1 Introduction

Many countries in the world have either banned or introduced strict guidelines and regulations towards the use of traditional antibiotic growth promoters (AGP) in livestock diets [1]. This is due to strong evidence suggesting repeated exposure to AGP induces elimination of sensitive bacteria, disrupt microbial barrier, generates selective pressure for resistant bacteria, as well as diminish restoration time of indigenous microflora [2, 3]. Consequently, the development of antibiotic-resistant strains (superbugs) of both commensal and pathogenic species bacteria such as Salmonella, Escherichia, Staphylococcus, Yersinia and Enterococcus species arises [4,5,6]. This pattern leads to potentially unsafe residues in meat and meat products that affect consumer health, safety and confidence, a challenge to the medical and wider scientific community [7, 8]. The results include diminished treatment options and therapeutic efficacy in human medicine [9]. Consequently, catastrophic effects have been recorded with fatalities projected at over 10 million by 2050 mainly in Africa and Asia [10, 11]. This has led to a European ban on sub-therapeutic use of antibiotics [12]. Subsequently, there has been burgeoning consumer consciousness, interest, demand, and expanding market for drug/antibiotic-free poultry products [13] and the consumer driven “no antibiotic ever-NAE” or “raised without antibiotics, RWA” initiatives [14]. To add to this pressure on producers, terminating use of AGP in the poultry immediately leads to gut ecosystem disruptions, diarrhoea, drop in growth performance and gradual extensive global economic losses, thus a sustainable balance is urgently needed [15]. All this comes against the backdrop of chicken supplying the highest animal protein in developing and rural based economies [16]. The United Nations notes that broiler production is key to sustainable production of high quality, nutritious, safe and affordable animal protein or attainment of the sustainable development goal of “food security/zero hunger” for the ever-growing world population, projected to be 9.6 billion by 2050 [1].

Concomitantly, research on strategies that focus on finding and developing natural and safe substitutes to antibiotics that can be used in both the small-scale and commercial broiler chicken production has grown tremendously over the last few decades [17]. From this premise, several options have been postulated to improve broiler chicken growth performance. These include use of nanoparticles [18], prebiotics [19], probiotics [20], postbiotics [21], and organic acids [22]. However, the efficacy and monetary cost of these commercially available technologies remain unclear due to disparities in experimental conditions, inconsistent experimental results and cost structure of poultry production [17, 23]. Interestingly, phytogenic compounds containing plants and herbs such as Moringa oleifera, have gained much of the favourable attention due to several perceived and research supported animal and human benefits [15, 24,25,26,27,28]. Indeed, phytogenic compounds have shown several biologically important activities, including anti-inflammatory, anticoccidial, antioxidant, antimicrobial, immunomodulating, antiviral, growth promoting effects, as well as improve feed palatability, endogenous enzyme secretion, gut function and health, and meat safety and quality, making them an ideal natural growth promoter [29,30,31,32,33]. This has fuelled the growth of phytogenic additives market segment to the value of USD 962.5 million in 2023, up from USD 586.8 million in 2017 [34], which is projected to rise reach USD 1,223 billion by 2027.

Moringa oleifera tree (drumstick) is widely grown in sub-Saharan Africa, and readily available for utilisation in the northern parts of South Africa. The South African climate supports prodigious growth rates, high biomass production and high crude protein content of 25–27% DM necessary to support broiler production [35], energy, vitamin (A, B and C), calcium, iron and phosphorus [36]. Also, the negligible tannins and other anti-nutritional compounds make it an ideal feed ingredient for broiler chickens [37, 38]. There are no documented toxic characteristics that may disturb the lymphoid/immune organ physiology and function or promote proliferation of pathogenic bacteria and toxins. Furthermore, PFAs should induce growth of beneficial indigenous microbes such as Lactobacillus species through the competitive exclusion, antagonism and compatibility with the gut microflora [39]. Based on results from in vivo studies, Moringa oleifera leaf meal powder (MO) has potential for use as a non-therapeutic phytogenic feed additive (PFA) and alternative to AGP [40,41,42]. This is supported by high levels of polyphenol and a host of bioactive compounds with comprehensive pharmacological and medicinal actions that promote growth rates, feeding conversion efficiency and animal gut health [43,44,45,46]. Specifically, supplementing with 1.2% MO led to improved body weight, duodenum, jejunum, ileum villus height and intestinal length [47]. Furthermore, higher E. coli inhibition with 12% M. oleifera extracts [48], and increased proliferation of probiotic P. pentosaceus, L. acidophilus and L. plantarum bacteria was observed in vitro [49]. These findings show a reduced predisposition of broilers to an increase in Clostridium perfringens that cause clinical and sub-clinical intestinal necrotic lesions and moderate to severe hemorrhaging and bloody/ diarrhea excreta. Indeed, PFA as a storehouse of several bioactive ingredients may offer protection to immune organs such as the thymus, spleen, and bursa of Fabricius against damage causing reactive oxygen species (ROS) production increases.

However, the effectiveness of PFA is affected by many factors including their functional groups, chemical composition, interactions (synergistic or antagonistic) with other feed components, as well as environmental and husbandry conditions [15]. In vivo results have been varied and rudimentary, limiting available literature to draw focused conclusions specific to the comparative effects of MO vs AGP broiler growth performance and health indicators. Available literature shows other MOLP inclusion levels, except 1, 3 and 5% of mature and undamaged (no visible leaf rot, damping off, rust or twig canker disease) leaf material without stem, stalks, twigs, roots or seeds. This necessitates further extensive scientific evaluation and validation of the potential of MO as a sustainable substitute to AGP. Also, given the higher efficacy (higher weight gain and better feed conversion), availability and low cost of available synthetic AGP, and the farmers’ aim at further optimising the feed conversion efficiency, identifying the greatest opportunities to yield sustainable productive, financial and economic benefits of withdrawal of AGP for PFA seems mandatory [50, 51]. Therefore, the objective of this study was to investigate the effects of feeding graded levels of MO as an additive on broiler chicken growth performance, carcass characteristics, lymphoid organ indices, intestinal lesions, bloody excreta, microbial population and mortality, and economic viability.

2 Materials and methods

The methods were carried out in accordance with relevant guidelines and regulations of the University of Fort Hare’s Research Ethics committee (UREC) approved guidelines on plant and animal welfare. An ethical clearance certificate number: MUC181SNAT01 was obtained, plants and animals were raised and used in accordance with the University of Fort Hare’s Research Ethics committee (UREC) approved guidelines on animal welfare.

2.1 Study site and feed material preparation

The feeding trial was conducted at the Fort Cox College of Agriculture and Forestry situated 32°43ʹ 48ʹʹ S 27° 1ʹ 32ʹʹ E in the Eastern Cape Province of South Africa. Mature, undamaged M. oleifera leaves with no visible leaf rot, damping off, rust or twig canker diseases or physical damage from healthy plants were selected. No stalks, twigs or roots were included. The material was collected from villages around Limpopo Province of South Africa. The leaves were sun dried, ground with a 4 mm sieve, sealed in polythene bags and stored in a cool and dry place. Proximate analysis was used to determine the nutrient composition of MO and other feed ingredients. Analyzed nutrient composition of Moringa oleifera leaf meal (MO) was presented in Table 1. Feed components measured include dry matter, ash, ether extract, metabolisable energy, crude protein, crude fibre and nitrogen free extract.

Table 1 Analyzed nutrient composition of Moringa oleifera leaf meal (MO) on as fed basis
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2.2 Experimental design, dietary treatments and management

Day old Cobb-500 (n = 1600) unsexed chicks, sourced from Tydstroom (SA), a commercial hatcher, were allotted to 4 dietary treatment groups, with 4 replicate pens. Each pen contained 100 birds in a completely randomised design. Birds were vaccinated for infectious bursal and Marek’s diseases at hatchery prior to collection, and no other vaccinations followed during rearing period. Fresh dry wood shavings were set as bedding at 10 cm depth on concrete floor pend and a stocking density of 10 birds/m2 was maintained throughout the experiment. The dietary treatments were as follows: positive control group/CON = standard basal diet containing 486 mg zinc bacitracin and 650 mg salinomycin per kg of feed; MO1 = Basal diet + 1% MO; MO3 = Basal + 3.0% MO; and MO5 = Basal diet + 5% MO. Moringa included as part of the 100% of the diet formulation during mixing of diet ingredients. Feed was prepared according to the NRC standards [52], designed to meet the bird’s nutrient requirements. The gross composition of the experimental diets is presented in Tables 2, and 3 depicts Analysed nutrient and mineral composition of the experimental diets on a dry matter basis. A 3-phase feeding programme consisting of starter (1–21 d), grower (22–28 d) and finisher (29–35 d) diets was formulated to be iso-caloric and iso-nitrogenous. Proximate analysis of feed was conducted using the methods outlined by the guidelines of AOAC [53]. The treatment groups were subjected to the same environmental conditions, with feed and clean water offered ad libitum. The ambient temperature in the experimental house was maintained at brooding temperature of 34 °C during the first week, and gradually reduced to 22 °C in keeping norms with standard brooding practices using an automated system. Continuous light was provided on first day, reduced to 22 h and 2 h darkness period on the next day. Thereafter, a 16-h light: 3-h dark: 2 h-light: 3 h-dark regime was followed. Principles of animal care and management were strictly adhered to during the entire experiment according to NRC [52]. Birds were monitored daily, and dead birds were weighed, and their data was used correction in the feed consumption and weight gain calculations.

Table 2 Gross composition of experimental diets on as fed basis
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Table 3 Analysed nutrient and mineral composition of the experimental diets on a dry matter basis
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2.3 Performance parameters

The chicks were weighed on the day of arrival and at each 7-day interval, throughout the experimental trial using a Z742838 Kern PCB precision balance, (Sigma Aldrich, SA, USA). Variables measured include day old chick weight before watering and feeding, average weekly weight, average daily feed intake (ADFI), average daily weight gain (ADG), cumulative feed intake (FI), feed conversion ratio (FCR) for each phase (starter, grower and, finisher) and the entire experimental period [54]. Mortality rates were calculated at the end of the experimental trial.

2.4 Carcass and internal organ characteristics

At 35 d of age, feed was withdrawn for 8 h to allow crop emptying. Birds had free access to water until slaughter. Thereafter, 20 birds per replicate pen were randomly sampled and processed. The birds were individually weighed, electrically stunned using 70 V for 5 s to render them unconscious and sacrificed by cervical dislocation using a sharp blade. Bleeding, soft scalding at 57 °C for 120 s, automated plucking and washing was followed by manual evisceration and separation of digestive tract, abdominal fat and other internal organs (liver, heart, gizzard, and spleen). The carcasses were placed in a chiller overnight for dripping and cooling at − 4 °C, pending further analysis. The cold carcass weight was obtained followed by dissection to separate breast (both Pectoralis major and Pectoralis minor), skeleton and skeletal muscle, thigh, drumstick, wing, feet, neck and head, according to the method developed by Ziolecki and Doruchowski, [55]. Leg and breast were trimmed of adipose tissue. All dissected components and internal organs were individually weighed using a Z757284 Kern ABT analytical balance (Sigma Aldrich, SA, USA). Dressed carcass weight, internal organs and abdominal fat were expressed in relation to preslaughter body weight. The weights of cut breast, backbone, thigh, drumstick was expressed as percentage of cold dressed carcass weight.

2.5 Immune organ index, gross lesion and bloody excreta scores

The thymus was separated by ventral neck dissection. The spleen, bursa of Fabricius and small intestine were collected by ventral abdominal dissection [56]. Excess fat was removed, and organs rinsed in saline solution and bloated with filter paper to remove extra moisture. The organs were weighed using a Z757284 Kern ABT analytical balance (Sigma Aldrich, SA, USA) and immune organ index for each individual organ calculated and expressed as percentage of live body weight [57] using the formula;

$${\text{Immune organ index}}\, = \,{\text{immune organ weight }}\left( {\text{g}} \right)/{\text{Live weight }}\left( {\text{g}} \right).$$

Intestinal contents were aseptically collected into sterile plastic bags (Sigma Aldrich, SA, USA) and stored appropriately for microbial flora enumeration as described later.

The intestinal mucosa was gently flushed with distilled water to remove digesta and non-adherent micro-organisms and excess moisture dripped on cotton swabs. Approximately 3 cm identical segments of the caecum, duodenum and jejunum were dissected and flushed with phosphate buffer saline before macroscopic observations (Sigma Aldrich, SA, USA). An independent panel, blinded to the experimental design, was tasked to assign gross lesion score using 5-point scale model [58]. In this model, 0 = normal status with no gross lesions, 1 = small, scattered petechiae, 2 = numerous lesions, 3 = extensive haemorrhage and 4 = dark coloured haemorrhaged intestine.

The presence of bloody excreta was determined daily in week 4 and 5 by observing freshly evacuated faeces on the ground litter. The extent of bloody diarrhoea was described using a 5-point scale method [58, 59]. The following scores were used; 0 = normal faeces with no bloody diarrhoea observed (normal status); 1 = less than 25%; 2 = between 26 and 50%; 3 = between 51 and 75% and 4 = above 75% bloody faeces in total faeces/profuse amount of blood.

2.6 Enumeration of bacterial population

Fresh digesta samples from the ileum and caecum were emptied into sterile containers immediately after slaughter for microbial population enumeration within an hour of collection. One-gram sample from each bird using a Z757284 Kern ABT analytical balance (Sigma Aldrich, SA, USA) and diluted 10 folds i.e., 1:9 wt/vol with physiological salt water and homogenised for 3 min. This was followed by serial dilution of digesta homogenate from 10–1 to 10–7 and coated onto the appropriate agar media [60]. Total aerobic bacteria (TAB) were inoculated on Nutrient agar (Merck, South Africa) plates after incubation at 37 °C for 48 h in aerobic conditions. E. coli was inoculated on MacConkey agar (Merck, South Africa) and incubated for 24 h at 37 °C under aerobic conditions. Lactobacillus spp was inoculated on de Man Rogosa Sharpe agar (Merck, South Africa) and incubated at 37 °C under anaerobic conditions for 72 h. Clostridium perfringens (C. perfringens) was inoculated on Clostridial Agar and incubated at 37 °C under anaerobic conditions for 48 h (black colonies). Bacterial colonies on plates were enumerated using a colony counter (Digital colony counter, LA663, HiMedia Lab. Pvt. Ltd., Mumbai India). Finally, the enumerated microbial colonies forming units were expressed as log10 CFU/g of ileal or caecal digesta [61, 62]. All microbiological analyses were performed in duplicate and the average values were used for statistical analysis.

2.7 Economic viability

The profitability indices calculated include the gross and net income, which were included in the calculated partial economic analysis. The economic viability of including MO in the diet was calculated based on animal performance data during the experimental period and according to the prices of feed ingredients at the time of the study, whilst all other management variables were kept constant across all dietary treatments [63].

2.8 Statistical analysis

An ANOVA was carried out using the general linear model procedure of SAS (Statistical Analysis System Institute, version 9.3, Cary, NC, USA) to analyse the effect of dietary treatment on overall growth performance, carcass and internal organ characteristics, mortalities and economics [64]. The Fisher’s least significant difference test was used to compare means between test subjects and were significant when P < 0.05. Data were expressed as mean and pooled SEM.

3 Results

3.1 Performance parameters

The results for growth performance parameters are presented in Table 4. Data on growth performance indicators showed significant weight gain and feed efficiency differences due to the effects of MO dietary supplements. The results presented show that MO supplementation did not affect ADFI (P < 0.05) but led to a significant increase in ADG (P < 0.05) in the starter phase. Interestingly, birds fed a diet with 5% MO exhibited the lowest FCR during this period. An increase in dietary MO led to a significant decrease in ADG, ADFI as well as FCR in the grower phase (P < 0.05). During this period, MO3 and MO5 exhibited the lowest FCR, whilst CON and MO1 had the highest FCR. An increase in MO supplementary levels led to a significant increase in ADFI and ADG in the finisher phase of the experiment (P < 0.05). Cumulative feed intake was significantly higher in CON group and decreased with incremental levels of MO for the 0–35 d period (P < 0.05). The FCR of birds in the MO5 was higher than in the CON, MO1, MO3 groups during this period. Birds on MO3 and MO5 exhibited the lowest FCR, whereas those in the MO1 exhibited the highest for the entire period (0–35 d) (P < 0.05).

Table 4 Effect of feeding graded levels of Moringa oleifera leaf meal (MO) on feed intake, body weight gains, and feed conversion ratio of broilers
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3.2 Carcass and internal organ characteristics

The effect of MO on the carcass and internal organ characteristics of broiler chickens was further explored, and the detailed results presented in Table 5. Birds fed the control diets were significantly heavier at slaughter with corresponding higher cold dressed carcass weights and dressing percentages (P < 0.05), whilst the lowest was recorded for those receiving MO5 diets. There was no significant effect of MO on breast, thigh and heart proportions (P > 0.05). The mean proportions of drumstick, wing, feet, neck, small intestine, liver, and gizzard (P < 0.05) improved with MO supplementation. The results showed that the MO5 group had significantly increased proportions of drumstick and wing compared to other treatment groups. The highest (P < 0.05) abdominal fat was observed in the control group. The MO5 group exhibited significantly higher liver, gizzard and intestinal weights than other groups (P < 0.05).

Table 5 Effect of graded levels of Moringa oleifera leaf meal (MO) on carcass characteristics and internal organ characteristics of broilers
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3.3 Immune organ index, macroscopic lesions and bloody diarrhoea scores

The effects of MO on immune organ indices, macroscopic lesions and bloody diarrhoea scores are presented in Table 6. The were no significant differences in thymus indices on day 28 (P > 0.05). The spleen, Bursa of Fabricius, liver, and intestine indices were highest in birds in MO5 group and comparable among other groups. This trend was observed amongst all organs on day 35, with the 5% MO feed group showing the highest indices (P < 0.05).

Table 6 Effect of Moringa oleifera leaf meal (MO) on live weight, immune organ indices, macroscopic lesion and bloody excreta scores and mortality of broilers
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Results show a reduction in necrotic scores with an increase in dietary MO levels as depicted in Table 6. The duodenum, jejunum and ileum necrotic scores in 1 and 3% MO feed groups were higher (P < 0.05) than that in the control and 5% MO group at days 28 and 35. The jejunum had significantly higher necrotic scores than duodenum and ileum at both days 28 and 35 (P < 0.05).

Bloody excreta scores observed were significantly higher in MO1 treatment groups compared to all other groups in weeks 4 and 5 (P < 0.05). Interestingly, the control and MO5 groups recorded the least bloody excreta scores among all treatment groups (P < 0.05).

3.4 Microbial population

From the experimental results, incremental levels of MO led to reduced total aerobic bacteria, E. coli, C. perfringens and lower Lactobacillus spp counts in all intestinal segments as depicted in Table 7. Specifically, higher E. coli and C. perfringens and lower Lactobacillus spp counts were observed in MO1 treatment group compared to control and MO5 groups (P < 0.05) in the duodenum and jejunum at day 28 of sampling. Interestingly, an increase in MO led to higher Lactobacillus spp counts in the ileum of MO5 than those observed in the control and MO1 group of birds.

Table 7 Effect of Moringa oleifera leaf meal (MO) on gut microbial population of broilers
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At day 35, TAB and E. coli in the duodenum and jejunum were higher in MO1 group of birds compared to those in control and MO5 groups (P < 0.05). The opposite was true for Lactobacillus spp. counts with the highest recorded in MO5 group. Lactobacillus spp in the ileum were significantly improved by increasing dietary MO (P < 0.05). Duodenum C. perfringens and ileum TAB and E. coli were not affected by dietary treatment. Generally, microbial counts of Jejunum are usually higher than duodenum and ileum.

3.5 Economic analysis

The economic analysis of using phytogenic MO in broilers diets at different substitution levels in terms of AGP, MO feed costs and, economic returns was analyzed and displayed in Table 8. Birds in the CON group had significantly higher mortality (2.00%), followed by MO5 (1.8%) and MO3 (1.6) and lowest in those receiving 1% MO (0.95). The feed costs were positively related to incremental levels of MO in broiler diets, guided by low cost of AGP per bird than that of PFA per bird. The economic analysis showed increases in the broiler diet cost with an increase in MO. Overall total cost of feed per bird was highest in birds in MO5 (39.97) followed by MO3 (30.58) and MO1 (20.90) and lowest in CON (20.42). Birds in the MO5 group attained the lowest weights, followed MO3 and MO1, with the highest final weights observed for CON group. The CON group obtained the highest gross and net revenue per bird, whereas the MO5 group recorded losses per bird. The highest economic efficiency (1,14) was achieved in birds on CON diets, and lowest in MO5 group (− 0.11) birds.

Table 8 Effect of graded levels of Moringa oleifera leaf meal (MO) on economic efficiency of broilers
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4 Discussion

The current study aimed to compare the effects phytogenic Moringa oleifera leaf powder (MO) vs antibiotic growth promoter on growth performance, carcass characteristics, lymphoid organ indices, intestinal lesions, bloody excreta, microbial population, and economic viability of broiler chicken grown for 35 d. To date, MO has been used in many feeding trials of monogastric animals. However, there are several inconsistencies in the available literature, due to variations in chicken strain, intestinal stability of the active compound, compositions of active ingredients and experimental conditions and duration [14]. Emerging scientific findings are starting to shed light on the responsible mechanisms of action of these natural PFA. In the current study, chickens did not reach the weights expected of the Cobb-500 strain, despite several studies showing maintained or improved FI, AGD and FCR [35, 47, 65]. The dietary inclusion of MO led to lower marginal ADFI, ADG, and final body weights but favourable FCR. These results are consistent with reports of a lowered feed intake but improved FCR when broiler chickens were supplemented with 2% MO as a substitute for antibiotics [66]. The trend is similar to the results of [67] who found lower body gains and better FCR when aqueous MO extracts were offered at 90, 120, and 150 ml/l to Hubbard broiler chickens. Aderinola et al. [68] and Younis et al. [69] observed reduced body weight gains and final weight in Cobb-500 strains supplemented with MO. Similarly, Oghenebrorhie and Oghenesuvwe [70] found a reduction in average daily gain at 6, 8, 10% MO inclusion levels. Two explanations could be attributed to depressed feed intake but improved FCR. Firstly, Toghyani et al. [71], described factors such as feed presentation or form to have an effect in stimulating appetite and intake. The mash, plus the size of powder grains used in the current study was characterised by a high volumetric density with dust particles accumulation which may have caused lower acceptability, selective feeding, longer feeding time and higher energy expenditure. It is possible that the, dark colour of MO of feeds from tannins in raw leaf meals may have acted as deterrent factors thus depressed intake [48, 72]. In addition, low digestibility associated with high fibre content (3.5%) could be another reason for reduced growth. Secondly, previous studies attribute low feed intake and improved FCR to adequate supply of minerals and vitamins [73]. Although the chemical concentration was not determined in our study, the pharmacological biochemical profile of MO nutrient reveals a plethora of bioactive compounds that have high nutritional quality (including adequate minerals and vitamins), antimicrobial, antioxidant properties and immune-stimulant activities [36, 37, 43, 45, 49, 74]. These compounds aid in suppression of free radical build-up, activate antioxidant enzymes, inhibit oxidases and increase protective effects on the cytoplasmic membrane allowance [48]. This in turn improves nutrient availability, digestion, absorption, metabolism, utilisation and therefore early nutrient satisfaction of birds on MO diets [67]. In addition, phytogenic and rare polyphenolic compounds; apigenin, quercetin, zeatin, and kaempferol limit growth and proliferation of pathogenic microbes, effect oxidase and activate antioxidant enzymes [46, 75], that in turn, reduces microbial utilisation of nutrients, and improves nutrient digestibility and mineral absorption allowing for efficient mobilisation for growth [41, 74, 76]. This is supported by Zhang et al. [65] who suggested that an addition of 5% MO in early life stages helps enhance the richness and diversity of the broiler intestinal flora, impacting future digestion, absorption, and health of the host. Also, derivatives from phytogenics facilitate increased production of lactic acid bacteria, thus increasing beneficial probiotic concentration and reducing pathogenic gram-negative bacteria [77]. Recent research suggests that phytogenics improve broiler feed efficiency via a decrease in feed intake by modulation of feed-related hypothalamic expression of muscle rapamycin (mTOR) pathway and peripheral intermediary hepatic lipogenic neuropeptides. These peptides are responsible for regulating muscle protein synthesis and lowering hepatic lipogenesis and adipose tissue lipolysis [34, 78,79,80]. These molecular pathways control appetite and energy balance, protein accumulation in the muscle and fat distribution and improved performance [81]. This is however contrary to the effects of flavonol glycosides, quercetin, kaempferol and alkaloid moringinine, predominant in MO that are synonymous with improved voluntary appetite improvement [82]. This remains debatable. Lastly, it is predicted that MO at a small dosage of 3–5% could be useful and safe as an AGP replacement. Furthermore, Khan et al. [47] dismisses higher dose in MO toxicity but notes the need for sensitive dose-dependent trials.

Result on carcass and internal organs of broiler chickens fed varying levels of MO indicate depressed carcass weight and yield. This is contradictory to previous studies [35, 65, 83] that revealed an improvement in both carcass weight and dressing percentages with feeding MO. Interestingly, drumstick, wing and internal organ gizzard, liver and intestine proportions increased with MO incremental levels in our study. Akande et al. [82] notes a direct relationship between improved FCR and better carcass and prime cuts proportion as noted in our study. Higher muscle accretion, dressing percentages and carcass proportion are important traits in achieving greater profitability of any broiler enterprise. Today’s broiler products are marketed as retail cuts or further processed foods in most retail. As such, increasing the amount of edible and saleable meat per carcass [50] and yields of items such as breast, wing, drumstick and thigh has become of key value to producers and processors as a strategy of maximising economic returns [84]. Therefore, if these production objectives are not achieved, feeding MO to broilers should then be for other additional parameters such as health performance, meat quality or reasons other than broiler weight improvement to justify the additional costs incurred with its inclusion [85]. Internal organ proportion changes are an indication of biotransformation function and presence of risk factors such as of anti-nutritional factors in the diet [73]. The promotion of digestive organ (intestine, liver, and gizzard) proportions may indicate improved intestinal development and efficient absorption of nutrients. The increase in liver size may be due to increased sugar storage and lipid metabolism in the body [86]. These changes lead to increased capacity for synthesis of accessory proteins [87] and the formation of lymphoid clusters integral factors in humoral immunity and continued protection against pathogenic organisms [88]. The gizzard weight is extremely sensitive, and increases with high dietary fibre intakes. Consistent with our results, Wang et al. [89] observed reduced intestinal length and weight when diets with a common combination of bacitracin and salinomycin antimicrobials were administered. The authors speculated that this is synonymous with energy partitioning towards growth rather than organ maintenance. Rather, Khan et al. [90] observed an increase in caecum weight and attributed heavier intestinal weights to longer retention times of digesta to high fiber content, as well as the potentiating effect on the gut.

Results from this study show a linear decrease in abdominal fat accumulation with incremental levels of MO. It is reported that 90% of de novo synthesis of fatty acids occurs in the chicken liver and is controlled by several enzymes including rate-limiting acetyl-CoA carboxylase alpha (ACCα) and sterol regulatory element binding protein(SREBP)-cleavage activating protein, (SCAP) gene responsible for activation of SREBP-1, a key transcription factor for lipogenesis [91]. Flees et al. [92] observed downregulation of SCAP and increased phosphorylation of ACCα leading to reduced de novo lipogenesis, as well as, mobilization of fat stores when two phytogenic water additives, AV/SSL12 (AVSSL) and Superliv Gold (SG) (Ayurvet Ltd. Kaushambi, Ghaziabad, India) were provided for broilers. This process is initiated by MO causing an increase in beneficial Lactobacillus that regulates acetyl-CoA carboxylase activity [93]. Another potential mechanism by MO is through the blockade of expressions of the genes, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR), peroxisome proliferator-activated receptor α1 (PPARα1), and peroxisome proliferator-activated receptor γ (PPARγ). Abdominal fat reduction was also observed when a mixture of phytogenic compounds with manganese (ValiMP,) was fed to broilers [94]. Similarly, a blend of Echinachea purpurea, Matricaria chalmomilla, Viola triocolor, Triticum repens, and garlic (Phyt Exponent®) reduced abdominal fat in broilers [95]. Finally, dietary inclusion of phytogenic pomegranate meal significantly decreased serum triglycerides, cholesterol and abdominal fat deposition [96]. These peripheral molecular mechanisms may explain the positive impact of MO as a multi-functional hypo-cholesterolemic agent inhibiting lipid deposition in adipocytes and reduction of abdominal fat accumulation in our trial. From a producer’s point of view, low abdominal fat level is an indication of efficiency of use of feed energy and improved carcass percentage and monetary value. From a marketing and health perspective, low levels of abdominal fat on a carcass and meat helps improve consumer acceptance and satisfaction levels [97]. Finally, lean meat consumption reduce incidences of cardiovascular diseases caused by excess lipid and cholesterol accumulation in the blood pathways [16].

The thymus and bursa are the lymphoid organs, whilst the spleen is a peripheral lymphoid organ involved in body immune system of poultry. In the presence of diseases or the invasion of pathogenic microorganisms, these organs produce and develop cell-mediated immune responses reflected by their histomorphology appearances. Immunomodulatory effects of PFA in augmenting a healthy immune system are well recognized and evidenced by increased thymus, small intestine, liver, and spleen weights [98]. The mechanism involves suppressing, stimulating, or modulating adaptive or innate parts of the immune response [99]. In our study, immune organ proportions increase with MO incremental levels, indicating activation and improved immune responses. This phenomenon can be explained by MO diets’ improved appetite, digestion stimulation, availability of energy and nutrients, and absorption-enhancing properties that trigger the growth and development of immune organs when stressful conditions arise [32, 57].

Regarding the excreta score and intestinal lesion scores, control and 5% MO diets were proven effective in restoring gut integrity and reducing intestinal lesion scores in our experiment. The gut-mucosa-lymphoid organ system acts as the first line of defense against any ingested pathogenic microbes. Any attack is reflected by textural and colour of excreta and post mortem macroscopic appearance of the gut mucosa lining. This improved gut lesion scores may be another possible reason for improved FCR in broilers on MO diets. This is in agreement with the results by Hussein et al. [100] who found reduced gross intestinal lesion scores when a phytobiotic Sangrovit (0.12 g kg−1) was offered to Clostridium perfringens-infected Ross-308 broiler chickens. Researchers have demonstrated that the intestinal mucosa layer is the site of the first crucial mechanical defense barrier dialogues with microorganisms and immune activation responses [101]. Induction of pathogenic microbes into epithelial cells disrupts the intestinal epithelial barrier integrity/function, and gut cellular junctions, finally causing lesions that reduce nutrient absorption and mortalities. However, this is improved by biophenols-rich PFA additive supplements that alleviate intestinal damage through the restoration of normal redox balance and induce the integrity of gut cell wall [19, 77, 81]. Observations recorded by authors indicate acceptable levels as they also corresponded with low mortality rates and low levels of pathogenic microorganisms of economic importance discussed below.

In terms of changes in the intestinal microbiota, as shown in Table 6, dietary inclusion of MO prevented proliferation of C. perfringens, E. coli and TAB and promoted growth of Lactobacillus spp. The antimicrobial efficiency of MO in the current study is supported by El Banna [102]. The antimicrobial effects may be ascribed to presence of active agents such as benzyl isothiocyanate, pterygospermin and aglycone of deoxy-Niazimicine [103]. These compounds penetrate and damage the bacterial cell wall. Also, these compounds have the potential to colonize the intestine and antagonize and cause competitive exclusion of pathogenic pathogenic bacteria and improve digestion and absorption [104]. The synergistic effect of active agents of MO on beneficial bacteria is described by Anonye [105] and supported by Alloui et al. [106]. This synergistic potential phenomenon confirms possibility of MO for use as a growth promoter in broiler diets. The results also indicate that microbial counts of jejunum are usually higher than those in the duodenum and ileum and especially higher for groups supplemented with 5% MO. Svihus [107] described the jejunum as the site of completion of digestion and absorption of major nutrients fat, starch and proteins. This is confirmed by an empty weight 20–50% higher than that of the ilium [108]. In addition, E. coli, TAB and C. perfringens decreased slightly with advanced age, whilst Lactobacillus spp microbial counts increased. This generally suggests better and continued protection of the broiler birds for better utilization of available nutrients.

Feed costs constitute about 70–80% of the production variable costs of broiler enterprises, thus many producers are continuously searching for cheaper feed ingredients that result in optimum feed efficiency and high production performance parameters [109]. Profitability indices are key in assessing the feasibility and sustainability of any production option of any broiler enterprise. As such, the use of MO as a feed ingredient was assessed in the current study and results show an increase total feed cost and lower weight gains of birds. This in turn resulted in lower gross returns per bird. Ayo-Ajasa et al. [110], found an increase in total feed cost when Moringa leaf meal was included in the diet. However, Juniar et al. [111] found no significant effect of feeding MO at up to 10% on income over feed cost. Similarly, Paguia et al. [112] did not observe any changes in feed cost and income over feed and chick costs when feeding MO at up to 0.5% inclusion level. However, Zanu et al. [113] found a reduction in feed cost with an increase in level of MO inclusion at up to 15% as a substitute for fishmeal. The increase in overall feed cost as MO inclusion increased may suggest that this ingredient could be far more expensive than that sourced elsewhere in other studies. As observed by Ayo-Ajasa et al. [110], the claims for health benefits in humans have increased the demand and prices for MO from the current source, thus making it more expensive and economically unsustainable to use as livestock feed. The cost of MO could be a game-changer in its application as an alternative to antibiotic in the poultry industry. In the past decade, low production levels and the use of MO in the treatment of many diseases in humans led to high market prices. However, current trends show an increase in MO production, availability, as well as a decrease in market prices. At the same time, there has been an increase in adoption of MO use in animal production systems as a protein source and phytogenics additive in areas where it is easily accessible[114].

5 Conclusion

Dietary inclusion MO at 1–3% as an alternative to antibiotics led to better feed conversion ratios, reduced abdominal fat, improved immune indicators, gut microbiota populations and low mortality. The high cost of MO reduces the economic efficiency and thus cheaper sources are necessary for sustainable small-scale communal and commercial enterprises. In recommendation, further studies to develop robust and novel techniques to quantify and improve bioactive ingredient bioavailability and bioaccessibility of AGP bioactive compounds, determine efficacy and effective doses of extracts, derivatives or AGP combinations at each phase feeding stage are necessary to improve economic efficiency. Finally, studies to determine expression of feeding-related hypothalamic neuropeptides as potential underlying mechanisms’ indicators of growth performance in relation to PFA would be appropriate.