Nutrient digestibility, gut microflora, carcass yield, and meat microbiology of broilers fed diets supplemented Ethiopian pepper (Xylopia aethiopica), cloves (Syzygium aromaticum), and their composite

The effect of dietary supplementation of Ethiopian pepper (Xylopia aethiopica) and clove (Syzygium aromaticum) and their composite on nutrient digestibility, gut microflora, carcass yield, and meat microbiology were investigated in a 52-day boiler feeding trial. Three hundred and sixty unsexed Ross broilers were used for the study. Four experimental diets were formulated for the starter (0–28 days) and finisher (29–56 days) phases: diet 1 was the control without phytogenic supplementation, diet 2 (supplemented with 1% Ethiopian pepper (EP)), diet 3 (supplemented with 1% clove (CL)), and diet 4 (supplemented with 1% mix of equal quantity (0.5% each) of Ethiopian pepper and clove (EPCL)). Each treatment was replicated 6 times with 15 birds per replicate. Nutrient digestibility was determined on days 28 and 56, gut microflora was determined for the small intestine and caecum on day 56, carcass yield, organ weights, and meat microbiology were also determined on day 56. Data obtained were subjected to a one-way analysis of variance using SAS 2000 and significant means were separated using Tukey’s test in the same software. At day 28, broilers fed the diet supplemented EPCL had the highest (P < 0.05) dry matter digestibility (DMD, 86.81%) crude protein digestibility (CPD, 71.28%), and ash digestibility (64.24%). Broilers fed EP supplemented diet had reduced (P < 0.05) DMD (70.50%). Increased (P < 0.05) ether extract digestibility (EED) was observed for broilers fed the diet supplemented CL (75.27%) and EPCL (76.43%). Ash digestibility (AD) was lowest (P < 0.05) for broilers fed control diet (50.30%). At day 56, broilers fed the diet supplemented CL and those fed the EPCL supplemented diet had higher (P < 0.05) CPD (78.07%, 79.35%) and EED (70.20%, 71.42%) than other treatments. Ash digestibility was higher (P < 0.05) for broilers fed diet supplemented EPCL (74.60%) than other treatments. Intestinal clostridium count reduced (P < 0.05) and lactobacillus count increased (P < 0.05) in the intestine and caecum of broilers fed the diet supplemented CL and those fed EPCL supplemented diet. Intestinal coliform and salmonella count reduced (P < 0.05) for broilers fed the diet supplemented EP and those fed the EPCL supplemented diet. Dietary supplementation of EPCL resulted in the highest (P < 0.05) body weight (BW, 2551.38 g), dressing percentage (DP, 81.68%), and percentage of breast muscle (20.01%). Supplementation of EP, CL, and EPCL in the diet of broilers resulted in higher (P < 0.05) spleen weight while dietary supplementation of EP and EPCL resulted in higher (P < 0.05) thymus weight. Clostridium count was reduced (P < 0.05) in the meat of broilers fed the diet supplemented CL and EPCL and the lowest (P < 0.05) salmonella count was observed in the meat of broilers fed the diet supplemented with EPCL. In conclusion, the current study reveals that dietary supplementation with EP and CL composite improves nutrient digestibility, gut microflora, and dressing percentage with reduced meat microbial load of broilers.


Introduction
In recent years, phytogenic feed additives have gained increasing interest as substitute feeding strategies for antibiotic growth promoters. These active additives are derived from plants and are added to feeds for improved performance of livestock (Windisch et al., 2008). Antibiotic growth promoters have been used for improved productivity of broilers under intensive production (Rehman and Munir, 2015); however, the usage of antibiotics results in the proliferation of a microbial population that exhibits resistance to medications used by humans and animals with negative consequential effect on the health of consumers (Rehman and Haq, 2014). Thus, the search for alternative feed additives that can replace antibiotic growth promoters becomes imperative.
Active components of phytogenic plants improve the performance of poultry birds through increased digestive enzymes secretion causing enhanced nutrient digestion and utilization from the phyto-stimulation effect (Geier and Ouster, 2001;Recoquillay, 2006). The basic mode of activity of growth-enhancing phytogenic feed additives is through gastrointestinal microbial stabilization with the selective elimination of harmful pathogens leading to a beneficial microbiota environment (Roth and Kirchgessner, 1998). This activity is of great importance during the challenging developmental stage of livestock when they are prone to diseases and digestive malfunction precisely in young poultry birds (Eckel et al., 1992). The research by Kroismayr et al. (2007) which compared the effect of dietary inclusion of essential oil blend (oregano, anise, and citrus peels) with antibiotics revealed a similar effect of both additives on ileum, cecum, and colon in terms of microbial properties. The penetration of microbial cell membranes and disintegration causing ion leakage are the modes in which phyto-additives exhibit antimicrobial effects through the activity of essential oils (Newton et al., 2002). Therefore natural feed additives in poultry nutrition may be of great benefit and value, especially for broilers.
Spices, herbs, and their extracts exhibit antimicrobial function against dangerous pathogens and they are also active against fungi (Ozer et al., 2007). Garlic (Allium sativum) is an example of a well-known herbal spice which have been reported to possess important bioactive compounds that are active against inflammation, and immune breakdown in addition to antiparasitic and antibacterial effect (Lewis et al., 2003). Ethiopian pepper (Xylopia aethiopica) and cloves (Syzygium aromaticum) belong to this group of plants that could serve as phytogenic feed additives due to their chemical properties and nutritive values. Cloves (Syzygium aromaticum) are known due to their usage to possess many medicinal properties such as ameliorating digestive disorders for improved nutrient digestion (Burt and Reinders, 2003), elimination of intestinal parasites and eradication of microbial agents (Shafi et al., 2002), and it has been reported to be active against inflammation (Muruganadan et al., 2001). Ethiopian pepper (Xylopia aethiopica) also possesses medicinal properties which are potent against different diseases which occur in humans as well as animals (Ogbonnia et al., 2008). Research studies have shown that clove's essential oils possess active components that inhibit the proliferation and growth of various bacteria and fungi strains (Asekun and Adeniyi, 2004). Fruit extract of cloves has been reported to be active against gram-negative and gram-positive bacteria (Tatsadjieu et al., 2003). However, dearth of information exists on the potency and efficacy of these phytogenic plants as additives in broiler nutrition.
Phytogenic plants are regarded to be a relatively new class of feed additives and information about some of them such as cloves and Ethiopian pepper is still rather limited in terms of application and their modes of action. Knowledge of the aspects of their utilization in broiler production is also limiting, as the activity of these phytogenic feed additives may vary widely with respect to the botanical origin, processing, and composition. A new strategy for the use of phytogenics as alternatives to antibiotics is their combination to examine the potency of their interactive effect (Bassole and Juliani, 2012). This new strategy has been reported to promote synergistic and additive effects of different bioactive compounds (Cetin et al., 2016). A combination of Bermuda grass and betel leaf extract exhibited improved antioxidant activity compared to individual plants (Thiruvengadarajan et al., 2011). Oso et al. (2019) also reported enhanced performance with 1% dietary supplementation of a blend of mountain knotgrass, betel, Bermuda grass, and black pepper. Therefore, this study investigates the nutrient digestibility, gut microflora, carcass yield, and meat microbiology of broilers fed diets supplemented with Ethiopian pepper (xylopia aethiopica), cloves (syzygium aromaticum), and their composite.

Test ingredients and processing
The test ingredients which are dried Ethiopian pepper (Xylopia aethiopica) and clove (Syzgium aromaticum) were purchased from Kuto market in Abeokuta Ogun State, Nigeria. The properly dried fruit of clove and Ethiopian pepper were milled to particle size 0.2 mm using a Kenwood blender and stored in plastic containers before diet formulation and commencement of feeding trial.

Chemical composition of test ingredients
Three grams ground samples of the test ingredients were taken and analyzed for their chemical composition (Table 1). The proximate composition was determined using standard method (AOAC, 2005). Gross energy was estimated using the adiabatic bomb calorimeter (Model 1261; Parr Instrument Co., Moline, IL, USA). Analysis of mineral nutrients was determined using the method by Kumari et al. (2017). The quantitative analysis was carried out by using inductively coupled plasma atomic absorption emission spectroscopy (ICP-AES). Samples were also analyzed for mineral contents using atomic absorption spectrophotometer (Perkin Elmer Optima 4300DV ICP spectrophotometer, UK). The phytochemical constituents were analyzed following standard procedures; total polyphenol (Wright et al., 2000), flavonoid (Arvouet-Grand et al., 1994), extractable tannin (Hoff and Singleton, 1977), alkaloids (Saddiqui and Ali, 1997), and saponin content (Edeoga et al., 2005).

Experimental birds and management
Three hundred and sixty (360) unsexed day old Ross broilers were purchased from Agric International Technology and Trade Limited (AGRITED) hatchery in Ibadan, Nigeria. The pens were washed and disinfected a week before the arrival of the birds. On arrival of the birds, they were unloaded from the boxes and distributed into pens that was preheated before arrival. Brooding temperature was controlled at 34 °C for the first 2 days and gradually reduced by 3 °C per week to a final ambient temperature of 28 °C after 2 weeks of brooding. Heating of the pen was done with the use of electric bulb and charcoal provided in the poultry pen. The chicks were reared on a deep litter system made of concrete floor and wood shavings provided as bedding for the birds. Newcastle disease and infectious bursa disease (Gumboro) vaccines were administered to the birds generally on 2nd and 7th day respectively. Feeding trays and water troughs were made available for the birds. Pen temperature and relative humidity were monitored using a thermo-hygrometer at 08:00, 14:00, and 19:00 daily. The average temperature was 29.56 ± 1.39°, and the relative humidity was 65.5 ± 2.7.

Experimental design and dietary treatment
The broilers were allotted on a weight equalization basis to four dietary treatments using a completely randomized design. The deep litter house was partitioned into 24 experimental pens (each measuring 2.5 × 3 m) using wire mesh.
Each pen was open-sided unit screened with wire mesh fastened on wood cemented on concrete walls. Each treatment has six replicates with 15 birds per replicate. The formulated diet consists of a control (diet 1) (without phytogenic supplementation), diet 2 (supplemented with 1% (10 g/kg) Ethiopian pepper (EP)), diet 3 (supplemented with 1% (10 g/ kg) clove (CL)), and diet 4 (supplemented with 1% mix of equal quantity (0.5% (5 g/kg) each) of Ethiopian pepper and clove (EPCL)). The phytogenic supplementation level of 1% (10 g/kg diet) were based on reports on dietary inclusion of phytogenic additives up to 1% (10 g/kg) (Al-Kassie et al., 2011;Ndelekwute et al., 2015). The phytogenic additive was added to the basal diet and thoroughly mixed to ensure uniform distribution. The diets were formulated (Table 2) for starter (0-28 days) and finisher phases (29-56 days) based on the nutrient requirement guide for Ross 308 (Ross Broiler Nutrition Specifications 2016). The premix used in feed formulation was purchased from Bio-Organic nutrient systems limited Nigeria. The experiment lasted for eight weeks and all birds were raised intensively on a deep litter system with a spacing of 1 square feet per bird while feed and water were supplied ad-libitum during the experimental period. No infeed antimicrobials and anti-coccidial drugs or antibiotics were fed to the birds throughout the duration of the study.

Apparent total tract nutrient digestibility
A digestibility trial was conducted on days 28 and 56 of the study to determine the apparent total tract nutrient digestibility. A bird per replicate (6 birds /treatment) was randomly selected and housed separately in appropriate metabolism cages fitted with individual feed troughs, water troughs and facilities for separate excreta collection. The birds were acclimatized for 2 days prior to the commencement of 5 days collection period. A known weight of feed (slightly above the respective daily requirements) with the addition of 0.5% titanium dioxide as an indigestible marker was fed to the birds housed in individual metabolic cages. Excreta collected per bird per day (collected twice daily) were stored in air-tight bags and stored at -18 °C till analyzed. The frozen excreta per bird were thawed, pooled for each treatment, oven dried (60 °C) for 48 h, and then ground to 0.5-mm size.
The concentration of titanium dioxide in diets and excreta was measured according to Short et al. (1999). The proximate composition of feed and dried excreta samples was determined using the standard method of AOAC (2005). The apparent nutrients digestibility was calculated according to Maynard et al. (1979) using the equation.

Gut microflora
At day 56, 1 bird per replicate was selected and slaughtered for the collection of two sets of intestinal contents. Fresh digesta from the small intestine (from the distal end of the duodenum to the ileo-cecal junction) were collected and emptied into labeled sterile bottles. Fresh cecal content collected from a pair of ceca of the selected broiler was also collected in a different labeled sterile bottle. Samples collected were analyzed for the estimation of gut microbiota according to the methods of Xia et al. (2004). One gram of the sample was dispersed in a 9 mL phosphate buffered saline solution with 0.5 g/L of Cys. HCl, and further diluted to a factor of 10-8. 0.1 mL of diluted sample was spread onto a Petri dish containing selective media for the enumeration of bacteria.
expressed as colony-forming units (CFU) of microorganisms per gram of fresh sample.

Carcass yield
At day 56, 1 bird per replicate whose weight is representative of the average weight of broilers in each replicate was selected (six birds per treatment), slaughtered, de-feathered, and eviscerated using standard procedures (Jensen, 1984). The body weight and dressed weights were measured, while the dressing percentage was calculated. The retail cut parts, which are breast, thighs, back, wings and drumsticks, were weighed and expressed as relative weights, i.e., each weight of the parts was divided by the body weight and multiplied by 100 (percentage of body weight). The organs which are the kidney, lungs, pancreas, liver, heart, thymus, bursa, and spleen were collected, weighed, and also expressed as percentages of body weight.

Meat microbiology
At day 56, 1 bird per replicate was selected and slaughtered for the collection of breast meat samples. The meat samples were stored in the refrigerator at 4 °C for 3 days. After 3 days, the meat samples were removed and allowed to thaw and meat samples were analyzed using methods by Domanska and Rozanska (2003). Using sterile spoons, 10 g of breast meat was weighed aseptically into a sterile blender jar. About 90 ml of sterile Butterfield's phosphate (BPD) diluent or buffered peptone water (BPW) was added and blended for 2 min. From the prepared homogenate, about 10 serial dilutions were prepared by diluting 1 ml of homogenate with 9 ml of 0.1% peptone water and spread plated (0.

Statistical analysis
Data obtained were subjected to one-way analysis of variance using a completely randomized design. The replicate served as the experimental unit for the statistical analysis. The data were analyzed using the ANOVA procedure of SAS (2000). Mean separation was done using the Tukey test of SAS to determine the effect of phytogenic supplementation. Significant differences were considered at P < 0.05.

Nutrient digestibility
The nutrient digestibility of broilers fed diets supplemented with EP, CL, and EPCL are shown in Table 3. At day 28, nutrient digestibility was significantly (P < 0.05) affected by phytogenic supplementation except for crude fiber digestibility (CFD) which was not significantly (P > 0.05) influenced. Broilers fed the diet supplemented EPCL had the highest (P < 0.05) dry matter digestibility (DMD) and those fed diet supplemented EP had the lowest (P < 0.05) DMD while those fed the control diet and those fed diet supplemented CL had intermediate DMD. Crude protein digestibility (CPD) increased (P < 0.05) for broilers fed the diet supplemented EPCL compared to other treatments. Supplementation of CL and EPCL resulted in higher (P < 0.05) ether extract digestibility (EED) compared to other treatments. Ash digestibility (AD) was highest (P < 0.05) for broilers fed the diet supplemented EPCL but lowest (P < 0.05) for broilers fed the control diet while those fed diets supplemented EP and CL had intermediate AD. At day 56, CPD, EED, and AD were significantly (P < 0.05) influenced by phytogenic supplementation while DMD and CFD were not significantly (P > 0.05) affected. Dietary supplementation of CL and EPCL resulted in higher (P < 0.05) CPD and EED than other treatments. The AD increased (P < 0.05) with EPCL supplementation for broilers while other treatments had reduced (P < 0.05) AD.

Gut microflora
Gut microflora of broilers fed diets supplemented with EP, CL, and EPCL are presented in Table 4. In the small intestine, except for total bacteria count (TBC), other parameters were significantly (P < 0.05) affected by phytogenic supplementation. Broilers fed the diet supplemented CL and those fed diet supplemented EPCL had lower (P < 0.05) clostridium count than those fed the control diet and those fed diet supplemented EP. The coliform count increased (P < 0.05) for broilers fed the control diet and those fed diet supplemented CL while those fed diets supplemented EP and EPCL had reduced (P < 0.05) coliform count. Dietary supplementation of CL and EPCL for broilers resulted in a higher (P < 0.05) lactobacillus count than other treatments. Broilers fed the control diet and those fed diet supplemented CL had increased (P < 0.05) salmonella count while those fed diets supplemented EP and EPCL had reduced (P < 0.05) salmonella count. In the caecum, only lactobacillus count was significantly (P < 0.05) affected by phytogenic supplementation. Broilers fed diets supplemented CL and EPCL had the highest (P < 0.05) lactobacillus count while those fed the control diet had the lowest (P < 0.05) count but those fed diet supplemented EP had intermediate lactobacillus count. Table 5 presents the carcass characteristics and relative organ weights of broilers fed diets supplemented with EP, CL, and EPCL at day 56. Higher (P < 0.05) body weight (BW) was observed for broilers fed the diet supplemented EPCL compared to other treatments. The highest (P < 0.05) dressed weight (DW), dressing percentage (DP), and breast weight were recorded for broilers fed the diet supplemented EPCL while those fed the control diet and those fed diet supplemented CL had the lowest DW, DP, and breast weight but those fed diet supplemented EP had intermediate weight for DW, DP, and breast weight. Thigh weight increased (P < 0.05) for broilers fed diets supplemented with CL and EPCL but reduced (P < 0.05) for other treatments. Higher (P < 0.05) wing weight was observed for broilers fed the control diet and those fed the diet supplemented CL compared to other treatments. Lower (P < 0.05) back weight was recorded for broilers fed the diet supplemented CL compared to other treatments. The highest (P < 0.05) liver weight was observed for broilers fed the control diet while those fed diet supplemented EP had the lowest (P < 0.05) liver weight but those fed diets supplemented CL and EPCL had intermediate liver weight. Broilers fed the control diet had lower (P < 0.05) spleen weight compared to other treatments. Supplementation of EP and EPCL in the diet of broilers resulted in higher (P < 0.05) thymus weight than other treatments. Drumstick, kidney, lungs, pancreas, bursa, and heart weight were not significantly (P > 0.05) by dietary supplementation of EP, CL, and EPCL.

Meat microbiology
The effect of EP, CL, and EPCL supplementation on the meat microbiology of broilers is presented in Table 6. The result shows a significant (P < 0.05) effect of phytogenic supplementation on other parameters except for TBC which was not significantly (P > 0.05) affected. Broilers fed diets supplemented CL and EPCL had lower (P < 0.05) clostridium count than other treatments. A lower (P < 0.05) coliform count was observed for broilers fed diets supplemented with EP and EPCL than other treatments. Higher (P < 0.05) lactobacillus count was recorded for broilers fed diets supplemented CL and EPCL while those fed the control diet and those fed diet supplemented EP had reduced (P < 0.05) lactobacillus count. Salmonella count was lowest (P < 0.05) for broilers fed diets supplemented EPCL while those fed control diet had the highest (P < 0.05) salmonella count but it was intermediate for broilers fed the diet supplemented EP and those fed diets supplemented CL.

Nutrient digestibility
The dietary supplementation of EPCL resulted in increased DMD higher than other treatments at day 28. The improved DMD obtained with EPCL supplementation suggests that  (Reddy et al., 2004). Clove contains molecules with the most important one being eugenol which has intrinsic effects on the physiology and metabolism of animals (Dalkiliç and Güler, 2009). The increased DMD observed could be attributed to the enhancement of digestion by the active constituents of the EPCL mixture. The findings of Hernandez et al. (2004) also indicated that blends of Salvia officinalis, Thymus vulgaris, and Salvia rosmarinus in the diet of broilers improved total tract and ileal digestibilities of the nutrients. The crude protein digestibility increased for broilers fed the diet supplemented EPCL. This is in agreement with the report of Jamroz et al. (2003), who reported improved nutrient digestibility of broilers fed a diet containing carvacrol, cinnamaldehyde, and capsaicin which are bioactive constituents of phytogenic plants. Phytoadditives promotes digestion by promoting the proliferation of beneficial microflora through binding and removing harmful microbes from the gastrointestinal tract (Dieumou et al., 2009;Shanoon et al., 2012). An increased EED was observed for broilers fed diets supplemented CL and those fed diet supplemented EPCL. This is in agreement with the report of Attia et al. (2017) who reported increased EED of broilers fed the diet supplemented blend of plant extract at 100 and 500 g/ kg compared to the control group. It is worthy of note that broilers fed the diet supplemented EP had similar EED with those of the control group which implies that the constituents of CL influenced EED which is a reflection of what was observed for broilers fed the diet supplemented EPCL. Ghasemi and Taherpour (2015) stated that differences in activity and health benefits with the use of spice products mostly rely on the dosage and the spice type. One of the mechanisms by which phyto-additives promote nutrient digestion is through the enhancement of intestinal mucus secretion and increased activity of the digestive enzyme (Williams and Losa, 2001;Jamroz et al., 2006). The increased ash digestibility observed for broilers fed diet supplemented EPCL suggests the positive synergistic effect of phytogenic mix supplementation in the diet of broilers. The Saponin content of EP and CL is reported to be effective in enhancing food breakdown due to its stimulatory activity on enzymes (Sauvaire et al., 2000;Bin-Hafeez et al., 2003). At day 56, the increased CPD observed for broilers fed the diet supplemented CL and EPCL shows that either CL or EPCL dietary supplementation was able to elicit improvement in CPD compared to the control at this phase. The use of phyto-additives has been reported to promote the flow of bile and stimulates the activity of feed-degrading enzymes arising from the pancreas and intestinal mucosa (Platel and Srinivasan, 2004;Jang et al., 2007). In this study, digestion stimulation and enhanced digestive enzyme activity could explain improved apparent nutrient digestibility. The increase in EED observed on day 28 following dietary supplementation of CL and EPCL for broilers was repeated on day 56 and this is associated with inherent active components of the phytogenic additive, especially clove. Cloves are among the group of spices that are most active having active bio components such as eugenol acetate, and β-caryophyllene (Jimoh et al., 2017). Higher digestibility for ash was observed for broilers fed diet supplemented EPCL. The exhibition of increased ash digestibility for the group of broilers suggests that improvement was achieved due to the combined effect of EP and CL components in the diet of broilers. Marcinčák et al. (2011) have earlier reported that the effect of phyto-additives is dependent on the used plants in feeding as well as the quantity. Alcicek et al. (2004) and Brenes and Roura (2010) suggested that this challenge could be addressed by supplementation of different herb combinations with varying constituents for a synergistic effect.

Gut microflora
In the small intestine, clostridium count reduced with the supplementation of CL and EPCL in the diet of broilers while those fed diet supplemented EP had clostridium count similar with that of the control group. This is an indication that CL has inhibitory effect on clostridium specie as observed in this current study. The disintegration of cell walls and membranes of microbes by penetration of the membranes of the cytoplasm resulting in halting of protein and deoxyribonucleic acid can be obtained with the use of cloves (Xu et al., 2016). The outcome of the research by Cui et al. (2010) is in agreement with our findings, the author found that clove extract had great antimicrobial activity against Clostridium botulinum. The reduced coliform count found with broilers fed diets supplemented EP and EPCL suggests active effect of EP against coliform bacteria. This is in consonance with the report of Isikwenu et al. (2014) who reported decrease in E. coli count of broilers fed diet containing Xylopia aethiopica as feed additive. Preliminary study by Ilusanya et al. (2012) shows that Xylopia aethiopica fruits contain bioactive constituents such as tannins and phlobatannins, flavonoids, and steroids. These bioactive compounds exerts bactericidal, pesticidal, and fungicidal effects (El astal et al., 2005). Lactobacillus count increased for broilers fed diet supplemented CL and those fed diet supplemented EPCL. This is in concordance with the report of Khaksar et al. (2012) who reported increased Lactobacillus count in the intestine of Japanese quail fed diet containing thyme essential oil. Dorman and Deans (2000) also reported increased number in gut Lactobacillus count of quails fed diet containing phytogenic feed additives. Lactobacillus is known to be one of the beneficial intestinal microbe and phytogenic compounds or their extracts is reported to increase the number of intestinal normal and beneficial microbes for improved intestinal performance (Liu et al., 2014). A decrease in salmonella count was observed in the intestine of broilers fed diet supplemented EP and those fed diet supplemented EPCL. The reduction in salmonella count observed implies that dietary EP supplementation was potent against intestinal salmonella multiplication. Ilusanya et al. (2012) earlier reported that EP had antibacterial properties that improves broiler productivity. This is also in agreement with the report of Usman et al. (2016) who reported that EP contained essential oils with bioactive compounds which exhibits inhibitory effect against gram negative bacteria.
In the caecum, only lactobacillus count was influenced by phytogenic supplementation and a similar trend observed for lactobacillus count in small intestine occurred in the caecum in which broilers fed diet supplemented CL and those fed diet supplemented EPCL had highest lactobacillus count. It is important to note that broilers fed control diet had the least lactobacillus count. This observation implies that dietary supplementation of either EP or CL was capable of influencing increase in caecal lactobacillus count. The potent compounds of phytobiotics inhibit intestinal pathogens and stimulates trypsin enzyme which deactivates toxins of harmful microbes (Windisch et al., 2008). The bioactive compounds contained in phytogenic plants helps to stabilize microflora environment and multiplication of beneficial gut microbe including lactobacillus specie (Cho et al., 2014). The preponderance of CL over EP in promoting increase in lactobacillus count could be associated to the essential oil of cloves which is reported to be a heterogeneous mixture of flavonoids, polyphenols, and tannins that exerts antioxidant, antibacterial, and antifungal properties (Bourgaud et al., 2001). Its potency could also be associated to the proportion of essential oil yield of cloves which is reported to be as much as 20% (Lukas et al., 2015).

Carcass yield and relative organ weights
The increased body weight of broilers fed the diet supplemented EPCL compared to other treatments suggests the positive synergistic effect of EP and CL on protein synthesis for increased tissue accretion resulting in increased body weight. This corroborates the report of Hernandez et al. (2004) who reported an increase in body weight of broilers fed the diet supplemented extract mix of Salvia officinalis, Thymus vulgaris, and Salvia rosmarinus at 500 g/kg. Barreto et al. (2008) also pointed out that the inclusion of only one plant extract into the feed or water may not favorably influence the weight gain of poultry. Dressing percentage (DP) and breast meat percentage were highest for broilers fed the diet supplemented EPCL. This is simply the reflection of what was observed for body weight as broilers fed diet supplemented EPCL had the highest body weight. This is in accordance with the report of Singh et al. (2009) who reported a better dressing percentage for broilers fed a diet containing polyherbal growth promoters. Oso et al. (2021) also observed increased DP for broilers fed a diet containing 1% phyto-supplement blend of mountain knotgrass, betel, Bermuda grass, and black pepper. However, dietary supplementation of EP resulted in intermediate DP and breast meat percentage which is an indication that better weight obtained for broilers fed the diet supplemented EPCL is associated with a positive interactive effect of constituents of both phyto-additive. The improvement observed with EPCL supplementation confirms the evidence that suggests that herbs, spices, and plant extracts possess properties that promote digestion and inhibits pathogenic microbes resulting in nutrient availability and utilization (Kamel, 2001). The group of broilers fed the control diet and those fed diet supplemented CL had increased wing weight. The broilers fed diet supplemented CL also had increased thigh weight. This corroborates the report of Marcinčák et al. (2011) who reported an increase in thigh weight for broilers fed the diet supplemented with 1% clove powder. The back weight was lower for broilers fed the diet supplemented CL compared to other treatments and this is associated with the relatively low body weight. The liver weight was lowest for broilers fed the diet supplemented with EP and those fed the control diet had the highest liver weight while the CL and EPCL supplemented group had intermediate liver weight. This suggests that EP supplementation was potent against inflammation of the liver which may result in fatty liver disease. An increase in liver weight has been connected with the occurrence of disorders in the metabolic process such as fatty liver (Skrivan et al., 2000). This is similar to the report of Amad et al. (2011) who reported reduced weight of the liver of broilers due to the dietary inclusion of a mixture of thymol and anethole essential oils. Oso et al. (2019) also reported a reduction in weight and relative weight of the liver after dietary supplementation with a phytogenic blend for broilers. Groups of broilers fed diets supplemented with EP, CL and EPCL had higher spleen weight than the control group. This agrees with the findings of Gandomani et al. (2014) who observed increased relative weight of the spleen in laying hens following dietary supplementation with clove bud powder. However, on the contrary, Chowdhury et al. (2018) reported no significant effect of clove supplementation on the relative weight of the lymphoid organs of broilers. Al-Mufarrej et al. (2019) also reported that supplementation of clove seed powder supplementation did not influence bursa, thymus and spleen weight. This inconsistency may be associated with differences in clove plant sources and levels of inclusion. The supplementation of EP and EPCL in the diet of broilers resulted in higher thymus weight compared to other treatments. This implies the good health status of the birds as the development of lymphoid correlates with the health status of animals. Kwak et al. (1999) established that the indicators of health and immunological stress are a measure of the thymus and spleen weight. The thymus weight is associated with the extent of T cell development and spleen weight is associated with the production of immune cells resident in the lymphoid tissue at times of invasion by foreign agents (Elmore, 2006;Pozo et al., 2009).

Meat microbiology
Clostridium count in broiler meat significantly reduced with supplementation of CL and EPCL in the diet of broilers. It was observed that meat from broilers fed the control diet and those fed diet supplemented with EP had increased clostridium count. This suggests that CL supplementation was capable of inhibiting clostridium development. Cloves possess a bioactive compound called eugenol which can deteriorate bacteria cell walls causing bacterial cell lysis (Burt, 2004). This bioactive constituent is effective by binding protein receptors making the substrate unavailable to resident pathogens (Hintz et al., 2015). This is also in agreement with the report of Cui et al. (2010) who reported that clove extract exhibited the greatest antimicrobial activity against Clostridium spp. among other plant extract. The reduced coliform count in the meat of broilers fed diets supplemented with EP and EPCL and the increased coliform count observed for broilers fed diet supplemented with CL implies that EP has constituents active against the growth of coliform bacteria. El astal et al. (2005) documented that the bioactive compounds like tannins and phenols present in EP are active against bacteria, pests or fungi. These active components are similar to quinone extracted from Pergularia daemia leaves which have been reported to inhibit the growth of microbes that deteriorates and contaminate food like Escherichia coli and Bacillus subtilis (Ignacimuthu et al., 2009). The increased lactobacillus count observed in the meat of broilers fed diets supplemented with CL and EPCL is indicative of the proliferation of the bacteria as stimulated by the phytogenic supplementation. Lactobacillus is known to produce lactic acid due to the degradation and utilization of sugar and it is one of the prominent bacteria found in fresh meat causing meat spoilage (Doulgeraki et al., 2012). Meat from broilers fed the diet supplemented EPCL had the least salmonella count while meat from the groups of broilers fed diets supplemented EP and CL had a lower salmonella count than that of the control group. The least salmonella count observed from broilers fed diet supplemented EPCL reveals the synergistic effect of EPCL in limiting the growth of salmonella on preserved meat. Shan et al. (2009) reported that raw pork treated with clove extract was found to have the lowest colonies of microorganisms such as Staphylococcus spp. that can accelerate meat spoilage. Bayoub et al. (2010) also found that the ethanol extract of cloves was the most potent against monocytogenes among other selected plant extracts. Akinduro et al. (2020) reported no presence of salmonella species in preserved broiler meat boiled with EP as spice after the third day of refrigeration. The report of Isikwenu and Udomah (2015) detailed that the dietary inclusion of EP reduces the bacterial load when added to broiler feed. This confirms that bioactive compounds contained in EP have an inhibitory effect against gram-negative bacteria such as salmonella and microbes that may cause diseases (Usman et al., 2016).

Conclusion
The present study confirms the preponderance of the positive synergistic effect of Ethiopian pepper and clove composite dietary supplementation on nutrient digestibility, gut microflora, and carcass characteristic of broiler chickens compared to supplementation of individual phytogenic plants. Furthermore, clove supplementation is potent against clostridium proliferation, while EP supplementation shows a high inhibitory effect against salmonella organisms. The supplementation of EPCL in the diet of broilers increased the weight of lymphoid organs (spleen and thymus) important in immune function and can serve as a replacement for in-feed antibiotics.