Medicinal plant is any plant which in one or more of its organs contains substances that can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs (Sofowora, 1993). Recently, there has been increasing demand for organic or natural products to reduce or eliminate the use of antibiotics as growth promoters due to its high cost and negative human health issue of antibiotic resistance (Zolikha Hassan, 2007). There have also been concern on the contamination of animal products which made the European Union in 2006 to ban the use of antibiotics as growth promoters in livestock feed. Plants (herbs and spices) are therefore botanical alternatives to antibiotics because they possess phytochemicals or secondary metabolites. Phytochemicals also regarded as phytobiotics are a natural bioactive compound that are derived from plants and incorporated into animal feed or water as extract to enhance productivity through the improvement of digestibility, nutrient absorption and elimination of pathogens resident in the animal gut (Rios and Recio, 2005; Sokovic et al., 2010; Levic et al., 2011; Rajasekaran et al., 2013; Gadde et al., 2017). Plant extract are used in animal nutrition as appetite and digestion stimulants, stimulants of physiological functions and treatment of certain pathological conditions, colourants and antioxidants (Tamara et al., 2009). According to Lina Šernaite (2017) plants have the ability to synthesize secondary metabolites such as phenol, phenolic acids, flavones, flavonoids, flavonols, quinones, tannins, saponins, alkaloids and phytate. Phenolic compounds have been reported to exhibit antimicrobial and antioxidant activity (Martins et al., 2015), steroids have been reported to have antimicrobial and antifungal activity (Martins et al., 2015) and stimulation of bone marrow and growth. Saponins have been reported to perform antioxidant role and are useful in vaccine production (Asl and Hosseinzadeh, 2008). Flavonoids and tannin have been suggested play a key roles as antibacterial, antifungal, antidiarrheal and antioxidant (Adisa et al., 2010). Alkaloids have been shown to have antipyretic activity (Faizi et al., 2008).
Previous studies has been carried out on the use of plant extracts and their level of inclusion in the water of broilers, for instance, citrus sinensis peel extract (Abbas Ebrahimi et al., 2014; Akbarian, 2013; Pourhossein et al., 2012), Morigold flower extract (Nuraini et al., 2017; Skrivan et al., 2016), Turmeric extract (Nuraini et al., 2019; green tea extract (Farahat, 2016; El-deek et al., 2012; Khalaji et al., 2011; Shahid et al., 2013), Neem leaf extract (Biu et al., 2009; Chakravarty and Pasad, 1991; Nagalakshmi et al., 1996), Moringa olifera leaf extract (Alabi et al., 2017; Khan et al., 2017; Faluyi and Agbede, 2018). yet there is a dearth of information on the use of Luffa aegyptiaca extracts on the heamato-biochemical and bacterial count of broilers.
There is a correlation between nutrition and immune response, an animal that is not properly managed will undergo stress, poor health and finally death during serious conditions. Hence, the present study is focused to investigate the effect of Luffa aegyptiaca leaf extracts on the haematology, serum biochemistry and bacteria count of broiler chicken.
The experiment was carried out at Division of Animal Nutrition, Sumitra Research Farm, Gujarat, India during the month of January to March, 2019.
Collection and processing of Luffa aegyptiaca leaves (LUF)
Luffa aegyptiaca leaf was identified and authenticated by a botanist on the research farm. Thereafter, fresh disease free leaves of L. aegyptiaca was harvested from the farm, the leaves were thoroughly washed with running tap water to remove the debris and allowed to dry under shade for 11 days, it was then hammer milled into L. aegyptiaca powder (LUF). The extract was prepared by soaking 200grams of LUF in one litre of water and kept in an air tight plastic container and the mixture kept in the refrigerator at 4oC for 48 hours and then sieved with a with cheese cloth, then with WhatMan No1 filter paper (24cm).
A deep litter poultry house was used for the experiment, the pen was swept, cleaned and well disinfected with Cid 2000, feed and water troughs were also washed. The electrical fittings (bulb) 200 watts were properly fixed and a vaccination programme was designed before the commencement of the study.
Animal management and Experimental set -up
One day old 200 (Ross 308) broilers of mixed sex were obtained from a commercial hatchery in India. The chicks were weighed individually at the beginning of the experiment and wing banded. They were assigned into five treatments, each group was further divided into five replicates each of ten (10) birds. Anti-stress was added in the drinking water of the birds. The light (electric bulb) was continuous and the initial brooding temperature was 34oC for the first week of age and it was gradually reduced by 2oC per week. Vaccines were administered according to the prevailing vaccination schedule in the environment. Vitamins (Miavit) was added in water a day before and after each vaccination. Clean feed and water was provided unrestricted throughout the experimental period which lasted for 3 weeks.
Diets were formulated to meet the nutritional requirements of birds according to NRC (1994).
Treatment 1: 1.20 g/ liter Neomycin (drinking water)
Treatment 2: 5.0ml/liter of LUF
Treatment 3: 10ml/liter of LUF
Treatment 4: 15ml/liter of LUF
Treatment 5: 20ml/liter of LUF
At the end of the experiment, ten (10) birds were randomly selected from each treatment, two from each replicate for blood analysis. Blood was collected from the wing vein with a syringe and needle. Samples meant for hematology was put in tubes containing EDTA to prevent coagulation while those for serum biochemical parameters was put in bottles without EDTA. Hematological parameters covered pack cell volume (PCV), red blood cell (RBC), hemoglobin concentration (Hb), white blood cell (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). Serum parameters included albumin, globulin, total protein, cholesterol, calcium, phosphorus, serum glutamic oxaloacetate transaminase (SGOT) and serum glutamic pyruvate transaminase (SGPT). PCV, RBC, Hb, MCV, MCHC, WBC and its differentials were determined using standard techniques as reported by Jain (1986). The serum total protein, Albumin and Globulin were computed according to (Doumas and Briggs, 1972), Glutamic oxaloacetate transaminase (SGOT), Glutamic phosphatase transaminase (SGPT) was determined according to Scott (1965).
Carcass evaluation and bacteria count evaluation
At the end of the 3rd week, five birds were randomly selected per treatment; they were fasted overnight and given only water, weighed and slaughtered. After evisceration, the weight of the visceral organs and other parts of the birds were recorded. One gram of each sample of the jejunal content was collected and transferred into the test tube and was then diluted with 9ml of 1% peptone broth and homogenized. Counts of bacteria and lactobacillus were determined according to Farmer (2003).
Phytochemical analysis was carried out on the plants leaf extract using standard methods Sofowora (1993) and AOAC (2000). Percentage composition of flavonoids, saponin, phytate, alkaloids, tannin and oxalate were carried out according to procedures outlined by Harbone(1984) and Boham and Kocipai-Abyazan (1974). Mineral analysis was carried out using Atomic Absorption Spectrophotometer (AAS). Proximate analysis of crude protein, ash, ether extract and crude fibre in the experimental diet were carried out in accordance with the Association of Official Analytical Chemists (AOAC, 2000).
All data collected were analysed using Snedecor and Cochran (1989). The difference in treatment means were separated using Duncan’s multiple range test as outlined by Obi (2002).
RESULTS AND DISCUSSION
Table 1 shows the percentage composition of experimental diet, the diets were formulated to meet the nutritional requirements of the animals according to NRC (1994) specifications. The result on the phytochemical analysis of LUF revealed the presence of several bioactive compounds at different concentrations as presented in Table 2. The phytochemical components are saponin, flavonoids, phytate, alkaloids, tannin, oxalate and phenol at 6.22%, 3.02%, 1.01%, 2.81%, 3.11%, 1.01% and 11.43% respectively. The result obtained in the current study was consistent with the findings of Shakeri et al (2012); Jong et al (2010); Mabhiza et al (2016). However, all values falls within the safe recommended limit as proposed by Vikas Kumar et al (2009). According to Igwe et al (2010), alkaloid help in repelling parasites and predators in plants and when ingested at high level could result in a negative effect on the thyroid stimulating hormone and some enzymes (Okaka et al., 1992). Flavonoids performs several functions such as antibacterial, antioxidant, anti-inflammatory, anti-viral and anti-carcinogenic (Middleton et al., 2000).
The mineral composition of LUF reveals that calcium (34.31mg/g), phosphorus (0.12mg/g), potassium (2.16mg/g), magnesium (7.44mg/g), sodium (0.31mg/g), manganese (0.01mg/g), zinc (0.001mg/g) and iron (0.03mg/g). Calcium had the highest quantity followed by magnesium, zinc had the lowest value, the result obtained in this experiment is contrary to the reports of Alagbe Seyi Valerie et al (2017) who recorded a higher value for calcium (58.6mg/g) and magnesium (12.4mg/g), this higher value can be attributed to differences in variety of L.aegyptiaca leaf used, environment and stage of growth of the plant before it was harvested. The presence of different minerals in LUF facilitates various physiological activities in animals. According to Murray et al (2000) minerals are structural components of the body tissues are involved in the maintenance of acid base balance, regulation of body fluids, transport of gases and in muscle contractions.
Calcium plays a vital role in providing rigidity and support to animals (Ibrahim et al., 2001). Magnesium, zinc, iron, manganese are important co-factors found in the structure of certain enzymes and are indispensable in numerous biochemical pathways (Soetan et al., 2010).
The relative organ weights of broilers given Neomycin and LUF is presented in Table 4. The relative weights heart, liver, pancreas, gizzard, proventriculus and intestine were not (P>0.05) significantly different among the groups. The weight of the heart ranged from 3.44 – 3.57g, liver (1.26-1.49g), pancreas (2.22 – 2.27g), gizzard (3.00 – 3.17g), proventriculus (1.41 – 1.52g) and intestine (79.3 – 91.9 cm). According to Madhusadha et al (1986) antinutrients are causes of internal organs enlargement in birds, the non -significant differences in the organs weight simply a reflection that the test materials are non-toxic. The results obtained in this experiment is in agreement with the findings of Bolu et al (2009) but contrary to the findings of Nderi et al (2014).
Table 5 revealed the bacteria and Lactobacillus count of broiler given Neomycin and LUF. The bacteria count ranged from 21.11- 22.11 (cfu/g) while those of lactobacillus ranged from 10.44 – 20.73 (cfu/g). There was a significant difference (p<0.05) in the E.coli count among the groups, the values slightly increased from group 1 to 2 before it eventually decreased though not at a significant rate. This was comparable to the findings of Hanan E. Al-Mashhadani (2014). There was no significant (p>0.05) difference in the lactobacillus count among the groups, thus acting as a prebiotics. The current study is also in line with the findings of Bird et al., 2002; Tiwari and Jyoti 2008 who reported that Lactic acid bacteria produce several bactericidal/antibiotic like substances, which have been found effective against enteric pathogens. These bacteriocins can kill pathogenic bacteria, prevent colonization actions and also performs competitive exclusion which refers to the physical blocking of opportunistic pathogen (Watkins and Kratzer, 1983).
Haematological parameters of broilers given Neomycin and LUF is presented in Table 6. The PCV values ranged from (29.22-31.71%), Hb (11.51-13.11g/dl), RBC (2.06-2.30 106/µl), MCV (103.5 -161.9 fl), MCH (48.34 – 53.33 pg), MCHC (39.67 – 42.01%), WBC (19.31-21.56 106/µl), heterophils (6.13 – 8.15 %), monocytes (0.89-1.12%), lymphocytes (9.04 – 9.85 %) and eosinophils (1.00 -1.12%). There was not significant (p>0.05) differences in all the haematological parameters measured, the haematological values slightly increased from group A to E but not at a significant level. The trend in the values could be as a result of intestinal microbial balance and a reflection that the animals were well nourished. However, all values are within the range reported by Talebi et al (2005); Islam et al (2004). Changes in the RBC, MCH, MCHC and PCV values have been attributed to adaptation to adverse weather condition (Minka and Ayo, 2007), PCV and Hb variations could also be linked to differences in the breed of animals (Adili and Melizi, 2013). The result obtained in the current study was consistent with the finding of Akintomide et al (2018) when neem leaf meal was fed to cockerels. Adeyinka and Bello (2013) also reported that WBC and its differentials helps to fight infections and produce antibodies to protect the body. Herbs, spices, and their extracts have high antioxidant capacity (Wojdylo et al, 2007).
The serum biochemical parameters of broiler chicken given Neomycin and LUF revealed that albumin values ranged from (1.83-1.95g/dl), globulin (1.62-1.83 g/dl), cholesterol (65.1 – 68.3mg/l), calcium (6.91-7.71mg/l), phosphorus (4.01 – 4.41 mg/l), SGPT ( 28.1-33.1mg/l) and SGOT (55.1 – 63.2 mg/l). All the serum biochemical traits were not significantly (p>0.05) among the groups. Albumin values in the blood can be easily influenced by protein shortage, the results obtained is an indication that the experimental diets contained enough protein to support the normal protein reserves across the group. The values for all the parameters fall within the normal range values established for birds by Ibrahim Albokhadaim (2012). Reports have also shown that when probiotic is supplemented in the diets broilers they tend to have lower total cholesterol, VLDL cholesterol and triglyceride concentrations in the serum.
It can be concluded from the experiment that LUF could be given to broilers at 20ml/litre without any deleterious effect on the blood profile and general performance of the animal. LUF has proven to be play a role of a competitive exclusion i.e. physical blocking of opportunistic pathogen, thus maintaining a balanced gut microbial ecosystem making it a botanical alternative to antibiotics for use in organic poultry production.