Primacs100 analyzer

Proximate composition of experimental diets and whole fish samples were measured. Moisture was analyzed by drying the samples to constant weight at 105 °C. Crude lipid was measured by petroleum ether using the Soxhlet method. Crude protein was determined using the Dumas Nitrogen method by Primacs 100 analyzer (Skalar, Dutch) and estimated by multiplying nitrogen by 6.25.

Following a feeding period of 56 days, the shrimp in each tank were subjected to 24 h fasting. The weight of the shrimp was measured and the survival rate was determined through calculations. To determine the moisture content, crude lipid, and crude protein levels, a random sample of five shrimp was chosen from a tank. The hemolymph of five shrimp per tank was collected and subsequently centrifuged at 4 °C and 3500 rpm for 15 min. After the hemolymph supernatant was obtained, it was carefully collected and immediately preserved at a temperature of −80 °C. Hepatopancreas of four shrimp were taken, washed in normal saline (0.9% NaCl solution), then transferred to liquid nitrogen, and then stored at −80 °C for subsequent analysis. The intestine and hepatopancreas were collected from four shrimp, kept in RNA later reagent (Ambion^®, ThermoFisher, Waltham, MA, USA), and stored at −80 °C.
The whole shrimps underwent a drying process in a 105 °C oven, which was carried out with the aim of assessing their moisture content. The determination of the crude protein content was carried out using the Dumas Nitrogen method with a Primacs100 analyzer (Skalar, Breda, Dutch). For the determination of the crude lipid, the XT15 extractor (Ankom, NY, USA) was utilized. Additionally, the amino acid content was analyzed in accordance with the standard GB/T 18246-2019.

After a 4-week feeding trial, shrimp in each tank were counted and weighed. Moisture of diets was determined by oven drying at 105°C: weight reduction of feed after drying. Crude protein of feces and diets was detected by Primacs100 analyzer (Skalar, Dutch): after full combustion of the feed, the nitrogen oxides are reduced to nitrogen (crude protein = Total − N × 6.25). Crude lipid was detected by an XT15 extractor (Ankom, USA): weight reduction of feed after extraction by petroleum ether. Ash was detected by burning at 550°C: weight reduction of feed after fully burning [32 , 33 ]. The amino acid compositions of ingredients were determined by an automatic amino acid analyzer 433D (Sykam, Germany) after hydrolysis in 6 M HCl for 24 h at 110°C. After being digested with nitric acid and hydrogen peroxide (6 mL 68% nitric acid and 1 mL 30% hydrogen peroxide) by microwave digestion (Anton Paar Multiwave PRO 41HVT56, Austria), samples were conducted in an inductively coupled plasma-mass spectrometer (ICP-MS, Agilent 7500cx, USA) to determine the phosphorus content. The nutrient levels and amino acid composition of diets are shown in Table 3.