Impact of dietary supplementation of Rhamnus prinoides leaf extract on the growth performance, nutrient retention and intestinal microbial count of “japanese quails”

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Introduction
Public concern about potential antibiotic resistance threats to human health has sparked research in poultry nutrition and the implementation of antibiotic-free feeding systems.This has resulted in the emergence of feed additives that can be used as in-feed antibiotic alternatives in quail feeding plans (Caroline et al., 2021;Musa et al., 2021).The use of medicinal plants has been proposed as one response to the increasing incidence of antibiotic resistance in animals (Alagbe, 2020).
Rhamnus prinoides are among the widely used medicinal plant with several therapeutic properties (Abebe et al., 2003;Alagbe et al., 2020).The plant belongs to the family Rhamanceae which is made up of about 150 species and are widely distributed in East Africa, Central Africa and some parts of Asia including India (Megersa et al., 2013).Rhamnus prinoides contains a variety of secondary metabolites including tannins, flavonoids, alkaloids, saponins, steroids, phenols and anthraquinones which have multiple pharmacological properties (Amabye, 2015;Bosire, 2003).
Traditionally, the extracts from the leaf of R. prinoides is used for the treatment of malaria, rheumatism, gastrointestinal disease, body pain, constipation, cold, tooth ache, sexually transmitted disease, brucellosis amongst others (Berhanu, 2014;Bitew et al., 2019).Root infusion can be used to treat cough, respiratory infection, stomach discomfort, joint pain and sore throat (Dzoyem et al., 2016).
Previous research has shown that plant extract can improve bird growth performance, nutrient digestibility, microbial population in the gastrointestinal tract, feed palatability, fatty acid composition, and blood parameters (Musa et al., 2020;Adewale et al., 2021;Agubosi et al., 2022).However, the majority of the results obtained were inconsistent, implying that processing or extraction methods, species, geographical region, plant age, and dosage supplied may all influence the outcome of the studies (Shittu;Alagbe, 2020).There is relatively limited evidence on the utilization of R. prinoides leaf extract with quails.
Given R. prinoides vast potential, there is a need to assess its efficacy and determine its tolerable level in birds.This will further contribute to increased poultry output and food security.As a result, the purpose of this study was to see how dietary supplementation with Rhamnus prinoides leaf extract affected "japanese quail" growth performance, nutrient retention, and intestine bacteria count.

Experimental location, ethical approval and preparation of Rhamnus prinoides leaf extract
The study was conducted at the Sumitra Institute's Livestock unit, which is located between 23o 13' N and 72o 41' E and it was conducted in accordance with the guidelines and requirements of procedures that had been authorized by the research ethics council of India's Sumitra Research Institute (AA/HJ/008C).
Rhamnus prinoides leaves were harvested from Lakadiya village, located in Bhachau, Takula of Kachchh district, Gujarat, India and sent to the department of Crop Protection where it was identified and authenticated before it was air dried for 11 days until a constant weight was achieved.Thereafter, it was powdered using an electric blender.200 grams of R. prinoides powder was immersed into 1000 mL of ethanol in a conical flask for 48 h.
The mixture was stirred intermittently every 5 h and filtered into a container to obtain R. prinoides extract.
Rhamnus prinoides extract was evaporated to dryness using a vacuum distillation unit before it was sent to the institute's laboratory for gas chromatography and mass spectrometry (GC-MS) analysis.Gas chromatography and mass spectrometry analysis of R. prinoides extract carried out using UV-Vis 230 UV-visible spectrophotometer which has an instant helium saver module which allows for an automatic reduction in Helium flow to enable sample transfer to the column.10 mL of R. prinoides extract is injected to the inlet of the machine and to ensure accuracy the kit is adjusted at a wavelength of 190-1100 nm, wavelength accuracy and reproducibility ± 2.0, 1.0 nm, monochromator (Single, C-T, 1200 L/mm -1 ) and photometric accuracy (± 0.5% T).

Animal management, experimental diet and design
In a trial carried out at Sumitra Research Institute in India, 300-2 weeks old "apanese quails" were allocated sixty birds per treatment and each treatment had four replicates (15 quails per replicate).On arrival, quails were unboxed, and stratification of their weights was taken into consideration before they were randomly distributed into a specially constructed all wired battery cage placed in a semi sided pen.
The cages measuring 80 cm by 60 cm by 40 cm (length, breath and height) disinfected with Aquaclean ® at the rate 5 mL to 5 L of water two weeks before the arrival of birds.Cages were equipped with automatic feeders and drinkers and 100-watt bulb was used to supply heat to birds.A mixture of Vitamix ® and glucose at 15 g to 10 L of water was given for 5 days and experimental diet (basal) were adequate in all nutrients according to the recommendation of NRC (1994).A completely randomized design was adopted, and birds were given unrestricted access to feed and clean water.The experiment lasted for forty-nine days under strict daily and routine management practices.Feed intake, daily gain and feed conversion ratio were measured.

Calculations
1 Final body weightinitial body weight.
2 Weight gain /number of experimental period.
4 Total feed intake/number of experimental period.
5 Average daily feed intake/average daily weight gain.
Nutrient Digestibility = Nutrient in feed -Nutrient in droppings/Nutrient in feed × 100

Experimental set-up
A basal diet containing Corn-soya meal was formulated and the treatment was set-up as follows: Group 1: A corn-soya meal (basal diet) without R. prinoides extract; group 2: basal diet with 0.2 mL/L R. prinoides extract; group 3: basal diet with 0.4 mL/L R. prinoides extract; group 4: basal diet with 0.6 mL/L R. prinoides extract and group 5: basal diet with 0.8 mL/L R. prinoides extract.

Nutrient retention trial (NRT)
Digestibility retention trial (NRT) was carried out at the end of the experiment.Four birds were selected from each of the replicate, weighed and transferred to a labeled battery cage.Birds were given two days as acclimatization period while five days were used for data collection.Total droppings voided from each replicate were weighed and recorded.Wet droppings were oven dried at 70 ºC for 24 h to determine the dry matter content.Record on daily feed intake was recorded and droppings from the same treatment were thoroughly bulked together and taken to the laboratory for analysis.Proximate analysis of droppings was carried out using near infra-red automated kit (NIR -7000, USA) which uses SensorVu windows ® based PC software allowing analyst to insert set up parameters, perform or adjust calibrator and examine diagnostic values.

Intestinal microbial examination
At the end of the experiment (49th day), four birds were selected from each of the replicate for intestinal microbial examination.Birds were slaughtered very early in the morning and their intestinal content was collected into a sterile sample bottle and transferred immediately to the department of microbiology, Sumitra Research Institute, India.Microbial analysis was carried out using Quantom TxTM (Model Q10002, South Korea) microbial cell counter equipped with automated fluorescence imaging for accurate and objective bacterial cell counts.For accuracy in the results sample volume is adjusted at loading volumes: 5-6 µL, measuring volume: 0.09 µL (10 images).

Statistical analysis
Data obtained from the experiment was subjected to analysis of variance (ANOVA) using the computer software package (SPSS version 25.0) differences among treatment means was compared with Duncan's multiple range test (Duncan, 1995).
Phytol has the potential to prevent against different stages arthritis and scavenge against the activities of free radicals (Ogunlesi et al., 2009).It also possesses antimicrobial, anti-inflammatory, antioxidant activity and are found to be present in leaf and stem extract of Moringa concanensis (Vadivel et al., 2015).4-Allyl-1,2diacetoxybenzene and Allylipo nitrite and emodinanthrone also exhibits antimicrobial, antidiarrheal and antifungal effects (Doughari et al., 2021).The result obtained in this study is in agreement with the findings of Gashew et al. ( 2021).Growth performance characteristics of "japanese quails" fed R. prinoides leaf extract (Table 3) shows that weight gain and average daily weight gain which ranged from (108.52-158.70 g) and (2.21-3.24g) in birds fed 0.4 mL/L R. prinoides extract (group 3) and 0.6 mL/L R. prinoides extract (group 4) were similar (P > 0.05) to those given 0.8 mL/L R. prinoides extract (group 5) but significantly higher (P < 0.05) than those fed 0 mL (group 1: G1) and 0.2 mL/L R. prinoides extract (group 2).The result demonstrates that feeding quails R. prinoides extract at 0.4 mL, 0.6 mL and 0.8 mL/L influenced the activities of endogenous enzymes thereby improving the digestion and absorption of nutrient for metabolism.
The presence of bioactive compounds in R. prinoides extract as presented in (Table 2) can exert a growth promoting effect on birds.It is clear that at 0.4 mL, 0.6 mL and 0.6 mL more nutrients are up taken and metabolized and then improved body weight was recorded relative to the other groups.According to Sandra (2020), phytogenic feed additives are known for their beneficial effects on animals, from flavouring and sensorial stimulation, antimicrobial, anti-inflammatory, antioxidant properties amongst others.The result obtained on weight gain agrees with the report of Shittu et al. (2022) when Sida acuta leaf extract was fed to 8 mL/L.Similar observation was made by Alagbe & Ushie (2022) who reported a significant (P < 0.05) difference in average daily weights of broilers fed Citrus aurantium stem bark extracts at 4 mL/L.Average daily feed intake which varied from 703.06-708.81g were not influenced (P > 0.05) by the treatment.
Conversely, feed conversion ratio and mortality rate were significantly (P < 0.05) different among the groups.
The result demonstrates that feeding quails up to 0.6 mL/L R. prinoides extract did not improve the palatability of the feed.Part of the explanation could be the presence of β-sorigenin which is responsible for the bitter taste in R. prinoides leaf (Gashew et al., 2021).Improved feed conversion ratio and no mortality was recorded among quails fed 0.4 mL, 0.6 mL and 0.8 mL/L R. prinoides extract.
This could be as a result of safe gastrointestinal tract morphology.The result obtained agrees with the findings of Alagbe et al. (2022) when Rubia cordifolia root extracts was fed to growing rabbits.Conversely, Alabi et al. (2017) reported that M. oleifera leaf extracts influenced the feed intake of Hubbard broiler chicken.This variation in result can be attributed to difference in phyto-constituents or bioactive compounds, dosage feed to birds, specie of plant, extraction techniques amongst others (Alagbe, 2021).Nutrient retention indices of "japanese quails" fed R. prinoides leaf extract in (Table 4) revealed that dry matter, crude protein, ether extract and nitrogen free extract values which varied from 70-86%, 58-68%, 38-46% and 50-66%.Quails fed 0.4 mL/L R. prinoides leaf extract (group 3); 0.6 mL/L R. prinoides leaf extract (group 4) were similar (P < 0.05) to those fed 0.8 mL/L R. prinoides leaf extract (group 5) but significantly higher (P > 0.05) than those in other groups.
Crude fibre values in quails fed 0.2 mL/L R. prinoides leaf extract (group 2) and 0.4 mL/L R. prinoides leaf extract (group 3) were similar (P < 0.05) to those fed 0.6 mL/L R. prinoides leaf extract (group 4) and 0.8 mL/L R. prinoides leaf extract (group 5) but significantly (P > 0.05) lower than those in fed 0 mL (group 1).The result obtained demonstrates that R. prinoides leaf extract has the potential to improve nutrient absorption and secretion of digestive juices in the gut of quails.
Higher dry matter recorded in G3, G4 and G5 suggests a significant improvement in the nutrient permeability of the gut wall of birds which is made possible as a result of bioactive compounds or phyto-constituents in R. prinoides extract.Result obtained in this study is in agreement with the reports of Alagbe et al. (2022) when Juniperus thurifera root extract was fed rabbits.Similar outcome was recorded by Esonu et al. (2002) when Microdesmis puberula leaf meal was supplemented in the diet of broiler chickens.However, Oloruntola et al. (2016), reported a non-significant (P > 0.05) difference in crude fibre and ether extract digestibility of broilers fed diet diets supplemented with Gliricidia sepium leaf meal.Conversely, Lactobacillus sp.count was higher (P < 0.05) in group 2, 3, 4 and 5 relatives to those fed in group 1.
According to Kogut & Arsenault (2016), a healthy gut is defined as the absence, prevention and/or avoidance of disease so that the animal is able to perform its physiological functions in order to withstand exogenous and endogenous stressors.The balance of the gut microflora plays an essential role in the health of the animal.Imbalances in the gut microflora will lead to mortality and decreased performance in general (Gwendolyn, 2020).
The result demonstrates that R. prinoides leaf extract can interfere with the colonization of harmful microbial species like E. coli, Salmonella sp. and Staphylococcus sp.This may be due to its content of bioactive compounds (Table 2) decreasing the number of available binding sites for pathogenic organisms.Lactobacillus sp. is a beneficial bacterium capable of stimulating the gut epithelial cells, reduction of pH, production of antimicrobial agents, inhibiting the growth of potential pathogen, stimulating gut immune functions which aid the absorption of nutrients and synthesize vitamins of the B group (Gwendolyn, 2020).Result obtained is in agreement with the reports of Alagbe et al. (2023) when P. africana oil was fed to birds at 800 mg/kg -1 .According to Goliomytis et al. (2014), dietary supplementation of quercetin in birds prevented dysbiosis in their gastro intestinal tract.

Conclusions
In conclusion, Rhamnus prinoides leaf extract contains several bioactive compounds with pharmacological properties (anti-helminthic, anti-cancer, anti-malarial, anti-hyperglycemic, anti-inflammatory, anti-rheumatic, immune-stimulatory and anti-pyretic activities amongst others).Feeding quails up to 0.8 mL/L with R. prinoides leaf extract has the potential to improve the growth performance, secretion of digestive enzymes, nutrient absorption, increase gut permeability as well as modulating the gastro intestinal tract thereby preventing dysbiosis.Supplementation up to 0.8 mL/L did not negatively affect the performance of quails and this can confer Rhamnus prinoides leaf extract as a natural alternative to antibiotics.

Table 1 .
Ingredients and chemical composition of experimental diets.
Note: (% area) = Expressed as a percentage of relative area.Source: Author, 2023.