Study site
The experiment was conducted at Welgevallen Experimental Farm (33° 56′ 33” S 18° 51′ 59” E; Stellenbosch University, Stellenbosch, South Africa) between June and July 2021.
Diets, experimental design and animal management
Hempseed cake (HSC; Cannabis Sativa L. Fedora 17) was sourced from a local hemp oilseed processing company. Five pelleted (5 mm × 30 mm) total mixed diets were formulated and manufactured by a local commercial company by substituting SBM with HSC at 0, 25, 50, 75, and 100% intervals to produce diets containing 0, 25, 50, 75, 100 g HSC per kg diet (as is basis; Table 1), respectively. The diets met the nutritional requirements for growing goats (National Research Council, 2007). Twenty-five Kalahari Red wether goats (27 ± 3 kg) of 4-5 months were sourced from a commercial goat farmer and housed in individual pens with a wooden slatted floor. Five goats were allocated to each dietary treatment using a completely randomized design. On arrival, goats were drenched with 2 mg/kg body weight Derquantel and 0.2 mg/kg body weight Abamectin (Startect®, Zoetis, South Africa) to control internal parasites. Goats were also dosed with 5 mL of Vitamin A, D and E (Embavit™®, Prima Vetcare Private Limited, India), then a 2 mL of a combined clostridial and pasteurellosis vaccine (Multivax P Plus®, MSD, South Africa) was administered subcutaneously on the upper inner thigh to prevent pulpy kidney, tetanus and pasteurellosis. Animals were adapted to the experimental diets for 21 days and data collection done on day 28. The animals were offered clean water ad libitum and fresh feed every morning at 0800h.
Sample collection
The goats were fitted with a strap-on canvas fecal collection bag and a funnel-shaped latex bag connected to a urinary tube 5 days before data collection. Feed offered, fecal and spot urine were sampled and stored at -20 °C pending analyses. Feed (72 h of drying in a forced air-oven at 60 °C) and freeze-dried fecal samples were milled (Hammer mill; Scientec RSA Hammer mill ser Nr 372, Centrotec) through a 1.5 mm sieve.
On day 28, blood was collected into 10 mL purple (Ethylenediaminetetraacetic acid; EDTA) and yellow (Serum-separating tube; SST) cap vacutainer tubes from the jugular vein 4 h after feeding and transported on ice. The purple tubes were centrifuged at 2500 × g for 15 min at 4 °C to obtain serum which was decanted and stored in cryotubes at -80 °C. Goat were transported to the abattoir 70 km away from the experimental farm and waited in the lairage for 16 h. The goats were stunned for 3 s with 220V and 1.4 amp before being slaughtered following procedure of South African Meat Safety Act (No. 40 of 2000). The carcasses were dressed before the liver was collected after 60 min post-slaughter, and 24 h later, a sample of the right longissimus thoracis et lumborum (LTL) muscle between the 9th to 13th ribs was excised and stored at -80 °C pending analyses.
Chemical analyses and computations
Proximate, fiber and polyphenolic composition of the feed ingredients and diets
The feed dry matter (DM), ash and ether extract were respectively evaluated using 934.01, 942.05 and 920.39 methods of the AOAC (2002). The total nitrogen content of feed was determined using a macro-Nitrogen analyzer (LECO® FP828, LECO Corporation, Miami, USA) with a Dumas method of AOAC (2002) and a factor of 6.25 was used to obtain the crude protein (CP) content. A commercial starch assay (Total Starch Megazyme kit KTSTA, Megazyme International Ireland Ltd., Wicklow, Ireland) was utilized to analyze the sample total starch content (Hall, 2009). The neutral detergent fiber (aNDFom), acid detergent fiber (ADFom) and lignin (sa.) were assayed using Ankom F57 filter bags and fiber analyzer 2000 (ANKOM Technology, New York, USA) (Ryan et al., 1990). The NDFom, ADFom and lignin (sa.) were determined without ash. Feed total phenols and tannins were measured using the Folin-Ciocalteu colorimetric method described by Makkar (2003). The results were expressed as gram gallic acid equivalents per kg DM of feed. Five replicates of each sample were used and all chemical analyses executed in duplicates.
Cannabinoids, tocopherols and bioactive minerals
Cannabinoids and tocopherols were determined as described in a companion paper by Semwogerere et al. (2023). For bioactive minerals, five grams of feed, liver, meat, urine and fecal samples were weighed into microwave digester Teflon vessels, followed by the addition of 6 mL ultra-pure HNO3 + 1 mL H2O2, prior to digestion using MARS microwave digester (CEM, Germany). The analysis was conducted using inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Sah and Miller, 1992). The microwave was powered at 1600 W at 100%, with 25 min ramp time and pressurized at 800 psi held for 10 min. The instrument radio frequency was 1600 W, argon was the carrier gas at 0.83 L/min, 10 mm sample depth, 0.15 L/min make-up gas, helium flow at 5 mL/min, hydrogen flow at 6 mL/min and 0.4 mL/min micro mist of nebulizer.
Blood samples (SST) were vortexed, then diluted 20× in an alkaline medium of NH4OH, EDTA and Triton-X before being analyzed with an Agilent 8800 QQQ Inductively Coupled Plasma-Mass Spectrometry (Agilent, USA). A blood reference material, Seronorm Trace Elements Whole Blood L-2 (SERO AS Stasjonsveien 44, NO-1396 Billingstad, Norway) was prepared in the same way and analyzed as quality control standard to verify the accuracy. Results for all analyzed elements were within 5% of the certified values and the analysis was done in duplicate.
Antioxidant assays
Feed ingredients and diets extracts were obtained by adding 40 mL of 80% aqueous methanol (v/v) to 5 g of the sample, vortex and extracted twice with ultrasonic extraction (Branson B-220H, SmithKline Co., USA). The extracts were stored at −80 °C pending analysis. The antioxidant activities of feed ingredients and diets were determined using ferric reducing antioxidant power (FRAP), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays described by Thaipong et al. (2006). For FRAP, 10 µL of the extract was mixed with 190 µL of FRAP reagent, incubated for 10 min at 37 °C and absorbance read at 593 nm using microplate reader (SPECTROstar Nano, BMG LABTECH, Germany). The results were expressed as µM Fe2+ (Fe2SO4·7H2O, Sigma-Aldrich, Germany) equivalents (Fe2+ eq)/g DM. For DPPH, 25 µL of the extract was mixed with 200 µL of 0.1mM DPPH solution, incubated for 30 min at room temperature in the dark and absorbance read at 517 nm using a microplate reader. The results were expressed as mM Trolox equivalent (TEq; Sigma-Aldrich, Germany)/g DM. For ABTS, 20 µL of the extract was mixed with 200 µL of ABTS working solution of 7.4 mM ABTS with 2.6 mM K2O8S2 diluted to obtain a 1.1 ± 0.02 optical density (OD) at 734 nm, incubated 7 min at room temperature in the dark and absorbance read at 734 nm using a microplate reader. The assay results were reported in mM TEq/g DM using Trolox standard curve.
The antioxidant activities of liver and meat were determined using FRAP, ABTS (Descalzo et al., 2007) and DPPH assays (Thaipong et al., 2006). Briefly, 1 g of meat or liver sample was homogenized for 2 min with IKA T18 digital ULTRA-TURRAX® at 3000 rpm in 5 mL of a potassium phosphate buffer (pH 7.2). The homogenate was centrifuged at 4024 × g for 30 min. For FRAP or ABTS, 20 µL of aliquot was mixed with 180 µL of FRAP reagent or ABTS working solution, incubated for 10 min at 37 °C or 7 min at room temperature in the dark and absorbance read at 593 nm or 734 nm using a microplate reader. Results for FRAP and ABTS were expressed as µM Fe2+ eq/g tissue or mM TEq/g tissue, respectively. For DPPH, 25 µL of aliquot was mixed with 200 µL of 0.1 mM DPPH solution, incubated for 30 min at room temperature in the dark and absorbance read at 517 nm using a microplate reader. The results were expressed as TE/g tissue.
For blood bioactivity, serum (EDTA tubes) was used directly for analysis of FRAP, ABTS and DPPH (Cecchini and Fazio, 2020). For FRAP, 10 µL of serum was added to 300 µL FRAP reagent in microplate, incubated for 5 min at 37 °C and absorbance read at 600 nm in a microplate reader. Results were expressed as µM Fe2+ eq/mL blood. For DPPH, 25 µL of serum were mixed with 475 µL of 10 mM phosphate buffered saline pH 7.4, and 500 µL of a 0.1 mM DPPH solution, incubated at room temperature in the dark for 30 min and absorbance read at 520 nm using a spectrophotometer. The results were expressed as TEq/mL blood using a Trolox standard curve. For ABTS, 10 µL of serum were added to 300 µL of ABTS working solution of 7.4 mM ABTS with 2.6 mM K2O8S2 diluted to obtain a 1.1 ± 0.02 OD at 734 nm, incubated 7 min at room temperature in the dark absorbance read at 734 nm using a microplate reader. The assay results were reported in mM TEq/g DM using Trolox standard curve. The antioxidant potency composite index (APC) was calculated as: antioxidant index score = [(sample score/best score) × 100] (Seeram et al., 2008). The scores were calculated for each assay then averaged per sample. All antioxidant assays were evaluated in duplicate.
Statistical analysis
All the data was analyzed using the GLIMMIX procedure of SAS (version 9.4; SAS Institute Inc. Cary, NC, USA) with animal and diet as a random and fixed factor, respectively. Orthogonal polynomials of SAS Institute Inc. (2012) were used to test the linear and quadratic effects of the treatment diet inclusion levels. Tukey’s test was executed to test for significant differences among least square means (LSMEANS), which were considered different at P ≤ 0.05 and tendency at 0.05 < P ≤ 0.10.