Anaesthesia and changes in parameters that reflect glucose metabolism in pigs – a pilot study

Animals

In total, five specific pathogen-free pigs (Yorkshire × Swedish Landrace × Hampshire) from the same litter, two males and three females (prepubertal), were obtained from the university herd (Swedish Livestock Research Centre, Lövsta, Sweden). One pig was difficult to ventilate during experiment 1; thus, it did not participate in experiment 2. Instead, a reserve sibling was used in experiment 2, together with the other three pigs. The animals were six weeks old upon arrival; after a two-week acclimatisation period their mean weight was 33.6 ± 1.6 kg. They were housed at the Department of Clinical Sciences in individual pens measuring 3 m², within sight and sound of one another. Straw and wood shavings were used as bedding. A 14:10 h light/dark schedule (lights on at 06:00 h) was used, and an infrared lamp (24 h) was provided in each pen. The room temperature was 16–18℃. The pigs were fed a commercial finisher diet without growth promoters (SOLO 330, Lantmännen, Sweden) twice daily (07:00 and 15:00), the amount of which was dependent on their body weight (BW) and according to the Swedish University of Agricultural Sciences regimen for growing pigs. ²⁰ Water was provided ad libitum. During the two-week acclimatisation period the animals were handled frequently, trained to be bottle-fed with glucose dissolved in water and to step onto an electronic scale. The pigs were bottle-fed every day during the experimental period.

Surgical implantation of an indwelling venous catheter

After the acclimatisation period, the pigs underwent surgery and an indwelling silicon catheter (SIL-C70 with rounded tip; Instech Solomon, Plymouth Meeting, PA, USA) was inserted into the vena jugularis dextra under general anaesthesia as previously described. ³ The surgical procedure was completed within 45 min. The catheters were flushed twice daily with heparinized saline 100 IE/mL (Heparin LEO, 5000 IE/mL; Leo Pharma, Ballerup, Denmark) and facilitated frequent blood sampling without stress.

In connection with the surgical procedure, venous blood samples were obtained. Blood preserved in EDTA was analysed for total and differential white blood cell (WBC) counts and haemoglobin (Hb) by an electronic cell counter validated for porcine blood (Advia 2120; Siemens, Erlangen, Germany). Serum samples were analysed for creatinine (CREA), and enzyme activities of aspartate amino transferase (ASAT), alanine amino transferase (ALAT), γ-glutamyltransferase (GT) and glutamate dehydrogenase (GLDH), by an automated equipment (Architect C4000; Abbott Diagnostics, North Ryde, Australia).

Experiment 1

Two anaesthetic protocols were evaluated for their effect on blood glucose levels. Four pigs were anesthetized using both protocols, with five days’ washout between the different protocols. Anaesthesia was induced in the pig’s home pen without any pre-anaesthetic treatment. After induction the pigs were placed on a stretcher and intubated while still in the stable, thereafter they were moved to the surgical ward once the endotracheal tube was in place. On the way to the surgical ward (1–2 min) the pigs were manually ventilated with a bag valve, if needed. In the surgical ward the endotracheal tube was connected to a pressure-controlled ventilator (FLOW-i; Maquet Critical Care AB, Solna, Sweden), and the airways were ventilated to maintain an end-tidal carbon dioxide (CO₂) of approximately 5 kPa. Ringer acetate (RAc) infusion (5–10 mL/kg/h) was provided as maintenance therapy. During anaesthesia, respiratory rate, respiratory minute volume, positive end expiratory pressure, peak inspiratory pressure, end-tidal CO₂, inspired/end-tidal oxygen (O₂), O₂ saturation, heart rate, non-invasive blood pressure (cuff placed on upper forelimb), RAc infusion rate, rectal temperature (measured repeatedly with a digital thermometer), palpebral reflex, nystagmus, shivering and anaesthetic infusion rate were monitored and noted every 15 min. The above-listed physiological parameters were measured with Datex Ohmeda S/5 monitors (Datex-Ohmeda, Inc, Madison, WI, USA). The depth of anaesthesia was assessed for adequacy by checking for absence of spontaneous movements, absence of palpebral reflex and absence of response to manipulation of the extremities and the endotracheal tube. Blood glucose was measured before anaesthesia, and at 5, 10, 20, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 and 180 min after the induction of anaesthesia.

Experiment 2

Four pigs underwent OGTT without anaesthesia, and subsequently OGTT with anaesthesia (protocol 2), which was induced after glucose intake. Oral glucose tolerance was tested after an 18 h overnight fast. Water was withheld one hour before the experiment. The pigs were bottle-fed with an oral glucose load (orally soluble glucose, Glukos; APL, Stockholm, Sweden) of 1.75 g/kg BW, maximum 75 g, mixed with 2 mL of water per gram of glucose. The glucose solution had to be consumed within 5 min.

Blood samples were collected before and at 5, 10, 20, 30, 45, 60, 90, 120 and 180 min after glucose intake. Blood samples were collected in BD P800 tubes (BD Diagnostics, Franklin Lakes, NJ, USA) containing protease-, esterase- and dipeptidyl peptidase-4 inhibitors, and EDTA for measurements of insulin and glucagon. Blood glucose was measured immediately; the blood samples were then centrifuged, and plasma was stored at –80℃ until analysis.

Glucose, insulin and glucagon analyses

Blood glucose concentrations were measured with a blood glucose meter (Accu-Chek, Roche Diagnostics, Basel, Switzerland; validated for porcine blood at the Department of Clinical Chemistry, SLU, Sweden). Plasma concentrations of insulin were determined by Porcine Insulin ELISA (Mercodia, Uppsala, Sweden). All samples were run in duplicate with a coefficient of variance (CV) of < 10%. Results were converted from ug/L to pmol/L using a conversion factor of 174 as recommended by the manufacturer. Plasma concentrations of glucagon were analysed by Glucagon ELISA (Mercodia, Uppsala, Sweden). All samples were run in duplicate with a CV of < 10%.

Euthanasia and post-mortem examination

The pigs were euthanized with an IV overdose of pentobarbital sodium (Euthasol vet, 400 mg/mL; Virbac) at the end of the experiment. The pigs underwent gross examination post-mortem.

Statistical analyses

All values are presented as mean ± standard error of the mean (SEM). Area under the curve (AUC) values are presented as the net area, and were calculated using the trapezoid rule with the 0 min value as baseline. Comparisons between groups were determined by Kruskal–Wallis one-way analysis of variance on ranks with Dunn’s post hoc test. P values of < 0.05 were considered to be statistically significant.

Experiment 1

Anaesthesia induction was smooth with both the anaesthetic protocols; the pigs lay down calmly and could be lifted onto the stretcher for prompt endotracheal intubation after drug administration. The time from induction until the endotracheal tube was connected to the ventilator in the surgical ward (approximately 5–10 min) was similar for both protocols. After TZB induction, the pigs continued to breathe spontaneously without any need for manual ventilation; while all pigs had to be manually ventilated after propofol induction until the endotracheal tube was connected to the ventilator. Initial tachycardia (Figure 1) and low blood pressure (data not shown) were observed with propofol induction. Two of the pigs shivered during both anaesthetic protocols while the other two pigs did not. Apart from the above-mentioned changes, no abnormal physiological parameters were noted. For the TZB protocol, doses of 5–6.25/0.2–0.25 mg/kg/h were sufficient to produce general anaesthesia, whereas doses of 13–20 mg/kg/h of propofol were needed to produce the same effect. OPEN IN VIEWER

During TZB anaesthesia blood glucose was within the reference range throughout the 180 min (range 3.6–6.8 mmol/L) while hyperglycaemia was observed in three out of four pigs during propofol anaesthesia within 5–10 minutes (peak levels range 8.3–15.4 mmol/L), see Figure 1. Blood glucose levels were within the reference range within 20–105 min after induction of anaesthesia. Due to the hyperglycaemia observed after propofol induction, TZB was chosen for further evaluation with OGTT.

Experiment 2

Blood glucose levels, plasma insulin and glucagon concentrations, and AUC values during OGTT are presented in Figure 2. During OGTT without anaesthesia, blood glucose levels increased, with peak levels at 30 min (7.1 ± 0.3 mmol/L). When anaesthesia was induced immediately after glucose intake, blood glucose increased by 0.6 mmol/L in one pig and none at all in a second pig. Consequently, a 15 min delay was introduced between glucose intake and anaesthesia in the remaining two pigs. In these two pigs, the initial increase in blood glucose was comparable to the values from OGTT without anaesthesia, with peak levels of 7.4 and 6.5 mmol/L at 20 min. However after 20 min, blood glucose concentrations fell more rapidly, and the AUC of glucose was lower than during OGTT without anaesthesia. OPEN IN VIEWER

During OGTT without anaesthesia, peak insulin levels (42.1 ± 5.9 pmol/L) and glucagon nadir (2.1 ± 0.3 mmol/L) were observed at the 20 and 30 min sampling periods, respectively. During OGTT with anaesthesia, peak insulin levels were also observed at 20 min after glucose intake; however, peak values were lower, both when anaesthesia was induced immediately (80% lower) and 15 min after glucose intake (25% lower). The AUC of insulin was lower with anaesthesia than without; this difference did not reach statistical significance, but the insulin AUC was more than 10 times higher during OGTT without anaesthesia. When anaesthesia was induced immediately after glucose intake, glucagon levels were suppressed by 0.9 mmol/L, while the glucagon suppression was 6.3 mmol/L during the first 30 min when anaesthesia was delayed, which is comparable to the suppression of OGTT without anaesthesia. With anaesthesia, glucagon levels increased in all pigs during the second half of the OGTT, especially between 120 and 180 min. In one pig, glucagon levels increased to 153.4 mmol/L at 180 min; in this pig the corresponding blood glucose concentration was 2.2 mmol/L.