International Journal of Aquaculture, 2024, Vol.14, No.1, 9-19 http://www.aquapublisher.com/index.php/ija 16 Experiment 2 determined the effect of varying storage temperature on survival at a constant storage density for 14 days. The varying temperature settings were (4, 8 and 12 °C) with each treatment comprising 42 glass jars (N=42). These temperature variations were chosen based on previous studies indicating that copepods can still survive at moderate temperatures (4 °C~15 °C) (Mäkinen et al., 2017; Werbrouck et al., 2017). The copepod density in all jars was 1 000 individuals/L. The jars were distributed in three separate fridges, each set at the target temperature. Each treatment had a total of three replicates and N=42. 3.4 Estimation of survival of Thermocyclop sp. The culture media from each specimen bottle was stained using intra-vitam staining with neutral red stain, a method for separating live and dead copepods in natural samples. Live Thermocyclop sp. stain red by filtering and ingesting the dye, whereas dead copepods remain unstained as they don’t ingest the dye (Drillet et al., 2015). Survival was estimated by computing the proportion of red-stained copepods (live) against the initial storage density and temperature in the preservation bottles over a 14–day experimental cycle. When using intra-vitam staining with neutral red stain for separating live and dead copepods in natural samples, it's essential to take certain precautions to ensure the reliability and accuracy of the results. For example, it ensures the quality and freshness of the neutral red stain as it can degrade over time, potentially leading to variations in staining intensity and affecting the accuracy of live/dead differentiation. 3.5 Fatty acid composition The fatty acid composition of the Cycloid copepods stored at 4, 8 and 12 °C was also analyzed. Thermocyclop sp. were collected on the 7th and 14th days of the experiment and stored at -80 °C for further analysis. Before extraction, individual Thermocyclop sp. were removed from the deep freezer (-80 °C) and thawed for at least one hour under standard laboratory conditions. Lipid extraction followed a protocol by (Evjemo et al., 2003). 0.5 ±0.1 g of each sample was weighed and then ground to get a homogeneous powder. It was then used for extraction with chloroform–methanol mixture (2:1, v/v). Each sample was then filtered and transferred into a separating funnel and added to a 0.2 volume of 0.9% sodium chloride. The samples were shaken to allow two distinct layers to separate; the lower chloroform phase was transferred into a pre-weighed tube and then evaporated using a stream of nitrogen gas until a constant weight of the lipid was achieved. The fatty acid composition was determined using gas chromatography–mass spectrometry analysis following (Kwetegyeka et al., 2011). Approximately 10 mg of the lipid extract was transferred into a reaction vial containing an internal standard, nano decanoic acid (C19:0), and 1.0 mL of acidified methanol. The vials were securely closed with Teflon-lined screw caps and placed in an oven for two hours at 90 °C to allow complete methanolysis. Finally, the vials were removed from the oven and then allowed to cool at room temperature. Subsequently, methanol was evaporated to half its original volume by a stream of nitrogen to make methyl esters less soluble in the methanol phase. Then, 0.5 mL distilled water, followed by 1 mL hexane, was added to the methanolized lipid fraction. The tubes were capped tightly and shaken at least 4 times to allow mixing, followed by centrifugation to separate the phases. The upper hexane layer containing fatty acid methyl esters (FAMEs) was carefully transferred to the vial using the pipette. The water–methanol phase was extracted twice using 1.0 mL n-hexane. The extracts were pooled and stored under refrigeration, awaiting GC–MS analysis. The samples were then quantitatively analyzed using GC–MS equipment (Agilent 6890-version N.05.05, GC-System, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, USA) fitted with an electronic pressure control and mass selective detection (ionizing energy, 70 eV; source temperature, 250 °C). The injector temperature was set at 260 °C while the detector was set at 330 °C. The oven program was set at 90 °C for 4 min, 30 °C/min to 165 °C, then 3 °C/min to 225 °C where it was left to isothermal for 10.5 minutes before cooling for the next run. Fatty acids were quantitatively identified in the samples by means of the standard mixture and mass spectrometry, quantified using internal standard C19:0. The peaks were further integrated using Chemstation software (Thermo Lab Systems), and the contribution of each of the fatty acids was calculated based on their relative retention times and peak areas. The relative amount of each common fatty acid is expressed as a percentage of the total fatty acids.
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