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The effect of symbiotic supplementation of nutmeg flesh extract (Myristica fragrans Houtt) and Lactobacillus plantarum on microbial and intestinal morphology of broiler chickens

Yusri Sapsuha, Said Hasan1 and Amran Nur2

Department of Animal Science, Faculty of Agriculture, Universitas Khairun, Ternate, North Maluku, Indonesia
yus_ara01@yahoo.co.id
1 Department of Biology, Faculty of Teacher Training and Education, Universitas Khairun, Ternate, North Maluku, Indonesia
2 Department of Pharmacology, Biomedicine, Medical Faculty, Universitas Khairun, Ternate, North Maluku, Indonesia

Abstract

This study investigated the effect of symbiotic from nutmeg flesh and L. plantarum extracts on performance, microbial population, and intestinal morphology in broiler chickens. A total of 250 Lohmann broilers (unsexed) were arranged in a completely randomized design. During the first 7 days of rearing, chicks were raised communally on commercial starter feeds. On day 8, broilers (weight 188 2.62 g) were randomly assigned to one of five treatment groups, each replicated five times. Symbiotics from nutmeg flesh and L. plantarum extracts were added to the feed from day 8 as follows, 0, 0.5, 1, 1.5, and 2 ml/kg of diet. The results showed that symbiotic supplementation from nutmeg flesh and L. plantarum extracts increased (p<0.05) performance, villi length in the duodenum, jejunum, and ileum, increased the ratio of villi length: crypt depth in the jejunum and ileum, and increased acid bacteria. lactate. The symbiotic supplementation from nutmeg flesh and L. plantarum extracts also significantly reduced (p<0.05) coliform bacteria in the ileum and cecum during rearing.

Keywords: broiler, microbiology, morphology, performance, symbiotics


Introduction

Broiler chicken meat is the most popular choice for meeting people's animal protein needs in Indonesia because it has a high content of essential nutrients, low levels of saturated fat, and a low price, making it a significant contributor to the Indonesian economy (Astuti et al 2022). However, with the ban on the use of antibiotic growth promoters (AGP) in feed, the broiler industry is beginning to face sustainability issues. The long-term use of AGP may result in the development of antibiotic resistance and a negative impact on consumer health (Sugiharto, 2016). According to the literature, adding feed additives to broiler feed can help the chickens grow faster and stay healthy. The use of probiotics as a feed additive reduces the number of pathogenic bacteria in broiler intestines and increases feed digestibility, which improves growth rates (Hegarty et al 2016; Sarangi et al 2016). Prebiotics are another feed additive that can assist probiotics in increasing the population of non-pathogenic bacteria in broiler chicken intestines (Basturk et al 2016).

L. plantarum is a gram-positive bacterium that is widely used as a probiotic for broiler chickens (Wang et al 2015). It can help to maintain microbial balance, improve digestive function, and increase absorption in broiler intestines. Furthermore, it can modulate broiler immune responses, decrease E. Coli content in the cecum, and increase the number of lactic acid bacteria in the cecum and ileum (Peng et al 2016). In addition to bioactive substances, the flesh of nutmeg (Myristica fragrans Houtt) contains oligo and polysaccharides, which can act as prebiotics (Abourashed and El-Alfy., 2016). Prebiotics have been shown to improve food safety by increasing body weight gain, carcass weight, and digestibility, as well as reducing pathogenic bacteria (Sugiharto, 2016; Mohammed et al 2019).

Symbiotic is a combination of prebiotics and probiotics that has been shown to improve probiotic survival in the digestive tract (Anandharaj et al 2014). When compared to a single supplementation, symbiotic supplementation in broiler feed increases body weight, average daily gain, feed efficiency, and carcass yield (Bogucka et al 2019; Yuanita et al 2019). Mohammed et al. (2018) and Abdel-Wareth et al. (2019) also found that symbiotic supplementation increases broiler weight gain more effectively than probiotics or prebiotics alone. According to Dibaji et al. (2014), symbiotic dietary supplementation can increase the amount of LAB in the chicken’s digestive tracts, which leads to an increase in the immune system, an increase in nutrient digestibility and feed utilization, and a decrease in production costs a decrease in mortality.

Based on the symbiotic potential, the use of nutmeg flesh extract combined with L. plantarum is expected to be a symbiotic that can play an important role in increasing broiler chicken growth previously played by AGP. However, no studies on the use of nutmeg flesh extract as a symbiotic with L. plantarum have been found in the literature. This study aimed to investigate the symbiotic effect of nutmeg and L. plantarum flesh extracts on the microbial population and gut morphology of broiler chickens.


Materials and method

Symbiotic Manufacturing

Nutmeg flesh extract was prepared as per method described by Sapsuha et al. (2021), in which 1 kg of nutmeg flesh was macerated by immersing it in a 4 L of 96% ethanol solution for 3 x 24 hours. Stirring was done twice during the maceration process (in the morning and evening). The immersion results in the form of filtrate were then filtered and evaporated using a rotary evaporator to produce nutmeg flesh extract. Symbiotic preparation was performed by mixing 20 ml/100 ml distilled water extract with 10 ml Lactobacillus plantarum (bacterial concentration 1 x 109 cfu/ml). Symbiotics were stored in the refrigerator until use.

In vivo Experiment

The Animal Research Ethics Committee, Faculty of Agriculture, Universitas Khairun, approved in vivo experiments with approval number 04/KEPH/PH/2022. A total of 250 Lohmann broilers (unsexed) were arranged in a completely randomized design. During the first 7 days of rearing, chicks were raised communally on a commercial starter feed, which, according to the feed label, containing 22-24% crude protein, no more than 5% crude fiber, 5% crude fat, and 7% ash. On day 8, broiler chickens (weight 188 2.62 g) were randomly assigned to five treatments and each treatment was repeated five times. The feed given was in the form of mash and formulated (Table 1) as starter feed (days 1-21) and finisher feed (days 22-35). Symbiotic extracts of nutmeg flesh and L. plantarum were added to the feed from day 8 in the amount of 0.5, 1, 1.5, and 2 ml/kg of diet. Meanwhile, symbiotic extracts of nutmeg flesh and L. plantarum were not added to the control feed (level 0 ml/kg of diet). Feed and drinking water were provided ad libitum until day 35. All chickens were vaccinated with the commercial Newcastle disease (ND) vaccine on day 4 through eye drops and on day 18 through drinking water. Then, the chickens were vaccinated with Gumboro vaccine on day 12 through drinking water. During the rearing period, the chickens were kept in a ventilated broiler cage with rice husks as the base for the cage.

Table 1. Feed ingredients and nutrient composition of the research ration

Items (%, except that otherwise mentioned)

Starter (1-21)

Finisher (22-35)

Yellow corn

56.25

64.10

Fine bran

20.18

15.17

Fish flour

16.73

13.89

Palm oil

2.38

2.38

DL-methionine

0.25

0.25

Bentonite

1.11

1.11

Limestone

1.32

1.32

MCP

1.25

1.25

premix1

0.18

0.18

Chlorine chlorite

0.06

0.06

NaCl

0.29

0.29

Nutrient content based on laboratory analysis:

ME (kcal/kg)

2,935

3,082

dry matter

85.62

86.43

Crude protein

21.14

19.05

Extract ether

4.46

4.78

Crude fiber

3.42

3.33

Ash

8.59

9.71

1Premix contained (per kg of diet) of vitamin A 7,750 IU, vitamin D3 1,550 IU, vitamin E 1.88 mg, vitamin B1 1.25 mg, vitamin B2 3.13 mg, vitamin B6 1.88 mg, vitamin B12 0.01 mg, vitamin C 25 mg, folic acid 1.50 mg, Ca-d-pantothenate 7.5 mg, niacin 1.88 mg, biotin 0.13 mg, Co 0.20 mg, Cu 4.35 mg, Fe 54 mg, I 0.45 mg, Mn 130 mg, Zn 86.5 mg, Se 0.25 mg, L-lysine 80 mg, Choline chloride 500 mg, DL-methionine 900 mg, CaCO3 641.5 mg, Dicalcium phosphate 1500 mg

Chicken body weight, feed consumption, and feed efficiency were measured at the end of rearing. Data on daily weight gain was obtained by weighing the chickens on day 35 (final weight) minus the initial weight (day 14), and then divided by the length of rearing (21 days). Daily feed consumption was determined by dividing the total feed consumption during the study by the length of the study (28 days). Feed efficiency was calculated by dividing body weight during the study by feed consumption and then multiplied by 100%.

On day 35, one rooster was taken with a body weight close to the average body weight of the cage. The rooster was chosen to avoid physiological errors due to sex variations. The chicken was slaughtered, and the internal organs were then removed. To measure the population of intestinal bacteria, digesta was taken from the ileum and cecum and put into a sterile sample container. Digesta was also collected from the duodenum, jejunum, ileum, and cecum for measurement of pH values (using a Thermo Fisher Scientific Inc. electronic pH meter). For the study of the morphology of the small intestine, the intestine was taken approximately 2 cm from each part of the small intestine and put into a sample tube containing 10% neutral formalin buffer solution.

Measurement of villi height and crypt depth was carried out with an optical microscope equipped with a camera. Total coliform bacteria were counted on MacConkey agar (Merck KGaA) medium and incubated at 38C for 24 hours under aerobic conditions. This is done so that the coliform bacteria that grow on the media become red, then the bacterial colonies are counted. Calculation of total lactic acid bacteria (LAB) was carried out on de Man, Rogosa, and Sharpe agar (MRS, Merck KGaA) medium. After that it was incubated anaerobically at 38oC for 48 hours (Sugiharto et al 2017).

This study was conducted based on a completely randomized design. The data obtained were analyzed using ANOVA (SPSS version 16.0) and if there was a significant effect, the treatment would be carried out with the Dancan test.


Results and discussion

The performance data of broiler chickens fed with symbiotic extract of nutmeg flesh and L. plantarum are shown in Table 2. During the rearing period, higher body weight gain (p<0.05) was seen in the level 0.5, level 1, level 1.5 and level 2 versus level 0. Meanwhile, there was no significant difference (p>0.05) in ration consumption between level 0.5, level 1, and level 1.5 treatments as well as between level 1.5 and level 2 treatments. It was seen that the supplemetation of symbiotic extracts from nutmeg flesh and L. plantarum increased ration consumption (p<0.05) compared to level 0. Meanwhile, between level 1, level 1.5, and level 2 treatments, no significant difference was observed (p>0.05). Supplementation of symbiotic extracts of nutmeg flesh and L. plantarum up to a level of 2 ml/kg ration could improve feed conversion (p<0.05) compared to level 0, level 0.5, and level 1. However, there was no significant difference (p>0.05) compared to level 1.5.

Table 2. Performance of broilers chickens

Items

Level of symbiotic, ml/kg of diet

SEM

p

0

0.5

1

1.5

2

Daily weight gain (g)

69.05a

75.70b

76.45b

78.38bc

79.45c

2.74

<0.01

Daily feed intake (g)

113.22a

117.07b

118.85c

118.95c

118.97c

2.48

<0.01

Feed efficiency (%)

60.98a

64.66b

64.32b

65.89b

66.78bc

3.07

0.02

a,b,c Means in the same row with different superscripts differ at p<0.05

Several studies have found that supplementation symbiotics to broiler chickens can improve body weight gain and feed efficiency (Sugiharto, 2016; Sunu et al 2019; Mangisah et al 2021). However, there are no studies in the literature that explain the effect of using symbiotic extracts of nutmeg flesh and L. plantarum on increasing body weight gain in broiler chickens. However, the symbiotic effectiveness of nutmeg and L. plantarum flesh extracts could most likely be attributed to the synergistic action of various phytochemicals present in nutmeg flesh (Zakaria et al. 2015; Vangoori et al 2019; Adu et al 2020) and the role of probiotics from L. plantarum which can maintain microbial balance, improve digestive function and intestinal absorption of broilers (Setyaningrum et al 2019) that in turn can improve physiological conditions in chickens. The latter condition has an impact on increasing the utilization and efficiency of feed so as to improve the growth performance of broiler chickens. In addition, previous studies have shown that nutmeg flesh has antibacterial, antiparasitic, antifungal, anti-coccidiotic, and hepatoprotective properties that can stimulate the growth of beneficial bacteria, inactivate pathogenic bacteria, and facilitate nutrient metabolism and absorption in the digestive tract which in turn can increase growth performance of broiler chickens (Sapsuha et al 2021). Improvements in feed conversion observed in broilers fed symbiotics with nutmeg flesh extract and Lactobacillus plantarum showed evidence of better feed utilization than the control group. Another study revealed that the use of symbiotics in broiler chicken feed can increase intestinal digestibility which in turn increases the growth of broiler chickens (Rehman et al 2020).

Figure 1. Effect of level of symbiotic on feed intake Figure 2. Effect of level of symbiotic on liveweight gain

Figure 3. Effect of level of symbiotic on feed efficiency

The average pH and gut microbes of broiler chickens treated with symbiotic extract of nutmeg flesh and L. plantarum are presented in Table 3. The results of this study showed that the supplementation of symbiotic extract of nutmeg flesh and L. plantarum in broiler chickens significantly (p<0.05) decreased pH in the ileum and cecum of broiler chickens. However, it had no significant effect (p>0.05) on the pH of the duodenum and jejunum. As shown in Table 3, the supplementation of symbiotic extracts of nutmeg flesh and L. plantarum significantly (p<0.05) increased lactic acid bacteria and decreased (p<0.05) coliform bacteria in the ileum and cecum.

The supplementation of symbiotic extracts of nutmeg and plantarum flesh can lower the pH in the ileum and cecum. This study is in line with Sunu et al. (2020) who reported that supplementation of symbiotic extracts of garlic and L. acidophilus could reduce pH in the ileum and cecum. Carbohydrate content, especially oligosaccharides found in almost all plants can be a good substrate for the fermentation process that supports the growth of beneficial microbes. The fermentation process of lactic acid bacteria produces a high concentration of lactic acid, so it will affect the decrease in pH and reduce the growth of harmful bacteria (Nkukwana et al 2015).

The decrease in pH in the ileum and cecum due to the supplementation of symbiotics was associated with an increase in the population of lactic acid bacteria in the ileum and cecum. Lactic acid is a metabolite of lactic acid bacteria and is one of the components of organic acids that play a role in lowering pH. The decrease in pH in the ileum and cecum due to the supplementation of symbiotics reduces the number of pathogenic bacteria in the intestine while increasing nutrient digestibility. For the record, high acidity reduces the growth of pathogenic bacteria (Song et al 2014) so that the intestines become healthier.

In this study, supplementation of symbiotic extracts of nutmeg flesh and L. plantarum reduced the number of coliforms in the digesta ileum and cecum of broiler chickens. This result is consistent with Setyaningrum et al. (2019), which showed a decrease in the population of E. coli in the digesta ileum and cecum of broilers when given the symbiotic inulin from sweet potato and L. plantarum. Dunislawska et al. (2017), showed that the supplementation of symbiotic L. salivarius with galactooligosaccarides (GOS) can reduce the number of coliform bacteria in the intestines of broiler chickens. According to Dibaji et al. (2014), adding symbiotics to the feed of broiler chickens results in an increase in the amount of LAB in the digestive tract of chickens, which leads to an increase in the immune system, a reduction in mortality, an increase in nutrient digestibility and feed utilization, which consequently reduces production costs. Acidic conditions in the digestive tract, especially the ileum and cecum, support the balance of the digestive tract microflora, where the population of lactic acid bacteria increases and the population of pathogenic bacteria decreases (Yadav and Jha, 2019). Most pathogens grow in pH close to 7 or tend to be neutral. On the other hand, beneficial microorganisms live in acidic pH (5.8-6.2) and compete with pathogenic bacteria which may lead to competitive exclusion (Rahmani et al 2005). Lactic acid bacteria produce lactic acid which is able to maintain the pH of the digestive tract of broiler chickens to be acidic. In addition, acidic conditions in the digestive tract can increase the effectiveness of the process of absorption of nutrients by the walls of the digestive tract, so that nutritional needs are fulfilled, health status is maintained, and productivity is increased (Mabelebe et al 2014; Sugiarto 2016).

Table 3. pH and selected bacteria population in the intestine of broiler chickens

Items

Level of symbiotic, ml/kg of diet

SEM

p

0

0.5

1.0

1.5

2.0

pH

Duodenum

6.24

6.45

6.31

6.12

6.15

0.23

0.61

Jejunum

5.78

5.89

5.76

5.53

5.52

0.43

0.64

Ileum

6.24b

6.21b

5.63a

5.35b

5.32b

0.79

0.02

Cecum

7.72c

7.61c

6.67ab

6.46a

6.39a

0.54

<0.01

Lactic Acid Bacteria (log cfu/g)

Ileum

9.38a

10.31b

11.10bc

11.13bc

11.22c

0.87

p<0.01

Cecum

9.76a

10.74b

11.33c

11.35c

11.38c

0.79

p<0.01

Coliform (log cfu/g)

Ileum

7.80b

6.75b

5.75a

5.66a

5.51a

1.37

<0.01

Cecum

8.77b

7.79b

7.62b

7.62ab

6.99a

1.21

0.03

a,b,c Means on the same row with different superscripts differ at p<0.05)

Data on the intestinal morphology of broiler chickens treated with symbiotic extract of nutmeg flesh and L. plantarum are presented in Table 4. The results of this study showed that the supplementation of symbiotic extract of nutmeg flesh and L. plantarum significantly (p<0.05) increased villi length in duodenum, jejunum, and ileum and the ratio of villi length: crypt depth in the jejunum and ileum. However, there was no significant effect (p>0.05) on the depth of crypts in all segments of the small intestine and the ratio of villi length : crypt depth in the duodenum of broiler chickens.

Supplementation of symbiotic extracts of nutmeg flesh and L. plantarum can increase the length of the villi in the duodenum, jejunum, and ileum as well as the ratio of villi length : crypt depth in the jejunum and ileum in broilers reared at high density. The results of this study are in line with Bogucka et al. (2019) which stated that the supplementation of symbiotics increased the length of the villi and the depth of the crypts in the duodenum, jejunum, and ileum. According to Sugiharto (2016), some factors like pathogenic bacteria and stress have a negative effect on intestinal microflora or intestinal epithelium, resulting in changes in cell permeability as the body's natural resistance. Thus, harmful compounds and pathogenic bacteria easily penetrate the cells of the small intestine, which will interfere with metabolism, digestion, and absorption of nutrients. This condition can cause chronic inflammation of the intestinal mucosa, which eventually leads to villi height, digestion, and absorption. The reason symbiotic extracts of nutmeg flesh and L. plantarum produced a better impact on intestinal morphology was because nutmeg flesh contains bioactive compounds (Sapsuha et al 2021) which play a role in stimulating the proliferation and growth of cells in the digestive tract. Thus, this can result in a greater villi height and a deeper crypts, while probiotics can reduce pathogenic bacteria, reduce mucosal infections, and maintain the structural integrity of the intestine for better nutrient absorption (Sethiya, 2016). The mechanism by which symbiotics provide benefits in improving intestinal morphology is that symbiotics in general can protect intestinal tissue from microbial attack (Sugiharto, 2016).

Table 4. Intestinal morphology of broiler chickens

Items

Level of symbiotic, ml/kg of diet

SEM

p

0

0.5

1.0

1.5

2.0

Duodenum

Villi height (m)

1232.85a

1276.86b

1278.26b

1314.15c

1314.17c

112.53

<0.01

Crypt depth (m)

111.34

111.23

110.48

110.11

109.87

12.92

0.23

VH/CD

11.08

11.48

11.58

11.94

11.96

1.78

0.62

Jejunum

Villi height (m)

1002.43a

1017.84b

1017.98b

1020.71c

1020.89c

87.34

<0.01

Crypt depth (m)

98.74

98.11

96.78

93.45

93.11

2.27

0.72

VH/CD

10.15a

10.38a

10.52a

10.92b

10.97b

1.27

0.03

Ileum

Villi height (m)

698.34a

705.67b

715.14c

718.98bc

718.98c

117.23

<0.01

Crypt depth (m)

80.87

80.21

78.22

72.75

71.23

3.67

0.41

VH/CD

8.64a

8.80a

9.14ab

9.87b

10.10b

1.32

0.02

a,b,c Means in the same row with different superscripts differ at p<0.05


Conclusion

The use of symbiotic extracts of nutmeg flesh and L. plantarum has a positive impact on performance, improves intestinal morphology, by increasing lactic acid bacteria and reducing coliform bacteria during rearing.


Acknowledgments

The project was financed by Ministry of Education, Culture, Research, and Technology, Republic of Indonesia, through “Penelitian Dasar Unggulan Perguruan Tinggi (PD-UPT)” scheme, year 2022 (Contract No. 112/E5/PG.02.00.PT/2022).


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