EFFECT OF DIETARY INCLUSION OF BIOCHAR ON GROWTH PERFORMANCE, HAEMATOLOGY AND SERUM LIPID PROFILE OF BROILER BIRDS

C.G


INTRODUCTION
With global human population projected to increase to 10 billion people by 2050 (FAO 2009a), there is the need for adequate and nutritious food supply for the teeming human population, through sustainable production systems.Poultry production is one sustainable means proposed for the attainment of this goal of global food sufficiency.Sustainable poultry production is also a key measure in alleviating poverty and addressing the menace of protein insufficiency among many households in sub-Saharan Africa, particularly, Nigeria (Ani, 2007a).Poultry are fast growing birds that yield products containing essential amino acids which the body requires for normal metabolic activities (Ibe, 2004).Poultry have potential to give high turnover rate on investment, and are also efficient converters of feed to animal protein (Oluyemi and Roberts, 2000).Over the last few decades, the use of dietary inclusions, majorly, antibiotic growth promoters have been explored with the aim of increasing efficiency of feed utilization in poultry (Hamasalim, 2016).Notwithstanding, numerous health complications are often associated with the use of antibiotic growth promoters in poultry nutrition.Thus, the search for and use of natural and locally available feed additives with no attendant health implication on humans is justified (Hajati and Hazaei, 2010;Saleha et al., 2009).In view of these, biochar is gaining attention as a locally sourced feed additive with capacity to improve digestibility, feed efficiency, weight gain, feed conversion ratio and dietary energy absorption in poultry birds (Gerlach and Schmidt, 2012).Inclusion of biochar in poultry nutrition has been reported to rapidly decrease the incidence of diarrhea, eliminate allergies and ameliorate the detrimental effects of mycotoxins in feed (Marie, 2013).
Please cite as.Dim, C.E., Akuru, E.A., Egom, M.A., Nnajiofor, N.W., Ossai, O.K., Ukaigwe, C.G. and Onyimonyi, A.E. ( 2018) Effect of dietary inclusion of biochar on growth performance, haematology and serum lipid profile of broiler birds Agro Science 17 (2), 8 -16 DOI: https://dx.doi.org/10.4314/as.v17i2.2 Biochar is a carbon-rich charcoal-like substance obtained through the processes of pyrolysis, gasification and hydrothermal carbonization.While pyrolysis which is the first step in biochar production, involves thermal decomposition of organic matter under limited oxygen supply and high temperatures ( < 700°C), gasification involves exposing biomass to high temperature coupled with oxygen supply, whereas hydrothermal carbonization (HTC) involves steaming biomass with different types of acids acting as catalysts (Harris, 1999;Almuth, 2011).Biochar production usually begins with biomass (which could be crop residue, wood and wood waste, certain animal manure and/or various other organic materials) being fed into a pyrolin kiln i.e. a furnace that burns with little or no oxygen.The process yields two main products, biochar, constituting 50% of the carbon content of the biomass, and biofuel, often syngas which can be employed in several energy application processes (Anita, 2009;Almuth, 2011).Gerlach et al. (2014b) and Van et al. (2006a) reported that dietary inclusion of biochar is capable of enhancing the immune status of animals.According to Gerlach and Schmidt (2012) inclusion of biochar in the diet of broiler birds deactivated toxins found in the digestive tract, helped activate intestinal flora and resulted in improved vitality of the birds.Teleb et al. (2004) found that supplementation of broiler diets with 0.5% biochar reduced the detrimental effects of aflatoxins, by lowering mortality and improving growth performance.Kutlu et al. (2000), Kana et al. (2010) and Prasai (2013) observed improved growth performance in broilers fed diets containing 2 and 6% inclusion levels of biochar obtained from oak, maize cob, canarium seed, coconut shell, and locally available wood.Odunsi et al. (2007) however reported than at an inclusion level of 7% or more, biochar depressed growth and final body weights in broilers.Doydora et al. (2011), Ritz et al. (2010) and Marie (2013) noted that dietary biochar obtained from pine chips, peanut hulls and locally available wood have potential to significantly reduce ammonia and phosphorus content of poultry droppings, reduce odour and water runoff from liter piles during rainfall.Apart from its relevance as litter and feed additive, biochar has other agricultural, environmental, electrical, industrial and textile applications, ranging from silage preparation, manure composting, slurry treatment, water treatment in fish farming, bioremediation, biogas production, insulator and air decontaminator in buildings, and a host of other uses (IUCN, 2012;Elad et al., 2010;Lin et al., 2008;Inthapanya et al., 2012).Based on the inherent potentials of biochar, the present study was therefore designed to investigate the effect of varying inclusion levels of biochar on growth performance, hematology and serum lipid profiles of broiler birds.

MATERIALS AND METHODS
The study was carried out at the Department of Animal Science Teaching and Research Farm, University of Nigeria, Nsukka.The duration of the study was eight weeks.Corn Stover served as the organic material used for biochar production through the process of pyrolysis, and was procured from Department of Crop Science, University of Nigeria Nsukka.The Stover was chopped into small pieces and sundried for two weeks, after which it was fed into the pyrolysis kiln, to undergo pyrolysis, under serious and careful precautionary measures.The resultant biochar product was then used for the study.

Management of experimental birds and data collection
A total of one hundred and twenty (120) unsexed day-old commercial broiler chicks were randomly divided into four groups of 30 birds each, replicated twice with 15 birds per replicate.The groups were randomly assigned to four isocaloric (2.80/27.2Mcal of ME/kg) and isonitrogenuos (23.08/20.10%crude protein/) diets at both starter (0-28 days) and finisher (28-56 days) phases in a completely randomized design with treatment as main effect involving 0%,(control), 2%, 4% and 6% dietary inclusion levels of biochar.The birds were placed in 2.6 × 3 m deep litter pens with fresh wood shavings which were changed on weekly basis.Feed and water were supplied ad libitum to the birds from 0 to 56 days of age.The birds were subjected to standard broiler management procedures.The birds were properly vaccinated as and when due following the vaccination protocol for broiler birds by the National Veterinary Research Institute Haematology and Serum Lipid Profile of Broiler Birds Fed Diets Containing Biochar (NVRI), Vom, Plateau State, Nigeria (unpublished).The birds were weighed at the beginning of the experiment to obtain their initial body weights, and subsequently on weekly basis to determine their growth performance.The birds were finally weighed at the end of the experiment to determine their final live weights.Feed conversion ratio was calculated from these data as quantity (gram) of feed consumed per unit (gram) weight gained over the same period.

Blood collection for haematological and lipid profile evaluation
At weeks 4 and 8 of the experiment, blood was collected with a sterile needle from the wing veins of two birds per replicate (32 birds in all for both phases).The blood was collected into properly labeled sterile bottles containing ethylene diamine tetra acetic acid (EDTA) and cooled at 4°C using icepacks.The samples were transferred to the Laboratory of Faculty of Veterinary Medicine, University of Nigeria Nsukka for determination of hematological parameters of the birds.The packed cell volume (PVC) was determined using the micro hematocrit centrifuge method (Coles, 1986).A 0.5 ml of blood was centrifuged at approximately 10,000 revolutions for 5 min in a micro hematocrit centrifuge.The PCV was subsequently determined by measuring the height of the erythrocyte column and expressing this as a fraction of the height of the total blood column.PCV -Height of packed cell column divided by height of whole blood column.Haemoglob in concentration was determined spectrophoto metrically (Perkin-Elmer) by the cyanomethaemoglob in method (Higgins et al., 2008).A 0.5 ml of blood was diluted in a buffered solution of potassium ferricyanide and potassium cyanide to yield the stable haemoglob in derivative cyanmethaemoglob in.The potassium ferricyanide converted the haemoglob in to methaemoglob in which was further converted to cyanmethaemoglob in by the action of potassium cyanide.Cyan-methaemoglob in produced a color which was measured in a spectrophotometer.The color relates to the concentration of hemoglobin in the blood.The absorbance of this solution was read in a colorimeter at a wavelength of 540 nm.Erythrocyte count (RBC) and leukocyte count (WBC) were determined using Neubarhemocy to meter after the appropriate dilution (Mitruka and Rawnsley, 1977;Thrall and Weiser, 2002).Blood samples for serum analysis were centrifuged at 3000×g for 15min and serum was separated.Determination of serum total triacylglycerol (TAG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) concentrations were done with Randox kits (BT294QY, Randox, United Kingdom).The lipoproteins LDL-C and HDL-C were fractionated by a dual precipitation technique (Jacob et al., 1990).After fractional precipitation, lipoprotein cholesterol was estimated.

Proximate and statistical analyses
Samples of diets were analyzed for proximate composition using the methods of AOAC (1990).Data collected were subjected to analysis of variance as described for completely randomized design (Steel and Torrie, 1980), and differences between treatment means were separated using Duncan's New Multiple Range Test (Duncan, 2003) as outlined by Obi (2002).

Growth performance of broilers fed varying dietary levels of biochar
Table 2 shows the proximate composition of experimental diets, while growth performance of broiler birds (starter and finisher phases) to the experimental diets is shown in Table 3. Treatments did not differ significantly (P >0.05) in final body weight, average daily weight gain, average daily feed intake and feed conversion ratio at the starter phase.However, at the finisher phase, significant differences (P<0.05) were observed among treatments in final body weight, average daily weight gain and feed conversion ratio.Broilers fed 4 and 6 % biochar kg-1 were similar in final body weight but heavier (P < 0.05) than those fed 2% biochar kg-1 and those on the control diet (0% biochar kg-1).For average daily weight gain, birds without biochar supplementation (control group) gained the least weight, and this was comparable to the weight gained by those on 2% biochar kg-1.Birds on 4 and 6% biochar kg-1 had the highest but similar weight gain.Similar trend was observed in feed conversion ratio (FCR), where birds on 4 and 6% biochar kg-1 had comparable but the least FCR.Birds fed 0% biochar kg-1 had the highest FCR, and this was comparable to the FCR of birds with 2% biochar kg-1 in their diets.As shown in Table 3, it is evident that at the starter phase, the effect of treatment on performance traits of chicks was not significant.This may be attributed to the fact that at this stage, the gut system of the birds was yet to adjust to the test ingredients.According to Kleyn, (2013), birds' digestive tract is usually not fully developed at an early stage of life.As the birds advanced in age (finisher phase), they became capable of absorbing nutrients effectively and thus competed equally.As a feed supplement, the use of biochar is capable of improving performance traits such as weight gain, nutrient digestibility and feed efficiency in broiler chickens and ducks (Gerlach and Schmidt, 2012;Kana et al., 2010;Ruttanavut et al., 2009).The significantly higher growth performance (final live weight, average daily weight gain and feed conversion ratio) observed for birds fed 4 and 6% dietary biochar kg-1 compared to those in the control suggest that dietary inclusion of biochar improved the performance of the birds.The result of the present study on growth performance are similar to the reports of Kutlu et al. (2000) Kana et al. (2010) Prasai ( 2013) and Jiya et al. (2013)whose studies showed that at 0.2-0.6%levels of inclusion, dietary biochar resulted in improved performance of broiler birds.Kana et al., (2014) also reported that dietary inclusion of born charcoal and canarium charcoal at 0.4% and 0.2% led to an improvement in body weight and weight gain of broilers fed diets containing aflatoxin (AFB1) contaminated diets.The results however contrast the findings of Odunsi et al. (2007) Kana et al. (2010) and Jiya et al. (2013) who reported that from 2% and higher levels of inclusion, dietary biochar is capable of depressing growth rates and final body weights of broiler chickens.The superior growth performance observed in the birds on biochar inclusions can be attributed to the adsorbent features of the biochar in the gut of inclusions can be attributed to the adsorbent features of the biochar in the gut of the birds, arresting toxins and anti-nutritional factors that will interfere with aborption of nutrients by the birds' intestinal walls.Hence, the birds had ample supply of nutrients from the feed with minimal obstruction in their absorption.Notable among the growth enhancing potentials of biochar in the present study is the fact that at 4 and 6% kg-1, the feed conversion ratio of birds were significantly improved.Prasai et al. (2016) noted that a probable mechanism of action by which biochar improves FCR is by changing the microbiota constitution in the digestive tract of birds.

Hematology and serum lipid profiles of broilers fed varying dietary levels of biochar hematological traits
The hematological traits and serum lipid profile of broilers fed the experimental diets are shown in Table 4.At the starter phase, treatment did not differ significantly (P > 0.05) in white blood cell count.There were however, significant differences (P < 0.05) among treatments in packed cell volume (PCV), haemoglobin concentration (HC) and red blood cell (RBC) count.Broiler chicks fed 6% biochar kg-1 had the highest PCV value, and this was comparable to the PCV values of chicks on 4% biochar kg-1 diets.Broiler chicks fed 0 (control dietary group) and 2% biochar kg-1 had similar PCV values.Broiler chicks fed 6% biochar kg-1 had the highest HC value, and this was statistically higher (P < 0.05) than the HC value of chicks on the control diet.Broiler chicks fed 2 and 4% biochar kg-1 had similar HC values.Broiler chicks fed 2% biochar kg-1 had the least RBC value, and this was statistically lower (P < 0.05) than the RBC values of chicks on other treatments.At the finisher phase, PCV and WBC values of broilers were not significantly (P > 0.05) affected by treatments.There were significant differences (P < 0.05) in haemoglobin concentration and red blood cell counts, however similar trend as was the case at the starter phase was also observed.Blood analysis is a readily available tool for assessing the clinical and nutritional status of animals on a feeding trial (Olabanji et al., 2009).Usually, animals that have good blood composition tend to possess records of improved performance Haematology and Serum Lipid Profile of Broiler Birds Fed Diets Containing Biochar (Isaac et al., 2013).The result of the present study on haematology (Table 4) shows that treatment significantly affected red blood cell count, packed cell volume and haemoglobin concentration at the starter phase, and only red blood cell count and haemoglobin concentration at the finisher phase.The result agrees with the findings of Iheukwumere et al. (2002) who reported significant differences in PCV, Hb concentration and red blood corpuscles values among birds who were feed restricted.However, broilers in the present study were not feed restricted.The physiological implication of the above result obtained in the present study can be attributed to the ability of the birds on biochar treatment to maximize the vitamin-mineral premix of the diet (especially iron and B-complex vitamins) better than birds on the control group.This is because of the probable binding of the biochar with toxins and anti-nutritional factors in the gut of the birds which would have impeded the utilization of the said vitamins and minerals for the production of the birds' RBC.This consequently influenced their Hb concentration.The results however contrasts the findings of Majewska et al. (2009) whose study showed that dietary supplementation of 0.3% charcoal did not have significant effect on hematological indices of turkey.Kana et al. (2014) reported that bio-charcoals had no significant effect on RBC, WBC, haemoglobin and hematocrit values of broilers fed aflatoxin B1-contaminated diets.The study of Boonanuntanasarn et al. (2014) also showed that dietary inclusion of activated charcoal (bioachar) did not significantly affect RBC, hemoglobin or hematocrit values of Nile tilapia in a 28-day feeding trial.Boonanuntanasarn et al. (2014) attributed the immune enhancing potentials of activated charcoal (biochar) to its role as a non-specific detoxifier, capable of improving overall health conditions of animals.From the study, it was observed that the RBC values of birds on the 2% biochar kg-1 diets were significantly decreased compared to those on other treatments.Low levels for haematological parameters as reported by Bawala et al. (2007) could be due to harmful effects of high dietary contents.Kana et al. (2014) also associated decrease in red blood cell count values with poor quality feeds.

Serum lipid profiles
As shown in Table 4, there were no significant differences (P > 0.05) observed for high density lipoprotein (HDL) and triacylglycerol (TAG) across the various treatment means at the starter phase.However, significant differences (P<0.01)existed among treatments in cholesterol and low density lipoprotein (LDL) values.Birds fed 6% biochar kg-1 had the least cholesterol value, and this was statistically different from the cholesterol values of birds fed 0 and 2% biochar kg-1.The cholesterol values of birds fed 0, 2 and 4% biochar kg-1 were comparable.Birds fed 0, 4 and 6% biochar kg-1 had comparable LDL values.Birds on 2% biochar kg-1 diets had the least LDL value.Treatment significantly (P < 0.01) affected cholesterol, TAG, and LDL values of finisher birds.Birds fed 6% biochar kg-1 had the least cholesterol value and this was statistically lower than the cholesterol value of birds on control diet.Birds fed 0, 2 and 4% biochar kg-1 had comparable cholesterol values.Birds fed 4 and 6% biochar kg-1 had similar LDL values, and these were significantly lower than the LDL of birds fed 0 and 2% biochar kg-1.For TAG values, birds fed 0 and 6% biochar kg-1 had similar values, and these were significantly higher than the TAG values of birds fed 2 and 4% biochar kg-1.The result of the present study on serum lipid profile is in tandem with the findings of Boonanuntanasarn et al. (2014) whose study showed that dietary supplementation of 20g/kg activated charcoal had a significant effect on blood cholesterol levels in 4-weeks old Nile Tilapia.The study of Yoo et al. (2005) also revealed that dietary inclusion of a mixture of charcoal and wood vinegar in the diet of flounders not only improved fatty acid composition, but also decreased saturated fatty acid levels.Earlier report (Neuvonen et al., 1989) has shown that intake of biochar (activated charcoal) has potential to interfere with the enterohepatic circulation of bile acids and cholesterol, thereby lowering serum cholesterol levels in hypercholesterolemic conditions.It was observed that there was a significant (P < 0.01) decrease in cholesterol levels of birds fed the highest inclusion levels of biochar (6% kg-1) compared to birds on other treatments at both the starter and finisher phases of the experiment.This finding is similar to the result of Boonanuntanasarn et al. (2014) who observed that significant differences existed in cholesterol values of Nile Tilapia fed dietary activated charcoal, which appeared to decrease as the level of activated charcoal increased in the diets.The result of the study however disagrees with the reports of Majewska et al. (2009) whose study showed that no significant differences existed in triglycerides and total cholesterol levels and other biochemical indices of 20-week old turkeys fed diets containing charcoal, silica grit and hardwood ash.Edrington et al. (1997) who fed broilers diets containing super activated carbon, and Majewska et al. (2002) who used charcoal feed additives also reported nonsignificant differences in the biochemical indices of the birds and turkeys.
It is also evident from the result of the study that dietary biochar significantly (P < 0.05) lowered the low density lipoprotein (LDL) levels of birds fed 6% biochar kg-1 at finisher Haematology and Serum Lipid Profile of Broiler Birds Fed Diets Containing Biochar phase.There was also a significant (P < 0.05) reduction in triglyceride levels of broilers fed 2 and 4% dietary biochar kg-1 at the finisher phase.Chu et al. (2013) reported that dietary supplementation of 0.6% bamboo charcoal decreased the concentration of LDH, triglyceride and bilirubin levels in fattening pigs.Shabani et al. (2010) opined that when plasma cholesterol and triglyceride levels are reduced, there is an accompanying reduction in lipogenesis and damaged lipid transport, and prevention of hepatic cholesterol biosynthesis in broiler chickens.

CONCLUSION
The result of the present study showed that up to 6% dietary inclusion of biochar kg-1 led to improvement in growth performance, haematology and serum lipid profiles of broiler birds.However, at 2% level of inclusion kg-1, dietary biochar may not have a lowering effect on cholesterol and triglyceride concentrations of broiler birds.

Table 3 :
Performance characteristics of broilers fed varying dietary levels of biochar

Table 4 :
Haematological and serum lipid profile of broilers fed varying dietary levels of biochar a, b, c, means on the same row with different superscripts are significantly different (P > 0.05).RBC: red blood cell; WBC: white blood cell; Conc.: Concentration; HDL: high density lipoprotein; LDL: low density lipoprotein.