Precellys evolution super homogenizer

In December 2021, a suspected outbreak of DPV occurred on a farm with 5000 hemp ducks at approximately 310 days of age in Qingyuan city, Guangdong, China. Clinical signs of depression, inability to stand, inappetence, white-green diarrhea, watery dejecta, corneal opacity, and sudden death were observed. The number of sick ducks increased daily by 15–30, with a mortality rate of about 30%. There was no obvious effect after treatment with antibiotic drugs. Ducks suspected of having died from DPV infection and those showing typical clinical signs of DP were collected. Tissues from the heart, liver, spleen, kidney, and pancreas (0.5 g) were collected in 1 mL of phosphate-buffered saline (PBS) (pH = 7.4). The tissues were then homogenized with glass beads for 2 cycles at 6000 rpm for 10 s using a Precellys Evolution Super Homogenizer (Bertin Technologies, France). The homogenates were centrifuged for 5 min at 12,000 rpm and the supernatant was collected and stored at −80 °C.

MFI was measured with reference to Hopkins [26 (link)] with some modifications. Exactly 0.3 g of raw meat was weighed and added with 4.5 mL of MFI buffer (0.1 M KCl, 7 mM KH₂PO₄, 18 mM K₂HPO₄, 1 mM EDTA·2Na, pH 7.0, 4 °C). The mixture was homogenized (Precellys Evolution Super Homogenizer, Bertin Technologies, Montigny-le-Bretonneux, France) twice at 10,000 rpm, 20 s each time. Then, the homogenate was filtered through two layers of medical gauze and centrifuged (Avanti J-E, Beckman Coulter, Inc., Brea, CA, USA) at 1000× g and 4 °C for 10 min. The centrifugal pellet was collected and added with 5 mL of MFI buffer and centrifuged twice. The precipitate was dissolved with 3 mL of buffer, and the myofibrillar protein (MP) solution for MFI was obtained and measured using biuret method with BSA as the standard protein. The protein concentration was adjusted to 0.5 mg/mL. The absorbance at 540 nm was determined immediately. MFI values were obtained by multiplying the absorbance at 540 nm by 200.

Tissues from left ventricles and left lobe of liver were selected. Long bones were flushed with ice-cold phosphate-buffered saline to remove residual cells within bone marrow spaces. Bone specimens were chopped into small pieces. Whole brain tissues were placed with dorsal surfaces up on the cutting block. Razor blades were placed in fixed position to obtain coronal sections. In each section, hypothalamus was located and dissected as previously described (Heffner, Hartman & Seiden, 1980 (link)). Specimens from the kidneys were obtained after the surrounding connective tissues and renal capsules were carefully removed, they were then chopped into small pieces. Heart, liver, hypothalamus, and kidney were homogenized for RNA isolation following a protocol previously described (Tiyasatkulkovit et al., 2019 (link)). Bone tissues were homogenized using a Precellys Evolution Super Homogenizer with ceramic beads according to the manufacturer’s protocol (Bertin Instruments, Montigny-le-Bretonneux, France). Total RNAs were extracted using TRIZol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA quality was assessed by measuring the absorbance at 260 and 280 nm using a NanoDrop-2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the 260/280 ratio ranged between 1.8 and 2.0.