Introduction

A minimum of 30g of animal and 40g of plant protein is recommended (FAO, 1994) and up till now, Nigeria (and most other developing countries) still battle with the problems of inadequate protein intake from animal sources. Campaign has been intensified in recent times, in countries beset with the problem of deficiency in dietary animal protein intake by encouraging micro–livestock production such as rearing of snails, porcupine, and cane rats and so on, with a view to further bridge the gap of protein deficiency. It is obvious that the production of these micro sources of animal protein cannot achieve much in solving the deficiency problem as a result of their size, slow growth rate and reproducibility. The turnover of protein derivable from micro livestock production is small compared with poultry, pig and ruminants. To meet this target, animals with short generation interval, high fecundity and prolificacy are the likely channels for providing the expected animal protein. Such animals include poultry, fish and swine and on the long run cattle, sheep and goats. Among the livestock enumerated, the major antidote to this protein deficiency is the development of poultry, which include table egg production. Their ability to utilize some non-conventional feedstuff and agro-industrial by-products of no dietary value to man. Cocoa bean shell (CBS) is a waste product from the cocoa industries. It is the thin husk surrounding the cocoa bean. These CBS is high in nutritive potential but it is of limited use in animal feeds because of its theobromine content (Olumide et al., 2015). Theobromine belongs to the same naturally occurring methylated xanthine group as caffeine (Olubamiwa and Hamzat, 2005). In Nigeria, large quantities of cocoa by-products (including discarded cocoa bean shell and husk) are produced annually by farmers and associated processing Industries. Cocoa bean shell (CBS), cocoa pod husk (CPH) and cocoa bean cake (CBC) could prove as animal feed ingredients. The purpose of investigating blood composition is to have a way of distinguishing normal states from states of stress. The stress factors could be nutritional, environmental or physical. Serum or plasma chemical values are useful in the assessment of the nutritional and health status of animals. Dietary components have measurable effects on blood components and such blood constituents are widely used in nutritional evaluation and survey of animals (Church et al., 1984; Veulterinora, 1991; Olorode et al., 1996). Blood variables most consistently affected by dietary influences include red blood cell counts, packed cell volume, plasma protein and glucose (Aletor and Egberongbe, 1992). Serum level detection tells the existence of liver cells damage (Aniket, 2005), for example if liver fails to deaaminate amino acids, nitrogenous wastes may accumulate in the body causing harm. Babatunde and Pond, (1987) have established that packed cell volume and hemoglobin were directly related to the nutritional balance of the diet fed to the animal and attributed decrease in total protein to inhibition of protein utilization. Hematological parameters usually studied include erythrocytes (RBC), packed cell volume (PCV, hematocrit), hemoglobin (Hb), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular, Hemoglobin Concentration (MCHC), leucocytes total count (WBC) thrombocytes (platelets) etc. (Jaini 1986). These parameters measure the condition of health of the animal under study. Serum biochemical metabolites include electrolytes, blood proteins, plasma lipids and blood glucose. Based on the previous work of Olumide et al., 2015, which concluded that broilers can effectively consume up to 10% inclusion of RCBS in their diets without compromising health status. This study investigated the effect of treated cocoa bean shell based-diets on serum biochemistry and hematological parameters of laying birds.

Materials and Methods

Experimental Site

This experiment was carried out at Kolmart Farm, Poultry Section, Erunmu, Ibadan, Oyo – State, Nigeria. The birds were housed in an open-sided building in a thoroughly cleaned, washed and disinfected two tier cage system of 32 x 38 x 42 cm dimension. The birds were caged individually.

Experimental Birds and Layout of the Experiment

A total of 140 laying hens at six week in lay were used in this experiment with 20 birds per treatment, replicated twice. The experiment lasted for eight weeks. The cocoa bean shell was sourced from cocoa industry in Lagos, while other feed ingredients were purchased from a commercial feed miller in Ibadan. Seven diets were formulated as shown in Table 1, Diet A (Control diet) contained no test ingredient (CBS). Diets B and C contained raw cocoa bean shell (RCBS) with a 5% and 10% maize replacement respectively. Diet D and E contained 5 and 10% CBS with the inclusion of Rovabio enzyme (ECBS) while diet F and G also contain 5 and 10% fermented cocoa bean shell (FCBS).

Table 1- Gross Composition (g/100gDM) of Cocoa Bean Shell-Based Layer Diets

Calculated Composition

*RCBS-Raw Cocoa Bean Shell, *ECBS-Enzyme-treated Cocoa Bean Shell, *FCBS-Fermented Cocoa Bean Shell **Composition Vitamin/Mineral Mix 1 kg (layers): Vitamin A 1000000IU, Biotin 40g, Vitamin B12 10mg.Folic acid 500mg, Manganese 4800MG, Zinc 58mg,Iron 5800mg, Selenium 120mg, Iodine 60mg, Cobalt 300mg. Composition of methionine 20,000mg, Butylated hydroxytolerance BHT 50,000mg.

Management of Experimental Birds

The experimental birds were raised until point of lay on commercial diet and to six weeks in-lay. The birds were given adequate medication and vaccination before the commencement of the experiment. The birds were weighed individually at the beginning of the experiment before they were placed on experimental diets. Feed and water were supplied adequately and other daily routine managements were provided. The performance records of daily feed intake, egg production and feed conversion ratio were monitored.

Hematology and Serum Biochemistry

At the end week 8, 2 birds per replicate and a total of 4 birds per treatment were randomly selected and bled by the jugular vein using needle and syringe. The blood was carefully labeled for hematological and serum biochemistry analysis. The blood samples for hematological parameters were collected into bottles pretreated with EDTA, an anti-coagulant. Blood samples for biochemical indices were collected into another sample bottle containing no anti-coagulant. The samples for serum biochemistry were kept in the refrigerator. The samples were spurned in the centrifuge at 3,000 rpm and the clearer portion decanted into small sample tubes stored in a freezer to assay for serum biochemical indices. The hematological indices monitored include Red Blood Cell Count (RBC), White Blood Cell Count (WBC), Packed Cell Volume (PCV) and Hemoglobin Concentration (Hb). Serum biochemical indices investigated were total protein, albumin, creatinine, globulin, albumin, globulin, cholesterol and glucose.

Analysis

Proximate analysis of the feed was carried out (AOAC, (1990). The anti – nutritional factor, theobromine, in the diet was also determined (AOAC, (1990).

Statistical Analysis

Data collected were analyzed using analysis of variance (ANOVA) and significant means where significant separated using Duncan Multiple Range Test (Gomez and Gomez, 1985). Statistical Analysis Software (SAS, 1999) computer package was used.

Results

The gross composition of the layer diets is presented in Table 1. Metabolisable energy decreased from 2609.96 kcal ME/kg in diet A (control diet) to 2566.39 kcal ME/kg in diet G (10% FCBS). Table 2 shows the determined analysis of experimental diets. Crude protein ranged between 17.00–17.40% (diet E). Crude fiber increased from 4.10% (diet A) to 4.8% (diet G). Ether extract values decreased from 3.49% (diet A) to 3.39% (diet G). The values obtained for ash ranged from 5.16 (diet G) – 10.00 (diet C).

Table 2 – Proximate Composition of Cocoa Bean Shell-Based Diet

No of observation (2)

Performance Characteristics

Table 3 shows the result of performance characteristics. The feed intake ranges from 89.40 for treatment G and 98.00 for treatment D. Treatment D had the highest percentage hen day production of 86.68, while the lowest 78.17 was obtained from treatment G. Although, differences in HDP of birds on diet D and E (ECBS) were not significant compared with control, the birds on diet D had the highest hen day production followed by those on diets A and E. The egg weight of birds on the RCBS was lower than all other treatments; this could be attributed to the presence of theobromine. Egg weight of birds on diet with enzyme CBS diets D and E (5 and 10%) has higher egg weight compared with the control diet. The FCR obtained in this study ranged from 2.43–2.65. The absence of mortality throughout the period of this experiment further attested to the suitability of CBS as a substitute for maize in layers diet. This also indicates that layer can tolerate CBS based diet than broilers.

Table 3 – Performance Characteristics of Layers Fed Variously Treated CBS Based Diet

a, b, c: Means along the same row with any identical superscripts are not significant (P > 0.05). No of Observation (2)

Serum Biochemistry and Hematological Characteristics of Layers Fed CBS -Based Diets

The results of the serum biochemistry and hematological parameters of birds fed CBS based diets are presented in Table 4. Variations observed in the total protein were significant, although as the level of CBS in the diet increased the total protein level in the blood also decreased. However, variations observed for serum biochemical indices like, albumen globulin and creatinine were not significantly different (P>0.05) among the dietary treatments. There were also significant differences in values obtained for Albumin: Globulin ratio, glucose and cholesterol value. The highest serum albumin value of 3.15 g/dl was obtained from birds on diet G while the least was obtained from diet E (2.16 g/dl). The globulin varied from 1.71–2.76 g/dl. Diet G has the highest value of albumin-globulin of 2.67 and diet A had the least value of 0.88. Variation observed in the glucose levels were significant (P < 0.05). Birds on diet C (10%RCBS) had the highest glucose concentration (mg/dl) of 136.45 while birds on diet G had the least 109.10. Although, the glucose concentration increased as the level of substitution increases for all the birds on the diets. The serum creatinine levels (mg/dl) were not significantly different, the value ranged from 1.19–1.40. Although as the level of CBS increases the creatinine level in the serum increases. Total cholesterol levels significantly decreased (P<0.05) as the level of substitution of CBS for maize increased. The results of the hematology of layers fed variously treated CBS based diet indicted that all hematological parameters (Red Blood Count, White Blood Count, Packed Cell Volume and Hemoglobin) monitored were similar across the dietary treatments. The red blood cells (mm³ x 10⁶) ranged between 2.45–2.93, while the white blood cells (mm³ x 10³) varied from 19.94–20.72 without following a particular trend among dietary treatments. The lowest packed cell volume of 21.25% was obtained from birds fed the control diet A and the highest value of 26.00% observed in birds fed diet B. The other PCV values were within these two values. The hemoglobin concentration (g/100ml) was highest (8.66) in birds fed diet B (5% RCBS) and lowest (7.34) in birds fed diet F (5% FCBS).

Table 4 – Serum Biochemistry and Hematological Characteristics of Layers Fed CBS–Based Diets

a,, b, c, d, e: Means along the same row with any identical superscripts are not significant (P>0.05).

Discussion

Proximate Composition of Experimental Diets

Birds on the control diet recorded the highest calculated metabolisable energy, ME (kcal/kg) and this decreased with increased contents of CBS in the diets. The analyzed proximate composition (g/100DM) of the experimental diets revealed that the percentage crude protein increases as the level of inclusion of various CBS increases in the diet. The crude fiber level in the diet also increases with increase in the level of CBS in the diet while the ether extract values reduce accordingly. No particular trend was observed in the ash value obtained. Nitrogen free extract did not follow any particular trend but the values obtained were close for all the dietary treatments. The highest values for crude protein was obtained for diet E (17.40%) while the least value was obtained for the control diet (17.00). The control diet (A) had the lowest crude fiber value (4.10%) while the highest value was obtained with diet C (4.82%). The highest ether extract value of 3.49% was obtained in the control diet. The metabolisable energy (kcal/kg) values of all the diets met the levels recommended by Olomu, (1979) and Fetuga (1984). Although the dietary crude protein levels in all the diets met the values recommended by National Agriculture Extension and Research Liaison Services NAERLS, (1990) and Olomu, (1995). The crude fibre falls within 3–5% recommended by NRC (1994) for layers. Bentil et al. (2015) also suggested that the nutritive value of cocoa bean shells for use as feed for animals through solid state fermentation.

Performance Characteristics

Variations observed in the feed intake (FI), hen day egg production (HDEP) and Egg Weight were significant except feed conversion ratio. The feed intake of the birds on the control diet was higher and similar to those on diet D and E (5 and 10%ECBS). This is in line with the findings of Iyayi and Okhankhuele, (2002) who reported that birds on enzyme supplemented diets performed significantly better than those on other diets, in cassava leaf meal based diets supplemented with enzyme. However, the average daily feed intakes of the birds on the RCBS were lower than those on the control diets A, D and E (ECBS). This could be as a result of the anti-nutritional factor theobromine in the diet. As the level of CBS in all the diet increased the feed intake declined. Several reports (Yeong et al., 1989; Odunsi et al., 1999; Olubamiwa et al., 2002) depicted reduced feed intake by laying birds fed CBS and cocoa bean cake based-diets due to the theobromine content of the diet. Reduced feed intake is believed to be due to destruction of the intestinal lining and severe indigestion in the birds (Yeong et al., 1989; Olubamiwa et al., 2000). Although, differences in HDP of birds on diet D and E (ECBS) were not significant compared with control, the birds on diet D had the highest hen day production followed by those on diets A and E. This apparent increase in egg production could be due to the enzyme added to diets D and E. Also, Olukosi and Adeola, (2007) reported that supplementation of wheat based diet with combination of xylanase and phytate improved growth performance of broiler chickens. However, the HDP and egg weight obtained in this study were higher than those reported elsewhere (Keshavarz and Nakajima, (1995). This could be due to the fact that the protein requirement of these birds was met at lower level of 10% CBS as noted (Junqueira et al., 2006. The slight decrease in metabolisable energy/ (kcal/kg) and ether extract, and increase in crude fibre with increased levels of substitution could be responsible for slight numerical decreases in HDP obtained with increased levels of substitution of CBS for maize. This is in line with the report (Reid et al., 1984) which observed an increase in egg production of single comb white leghorns pullets fed 16% dietary protein. As metabolisable energy increased from 2.42 to 2.64 and from 2.68 to 3.08 kcal ME/g diet there was a 2.25% increase in egg production for every 0.22 kcal ME/g increase in the diet. Egg production was however not affected by dietary energy level (Harms et al., 2000). The values of ADFI (87.00–98.80g/bird/day) and HDEP (78.17–86.68%) obtained in this study were higher than the corresponding values with spent sorghum grain and whole cassava meal (Aderemi et al., 2006). The HDEP of 77– 78% obtained at the peak production (9–10 weeks) of Nera (Majaro, 1999) were lesser than those obtained in this study. However, while higher ADFI was obtained for birds fed kola pod-based meal by Olubamiwa et al., (2000), the percentage HDEP (64.5–71.3) recorded by these authors were far below the values obtained in the present study. The egg weight of birds on the RCBS was lower than all other treatments; this could be attributed to the presence of theobromine. Previous studies have shown that theobromine reduces feed intake which leads to lower laying rate and egg size (Hatagalung and Chang, 1978, Odunsi et al., 1999, Olubamiwa et al., 2002). Egg weight of birds on diet with enzyme CBS diets D and E (5 and 10%) has higher egg weight compared with the control diet. This shows that addition of enzyme Rovabio effectively reduce the anti-nutritional factor in cocoa bean shell. According to Viveros et al., (2002) effect of anti-nutrients is markedly reduced when diets are supplemented with enzymes by interfering with the antinutrients-protein complexes and thereby releasing these proteins for digestion. This improved performance of birds on enzyme-supplemented diets is brought about by improved absorption of fats and fatty acids as well as fat-soluble micronutrients contained in the diet (Danicke et al., 1999).

The FCR obtained in this study 2.43–2.65 was lower when compared with that recorded by Kwari et al (1999) 4.14–4.92 but fall with the same range with that of Fasuyi et al (2005) 2.38–2.76. The absence of mortality throughout the period of this experiment further attested to the suitability of CBS as a substitute for maize in layers diet. This also indicates that layer can tolerate CBS based diet than broilers. When all the performance of the laying birds fed 5% of the various forms of CBS based diet was compared, there was no significant difference in value of ADFI, HDP, egg with and FCR but when compared at 10%, significant difference (P > 0.05) was observed in the ADFI, HDP and FCR.

Serum Biochemistry and Hematological Characteristics of Layers Fed CBS -based Diets

The values obtained for the total protein were significantly different from each other. As the level of CBS in the diet increases, the serum protein level reduces for the RCBS and ECBS except for that of FCBS that increases. This significant variation observed in the values of total protein, albumin: globulin was an indication of bad quality protein but contrarily the values obtained for the albumin and Globulin shows that there are no significant variations observed, this was an indication of the good quality protein and other nutrients fed to these birds as well as adequate metabolism of these nutrients to meet the requirement of these birds. Olorode et al., 1996 confirmed that the significant (P < 0.05) reduction in total protein of birds fed 10 and 15% shea butter cake was an indication of inferior protein quality and/or nutrition. The values of total protein obtained for all the diets were lesser than that recommended by Mitruka and Rawnsley (1977) except for the control. This could be as a result of the anti-nutritional factor theobromine in the diets. While the values obtained for the albumin, glucose, creatinine fell within the ranges 2.10–3.45 g/dl, 152–182 mg/dl and 0.90 – 1.85 mg/dl respectively for normal chickens by Mitruka and Rawsley (1977). It was observed that as the level of inclusion of variously treated CBS in the diets increases the total albumin level reduces except for the FCBS which increases as the level of replacement increases for maize. This could be as a result of the theobromine content. The significant increases obtained in the serum glucose level with increasing levels of variously treated CBS in the diets is desirable especially when the highest content of 136.45mg/dl obtained from treatment C was far below 152mg/dl recommended by Mitruka and Rawsley (1977) for normal chickens. The author has recommended a range of 152–182 mg/dl for normal chicken. The differences in the result of this study compared to the recommendation of these authors could have been caused by differences in factors like diets among others. Although, no significant difference was observed in the values of creatinine obtained in this study. It was observed that as the level of variously treated CBS in the diets increased the level of creatinine also increased although the highest value was obtained from the serum of the birds fed RCBS based diet (1.40 mg/dl). This may point to slight tissue damage in birds fed 10% RCBS based diets. The highest serum cholesterol was obtained from the control and the values significantly (P<0.05) decreased with increasing levels of substitution of RCBS and ECBS for maize in the diets, except for the FCBS that was slightly reduced. The consistent reduction in the serum cholesterol obtained with the increasing substitution of variously treated CBS for maize in the diets could be explained by the increasing contents of anti-nutritional factor and fibers that was associated with the increased levels of substitution. Anti-nutritional factor like saponins have been found to assay a physiological effect on lowering the level of plasma cholesterol concentration in experimental animals (Potter et al., 1979; Oakenful, 1981). Results of past research works have suggested that lowering effect of fibre on liver lipid in poultry (Patel et al., 1979; Longe, 1984; Scheeman, 1990) Dietary fiber has the ability to bind nuclear components like bile acids and phospholipids, thus slowing the absorption of lipid from the small intestine and so lowering the plasma cholesterol and liver fat accumulation (Schneeman, 1990). Also, in the light of this, Donnica (2000) and Lazaro et al (2003) recommended the consumption of soluble fibers’ to lower blood cholesterol. The globulin values obtained in this study, although significantly different from each other, but they were higher than the recommended values e.g. 0.45–0.96 g/dl by Mitruka and Rawsley, (1977). Although no particular trend was followed with the increasing level of substitution of RCBS, ECBS and FCBS. Albumin: Globulin obtained was lesser than the values recommended by the previous authors for albumin globulin ratio (2.10–3.45) except for those obtained by diet B and G. The non-significant differences in all the hematological parameters investigated in the birds fed the control and variously treated CBS–based diets could explain absence of deleterious effect that could be linked with the diets fed to these birds. No particular trend was observed in the hematological parameters with the increase in CBS based diets. All the parameters falls with those recommended by Mitruka and Rawsley, (1977) 1.58-3.82 10^c1mm³, 7.4–12.2 g/dl, 9.2–28.610⁶mm³ for RBC ,Hb and WBC while some of the PCV values falls below the recommend value of the above authors (24.9–40.7) for normal chickens. Oladele et al., (2001) attributed the lower values of packed cell volume, hemoglobin and total protein of extensively managed indigenous diet recorded during dry season to low level of nutrition especially protein deficiency, due to scarcity of vegetables and farm left over’s to forage on. Ikhimioya et al., (2000) have suggested that the low erythrocytes’ parameters (PCV and RBC) an anemic condition recorded in scavenging Nigerian indigenous chicken was caused by poor nutrition resulting from their ability to ingest enough of highly nutritious feeds while scavenging for daily nutrient need. Serum and hematological indices such as album, globulin, creatinine, red blood cell count, blood cell count, packed cell volume and hemoglobin were not significantly (P >0.05) affected by dietary treatments. Creatinine and glucose levels in the blood increased while cholesterol decreased significantly (P <0.05). The creatinine and cholesterol levels of the birds fed variously treated CBS based diets were still within the value recommended for chickens. The increase in values with increasing contents of various forms of CBS based diets was desirable since the maximum content (136.45 mg/dl) at 10% RCBS was the closest to the minimum value in a range of 152-182 mg/dl recommended for normal chicken by these authors. The increased serum glucose content with increasing dietary forms of CBS-based diets was desirable, since the maximum content (136.45mg/dl) at 10% RCBS was the closest to the minimum value in a range of 152-182mg/dl recommended for normal chicken.

Conclusion

Results of various investigations carried out with CBS showed that it is a suitable alternative for maize in the diets of layers. Its nutritional qualities could further be enhanced to allow for incorporation into the diets of poultry at higher levels than adopted in the present study. It is therefore concluded that cocoa bean shell can be used to replace maize up to 10% in the diet of commercial layers without any deleterious effects on the performance, hematology and egg quality parameters.

References

1. A.O.A.C. 1990. Association of Official Analytical Chemists 15^th ed., Washington DC Analytical System Institute Inc. Cary NC.
2. Aderemi, F.A., Alabi, O.M. and Lawal, T.E. 2006. Utilization of Whole Cassava meal by egg type chicken. Proceedings of the 11^th Annual Conference of Animal Science Association of Nigeria (ASAN) Ibadan pg. 73-75.
3. Aletor, V.A. and Egberongbe, O. 1992. Feeding differently processed Soybean – Part 2: An Assessment of the Hematological Response in the chicken Diet. Nahrungi 36(4) 364-369.
4. Aniket, R. 2005. Medicine plus Medical Encyclopedia .Total Protein Division of Endocrinology and metabolism. John Hopkins University Baltimore M.D.
5. Babatunde G.M. and Pond, W.G. 1987. Nutritive value of Nigeria Rubber Seed (Heavea Brasiliensis) 1-Rubber seed meal. Nutri. Rep. Int., 36:617-630.
6. Bentil, J.A, Dzogbefia, V.P and Flemawor, F. 2015. Reduction in the lignocellulos content and the theobromine content of cocoa bean shell fermented with A.niger and P.Ostreatus respectively. International Journal of Applied Microbiology and Biotechnology Research. IJAMBR (3) 2015(20-30)
7. Church, J.P., Judd, J.I. Yong, C.W. Kebay, J.L., and Kin W.W. 1984. Relationship among dietary constituents and specific serum clinical components of subject eating self-selected diets. Amer. J. Clin Nutr. 40:1338-1344.
8. Donnica Moore, M.D. 2000. Lower Your Cholesterol and Triglycerides http://wwwdrdonnica.com/toptins/00000400.htm.
9. Danicke, S., Jeroch, H. Bottcher, W. Belford, M.R. and Ortwin S. 1999. Effects of Dietary Fat Type, Pentose Level and xylanase On High Fibrous Diets Supplemented with Roxazyme G enzyme. In food, lands and likelihood; Setting Research Agendas for Animal Science. Proceedings of BSAS/KARl/ILRI International Conference, KARI Conference Centre Nairobi, Kenya January 27-30.
10. F.A.O. 1994. Medium Term Prospect for Agricultural Commodity, Projection to year 2000, FAO Economic and Social Development Paper No. 120. Rome.
11. Fasuyi. A.O. Fajemilehin, S.O.K. and Omojola A.B. 2005. The Egg Quality characteristics of layers fed varying Dietary Inclusion of Siam Weed (Chromo laena odorata (Leaf Meal (SWLM). International Journal of Poultry Science 4(10) 752-757.
12. Fetuga, B.L. 1984. Techniques in Feed formulation. Paper Presented at the Feed Mill Management Training Workshop, Department of Agric. Econs. University of Ibadan, Ibadan Nigeria.
13. Gomez, A.K. and A.A. Gomez.1985. Statistical procedures for agricultural research Wiley, New York.
14. Harms, R.A., G.B Rissi; and D.R. Sloan 2000. Performance of four strains of commercial layers with major changes in dietary energy J. Appl. Poultry Res 9:535-541.
15. Hatagalung R.L. and Chang, C.C. 1978. Utilization of cocoa by products as animal feed. Proc. Int. Conf. on cocoa and coconut. Kaula Lumpur, Incorporated society of planters. Pg. 442 – 456.
16. Ikhimioya, I; Ariyeniwa, A, Oteku, I.I and Ahmed, A. 2000. Preliminary investigation on the Hematology of the Nigerian Indigenous chicken. Proceedings of 5^th Annual Conference of animal Science Association of Nigeria (ASAN), PortHarcout, Nigeria Pg. 10-12.
17. Jain, N.C. 1986. Schalms Veterinary Hematology, 4^th Ed Lea and Fibiger, Philadelphia U.S.A.
18. Lazaro, R., Garcia M., Aranibar, M.J. and Mateus, G.G. 2003. Effect of enzyme addition to wheat –Barley and Rye-based diets on Nutrient digestibility and performance of laying Hens. Brit. Poultry. Sci. 44(2):256-265.
19. Longe, O.G. 1984. Effects of Increasing the dietary fibre content of A layer’s diet Br. Poultry Sci. 25: 187-193.
20. Junqueira, O. M., De Laurentiz, A.C; Da Silva, Filardi, Rodriguez, E.A. and Casertelli, E.M. 2006. Effects of Energy and Protein Levels on Egg Quality and Performance of Laying Hens of Early Second Production Cycle. J. Appl. Poultry Res. 15:110-115.
21. Kwari, I.D. Igwebuike, J.V. and Kwada, M.V. 1999. Effect of Replacing Maize with Spent Sorghum Grain on Performance of Laying Hens. Journal of Sustainable Agriculture and environment 1: 25-31.
22. Keshavarz, K. and Nakajima, S. 1995. The Effects of Dietary Manipulations of Energy Protein and Fat during the Growing and laying periods on early egg weights and egg components. Poult .Sci. 74(1) 50-61.
23. Iyayi, E.A. and Okhankhuele, D.O. 2002. Cassava leaf meal and exogenous enzyme as supplements in broiler finisher diets. Tropical Vet 20(3):1 72-180.
24. Majaro, O.M. 1999. Performance Characteristics of Commercial laying Fowls in Nigeria Trop. Anim. Prod. Invest. 2:131-134.
25. Mitruka, B.M. and Rawnsley, H.M. 1977: Clinical Biochemical and Hematological reference values in normal experimental animal Masson Publishing USA Inc. New York.
26. National Agricultural Extension and Research Liaison Services (NAERLS). 1990: Federal Ministry of Science & Technology, Ahmadu Bello University, Samaru-Zaria.Guides to Poultry Feeds And Feeding, Extension Guide No 32, Poultry Series No 2, Pg. 13.
27. National Research Council. 1994. Nutrient Requirements of Poultry, 9^th Revision Ed National Academy Press Washington DC.
28. Oakenful, D. 1981. Saponins in Food. A Review Food Chem. 6: 19-28.
29. Odunsi A.A., Sobamiwa O, and Longe O.G. 1999. Comparative utilization of alkali treated and untreated cocoa bean cake in diets of egg type chickens. Tropical Journal of Animal Science 2(1) 63-68.
30. Oladele, S.B., Ayo, J.O. Esiero, K.A.N. and Ogundipe, S.O. 2001. Seasonal and sex Variations in Packed Cell Volume, Hemoglobin and Total Protein of Indigenous Ducks in Zaria Nigeria. Journal of Tropical Biosciences 1(1): 84-88. Olubamiwa O., Balogun-Kuku, O.I. Longe, O. Iyayi, E.A. and Oguntade, J.A. (2000). Cocoa Husk /Cassava Leaf Mixtures in Layers Mash. Proceedings of 5^th Annual Conference of Animal Science Association of Nigeria (ASAN), Port Harcourt Nigeria pg. 10-12.
31. Olorode, B.R.; Onifade, A.A., Okpara, A.O. and Babatunde, G.M. 1996. Growth, Nutrient Retention, Hematology and Serum chemistry of Broiler Chickens fed Shea butter cake or Palm Kernel Cake in the Humid Tropics J. Appl. Anim. Res. 10”173-180.
32. Olubamiwa O., O.A. Soetan, O.A. Olamijulo, R.A. Hamzat and O.G. Longe, 2002. Utilization of variously treated cocoa bean shells in layers mash. Proc. 27^th Annual Conf. Nig. Soc. For Animal Prod. (NSAP) March 17-21 2002, Fed University of Technology, Akure, Nigeria pp 267-269.
33. Olukosi, O.A and Adeola .O. 2007. Age related influence of a cocktail of xylanase, amylase and protease or phytase individually or in combination in broilers. Journal of Poultry Science 45:192-198.
34. Olomu J.M. 1995. Monogastric Animal Nutrition, Principles and Practice. A. Jachem Publication, pp. 1-5.
35. Olomu, J.M. 1979. Poultry Nutrition Research. Its contribution to National Poultry Industry. Nutrient Requirement, sources and nutrient content of feed ingredients. Poultry production: In Nigeria. Proceedings of the 1^st National Seminar on Poultry Production Held at ABU, Zaria: 241-268
36. Olorode, B.R.; Onifade, A.A., Okpara, A.O. and Babatunde, G.M. 1996. Growth, Nutrient Retention, Hematology and Serum chemistry of Broiler Chickens fed Shea butter cake or Palm Kernel Cake in the Humid Tropics J. Appl. Anim. Res. 10”173-180.
37. Olumide, M. D., Hamzat, R. A., Bamijoko, O. J. and Sabo, M. N. 2015. Evaluation of performance, serum biochemistry and hematological parameters of broilers fed graded levels of raw cocoa bean shell based diets. FUDMA J. Agric and Agric. Tech., 2015, Vol. 1: Pg. 105 -111.
38. Patel, M.B., McGinnis, J., and Pubolis, M.H. 1979. Effect of Cereal Grains and Added Citrus Pectin on Liver Content of Chicks and Laying Hens. Poultry Sci 58: 1092
39. Potter, J.D., Toppin, D.L. and Oakenful, D. 1979. Soya Products Saponins and Plasma, Cholesterol lancet 1:223-227.
40. Reid, B.L. Galaviz – Moreno, S. and Maiorino, P.M. 1984. A comparison of Glandless and Regular Cotton seed meals for laying hens. Poult. Sci. 63:1803-1809.
41. SAS Institute, 1999. SAS START ® Users Guide. Statistics Version 9 Edition SAS Institute Inc., Cary, North Carolina, U.S.A.
42. Scheeman, B.O. 1990. Gastrointestinal Responses to Dietary Fibre. In New Developments in Dietary Fibre (eds) I. Furda and C.J. Brine, Plennum Press, New York Pg. 37-41.
43. Veulterinora, M. (1991). Nutrition and Erythropoiesis. In CRC Handbook of Nutritional Requirements in Functional Context. M. Rechcizi (Ed) Boca Ration CRC press pp 65-74.
44. Yeong, S.W. Lim, F., and Aziza, A. 1989. The Nutritive value of cocoa bean shell for broiler and peckin chicks. Mardi Res. Journal. (Malaysia) Vol. 17 (2) pg. 258-264.