Effects of dietary supplementation of waste date’s vinegar on performance and improvement of digestive tract in broiler chicks

Document Type: Original Article

Authors

1 Department of Animal Science Researches, Agriculture and Natural Resources Education and Research Center of Kerman, Kerman, Iran

2 Graduate Student, Department of Animal Science, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

Abstract

Two hundred 1-day-old commercial broilers (Ross 308) were used to determine the effects of diets supplementation with waste date’s vinegar (WDV) on the growth and performance of digestive tract over a 42-days growing period. Chicks were randomly allocated to one of five experimental diets supplementing as 0 (control), 1, 2, and 3% of WDV and 2% industrial vinegar (IV). Broilers and their feed consumptions were weighed at the trial beginning and days 10, 21, 35 and 42 of experimental period. Moreover, one chick from every replicate was killed at days 21 and 42 to measure development of digestive tissues and morphology and microbiology of small intestine. Although the final body weight was higher following IV and 1% WDV usage, feed conversion ratio was negatively affected by IV usage compared to control (p < 0.05). Relative weight of different parts of small intestine was not affected by experimental diets. Villus height and width were reduced linearly in WDV, IV and control groups (p < 0.05), but crypt depth was not different among experimental diets. Also, ileum microbiota was not affected by treatments. Results indicated that diet supplementation with WDV has positive effects on growth performance and histomorphology of jejunum in broilers.

Keywords

Main Subjects


Introduction

 

Nowadays, efforts have been made to produce high quality animal products without using medicines and reduce environmental contamination by efficient utilization of natural substances. Researchers worldwide are working on organic alternatives due to the ban of a wide range of drugs for animal production. Probiotics consisting of live or dead organisms and spores,1 non-traditional chemicals,2 bacteriophages,3 acidifiers, enzymes and others have emerged in the last decades as some of the tools that could be potentially useful in the near future for pathogen control and poultry performance improvement. Some of these natural substances are not cited in the scientific literature, but are used locally. Such one application in poultry diets, is waste date’s vinegar (WDV) inclusion to diets in south of Iran. Annual production of waste date in Iran, which is not used by human, is 160,000 tones that could be used in poultry diets after removing the kernels. On the other hand, after some processing, WDV could be produced that its main component is acetic acid. Acetic acid is one of the main short chain fatty acids produced by intestinal microbes, which can affect intestinal functions and metabolism.4-6 In addition to these effects of WDV; it includes some beneficial bacteria such as lactobacillus spp. that can improve performance, immunity and digestive tract in broiler chicks. A strikingly crucial event in the development of probiotics was the finding that newly hatched chicks could be protected against colonization by Salmonella enteritidis throughdosing a suspension of gut contents derived from healthy adult chickens which is called competitive exclusion. Also, use of probiotics containing lactobacilli offers S. enteritidis and E. coli growth inhibition in broilers.7 Waste date’s vinegar’s lactobacillus as a probiotic could be settled in small intestine and as a result, causes infectious bacteria such as Salmonella and E. coli reduction. The intestine seems to be the most fundamental organ for improving animal products. Activation of intestinal function of broilers might increase the meat products in response to an increasing demand for animal protein.8 Therefore, it was interesting to investigate how intestinal histology would be affected after WDV feeding. In this study, effects of dietary WDV on body weight gain (BWG) and feed intake (FI) and efficiency were examined in broiler chicks. Then, weight and length of different tissues of digestive tract were measured. Also, jejunum villus height and crypt depth were measured and amounts of ileum lactobacillus and E. coli were counted.

 

Materials and Methods

 

The experiment was conducted at a commercial broiler farm in Kerman, Iran. Kerman has located at dry and arid area with average annual rainfall of 200 mm and maximum annual temperature of 40 ˚C with height of 1500 to 2000 m. The experimental protocols were reviewed and approved by the Animal Care Committee of Research Institute of Animal Science, Karaj, Iran.

Preparation of WDV. Almost one ton of fresh waste date in a commercial vinegar-making workshop in Kerman province was soaked in water, reduced to pulp; the kernels were removed and then, combined with water in ratio of approximately 1:3 waste date and water respectively to produce WVD before being used in the present study.

Chemical and microbial analyses. Representative samples of WDV and industrial vinegar (IV) were analyzed for percentages of acetic acid (titration method with a colored pH indicator) which were 2.60 and 10.40, respectively. Also, samples of WDV were tested for microorganism existence (microbial culture method) which showed lactobacillus, bacillus and mold existence with total account of 4.50 × 105 cfu. Table 1 shows the chemical and microbial composition of IV and WDV. Also, it is noticeable that to ignore the effects of different percentages of acetic acid in two vinegars,9 during all experiment, IV was four times diluted with distilled water before use.

Animals, diets and experimental design.Two hundred one-day-old mixed sex broilers (Ross 308) weighing 40.00 ± 1.50 g were allocated to five experimental diets in a balanced completely randomized design (n = 4) with 20 pens (1.50 × 0.70 m2 each) and 10 chicks in each pen. Experimental diets were supplemented using incremental levels of WDV including 0 (control), 1, 2 and 3% of diet and 2% of IV. Table 1 presents the chemical composition of diet10 and all chicks had free access to feed and water ad-libitum. Chicks were raised under similar environmental conditions based on Ross 308 management recommendations for 42 days.11

Table 1. Ingredients and chemical composition of diets and chemical and microbial composition of vinegars.

Ingredients of diet (%)

Diets per days

1-10

11-21

22-35

36-42

Corn meal

58.40

60.40

64.70

66.20

Soybean

35.50

33.50

30.20

28.70

Commercial concentrate1

6

6

5

5

Salt

1

1

1

1

Chemical composition of diet (%)

ME (Kcal kg-1)

2875

2900

2950

2963

Crude protein

21.80

21.10

19.80

18.70

Calcium (%)

1.25

1.25

1.05

1.05

Absorbable phosphorous

0.57

0.56

0.49

0.49

Sodium

0.18

0.18

0.16

0.14

Lysine

1.30

1.25

1.14

1.10

Methionine

0.49

0.48

0.43

0.43

Methionine-Cysteine

0.85

0.80

0.75

0.72

Tryptophan

0.26

0.26

0.25

0.25

Chemical and microbial composition of vinegars

Kind of vinegars

Acid acetic (g 100 mL-1)

 

IV

10.40

 

Acid acetic (g 100 mL-1)

 

WDV

2.60

 

Total Count (cfu g-1)

 

WDV

4.50 × 105

 

1 Made by Arshia Sepehr Co. in Tehran, Iran; IV: Industrial vinegar; WDV: Waste date’s vinegar.

 

Experimental period lasted 42 days. The feed amounts offered and refused were measured periodic at days 10, 21, 35 and 42 for each pens to calculate FI. Moreover, the chicks were weighed at these days after 2 hr of fasting to reduce the disputes arising from feed consumption and these weights were used to calculate the body weight (BW) changes and average periodic weight gain of chicks over the experimental time. By having these two measure-ments, feed conversion ratio (FCR) was calculated as feed consumed per unit of gain. Before the beginning of experiments, all animals were vaccinated for bronchitis and routine vaccinations including Newcastle (at days 8, 17 and 28) and Gambro (at days 13 and 24) were done during the growing period. Chicks were visited daily in a regular program for general health and some individual behaviors including illness, breath and anorexia.

Digestive tract sampling and analysis. At days 21 and 42 of experimental period, four chicks from every treatment (from two pens: male, and from two pens: female, with average weight about 20 g) were sacrifices by cervical dislocation to measure relative weight of different parts of small intestine including duodenum, jejunum and ileum as organ weight (g) / live body weight (g) as well as morphology and microbiology of jejunum and ileum.

Jejunum morphology and analysis. For histo-pathological and morphometric analysis, 0.50 cm tissue samples from the jejunum of above-mentioned chicks were obtained and fixed in 10% buffered formalin (100 mL of 40% formaldehyde, 4 g phosphate, 6.50 g dibasic sodium phosphate and 900 mL of distilled water) for 24 hr and then, 10% buffered formalin was renewed. Tissues were dehydrated by transferring through series of alcohols with increasing concentrations, placed into xylene and embedded in paraffin. A microtome was used to make five cuts that were 5 μm. The paraffin sections were stained with hematoxylin and eosin.12 The values were measured with a light microscope (model DM 1000 LED; Leica Microsystems GmbH, Wetzlar, Germany) using a software (LEICA Queen 550; Leica Microsystems GmbH). Measurements of villus height and width and crypt depth were determined at a magnification of 10×. A minimum of five measurements per slide were made for each parameter and averaged into one value.

Analysis of ileum microflora. Digesta were obtained from ileum of above-mentioned chicks and collected in sterile bags to count lactobacillus and E. coli. Digesta samples were homogenized with 1 mL normal saline. Aliquots (5 μL) were mixed with blood agar and eosin methylene blue and incubated at 37 ˚C for 24 hr. Then, bacteria colonies were counted in selective agar media for enumeration of target bacterial groups. The microbial counts were determined as cfu per gram of wet samples.13

Statistical analysis. Data obtained from performance (BWG per period, FI per period and FCR) were analyzed using repeated measurements model in which the time series (1-10, 11-21, 22-35 and 36-42) covariance structure was modeled by using four different covariance structures for each variable tested and the means were compared using Tukey’s multiple comparisons procedure. Other variables based on a completely randomized design were statistically analyzed using GLM procedure in SAS (version 9.1; SAS Institute, Cary, USA) and the means were compared using Duncan’s multiple comparisons procedure. Statistical models were as follows:

Dependent variable (Yijk) = μ+Ti+Pj+Ti×Pj+eijk

and Completely randomized design:

Yij = μ+Ti+eij

where, μ is overall mean, Ti is treatment, Pj is period effect and eij is random error.

 

Results

 

Animal performance. Data of performance variables are presented in Table 2. The final BW was increased by usage of IV and 1% WDV in comparison to control (p < 0.05) but average periodic weight gain was not different between treatments. Feed intake was reduced in control group as compared with 1% WDV treatments (p < 0.05) but FCR was negatively affected by use of IV in the diets as compared with control (p < 0.05).

 

Table 2. Effect of industrial vinegar (IV) and different levels of waste date’s vinegar (WDV) on performance of broilers.

Parameters

Groups

p-value

SEM

Control

2%IV

1%WDV

2%WDV

3%WDV

Treat

Period

Treat × Period

 

Final BW

2128.40b

2226.00a

2250.20a

2201.40ab

2161.40ab

0.050

28.546

BWG

521.53

545.96

552.13

539.92

529.66

0.053

< 0.0001

0.006

10.079

FI

945.40b

1023.81ab

1037.58a

991.95ab

997.19ab

0.055

< 0.0001

0.012

29.194

FCR

1.84b

1.97a

1.91ab

1.87ab

1.92ab

0.040

< 0.0001

0.018

0.039

BW: body weight; BWG: body weight gain per period; FI: feed intake per period; FCR: feed conversion ratio.

ab Means within a row with different subscripts differ significantly (p < 0.05).

 

Small intestinal growth. Table 3 shows growth (relative weight) of different parts of small intestine. All data (relative weights of duodenum, jejunum and ileum)  showed no significant difference between treatments.

Table 3. Effect of industrial vinegar (IV) and different levels of waste date’s vinegar (WDV) on relative weight (g/g) × 100 of different parts of small intestine of broilers.

Parameters

Treatments

p-Value

SEM

Control

2%IV

1%WDV

2%WDV

3%WDV

21st day

Relative weight of duodenum

1.38

1.59

1.41

1.33

1.31

0.38

0.0010

Relative weight of jejunum

2.61

2.69

2.53

2.48

2.23

0.60

0.0020

Relative weight of ileum

2.08

2.27

1.96

2.11

1.82

0.23

0.0013

42nd day

Relative weight of duodenum

0.82

0.89

0.72

0.72

0.70

0.16

0.0006

Relative weight of jejunum

1.75

1.94

1.66

1.87

1.60

0.25

0.0011

Relative weight of ileum

1.44

1.46

1.28

1.46

1.26

0.48

0.0010

ab Means within a row with different subscripts differ significantly (p < 0.05).

 

Jejunum histomorphology. Table 4 shows the intestinal morphology characteristics of broilers including villus height and width, crypt depth and ratio of villus height to crypt depth. Villus height and width increased linearly in control, IV and WDV treatments at 21st and 42nd day of growing period (p < 0.05) which showed the beneficial impression of WDV use on villus height and width of broilers. Crypt depth and ratio of villus height to crypt depth were not affected by feeding of experimental diets.

Ileum microbiota. According to Table 4, none of ileum microflora variables (including lactobacillus and E. coli spp. count) were affected by feeding of experimental diets, but WDV and IV supplementation maintained populations of unprofitable bacteria or potential pathogens (E. coli) at relatively low levels (numerically) in the ileum’s digesta. Furthermore, Salmonella spp. were not detected in content of ileum of broilers while sampling days over the entire of the experimental period.

 

Table 4. Effect of industrial vinegar (IV) and different levels of waste date’s vinegar (WDV) on jejunum morphology and ileum microflora of broilers.

Parameters

Treatments

p-Value

SEM

Control

2%IV

1%WDV

2%WDV

3%WDV

21st day

Villus height (µm)

989.53b

1070.11b

1199.80a

1268.00a

1177.96a

0.0002

32.873

Villus width (µm)

125.96c

176.53b

199.42a

200.32a

195.52ab

0.0001

6.937

Crypt depth (µm)

193.80

197.63

212.23

197.95

219.96

0.87

20.462

Villus height : Crypt depth

5.15

5.53

5.80

6.62

5.72

0.60

0.646

42nd day

Villus height (µm)

1188.16b

1248.26ab

1293.70a

1331.69a

1258.91ab

0.049

30.587

Villus width (µm)

175.25b

207.65a

213.09a

225.62a

200.87ab

0.018

9.381

Crypt depth (µm)

246.31

228.55

233.97

239.06

273.77

0.47

18.438

Villus height : Crypt depth

4.96

5.63

5.48

5.69

4.72

0.53

0.477

21st day

Lactobacillus (Log cfu g-1)

4.61

4.69

4.81

4.88

4.53

0.42

0.139

E. coli (Log cfu g-1)

4.69

4.17

3.97

3.84

4.42

0.30

0.295

42nd day

Lactobacillus (Log cfu g-1)

4.46

4.56

4.70

4.84

4.34

0.42

0.193

E. coli (Log cfu g-1)

4.73

4.22

4.05

3.97

4.47

0.39

0.299

abc Means within a row with different subscripts differ significantly (p < 0.05).

 

 Discussion

 

The performance data are in line with results which reported significantly increase in final BW and improve-ment of FCR by feeding vinegar (including 5% acetic acid) and probiotics in broilers14 and better BW and FCR in broilers fed organic acids,15 but some have reported that supplementing diet by bamboo vinegar solution does not affect the final BW, FI and FCR of ducks.8 Feed intake in chicken is a function of nutritive requirements but on the other hand, supplementing diet by both industrial and waste date vinegar causes the incensement of palatability of feed which may lead to increment of FI.16,17 Based on this theory, in this experiment, all groups containing vinegar have increased FI and positively affected final BW.

Acetic acid in vinegar reportedly increases gastric proteolysis and improves digestibility of proteins and amino acids.18 In addition, this acid inhibits growth of harmful intestinal bacteria which compete with the host animal for available nutrients.19 The combination of acetic acid and probiotic in WDV is the main reason which controls the balance of intestinal microflora and positively affects intestinal functions and metabolism compared with IV.

The results of small intestine growth are in line with the findings observed no changes in relative weight of different parts of small intestine of broilers derived from probiotic supplementation in diet.20 On the other hand, use of butyric acid21 and acetic acid22 in broiler diet caused an increase of relative weights of jejunum and ileum of broilers.

Jejunum histomorphology analysis results are in agreement with an increase of jejunum villus height and area on male broilers fed bamboo vinegar liquid.8

It is well demonstrated that organic acid in vinegar increases the solubility of nutrients and improves the gastric proteolysis which develops digestibility of proteins and amino acids18 and probiotic produces antimicrobial substances and protects the villi and absorption surface against toxins23 and mainly promotes secretion of digestive enzymes24 that all of these reasons lead to increase in the absorption of available nutrients, a mechanism that directly affects the recovery of the intestinal mucosa, increasing villus height and better intestinal function.23

Our findings about the ileum microbiota were not confirmed with the results reported decrease of adverse bacteria in gut microflora of broilers by use of probiotics25,26 and decrease of gram negative bacteria following probiotic and organic acid usage in broiler chicks.27 It is generally documented that there are two basic mechanisms by which probiotics act to maintain a beneficial microbial population including competitive exclusion and immune modulation. Competitive exclusion involves competition for substrates, production of antimicrobial metabolites that inhibit the pathogens and competition for attachment sites.28 Also, through direct interaction with gut mucosal immune system, probiotics can modulate either innate or acquired immunity, or both to protect the increase in amount of pathogens in gut.29

In conclusion,results from current experiment indicate that usage of 1%WDV and IV increases final BW of broiler chicks but IV has adverse effect on FCR. In addition, intestinal morphology is improved by adding WDV to diet of broilers. In conclusion, WDV can be supplemented to diets of broilers to improve growth performance.

 

Acknowledgements

 

The authors gratefully appreciate Dr. Mohammad Mahdi Arabnejad (the producer of WDV and the financial provider of this study) for his support and cooperation.

 

 

  1. Patterson JA, Burkholder KM. Application of prebiotics and probiotics in poultry production. Poult Sci 2003; 82(4): 627-631.
  2. Moore RW, Byrd JA, Knape KD et al. The effect of an experimental chlorate product on Salmonella recovery of turkeys when administered prior to feed and water withdrawal. Poult Sci 2006; 85: 2101-2105.
  3. Higgins JP, Higgins SE, Guenther KL, et al. Use of a specific bacteriophage treatment to reduce Salmonella in poultry products. Poult Sci 2005; 84: 1141-1145.
  4. Bergman EN. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Phys Rev 1990; 70: 567-590.
  5. Kishi M, Fukaya M, Tsukamoto Y, et al. Enhancing effect of dietary vinegar on the intestinal absorption of calcium in ovariectomized rats. Biosci Biotech Biochem 1999; 63: 905-910.
  6. Lutz T, Scharrer E. Effect of short-chain fatty acids on calcium absorption by the rat colon. Exp Physiol 1991; 76: 615-618.
  7. Murry AC, Hinton A, Buhr RJ. Effect of botanical probiotic containing lactobacilli on growth performance and populations of bacteria in the ceca, cloaca, and carcass rinse of broiler chickens. Int J Poult Sci 2006; 5(4): 344-350.
  8. Ruttanavut, J, Yamauchi K, Goto H, et al. Effects of dietary bamboo charcoal powder including vinegar liquid on growth performance and histological intestinal change in Aigamo ducks. Int J Poult Sci 2009; 8 (3): 229-236.
  9. Scharf W, Malerich C. Determination of acetic acid content of vinegar. New York, USA: Natural Sciences 2010: 10010.
  10. NRC. Nutrient requirements of poultry. Washington, USA: National Academy of Science Press 1994:19.
  11. Aviagen. Ross broiler management manual. Scotland. 2009. Available at: www.aviagen.co. Accessed:
    23 Feb, 2015.
  12. Thompson KL, Applegate TJ. Feed withdrawal alters small-intestinal morphology and mucus of broilers. Poult Sci 2006; 85(9): 1535-1540.
  13. Boyd DA, Mulvey MR. CNPT protocol presented at the CACMID (Canadian association for clinical micro-biology and infectious disease). Meeting by Public Health Agency of Canada. Toronto: Canada, 2013.
  14. Kral M, Angelovicova M, Mrazova L, et al. Probiotic and acetic acid effect on broiler chickens performance. Anim Sci Biotech 2011; 44 (1): 62-64.
  15. Kamal AM, Ragaa, NM. Effect of dietary supplementation of organic acids on performance and serum biochemistry of broiler chicken. Nat Sci 2014; 12(2): 38-45.
  16. Esmail SH. Factors affecting feed intake of chickens. Available at: http://www.poultryworld.net /Broilers/ Nutrition. Accessed 29 March, 2013.
  17. Jeffri D, Firman H, Kamyab A. Comparison of soybean oil with an animal/vegetable blend at four energy levels in broiler rations from hatch to market. Int Poult Sci 2010; 9: 1027-1030.
  18. Samanta S, Haldar S, Ghosh TK. Comparative efficacy of an organic acid blend and bacitracin methylene disalicylate as growth promoters in broiler chickens: effects on performance, gut histology and small intestinal milieu. Vet Med Int 2010; doi: 10.4061/ 2010/645150.
  19. Dhawale A. Better eggshell quality with a gut acidifier. Poult Int 2005; 44: 18-21.
  20. Seifi S. An investigation of the effects of using an enzyme-probiotic combination on broilers performance. Iran J Vet Med 2013; 7(4): 299-304.
  21. Mahdavi R, Torki M. Study on usage period of dietary protected butyric acid on performance, carcass characteristics, serum metabolite level and humoral immune response of broiler chickens. Anim Vet Adv 2009; 8(9):1702-1709.
  22. Furuse M, Yang SI, Niwa N, et al. Effect of short chain fatty acids on the performance and intestinal weight in germ-free and conventional chicks. Br Poult Sci 1991; 32(1):159-165.
  23. Pelicano ERL, Souza PA, Souza HBA, et al. Intestinal mucosa development in broiler chickens fed natural growth promoters. Braz J Poult Sci 2005; 7(4):221-229.
  24. Ledezma-Torres R, Posadas-Cantu A, Espinosa-Leija R, et al. Effect of adding different levels of probiotics to broilers’ diets on gastrointestinal tract development and production performance. Afr J Microbiol Res 2015; 9(12): 892-897.
  25. Biernasiak J, Slizewska K. The effect of a new probiotic preparation on the performance and faecal microflora of broiler chickens. Vet Med 2009; 54 (11): 525-531.
  26. Decroos K, Vercauteren T, Werquin G, et al. Repression of clostridium population in young broiler chickens after administration of a probiotic mixture. Commun Agri Appli Biol Sci 2004; 69: 5-13.
  27. Gunal M, Yayli G, Kaya O, et al. The effects of antibiotic growth promoter, probiotic or organic acid supple-mentation on performance, intestinal microflora and tissue of broilers. Int J Poult Sci 2006; 5 (2): 149-155.
  28. Yang Y, Iji PA, Choct M. Dietary modulation of gut microflora in broiler chickens: a review of the role of six kinds of alternatives to in-feed antibiotics. World Poult Sci 2009; 65: 97-114.
  29. Dugas B, Mercenier A, Lenoir-Wijnkoop I, et al. Immunity and prebiotics. Immunol Today 1999; 20: 387-390.