Effects of inclusion of herbal extracts in drinking water on growth performance and fecal E. coli counts in growing Japanese quails

Nguyen Vu Thuy Hong Loan¹, Pham Luong Thuc Nhi¹, Truong Minh Loc¹ and Trinh Thi Lan Anh¹

¹ HUTECH University, Faculty of Veterinary and Animal Science, P25, Binh Thanh, Ho Chi Minh City 717.000, Viet Nam
nvth.loan@hutech.edu.vn

Abstract

The study aimed at evaluating the effect of inclusion of herbal extracts in drinking water on growth performance and fecal E. coli counts in growing quails. A total 270 growing quails of 3 old weeks were randomly located to 3 treatments with 3 replicates. In treatment 1 as control (CT), the birds were fed basal diet and ad lib drinking water; meanwhile, the birds in treatments 2 and 2 were given guava leaf extract (3 g/L) and sapodilla leaf extract (4 g/L) namely GT and ST, respectively. The experiment lasted 3 weeks. The results show that final weight and average daily gain (ADG) in the GT and ST are higher than in the CT. The ADG values in GT and ST range 2.45-2.51 g and are higher in the CT (2.13 g). Fecal E. coli count at 10 ^-5 is lowest in the GT (2 CFU/g) and highest in the CT (22.4 CFU/g). In conclusion, the inclusion in drinking water of guava and sapodilla leaf extract improved efficiently growth performance, and significantly reduced E. coli count in feces of growing quails of 3-6 weeks of age. The extracts from guava and sapodilla leaves could be used as E. coli anti-bacterial and growth stimulates, however, guava leaf extract is better than sapodilla one.

Keywords: guava leaf extract, E. coli count, sapodilla leaf extract

Introduction

In Viet Nam, Japanese quail production has been rapidly developing (Nguyen Hai Quan et al 2023). During 2015-2020, quail population and meat production annually increased 11% and 37.9%, respectively. In 2020, quail population and meat production were approximate 31,410 thousand heads and 8,650 tones, respectively. However, some diseases including intestinal once caused by E. coli and Salmonella are main health problems in quail production.

Escherichia coli (E. coli) and Salmonella are common Gram-negative bacteria that cause gastrointestinal diseases in poultry causing significant economic losses to the livestock industry. In particular, these are diseases that can be transmitted from animals to humans (zoonoses), mainly through contaminated food, causing public health problems (Newman et al 2021). Currently, there is no vaccine that is directly effective in protecting chickens from infection, mainly due to the wide variety of bacterial strains and lack of cross-protection (Mehat et al 2021). In fact, both E. coli and Salmonella have been listed by the WHO as among the pathogens requiring antibiotic susceptibility determination before treatment (WHO 2021). The problem of antibiotic resistance is increasing due to overuse of antibiotics in livestock farming, causing many common antibiotics to be resistant to E. coli and Salmonella spp., reducing the effectiveness of antibiotics and changing the microbiome in intestinal tract and negatively affects health (Farhat et al 2023). Therefore, it is necessary to look for antibiotic alternatives such as prebiotics or/and probiotics that include live microorganisms and their metabolites, which when administered orally will bring health benefits to the host (Shim et al 2012). 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 (Sarangi et al 2016; Sapsuha et al 2023). 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). Nguyen Thi Thuy et al (2022) indicated that, inclusion of garlic and green onion aqueous extract at the level of 1% in drinking water could be reduced Salmonellaspp.; E. coli and Clostridium perfringens in the feces of local Noi chickens.

Guava plant (Psidium guajava L.) is rich in antioxidants, vitamins C, potassium and fiber. The extracts from parts of guava plant had the antibacterial ability, healing the mucosa and stopping diarrhea. The extract ethyl acetate from guava leaves had the highest anti-bacteria and resistance against Salmonella aureus. Ethylacetate extract had an inhibitory effect on S. aureus, E. coli and Salmonella typhi(Linh et al 2019). In addition, guava (Psidium guajava) extract against Aeromonas schubertii causing the internal white-spot disease in snakehead (Khoi et al 2018). Nguyen Thi Thuy and Pham Cong Ha (2023) concluded that, supplementation of 2g/kg feed of garlic or guave leaf powders in the diets for weaning piglets trend to improve average daily gain, feed conversion ratio, reduced diarrhoea incidence, faecal score and E.coli in feces of piglets.

Sapodilla (Manilkara zapota L.) has been cultivated mainly in tropical areas including Vietnam. Sapodilla leaves aqueous extract exhibited the highest content of total phenolic and total flavonoids, and antioxidant capacities and therefore selected for phytoconstituents identification which could be useful for medicinal and nutritional functions (Tamsir et al 2020). Mehedi et al (2023) reported that Manilkara zapota (L.) seed oil against bacteria and fungi were also evaluated. Both Gram-positive and Gram-negative bacteria were resistant to the oil. It has a strong reaction to S. typhi while showing no reaction to E. coli. When the oil's antifungal activity was evaluated against Aspergillus niger and Aspergillus flavum, excellent inhibitory results were obtained. The derived oil has significant antibacterial and antifungal properties, according to Mehedi et al (2023) study.

Materials and methods

The experiment was carried out at the Quail Farm in Nha Be district, Ho Chi Minh City during March 2023-May 2024.

Herbal extract preparation

Leaves of guava and sapodilla were collected and dried under sunshine for 3-4 hours. The leaves were extracted in 70% ethanol (guava) and 50% ethanol (sapodilla) for 3-4 hours, and concentrated by steaming at 60 ^oC to a thick consistency. In order to increase the osmotic pressure of  the extract and prevent the growth of microorganisms, added 800 g sugar to 1L herbal extract and boil over low heat and carefully mixed until the herbal extract to form a condensed homogeneous solution. The condensed herbal extracts were preserved in the refrigerator.

Animals and housing

The quails were purchased at 1 day old and were nursed on a rice husks floor with a heating lamp system until 14^th day old. At the 15^th day, the birds were moved to cages and kept at temperatures of 36 – 38^oC and humidity 65% (Photo 2) to the end of 6 weeks of age.

Experimental design

A total 270 quails of 3 weeks of age were randomly allocated into 3 treatments and 3 replicates per treatment. In the 1^st treatment, the birds were fed a basal diet (Table 1) without herbal extracts (CT), and the birds in the 2^nd and 3^rd treatments were supplemented guava leaf extract in drinking water at 3 g/L (GT) or sapodilla leaf extract at 4 g/L (ST). The experiment was prolonged for 3 weeks.

Measurements

Growth performance: In each replicate, five birds were numbered and weighted one week-interval. Average daily gain (ADG) was accordingly calculated.

Anti-microbial susceptibility of the herbal extracts:The anti-microbial susceptibility of the extract was determined by the agar disc diffusion method (Hadacek and Greger, 2000). Accordingly, the diameter of the inhibition growth zones around each antibiotic disc is also included in the result. This is performed manually using a sliding ruler which is held on the black of the inverted agar plate. According to Celikel and Kavas (2008), the susceptibility of anti-microbial of the herbal extracts is classified based on the BK (BK = bactericidal kill):

- BK < 8 mm: not susceptible;

- BK = 9 - 14 mm: susceptible;

- BK = 15 - 19 mm: very susceptible; and

- BK > 20 mm: extremely susceptible

In the experiment, the dilution method was also used for the comparative testing of anti-microbial herbal extracts, after equivocal results of disc tests. The herbal extracts were diluted at 1, 2 and 3 g per liter of water.

E. coli isolation and colony counting: At the end of the experiment (birds at 6 ^th weeks of age), fecal samples of 5 birds/replicate were collected directly from cloaca and stored in cold storage for E. coli isolation and counting the colony. The MacConkey agar medium was used in isolating and counting E. coli bacteria. Fecal samples were diluted in NaCl 9‰ at 10^-5, 10^-6, 10^-7.

Statistical analysis

The data was analyzed by ANOVA using the General Liner Model (GLM) of Minitab Software Version 16.2 (2010). Tukey pair-wise comparisons were used to determine differences between treatment means at p<0.05. The statistical model used is as follows:

Y _ij = µ + T_i+ e _ij

In which, Y _ij is growth performance or fecal E. coli counts; µ is overall mean averaged over all treatments; T _i is effect of treatment; e _ij is random error associated with treatment and replicate within treatment.

Results and discussion

Antibacterial characters of the herbal extracts as compared with some common antibiotics

The diameters of the inhibition zone of guava leaf extract range 18-24 mm and highest in 2 g/mL (p<0.05); meanwhile, these values in sapodilla extract are 18-23 mm, and higher in 1 g/mL than in 2 and 3 g/mL (p<0.05). However, the BK of some common antibiotics are low or none.

Growth performance

The effect of inclusion of the herbal extracts in drinking water on growth performance of growing quails present in Table 3. After 3 weeks of the experiment, the final body weights (BW) of quails range 147.7-156.8 g, and the BW in the GT and ST are higher than in the CT (p<0.05). The difference in the BW between birds fed a basal diet and with inclusion of herbal extract started at the first week to the end of the experiment (p<0.05). Live weight of quails at the end of experiment ranges 154.6 g in ST and 156.8 g in GT and is higher in CT. Similarly, the ADG values of animals in the GT and ST are higher than in the CT (p<0.05). It means that the inclusion of the extracts of guava and sapodilla leaves in drinking water could improve growth performance of growing quails at 3-6 weeks of age.

Our findings are similar to previous studies. Nguyen Thi Thuy et al (2022) reported that the use of green onion and garlic aqueous extracts had significant improved in final weight and FCR of local Noi chickens. Pham Tan Nha and Le Thu Thuy (2023) reported that the supplementation of both green onion and garlic aqueous extract in diets improved final weigh of local Ac chickens and garlic aqueous extract effected more than green onion. These positive effects may be because both garlic and onion are among the common medicinal plants used as growth promoters, and the most important chemical constituents reported from Alliumsare the sulfur compounds (Ali and Zahran 2010). Garlic has antimicrobial, antioxidant as well as antihypertensive properties, these functions were attributed to bioactive components present in garlic (Amagase et al 2001), and one of which is a sulphur-containing organic compound that could be responsible for the growth promoting effect of garlic. In addition, Nguyen Thi Thuy and Pham Cong Ha (2023) found that, supplementation of 2 g/kg feed of garlic or guave leaf powders in the diets for weaning piglets improved ADG and FCR.

To our knowledge, there is not many studies on the use of guava and sapodilla leaf extracts in quails production. Therefore, this leads to limitation of discussion on this paper.

E. coli counts

The effect of herbal extracts in drinking water on E.coli count in quail feces presents in Table 4. At the 10^-5 – 10^-6 dilution, the E. coli count in the CT is highest and lowest in the GT (p<0.05), but at 10^-7 dilution, the values of E. coli count in three diets are similar (p>0.05).

The findings in our study are agree with previous results. Pham Tan Nha and Le Thu Thuy reported that the inclusion of green onion and garlic aqueous extracts in diets decreased total fecal E.coli. Similarly, Nguyen Thi Thuy et al (2022) indicated that the inclusion of green onion or garlic aqueous extracts in drinking water reduced Salmonella.spp; E. coli and Clostridium perfringens in the feces of local Noi chickens. In addition, Nguyen Thi Thuy and Pham Cong Ha (2023) concluded that, supplementation of 2g/kg feed of garlic or guave leaf powders in the diets reduced diarrhoea incidence, faecal score and E.coli in feces of piglets. It probably dues to the garlic extract contained organic compound which was transformed into allicin by the enzyme allinase, and can improve chicken health (Olobatoke and Mulugeta 2011), and these natural additives has prevented the development of harmful microorganisms in the intestines. The anti-microbial susceptibility of garlic allows classifying it among plants currently defined as phytobiotics to be used as potential feed additives (Diaz-Sanchez et al 2015).

Conclusion

Inclusion in drinking water of guava leaf extract (3 g/L) and sapodilla leaf extract (4 g/L) improved efficiently growth performance, and significantly reduced E. coli count in feces of growing quails of 3-6 weeks of age. In recommendation, the extracts from guava and sapodilla leaves could be used as E. coli anti-bacterial, however, guava leaf extract is better than sapodilla one, and growth stimulates.

Acknowledgement

The authors acknowledge the final support of HUTECH University.

References

Ali FH and Zahran DA 2010 Effect of growth enhancers on quality of chicken meat during cold storage. Advance Journal of Food Science and Technology, 2(4): 219-226. https://maxwellsci.com/print/ajfst/v2-219-226.pdf

Amagase H, Petesch BL, Matsuura H, Kasuga S and Itakura Y 2001 Intake of garlic and its bioactive components. Journal of Nutrition, 131: 955-962; 10.1093/jn/131.3.955S

Basturk A H M E T, Artan R and Yilmaz A 2016 Efficacy of symbiotic, probiotic, and prebiotic treatments for irritable bowel syndrome in children: a randomized controlled trial. Turkish Journal of Gastroenterol, 27(5): 439-443; https://doi.org/10.5152/tjg.2016.16301

Celikel N and Kavas G 2008 Antimicrobial properties of some essential oils against some pathogenic microorganisms. Czech Journal of Food Sciences, 26, 174-181. DOI: 10.17221/1603-CJFS

Diaz-Sanchez S, D’Souza D, Biswas D and Hanning I 2015 Botanical alternatives to antibiotics for use in organic poultry production. Poultry Science, 94:1419–1430; 10.3382/ps/pev014

Farhat M, Khayi S, Berrada J, Mouahid M, Ameur N, El-Adawy H and Fellahi S 2023 Salmonella enterica serovar gallinarum biovars pullorum and gallinarum in poultry: review of pathogenesis, antibiotic resistance, diagnosis and control in the genomic era. Antibiotics (Basel), 13(1): 1–20. https://doi.org/10.3390/antibiotics13010023

Hadacek Fand Greger H 2000 Testing of antifungal natural products: methodologies, comparability of results and assay choice. Phytochemical Anal., 11, pp 137-147 https://doi.org/10.1002/(SICI)1099-1565(200005/06)11:3<137::AID-PCA514>3.0.CO;2-I

Khoi L M, Dung L N T, Tu H H C, Trung N B and Dung T T 2018 The effect of extracts from guava (Psidium guajava) leaves and false daisy (Eclipta alba) against the internal white spot disease of snakehead (Channa striata).Cantho University Journal of Science. 54(Special Issue: Fishery) (2): 177-186. DOI:10.22144/ctu.jsi.2018.051

Linh D N, Phat NT, Lien N D T, Ni T T A, Duy N T and Minh N V 2019 Evaluating the potential antibacterial activities of extracts from the leaves of (Psidium guajava L.). Ho Chi Minh City Open University Journal of Science 14(1): 84-94; http://dx.doi.org/10.46223/HCMCOUJS.tech.vi.14.1.2162.2019

Mehat J W, van Vliet A H and La Ragione R M 2021 The Avian Pathogenic Escherichia coli (APEC) pathotype is comprised of multiple distinct, independent genotypes. Avian Pathology, 50(5), 402–416. https://doi.org/10.1080/03079457.2021.1915960

Mehedi A, Arabi II, Islam Z, Das S and Mannan A 2023Extraction, physico-chemical characterization and antimicrobial studies of seed oil of Sofeda (Manilkara zapota). Journal of Siberial Federal University Chemistry, 16(4): 485–497. EDN: VATUWQ; https://www.researchgate.net/publication/376557025_Extraction_Physico-Chemical_Characterization_and_Antimicrobial_Studies_of_Seed_Oil_of_Sofeda_Manilkara_zapota

Newman D M, Barbieri N L, de Oliveira A L, Willis D, Nolan L K and Logue C M 2021 Characterizing avian pathogenic Escherichia coli (APEC) from Colibacillosis cases, 2018. PeerJ, 9, e11025. https://doi.org/10.7717/peerj.11025

Nguyen Hai Quan, Vo Thi Minh Tam, Le Duc Thao and Le Duc Ngoan 2023 Effect of inclusion of black soldier fly larvae (Hermetia illucens) meal in diets on growth performance and meat quality of growing quail. Livestock Research for Rural Development. Volume 35, Article #23. Retrieved September 3, 2024, from http://www.lrrd.org/lrrd35/3/3523nhqu.html

Nguyen Thi Thuy and Nguyen Cong Ha 2023 Effect of adding guava leaf (Psidium guajava) and garlic (Allium sativum) powders in diets on growth performance and diarrhea incidence of weaned piglets. Livestock Research for Rural Development. Volume 35, Article #12. Retrieved September 3, 2024, from http://www.lrrd.org/lrrd35/2/3512nthi.html

Nguyen Thi Thuy, Nguyen Cong Ha and Le Thanh Phuong 2022 Effect of green onion (Allium fistulosum) and garlic (Allium sativum) aqueous extracts in drinking water on growth performances and intestinal microflora of local chickens in the south of Vietnam. Livestock Research for Rural Development. Volume 34, Article #31. Retrieved September 3, 2024, from http://www.lrrd.org/lrrd34/4/3431nthi.html

Olobatoke R Y and Mulugeta S D 2011 Effect of dietary garlic powder on layer performance, fecal bacterial load, and egg quality. Poult Sci. 90(3):665-70.

Pham Tan Nha and Le Thu Thuy 2023 Effects of garlic and turmeric supplements in diets on the productivity and quality of Ac chickens eggs in Vietnam. Livestock Research for Rural Development. Volume 35, Article #104. Retrieved September 3, 2024, from http://www.lrrd.org/lrrd35/11/35104ptnh.html

Sapsuha Y, Hasan S and Nur A 2023 The effect of symbiotic supplementation of nutmeg flesh extract (Myristica fragrans Houtt) and Lactobacillus plantarum on microbial and intestinal morphology of broiler chickens. Livestock Research for Rural Development. Volume 35, Article #4. Retrieved September 3, 2024, from http://www.lrrd.org/lrrd35/1/3504jus.html

Sarangi N R, Babu L K, Kumar A, Pradhan C R, Pati PK and Mishra J P 2016 Effect of dietary supplementation of prebiotic, probiotic, and symbiotic on growth performance and carcass characteristics of broiler chickens. Veterinary world, 9(3): 313. https://doi.org/10.14202%2Fvetworld.2016.313-319

Shim Y H, Ingale S L, Kim J S, Kim K H, Seo D K, Lee S C, Chae B J and Kwon I K 2012 A multi-microbe probiotic formulation processed at low and high drying temperatures: Effects on growth performance, nutrient retention and caecal microbiology of broilers. British Poultry Science, 53(4): 482–90. https://doi.org/10.1080/00071668.2012.690508

Tamsir N M, Esa N M, Omar S N C, Shafie N H 2020Manilkara zapota (L.) P. royen: Potential Source of Natural Antioxidants. Malaysian Journal of Medicine and Health Science. 16(SUPP6): 196-204; https://medic.upm.edu.my/upload/dokumen/2020081208335426_MJMHS_0716_L.pdf