Enzyme linked immunosorbent assay kit

Fasting blood samples (5 ml) were collected from all participants at the beginning and end of the study and were immediately centrifuged (3000×g, 10 min, 4°C). Blood samples were poured into anticoagulant tubes to extract serum samples and were sent to the laboratory in cool boxes. All samples were stored at −70°C until biochemical analyses. Serum glucose, triglyceride (TG), HDL and TC was measured by standard enzymatic methods using the Pars Azmoun kit (Tehran, Iran). Serum insulin was measured by a human insulin enzyme-linked immunosorbent assay kit (Monobind Inc.). Insulin resistance was estimated according to the homeostatic model assessment of insulin resistance (HOMA-IR), calculated as: HOMA-IR = fasting concentrations of glucose (mg/dl) × fasting insulin (μU/ml) / 405 [22 ]. The Friedewald formula was used for the calculation of LDL-C [23 ]:
LDL-C (mg/dl) = TC (mg/dl) − HDL-C (mg/dl) − TG (mg/dl)/5, where TG/5 is used to represent very-low-density lipoprotein cholesterol.
Serum markers of oxidative stress such as total antioxidant capacity (TAC) and malondialdehyde (MDA) were measured by reliable spectrophotometric methods using Zell Bio GmbH kit (Germany). Serum levels of high-sensitivity C-reactive protein (hs-CRP) were measured by enzyme-linked immunosorbent assay kits (Monobind Inc.).

Following cardiac functional recordings on closed-chest animals, a left thoracotomy exposed the heart and blood was obtained from the right ventricle. Serum was collected, aliquoted and stored at − 20 °C until it was analyzed for free T3, total T3, total T4 and TSH using enzyme-linked immunosorbent assay kits (Monobind Inc., Lake Forest, CA) following the manufacturer’s recommended protocol.

Preprandial blood samples were collected from the jugular vein of each doe on days −21, −14, −7, 0, 7, 14, and 21 relative to parturition. The blood sample was collected into an evacuated heparinized tube, and plasma was separated, followed by centrifugation at 3000× g for 15 min; it was then stored (−80 °C) until analysis.
Plasma glucose was measured by an autoanalyzer (BT 1500, Biotecnica SpA, Rome, Italy) using commercial kits (Pars Azmoon Co., Ltd., Tehran, Iran) according to the manufacturer’s instructions. BHBA and NEFA were determined by commercial colorimetric kits (Randox Laboratories Ltd., Ardmore, UK) using the same autoanalyzer. The serum insulin level was measured using an enzyme-linked immunosorbent assay kit (Monobind Inc., Lake Forest, CA, USA). Intra- and inter-assay coefficients of variation for measuring insulin were 6.9 and 8.2%, respectively.
The revised quantitative insulin sensitivity check index (RQUICKI) and RQUICKI including BHBA, relative insulin sensitivity measures, were estimated as RQUICKI = 1/[log (glucose) + log (insulin) + log(NEFA)], and RQUICKI_BHB = 1/[log glucose + log insulin + log NEFA + log BHB] according to Abuelo et al. [36 (link)].

Blood (10 mL/goat) was collected from each animal after overnight fasting, once weekly from 21 days before expected kidding, up to 21 days of lactation. Blood was harvested from the jugular vein using heparinized blood collection tubes (RotexMedica, Trittau, Germany). Blood samples were centrifuged at 3000× g at room temperature for 15 min. Plasma was obtained and stored at −80 °C until analyzed. Plasma samples were analyzed for concentrations of albumin, cholesterol, glucose, and bilirubin using commercial kits (Pars Azmun Co., Ltd., Tehran, Iran) by an autoanalyzer (BT 1500, Biotecnica SpA, Rome, Italy). Plasma haptoglobin and ceruloplasmin were determined using commercial test kits (Randox Laboratories Ltd., Ardmore, UK). Plasma paraoxonase (PON) activity was measured by adapting the method of Ferre´ et al. [24 (link)]. Serum insulin level was measured using enzyme linked immunosorbent assay kit (Monobind Inc., Lake Forest, CA, USA). In addition, BHBA and NEFA were measured using commercial kits (Randox Laboratories Ltd., Ardmore, UK).