The effects of Myristica fragrans seed meal and Syzygium aromaticum leaf meal dietary supplementation on growth performance and oxidative status of broiler chicken

The study examined the effects of Myristica fragrans seed meal (MSM) and Syzygium aromaticum leaf meal (SLM) dietary supplementation on the growth performance, health status, and meat quality of broiler chicken. A total of two hundred and forty 1-day-old broiler chickens were assigned the four dietary treatments: Diet 1/control (no supplementation), Diet 2 (0.25% MSM), Diet 3 (0.25% SLM), and Diet 4 (0.25% MSCM, i.e., MSM + SLM 1:1). The birds were fed ad libitum throughout the feeding trial. The dietary supplementation of 0.25% MSCM resulted in higher body weight gain and lowered feed conversion ratio (p < 0.05) at the finisher stage and overall period, while feed intake was not statistically affected (p > 0.05) across the treatments. The dietary inclusion of MSM, SLM, and MSCM did not significantly influence carcass traits, relative internal organ weights, and haematological indices of the broiler chicken (p > 0.05) except mean cell volume and mean cell haemoglobin content (p < 0.05). The result further revealed that broilers fed diets supplemented with MSM, SLM, and MSCM had higher (p < 0.05) serum catalase, glutathione peroxidase and lowered serum cholesterol compared with control. The meat cholesterol levels and lipid peroxidation of the birds were significantly (p < 0.05) reduced, while glutathione peroxidase was increased by dietary MSM, and MSCM supplementation. The MSCM supplementation at 0.25% enhanced the body weight gain, improved serum, and meat glutathione peroxidase and catalase but reduced the broiler’s meat cholesterol level and lipid oxidation.


Background
The recognition and utilization of herbs, spices, and their extracts as natural feed additives in poultry diets have increased in recent years due to their inherent multi-bioactive properties and ability to enhance performance traits, reduce pathogenic bacteria, and decrease antibiotic residues in meat and egg products (Dhama et al. 2015). In comparison to antibiotic growth promoters, herbs and spices are economical and readily available, edible and low in toxicity, rich in nutrients, and useful in improving the health of animals and consumers (Dhama et al. 2015). Applications of herbs in poultry diets have been reported to stimulate endogenous antioxidants, facilitate nutrient metabolism and improve meat quality by lowering cholesterol content and inhibiting peroxidation (Oloruntola et al. 2018;Oloruntola et al. 2019). Interestingly, Myristcan fragrans and Syzygium aromaticum plants are among the herbs and spices with a lot of potentials that can be utilized as feed additives in poultry production.
Myristica fragrans, also known as nutmeg, is an aromatic perennial tropical plant with a characteristic scent that possesses multiple medicinal and antioxidant properties (Periasamy et al. 2016). Its seed is widely used as herb and spice in many traditional and conventional food industries around the world (Olaleye et al. 2006) The seed contains phenolic, flavonoid, and other important bioactive compounds (Zakaria et al. 2015;Yakaiah et al. 2019). Nutritionally, nutmeg is rich in energy, carbohydrates, proteins, dietary fibre, vitamins (Vitamins A, C, and E) and minerals (Agbogidi and Azagbaekwe 2013). The potential phytochemicals in Myristica fragrans plant have been reported to exert antioxidant, antimicrobial, antipain, antiobesity, and hepatoprotective activities in biological systems (Morita et al. 2003;Olaleye et al. 2006;Agbogidi and Azagbaekwe 2013;Yakaiah et al. 2019). The dietary administration of Myristica fragrans extracts was reported to inhibit lipid digestion, absorption and accumulation of adipose tissue in the rat (Yakaiah et al. 2019), reduce cholesterol level in rabbits (Sharma et al. 1995), and prevent liver damage in the rat (Morita et al. 2003). Inclusion of Myristica fragrans seed extracts at 1.25, 2.50, and 5.00% (g/ml) has been demonstrated to improve physicochemical properties and oxidative stability of raw beef during 3-week cold storage at − 18°C compared with control (Zakaria et al. 2015). Despite the efficacy of Myristica fragrans seed in muscle food preparation, little or no work has been done on its dietary application in poultry production.
Syzygium aromaticum (clove) is an aromatic evergreen plant commonly used in the preparation of curry powder. It is very rich in many bioactive compounds including eugenol (72-90%), flavonoids, and triterpenoids (Bhowmik et al. 2012). Nutritionally, it contains vitamin A (retinol); beta-carotene; and vitamins K, B6, B1, and C (Nadkarni, 2000;Dorman et al., 2000). Syzygium aromaticum has been used to treat indigestion, parasitic infestations, cough, and blood impurities (Bhowmik et al. 2012). Many reports have shown that it contains antimicrobial, antioxidant, antiviral, and anticancer agents (Bhowmik et al. 2012;Sulieman et al. 2007). Application of Syzygium aromaticum at a high concentration as a growth promoter in the animal diet has been successfully reported although with contrasting results particularly on growth performance. Mahrous et al. (2017) in a study found that supplementation of Syzygium aromaticum powder at 1.0 and 1.5 g/kg diets significantly decreased body weight gain but increased feed conversion ratio and protein efficiency ratio compared with the control treatment. Similarly, Al-Mufarrej et al. (2019a, 2019b reported that dietary inclusion of Syzygium aromaticum powder at 30, 40, 50, and 60 g/kg significantly reduced the body weight gain and increased necrotic enteritis of broiler chickens compared with control. On the other hand, Mahrous et al. (2017) found that the inclusion of clove powder at 1.0 and 1.5 g/kg diets did not affect growth performance parameters but improved the health and meat qualities of broiler chickens. It is therefore important to examine the supplementation of Syzygium aromaticum at low concentration on broiler chicken performance. The objective of this study was to determine the effects of Myristica fragrans seed meal and Syzygium aromaticum leaf meal dietary supplementation on broiler chicken.

Collection and processing of plant samples
The dry seeds of Myristica fragrans seeds and dry Syzygium aramaticum leaves were procured from the local markets in southwestern Nigeria, ground to about 100 μm with a hammer mill to produce Myristica fragrans seed meal (MSM) and dry Syzygium aramaticum leaf meal (SLM), respectively. Equal quantity (1:1) of MSM and SLM were mixed to form Myristica fragrans seeds and Syzygium aramaticum leaf meal composite mix (MSCM).

Animal management and experimental design
The broiler chickens' basal diet was prepared for the starter phase and finisher phase following the NRC (1994) nutritional recommendations (Table 1). At each of the production phases, the basal diet was divided into four equal parts and named diets 1 to 4. Diet 1 (control) has 0% supplementation, while diets 2, 3, and 4 were supplemented with 0.25% MSM, SLM, and MSCM, respectively.
Two hundred and forty 1-day-old Cobb 500 broiler chickens were randomly assigned to four experimental diets (60 birds/experimental diet; 10 birds/replicate) using a completely randomized design. The experimental pen temperature was regulated and maintained at 31°C ± 2 for the 14 days and gradually reduced by 2°C after each consecutive 7 days until the experimental house temperature was 26°C ± 2. The lighting duration was 23 h per day. The birds were fed ad libitum.
Growth performance, slaughtering procedures, collection of blood samples, and carcass analysis The experimental birds' body weight and feed intake were determined and recorded on a weekly interval. The feed conversion ratio was calculated as the ratio of feed intake by the birds to their weight gain. On day 56 of the experiment, 3 experimental birds per replicate were labelled and sacrificed as earlier described by Oloruntola et al., (2019). The jugular veins at the birds' neck region were cut with a clean and sharp stainless knife. Blood was collected into a plain blood sample bottle for serum antioxidant enzymes and serum enzymes and also into EDTA bottle haematological studies, respectively. The blood in the plain bottles was spined and its serum decanted into another set of plain well-labelled bottles and frozen at − 20°C prior analysis. The haematological indices were determined within 120 min of post-collection (Shastry 1983). The concentrations of serum enzymes were analyzed on a Reflectron ®Plus 8C79 (Roche Diagnostic, GombH Mannheim, Germany), using kits. The serum antioxidant enzymes-i.e. superoxide dismutase (Misra and Fridovich 1972), catalase (Aebi 1974), and glutathione peroxidase (Rotruck et al. 1973)-were determined. The selected slaughtered birds were dressed and weighed. The dressed percentage of the birds was calculated as a percentage of the slaughtered weight. About 100 g of the breast meat was cut for determination of the level of the lipid peroxidation (Botsoglou et al. 1994), meat cholesterol (Allain et al. 1974), catalase activity (Hadwan and Khabt 2018), and glutathione peroxidase activity (Cichoski et al. 2012).

Data analysis
The model, Tsm = μ + αs + βsm, was used in this experiment, where Tsm is any of the response variables; s is the overall mean; αs is the effect of the sth treatment (T = diets 1, 2, 3, and 4); and βsm is the random error due to experimentation. The data were subjected to oneway ANOVA using SPSS version 20. The differences among the experimental treatment means were determined (P < 0.05) by the Duncan multiple range test of the same package.

Results
Antioxidant activity and phytochemical composition of Myristical fragrans seed meal (MSM) and Syzygium aromaticum leaf meal (SLM) Table 2 shows the result of the phytochemical composition and antioxidant activity of Syzygium aromaticum leaf meal (SLM) and Myristica fragrans seed meal (MSM). The phytochemical composition analysis of MSM revealed higher saponin (43.35 ± 0.72 mg/g), flavonoid (2.06 ± 0.05 mg/g), phenol (26.27 ± 0.03 mg/g), and terpenoid (107.85 ± 0.75mg/g) contents than SLM. However, SLM had higher antioxidant (2,2-diphenyl-1picryl-hydrazyl-hydrate, DPPH) free radical scavenging activity (80.55 ± 0.07 %) than MSM (21.58 ± 0.42%). Table 3 shows the effects of dietary supplementation of Syzygium aromaticum leaf meal (SLM), Myristica fragrans seed meal (MSM), and their composite mix (MSCM) on the growth performance of broiler chickens at starter and grower phases and overall period. At the starter phase, the study revealed no significant difference  (P > 0.05) in the initial body weight, body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) across the treatments. However, at the grower phase and overall period, broiler chickens fed diets supplemented with MSCM had higher BWG and better FCR (P < 0.05) than the control group, while there was no significant difference in feed intakes (P > 0.05) across the treatments. In general (during the overall period), birds on diet 0.25% MSCM had the highest BWG (3516.05 g) followed by those on diets 0.25% MSM (2875.27 g) and 0.25% SLM (2845.00 g) and least for the bird on control diet (2815.40). The value of FCR was least in birds fed with diet containing 0.25% MSCM (1.39) and highest in the control group (1.78).

Carcass traits and relative internal organs of broiler chickens
As presented in Table 4, supplementation of MSM, SLM, and MSCM did not significantly influence the carcass traits (slaughtered weight, dressed weight, and dressing percentage) and relative internal organ weights (liver, heart, lung, pancreas, gall bladder, spleen, gizzard, and proventriculus) of the broiler chicken (P > 0.05).

Haematological indices of broiler chickens
The haematological indices of the broiler chickens fed with diet containing Myristica fragrans seed meal (MSM), Syzygium aromaticum leaf meal (SLM), and their composite mix (MSCM) are shown in Table 5. The packed cell volume, red blood cells, haemoglobin concentration, mean cell haemoglobin concentration, and white blood cells of broiler chickens were not significantly (P > 0.05) affected by MSM, SLM and MSCM supplementation compared with control. However, the mean cell volume and mean cell haemoglobin content of the broiler chicken were significantly influenced (P < 0.05) by MSM, SLM, and MSCM supplementation, with broiler chickens fed with diet containing 0.25% SLM having the highest mean cell volume (161.81 fl) and mean cell haemoglobin (53.93 pg), while birds on control diet had the lowest mean cell volume (112.50 fl) and mean cell haemoglobin (36.96 pg).
The serum metabolites and serum antioxidant enzyme of the broiler chickens Table 6 shows the serum metabolites and antioxidant enzymes of the broiler chickens fed with diet containing MSM, SLM, and MSCM. The result of serum metabolites revealed that creatinine, aspartate aminotransferase, and alanine aminotransferase concentrations of the broiler chicken were not significantly (P > 0.05) influenced by MSM, SLM, and MSCM supplementation compared with control. The serum cholesterol level was significantly (P < 0.05) lower in birds receiving diets supplemented with MSM, SLM, and MSCM compared with the bird receiving control diet (7.03 μmol/l). The concentration of serum catalase and glutathione peroxidase was significantly (P < 0.05) higher in birds fed with diet containing MSM, SLM, and MSCM than the control group, while there was no significant (P > 0.05) effect of MSM, SLM, and MSCM supplement on serum superoxide dismutase concentration of broiler chickens compared with control.

Meat lipid peroxidation, antioxidant enzymes, and cholesterol
The result of the effect of SLM, MSM, and MSCM supplement on meat quality of broiler chicken is presented in Table 7. The result revealed significant (P < 0.05) differences in lipid oxidation, glutathione peroxidase, and cholesterol content of breast muscle of broiler chicken across treatments, while the concentration of catalase was statistically different (P > 0.05) across treatments. The meat lipid peroxidation was significantly (P < 0.05) lower in broiler chickens fed with diets supplemented with 0.25% MSM (9.43 mg MDA/100 g), 0.25% SLM (4.48 mg MDA/ 100 g), and 0.25% MSCM (4.13 mg MDA/100 g) compared with broiler chickens on the control diet ( Intestinal bacteria of the broiler chickens Table 8 shows the result of the intestinal bacteria of the broiler chicken. The analysis revealed no significant difference (P > 0.05) in aerobic bacteria, lactic acid-producing bacteria, and intestinal negative bacteria counts except coliform bacteria (P > 0.05) counts. Supplementation of 0.25% SLM significantly decreased coliform bacteria compared with control, while the result of coliform bacteria count of 0.25% MSM and 0.25% MSCM was comparable to that of control.

Discussion
Phytochemical composition and antioxidant activity of Myristica fragrans seed meal and Syzygium aromatic leaf meal Herbs and spices are considered an important source of phytochemicals that possessed strong antioxidant activity

Performance of broiler chickens
The use of herbs, spices, and other phytogens as a feed additive has been demonstrated as an excellent growth enhancer in poultry production. The significant increase in body weight gain (BWG) and feed conversion ratio (FRC) of broilers fed with diets supplemented with MSCM compared with control during finisher stage and overall feeding period could be attributed to the synergetic action of different phytochemicals present in plants as shown in Table 2 above which in turn may have induced greater diet utilization and efficiency (Dhama et al. 2015) thereby promoting growth performance of the experimental birds. Phytochemicals such as flavonoid, phenol, and saponin have been reported to promote higher growth rate and better feed efficiency in domestic birds due to their ability to scavenge free radicals and maintain the integrity of the intestinal mucosa (Oloruntola et al. 2018(Oloruntola et al. , 2019. Also, earlier reports had shown that SLM and MSM possessed antibacterial, antiparasite, antifungal, anti-coccidiosis, and hepatoprotective properties (Cortes-Rojas et al. 2014;Olaleye et al. 2006;Panggabean et al. 2019) which are capable of stimulating the growth of beneficial bacterial, inactivate pathogenic bacteria, and facilitate nutrient metabolism and absorption in the gastrointestinal tract (Dhama et al. 2015) to enhance growth performance of broiler chickens. Additionally, the improved FCR observed in broiler chickens fed with diet containing MSCM indicate evidence of better feed utilization than the control group which may be due to increase in secretions of endogenous gastric   (Amad et al. 2011). Conversely, reports on the effect of MSM supplementation in poultry diet on growth parameters are very rare.

Carcass traits and relative internal organs of broiler chickens
Apart from impacting changes on growth performance, herbs and phytogens have been reported to exert positive influences on the development of carcass traits, internal organs, and meat qualities of broiler chickens (Oloruntola et al. 2018). Results on carcass traits and relative internal organs weights of broiler chickens were not significantly affected by the treatments indicating that supplementation of SLS, MSM, and MSCM did not influence the development of muscle and organ compared with control. A similar result has been reported by Tariq et al. (2015) who found no significant effect of supplementing clove powder at 0.5% relative organ weight of quail birds. On the contrary, Al-Mufarrej et al. (2019a, 2019b reported higher liver percentage and lower intestine weight of broiler chickens fed a diet containing clove powder compared with control.

Haematological indices of broiler chickens
Application of herbs has been observed to improve health and well-being of the animal because of their ability to acts as immune stimulants by stimulating and modulating the production of blood cells and other haematological indices and immune responses to alleviate diseases (Dhama et al. 2015;Anjusha et al. 2019).
The results on packed cell volume, red blood cells, haemoglobin, mean cell haemoglobin concentration, and white blood cells were not significantly different across treatments, although were numerically higher in birds supplemented with SLS, MSM, and MSCM compared with control. This observation, however, confirms the nutritional adequacy and safety of the diets supplemented with SLM, MSM, and MSCM in supporting the normal haemapoietic process of the broiler chickens (Oloruntola et al. 2019). Contrary to the present findings, Oloruntola et al. (2019) reported significant variation in packed cell volume, red blood cells, haemoglobin, mean cell haemoglobin concentration, and white blood cells of broiler chickens fed with different phytoadditives. Conversely, significant differences were observed in the values obtained for mean cell volume and mean cell haemoglobin across the treatments. The significant increase in mean cell volume and mean cell haemoglobin contents recorded for broiler chickens fed a diet containing SLM and MSM compared with control is evidence of the production of more oxygenated red blood cells in the tissue since haemoglobin is found in red blood cells as the protein that is responsible for the delivery of oxygen to the tissues. Herbs and phytogens that can exert hepato-stimulatory and hepatoprotective effects have been reported to stimulate syntheses of more hematoglobin in the bone marrow, thereby increasing the hematoglobin circulation in the tissue (Dhama et al. 2015) as seen in the present study.

The serum metabolites and serum antioxidant enzyme of the broiler chickens
Findings from this study show no significant influence of SLS, MSM, and MSCM supplements on serum creatinine, aspartate aminotransferase, alanine aminotransferase, and superoxide dismutase of the broiler chicken. In comparison with the control group, the inclusion of SLM, MSM, and MSCM in the broiler diets significantly lowered the serum cholesterol level, but increased the catalase, and glutathione peroxidase content. Cholesterol is a type of lipid that is found in the body as part of lipoproteins. Excessive accumulation of cholesterol in the body has been associated with cardiovascular disease, causing the blockage of artery walls and reduction in blood flow to the heart (Oloruntola et al. 2018). The observed decrease in serum cholesterol level of broiler chicken supplemented with SLS and SMS suggests their hypo-cholesterolemic activity which could be due to their ability to delay intestinal absorption of dietary fat by inhibiting pancreatic lipase activity when applied in poultry diet. Gaurav (2015) in their study have reported a reduction in serum total cholesterol level of broiler fed with diet containing herbs (Chlorophytum root and Camellia seed). Similarly, catalase has been reported as one of the important antioxidant enzymes that inactivate, scavenge, and remove free radical and other reactive species in the tissue to protect the body against oxidative stress (Delles et al. 2014). The observed increase in serum catalase and glutathione peroxidase content of broiler chicken supplemented with SLS, MSM, and MSCM indicate their ability to stimulate the production of more endogenous antioxidant enzymes to fight free radicals and protect the cell molecules against hydrogen lipid peroxidation and peroxide toxicity (Oloruntola et al. 2018). These findings are in agreement with the report of Kostadinović et al. (2015) and Daramola (2019) who found that the addition of herbal plants in the boiler diet significantly enhanced the concentration of catalase compared with control group.

Meat lipid peroxidation, antioxidant enzymes, and cholesterol
Herbal plants and other phytogens have been demonstrated as a natural source of antioxidant in poultry diets. As a natural antioxidant, they can donate hydrogen ions to inhibit the free radical formation and/or interrupt propagation of lipid oxidation in the muscle (Falowo et al. 2014) or increase the production of different endogenous enzymes such as catalase, superoxide dismutase, and glutathione peroxidase (Dhama et al. 2015). Results from this study revealed a significant decrease in lipid peroxidation and cholesterol levels and a surge increase in glutathione peroxidase concentrations in breast meat of broilers supplemented with SLM, MSM, and MSCM compared with control. The potential of SLM, MSM, and MSCM to decrease the level of lipid oxidation in meat samples could be attributed to the presence of inherent phytochemicals and antioxidant activity (Table 1) to inhibit the production of free radicals and prevent oxidative damages. Besides, the higher amount of glutathione peroxidase concentration of broiler chickens receiving SLM and MSCM could be responsible for the decrease in lipid peroxidation recorded in this study. The glutathione peroxidase is an important antioxidant enzyme that inactivate, scavenge, and remove free radical and other reactive species in the tissue to protect the body against peroxidation and oxidative damages. Another study has reported the capacity of herbal plants to reduce lipid oxidation and increase endogenous antioxidant enzymes in muscle food due to inherent bioactive components and antioxidant activity (Hashemi and Davoodi 2010;Valenzuela-Grijalva et al. 2017). The amount of total cholesterol in the breast muscle was significantly lowered by the supplementation of SLM, MSM, and MSCM compared with control. It is known that regular consumption of food products that contain high cholesterol content can lead to hypercholesterolemia which has been implicated in causing atherosclerosis and coronary heart diseases (Walker et al. 2005). This result is in line with other evidence that has shown that the addition of phyto-additives in broiler diets can significantly inhibit hypercholesterolemia and lower cholesterol level in muscle food due to the inherent bioactive compounds (Onyimonyi et al. 2012;Nabil Alloui et al. 2014;Yilkal 2015;Puvaca et al. 2015).

Intestinal bacteria of the broiler chickens
The result of the intestinal bacteria count revealed that the inclusion of SLM significantly reduced the coliform bacteria count, while the dietary effect of MSM and MSCM on coliform bacteria counts was comparable to that of control. On the other hand, the result of the total aerobic bacteria, lactic acid-producing bacteria, and intestinal negative bacteria counts was similar across the treatments thereby suggesting that SLM and MSM dietary supplementation could maintain or preserve the intestinal microflora throughout the feeding trial. Several studies have shown that maintenance of non-pathogenic intestinal bacteria is essential for the inhibition of pathogen proliferation, enhancement of growth performance, and reduction of morbidity and mortality of broiler production (Seidavi and Simoes 2015;Oloruntola et al. 2019). This result is consistent with the report of Oloruntola et al. (2019) who found that the addition of phyto-additive did not significantly influence the caecum microflora of the boiler chicken.

Conclusions
Findings of the present study reveal the potential of Myristica fragrans seed meal (MSM) and Syzygium aromaticum leaf meal (SLM) to enhance growth performance, health, and meat quality of broiler chickens when used as a phytogenic feed supplement. It was shown that the inclusion of MSCM at 0.25% can improve body weight gain and endogenous antioxidant enzymes, maintain carcass traits, organ weight, and gut microflora and reduce meat cholesterol and lipid oxidation of broiler chickens. Further research is required to assess MSM and SLM effects on immune responses and the meat fatty acid profile of broiler chickens.