Effects of Chito-oligosaccharide supplementation on egg production, nutrient digestibility, egg quality and blood profiles in laying hens.
In the past four decades, antibiotics have been used as feed additives to improve growth and egg production, and to protect animals from pathogenic microorganisms. The general ban on the use of feed antibiotics as microbial performance promoters that is expected to be introduced more widely throughout the world in the coming years, has increased the urgency for research on botanical feed additives (Best, 2000). Therefore, various oligosaccharides are now being added to livestock feed as prebiotics to improve animal health, production, and immune ability and to influence the gut microbiota (White et al., 2002; Lemieux et al., 2003). Certain oligosaccharides are considered to be prebiotic compounds because they are not hydrolyzed in the upper gastrointestinal tract and are able to favorably alter the colonic microflora (Biggs et al., 2007).
Chito-oligosaccharide (COS) is easily obtained by chemical and enzymatic hydrolysis of poly-chitosan which is the second most abundant carbohydrate polymer found in nature (Knaul et al., 1999). However, its application as a nutrient source for animals has been limited due to its insolubility and high viscosity. In contrast, COS has low molecular weight, good solubility and low viscosity (Chae et al., 2005).
COS has been shown to reduce the establishment of pathogens in the intestine (Li et al., 2006) and to enhance immune function in broilers (Wang et al., 2003). In addition, feeding COS to pigs has been found to increase nutrient digestibility (Rozeboom et al., 2005). However, data on the effect of COS in laying hens is still limited; only Spring et al. (2000) suggested that mannan-oligosaccharides (MOS) reduced intestinal Salmonella concentrations by 26% in broiler chicks compared with a control diet and subsequently improved growth performance from 0 to 21 d of age (Pelicano et al., 2004). Therefore, this study was conducted to further explore the effects of COS on egg production, nutrient digestibility, egg quality and blood profiles in laying hens.
MATERIALS AND METHODS
Preparation of COS
The COS used in this study, which was prepared and supplied by Easy Bio System, Inc. (Korea), was comprised of: 18.6% crude protein, 14.5% crude fat, 10.7% crude fiber, 22.6% crude ash, 9.0% moisture, 4.3% calcuim, 1.7% phosphorus, 4.0% chitin chitosan, and 3% chitosan oligosaccharide. The product was produced by microbial fermentation by Aspergillus, Aspergillus oryzac, Bacillus subtillus, Saccharomyces cerevisiae and Lactobacillus acidophilus.
Experiment design, animals and diets
A total of 240 Hy-line Brown laying hens (28-wk-old) were selected for a 6-wk feeding trial. Hens were randomly allocated into 5 dietary treatments with 4 replications per treatment according to a completely randomized block design. Each replication of 12 hens was assigned to six adjacent cages (38.1-cm widthx50-cm lengthx40-cm height) providing two hens per cage. Therefore, each replication represented six cages in which the hens were fed from the same feed trough. The experimental diets were as follow: i) CON, basal diet; ii) ANT, basal diet mg/kg avilamycin; iii) COS0.2, basal diet+ mg/kg COS; iv) COS0.4, basal diet+ mg/kg COS; v) ANTCOS, basal diet+ mg/kg COS mg/kg avilamycin. Hens were housed in a three-tier cage system. An environmentally controlled room was maintained at 21[degrees]C by a sensor which monitored the inside temperature and adjusted ventilation fans accordingly to control the temperature. Hens were maintained on a 17 h:7 h light:darkness photoperiod following light stimulation. Feed and water were provided ad libitum. All diets were formulated to meet or exceed the NRC (1994) requirements for laying hens. The composition of the experimental diet is shown in Table 1. The Animal Welfare Committee of Dankook University approved the animal care protocol used for this experiment.
Sampling and measurements
At the initial of the experiment, 8 laying hens were randomly selected from each treatment (two hens in each replication) and blood samples were collected from the wing vein using a sterilized injector. Then, samples were transferred into either vacuum or [K.sub.3]EDTA vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA). The same laying hens were also sampled at the end of the experiment. For serum analysis, blood samples were centrifuged at 2,000xg at 4[degrees]C for 20 min within 1 h of collection to separate the serum. The total protein and albumin in the serum were analyzed using an automatic biochemistry blood analyzer (HITACHI 747, Hitachi, Japan). The concentrations of white blood cells (WBC), red blood cells (RBC) and lymphocytes in the whole blood samples were determined using an automatic blood analyzer (Advia 120, Bayer, Tarrytown, NY, USA).
Daily records of egg production and weekly records of feed consumption were maintained. Egg production was expressed as average hen-day production. A total of 30 salable eggs (no shell defects, cracks or double yolks) were randomly collected at 17:00 h from each treatment (5 per replicate, n = 30) on a weekly basis and used to determine the egg quality at 20:00 h at the same day. Eggshell breaking strength was evaluated using an eggshell force gauge, model II (Robotmation Co. Ltd., Japan). Eggshell thickness was measured on the large end, equatorial region, and small end using a dial pipe gauge (Ozaki MFG. Co. Ltd., Japan). Finally, egg weight, egg yolk color, and Haugh unit (HU) were evaluated using an egg multi-tester (Touhoku rhythm Co. Ltd., Japan).
After the conclusion of the feeding trial, six birds per treatment were randomly chosen for metabolic trials. The selected birds were individually housed in metabolic cages to determine the digestibility of nutrients. Laying hens were fed their respective diets containing chromic oxide ([Cr.sub.2][O.sub.3] at 0.20% level) for 4 d prior to the collection period. All excreta of the birds were collected daily for 3 d. All the fecal samples along with feed samples, were then analyzed according to AOAC procedures (AOAC, 2000).
Statistical analysis was performed by using the GLM procedure in a completely randomized block design with the SAS software program (SAS Institute, 1996). Differences among all treatments were separated by Duncan's multiple range test. Results were expressed as the least squares means and SEM. Probability values less than 0.05 were considered significant.
The effects of COS supplementation on egg production in laying hens are presented in Table 2. During weeks 4-6, ANTCOS treatment improved the egg production (p<0.05) compared to that of birds fed the control diet. In addition, no effects on egg weight were observed among dietary treatments.
The effects of COS supplementation on egg characteristics in laying hens are shown in Table 3. At the end of the 6th wk, birds in COS0.2, COS0.4 and ANTCOS groups had higher (p<0.05) HU as well as larger differences between the beginning and end of the experiment than those in CON and ANT. However, other criteria were unaffected by treatments through the experiment.
COS0.4 and ANTCOS treatments significantly improved DM digestibility (p<0.05) compared to CON treatment (Table 4). The digestibility of N in CON group was lower (p<0.05) than other treatments.
The effects of COS supplementation on blood characteristics in laying hens are shown in Table 5. RBC, lymphocytes and albumin were not influenced by the dietary treatments. However, the WBC concentration of birds in the COS0.4 and ANTCOS group were higher (p<0.05) than that of birds in other groups at the end of the 6th wk. In addition, the difference in WBC counts between the beginning and end of the experiment in COS0.4 and ANTCOS groups were significantly higher (p<0.05) than that of CON and ANT groups. At the end of the experiment, the birds fed ANTCOS diet showed higher (p<0.05) total protein concentration than birds fed CON or ANT diets.
Egg production, egg weight and nutrient digestibility
Various oligosaccharides have been classified as prebiotics because of their beneficial effects on gut microflora, which improves health condition and product performance (Stanley et al., 1999; Berry and Lui, 2000). Similarly, previous studies also suggested that COS have antifungal (Hirano and Nagao, 1989) and antimicrobial (Jeon et al., 2000) activities that improved gut health in 196 day-old male broiler chicks (Li et al., 2007), which may promote nutrient digestibility and improve growth performance. In the present study, egg production was increased by the COS supplementation, which may be attributed to the observed improvement of DM and N digestibility in response to COS supplementation. Other study in our laboratory has also reported that DM and N digestibility was improved by COS supplementation in weanling pigs (Chen et al., 2009). Moreover, a considerable improvement in egg production in mannan-oligosaccharide (MOS)-fed broiler breeder hens was observed, which may suggest that COS is beneficial in chickens because of its similar structure to MOS. However, knowledge of the influence of COS supplementation on the laying hen appears limited and we have been unable to find any other study to confirm this result. Further study is needed to demonstrate the effect of COS supplementation in the laying hen.
HU is the measure used most commonly today (Williams, 1992) to measure albumen quality, and, consequently, to judge the freshness of an egg (Eisen et al., 1962). Results from the current study showed greater HU when laying hens were fed diets supplemented with COS, meanwhile, albumin height was also numerically increased in COS groups, which may indicate COS contributes to egg freshness.
Blood profiles of animals reflect the physiological disposition of their nutrition according to their internal and external environments. Therefore, we measured these characteristics to determine the response by which COS influenced the laying hens. The only change in blood profiles observed in the current study was an increased concentration of WBC when laying hens were fed COS0.4 and ANTCOS diets. However, Chen et al. (2009) reported that 5 g/kg of COS supplementation added in the diet did not affect the concentration of WBC, RBC and lymphocyte as well as total protein in weaning pigs, but obvious effects were observed in the COS group when pigs were challenged with lipopolysaccharide. The variation between the aforementioned studies may also be ascribed to different aspect as well as the environmental conditions employed in each study, as highly healthy conditions can significantly affect the response to supplemented antibiotics. Therefore, the current study may indicate that COS supplementation at 0.4% level could improve the health of laying hens.
Furthermore, significant differences in total protein were observed in the ANTCOS treatment compared with the CON and ANT treatments at the end of the 6th wk, but total protein was not affected by COS supplementation in the current study. This is partially in agreement with Li et al. (2007), who suggested that dietary COS increased the serum total protein in broiler chicks. Moreover, study conducted by Zhou et al. (2009) also reported inclusion of COS increased the RBC concentration in broiler chicks. Therefore, COS supplementation indeed increased the immune related blood cell content in the current study. Meanwhile, no significant difference in total protein was observed in chickens fed the COS diet compared with the CON group. Therefore, the reason for the significant improvement detected in the ANTCOS may be due to the synergestic effect of COS with antibiotics.
In conclusion, the results from the current study indicated that dietary supplementation of COS at 200 or 400 mg/kg increased egg production, the immune blood cell counts as well as nutrient digestibility, indicating the COS can be used as a potential alterative to antibiotics in laying hens. However, it is suggested that further research is warranted to elucidate the mechanism by which COS improves the nutrient digestibility, egg quality and production in laying hens.
AOAC. 2000. Official methods of analysis. 17th ed. Assoc. Off. Analysis Chemistry, Gaithersburg, MD.
Berry, W. D. and P. Lui. 2000. Egg production, egg shell quality and bone parameters in broiler breeder hens receiving BioMos and Eggshell. Poult. Sci. 79(Suppl. 1):124(Abstr.).
Best, P. 2000. Health booster from botany. Feed Int. 6:15-16.
Biggs, P., C. M. Parsons and G. C. Fahey. 2007. The effects of several oligosaccharides on growth performance, nutrient digestibilities, and cecal microbial populations in young chicks. Poult. Sci. 86:2327-2336.
Chae, S. Y., M. Jang and J. Nah. 2005. Influence of molecular weight on oral absorption of water soluble chitosans. J. Control. Release 102:383-394.
Chen, Y. J., I. H. Kim, J. H. Cho, J. S. Yoo, Y. Wang, Y. Huang, H. J. Kim and S. O. Shim. 2009. Effects of chitooligosaccharide supplementation on growth performance, nutrient digestibility, blood characteristics and immune responses after lipopolysaccharide challenge in weanling pigs. Livest. Sci. 124: 255-260.
Eisen, E. J., B. B. Bohren and H. E. McKean. 1962. The Haugh Unit as a measure of egg albumen quality. Poult. Sci. 41:1461-1468.
Hirano, S. and N. Nagao. 1989. Effects of chitosan, pecticacid, lysozyme and chitinase on the growth of several phytopathogens. Agric. Biol. Chem. 53:3065-3066.
Jeon, Y. J., F. Shahidi and S. K. Kim. 2000. Preparation of chitin and chitosan oligomers and their applications in physiological functional foods. Food Rev. Int. 61:159-176.
Knaul, J. Z., S. M. Hudson and K. A. M. Creber. 1999. Crosslinking of chitosan fibers with dialdehydes: Proposal of a new reaction mechanism. J. Polym. Sci. Part B: Polym. Phys. 37:1079-1094.
Lemieux, F. M., L. L. Southern and T. D. Bidner. 2003. Effect of mannan oligosaccharides on growth performance of weanling pigs. J. Anim. Sci. 81:2482-2487.
Li, C. H., Y. L. Dong, Y. M. Zhang, Z. J. Song and S. G. Qi. 2006. The chitosan and chitin and the application on chicken production. Jinri Xumu Shouyi. 7.
Li, X. J., X. S. Piao, Kim, S. W. Liu, P. Wang, L. Shen, Y. B. Jung, S. C. and H. S. Lee. 2007. Effects of Chito-oligosaccharide supplementation on performance, nutrient digestibility and serum composition in broiler chickens. Poult. Sci. 86:11071114.
NRC. 1994. Nutrient requirements of poultry. 9th rev. ed. National Academy Press, Washington, DC.
Pelicano, E. R. L., P. A. deSouza, H. B. A. de Souza, F. R. Leonel, N. M. B. L. Zeola and M. M. Boiago. 2004. Productive traits of broiler chickens fed diets containing different growth promoters. Rev. Bras. Cienc. Avic. 6:177-182.
Rozeboom, D. W., D. T. Shaw, R. J. Tempelman, J. C. Miguel, J. E. Ettigrew and A. Connolly. 2005. Effects of mannan oligosaccharide and anantimicrobial product in nursery diets on performance of pigs reared on three different farms. J. Anim. Sci. 83:2637-2644.
Spring, P., C. Wenk, K. A. Dawson and K. E. Newman. 2000. The effects of dietary mannanoligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of Salmonella-challenged broiler chicks. Poult. Sci. 79:205-211.
Stanley, V. G. and A. E. Sefton. 1999. Egg and serum cholesterol as influenced by mannan oligosaccharide and anatoxin. In Egg Nutrition and Biotechnology (Ed. J. S. Sim, S. Nakaiand and W. Guenter). CABI Publishing, Wallingford, UK. pp. 441-443.
Wang, X. W., Y. G. Du, X. F. Bai and S. G. Li. 2003. The effect of oligochitosan on broiler gut glora, microvilli density, immune function and growth performance. Acta Zoonutrimenta Sinica. 15(4).
White, L. A., M. C. Newman, G. L. Cromwell and M. D. Lindemann. 2002. Brewers dried yeast as a source of mannan oligosaccharides for weanling pigs. J. Anim. Sci. 80:2619-2628.
Williams, K. C. 1992. Some factors affecting albumen quality with particular reference to Haugh unit score. World Poult. Sci. J. 48:5-16.
Zhou, T. X., Y. J. Chen, J. S. Yoo, Y. Huang, J. H. Lee, H. D. Jang, S. O. Shin, H. J. Kim, J. H. Cho and I. H. Kim. 2009. Effects of chito-oligosccharide supplementation on growth performance, blood characteristics, relative organ weight and meat quality in broilers. Poult. Sci. 88:593-600.
Q. W. Meng, L. Yan, X. Ao, H. D. Jang, J. H. Cho and I. H. Kim *
Department of Animal Resource and Science, Dankook University, No.29 Anseodong, Cheonan, Choongnam 330-714, Korea
* Corresponding Author: I. H. Kim. Tel: 41-550-3652, Fax: 41-565-2949, E-mail: firstname.lastname@example.org
Received January 20, 2010; Accepted March 24, 2010
Table 1. Diet composition (as-fed basis) Ingredients % Corn 50.40 Soybean meal (CP 46%) 18.70 Wheat grain 10.00 Corn gluten meal 2.00 Wheat bran 5.00 Animal fat 4.40 Limestone 7.50 Tricalcium phosphate (P 18%) 1.40 Salt 0.30 DL-methionine (50%) 0.10 Vitamin premix (1) 0.10 Mineral premix (2) 0.10 Chemical composition (3) ME (kcal/kg) 2,904 Crude protein (%) 15.45 Lysine (%) 1.80 Methionine (%) 0.32 Calcium (%) 3.25 Phosphorus (%) 0.61 (1) Provided per kg of complete diet: 125,000,000 IU vitamin A 2,500,000 IU vitamin [D.sub.3] 10,000 mg vitamin E 2,000 mg vitamin [K.sub.3] 1,000 mg vitamin [B.sub.1] 5,000 mg vitamin [B.sub.2] 1,000 mg vitamin [B.sub.6] 15 mg vitamin [B.sub.12] 500 mg folic acid 35,000 mg niacin 10,000 mg Ca-Pantothenate and 50 mg biotin. (2) Provided per kg of complete diet: 8,000 mg Mn 60,000 mg Zn 25,000 mg Cu 40,000 mg Fe; 300 mg Co; 1,500 mg I and 150 mg Se. (3) Calculated values. Table 2. Effects of chito-oligosaccharide on egg production in laying hens (1) Items CON ANT COS0.2 Egg production (%) 0-3 wk 81.2 83.3 81.6 4-6 wk 81.5 (b) 83.8 (ab) 82.1 (ab) Egg weight (g) 0 wk 57.1 56.5 58.1 3 wk 58.9 58.7 61.5 6 wk 58.7 58.5 61.6 Items COS0.4 ANTCOS SEM (2) Egg production (%) 0-3 wk 83.5 84.1 1.68 4-6 wk 82.6 (ab) 85.4 (a) 1.72 Egg weight (g) 0 wk 56.8 57.4 1.42 3 wk 59.2 60.4 1.72 6 wk 59.8 61.7 1.68 (1) CON = Basal diet; ANT = Basal diet mg/kg avilamycin; COS0.2 = Basal diet+ mg/kg chito/oligosaccharide; COS0.4 = Basal diet+ mg/kg chito/oligosaccharide; ANTCOS = Basal diet+ mg/kg chito/oligosaccharide mg/kg avilamycin. (2) Standard error of the means. (a, b) Means in the same row with different superscripts differ (p<0.05). Table 3. Effects of chito-oligosaccharide on egg quality in laying hens (1) Items CON ANT COS0.2 Egg shell breaking strength (kg/[cm.sup.2]) 0 wk 3.24 3.47 3.34 3 wk 3.52 3.29 3.45 6 wk 3.31 3.52 3.80 Difference (0-6 wk) 0.07 0.05 0.46 Egg shell thickness ([10.sup.-2] mm) 0 wk 35.7 35.1 35.4 3 wk 35.8 35.7 35.3 6 wk 36.1 36.6 36.2 Difference (0-6 wk) 0.40 1.50 0.80 Yolk color unit 0 wk 8.68 9.00 8.70 3 wk 8.54 8.79 8.87 6 wk 8.75 8.91 9.01 Difference (0-6 wk) 0.07 -0.09 0.31 Albumin height (mm) 0 wk 8.54 8.68 8.26 3 wk 8.31 8.71 8.56 6 wk 8.64 8.75 8.81 Difference (0-6 wk) 0.10 0.07 0.55 Haugh unit 0 wk 89.7 90.1 88.6 3 wk 90.1 90.5 91.4 6 wk 90.5 (b) 90.2 (b) 92.7 (a) Difference (0-6 wk) 0.80 (b) 0.10 (b) 4.10 (a) Items COS0.4 ANTCOS SEM (2) Egg shell breaking strength (kg/[cm.sup.2]) 0 wk 3.56 3.51 0.24 3 wk 3.46 3.53 0.26 6 wk 3.85 3.56 0.24 Difference (0-6 wk) 0.29 0.05 0.41 Egg shell thickness ([10.sup.-2] mm) 0 wk 35.5 35.2 0.46 3 wk 35.6 36.1 0.44 6 wk 36.3 36.5 0.32 Difference (0-6 wk) 0.80 1.30 0.76 Yolk color unit 0 wk 8.79 8.90 0.26 3 wk 9.10 9.00 0.22 6 wk 9.11 8.98 0.29 Difference (0-6 wk) 0.32 0.08 0.07 Albumin height (mm) 0 wk 8.51 8.36 0.41 3 wk 8.64 8.47 0.36 6 wk 8.77 8.56 0.47 Difference (0-6 wk) 0.26 0.20 0.10 Haugh unit 0 wk 89.4 90.5 1.89 3 wk 91.6 92.6 1.57 6 wk 92.9 (a) 93.6 (a) 1.28 Difference (0-6 wk) 3.50 (a) 3.10 (a) 1.09 (1) CON = Basal diet; ANT = Basal diet mg/kg avilamycin; COS0.2 = Basal diet+ mg/kg chito/oligosaccharide; COS0.4 = Basal diet+ mg/kg chito/oligosaccharide; ANTCOS = Basal diet+ mg/kg chito/oligosaccharide mg/kg avilamycin. (2) Standard error of the means. (a, b) Means in the same row with different superscripts differ (p<0.05). Table 4. Effects of chito-oligosaccharide on nutrient digestibility in laying hens (1) Items CON ANT COS0.2 Dry matter 72.1 (b) 74 9 (ab) 74.6 (ab) Nitrogen 61.7 (b) 64.3 (a) 63.9 (a) Items COS0.4 ANTCOS SEM (2) Dry matter 76.8 (a) 77.4 (a) 1.25 Nitrogen 65.7 (a) 67.1 (a) 2.19 (1) CON = Basal diet; ANT = Basal diet mg/kg avilamycin; COS0.2 = Basal diet+ mg/kg chito/oligosaccharide; COS0.4 = Basal diet+ mg/kg chito/oligosaccharide; ANTCOS = Basal diet+ mg/kg chito/oligosaccharide mg/kg avilamycin. (2) Standard error of the means. (a, b) Means in the same row with different superscripts differ (p<0.05). Table 5. Effects of chito-oligosaccharide on blood profiles in laying hens (1) Items CON ANT COS0.2 WBC (x [10.sup.4]/[mm.sup.3]) 0 wk 26.8 28.3 26.9 6 wk 28.7 (b) 29.1 (b) 30.2 (b) Difference (0-6 wk) 1.90 (b) 0.80 (b) 3.30 (ab) RBC (x [10.sup.6]/[mm.sup.3]) 0 wk 2.38 2.24 2.17 6 wk 2.41 2.19 2.28 Difference (0-6 wk) 0.03 -0.05 0.11 Lymphocyte (%) 0 wk 66.9 65.8 67.4 6 wk 68.9 69.1 68.7 Difference (0-6 wk) 2.00 3.30 1.30 Total protein (g/dl) 0 wk 5.36 5.12 5.24 6 wk 5.46 (b) 5.50 (b) 5.71 (ab) Difference (0-6 wk) 0.10 0.38 0.47 Albumin (g/dl) 0 wk 2.03 2.18 2.21 6 wk 2.19 2.29 2.26 Difference (0-6 wk) 0.16 0.11 0.04 Items COS0.4 ANTCOS SEM (2) WBC (x [10.sup.4]/[mm.sup.3]) 0 wk 27.1 28.4 1.49 6 wk 32.7 (a) 32.9 (a) 1.89 Difference (0-6 wk) 5.60 (a) 4.50 (a) 0.67 RBC (x [10.sup.6]/[mm.sup.3]) 0 wk 2.23 2.26 0.08 6 wk 2.31 2.30 0.10 Difference (0-6 wk) 0.08 0.04 0.04 Lymphocyte (%) 0 wk 67.1 66.9 4.24 6 wk 70.2 71.4 4.98 Difference (0-6 wk) 3.20 4.50 0.89 Total protein (g/dl) 0 wk 5.31 5.29 0.34 6 wk 5.68 (ab) 5.91 (a) 0.56 Difference (0-6 wk) 0.37 0.62 0.07 Albumin (g/dl) 0 wk 2.19 2.09 0.12 6 wk 2.46 2.51 0.11 Difference (0-6 wk) 0.27 0.42 0.04 (1) CON = Basal diet; ANT = Basal diet mg/kg avilamycin; COS0.2 = Basal diet+ mg/kg chito/oligosaccharide; COS0.4 = Basal diet+ mg/kg chito/oligosaccharide; ANTCOS = Basal diet+ mg/kg chito/oligosaccharide mg/kg avilamycin. (2) Standard error of the means. (a, b) Means in the same row with different superscripts differ (p<0.05).
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|Author:||Meng, Q.W.; Yan, L.; Ao, X.; Jang, H.D.; Cho, J.H.; Kim, I.H.|
|Publication:||Asian - Australasian Journal of Animal Sciences|
|Date:||Nov 1, 2010|
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