2.1 Ethical approval
The handling and the use of the animals was approved by University of Fort Hare, Animal ethics and Use Committee [Approval number (MPE041IKU01)].
2.2.Study site description
The experiment was conducted at the small ruminant unit of the University of Fort Hare teaching and research farm, Alice, Eastern Cape, South Africa. The research farm lies at longitude 26º 50' E and latitude of 32°46' S. The annual rainfall is between 480-490 mm and temperature range between 24.6 º C and 11.1 º C (average is 17.8 º C) at an altitude of 535 meters above sea level.
2.3. Sheep and housing
Twenty-four Dohne Merino wethers were selected from a commercial farm in Mitford village, Tarkastad, Eastern Cape of South Africa. The wethers were about 5 months old and weighed 20 ± 1.5 kg on average. The wethers were used in a completely randomized design of four treatments with six wethers per treatment. All the 24 wethers were raised at the same housing and equipment facilities in the same area under the same average environmental condition. The pens had similar temperature (23.29 °C), relative humidity (76.75%), dry bulb temperature (24.82 °C) and wet bulb temperature (21.15 °C). They were individually housed in 1.5 m × 1.5 m well-ventilated roofed building with concrete flooring for each sheep. The experiment lasted for 105 days excluding 14 days of adaptation period. The sheep had access to clean and fresh water ad libitum.
2.4 Experimental Diets
The diets for the wethers consisted of concentrate and hay at 40:60 ratio. The concentrate was made up of maize (8%), sunflower oil cake (10 %), molasses (5%), wheat offal (15%), limestone (1.5%), salt 0.3% and sheep mineral-vitamin premix (0.2%), whereas the hay consisted of 30 % teff and 30 % Lucerne. The ingredients for concentrate were purchased from Monti Feeds (pty) Ltd, East London, South Africa while the teff and Lucerne were purchased from Umtiza Agricultural products (Pty) Ltd, Kwantu shopping mall, Alice, South Africa. All ingredients were thoroughly milled and mixed evenly together to form the basal diet. The feed was formulated to meet the nutritional (energy and protein) requirements of the used sheep (NRC, 2007). The four dietary groups were: basal diet (0%); basal diet +2% FSF; basal diet +4 % FSF and basal diet + 6% FSF. The wethers were fed at 8:00h and 15:00h at 4 % of the body weight (on dry matter (DM) basis). The food-grade Fossil shell flour was purchased from Eco-Earth (Pty) Ltd, Port Elizabeth, South Africa which produces this product under a license by Department of Agriculture, Forestry and Fisheries of South Africa.
2.5 Analytical procedures
2.5.1 Proximate analysis of the experimental diets, orts and fecal sample
The proximate composition of the experimental diet is presented on Table 3.1. Dry matter content of the diets, orts and fecal samples was measured by drying samples in an air-forced oven at 135°C for 24 h (method 930.15; AOAC 2005). Ash content was measured by placing samples into a muffle furnace at 550°C for 5h (method 938.08; AOAC 2005). Organic matter (OM) was calculated as the difference between DM and the ash content. Nitrogen (N) was measured by the Kjeldahl method using Se as a catalyst and crude protein (CP) was calculated as 6.25 × N. Gross energy (GE) was measured using a bomb calorimeter (C200, IKA Works Inc., Staufen, Germany). Ether extracts (EE) were measured by weight loss of the DM on extraction with diethyl ether in Soxhlet extraction apparatus for 8h (method 920.85; AOAC 2005). Crude fibre was determined by allowing the sample to boil with 1.25% dilute H2SO4, washed with water, further boiled with 1.25% dilute sodium hydroxide and the dried residue (65 º C for 3 hours) after digestion was taken as crude fibre (method 978.10) as described by Thiex (2009).
2.5.2. Mineral analyses
The mineral composition of the dietary FSF used is shown in Table 2. In determination of mineral content of the FSF, 5.0 g of the sample was weighed in triplicate, and burnt at 550 º C in a muffle furnace for 5.5 hours. The residues were cooled in desiccator, before dissolving in 100 ml of deionized water. Suitable salts of the elements were used to make their standards. The standard mineral solutions were injected into atomic absorption spectrophotometer (Jenway, FPSP 210 model 6305, United Kingdom) and concentration obtained. These standards were used to determine Mg, Zn, Fe, Cd, Ca, Al, Mn and B in an unknown feed sample. The concentration of Na and K were determined using flame photometer (Jenway Models PFP7 and PFP7/C, Cole-Parmer, United Kingdom).
2.6.1. Wool samples collection and measurement
An area of approximately 10 cm × 10 cm was shorn on the mid-side left of each wether with a clipper (Oster clippers, No 47cutting head, Germany) on day 0 of the experiment. At the end of the experiment (day 105), a second patch was clipped and wool removedfrom the same spot. Heron’s formula was used to measure and determined the patch area as described by . The formula stated as: that area of triangle=√(s-a) (s-b) (s-c), where a, b and c are the sides of the triangle. S is the semi perimeter of the triangle, calculated as S=a+b+c/2. The clipped wool was dried in an oven for 48 h at 60 °C and weighed to determine the total wool weight. Wool growth was determined by the equation: greasy weight (mg)/ area (10×10 cm) divided by number of experimental days. Greasy wool is the wool in its natural state, after removing from the sheep, before any commercial processing. It contains vegetable matters, extraneous soil, yolk, moisture and suint. Greasy weight was determined by near infrared method (NIR). The NIR is a spectrophotometer, which uses near infrared energy to measure the amount of grease remaining in a sample of scoured wool. Samples were then washed at 90 °C with Clean Plus (a mild soap produced by Clean Plus Chemical, Australia), and rinsed twice with cold distilled water to remove impurities and wool grease. This was followed by oven drying at 110 °C for 4 h. Samples were then reweighed at relative humidity of 65% to calculate the clean wool weight . Wool yield was then calculated by determining the percentage of clean wool weight relative to greasy fleece weight, after which they were packed in labelled plastic bag and sent to a commercial laboratory (BKB Wool (PTY) Ltd, Port Elizabeth, South Africa) where fibre diameter, SD, FDCV, CEM, comfort factor, staple length, FD Dev and number fibre <15% were measured according to IWTO 12 norms as described in detail by.
2.6.2. Body condition score
The BCS was carried out on weekly basis before the morning feeding by a trained person. The BCS chart was developed based on the system described by . In this method, there is 0.5 intervals for scores ranging from 1 (emaciated) to 5 (obese). Scoring was done by touching redundancy and crossing the amount of muscling and fat deposition over and around the vertebrae in the loin region.
2.6.3. Feed preference
For feed preference test, twenty-four wethers from section were individually housed in pens (5 × 7 m) having four feeding troughs with identification in a completely randomized design with six wethers per diet. The feeding troughs were distributed at equal distance of 0.7 m from one another, and positioned beside one facing the entrance of the pens. Prior to the commencement of the trial, all the wethers were allowed to acclimatize themselves to the pen and feeders for 5 days. After the adaptation period and sequel to an overnight fast, 200 g of each diet 0%, 2%, 4% and 6% FSF was put into the four different feeding troughs in each pen. Each wether in each pen with four different feeding troughs containing four different diet was allowed to access the feeding troughs for ten minutes daily for a period of 7 days. Daily, feed was rotated to a different feeding trough, and each wether was introduced into the pen for 10 minutes, and the order they accessed the various feeding trough was noted. The number of times the wether visit each feeding trough within the stipulated period were determined by counting and recorded as number of visit (NV), number of bites per visit by wether for each diet were determined by counting and recorded as number of bites per visit (NBV). Time spent on visit by each wether on each diet was determined using stop watch (EMC, Zhejiang, China) and recorded as time spent visit (TSV), and the dry matter intake (DMI) for each diet were calculated by multiplying percentage dry matter of the feed with the average daily feed intake. At the end of the 10 minutes, the feed refusal for each feeding trough was weighed to enable the determination of feed consumed. At the end of the preference test, animals received 1 kg of mixture of leucine and teff hay.
2.6.4. Feed intake
Feed intake was determined by weighing the feed leftover in feed troughs, including feed refusals every day at 0800 h. Amounts of feed disappeared were considered to be feed ingested by the wethers. The feeding troughs were design to reduce feed spillages. Weights of feed leftover were subtracted from the total weight of the feed allocated to each wether and divided by 7 to determine average daily feed intake (ADFI) . Dry matter intake was determined by multiply percentage dry matter of the feed with the average daily feed intake.
2.6.5. Average daily gain
Average daily gain (ADG) was measured by weighing the wethers every week throughout the experiment period. The difference in weight of wethers at the beginning and end of each week divided by 7 determined the ADG . To determine body weight gain (BWG), the wethers were weighed weekly, over 14 weeks using RUUDWEIGH, KM-2E electronic weighing system with 0.05 precision (RUUDSCALE, Durbanville, South Africa).
2.7. Statistical analyses
General linear model of SAS 9.4 (SAS Institute, 2012) was used to analyze the effect of FSF inclusion levels on body condition scores, average daily weight gain, fibre diameter, staple length, comfort factor, SD, FDCV, CEM, wool growth, number of visits, number of bites per visit, time spent per visit, and dry matter intake. The model included different inclusion levels of FSF as fixed effects for body condition score, wool parameters and feed preference. The following model was used:
Yij= µ+βi+Wj + (WxB)ij+ Eij. Where:
Yij is the dependent variable (BCS, ADG, FD, SL) μ is the overall mean
βi the effect different inclusion levels of FSF (j = 0 % ,2 % ,4 % and 6 %) Wj is the effet of the jth week;
(WxB)ij is the interaction between week and inclusion levels of FSF; Eij is residual error ~ N (0; Iσ2).
Turkeys’ studentized range test was used to test the significant differences between means.