Handling of experimental animals
Animal experiments for this study were conducted at the Animal Experimentation Unit of Centre for Plant Medicine Research (CPMR), Mampong-Akwapim, Ghana using outbred Sprague–Dawley rats purchased from the same Institution. The study received clearance from the Ethical Clearance Committee of CPMR through the Animal Experimentation Unit. All experimental protocols and procedures were approved by the Ethical Clearance Committee of CPMR and were performed according to standard operating procedures and guidelines of the Unit. Throughout the period of experimentation, the animals were handled in accordance with internationally accepted standards and principles of laboratory animal use and care (EEC Directive 2010/63/EU)17. During a 2-week acclimatization period, sterilized drinking water and regular rat chow, used as control diet in this study, were provided ad libitum. Formulation of the control diet (Table 1) was based on National Research Council, USA recommendations for rats and mice18.
Preparation of cocoa pod husk
Fresh husks of Theobroma cacao (cocoa) pods were collected from the Cocoa Research Institute of Ghana (CRIG), New Tafo-Akim, Eastern Region, Ghana. Cocoa pod husks were dried in an indirect solar dryer until moisture content was 9.8 ± 1.2%. The dried husks were milled to approximately 2 mm particle size and sterilized (121 °C and 15 psi for 20 min). Sterilization was to ensure that CPH substrate would be colonized only by the inoculum for biodetheobromination. Milled, sterilized CPH is subsequently referred to as substrate or untreated CPH.
Reagents and chemicals
Kits for assays of enzyme activities, albumin, creatinine, total and direct bilirubin were procured from Cypress Diagnostics (Langdorp, Belgium). Theobromine, as well as reagents and chemicals for histopathological tissue processing, were procured from Sigma-Aldrich (St. Louis, MO).
Bio-tool for detheobromination of cocoa pod husk
Detheobromination of CPH was by treatment with Talaromyces verruculosus KY697103 (TvTD), a filamentous fungus previously shown to be capable of theobromine-degradation15 and potentially useful as a bio-tool for detheobromination of CPH for animal feed16.
Bio-detheobromination of cocoa pod husk
Treatment of cocoa pod husk
Spore suspensions of TvTD were prepared in sterile distilled water19 from 14-day-old cultures on theobromine-sucrose agar slants at room temperature (25–29 °C)15. Detheobromination of CPH was done using 1 kg of milled CPH per batch of treatment. Fungal seed culture for treatment was prepared in 200 mL theobromine liquid medium (TLM) inoculated to contain of 2 × 104 spores/mL, and incubated with agitation at 90 strokes/min for 24 h at room temperature. Theobromine liquid medium15 was constituted with 0.2 mg/mL sucrose and 0.01 mg/mL theobromine. Theobromine was absent from control TLM. After the incubation period, seed cultures in control TLM were autoclaved to inactivate the inoculum15. Each seed culture was topped-up with distilled water to make 3 L of suspension and the entire volume was evenly mixed with 1 kg untreated CPH. Treatment vessels were loosely covered and left to stand for 10 days at room temperature.
Determination of theobromine content in cocoa pod husk
After the treatment period, CPH samples were autoclaved and dried to constant weight, for up to 5 days in an oven at 50 °C. Dried CPH, untreated or treated, was analyzed for theobromine content by a modification of the AOAC method for extraction of theobromine from cocoa beans20. Briefly, approximately 5.0 g of milled CPH were defatted with two portions of 30 mL petroleum ether (BDH, Poole, England) at room temperature, and left overnight in a fume hood to dry. The dried material was boiled in 100 mL distilled water for 20 min. After the period, an extra 50 mL distilled water was added twice, each time followed by boiling for 20 min. Between 70–90 mL of supernatant from each sample were concentrated by rotary evaporation under reduced pressure at 50 °C, filtered through a Whatman Spartan syringe filter with a 0.45 µm membrane (Sigma Aldrich; St. Louis, MO, USA) and analyzed for theobromine by high performance liquid chromatography (HPLC).
Chromatographic separation was done using a 20-µl sample volume at 27 °C on an Atlantis dC18 5 µm column (150 mm × 4.6 mm) fitted to a Waters HPLC unit consisting of a Waters In-Line degasser AF, Waters 1525 binary HPLC pump and Waters 2487 dual wavelength absorbance detector at 270 nm. Elution was by a solvent system of acetonitrile (ACN) and 0.1% formic acid adjusted to pH 3.75 with ammonia (AF), pumped for 20 min according to a gradient profile, with ACN:AF ratios starting from 2:98 through 12:88 and ending with 2:9816. Calibration plots were prepared from dilutions of a 0.1 mg/mL solution of theobromine. Internal theobromine controls were used to check for loss of theobromine during extraction from CPH. Also, analytical samples were spiked with theobromine for HPLC analysis, to confirm identity of the theobromine peak and also to validate quantitative deductions that were made from calibration plots.
Experimental feeds were formulated either with maize (control diet) or CPH replacing maize (substitution diets). Feeds were in the form of coarse powder. For the substitution diets, TvTD-treated or inactivated TvTD-treated CPH was included at 30% or 50% of the total diet (Table 1). Nutrient values for CPH used in feed formulations were based on values obtained from proximate analyses of untreated and TvTD-treated CPH16. Control and experimental diets were formulated to be isonitrogenous and isocaloric, estimated by calculation (Table 1).
Animal selection and treatment
A total of 48 male Sprague–Dawley rats (180–255 g) were selected for the study. For mating, 18 non-pregnant, nulliparous, female Sprague–Dawley rats (160–180 g) were selected. The male rats were assigned to 6 feeding groups of 8 animals each. After acclimatization, baseline values for body weight, full blood count and blood biochemistry were recorded. The designations and treatment regimen of the 6 feeding groups were as follows: Group 1 (CF), control diet (regular rat chow); Group 2 (CT), control diet + theobromine (300 mg/kg BW); Group 3 (ITC-30), base diet substituted with 30% inactivated TvTD-treated CPH; Group 4 (TC-30), base diet substituted with 30% TvTD-treated CPH; Group 5 (ITC-50), base diet substituted with 50% inactivated TvTD-treated CPH; Group 6 (TC-50), base diet substituted with 50% TvTD-treated CPH. Excess feed and drinking water were available to the rats for a period of 12 weeks.
Throughout the 12-week period, mortality of any animal in the treatment groups was recorded. Only surviving animals were used as statistical units for subsequent measurements and analyses.
Water and feed intake
Three rats from each feeding group were housed individually in metabolic cages and had free access to measured excesses of feed and water daily for 12 weeks. Quantities of feed and water consumed over the first 7 days after the start of animal feeding were used to establish baseline values. The weekly changes in feed and water intakes for each feeding group were expressed as percentages of their respective baseline values. Total feed and water intakes over the period were determined by area under curve (AUC) analysis of the mean weekly intake values for each feeding group.
Body weight of all the animals in each feeding group was determined before the start of feeding and then weekly thereafter over the experimental period. Total percentage changes in body weight were determined by AUC analysis of the mean weekly values for each feeding group.
All rats were sampled for baseline hematology values. Subsequently, the rats were sampled for blood at 4-week intervals from start until termination of feeding trial. Blood, approximately 1 mL, for hematological analyses was drawn by tail snip, collected into EDTA-coated tubes and analyzed within 24 h. Full blood counts were measured from the uncoagulated blood samples using a Sysmex KX-21N Automated Haema Screen (Ontario, Canada).
Blood for biochemical analyses (3 mL) was drawn from all rats into tubes on ice, allowed to clot and centrifuged at 4,000×g and 10 °C for 10 min. The supernatant serum was collected and used for analyses. For storage, samples were kept at − 20 °C. Biochemical analyses for heart, liver and kidney functions were conducted. The marker used for assessing cardiac function was creatine kinase MB (CK-MB) isoenzyme. For assessing liver function, albumin, total and direct bilirubin concentrations were determined. Creatinine levels were measured for kidney function.
At termination of feeding trial, 3 male rats from each feeding group were introduced to 3 female rats each. Number of animals used for mating was limited by mortalities in some treatment groups. After allowing 5 days for mating, the males were removed, weighed and sacrificed. Testes and thymus were excised, weighed and processed for histological analyses. Females were observed throughout parturition and upon delivery of pups, litter characteristics were recorded.
Organ wet weights
After the feeding trial and mating, 5 animals from each feeding group (3 animals from the CT group) were sacrificed by cervical dislocation. Selected organs (thymus, liver, kidney, heart, spleen, lungs and testes) were excised, blotted dry and weighed to investigate the effect of the different feeds on mean organ wet weight expressed as a percentage of body weight.
Histology of excised organs (liver, testes and thymus) was carried out in accordance with established protocol at the Electron Microscopy Unit of Noguchi Memorial Institute for Medical Research, University of Ghana, and as described in the Manual of Histological Staining Methods21 using hematoxylin and eosin. Stained tissues were observed under a light microscope at 100 × or 200 × magnification, and pictures were taken by a camera attached to an Olympus BH-2 photomicroscope (New York Microscope Co., Hicksville, NY, USA).
All statistical analyses were performed with data analyses tools in Microsoft Excel version 14, Microsoft Office Professional Plus 2010. For determining significance of differences, data were analyzed by one-way ANOVA and post hoc by Tukey’s multiple comparison test on GraphPad Prism version 8. Analysis of trends for changes in growth parameters was by EpiTools Epidemiological Calculators22. All analyses were conducted at α = 0.05. Individual rats were used as statistical units for analyses. Except for the CT group which had n = 3 for statistical analyses, all other groups had n ≥ 5 unless otherwise indicated.