Chapter 4 The effect of Hermetia illucens (black soldier fly) larvae on the blood haematology and
4.3 Research design and methodology
4.3.3 Data collection and analyses
Blood withdrawals were performed at 9 (before treatment diets were administered), 18 (middle of feeding scheme) and 26 days of age (end of trial). The veterinary services generously offered by the Elsenburg experimental farm, Western Cape were utilized for blood collection.
NOTE: In the current trial, great difficulty was experienced by the Animal Technician at each
withdrawal period, where in the attempt to withdraw blood samples from the animals there were multiple punctures required for sample collection. This also leading to the prolonged handling of each piglet, which in respect put a great amount of stress on the animal. Initially the use of a 21 gauge needle and syringe was to be utilized and blood injected into the microtainer tubes, but due to difficulty of withdrawal, the Animal Technician made an onsite decision to use 4 mL vacutainer tubes and to transfer the blood samples from these tubes to the microtainer tubes. The Animal Technician stated that there should not be any significant influences on the blood cell count. Animals also received an iron injection at 3 days of age (normal farming practises).
Assumptions
I. Normality between the piglets within a litter. II. Normality between the litters.
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4.3.3.1 Samples collected in EDTA
Samples were collected utilizing 21 gauge needles and 4 mL K3EDTA vacutainer tubes and blood
was transferred to two 0.5 mL K3EDTA microtainer tubes, as to achieve duplicate samples for testing
(Method adopted by Animal Technician). Samples were placed onto ice until testing was conducted, which was achieved within 5 h after withdrawal before any deterioration was to occur. Tests were performed utilizing a Cell-Dyn 3700 Haematology Analyzer (Abbott Diagnostics, USA) at the Animal Science department, Stellenbosch University, Western Cape, South Africa. These tests included the counts for White Blood Cells (WBCs) and differential WBCs (Neutrophils, Lymphocytes, Monocytes, Eosinophils and Basophils), Red Blood Cells (RBCs), Haemoglobin (HGB), Haematocrit (HCT), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), Red Blood Cell Distribution Width (RDW), Platelets (PLT) and Mean Platelet Volume (MPV).
Cell-Dyn 3700 Haematology Analyzer protocol
The mode of aspiration was done by means of the Open Sampler Mode, which was used to aspirate the sample from a collection tube that had been opened and held under the Open Sample Aspiration Probe. The aspiration volume for this particular mode was 130 µL ± 5%, where the sample was aspirated into the Analyzer by the Aspiration Peristaltic Pump, through the Shear Valve.
The white blood cell (WBC) analysis was performed by taking two measurements, namely the WBC optical count (WOC) and WBC impedance count (WIC). The WOC Sheath Syringe dispensed 1.6 mL of sheath reagent through the Shear Valve, picking up 32 µL of the sample. The sample segment and sheath were then routed to the Mixing Chamber where the dilution was bubble mixed with a final dilution of 1:51. The Peristaltic Pump transfered the dilution from the Mixing Chamber to the Sample Feed Nozzle in the Flow Cell and a stream of sheath reagent was directed through the cell. A metering syringe injected 78 µL of the dilution into the Flow Cell-sheath stream and a laser beam was focused on the Flow Cell and as the sample stream intersected the laser beam, the light scattered by the cells was measured for different angular intervals.
The WIC Diluent Syringe dispensed 5.25 mL of diluent through the Shear Valve, picking up 20 µL of the sample segment. The segment and diluent was routed to the Mixing Chamber in the von Beherns WIC/HGB Transducer and the Lyse Syringe delivered 0.75 mL of the Lyse to the Mixing Chamber simultaneously. The dilution was bubble-mixed (final dilution is 1:301) and pulled through the aperture by vacuum, where the Volumetric Meter ensured that 200 µL was utilized for measurement. The Electrical Impedance was then used to count the WBCs as the dilution passed through the aperture. The remaining dilution was transferred to the Haemaglobin (HGB) Flow Cell and the HGB concentration was measured spectrophotometrically.
The red blood cell (RBC) and platelet (PLT) analysis were also performed, where the diluent syringe dispensed 7.2 mL of the diluent through a Shear Valve, picking up 0.74 µL of the sample segment. The sample segment and diluent was routed to the Mixing Chamber of von Behrens RBC/PLT Transducer where the dilution was bubble-mixed (final dilution is 1:9760). The dilution was pulled through the aperture by vacuum and the Volumetric Meter ensured that 100 µL of the dilution was measured. An Electrical Impedance was used to count the RBCs and PLTs as they passed through the aperture.
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4.3.3.2 Samples collected in serum
Samples were collected utilizing 21 gauge needles and 4 mL Serum vacutainer tubes and blood was transferred to two 0.6 mL Serum microtainer tubes, as to achieve duplicate samples for testing. Samples were placed onto ice and centrifuged within 5 h at 3500 rpm at 4°C for 10 min utilizing a Spectafuge 24D (Labnet) centrifuge. The serum was separated by pipet from other blood components and frozen in 0.5 mL Eppendorf tubes at -20°C within 5 h after withdrawal before any deterioration of blood samples was to occur. Tests were performed utilizing a Siemens Advia 2120 Analyzer at the Haematology department, Tygerberg, National Health Laboratory Service (NHLS), Western Cape, South Africa. These tests included the concentrations of albumin, calcium, phosphorus and iron, as well as the concentration of the major serum constituents of antibodies which are IgA, IgG, and IgM. Albumin was tested as to calculate the corrected calcium concentration of the blood samples.
Note: The Siemens Advia 2120 Analyzer is calibrated specifically for human blood samples,
however, piglet samples were tested by before the onset of the trial. Duplicate samples were tested by the analyser and then also microscopically, and proved to provide accurate results.
Siemens Advia 2120 Analyzer protocol I. Calcium
The calcium ions form a coloured complex with Arsenazo III, which was measured at 658/694 nm. The amount of calcium present in the sample was directly proportional to the intensity of the coloured complex formed.
Equation 4:1
Ca2+ + Arsenazo III Ca-Arsenazo III Complex (purple)
II. Albumin
Serum or plasma albumin quantitatively binds to Bromocresol Green (BCG) to form an albumin-BCG complex that was measured as an endpoint reaction at 596/694 nm.
Equation 4:2
BCG + Albumin BCG Albumin complex
III. Corrected Calcium
The corrected calcium was a calculated value based on the total measure of calcium (mmol/L) and albumin (g/L). The equation was as taken from Burtis et al. (2012).
Equation 4:3
Corrected calcium (mmol/L) = 0.02 [40 – Albumin (g/L)] + Calcium (mmol/L)
IV. Phosphorus
Inorganic phosphorus reacted with ammonium molybdate in the presence of sulfuric acid to form an unreduced phosphomolybdate complex, which was measured as an endpoint reaction at 340/658 nm.
Equation 4:4
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V. Iron
Ferric iron was separated from its carrier protein, transferrin, in an acid medium and simultaneously reduced to the ferrous form. The ferrous iron was then complexed with ferrozine, a sensitive iron indicator, to produce a coloured chromophore, which absorbs at 571/658 nm.
Equation 4:5
Transferrin (Fe3+) Apotransferrin + Fe3+
Fe3+ +Ascorbic Acid Fe2+
Fe2+ + Ferrozine Fe2+/Ferrozine Complex
VI. Immunoglobulins
A PEG-enhanced immunoturbidimetric method was used for blood IgM and IgA, where they were diluted and then reacted with a specific antiserum to form a precipitate that can be measured turbidimetrically at 340/694 nm. A standard curve was constructed for each from the absorbencies of standards and the concentration of both IgM and IGA was determined.
IgG was determined utilizing a PEG-enhanced immunoturbidimetric method, where it was diluted and then reacted with a specific antiserum to form a precipitate that can be measured turbidimetrically at 340/694 nm. A calibration curve was constructed from the absorbencies of calibrators and the concentration of IgG was determined.
4.3.3.3 Statistical analysis
As there were a number of variables experienced when recording the data, an exploratory analyses was done to determine whether or not their influence was sufficient to warrant their inclusion in the final model for analyses.
The exploratory analyses included fitting the full model and combination of variables using the PROC GLM of SAS for Windows Version 9.3 (statistical software). A probability of P<0.05 was used to determine significance. The statistics were done using analysis of covariance with least square means (LSM) calculated with Bonferroni post hoc test. First, a full model was analysed with the main factor of parities being tested, with farrowing dates included as blocks. Age (included as blood sampling periods) and number of piglets per litter were also included as co-variables in the different models used for analysis. Due to parities, blocks and number of piglets per litter having no significant effects, these were excluded from the models. Breed was also excluded due to the very few Landrace animals included in the experiment.
After exclusion of all these variables, the final model included the variables of age (included as blood sampling period) and treatment. The piglet blood metabolite concentrations were analysed by means of PROC GLM for treatment with each withdrawal period being analysed separately. Descriptive statistics and graphs of treatments and age interaction for each blood metabolite are included to provide a graphical representation of the change in concentrations over time.
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