0.4386 0.1134 0.2904 0.1862 0.4558 0.6424 0.0056 0.1299 0.0284 0.7170 0.3722 0.0008 0.0024 0.0079 0.0533 0.7933 0.0834 0.4538 0.1236 0.0097
0.7050 0.8978 0.8265 0.8126 0.8630 0.9522 0.3079 0.6522 0.3764 0.9506 0.9271 0.5949 0.8166 0.5322 0.8873 0.3616 0.7340 1.0000 0.5971 0.3688
0.8303 0.6299 0.9695 0.7008 0.8656 0.9419 0.3551 0.5638 0.8588 0.9669 0.8382 0.5573 0.6710 0.8097 0.5480 0.3616 0.2955 0.8079 0.8376 0.5531
0.6648 0.4637 0.5218 0.5318 0.7084 0.8840 0.1993 0.4400 0.2703 0.9422 0.8012 0.1488 0.1108 0.0892 0.8317 0.3616 0.4978 0.8121 0.1146 0.2299
0.7878 0.2798 0.6450 0.4405 0.7108 0.8738 0.2342 0.3694 0.6893 0.9587 0.7156 0.1344 0.0756 0.1828 0.8042 0.3616 0.4767 0.6310 0.3862 0.3688
1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean from five replicates with 10 chicks/replicate (total of 250 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b Means in the same row with different superscripts differ (P < 0.05).
Table 5. Effect of dietary supplementation with sea plants on the excreta microflora of broilers.1,2 P-value Items, log10 cfu/g
NC
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
Lactobacillus E.coil
7.57 6.49
7.64 6.45
7.61 6.41
7.61 6.39
7.63 6.40
0.03 0.07
0.3973 0.4144
0.1964 0.3747
0.5213 0.6484
0.6485 0.9009
0.5213 0.5144
0.6485 0.9421
1 Abbreviation: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean of 5 replicates with 15 chicks/replicate (total of 375 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means.
normal water, and the feed was analyzed according to the AOAC method.
RESULTS Growth Performance
Statistical Analysis All data were analyzed in SAS (SAS Institute Inc., Cary, North Carolina, USA) using the General Linear Model procedure for analysis of variance. Duncan’s multiple range tests (Duncan, 1955) were employed to process the average difference, and <0.05 was considered statistically significant. To evaluate the effect on the nutrient and additive contents, we used contrast statistical methods.
At 21 to 35 d old, the broilers fed the PC2 treatment group (P < 0.05) exhibited higher BWG than in the primary NC treatment group (Table 2), which showed increased BWG by 2.70%. During the total experimental period, the weight of the broilers in the PC2 and T2 treatment groups (P < 0.01) was significantly higher than that of those in the NC treatment group, which showed increased BWG by 3.00 and 2.55%, respectively. The induced BWG of the broilers was reflected in increased FCR, with the broilers in the PC2 (P < 0.05) and T2 treatment groups (P < 0.05) exhibiting FCR values that were 4.32 and 2.76% lower,
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NC
Items
5
EFFECT OF RECOMBINANT CHLORELLA IN CHICKEN Table 6. Effect of dietary supplementation with sea plants on excreta noxious gas emission in broilers.1,2 P-value
Items, ppm NH3 H2 S R.SH
NC
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
31.10a 2.37 1.47
30.78a,b 2.36 1.32
30.56a,b 2.31 1.45
29.35b,c 2.11 1.52
28.89c 2.27 1.25
0.55 0.22 0.12
0.4920 0.8449 0.9072
0.0074 0.7444 0.2050
0.7789 0.8705 0.4506
0.5580 0.6024 0.1219
0.0743 0.4169 0.2483
0.0386 0.8962 0.2483
Table 7. Effect of dietary supplementation with sea plants on nutrient digestibility in broilers.1,2 P-value
Items, % Dry matter Nitrogen Energy
NC
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
69.74 66.06 72.85b
69.90 66.42 73.00a,b
70.21 66.71 73.60a
69.98 66.32 73.20a,b
70.11 66.57 73.35a,b
0.21 1.16 0.23
0.1194 0.6983 0.0270
0.2176 0.7591 0.1296
0.3035 0.8633 0.0712
0.6702 0.8787 0.6304
0.7871 0.9508 0.5429
0.7325 0.9353 0.4460
1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean of 5 replicates with 15 chicks/replicate (total of 375 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b Means in the same row with different superscripts differ (P < 0.05).
respectively, compared with those in the NC treatment group.
Meat Quality At 7 d olds, the T2 treatment group (P < 0.05) resulted in significantly lower drip loss in the breast than the NC treatment group (Table 3), which showed decreased drip loss by 2.26%. The breast organ weight in the PC2 treatment group (P < 0.01) was significantly greater than that in the NC treatment group in the proportions of 19.13 and 20.07 for NC and PC2 treatment groups, respectively. No significant difference was found among the treatments in terms of pH, water-holding capacity, and meat color (P > 0.05).
23.07, and 29.72%, respectively, whereas ALT concentration (P < 0.01) decreased by 23.23%, compared with the NC treatment group.
Excreta Microbial Shedding and Excreta Noxious Gas Emission No significant difference in the excreta microflora composition (P > 0.05) was found among the treatments involving Lactobacillus and E. coli (Table 5). The amount of fecal ammonia generated with the T2 treatment group was significantly lower by 1.73 and 7.10% compared with the PC2 (P < 0.05) and NC treatment groups (P < 0.01), respectively (Table 6).
Nutrient Digestibility Blood Profile The total cholesterol content in the blood of the broilers in the T2 treatment was significantly higher (P < 0.01) than that found in the broilers in the NC treatment group (Table 4), which showed increased total cholesterol content by 13.51%. However, the LDL content was significantly lower in the broilers in the T2 treatment group (P < 0.05) than in those in the NC group, which showed decreased LDL content by 18.95%. The T2 treatment group increased the BUN, creatinine, and IgA concentration (P < 0.01) by 28.12,
The PC2 treatment (P < 0.05) led to a significantly higher energy digestibility than the NC treatment, which showed increased energy digestibility by 1.29% (Table 7). The dry matter digestibility and nitrogen digestibility presented no significant differences among the treatments (P > 0.05).
DISCUSSION Several researchers have reported that the use of chlorella as a prebiotic has a positive effect on the
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1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean of 5 replicates with 15 chicks/replicate (total of 375 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b,c Means in the same row with different superscripts differ (P < 0.05).
6
CHOI ET AL.
ACKNOWLEDGMENTS This study was supported by the 2015–2019 Postdoctoral Fellowship Program of National Insti-
tute of Animal Science, Rural Development Administration, Republic of Korea. This work was conducted with the support of the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01020101)” of the Rural Development Administration, Republic of Korea.
REFERENCES Baltazar, M. T., R. J. Dinis-Oliveira, A. Martins, L. Bastos Mde, J. A. Duarte, L. Guilhermino, and F. Carvalho. 2014. Lysine acetylsalicylate increases the safety of a paraquat formulation to freshwater primary producers: A case study with the microalga Chlorella vulgaris. Aquat. Toxicol. 146:137–143. Berri, C., J. Besnard, and C. Relandeau. 2008. Increasing dietary lysine increases final pH and decreases drip loss of broiler breast meat. Poult. Sci. 87:480–484. Cho, J. H., and I. H. Kim. 2014. Effects of lactulose supplementation on performance, blood profiles, excreta microbial shedding of Lactobacillus and Escherichia coli, relative organ weight and excreta noxious gas contents in broilers. J. Anim. Physiol. Anim. Nutr. (Berl.) 98:424–430. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics. 11:1–42. Kang, H., H. Salim, N. Akter, D. Kim, J. Kim, H. Bang, M. Kim, J. Na, J. Hwangbo, and H. Choi. 2013. Effect of various forms of dietary Chlorella supplementation on growth performance, immune characteristics, and intestinal microflora population of broiler chickens. J. Appl. Poult. Res. 22:100–108. Kim, Y. R., J. S. Kim, S. H. Lee, W. R. Lee, J. N. Sohn, Y. C. Chung, H. K. Shim, S. C. Lee, M. H. Kwon, and Y. S. Kim. 2006. Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity. J. Biol. Chem. 281:15287–15295. Kotrb´ aˇcek, V., J. Doubek, and J. Doucha. 2015. The chlorococcalean alga Chlorella in animal nutrition: A review. J. Appl. Phycol. 27:2173–2180. Kotrbacek, V., R. Halouzka, V. Jurajda, Z. Knotkova, and J. Filka. 1994. Enhancement of defense-mechanisms in broilers after administration of biological feed supplements. Vet. Med. (Praha) 39:321–328. Nakano, S., H. Takekoshi, and M. Nakano. 2007. Chlorella (Chlorella pyrenoidosa) supplementation decreases dioxin and increases immunoglobulin a concentrations in breast milk. J. Med. Food. 10:134–142. Pant, N., A. Hultberg, Y. Zhao, L. Svensson, Q. Pan-Hammarstrom, K. Johansen, P. H. Pouwels, F. M. Ruggeri, P. Hermans, L. Frenken, T. Boren, H. Marcotte, and L. Hammarstrom. 2006. Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirusinduced diarrhea. J. Infect. Dis. 194:1580–1588. Patel, S., R. Shukla, and A. Goyal. 2015. Probiotics in valorization of innate immunity across various animal models. J. Funct. Foods. 14:549–561. Pugh, N., S. A. Ross, H. N. ElSohly, M. A. ElSohly, and D. S. Pasco. 2001. Isolation of three high molecular weight polysaccharide preparations with potent immunostimulatory activity from Spirulina platensis, aphanizomenon flos-aquae and Chlorella pyrenoidosa. Planta Med. 67:737–742. Song, Z., X. Dong, J. Tong, and Z. Wang. 2012. Effects of waste vinegar residue on nutrient digestibility and nitrogen balance in laying hens. Livest. Sci. 150:67–73. Vicente, J. L., A. Torres-Rodriguez, S. E. Higgins, C. Pixley, G. Tellez, A. M. Donoghue, and B. M. Hargis. 2008. Effect of a selected Lactobacillus spp.-based probiotic on Salmonella enterica serovar enteritidis-infected broiler chicks. Avian. Dis. 52:143–146. Wang, J. P., Z. F. Zhang, L. Yan, and I. H. Kim. 2016. Effects of dietary supplementation of emulsifier and carbohydrase on the growth performance, serum cholesterol and breast meat fatty acids profile of broiler chickens. Anim. Sci. J. 87:250–256.
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growth performance and immune characteristics of chickens and pigs (Yan, Lim and Kim, 2012; Kang et al., 2013). In the present study, the broilers in the PC2 and T2 treatment groups exhibited a greater BWG and FCR than those in the control NC treatment group (Table 1). It has been suggested that prebiotics generally modulate the gut environment by increasing beneficial microorganisms and inhibiting the proliferation of pathogens in the intestine (Cho and Kim, 2014; Kotrb´ aˇcek et al., 2015; Wang et al., 2016). The drip loss from the breast measured under the T2 treatment was significantly lower than for the NC treatment (Table 2). In contrast, the PC2 treatment resulted in a significantly greater breast organ weight than the NC treatment (Table 2). It was previously suggested that increasing dietary lysine decreases drip loss in broiler breast meat (Berri et al., 2008). Therefore, we hypothesized that the reason for the decreased drip loss in relation to broiler breast meat performance was likely due to the very large amount of lysine in chlorella proteins (Baltazar et al., 2014). The blood contents of BUN, creatinine, and IgA in the T2 treatment group were significantly higher than in the NC treatment group (Table 3). In contrast, LDL/C in the blood of the animals in the T2 treatment group was significantly lower than in NC (Table 3). A previous study found that dietary chlorella supplementation significantly increased the IgA concentration in the breast milk of pregnant women (Nakano et al., 2007). Additionally, previous reports have shown that dietary fiber enhances IgA production in the mesenteric lymphocytes (Yamada et al., 2003). Therefore, we hypothesized that the reason for the increased IgA concentration in the plasma of the broiler chickens was likely the increased production of B cells due to stimulating immunoglobulin in the autoassociated lymphoid tissue (Pugh et al., 2001; Kang et al., 2013). The amount of fecal ammonia generated in the T2 treatment was significantly lower than that in the NC treatment (Table 5). A previous study showed that supplementation with prebiotics could reduce fecal ammonia gas emission in broilers (Cho and Kim, 2014). It has been suggested that the N from ammonia is generally derived from the fermentation of unabsorbed N entering the large intestine (Song et al., 2012). Therefore, the reason for the decreased NH3 emission from the broilers fed chlorella was likely the increase in apparently metabolizable N compared with the control group. In conclusion, the results of this study indicated that dietary supplementation of broilers with recombinant chlorella could improve growth performance and increase the concentration of IgA and apparently metabolizable nitrogen in the blood, in addition to decreasing ammonia emissions.
EFFECT OF RECOMBINANT CHLORELLA IN CHICKEN Yamada, K., Y. Tokunaga, A. Ikeda, K. Ohkura, S. Kaku-Ohkura, S. Mamiya, B. O. Lim, and H. Tachibana. 2003. Effect of dietary fiber on the lipid metabolism and immune function of aged Sprague-Dawley rats. Biosci. Biotechnol. Biochem. 67:429–433. Yan, L., S. U. Lim, and I. H. Kim. 2012. Effect of fermented chlorella supplementation on growth performance, nutrient digestibility, blood characteristics, fecal microbial and fecal noxious gas content in growing pigs. Asian-Australas. J. Anim. Sci. 25:1742–1747.
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Zhao, P. Y., H. L. Li, M. Mohammadi, and I. H. Kim. 2016. Effect of dietary lactulose supplementation on growth performance, nutrient digestibility, meat quality, relative organ weight, and excreta microflora in broilers. Poult. Sci. 95:84–89. Zhao, P. Y., J. P. Wang, and I. H. Kim. 2013. Effect of dietary levan fructan supplementation on growth performance, meat quality, relative organ weight, cecal microflora, and excreta noxious gas emission in broilers. J. Anim. Sci. 91:5287–5293.
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the control NC treatment group. Moreover, the blood contents of blood urea nitrogen (BUN), creatinine, and IgA in the broilers of the T2 treatment group were significantly increased by 28.12, 23.07, and 29.72%, respectively —more than those found in the broilers of the NC treatment group (P < 0.01). In contrast, the LDL/C in the blood from the animals in the T2 treatment group was significantly decreased by 23.23% — more than that in the blood from the NC broilers (P < 0.05). Based on these results, we suggest that the dietary supplementation of broilers with recombinant chlorella could improve their growth performance, increase the concentration of IgA and apparently metabolizable nitrogen in the blood, and decrease ammonia emissions. Therefore, our findings have important implications for the effect of recombinant chlorella supplementation through increasing the concentration of IgA and the level of metabolizable nitrogen.
ABSTRACT The use of chlorella as an immune stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported. Thus, the aim of the present study was to clarify the effect of recombinant chlorella supplementation on growth performance, meat quality, and the blood profile, excreta microflora, and nutrient digestibility in broilers. A total of 375 one-day-old ROSS 308 broilers (male and female) were allotted to 5 dietary treatments using 5 cages with 15 chicks per cage. Treatments were: 1) NC, basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) PC1, 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) T1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). The broilers in the T2 treatment groups showed higher body weight gain (BGW) by 2.55% (P < 0.01) and lower feed conversion ratio (FCR) by 2.75% (P < 0.05) compared with those fed
Key words: recombinant chlorella, growth performance, blood profiles, broilers 2016 Poultry Science 0:1–7 http://dx.doi.org/10.3382/ps/pew345
INTRODUCTION
of broiler chicken leukocytes in response to feed enrichment with chlorella (Kotrbacek et al., 1994). Moreover, a study was recently conducted to determine the effect of dietary supplementation with chlorella on the growth performance of broiler chickens (Kang et al., 2013). However, the mechanisms through which recombinant chlorella positively affects broilers have not yet been investigated in detail. The 3D8 single-chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can bind to and hydrolyze nucleic acids without sequence specificity (Kim et al., 2006). Production and local delivery of genetically engineered antibody fragments by bacteria in the gastrointestinal tract could provide efficient therapy at a low cost (Pant et al., 2006). In this study, we investigated recombinant chlorella as a delivery system for 3D8 scFv as an immune stimulant. Thus, the aim of the present study was to clarify the effect of recombinant chlorella supplementation on growth performance, meat quality, blood profile, excreta microflora, and nutrient digestibility in broilers.
The beneficial effects of prebiotics are established, and prebiotics are widely used as an alternative to antibiotics in swine and poultry (Zhao et al., 2013; Zhao et al., 2016). Researchers have demonstrated that the dietary supplementation of broiler chickens with prebiotics leads to improved performance through enhancing growth performance and stimulation of the immune system (Vicente et al., 2008; Patel et al., 2015). The use of chlorella as an immune stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported (Yan et al., 2012). In addition, it has previously been shown that there is a significant increase in the phagocytic activity
C 2016 Poultry Science Association Inc. Received June 2, 2016. Accepted August 11, 2016. 1 Corresponding author: [email protected]
1
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Animal Biotechnology Division, National Institute of Animal Science, RDA, 1500, Kongjwipatjwi-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea
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CHOI ET AL. Table 1. Composition of basal broiler chicken diets (as-fedbasis).1 Items
Finisher
56.95 29.25 4.44 3.61 0.91 2.07 0.32 0.33 1.68 0.18 0.10 0.06 0.10
60.44 25.33 3.83 5.00 1.02 1.93 0.37 0.37 1.28 0.18 0.10 0.05 0.10
3,050 21.00 1.40 0.90 0.71
3,200 19.00 1.20 0.90 0.66
1 Provided starter diets from wk 1 to 3 and finisher diets from w 4 to 5. 2 Provided per kg of complete diet: vitamin A, 11,025 IU; vitamin D3, 1,103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, 4 mg; pantothenic acid, 29 mg; choline, 166 mg; and vitamin B12, 33 μ g. 3 Provided per kg of complete diet: Cu, 12 mg; Zn, 85 mg; Mn, 8 mg; I, 0.28 mg; and Se, 0.15 mg.
MATERIALS AND METHODS Animals, Diets, and Design This test was performed on one-day-old ROSS 308 broilers (male and female). In the 5-week trial, a total of 375 broiler chicks with an average body weight of 45 ± 0.39 g were allotted to one of 5 groups and given one of the experimental diets according to their initial body weight. The test design was as follows: 1) negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). The use of chlorella as an antiviral agent constitutes the production and local delivery of genetically engineered 3D8 fragments by bacteria in the gastrointestinal tract. Five replicates were performed per treatment, with 5 and 15 per iteration, in a completely random arrangement. The test was conducted on the Dankook University research farm, with the tested feed corn being formulated according to National Research Council (NRC) nutrient requirements, and soybean meal diets were provided (Table 1). All birds were housed in stainless-steel cages (1.75 m × 1.55 m) with concrete floors covered with clean rice bran, and continuous light was provided. The temperature of the room was maintained at 33 ± 1o C for the first 3 d and then decreased to 24o C throughout the rest of the experiment. The diets were fed in 2 phases: a starter phase, which was administered for the first 3
Feeding, Sampling, and Measurements Weight gain from the beginning of the experiment was determined at 3 wk and at the end of the experiment (5 wk). The feed intake was calculated by measuring the weight of the feed, and the feed conversion ratio was calculated by dividing this value by the weight gain. To determine when the experiment should be terminated (5 wk), 10 animals per treatment were randomly selected for measurements, and the tibia, breast, liver, F sac, abdominal fat, and proximal splenic weights were then measured at slaughter, after which the percentage of the live weight was calculated. The pH was measured with a pH meter (Testo 205, Testo AG, Lenzkirch, Germany), and a meat color colorimeter (CR-410, Minolta Co., Osaka, Japan) was used to measure each breast sample in the second iteration. At this time, the standard was L ∗ = 89.2, a ∗ = 0.921, b ∗ = 0.783. Storage loss (drip loss) was measured after holding the samples in polyethylene bags in a 4◦ C cold room for 7 d, with each sample being shaped to a constant shape with a thickness of 2 cm at one d, 3 d, and 5 d; finally, the loss generated after 7 d was measured. Blood was collected through a random selection process of 10 animals from each treatment at the end of the experiment (5 wk). Intravenous (jugular) blood was collected in a 2 mL K3EDTA tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and was then processed using an automatic blood analyzer (ADVIA 120, Bayer, NY, USA) to determine WBC, RBC, lymphocyte, heterophil, basophil, and platelet counts. For serum biochemical tests, blood (5 mL) was collected in vacuum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and then centrifuged at 4◦ C at 4,000 rpm for 15 min to obtain serum for analysis. A separate sample for the determination of serum total cholesterol was obtained, which was then analyzed using an HDL/C, LDL/C, triglyceride, AST, ALT, BUN, creatinine, total protein, albumin, globulin, IgG, IgM, and IgA content nephelometer (Behring, Germany). After harvesting for one min at the termination of the experimental treatment (at 5 wk), the obtained sample was suspended in sterile saline and homogenized. The viable cell count was then determined by dilution in steps from 10−3 to 10−6 . The experimental treatment of the broilers was performed in a matter of min on the broilers’ Lactobacillus and E. coli bacteria. The number of bacteria was measured using Lactobacillus MRS agar and E. coli MacConkey agar (Difco Laboratories, Detroit, MI) at 38 to 37◦ C. The Hennessey gas composition of the broilers was analyzed by obtaining a minute sample of excretory ammonia, hydrogen sulfide, and total mercaptan at the
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Ingredients (%) Corn Soybean meal, 45% CP Corn gluten meal, 60% CP Tallow Limestone Dicalcium phosphate Salt DL-Methionine L-Lysine-HCI L-Threonine Choline chloride Vitamin premix2 Trace mineral premix3 Calculated composition Metabolizable energy, kcal/kg Crude protein, % Lysine, % % Calcium, % Total Phosphorus, %
Starter
wk, and a finisher phase, which was administered during wk 4 and 5. The chicks were given free access to water and mash feed throughout the entire experiment.
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EFFECT OF RECOMBINANT CHLORELLA IN CHICKEN Table 2. Effect of dietary supplementation with sea plants on the growth performance of broilers.1,2 P-value
Items
NC
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
728 1045 1.435
745 1020 1.369
734 1032 1.405
741 1034 1.396
14 8 0.033
0.2252 0.1179 0.1280
0.3141 0.6465 0.3148
0.4091 0.0533 0.1838
0.7660 0.8954 0.6505
0.7509 0.3161 0.7235
0.8271 0.2532 0.5782
1011a,b 1502 1.486
1026a 1490 1.452
1016a,b 1495 1.471
1023a,b 1495 1.462
8 21 0.022
0.0286 0.6199 0.0908
0.0528 0.7320 0.1569
0.1929 0.7119 0.2907
0.5848 0.9839 0.7429
0.6322 0.8140 0.6623
0.7578 0.8771 0.7575
1739b,c 2546 1.464a,b
1771a 2510 1.417c
1751a,b 2527 1.443a,b,c
1763a,b 2529 1.434b,c
9 20 0.013
0.0010 0.2269 0.0032
0.0032 0.5736 0.0218
0.0248 0.2097 0.0208
0.3469 0.9380 0.6078
0.3620 0.4917 0.2824
0.5771 0.5049 0.3677
1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean from 5 replicates with 10 chicks/replicate (total of 250 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b,c Means in the same row with different superscripts differ (P < 0.05).
Table 3. Effect of dietary supplementation with sea plants on the carcass quality of broilers.1,2 P-value
Items
NC
pH value 5.47 Breast muscle color Lightness (L∗ ) 52.78 10.52 Redness (a∗ ) ∗ Yellowness (b ) 8.55 WHC, % 56.87 Drip loss, % d1 5.53 d3 9.04 d5 11.32 d7 13.23a Relative organ weight, % Breast muscle 19.13b Liver 3.02 Bursa of Fabricius 0.15 Abdominal fat 1.74 Spleen 0.11 Gizzard 1.24
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
5.45
5.42
5.37
5.41
0.04
0.3869
0.2632
0.6441
0.4721
0.1547
0.7957
52.76 10.69 8.51 56.82
52.46 10.46 8.43 56.64
52.91 10.28 8.58 56.91
52.77 10.34 8.27 56.83
0.68 0.18 0.21 0.67
0.7367 0.8195 0.6913 0.8113
0.9901 0.5044 0.3525 0.9733
0.7547 0.3923 0.7715 0.8480
0.8819 0.7930 0.2919 0.9399
0.8729 0.1241 0.8152 0.9290
0.7460 0.6594 0.5915 0.8373
5.56 8.93 11.16 13.08a,b
5.51 8.71 11.20 13.03a,b
5.47 8.81 11.16 13.00a,b
5.48 8.82 11.05 12.93b
0.40 0.14 0.14 0.09
0.9735 0.1157 0.5415 0.1531
0.9316 0.3029 0.1854 0.0304
0.9275 0.2853 0.8536 0.7215
0.9832 0.9259 0.5871 0.5881
0.8693 0.5448 1.0000 0.5481
0.9581 0.5743 0.4678 0.4327
19.50a,b 3.04 0.14 1.70 0.11 1.20
20.07a 2.94 0.15 1.71 0.12 1.27
19.69a,b 2.97 0.14 1.68 0.11 1.22
19.72a,b 3.02 0.13 1.72 0.12 1.25
0.68 0.11 0.01 0.12 0.01 0.04
0.0052 0.6135 0.5484 0.8569 0.3300 0.4978
0.0666 0.9896 0.1976 0.8758 0.2739 0.8542
0.0813 0.5219 0.4245 0.9186 0.5410 0.1574
0.9095 0.7361 0.4245 0.8381 0.7133 0.5821
0.5558 0.6456 0.7637 0.9377 0.7133 0.6998
0.2855 0.6045 0.0630 0.9808 0.9024 0.6203
1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean from 5 replicates with 10 chicks/replicate (total of 250 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b Means in the same row with different superscripts differ (P < 0.05).
same time at the end of the test (5 wk) for each treatment group. The ammonia, hydrogen sulfide, and total mercaptan contents were measured while holding the samples at room temperature for 5 d; 300 g of the sample was placed in a sealed plastic container with a 2,600 mL volume for 24 h of fermentation in a Gastec (Model GV - 100, Gastec Corp, Kanagawa, Japan) with ammonia and hydrogen sulfide, and measurements were
performed using a total detecting tube (No. 3 L, No. 4LT and No. 70 L, Gastec, Japan). Nutrient digestibility was assessed at the end of the experiment (5 wk), after 6 d in 0.2%, by adding fodder chromium oxide (Cr2 O3 ) as an indicator of water. The sample was collected after 72 h, and after drying in a dryer at 60◦ C, it was subjected to analysis via pulverization in a Willey mill. Cr was then mixed with
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d 1-21 BWG, g 720 FI, g 1040 FCR 1.444 d 21 to 35 BWG, g 999b FI, g 1506 FCR 1.508 Overall BWG, g 1719c FI, g 2545 FCR 1.481a
PC1
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CHOI ET AL. Table 4. Effect of dietary supplementation with sea plants on the blood profile of broilers.1,2 P-value
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
WBC, 103 /μ RBC, 106 /μ Lymphocyte, % Heterophil, % Basophil, % Platelet, 109 / Total Cholesterol, mg/dL HDL/C, mg/dL LDL/C, mg/dL Triglyceride, mg/dL AST, U/L ALT, U/L BUN, mg/dL Creatinine, mg/dL Total Protein, g/dL Albumin, g/dL Globulin, g/dL IgG, g/dL IgM, g/dL IgA, g/dL
22.9 2.81 61.8 31.3 2.87 21.78 111b 82 26.9a 69 187.2 14.2a 3.2b 0.26b 2.7 1.2 1.5 234 154 37b
23.1 2.91 63.3 31.9 2.93 21.97 115b 83 24.7a,b 67 183.7 12.8a,b 3.6a,b 0.28a,b 3.1 1.3 1.9 239 155 40a,b
23.9 2.94 64.1 32.4 2.98 22.02 120a,b 87 22.7a,b 66 183.0 12.3a,b 3.6a,b 0.29a,b 3.2 1.4 1.8 239 158 44a,b
24.0 3.10 65.8 33.1 3.04 22.08 121a,b 90 22.2a,b 66 181.7 11.5b 4.0a 0.31a 3.1 1.4 1.7 244 164 45a,b
24.5 3.22 65.9 33.8 3.10 22.14 126a 95 21.8b 66 180.1 10.9b 4.1a 0.32a 3.3 1.3 2.0 248 163 48a
1.4 0.2 2.8 1.30 0.22 0.54 4 6 2 7 5.6 0.6 0.2 0.01 0.2 0.1 0.2 13 4 3
0.6117 0.6027 0.5468 0.5738 0.7060 0.7597 0.0910 0.5256 0.0681 0.7560 0.5950 0.0406 0.1584 0.1542 0.0890 0.2426 0.2955 0.7865 0.4958 0.0771
0.4386 0.1134 0.2904 0.1862 0.4558 0.6424 0.0056 0.1299 0.0284 0.7170 0.3722 0.0008 0.0024 0.0079 0.0533 0.7933 0.0834 0.4538 0.1236 0.0097
0.7050 0.8978 0.8265 0.8126 0.8630 0.9522 0.3079 0.6522 0.3764 0.9506 0.9271 0.5949 0.8166 0.5322 0.8873 0.3616 0.7340 1.0000 0.5971 0.3688
0.8303 0.6299 0.9695 0.7008 0.8656 0.9419 0.3551 0.5638 0.8588 0.9669 0.8382 0.5573 0.6710 0.8097 0.5480 0.3616 0.2955 0.8079 0.8376 0.5531
0.6648 0.4637 0.5218 0.5318 0.7084 0.8840 0.1993 0.4400 0.2703 0.9422 0.8012 0.1488 0.1108 0.0892 0.8317 0.3616 0.4978 0.8121 0.1146 0.2299
0.7878 0.2798 0.6450 0.4405 0.7108 0.8738 0.2342 0.3694 0.6893 0.9587 0.7156 0.1344 0.0756 0.1828 0.8042 0.3616 0.4767 0.6310 0.3862 0.3688
1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean from five replicates with 10 chicks/replicate (total of 250 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b Means in the same row with different superscripts differ (P < 0.05).
Table 5. Effect of dietary supplementation with sea plants on the excreta microflora of broilers.1,2 P-value Items, log10 cfu/g
NC
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
Lactobacillus E.coil
7.57 6.49
7.64 6.45
7.61 6.41
7.61 6.39
7.63 6.40
0.03 0.07
0.3973 0.4144
0.1964 0.3747
0.5213 0.6484
0.6485 0.9009
0.5213 0.5144
0.6485 0.9421
1 Abbreviation: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean of 5 replicates with 15 chicks/replicate (total of 375 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means.
normal water, and the feed was analyzed according to the AOAC method.
RESULTS Growth Performance
Statistical Analysis All data were analyzed in SAS (SAS Institute Inc., Cary, North Carolina, USA) using the General Linear Model procedure for analysis of variance. Duncan’s multiple range tests (Duncan, 1955) were employed to process the average difference, and <0.05 was considered statistically significant. To evaluate the effect on the nutrient and additive contents, we used contrast statistical methods.
At 21 to 35 d old, the broilers fed the PC2 treatment group (P < 0.05) exhibited higher BWG than in the primary NC treatment group (Table 2), which showed increased BWG by 2.70%. During the total experimental period, the weight of the broilers in the PC2 and T2 treatment groups (P < 0.01) was significantly higher than that of those in the NC treatment group, which showed increased BWG by 3.00 and 2.55%, respectively. The induced BWG of the broilers was reflected in increased FCR, with the broilers in the PC2 (P < 0.05) and T2 treatment groups (P < 0.05) exhibiting FCR values that were 4.32 and 2.76% lower,
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NC
Items
5
EFFECT OF RECOMBINANT CHLORELLA IN CHICKEN Table 6. Effect of dietary supplementation with sea plants on excreta noxious gas emission in broilers.1,2 P-value
Items, ppm NH3 H2 S R.SH
NC
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
31.10a 2.37 1.47
30.78a,b 2.36 1.32
30.56a,b 2.31 1.45
29.35b,c 2.11 1.52
28.89c 2.27 1.25
0.55 0.22 0.12
0.4920 0.8449 0.9072
0.0074 0.7444 0.2050
0.7789 0.8705 0.4506
0.5580 0.6024 0.1219
0.0743 0.4169 0.2483
0.0386 0.8962 0.2483
Table 7. Effect of dietary supplementation with sea plants on nutrient digestibility in broilers.1,2 P-value
Items, % Dry matter Nitrogen Energy
NC
PC1
PC2
T1
T2
SEM3
NC VS PC2
NC VS T2
PC1 VS PC2
T1 VS T2
PC1 VS T1
PC2 VS T2
69.74 66.06 72.85b
69.90 66.42 73.00a,b
70.21 66.71 73.60a
69.98 66.32 73.20a,b
70.11 66.57 73.35a,b
0.21 1.16 0.23
0.1194 0.6983 0.0270
0.2176 0.7591 0.1296
0.3035 0.8633 0.0712
0.6702 0.8787 0.6304
0.7871 0.9508 0.5429
0.7325 0.9353 0.4460
1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean of 5 replicates with 15 chicks/replicate (total of 375 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b Means in the same row with different superscripts differ (P < 0.05).
respectively, compared with those in the NC treatment group.
Meat Quality At 7 d olds, the T2 treatment group (P < 0.05) resulted in significantly lower drip loss in the breast than the NC treatment group (Table 3), which showed decreased drip loss by 2.26%. The breast organ weight in the PC2 treatment group (P < 0.01) was significantly greater than that in the NC treatment group in the proportions of 19.13 and 20.07 for NC and PC2 treatment groups, respectively. No significant difference was found among the treatments in terms of pH, water-holding capacity, and meat color (P > 0.05).
23.07, and 29.72%, respectively, whereas ALT concentration (P < 0.01) decreased by 23.23%, compared with the NC treatment group.
Excreta Microbial Shedding and Excreta Noxious Gas Emission No significant difference in the excreta microflora composition (P > 0.05) was found among the treatments involving Lactobacillus and E. coli (Table 5). The amount of fecal ammonia generated with the T2 treatment group was significantly lower by 1.73 and 7.10% compared with the PC2 (P < 0.05) and NC treatment groups (P < 0.01), respectively (Table 6).
Nutrient Digestibility Blood Profile The total cholesterol content in the blood of the broilers in the T2 treatment was significantly higher (P < 0.01) than that found in the broilers in the NC treatment group (Table 4), which showed increased total cholesterol content by 13.51%. However, the LDL content was significantly lower in the broilers in the T2 treatment group (P < 0.05) than in those in the NC group, which showed decreased LDL content by 18.95%. The T2 treatment group increased the BUN, creatinine, and IgA concentration (P < 0.01) by 28.12,
The PC2 treatment (P < 0.05) led to a significantly higher energy digestibility than the NC treatment, which showed increased energy digestibility by 1.29% (Table 7). The dry matter digestibility and nitrogen digestibility presented no significant differences among the treatments (P > 0.05).
DISCUSSION Several researchers have reported that the use of chlorella as a prebiotic has a positive effect on the
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1 Abbreviations: Negative control (NC), basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) positive control (PC1), 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) treatment (T) 1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). 2 Each value represents the mean of 5 replicates with 15 chicks/replicate (total of 375 one-day-old ROSS 308 broilers (male and female) with an initial body weight of 45 ± 0.39 g). 3 Standard error of means. a,b,c Means in the same row with different superscripts differ (P < 0.05).
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CHOI ET AL.
ACKNOWLEDGMENTS This study was supported by the 2015–2019 Postdoctoral Fellowship Program of National Insti-
tute of Animal Science, Rural Development Administration, Republic of Korea. This work was conducted with the support of the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01020101)” of the Rural Development Administration, Republic of Korea.
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growth performance and immune characteristics of chickens and pigs (Yan, Lim and Kim, 2012; Kang et al., 2013). In the present study, the broilers in the PC2 and T2 treatment groups exhibited a greater BWG and FCR than those in the control NC treatment group (Table 1). It has been suggested that prebiotics generally modulate the gut environment by increasing beneficial microorganisms and inhibiting the proliferation of pathogens in the intestine (Cho and Kim, 2014; Kotrb´ aˇcek et al., 2015; Wang et al., 2016). The drip loss from the breast measured under the T2 treatment was significantly lower than for the NC treatment (Table 2). In contrast, the PC2 treatment resulted in a significantly greater breast organ weight than the NC treatment (Table 2). It was previously suggested that increasing dietary lysine decreases drip loss in broiler breast meat (Berri et al., 2008). Therefore, we hypothesized that the reason for the decreased drip loss in relation to broiler breast meat performance was likely due to the very large amount of lysine in chlorella proteins (Baltazar et al., 2014). The blood contents of BUN, creatinine, and IgA in the T2 treatment group were significantly higher than in the NC treatment group (Table 3). In contrast, LDL/C in the blood of the animals in the T2 treatment group was significantly lower than in NC (Table 3). A previous study found that dietary chlorella supplementation significantly increased the IgA concentration in the breast milk of pregnant women (Nakano et al., 2007). Additionally, previous reports have shown that dietary fiber enhances IgA production in the mesenteric lymphocytes (Yamada et al., 2003). Therefore, we hypothesized that the reason for the increased IgA concentration in the plasma of the broiler chickens was likely the increased production of B cells due to stimulating immunoglobulin in the autoassociated lymphoid tissue (Pugh et al., 2001; Kang et al., 2013). The amount of fecal ammonia generated in the T2 treatment was significantly lower than that in the NC treatment (Table 5). A previous study showed that supplementation with prebiotics could reduce fecal ammonia gas emission in broilers (Cho and Kim, 2014). It has been suggested that the N from ammonia is generally derived from the fermentation of unabsorbed N entering the large intestine (Song et al., 2012). Therefore, the reason for the decreased NH3 emission from the broilers fed chlorella was likely the increase in apparently metabolizable N compared with the control group. In conclusion, the results of this study indicated that dietary supplementation of broilers with recombinant chlorella could improve growth performance and increase the concentration of IgA and apparently metabolizable nitrogen in the blood, in addition to decreasing ammonia emissions.
EFFECT OF RECOMBINANT CHLORELLA IN CHICKEN Yamada, K., Y. Tokunaga, A. Ikeda, K. Ohkura, S. Kaku-Ohkura, S. Mamiya, B. O. Lim, and H. Tachibana. 2003. Effect of dietary fiber on the lipid metabolism and immune function of aged Sprague-Dawley rats. Biosci. Biotechnol. Biochem. 67:429–433. Yan, L., S. U. Lim, and I. H. Kim. 2012. Effect of fermented chlorella supplementation on growth performance, nutrient digestibility, blood characteristics, fecal microbial and fecal noxious gas content in growing pigs. Asian-Australas. J. Anim. Sci. 25:1742–1747.
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Zhao, P. Y., H. L. Li, M. Mohammadi, and I. H. Kim. 2016. Effect of dietary lactulose supplementation on growth performance, nutrient digestibility, meat quality, relative organ weight, and excreta microflora in broilers. Poult. Sci. 95:84–89. Zhao, P. Y., J. P. Wang, and I. H. Kim. 2013. Effect of dietary levan fructan supplementation on growth performance, meat quality, relative organ weight, cecal microflora, and excreta noxious gas emission in broilers. J. Anim. Sci. 91:5287–5293.
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