The experiment was executed in accordance with the approved guidelines for Animal Research by Nigeria Institute of Animal Science in Nigeria (NIAS).
Experimental site
The experiment was carried out at the piggery unit of the Directorate of University Farms, (DUFARMS) Federal University of Agriculture, Abeokuta, Alabata, Ogun State, Nigeria from March 2019 to July 2019. The site is situated in the derived savanna zone of Southwestern Nigeria on Latitude 7°9'39N and 3°20'54E and 76 m above sea level. The mean annual rainfall is 1040 mm and occurs from March to October, the temperature average is 34°C throughout the year.
Processing of test ingredients
Cassava peel meal (CPM) and cassava leaf meal (CLM)
Dried cassava peels were obtained from the cassava processing plant in Igbo-ora, Oyo state, Nigeria. The dried peels were subsequently hammer milled (2 mm sieve) to yield cassava peel meal (CPM) and stored in bags. Fresh cassava leaves without petioles were manually plucked from an established cassava farm (Odeda, Ogun State, Nigeria). The leaves were evenly spread on the concrete floor and sun-dried for 2-3 days until the dried leaves became crispy while still retaining the greenish colouration. The dried crispy leaves were milled (2 mm sieve) to yield cassava leaf meal (CLM) which was stored in plastic bags under room temperature.
Unfermented cassava peel-leaf blend (UCPLB)
A blend of cassava peel meal (CPM) and cassava leaf meal (CLM) was prepared using the Pearson Square method according to
Adeyemi et al., (2014) by mixing at a ratio of 5: 1 (5 parts CPM: 1 part CLM) to form an unfermented cassava peel-leaf blend (UCPLB) with a protein content of 8.83%. Crude protein contribution from individual components in the mix is 81.26% and 18.74% from CPM and CLM respectively.
Water fermented cassava peel-leaf blend (WCPLB)
Prepared by mixing dried CPLB (5:1) with water (in ratio 1:1, kg: Lt) in plastic drums. The blend was mixed thoroughly to ensure all portions of the blend come in contact with water. After mixing, the wet blend was placed in black polythene bags and tied properly to create an anaerobic environment within the bags. The bags were left for 7 days to ensure proper fermentation of the contents. On the seventh day, the bags were opened and the ingredients were sundried and stored before diet formulation.
Microbial (Aspergillus tamarii) fermented cassava peel-leaf blend (MCPLB)
Pure strains of
Aspergillus tamarii obtained from the Culture Collection Unit of the Department of Microbiology, Federal University of Agriculture, Abeokuta was used as inoculums in this processing method. Spores of
Aspergillus tamarii used for fermentation of the CPLB was prepared by following standard protocols described by
Murray et al., (2003). Spore suspension (inoculum) of
Aspergillus tamarii was prepared by washing spores from Petri dishes into clean water at an inoculum size of 10.5 x 108 spores/g of CPLB. The wet blend was mixed properly and placed into black polythene bags which were tied properly to create an anaerobic environment within the bags. The bags were stored and left for 7 days to ensure proper fermentation of the contents. On the seventh day, the bags were opened and the ingredients were sundried and stored before diet formulation.
Chemical composition of test ingredients
Proximate composition of samples from CPM, CLM, UCPLB
, WCPLB and
MCPLB was determined using standard method by Association of Official Analytical Chemists (AOAC) according to
Nochera and Ragone (2016) and fibre fractions were carried out according to the standard method by
McCleary (2007) respectively. All analysis done was on a dry matter basis. NDF (assayed without a heat-stable amylase and expressed inclusive of residual ash), ADF (expressed inclusive of residual ash) and Lignin (determined by solubilisation of cellulose with sulphuric acid) and crude protein (total nitrogen x 6.25). Gross energy was estimated using the adiabatic bomb calorimeter (Model 1261; Parr Instrument Co., Moline, IL, USA) while digestible energy was calculated according to
Adeola (2001). The cyanogenic glycosides of the samples were done using the method described by
Vetter (2000).
Experimental animal, design and dietary treatments
Twenty four (24) crossbred (Large white x Landrace) male pigs (15 wks old) with average weight (20-22 kg) purchased from reputable pig farm at Iperu Remo, Ogun State were randomly assigned on a weight equalization basis to four dietary treatments. Pigs were housed individually in 24 pens (0.5 m x 0.25 x 0.3 m). Six pens were assigned to each treatment. A standard soybean-maize based diet (control; Diet 1) was formulated following the National Research Council (NRC) requirement for growing pigs according to
Nyachoti et al., (2005). Three additional experimental diets were formulated such that UCPLB (Diet 2), WCPLB (Diet 3) and MCPLB (Diet 4) replaced maize (weight for weight) in the control diet (Table 1). Pigs in each treatment group were fed with their respective experimental diets. Feed was offered to the animals during the trial which lasted for 16 weeks based on the NRC recommended intake partitioned for each body weight range. Experimental diets were fed twice daily (8:00 and 18:00 hr) while clean water was supplied
ad-libitum.
Growth performance
The initial body weight of the pigs were taken and subsequent weight per pen was measured weekly, while the gain in weight was calculated. Daily feed intake was also measured as the diûerence between the feed offered and leftovers, while feed conversion ratio (FCR) was also calculated as Feed consumed/Weight gain.
Blood collection
Blood samples were collected from 3 randomly selected pigs per treatment at the end of 8th and 16th week of the study. This was done through the culinary vein using disposable syringes with 20 x 100 mm metallic needles. 2.5 ml blood was collected from each pig; it was collected in plain bottles (without EDTA) for serum parameters. Blood samples were centrifuged (1200 rpm for 15 min) for separation of plasma. Aliquots of plasma was taken and frozen at -20°C until further analysis.
Serum parameters
The total serum protein, albumin and globulin were determined using bromocresol purple method
(Varley et al., 1980), serum creatinine according to
Bonsness and Taussky (1945). Serum enzymes (alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST)) were analysed using the commercial kits (Qualigens India. Pvt. Ltd., Catalogue number 72201-04). The serum cholesterol was estimated using the enzymatic colorimetric methods (according to the manufacturer’s manual) using Randox ® diagnostic cholesterol kit.
Statistical analysis
All data obtained were subjected to a One-way analysis of Variance using Statistical Analysis System Software (SAS) and mean separation was done using Tukey test of SAS as described by
Ramatsoma et al., (2015) while significant differences were considered at p<0.05.