SOYBEAN MEAL REPLACEMENT WITH CASSAVA LEAF:BLOOD MEAL MIX WITH OR WITHOUT ENZYME IN BROILER DIETS

Two hundred and sixteen unsexed day-old Cobb broilers of a commercial strain were used to study the effect of replacement of soybean meal (SBM) by cassava leaf:blood meal mix (CLM:BM) with or without enzyme supplementation on broiler growth and performance. The birds were divided into 6 groups of 36 birds each after balancing for live weight. Each group was further sub-divided into 6 replicates of 6 birds each. CLM:BM replaced SBM at 0, 25 and 50 % on weight for weight basis. The resulting three dietary combinations were supplemented with a commercial enzyme (Maxigrain®) at the rates of 0 and 100 ppm thus resulting in 6 dietary treatments. The six groups of 36 birds were randomly allotted to the six experimental diets. Feed and water were provided free choice during the 56 days experimental period during which data were collected on performance, nutrient retention, feed transit time and carcass characteristics of birds. Replacement of SBM with CLM:BM mix had no effect (p>0.05) on weight gain (WG), feed intake (FI), feed: gain ratio (F:G), crude protein (CP), ether extract (EE) and crude fibre retention (CF), dressing percentage, breast cut, drumstick, thigh liver and kidney weights but significantly (p 0.05) nutrient retention, dressing and abdominal fat percentage but had no effect on FI. No mortality was recorded in the course of this study. Enzyme supplementation significantly (p<0.05) reduced gizzard and caecum weights. It was concluded that in broiler feeding CLM:BM mix can effectively replace 50 % SBM without adverse effect on the birds. Supplemen-tation of diets with enzyme led to further impro-vement in performance of broilers.


SUMMARY
Two hundred and sixteen unsexed day-old Cobb broilers of a commercial strain were used to study the effect of replacement of soybean meal (SBM) by cassava leaf:blood meal mix (CLM:BM) with or without enzyme supplementation on broiler growth and performance. The birds were divided into 6 groups of 36 birds each after balancing for live weight. Each group was further sub-divided into 6 replicates of 6 birds each. CLM:BM replaced SBM at 0, 25 and 50 % on weight for weight basis. The resulting three dietary combinations were supplemented with a commercial enzyme (Maxigrain ® ) at the rates of 0 and 100 ppm thus resulting in 6 dietary treatments. The six groups of 36 birds were randomly allotted to the six experimental diets. Feed and water were provided free choice during the 56 days experimental period during which data were collected on performance, nutrient retention, feed transit time and carcass characteristics of birds. Replacement of SBM with CLM:BM mix had no effect (p>0.05) on weight gain (WG), feed intake (FI), feed: gain ratio (F:G), crude protein (CP), ether extract (EE) and crude fibre retention (CF), dressing percentage, breast cut, drumstick, thigh liver and kidney weights but significantly (p<0.05) reduced intestinal transit time (ITT) and abdominal fat while increasing gizzard and caecum weight. Enzyme supplementation however significantly (p<0.05) improve WG and reduced F:G, improved (p>0.05) nutrient retention, dressing and abdominal fat percentage but had no effect on FI. No mortality was recorded in the course of this study. Enzyme supplementation significantly (p<0.05) reduced gizzard and caecum weights. It was concluded that in broiler feeding CLM:BM mix can effectively replace 50 % SBM without adverse effect on the birds. Supplementation of diets with enzyme led to further improvement in performance of broilers.

INTRODUCTION
The potentials of poultry and poultry products as a panacea to insufficient animal protein intake of Nigerians has continued to be a mirage principally because of astronomical increase in feed cost. The main reason for this being the competition among man, industry and livestock for grains and grain legumes.
This perennial problem has necessitated the search for alternatives to the expensive grains and protein concentrates (Adeyemi, 2005). Agunbiade (2009) surmised that the replacement of expensive conventional feed ingredients with cheap and available substitutes represents a suitable strategy for reducing feed cost and encouraging production. Among possible sources of cheap protein are leaf meal from some plants and processed animal by-products such as blood meal, epithelium scrappings, etc.
Cassava is traditionally grown for the production of roots. It yields about 10 -30 t ha -1 of leaves that is usually wasted or used as manure (Bokanga, 1994). Cassava leaves are rich in protein but they are low in sulfur amino acids (Phuc et al., 2000 andAyasan, 2010). In an earlier study (Adeyemi et al., 2012, we observed that the combination of blood meal and cassava leaf meal gave a good protein concentrate with a good balance of amino acids which can be useful in monogastric animal feeding. The high fibre level of cassava leaf meal portends a limitation to its utilisation in poultry feed, however, enzyme supplementation may provide a solution to this limitation.
This study is designed to determine the performance of broiler chicks fed diets containing CLM:BM mix as partial replacement for soybean meal with or without enzyme supplementation.

STUDY AREA/SITE PREPARATION
The experiment was carried out in the Poultry Unit of the College of Agricultural Sciences Olabisi Onabanjo University, Yewa Campus, Ayetoro, Ogun State. A week before the birds were moved into the experimental pen, they were thoroughly cleaned, washed and disinfected with a strong solution of IZAL ® .

TEST MATERIALS AND EXPERIMENTAL DIETS
Cassava leaves (variety TMS30572) without petioles were harvested, wilted overnight and air-dried under shade for five days, milled using hammer mill and bagged as cassava leaf meal (CLM). Blood was collected from the Central Abattoir in Ayetoro immediately after cattle were slaughtered. After collection, the blood devoid of extraneous materials was boiled immediately in a cask for 60 minutes in order to let water evaporate and destroy any parasites. After boiling it was then sundried on a concrete platform with screened mesh for 5 days, and then ground into flour (Blood meal, BM) using an attrition mill.
Samples of the prepared CLM and BM were analysed for proximate composition using the standard methods of AOAC (1995). CLM and BM were mixed together in such a manner that the protein value of the mix equaled that of soybean meal (44 %) using the Pearson square method. The resulting mix is in the ratio 1.5 parts CLM: 1 part BM and is termed cassava leaf: blood meal mix (CLM:BM).
Six experimental diets were formulated; three of the diets were formulated with enzymes supplementation while the remaining three are without enzyme Maxigrain ® supplementation (table I). The test ingredient (CLM:BM) replaced 0, 25 and 50 % of dietary soybean meal on a weight for weight basis.
Two hundred and sixteen unsexed dayold Cobb broilers of a commercial strain (initial weight 50-52 g), were selected from a larger pool purchased from a reputable hatchery. The birds were divided into 6 groups of 36 birds each after balancing for live weight. Each group was further subdivided into 6 replicates of 6 birds each and randomly allotted to the six experimental diets. The birds were floor brooded on wood shaving in an open sided poultry with facilities for tarpaulin curtains.
Feed and water were provided free choice during the 56 days experimental period. The birds in each replicate were weighed at the commencement of the experiment and weekly thereafter. Feed consumption records were kept on weekly basis. From these measurements the feed: gain ratios were determined using the equation: Feed:Gain ratio = Average feed intake (g/ bird)/Average daily weight gain (g/bird)

NUTRIENT RETENTION TRIAL
A nutrient retention trial was carried out mid way into the experiment. Three birds whose weights were close to the mean replicate weights were selected and transferred into metabolic cages. Weighed quantity of feed and unlimited supply of water was offered to the birds in the cage. The birds were acclimatized to the new environment for three days followed by a four day total excreta collection period. Feed transit time through the gastrointestinal tract was determined by including 0.3 % Ferric oxide as a marker at the age of 35 days. Transit time was determined as the period between the feeding of the marked diets and the appearance of the marker in the excreta (Cullison, 1982). Excreta samples were dried in a Gallenkhamp ® drying cabinet at 70 °C and ground prior to chemical analysis.

CARCASS QUALITY EVALUATION
At the 56 th day of age, 6 birds (1 from each replicate) were randomly selected from each treatment, fasted overnight, weighed the following morning and slaughtered. Dressed carcass and relative organ weights (expressed as percentage of live weights) were recorded.

CHEMICAL AND STATISTICAL ANALYSIS
Cassava leaves, blood meal, test ingredients, experimental diets as well as excreta samples were analysed for proximate fractions according to standard methods of AOAC (1995). Data collected during the experiment were subject to statistical analysis using a 2 (enzyme supplementation) by 3 (level of test ingredients) factorial experimental design. Significant differences were determined using the Duncan's multiple range test (Duncan, 1955).

RESULTS AND DISCUSSION
The main effects of CLM:BM mix on performance indices of broiler chickens is shown in table II. Replacement of soybean meal with CLM:BM mix at 0, 25 and 50 % had no effect on weight gain, feed intake, feed: gain ratio. Enzyme supplementation however significantly (p<0.05) improve weight gain and reduced the quantity of feed required for a unit weight gain although it has no effect on feed intake. Table III shows the interaction effect of CLM:BM mix level and enzyme supplementation. Enzyme supplementation improved weight gain on 0, 25 and 50 % replacement level of soybean meal by CLM:BM mix. The supplementation of the diets with enzyme led to an improvement in weight gain to the magnitude of 10.89, 10.43 and 21.44 % respectively over the non supplemented diets. These improvement in weight gain with enzyme supplementation in the presence of a relatively same feed intake across dietary treatments resulted in significant (p<0.05) reduction in the quantity of feed required to gain a unit weight by broilers. The feed: gain values were similar for enzyme supplemented diets and significantly lower than values obtained on the non enzyme supplemented diets. No mortality was recorded in the course of this study. The use of enzymes in poultry feeds has predominantly been related to the hydro- lysis of fiber or non-starch polysaccharide (NSP) fractions in cereal grains. These NSPs cannot be digested by the endogenous enzymes of poultry and can have antinutritive effects. The better growth rates as a result of enzyme supplementation found in this study is in consonance with previous findings (Biswas et al., 1999;Swain and Johri, 1999;Midau et al., 2011). These authors concluded that improved feed utilization due to enzyme supplementation was responsible for the increased live weight gain in broilers on similar levels of dietary nutrient concentration. The observation was however contrary to the report of Omojola and Adesehinwa (2007) that the inclusion of the exogenous enzymes did not significantly (p>0.05) improve body weight of broiler chicks. Divergent reports exist in literature on the relationship between endogenous enzyme supplementation and feed intake. Adrizal and Ohtani, 2002;Rahman et al., 2005 reported that enzymes have no effect on feed intake while Kadam et al., 1991 andSamarasinghe et al., 2000 reported reduction in feed intake as a result of enzyme supplementation. Lesson et al. (1996); Augelovicova and Michalik (1997) on the other hand reported increases in feed intake as a result of enzyme supplementation due to increased nutrient digestibility. The improved feed conversion due to enzyme supplementation in the present study is in agreement with earlier findings (Wang et al., 1997;Huazhong et al., 1999;Jackson et al., 2004;Rahman et al., 2005;Zou et al., 2006;Onu et al., 2011). These researchers reported that exogenous enzymes greatly improved the feed conversion ratio of broiler chicks fed enzyme supplemented diet.  Increasing concentration of CLM:BM mix in the diet had no effect on crude protein, ether extract and crude fibre retention (table  IV), while enzyme supplementation resulted in improvement (p<0.05) in retention of these nutrients. Intestinal transit time reduced with increasing concentration of CLM:BM mix in the diet. Enzyme supplementation reduced the feed intestinal transit time by about quarter of an hour. The interaction effect of level of CLM:BM mix and enzyme supplementation on nutrient retention in table V indicate that crude protein, ether extract and crude fibre retention decreased with increasing concentration of CLM: BM mix in the diet. Significant (p<0.05) interaction between CLM:BM mix and enzyme supplementation was observed for crude protein, ether extract and crude fibre retention. Exogenous enzymes increased the digestibility of fibrous feed ingredients through the disruption of plant cell walls thereby enhancing nutrients absorption (Acamovic, 2001). The use of enzymes in poultry feeds has predominantly been related to the hydrolysis of fiber or nonstarch polysaccharide (NSP) fractions in cereal grains. NSPs bind to large amounts of water and as a result, the viscosity of fluids in the digestive tract is increased. Danicke et al. (2000) explained that increased viscosity causes problems in the small intestine as it reduces the substrate-enzyme interaction, which reduces nutrient availability. According to Vukic-Vranjes and Wenk (1996) and Zanella et al. (1999), the supplementation of broiler diet with exogenous enzyme improved starch digestibility and consequently dry matter, crude protein and energy digestibilities. Ritz et al. (1995), opined that enzyme supplementation increased the length of villi within the jejunal and ileal sections of 3 weeks old turkey pullets fed corn soybean meal diets. Caspary (1992) explained that the increase in surface area suggested by the increased villus length might enhance nutrients absorption and improve nutrient digestibility.
The reduction in intestinal transit time with enzyme supplementation is in agreement with the report of Danicke et al., (1999) that a decrease in transit time with enzyme supplementation reduces the growth of microflora in the intestine. According to Lazaro et al. (2003), enzyme supplementation reduced the viscosity of the jejunum content and accelerates the rate of passage of digesta through gastrointestinal tracts of birds thus reducing the digestive transit time of broilers.
Results of the main effects of replacement level and enzyme supplementation on prime cuts and abdominal fat of broilers are presented in table VI. Abdominal fat content was significantly reduced as level of inclusion of CLM:BM mix increased in the diet. Enzyme supplementation of diets Table V. Interaction effects of Cassava leaf: blood meal mix and enzyme supplementation on nutrient retention of broilers. (Efectos de la interacción de la hoja de yuca: la mezcla de sangre comida y suplementos de enzimas en la retención de nutrientes de los pollos de engorde). resulted in increases in dressing percentage and abdominal fat content of chickens.The indices for which significant replacement level by enzyme supplementation interaction occurred are shown in table VII.

Replacement
Increasing the replacement level of CLM: BM for soybean had no effect (p>0.05) on dressing percentage, breast cut weight, drumstick and thigh weights. Abdominal fat was however significantly (p<0.05) reduced with increasing concentration of CLM:BM. Enzyme supplementation also had no effect on breast cut weight, drumstick and thigh weights but significantly (p<0.05) influenced dressing and abdominal fat percentage. The significant increase in dressing percentage observed with enzyme supplementation in this study agreed with the report of Lesson et al. (1996). They reported increased dressing yield for the addition of enzyme due to higher fat deposition in the carcass. Similar report was given by Abbas et al. (1998) that enzyme supplementation to fibrous diet improved the growth rate, thereby  increasing the dressing percentage. Bharathidhasan et al. (2009) also reported a marginal increase in dressing percentage and carcass yield in birds fed enzyme supplemented reduced energy and protein diets. However, Café et al. (2002) reported that addition of a commercial multi enzyme to corn-soybean meal-based diets did not improve dressing percentage, yield of breast, thigh and wing component, but resulted in a significant increase in abdominal fat. A similar result of no influence on dressing percentage was presented by Rahmatnejad et al. (2011) reported the supplementation of dried tomato pomace with Rovabio Excel™ significantly increased carcass weight and percentage of abdominal fat but did not affect carcass yield and breast, thigh, and visceral organs percentage. On the other hand, Yamazaki et al. (2007) reported that cellulase supplementation of a low-CP diet slightly lowered abdominal fat deposition. Table VIII indicated that increasing concentration of CLM:BM had no effect (p>0.05) on weight of liver and kidney but significantly (p<0.05) increase gizzard and caecum weight. Birds on 0 % replacement of CLM:BM for soybean meal had the least weight for gizzard and caecum. Enzyme supplementation significantly reduced gizzard and caecum weights but had no effect on liver and kidney weights. Significant interaction effects of dietary level of CLM:BM by enzyme supplementation was observed on gizzard and caecum weight. The pattern of the interaction is shown in table IX.
The gizzard breaks down ingested feed by muscular action. Higher dietary fibre would promote higher thickening of the  muscles (Onibi et al., 1999). Higher gizzard size in broiler diets may be related to higher dietary fiber content (Adeyemi, 2005). Viveros et al. (1993) reported that addition of enzyme to barley-based diets also produced an effect on digestive tract of the bird, reducing the relative weight of upper tract (mainly proventriculus and gizzard), and the size of the small intestine and colon of chicks. The findings of this study that enzymes supplementation reduced gizzard weight is however contrary to the findings of Omojola and Adesehinwa (2007) reported that the inclusion of the exogenous enzyme to broiler diets did not affect the relative weights of kidney, gizzard, heart and liver.
In an earlier study Baiao et al. (1997) reported that enzyme supplementation to rye diets reduced viscosity of intestinal content and the weight of digestive organs. Augustine et al. (2011) also reported a similar finding that internal organs decreased with increasing level of enzyme supplemented CPM.
Since no significant differences were recorded in the performance of broilers on CLM:BM mix compared to SBM based control diets up to 50 % of this mix can be used to replace SBM without any detrimental effects. However enzyme supplementation did bring about significant improvement in the indices of performance.