Vernonia amygdalina (VA) is a shrub or small tree that grows throughout tropical Africa. It is popularly called bitter leaf because of its abundant bitter taste (Ekpo et al., 2007). The leaves contain a considerable amount of anti-nutritional factors like high level of tannic acid and saponin (Charles and Boulevard, 2012). The findings by Akwaowo et al. (2000) reported that the young leaves often preferred for human consumption contain high cyanide (60.1 mg 100^-1g DM) and tannin content (40. 6 mg 100^-g DM) than older ones. Furthermore, V. amygdalina has also been used as feed for broilers, where it was able to replace 300g kg^-1DM of maize based diet feed efficiency (Bonsi et al., 1995). Research has shown that V. amygdalina have some beneficial effect in disease management of poultry (Dakpogan, 2006) such as anti-bacterial and anti-parasitic and anti-oxidant (Erasto et al., 2009) and as growth promoter by enhancing the gastro intestinal enzymes thus increasing feed conversion efficiency (Huffinan et al., 1996; Olabatoke and Oloniruba, 2009).

The effects of any feed ingredient on the haematological factors of the chicken are of immense assistance in deciding whether or not such a feed ingredient will be used as poultry feedstuff (Mitruka and Rawsley, 1977). Certain heamatological factors such as packed cell volume, red blood cell, hemoglobin etc can be associated with certain production traits and serve as means of assessing clinical and nutritional health status of animals. For example it has been established that high percentage white blood cells especially lymphocytes are associated with the ability of the chicken to perform well under stressful conditions.

Coccidiosis is one of the most common diseases of poultry production systems in spite of advances in chemotherapy, management, nutrition and genetics (MC Dougald et al., 1997). It remains a big concern to the commercial chicken production because of the high cost involves in the control of disease. Coccidiosis may strike any type of poultry in any type of facility (MC Dougald, 2003).The routinely use of these drugs on one  hand  has led to strains of parasites which are drug resistance (Long, 1986) and on the other hand, prejudicial to consumer health because of drug or antibiotic residue in poultry products (Youn and Noh, 2001). In recent years, interest has developed in many countries in the collection and extended use of medicinal plant extract for an alternative production purposes (Griggs and Jacob, 2005). The emergence of drug resistance strain of coccidial has made currently available anti coccidial less effective and this has threatened the economy of the country especially in developing countries where the problem has become a major concern to poultry farmers. However, this study has provided another alternative treatment of using V. amygdalina for coccidiosis which is targeted at solving farmers’ problem. Therefore, the objective of the study is to evaluate the effects of varying levels of V. amygdalina leaf meal on growth, hematological parameters and as anticoccidial for broiler chickens.

Determination of coccidial infection

At ten day of post infection, 5 g of feacal sample was randomly selected from each treatment and taken to the laboratory for feacal parasite screening using floatation method.

Management of experimental birds

The cages were washed and disinfected using Morigad disinfectant and left to rest for two weeks prior to the arrival of the chicks. Before the arrival of the birds, the brooding cages were ready and water was waiting for them upon arrival. The birds were raised on a normal starter diet for 4 weeks and finishers’ diet for four weeks. Vaccination was carried out as at when due, while routine medication was done to keep the birds healthy except giving them Coccidiostat. Water and feed were served at ad-libitum before the birds were separated into different treatments with their feeds weighed out on daily basis. Leftovers were weighed and recorded and subtracted from the feed giving to determine the feed intake.

Blood collection and haematology analysis

At the end of sixth week of feeding trials, two birds from each dietary replicate were randomly sampled to determine haematological responses. 5 ml of blood was taken from the jugular vein of randomly selected birds per replicate. 2.5 ml of sampled blood was put into labelled blood sample bottles containing anti-coagulant (Ethyl Diamine-Tetra-Acetate powder (EDTA)) to determine haematological parameters. Parameters analysed include; Packed Cell Volume (PCV)(%), Haemoglobin (g/l), Red Blood Cells (RBC)(10⁶ul), White Blood Cell (WBC)(10³ul), Lymphocytes (%), Neutrophils (%), Monocytes (%), Eosinophil (%) and Basophils (%) according to the procedure of Howlett and Jamie (2008).

Statistical analysis

All data collected were subjected to analysis  of  variance  (ANOVA) according to the procedure of SAS (2002). Significant differences between the treatments means were separated using Duncan multiple range test (Duncan, 1955).

Data collection

Initial weight of birds was taken before they were distributed into various treatments. Experimental diet containing V. amygdalina was served over a period of 7 weeks. Thereafter, final weights were determined using the kitchen weighing scale. The total feed intake was calculated over a period of 7 weeks. Feed intake (g) is the daily feed consumed which was obtained by deducting the weight of remnant feed from the feed offered the previous day. Body weight gain (g) was done on weekly basis. It was obtained by deducting the previous week’s body weight from the subsequent week’s body weight.

Tables 1 and 2 show the proximate analysis of the experimental broiler starter and finisher diets. Both starter and finisher are isocaloric and isonitrogenous in nature. Table 3 shows the phytochemical constituents of the test ingredients.  The levels of tannin, phenol, steroid, Phytate and oxalate levels are moderately positive while that of alkaloid, saponin, flavonoids and cyanide are strongly positive. Table 4 shows the performance characteristics of broiler fed diet with varying levels of V. amygdalina. The highest final weight was recorded in T2 (1591.17 g) and lowest in T5 (1196.83 g). The highest final weight gain was observed in T2 (1466.40 g) and the lowest in T5 (1062.40 g). For average daily weight gains, highest value was recorded in T2 (29.92) while the least value was obtained in T5 (21.68). The result for feed intake ranges   from    highest    T1   (3362.00 g)   to   lowest  T5 (2876.34 g). For feed conversion ratio, lowest value was recorded in T2 (1.99) to the highest of (2.40) T5. The mortality percentage reveals that T2 had the highest percentage of 20% while T5 has 6% mortality while other treatment had no mortality.

The result of the hematological parameters of broiler chicken fed varying levels of V. amygdalina based diet is as shown in Table 5. The result of hematological indices shows that there were no significant differences across the treatment except for hemoglobin in T2 that had the lowest value (8.04 × g/dl) while T4 had the highest (12.03 × g/dL). Eosinophil in T4 has the highest value (4.00%) and lowest in T5 (2.00%) and T2 (2.00 %). Packed cell volume in T4 has the highest value (38.00 %) and lowest in   T2   (25.00%).   Red   blood   cell   was   higher  in  T3 (3.57×10¹²/L) and lower in T2 (2.43×10¹²/L). However, all other parameters were within the normal range recommended by Merck (2011).

The result shows the performance characteristics of finisher broiler diet containing varying levels of V. amygdalina based diet. From this result it was observed that birds in T₂ (negative control) and T₁ (positive control)  had the best result in terms of feed intake, final weight, weight gain, average daily weight gain and the best feed conversion ratio. This could be as a result of non - inclusion of V. amygdalina leaf meal in  the  diet  of  these treatments and this might have resulted in better performance observed. Highest mortality ratio was recorded in T₂, which must have be as a result of non -  inclusion of V. amygdalina  or the use of anticoccidial drugs which must have led to outbreak of coccidiosis. Comparing the varying levels of inclusion, in terms of final weight, weight gain, average daily weight gain and feed intake, T₃ (200g) has the best result. T₃ (200g) also has the lowest feed conversion ratio value among the three inclusions levels and hence the treatment with best feed conversion. This could be as the result of the low concentration of the leaf meal which might have improved the performance. Lowest feed intake, average daily weight gain, weight gain and final weight were recorded in T₅ (600g). This might be as a result of higher concentration of anti-nutritional factors such as alkaloid, saponin, tannin and glycoside in V. amygdalina as reported by Arhoghro et al., (2009). No mortality was observed from the birds on T1, T3 and T4 while 6% mortality rate was recorded in T₅ (600g). This indicates that as the level of inclusion of bitter leaf increased the mortality rate increased. This could be as a result of the anti - nutritional factors in the test ingredient (Saponin, Alkaloids and Tannin).

The result of hematological parameters of broilers birds fed diet with varying inclusion levels of V.amygdalina shows that bird fed with inclusion level of 400g (T₄) has highest packed cell volume. For hemoglobin, T₃ and T₄ with inclusion level of 200 and 400 had the highest value also. For red blood cell, white blood cell T₃ with inclusion level of 200 g has higher value.  Owen and Amakiri (2011) made similar observation, with the exception of White blood cell. All other hematological indices measured were influenced by increasing levels of V. amygdalina. Osho et al. (2014) demonstrated in his own experiment that oral adminstation of bitter leaf extract on broiler chickens did not have a significant effect on the Hb and RBC in treated birds. Increase in White blood cell in T₄ could be attributed to the presence of anti-nutritional compounds of V. amygdalina and presence of infection. This is in line with Aregheore et al. (1998) who suggested that the presence of some phytochemicals in bitter leaf extract allows the animal to respond to infection. According to Isaac et al. (2013), packed cell volume is involved in the transport of oxygen and absorbed nutrients. Increased packed cell volume shows a better transportation and thus prevents anaemia (Coles, 1986). The result of packed cell volume and haemoglobin  which increases as the inclusion level of bitter leaf increased in the diets is in accordance with Adejumo (2004) who reported that packed cell volume and haemoglobin were positively correlated with the nutritional status of the animal. This observation however implies that the diets supported haemopoictic tissue with production of adequate white blood cells.   Thus result indicated that the immune system of the birds was not compromised because the White Blood Cells function primarily as defence system in the body (Eroschenko, 2000). Values obtained for Neutrophils, Monocytes Eosinophils and basophiles are within the normal range for healthy birds (Mitruka and Rausley, 1977; Archetti et al., 2008).

It was reported by Frandson (1986) that the number of neutrophils in the blood increases rapidly when acute infection is present; hence a blood count showing this increase is useful in diagnosis of infections, which is contrary to this result. The low values of monocytes and basophils agreed with the statement that basophils and monocytes are normally present in small to moderate number in the blood system. For Eosinophil, T₄ has the highest value, and eosinophil is known to protect animal against infection. This shows birds on this treatment were able to fight against the infection. Other treatments were also high but not as high as T₄.

Based on the study, it can be concluded that inclusion of V. amygdalina at the rate of 200 g - 400 g/ 150 kg feed had no adverse effect on the growth and hematological parameters measured; it also prevented the occurrence of coccidiosis in broiler chicken. Hence, the addition of this natural herb at this level, 200 g - 400 g/150 kg of feed can be used as anticoccidial in broiler chickens’ diet without any delecterious effect on the health status of the birds.

The authors have not declared any conflict of interests.