Garra-based poultry feed formulation
The experimental feed formulation and feeding trials were carried out at Mekelle University, Ethiopia. Garra (G. dembecha) fish were caught from several water bodies and reservoirs of the Tigray region, Northern Ethiopia. Whole fish were uniformly distributed for drying on a triangular shaped entirely mesh-enclosed drier stand (2 m x 12 m x 2.5 m) established on the roof of a 15 m high building and sun-dried for a fixed time period of 5 ± 1 d. The drying environment was with maximum average temperature of 28 ± 1.4 oC and wind speed at 2.72 m/s across the drying periods, December, January and February 2019/20. During the drying period, whole fish were uniformly turned over using a brush-ended clean stick to facilitate drying. Dried garra meal was collected and milled to acceptable smaller size using a grinder machine and made ready as feed substituent. Commercially available formulated broiler starter (d 0-21) and finisher (d 22-42) feeds were purchased from EthioChicken Plc. For both growth phases, a 10, 20, and 30% garra meal addition to the formulated commercial diets were prepared. We deliberately chose for addition of the garra meal instead of substitution with other ingredients to obtain the same nutrient profile. Both scenarios inevitably lead to confounding factors, since we wanted to know the pure effect of garra meal, we opted for addition, in the awareness that this implied different dietary nutrient concentrations. The macronutrient compositions of the garra meal, broiler starter and finisher basal diets were determined by proximate analysis (AOAC 1990) (Table 1).
Similarly, as a starting protocol for the experiment, garra meal, broiler starter and finisher basal diets were further evaluated for amino acid, minerals, and fatty acid contents. Amino acids assay was done using reversed-phase high-performance liquid chromatography analysis at feed & food quality laboratory, Belgium (Table 2). The total ash content was determined following the ISO method (ISO 936, 1998) and the mineral (Ca, P, Mg, K, Na, Br, Cu, Fe, Mn and Zn) concentrations of the garra meal and feed samples were evaluated after dry ashing mineralization according to ISO method (ICP-AES: ISO 11885) as per the procedures described by Gorsuch, (1970) at the Chemistry laboratory, Ghent University (Table 3) and the fatty acid composition at Lanupro, Ghent University (Table 4).
Experimental animal set up and feeding protocol
The experiment involved 320 day-old broiler chickens, randomly divided over 20 indoor floor pens with 16 chickens each (at a density of 0.28 m²/chicken). Each pen was randomly attributed to one of four dietary treatments, i.e. 0, 10, 20 and 30% garra meal additions on top of the broiler starter and finisher diets. Broiler chickens (Sasso-C44 breed) were purchased from hatchery supply of EthioChicken Plc.
Pens were considered as experimental units. All pens were designed to be similar in all outlines, with concrete floor having wood shavings as bedding at a depth of 5-10 cm, and built in accordance with the international and institutional research ethics and welfare requirements for experimental animals. Each pen was set with separate feeding and watering troughs, and an electrically controlled warming 200 W bulb lamp hung from the top of each pen. Indoor heating temperature was controlled using wall-fixed digital thermometers and maintained at 30 ± 1.0 °C across the starter periods of the experiment. All equipments were properly cleaned and disinfected using detergents and the entire indoor pens were fumigated with a mix of potassium permanganate and formaldehyde (500 g : 250 ml) 10 d before stocking of the experimental chickens.
All experimental chickens were procedurally vaccinated against Infectious bursal (Gumboro) disease, Newcastle disease and Fowl typhoid as per the manufacturer’s recommended vaccination protocols for day-old chickens. The experiment lasted for 42 d and individual chickens were fed on experimental unit bases. Weighed amounts of feed were given on daily basis and feed leftovers were collected, cleaned, air-dried, and then weighed and recorded daily before the next morning feeding, at 08:00am. Feed and clean water were given ad libitum and chickens were allowed to stay indoors throughout the experimental period.
Measurement of growth performance indicators
Performance indicating factors were assessed for chickens in each experimental unit. Following fasting for 12 h, chickens were weighed on weekly basis on d 0, 7, 14, 21, 28, 35 and 42 in the morning before feeding starts at 08:00am. The average weekly feed intake (g/wk), body weight gain (g/wk) and feed conversion ratio (FCR, g/g) were evaluated at each sampling time point following standard procedures. Mortality cases were checked and recorded from every replicate of each treatment group (n = 80) on a daily basis and correction factors were made for all dependent parameters across the study period. Mortality was calculated as follows:
Where: N stands for the number of dead chicken in every replicate of each treatment group, 80 is the total number of chickens per group.
Evaluation of apparent nutrient digestibility using titanium (IV) oxide as a biomarker
Marker-feed preparation and feeding
Starting from d 22 onwards, titanium (IV) oxide (VWR Chemicals®, Belgium, PN84853, > 99.0% purity) was supplemented into each experimental diet of grower chickens at 0.4% addition. To assure homogenous mixing of the marker, premixing was applied on a small amount of diet (1% of the total diet). Feeding with the marker diet started on the first day of the growth phase (d 22) and continued till d 42 of the experimental period.
Excreta collection and processing
Prior to sampling, all wood shavings were removed and replaced with plastic mats for easier collection of excreta. Excreta sampling from each experimental unit was done for six consecutive days of the last week of the experimental period, d 37-42. Excreta were collected using a blunt-flat scraper from the floor quantitatively for 24 h at 2x/day (06:00 and 18:00), pooled and weighed daily and kept frozen (-20 ˚C) during the sampling days. Only clean and uncontaminated excreta were collected from several predetermined locations of each pen. Finally, samples were pooled per pen, homogenized with a mixer (1-2 min), and sample of known weight (about 300 g/pen) was taken in plastic bags from each experimental unit. Excreta samples were then freeze-dried to stable weight and kept at -20 ˚C till analysed for their nutrient and marker concentrations (Schaafstra et al. 2017). Feed and water were given ad libitum across the collection period.
Measurement of apparent digestibility coefficients
Freeze-dried excreta samples and the experimental diets, 0, 10, 20 and 30% garra meal formulations, were all analysed for their individual nutrients. The DM, CP and EE contents were evaluated according to AOAC (1990). The CP contents were evaluated using the Kjeldahl method as described by AOAC (2001) and the TiO2 contents as per the procedures stated by Myers et al. (2004). All samples were evaluated in duplicates at Ghent University, Belgium. Obtained mean values were used for the calculation of apparent digestibility coefficients of individual nutrients using the biomarker and the following formula:
Evaluation of chicken blood biochemical profiles
Blood sample collection and processing
Blood samples (5 mL/chicken) were drawn from wing veins of three randomly selected chickens from each experimental unit at the end of the starter (d 22) and grower phases (d 43). Blood samples were allowed to coagulate at room temperature for 15 min, then centrifuged (1,500 × g for 10 min) and serum were collected in Eppendorf tubes and stored at -20 °C until analyses. The same chickens were traced and sampled for their serum in round two blood sampling.
Measurement of biogenic amines and oxidative stress biomarkers
Serum samples (100 µL) were evaluated for histamine concentration. Following sample preparation and acylation, histamine quantification was performed using Enzyme-Linked Immunosorbent Assay (ELISA) following the manufacturer’s kit protocol (Ridascreen® Histamine (enzymatic), Art No. R1605) at Ghent University, Belgium. Serum (150 µL) malondialdehyde concentrations were analysed as thiobarbituric acid reactive substances (TBARS) according to the method of Grotto et al. (2007) at Lanupro, Ghent University.
For the evaluation of shifts in nutrient metabolism, profiling of acylcarnitine concentrations were done. A drop (50 µL) of blood was taken from each of the whole blood samples collected and placed on circular specimen collection papers (Whatman protein saver cards, 903™, UK). Each specimen kit was allowed to dry, and later sent to Ghent University Hospital (Laboratory for Clinical Chemistry) for their biochemical assay.
Evaluation of intestinal histo-morphological changes
Intestinal sample collection
At the end of the experimental feeding period, following 12 h fasting, the traced 3 chickens from each experimental unit were sampled for their intestines on d 42. Chickens were weighed and then euthanized humanely using sodium pentobarbital (IP, 28 mg/kg BW) to death. The entire small intestine segment was exposed under aseptic post-mortem examination.
A fixative solution (mixture of glutaraldehyde [3%] and paraformaldehyde [4%] fixative solution in 0.1 mol/L cacodylate buffer [pH 7.4]) was prepared and injected into the intestinal lumen of the middle part of each intestinal segment. The entire small intestine spanning from the gizzard to pancreatic and bile duct (duodenum), from bile duct to Meckel’s diverticulum (jejunum), and from diverticulum to the ileo-caecal-colonic junction (ileum) was then excised and placed into separate bottles containing the same fixative solution. Similarly, the cecum part were also separately removed and placed in the same fixative solution until morphologically evaluated.
Gut tissue samples (2cm long) were collected from the middle part of each intestinal segment, dehydrated under graded alcohols and xylene, and then embedded in paraffin. Serial paraffin sections (5 μm) were made from samples and kept at 37 °C for 12 h or more. Then after, sample sections were prepared in slides and stained with haematoxylin-eosin stain following a routine protocol. Finally, the mucosal structures were observed under a Nikon phase-contrast microscope and examined using MicroComp integrated digital image-analysis system with the help of Microscope camera. The villous height and crypt depth of 10 well-oriented villi were measured per section. The average value for each tissue was obtained from 3 sections per tissue sample. That is, 10 villi/crypt per section and 3 sections per sample were measured and the average value used for statistical analysis (Fan et al. 1997). Furthermore, the villus height to crypt depth ratio was also calculated for each value obtained.
Evaluation of intestinal permeability using the everted sac test (ex vivo)
The effect of garra meal supplements in the intestinal permeability were evaluated. For this, 20 cm segment of the hind intestine (ileum part) was cut and washed in phosphate buffer saline (PBS) solution along the hollow, weighed, and then kept in an ice water mounted petri dish. The hollow segment was then inverted using a small crochet needle inserted into the lumen. One part of the lumen was ligated while filled with the PBS solution from an elevated container through the other end of the sac and then ligated on either side. The sacs were fully immersed and incubated in a glass beaker filled with PBS solution containing a cobalt marker with a constant oxygenation through aqua-aerators. The glass beakers were kept in an open water bath set up at 39.3 ± 0.8 °C for an hour. Then after, sacs were taken out, rinsed with water, and 10 ml of the intestinal fluid contents were collected with syringe into a tube and stored at until analysed. Similar 10 mL sample were collected from the ringers’ solution with markers at the start of the experiment for conjoint evaluation. Spectrophotometric reading of cobalt concentration was done at 640 nm (Hamilton 2014).
Data management and analysis
The different data obtained from performance indicator parameters, blood biochemical profiles, and intestinal measurements were entered into a spreadsheet Excel separately and analysed accordingly. Data from experimental diets (four percent levels) were subjected to one-way analysis of variance (ANOVA) and analysed using SPSS software (version 27.0). Every replicate was considered as the experimental unit for all statistical analyses. In either case, results were expressed as treatment means with their pooled standard error of the mean (SEM). A P < 0.05 probability value was described to be the level of significance and the notable differences between the treatment groups were determined via Tukey multiple comparisons test.