Alquremold Natural (AMN) for Growth Performance, Carcass Characteristics and Bacterial Growth Inhibition of Commercial Broilers

An experiment was conducted with 648-day-old Cobb 500 broiler chicks to evaluate the effects of Alquermold natural (AMN) on overall growth performance, carcass characteristics and inhibition of enteric bacterial growth of commercial broilers. The birds were randomly assigned to four different treatments: T0 (Control), T1 (0.5 g/kg AMN), T2 (1.0 g/kg AMN) and T3 (1.0 g/kg Mold Inhibitor (MI)), respectively. They were reared for up to 28 days. At the end of experiment, the birds’ growth performances in terms of body weight (BW), body weight gain (BWG), feed intake (FI), feed conversion ratio (FCR), survival, and meat production characteristics were recorded and calculated. Weekly fecal sample collection and microbiological count were performed to determine the bacterial load, specifically Escherichia coli and anaerobic bacteria in the broiler feces. The results showed that birds receiving 1.0 g/kg AMN had significantly higher BW and BWG (p0.01) than the control, 0.5 g/kg AMN, and MI groups. The FI and FCR of broilers were comparable in all treatment groups. There were no significant (p > 0.05) changes in meat yield parameters such as dressing percentage, neck, shank, drumstick, breast meat, thigh meat, and wing meat. However, when compared to the other dietary treatments, the bacterial load (E. coli and anaerobic bacteria) was significantly lower in the birds fed 1.0 g/kg AMN. Based on the findings, it can be concluded that feeding AMN at a rate of 1.0 g/kg may be considered for improving growth performance, maximizing feed utilization, and inhibiting bacterial load in the intestine of commercial broiler chickens.

Introduction

Drug-resistant illnesses are expected to kill 10 million people annually by 2050 with Asia and Africa accounting for 90% of the predicted deaths, according to recent estimates, which show that antibiotic resistance is a worldwide health catastrophe with wide-reaching effects [1], [2]. In 2010, Bangladesh's Animal Feed Act also prohibited the use of antibiotics in feed (Fish Feed and Animal Feed Act, 2010) [3]. Under such circumstances, poultry nutritionists and the poultry industry have made various attempts to produce safe and high-quality broiler meat by supplementing it with natural growth promoters–such as prebiotics, probiotics, synbiotics, acidifiers, enzymes, medicinal plants, plant extracts, herbs, and other natural ingredients as an alternative to traditional antibiotics [4], [5]. Plant extracts are being studied as alternatives to antibiotics as growth promoters due to their antioxidant, antibacterial, antiviral, anticoccidial, and anthelmintic effects. Various types of plant extract products have recently become available on the market. Alquermold natural (AMN) is one such plant extract-based product developed by Biovet S. A. Laboratories (Constanti, Tarragona, Spain). The cimenol ring, found in plant extracts such as Thymus vulgaris, Origanum majorana, Mentha piperita, Ocimum basilicum, Salvia officinalis, and others, is the principal active compound of Alquermold natural [6]. Chemically, the AMN combines its cimenol ring (active ingredient of botanical origin) with citric acid to produce a broad-spectrum bactericidal and fungicidal activity that enhances the performance of broilers. It also limits the proliferation of pathogenic flora in the digestive system. Numerous studies have been carried out abroad on the antimicrobial properties and potential effects of AMN on broiler production performance, its effectiveness in broad-spectrum antimicrobial and fungicidal activities, and its capacity to inhibit mold growth in poultry feed. However, no research had yet been done that took into account the Bangladeshi environment. Therefore, the present feeding trial was carried out at Bangladesh Agricultural University (BAU) Poultry Farm before the product was used commercially. Its goals were to first determine the effects of AMN on the growth performances of broilers, then to observe the meat yield parameters after including AMN with the base diet, and finally to ascertain the effects of AMN on the E. coli and anaerobic bacterial count in the feces of experimental birds.

Materials and Methods

Ethical Approval

The Animal Welfare and Experimentation Ethics Committee of the Bangladesh Agricultural University, Mymensingh-2202, Bangladesh (Ref. No. AWEEC/BAU/2023 (27)) approved all methodological protocols and experimental designs used for the trial.

Experimental Design, Chick Collection and Distribution

A total of 648 unsexed Cobb 500 commercial broiler chicks were purchased from a nearby hatchery, weighed, and randomly divided into four nutritional treatment groups having 9 replications in each treatment and 18 birds per replication. The treatments were: T₀ = Standard basal diet (SBD) or Control, T₁ = SBD + 0. 5 g/kg AMN, T₂ = SBD + 1.0 g/kg AMN, T₃ = SBD + 1.0 g/kg Mold Inhibitor (MI). Experimental birds were reared for 28 days.

Brooding, Experimental Diet Preparation and Managemental Practices

The chicks were brooded in their designated pens with one 200-watt electric bulb in each pen, where a temperature of 36 °C was maintained for the first week before gradually dropping by 3 °C per week until they were two weeks old. Diets were formulated using widely used, high-quality feed items that were consistently fresh and readily available. Both crumble and pellet forms of diets were formulated for two phases, namely starter and grower, where the starter diet was provided for 1 to 15 days and the grower diet for 16 to 28 days. Nutrient requirements (ME, CP, CF, EE, Ca, P, Lysine and Methionine) were satisfied as recommended for Cobb-500 broiler’s nutrient specifications and similar for all the treatment groups (Table I). Followed by formulation, the diets of each treatment were stored in separate gunny bags. For the remaining dietary treatment groups, the standard diet was supplemented with a specific amount of AMN and MI (50 g AMN in T₁, 100 g AMN in T₂, and 100 g MI in T₃ per 100 kg was added to feed) by removing the corresponding amount of soybean meal from each diet. The experimental broilers were always given ad libitum feeds twice a day (morning and afternoon). There was fresh, clean water available for birds to drink. For the first three days of the brooding period, birds were exposed to 24 hours of lighting; after that, the amount of light was gradually reduced to 20 hours for the next seven days, followed by 16 hours of lighting for the remaining days of rearing (both natural and artificial lighting). As litter material, fresh and dried rice husk was spread across the floor at a depth of around 3 cm. The upper portion of the litter was rotated after the first two weeks, and the paper sheets covering the litter were taken off After 14 days, the litter was stirred on every alternate day to prevent it from becoming too compact, to keep the proper moisture levels, and to help it dry out quickly to release dangerous gases.

Table I. Standard Basal Diet (SBD) for Starter and Grower are Given Below

Feed Ingredients	Amount (kg/100 kg feed)
	Starter (0–15 days)	Grower (16–28 days)
Corn	52.8	52.8
Corn gluten meal	2.0	2.0
Soybean meal	32.72	32.72
MBM	3.0	3.0
Poultry meal	3.0	3.0
Soybean oil	3.0	3.0
Limestone (fine powder)	0.80	0.80
MCP	0.97	0.97
Broiler Vitamin	0.05	0.05
Mineral	0.10	0.10
Methionine	0.32	0.32
Lysine Sulphate	0.57	0.57
Threonine	0.11	0.11
Valine	0.08	0.08
Salt	0.25	0.25
Sodium bi carbonate	0.08	0.08
Choline Chloride	0.15	0.15
Alquermold natural	00	00
Total	100	100

Vaccination, Sanitation and Bio-Security

Newcastle disease and infectious bronchitis vaccinations were given to birds on the fourth day of raising, with a booster shot given on the nineteenth day. Through the intra-ocular route (one drop of vaccine in one eye/bird), birds were also immunized against Gumboro disease on the 14^th day of the experiment, followed by a booster dose on the 21^st. At the entrance to the experimental home, a foot bath containing potassium permanganate (KMnO₄) was set up for sanitation and hygiene purposes. The study shed was kept under stringent bio-security both inside and out. Dead birds were disposed of properly for biosecurity management.

Collection, Processing and Preparation of Feces Sample from Experimental Birds

The whole amount of fresh droppings from a single bird was collected aseptically into an Eppendorf tube. Throughout the entire experimental period (0–28 days), a total of 20 samples were randomly collected from four different groups (5 samples/treatment group). The feces were moved as quickly as possible into the bacteriology laboratory of the Department of Microbiology and Hygiene for additional bacteriological processing. Electric balance was used to weigh one gram of fresh feces, which was then properly mixed with 10 ml of PBS using a vortex. Then tenfold serial dilution was done within the Eppendorf tube up to 10⁶ dilutions. Afterward, 50 μl samples from each dilution were poured into Petri dishes containing different media. Then the samples were spread by sterile glass spreader onto the bacteriological media such as Nutrient agar, MacConkey agar for E. coli and Blood agar for anaerobic bacteria determination. Sample inoculated Nutrient agar and MacConkey agar plates were incubated overnight at 37 °C and Blood agar plates were incubated anaerobically at 42 °C. After overnight incubation, the bacterial growth colony was counted, followed by a morphological characterization by Gram’s staining. Finally, the determination of CFU (Colony forming unit) of E. coli and anaerobic bacteria in feces samples was conducted. The following formula was applied for the calculation of the total number of colonies:

Data Collection and Record Keeping

Body weight and feed consumption were recorded at the age of 1^st, 2^nd, 3^rd and 4^th weeks of age. By subtracting the beginning body weight from final body weight, the body weight gain of experimental birds was computed. The feed conversion ratio (FCR) was then determined each week by dividing the feed intake by the increase in body weight. At the end of the experiment, two birds with pen average body weight from each replicate group were randomly selected and processed further to observe the carcass features. An automatic thermo-hygrometer was used to measure temperature and relative humidity (%) four times per day during the duration of the experiment. Total expenses and profit were computed in order to conduct the cost-benefit analysis.

Statistical Analysis

In line with the principles of Completely Randomized Design (CRD), a thorough statistical analysis was conducted on all recorded and calculated data. One-way ANOVA technique was employed by using SPSS Computer Package Program 20.00 [7] (SPSS Inc., Chicago, IL) to ensure accurate statistical analysis. In instances where ANOVA revealed significant differences, Duncan Multiple Range Tests (DMRT) were performed to further investigate and compare significant variations among the treatment groups.

Results and Discussion

Growth Performance of Broilers

Table II displays the overall growth performance of the experimental broiler chickens, including BW, BWG, FI, and FCR, under various nutritional regimens. The initial BW of experimental birds was almost equal to approximately 47.0 g/bird. After analyzing data, birds receiving 1.0 g/kg AMN exhibited considerably (P < 0.05) higher final body weight (1751.71 g/bird) and weight gain (1642.89 g/bird) compared to the other experimental groups. On the contrary, feed intake and FCR values showed no significant differences; however, numerical data exerted lower and better FCR (1.352) in birds receiving 1.0 g/kg AMN.

Table II. Overall Growth Performance of Broiler Chickens after Supplementation of AMN at Different Levels

Parameters	T0 (SBD)	T1 (0.5 g AMN/kg Feed)	T2 (1.0 g AMN/kg Feed)	T3 (1.0 g MI/kg Feed)	P-value
Initial weight (g/bird)	47.50 ± 0.14	47.44 ± 0.15	47.37 ± 0.14	47.63 ± 0.17	0.663
Final weight (g/bird)	1661.16b ± 15.91	1690.33b ± 18.36	1751.71a ± 9.78	1675.13b ± 15.77	0.001
Total weight gain (g/bird)	1613.64b ± 15.88	1704.32b ± 9.75	1642.89a ± 18.81	1627.49b ± 15.71	0.001
Feed intake (kg/bird)	2.268 ± 0.02	2.304 ± 0.01	2.284 ± 0.02	2.276 ± 0.01	0.617
FCR	1.406 ± 0.01	1.391 ± 0.02	1.352 ± 0.01	1.401 ± 0.01	0.135

Previous report of Hernandez et al. [8] suggests that the supplementation of 5000 ppm labiatae extract (LE) from sage, thyme and rosemary have significant impact on body weight (2461.6 g) and body weight gain of broiler chickens consuming an almost similar amount of feed (non-significant; P > 0.05) at 35 days of age. Further, Williams et al. [9] recently delineated that the dietary supplementation of EPCL (1% mix of equal quantity (0.5% each) of Ethiopian pepper and clove) resulted in the highest body weight (BW, 2551.38 g) of broilers at day 52. Al-Kassie [10] investigated the effects of thyme and cinnamon plant extracts on the performance of broiler birds and revealed that the supplementation groups receiving 200 ppm of the extracts saw considerably higher body weight (2882 g and 2866 g/bird, respectively) during 42-days of the experimental period. He did, however, note significant variations in feed consumption and FCR in the birds fed plant extracts, which don't line up with our current findings. On contrary, several published references demonstrate the positive impacts on the use of AMN in broilers. However, there were no significant variations in feed intake and FCR when broiler chickens were fed an aqueous extract of ginger (Zingiber officinale) at various concentrations [11]. Significant improvement in final live weight (1660 g/bird) and live weight gain (1550 g/bird) were also observed when the birds were supplemented with 1 ml polyherbal extract with 1 L of drinking water from 2 to 6 weeks of age [12]. According to Jamroz and Kamel [13], the addition of a plant extract containing capsaicin, cinnamaldehyde, and carvacrol at a rate of 300 mg/kg feed resulted in considerably higher daily weight gain (8.1%) and FCR (7.7%). The herbal preparation of Moringa olifera leaf extract can be utilized as an effective feed supplement due to its beneficial effects on the average daily weight gain (52.57 g) and FCR (1.718) in broiler chickens [14]. Therefore, it is likely that the herbal and plant extract natural products have the potential to improve body weight, body weight gain, and FCR in broiler chickens. If added at the recommended levels, they can also be used to replace antibiotic growth promoters. In recent published report displayed the average live weight and body weight gain of commercial boilers were notably increased along with significantly improved FCR when basal diet was supplemented with 2.0% mint leaf [15]. Black cumin seed meal supplementation (BCSM) at levels of 40 and 60 g per kg diet significantly improve the body weight gain, FCR and survivability of commercial boilers compare with control and 20 g supplemented diet during a rearing period of 42 days [16]. In contrast, Cristo et al. [17] reported that vegetable extract (carvacrol, cinnamaldehyde, and eugenol) from oregano, cinnamon, and cloves did not affect broiler chickens’ productive performance or carcass yield. Finally, it is likely that the addition of diverse plant extracts, such as our test component AMN, may significantly boost bird’s performance due to the bioactive compound present in a particular plant.

Meat Yield Characteristics of Broilers

Table III displays the meat yields of experimental broilers among the treatment groups fed different diets. Although there were no statistically significant (P > 0.05) differences in the meat yield parameters among the treatment groups, we can see an intriguing pattern of improvement when we used AMN in the diet and found highest numerical value in meat yield parameters in the birds receiving 1.0 g/kg AMN (DP = 76.03%).

Table III. Meat Yield Characteristics of Broiler Chickens after Supplementation of AMN at Different Levels

Variable (%)	T0 (SBD)	T1 (0.5 g AMN/kg Feed)	T2 (1.0 g AMN/kg Feed)	T3 (1.0 g MI/kg Feed)	P-value
DP	72.262 ± 0.803	74.362 ± 1.292	76.031 ± 0.552	73.248 ± 0.849	0.066
Thigh	20.858 ± 0.401	21.425 ± 0.721	22.789 ± 1.141	19.553 ± 0.811	0.152
Drumstick	12.446 ± 0.507	13.427 ± 0.550	13.555 ± 0.250	12.927 ± 0.367	0.302
Breast meat	35.343 ± 0.513	35.813 ± 0.514	36.245 ± 0.384	35.232 ± 0.707	0.550
Wing	11.742 ± 0.243	11.958 ± 0.316	12.324 ± 0.215	11.359 ± 0.373	0.185
Head	1.616 ± 0.450	1.604 ± 0.304	1.711 ± 0.215	1.545 ± 0.350	0.340
Liver	3.674 ± 0.291	3.242 ± 0.089	3.551 ± 0.285	2.998 ± 0.131	0.214
Gizzard	2.174 ± 0.213	2.254 ± 0.330	2.447 ± 0.114	2.186 ± 0.106	0.074
Heart	0.738 ± 0.240	0.817 ± 0.261	0.850 ± 0.018	0.735 ± 0.376	0.066
Giblet	6.570 ± 0.286	6.154 ± 0.174	6.838 ± 0.212	6.023 ± 0.135	0.111

According to earlier research, dressing and giblet yields were altered significantly in broiler chickens fed garlic and neem leaf powder [18]. Broilers fed a diet containing polyherbal extract showed no discernible variations in their relative gizzard and spleen weights, DP%, or other organ weights [12]. Garcia et al. [19] again reported that the use of formic acid and plant extract had no impact on meat yield indices. Recent reports clearly revealed that the supplementation of 1% mixture of Ethiopian pepper and Clover (EPCL) with basal diet significantly improved the dressing percentage (81.68%) and breast meat percentage (20.01%) of broiler chicken [9]. On the other hand, Akter and Asaduzzaman [15] proclaimed that broilers fed with 2.0% mint leaf have significant effects on the thigh, wing, back, liver, neck, heart, gizzard intestine, spleen and bursa while it appeared insignificant on dressing percentage, breast and drumstick. In summary, the features of broiler chickens have remained essentially intact, despite the fact that diverse herbal plant extract products function on numerous group parameters in broiler chickens.

Bacterial Load in Feces of Broilers

Tables IV and V demonstrate the E. coli and anaerobic bacterial counts (log cfu/g) in the feces of experimental broiler chickens fed various experimental diets. The E. coli levels in bird’s droppings were about incomparable in all treatment groups at the first week of age, according to the results; however, AMN started to have an effect on E. coli counts from the second week through the remainder of the raising period. Although the E. coli bacterial load was notably higher in mold inhibitor group even when compared to SBD. On contrary, E. coli loads in droppings were significantly lower in the birds supplemented with 0.5 g and 1.0 g AMN/kg feed from the second to fourth weeks of age. For the second, third, and fourth weeks of age, anaerobic bacteria in droppings were counted, where the anaerobic bacterial load was again significantly lower in the birds fed 0.5 g and 1.0 g AMN/kg feed as compared to SBD and MI groups. These results are consistent with a number of previous research findings utilizing AMN in poultry [6].

Table IV. Bacterial Load (E. Coli) in Feces of Broiler Chickens after Supplementation of AMN at Different Levels

Age of Birds	Bacterial load (log CFU/g ± SE) in feces of experimental birds
	T0 (SBD)	T1 (0.5 g AMN/kg Feed)	T2 (1.0 g AMN/kg Feed)	T3 (1.0 g MI/kg Feed)	P-value
1st week	5.794 ± 0.158	5.794 ± 0.195	5.262 ± 0.126	5.550 ± 0.196	0.131
2nd week	6.028a ± 0.334	5.370b ± 0.074	5.140b ± 0.050	5.272b ± 0.065	0.012
3rd week	5.922b ± 0.232	5.822b ± 0.154	5.784b ± 0.078	6.978a ± 0.152	0.000
4th week	6.276b ± 0.189	5.890b ± 0.247	5.904b ± 0.058	7.032a ± 0.073	0.000

Table V. Bacterial Load (Anaerobic Bacteria) in Feces of Broiler Chickens after Supplementation of AMN at Different Levels

Age of Birds	Bacterial load (log CFU/g ± SE) in feces of experimental birds
	T0 (SBD)	T1 (0.5 g AMN/kg Feed)	T2 (1.0 g AMN/kg Feed)	T3 (1.0 g MI/kg Feed)	P-value
2nd week	3.36 ± 0.076	3.00 ± 0.282	3.01 ± 0.211	3.47 ± 0.128	0.231
3rd week	3.28ab ± 0.051	2.94b ± 0.074	2.96b ± 0.120	3.58a ± 0.151	0.012
4th week	3.27ab ± 0.055	3.10b ± 0.173	3.09b ± 0.105	3.56a ± 0.118	0.047

A recent article [20] on the supplementation of plant extracts as a replacement for antibiotics amply demonstrated the strong antibacterial effect on coliform and total anaerobic bacteria. Ramiah et al. [21] investigated how dietary herbal extract supplementation affected broilers and found that the E. coli count was considerably reduced in the intestine of experimental birds. Sakthi Priya et al. [22] postulated that layer chickens fed various doses of herbal supplementation had significantly lower total fecal bacterial counts and fecal coliform counts (P = 0.01) than the control group, and E. coli was present in the control but absent in the supplemented groups. With the addition of various phytogenic feed additives, Murugesan et al. [23] found a significantly lower total anaerobic bacterial and coliform count (cfu/g). Furthermore, addition of 2.0% mint leaf in the broiler diet results in a significant lower amount of Escherichia coli in caeca than in the control group [15]. Again, Williams et al. [9] described that intestinal coliform count reduced in broilers fed the diet supplemented EP (Ethiopian pepper) and those fed the EPCL (mixture of Ethiopian pepper and Clover) supplemented diet. Black cumin seed meal supplementation (BCSM) at levels of 40 and 60 g per kg diet also effectively decreased the caecal microbial counts in broilers [16]. All these findings suggest the reduction of bacterial load in guts after inclusion of plant extracts in diets.

Cost-Benefit Analysis

Table VI shows the cost of production for various commodities in four distinct treatment groups. The cost per kg feed formulation in the 1.0 g/kg AMN group was a little bit higher (BDT: 42.30/) as compared with the others. In comparison to the control and MI dietary groups, the total cost of production per kg of live birds was lower in the 0.5 g/kg and 1.0 g/kg AMN groups. Furthermore, when selling live broiler birds for BDT:125/- per kg at the local market, it was found that 1.0 g/kg AMN (BDT: 26.57/- per kg) and 0.5 g/kg AMN group (BDT: 23.66/- per kg) showed higher profit/kg live bird compared to control group (BDT: 22.5 tk/- per kg) and MI group (BDT: 22.89/- per kg). The benefit-cost ratio of supplemented AMN group was close to the control group.

Table VI. Cost-Benefit Analysis of Broiler Chickens after Supplementation of AMN at Different Levels

Parameter	Treatments
	SBD	0.5 g AMN/kg Feed	1.0 g AMN/kg Feed	1.0 g MI/kg Feed
Feed intake (g/broiler)	2268	2284	2304	2276
Final body weight (g/broiler)	1661.15	1690.33	1751.70	1675.13
Feed price (tk/kg)	42.00	42.00	42.00	42.00
Mold Inhibitor (MI) cost	–	–	–	0.20
Cost for AMN	–	0.15	0.30	95.256
Total feed cost (with or without additives)/kg	42.00	42.15	42.3	42.20
Feed cost/bird	95.26	96.27	97.46	96.04
Chick cost tk/bird	55.00	55.00	55.00	55.00
Miscellaneous (vaccines, disinfectants, transport, bedding materials, labour etc.)	20.00	20.00	20.00	20.00
Total cost of production/live bird	170.26	171.27	172.46	171.04
Total cost of production tk./kg live bird	102.5	101.34	98.43	102.11
Total income/bird (sales price tk.125/per kg live bird)	207.62	211.25	219	209.38
Profit tk./live bird	37.36	39.98	46.54	38.34
Profit tk./kg live bird	22.5	23.66	26.57	22.89
Benefit-cost ratio	1.22	1.23	1.26	1.22

Conclusion

Based on the findings presented above, it is possible to conclude that inclusion of AMN in the broiler diets not only improves feed conversion ratio and growth parameters, but also reduces bacterial burden, particularly E. coli and other anaerobic bacteria. Therefore, it is recommended that 1.0 g/kg AMN may be added to the broiler diet in order to produce broilers in an economical and effective manner. Future research can be planned and carried out with a focus on how AMN affects the immune system, microbial regulation, and histological changes in the guts of commercial broilers.