Effect of the supplementation of pig skin collagen on growth performance, organ weight, blood characteristics and intestinal microbiota in broilers

Korean Journal of Agricultural Science
Ji Seon An1Won Yun1Ji Hwan Lee1Han Jin Oh1Young Gwang Kim1In Kyu Bae1Kwon Jung Kim1Ju Ho Lee2Gok Mi Kim3Yang Il Choi1Jin Ho Cho1*

Abstract

This experiment was conducted to investigate the effects of pig skin collagen supplementation on growth performance, organ weight, blood characteristics, and intestinal microbiota in broilers. A total of 50 Ross 308 broilers were used for 2 weeks. The five dietary treatments were as follows: NC) basal diet, PC) NC + fish collagen powder 0.1%, T1) NC + pig skin collagen 0.1%, T2) NC + pig skin collagen 0.5%, and T3) NC + pig skin collagen 1.0%. The body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were not affected (p > 0.05) by the dietary treatments in this experiment. Additionally, there were no significant differences (p > 0.05) in the organ weights among the treatments. Broilers fed T1, T2 and T3 diets had higher (p < 0.05) white blood cell (WBC) counts than the broilers fed the NC and PC diets. The Lactobacillus counts in the excreta were improved (p < 0.05) in the broilers fed the T1 and T2 diets. Moreover, the Salmonella counts in the excreta were decreased (p < 0.05) in the broilers fed the PC and T1 diets. In conclusion, supplementation of pig skin collagen in diets improved the white blood cells (WBCs) in the blood and Lactobacillus counts in the excreta, and reduced the Salmonella counts in the excreta. However, when pig skin collagen was increased in the diets, there were no significant differences (p > 0.05). Therefore, the addition of 0.1% pig skin collagen in the feed provided beneficial effects on the blood characteristics and the intestinal microbiota environment.

Keyword



Introduction

Collagen is a fibrous substrate protein that is high in animal skins and accounts for about 30% of the total weight of biocompatible proteins (Oikarinen et al., 1992). Collagen peptide consists of proline, glycine and hydroxyproline. Also, it has functions such as physiological control function, superior biocompatibility, absorbability and low side effects (Iwai et al., 2005). Also, many studies have been conducted because collagen has safe biocompatibility due to its biological properties, such as biodegradability and low antigenicity (Maeda et al., 1999). Studies in diabetic rats showed the effect of preventing hyperlipidemia in blood lipid metabolism and lowering blood glucose (Kim et al., 2009b). And for collagen extract from shark skin, showed high antibacterial properties against S. enteritidis and E. coli (Kim et al., 2009d). The low-molecular enzyme decomposition extract from pig skin collagen also showed high antioxidant activity and the effect of protecting nerve cells against oxidative stress (Kim et al., 2013a). Like this, collagen has many characteristics such as arthritis treatment effects, lipid metabolism, antibacterial properties, and antioxidant effects, so many studies are conducted on humans and rats.

Collagen is especially high in fish, pigs and chicken. Among them, pig skin collagen has more stable properties and more year-round supply than any other collagen, but its molecular weight is about 300,000 Dalton. So, absorption in the body is relatively limited compared to other animal originated collagen (Jeon et al., 2016). For this reason, the demand for fish collagen, which is relatively small in molecular weight, has recently increased, and has been used for skin care, arthritis treatment (Yoo et al., 2008). But it is difficult to extract large amounts of fish collagen (Jeon et al., 2016). For the efficient use of pig skin collagen, a variety of processing methods are being studied, including enzyme treatment and radiation treatment for low molecularization (Cho et al., 2006; Yang and Shu, 2014). Accordingly, many studies have been conducted in humans and rats on skin anti-aging effects (Kim et al., 2009c), skin wrinkle betterment effects (Kang and Jeon, 2009), and skin barrier protection effects (Kim et al., 2011) using low-molecular pig skin collagen. However, research on livestock such as pigs and chickens is still scarce. This study was conducted to investigate the effects of pig skin collagen supplementation on growth performance, organ weight, blood characteristics, and intestinal microbiota in broilers.

Materials and Methods

The experimental protocol was approved and conducted under the guidelines of the Animal Care and Use Committee of Chungbuk National University.

Pig skin collagen

The experiment was conducted with pig skin collagen made by Chungbuk National University's Department of Animal Science. 6 Liter of distilled water and 3 kg of pig skin were put into an electronic pressure extractor (KS 220S, Kyungseo E&P, Korea) under 80℃ for 5 hours. After heating, insoluble collagen was ejected through the gauze. The collagen extract (CE) was adjusted to pH 3.0 by adding citric acid. CE was hydrolyzed at 35℃ for 5 hours using protease (Love me tender, H GROUP USA LLC, USA). CE was concentrated at 80℃ for 12 hours. CE was cooled down at room temperature for 20 minutes and was filtered to 9,450 Dalton using a 10,000 Dalton filter (Multi-Angle Light Scattering, Korea Basic Science Institute, Korea). Finalized extracted collagen from pig skin was stored at 4℃ for 24 hours and used immediately for feed additive.

Experimental design and animals

A total of 50 Ross 308 broilers (BW, body weight = 322.5 ± 0.3 g) were used in 2 weeks. Broilers were assigned to 5 treatments (2 replicate pens per treatment and 5 broilers per pen) in a randomized complete block. The experiment lasted for 2 weeks. The dietary treatments were as follows: (1) negative control, basal diet (NC), (2) positive control, basal diet + 0.1% fish collagen powder), (3) basal diet + 0.1% pig skin collagen (T1), (4) basal diet + 0.5% pig skin collagen (T2), (5) basal diet + 1.0% pig skin collagen (T3). The basal diets were formulated to meet or exceed the NRC (1994) requirements (Table 1). All broilers were allowed to consume feed and water ad libitum.

Table 1. Compositions of the basal diets (as-fed basis).

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y Contained per kg of diet: vit A, 10,000 IU; vit D3, 2,000 IU; vit E, 421 IU; vit K, 5 mg; riboflavin, 2,400 mg; vit B2, 9.6mg; vit B6, 2.45 mg; vit B12, 40 ug; niacin, 49 mg; pantothenic acid, 27 mg, biotin, 0.05 mg.

z Contained the mg per kg of diet: Cu 140 mg, Fe 145 mg, Zn 179 mg, Mn 12.5 mg, I 0.5 mg, Co 0.25 mg, Se 0.4 mg.

Sampling and measurements

The broilers were weighed individually, and body weight was recorded initially and end of the experimental period (2 weeks) to calculate body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR). At the end of the experiment, five broilers per pen were bled via the wing vein using a sterilized syringe, and blood samples were collected in tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, USA) immediately. Samples were centrifuged at 3000 × g for 15 min at 4℃ and plasma was stored at - 20℃. The concentrations of white blood cell (WBC), and red blood cell (RBC) were analyzed by using an automatic blood analyzer (ADVID 120, Bayer, USA). Immunoglobulin G (IgG), glucose, and blood urea nitrogen (BUN) were determined by using an automatic biochemistry analyzer (Hitachi 747, Tokyo, Japan). After blood collection, the same broilers were weighed individually and slaughtered by approved methods. The liver, spleen, and bursa of Fabricius removed and weighed. Organ weight was expressed as a percentage of BW.

Excreta samples were collected at the end of the experiment (2 weeks). The samples were stored at -20℃ until the experiment. Also, serial dilution (10-1 to 10-6) of samples was made using anaerobic diluents and placed on MacConkey agar plates (BD BBL, Maryland, USA), Lactobacilli MRS agar plates (Difco, Maryland, USA) and Salmonella shigella agar plates (BD BBL, Maryland, USA) to isolate the Escherichia coli (E. coil), Lactobacillus and Salmonella, respectively. The Lactobacilli MRS agar plates were then incubated for 48 h at 39℃ under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37℃ and the Salmonella shigella agar plates were incubated for 36 h at 37℃. The E. coli, Lactobacillus and Salmonella colonies were counted immediately after removal from the incubator.

Statistical analysis

Data were statistically analyzed by ANOVA using the GLM procedure SAS ver 9.4 (SAS Institute Inc., Cary, USA), with each pen being used as the experimental unit. Differences among all treatments were separated by Duncan’s multiple range tests. Variability in the data is expressed as the standard error (SE) and a probability level of p < 0.05 was considered to be statistically significant.

Results and Discussion

Growth performance

The effect of the supplementation of pig skin collagen in broiler feed on the growth performance is shown in Table 2. There were no significant differences (p > 0.05) among the NC and the other treatments at both in BWG, FI, and FCR.

Table 2. Effect of supplementation of pig skin collagen on growth performance in broilers.

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NC, basal diet; PC, basal diet + 0.1% fish collagen powder; T1, basal diet + 0.1% pig skin collagen; T2, basal diet + 0.5% pig skin collagen; T3, basal diet + 1.0% pig skin collagen; SE, standard error; BW, body weight; BWG, body weight Gain; FI, feed intake; FCR, feed conversion ratio.

The growth performance of broilers is always very important in the poultry industry (Liu et al., 2019). In an experiment by Park et al. (2012), the effect of collagen supplementation in 4-week-old rats showed significantly increased in BWG and FI compared to CON. However, in a study of SPF (special pathogen free) rats aged 8 weeks old, the results of direct oral administration of collagen showed no significant difference in the BWG and FI (Kim et al., 2009c). With a similar study, Matsuda et al. (2006) reported that the supplementation of pig skin collagen in weaning pigs was no significant difference in the BWG. This experiment has shown similar results in BWG, FI, and FCR. The collagen sample of this study is directly extracted and hydrolyzed collagen from pig skin, and since it is in liquid form, the size of the feed particles may increase due to lumps when adding collagen to feed. So, when the broilers fed diets, the feed intake may vary as the size of the larger particles makes it difficult to intake. As FI difference, the FCR may also increase the likelihood of error. Also, there may be differences follow as absorption levels of animals and the method of collagen supplementation such as oral administration or addition to feed.

Organ weight

The effect of the supplementation of pig skin collagen in broiler feed on the organ weight is shown in Table 3. There was no significant difference (p > 0.05) on organ weight among dietary treatments during the experiment.

Table 3. Effect of supplement of pig skin collagen on organ weight in broilers

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NC, basal diet; PC, basal diet + 0.1% fish collagen powder; T1, basal diet + 0.1% pig skin collagen; T2, basal diet + 0.5% pig skin collagen; T3, basal diet + 1.0% pig skin collagen; SE, standard error.

The liver, spleen, and bursa of Fabricius are the major immune-related organs of broilers, and as the weight increases, the immune function improves (Rivas and Fabricant, 1988). Until 35 days after hatching, bursa of Fabricius is typically larger than the spleen. However, if the spleen is larger than the bursa of Fabricius, it usually indicates a complex vaccine response and an immune suppression condition and is prone to outbreaks such as respiratory infections (Kim et al., 2013b). The results of this study showed that there was a tendency to increase the weight of the liver as collagen was added, but there were no significant differences among dietary treatments. Moreover, the weight of the bursa of Fabricius is greater than the weight of the spleen, so the addition of the pig skin collagen doesn't appear to affect the organ weight and immune organs of the broilers.

Blood characteristics

Table 4. showed the effect of pig skin collagen on blood characteristics throughout the experiment. The concentration of WBC in the blood of the broilers was significantly higher (p < 0.05) in T1, T2, and T3 treatments than NC treatment. However, the results of the concentration of RBC, glucose, BUN, and IgG were unaffected by pig skin collagen supplementation in this study.

The WBC, which responds to pathogens or stress environments and is involved in innate immune responses according to non-specific and specific immune responses (Paul, 1998) showed significantly high levels at T1, T2, and T3 treatments in this study. This can be attributed to increased WBC in the broilers blood, resulting in an immune control effect, but due to a lack of prior research, a precise study is needed. In a study by Lee et al. (2018), oral administration of collagen in 7-week-old rats showed no significant difference in glucose, which was consistent with the results of this study. BUN (blood urea nitrogen) is the final product of protein metabolism and is released into the blood through the liver (Cho et al., 2010). In this study, the addition of collagen does not appear to affect BUN, given that there is no significant difference among the dietary treatments. IgG, produced at B-cells in chicken bone marrow, is the highest concentration of immune proteins and is mainly responsible for in vivo immunity (Kim et al., 2009a). There are reports that higher IgG levels in the blood improve growth performance (Cetin et al., 2005), but there were no significant differences on IgG in the present study.

Table 4. Effect of supplementation of pig skin collagen on blood characteristics in broilers.

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NC, basal diet; PC, basal diet + 0.1% fish collagen powder; T1, basal diet + 0.1% pig skin collagen; T2, basal diet + 0.5% pig skin collagen; T3, basal diet + 1.0% pig skin collagen; SE, standard error; RBC, red blood cell; WBC, white blood cell; BUN, blood urea nitrogen; IgG, immunoglobin G.

a, b: Means in a row with different letters are significantly different (p < 0.05).

Intestinal microbiota

The results of the intestinal microbiota are presented in Table 5. The Lactobacillus counts in the excreta of broilers were significantly higher in T1 and T2 treatments (p < 0.05) and the Salmonella counts were higher in PC and T1 treatments (p < 0.05). However, there were no significant differences in E. coli counts in excreta among treatments (p > 0.05).

Table 5. Effect of supplementation of pig skin collagen on intestinal microbiota in broilers.

http://dam.zipot.com:8080/sites/kjoas/images/N0030460314_image/Table_kjoas_46_03_14_T5.jpg

NC, basal diet; PC, basal diet + 0.1% fish collagen powder; T1, basal diet + 0.1% pig skin collagen; T2, basal diet + 0.5% pig skin collagen; T3, basal diet + 1.0% pig skin collagen; SE, standard error; RBC, red blood cell; WBC, white blood cell; BUN, blood urea nitrogen; IgG, immunoglobin G.

a, b: Means in a row with different letters are significantly different (p < 0.05).

Kim et al. (2009d) reported that as a result of studies using collagen extracted from shark shells to experiment with antibacterial properties for S. enteritidis and E. coli, the size of the clean zone increased as the concentration of collagen increased and accordingly collagen causes to have the antimicrobial ability. In this study, Salmonella counts were significantly lower in the PC and T1 treatments, and this study agrees with precedent research. However, there was no significant difference in the concentration of E. coli, and it is considered to require a more precise study because it showed different results from the preceding study. For Lactobacillus, in the study of Kim et al. (2017), the mixed low-molecular collagen and L. brevis showed significantly higher levels in viable cell counts than the single-cultured treatment of L. brevis and were consistent with the results of this experiment.

However, the effect of T3 treatment with 1% collagen added in this study was not significant, which is estimated to be due to increased harmful bacteria resulting from the fermentation of high protein in the intestinal tract. Previous studies report that if protein content in feed increases above a certain level, intestinal pathogenic microorganisms (E. coli, Clostridium and Enterobacteriaceae) increase and viable cell counts of Lactobacillus decrease (Heo et al., 2013; Rist et al., 2014). In an experiment in broilers, the supplementation of high-level glycine, which a constituent amino acid of collagen, was significantly reduced the Lactobacillus counts of the extract and the Clostridium counts was significantly increased (Dahiya et al., 2005). This was similar to the results of this study, which 0.1% collagen supplementation in feed has been effective in improving the microbial environment, but 1% collagen addition has not been effective.

Conclusion

In this experiment, the addition of 0.1% pig skin collagen in the feed provided beneficial effects on blood characteristics and the intestinal microbiota environment. However, due to the lack of research on the appropriate level of addition of collagen, it is necessary to investigate the level of collagen addition in broiler feed.

Acknowledgements

This work (Grants No. C0505236) was supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Small and Medium Business Administration in 2017.

Authors Information

Ji Seon An, https://orcid.org/0000-0002-9205-8095

Won Yun, https://orcid.org/0000-0002-1835-2640

Ji Hwan Lee, https://orcid.org/0000-0001-8161-4853

Han Jin Oh, https://orcid.org/0000-0002-3396-483X

Young Gwang Kim, https://orcid.org/0000-0003-2093-695X

In Kyu Bae, https://orcid.org/0000-0001-6088-3624

KwonJung Kim, https://orcid.org/0000-0001-7873-3747

Ju Ho Lee, https://orcid.org/0000-0003-2222-5561

Gok Mi Kim, https://orcid.org/0000-0003-1053-4535

Yang Il Choi, https://orcid.org/0000-0002-3423-525X

Jin Ho Cho, https://orcid.org/0000-0001-7151-0778

References

1  Cetin N, Guclu BK, Cetin E. 2005. The effect of prebiotics and mannan-oligosaccharide on some haematological and immunological parameters in Turkey. Journal of Veterinary Medicine Series A 52:263-267.  

2  Cho SK, Jo CR, Jung SME, Kim MK, Oh HM, Lee BD, Lee SK. 2010. Effects of dietary quercetin on the feed utilization, blood parameters, and meat quality in Korean native goats. Journal of Animal Science and Technology 52:297-304. [in Korean]  

3  Cho YJ, Seo JE, Kim YJ, Lee NH, Hong SP, Kim YH. 2006. Study on the degradation of pigskin collagen using irradiation technique. Journal of the Korean Society of Food Science and Nutrition 35:588-593. [in Korean]  

4  Dahiya JP, Hoehler D, Wilkie DC, Van Kessel AG, Drew MD. 2005. Dietary glycine concentration affects intestinal Clostridium perfringens and lactobacilli populations in broiler chickens. Poultry Science 84:1875-1885.  

5  Heo JM, Opapeju FO, Pluske JR, Kim JC, Hampson DJ, Nyachoti CM. 2013. Gastrointestinal health and function in weaned pigs: A review of feeding strategies to control post‐weaning diarrhoea without using in‐feed antimicrobial compounds. Journal of Animal Physiology and Animal Nutrition 97:207-237.  

6  Iwai K, Hasegawa T, Taguchi Y, Morimatsu F, Sato K, Nakamura Y, Higashi A, Kido Y, Nakabo Y, Ohtsuki K. 2005. Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates. Journal of Agricultural and Food Chemistry 53:6531-6536.  

7  Jeon KH, Hwang YS, Kim YB, Choi YS, Kim BM, Kim DW, Jang AR, Choi JY. 2016. Quality characteristics of pork skin collagen with enzyme treatments. The Korean Society of Food and Nutrition 29:760-766. [in Korean]  

8  Kang SM, Jeon YS. 2009. Influence of collagen intake upon facial-skin wrinkles. The Korean Society for Aesthetics and Cosmetology 7:79-94. [in Korean]  

9  Kim CH, Shin KS, Woo KC, Paik IK. 2009a. Effect of dietary oligosaccharides on the performance, intestinal microflora and serum immunoglobulin contents in laying hens. Korean Journal of Poultry Science 36:125-131. [in Korean]  

10  Kim D, Park KM, Ha G, Jung JR, Chang O, Ham JS, Jeong SG, Park BY, Song J, Jang A. 2013a. Anti-oxidative and neuroprotective activities of pig skin gelatin hydrolysates. Korean Journal for Food Science of Animal Resources 33:258-267. [in Korean]  

11  Kim HS, Yoon HD, Seong JH, Lee YG, Xie CL, Kim SH, Choi WS. 2009b. Effects of soluble collagen peptides extract derived from Mugil cephalus scale on the blood glucose and lipid metabolism in diabetic rats. Journal of Life Science 19:1794-1801. [in Korean]  

12  Kim JK, Lee JH, Bae IH, Seo DB, Lee SJ. 2011. Beneficial effect of a collagen peptide supplement on the epidermal skin barrier. Korean Journal of Food Science and Technology 43:458-463. [in Korean]  

13  Kim JK, Lee JH, Yang MS, Seo DB, Lee SJ. 2009c. Beneficial effect of collagen peptide supplement on anti-aging against photodamage. Korean Journal of Food Science and Technology 41:441-445. [in Korean]  

14  Kim JW, Kim DK, Park JS, Lee YK, Beik KY, Kim SD. 2009d. Antioxidant and antimicrobial activities of shark collagens, and inhibitory actions on elastase and tyrosinase. Korean Journal of Food Preservation 16:419-426. [in Korean]  

15  Kim SC, Kim JW, Kim JU, Kim IH. 2013b. Effects of dietary supplementation of bacteriophage on growth performance, nutrient digestibility, blood profiles, carcass characteristics and fecal microflora in broilers. Korean Journal of Poultry Science 40:75-81. [in Korean]  

16  Kim SY, Oh DG, Kim KY. 2017. Preparation and characterization of double-layered cCoated capsule containing low molecular marine collagen and gamma-aminobutyric acid producing Lactobacillus brevis CFM20. Journal of The Korean Society of Food Science and Nutrition 46:857-867. [in Korean]  

17  Lee HJ, Woo M, Song YO, Noh JS. 2018. Inhibitory effect of skate skin collagen on hepatic lipid accumulation through regulation of lipid metabolism. Journal of The Korean Society of Food Science and Nutrition 47:235-242. [in Korean]  

18  Liu SD, Song MH, Yun W, Lee JH, Cho SY, Kim GM, Kim HB, Cho JH. 2019. Effects of a mixture of essential oils and organic acid supplementation on growth performance, blood profiles, leg bone length, and intestinal morphology in broilers. Korean Journal of Agricultural Science 46:285-292.  

19  Maeda M, Tani S, Sano A, Fujioka K. 1999. Microstructure and release characteristics of the minipellet, a collagen-based drug delivery system for controlled release of protein drugs. Journal of controlled release 62:313-324.  

20  Matsuda N, Koyama YI, Hosaka Y, Ueda H, Watanabe T, Araya T, Irie S, Takehana K. 2006. Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in the dermis. Journal of Nutritional Science and Vitaminology 52:211-215.  

21  NRC (National Research Council). 1994. Nutrient requirements of poultry, 9th. ed. National Academy Press, Washington D.C., USA.  

22  Oikarinen A, Autio P, Kiistala U, Risteli L, Risteli J. 1992. A new method to measure type I and III collagen synthesis in human skin in vivo : Demonstration of decreased collagen synthesis after topical glucocorticoid treatment. Journal of Investigative Dermatology 98:220-225.  

23  Park JE, Ham JS, Kim HK, Lee CH, Kim DW, Seol KH, Oh MH, Kim DH, Jang AR. 2012. Effect of pig skin gelatin hydrolysates on the bone mineral density of ovariectomized rats. Korean Journal for Food Science of Animal Resources 32:234-240. [in Korean]  

24  Paul WE. 1998. Fundamental immunology, 4th. Raven Press, New York. USA.  

25  Rist VT, Weiss E, Sauer N, Mosenthin R, Eklund M. 2014. Effect of dietary protein supply originating from soybean meal or casein on the intestinal microbiota of piglets. Anaerobe 25:72-79.  

26  Rivas AL, Fabricant J. 1988. Indications of immunodepression in chickens infected with various strains of Marek's disease virus. Avian Diseases 32:1-8.  

27  Yang H, Shu Z. 2014. The extraction of collagen protein from pigskin. Journal of Chemical and Pharmaceutical Research 6:683-687.  

28  Yoo SJ, Cho SM, Woo JW, Kim SH, Han YN, Ahn JR, Kim SY, Kim YW, Kim SB. 2008. Processing and physicochemical properties of collagen from yellowfin tuna (Thunnus albacares ) abdominal skin. Korean Journal of Fisheries and Aquatic Sciences 41:427-434. [in Korean]