Effects of Three Anesthesia Drug Combinations in Miniature Pigs.

Background To compare the effects of three anesthesia drug combinations in miniature pigs. Methods Guizhou miniature pigs (cid:0) 15 male, 15 female, 22-24 months (cid:0) were randomly divided into three groups (n=10, 5 male, 5 female) and anesthetized intramuscularly (neck) with sumianxin (cid:0) (0.1 ml/kg) and 3% pentobarbital (0.8 ml/kg; Group A); ketamine (10 mg/kg) and 3% pentobarbital (0.8 ml/kg; Group B); or ketamine (10 mg/kg) combined with sumianxin II (0.1 ml/kg; Group C). Induction time, maintenance time, recovery time, and mortality were recorded. Heart rate, respiration, mean arterial pressure, and body temperature were analyzed pre-anesthesia (T0), after induction (T1), 30 min after operation (T2), 60 min after operation (T3), and at the end of anesthesia (resuscitation, T4). Results There were no signicant differences in body weight, length, or experimental period between animals in the three treatment groups. The induction times of groups B and C (2-3 min) were signicantly shorter than that of group A (7.4±1.1 min). The maintenance time of group B (136±11.4 min) was signicantly different from those of groups A and C. The recovery time of group C was 4±1.0 min, compared with 9±0.6 min in group A and 10±1.5 min in group B. In group A, ve pigs had adverse reactions (nausea and vomiting), and one pig died. All animals in group B had adverse reactions, and one pig died. The pigs in group C did not experience adverse reactions. The heart rates and respiration of the animals in group C were decreased at T2 and T3 compared with those of group A and B; there were no differences in heart rate and respiration between groups A and B. There were no differences in body temperature or mean arterial pressure of the pigs in the three treatment groups.

animals in the three treatment groups. The induction times of groups B and C (2-3 min) were signi cantly shorter than that of group A (7.4±1.1 min). The maintenance time of group B (136±11.4 min) was signi cantly different from those of groups A and C. The recovery time of group C was 4±1.0 min, compared with 9±0.6 min in group A and 10±1.5 min in group B. In group A, ve pigs had adverse reactions (nausea and vomiting), and one pig died. All animals in group B had adverse reactions, and one pig died. The pigs in group C did not experience adverse reactions. The heart rates and respiration of the animals in group C were decreased at T2 and T3 compared with those of group A and B; there were no differences in heart rate and respiration between groups A and B. There were no differences in body temperature or mean arterial pressure of the pigs in the three treatment groups.
Conclusions Intramuscular injection of ketamine combined with sumianxin is a suitable anesthesia therapy in miniature pigs because of short induction and recovery times, long maintenance time, and effectiveness.

Background
Large animals such as sheep, dogs, and pigs are used often for experimental surgeries. Guizhou miniature pigs have the advantages of small size, stable physiological and biochemical indexes similar to humans, and rough feed tolerance. Thus, Guizhou miniature pigs are used widely in diabetes, dentistry, and orthopedic surgery (1). Safety and reliability of anesthesia during experimental surgery are crucial (1). General anesthetic drugs should provide analgesia, deep hypnosis, and muscle relaxation with minimal side effects. A single anesthetic drug usually does not ful ll all these criteria. High anesthesia doses may cause deep anesthesia and death, and low doses lead to shallow anesthesia and strong, painful stimuli that directly affect surgical outcome (2).
However, sumianxin II has a long, unsatisfactory induction time (3,4). As a moderate sedative, pentobarbital can induce hypnosis, but the dose required for a typical analgesic effect is toxic (5,6).
Additionally, pentobarbital must be dissolved and diluted for drug preparation, which is a relatively timeconsuming activity (6). Ketamine, a non-barbiturate anesthetic, provides intense analgesia with small circulation effects and a short maintenance time in large animals (7). Therefore, combined anesthetic drugs may enable investigators to use the different properties of the drugs to obtain balanced anesthesia and avoid the side effects of any one drug. So far, few studies have been conducted to compare the anesthesia effects of sumianxin II-pentobarbital, ketamine-pentobarbital, and ketamine-sumianxin II in miniature pigs. Thus, we compared these three anesthetic formulations in miniature pigs to nd the safest, most effective, and reliable combination.

Study design
Thirty Guizhou miniature pigs (15 male, 15 female, 22-24 months) were randomly divided into three groups (A, B, and C; n = 10, 5 male, 5 female) and fasted for 12 h before treatment. Randomization of pigs between treatment groups was performed using Research Randomizer (http://www.randomizer.org).
Anesthetics were introduced via intramuscular injection as follows: Group A, sumianxin II (0.1 ml/kg) and pentobarbital (0.8 ml/kg); Group B, ketamine (10 mg/kg) and pentobarbital (0.8 ml/kg); Group C, ketamine (10 mg/kg) and sumianxin II (0.1 ml/kg). All injections were in neck muscles caudal to the base of the ear (splenius and brachiocephalic muscles). No more than 10 mL was injected at any one site to avoid tissue irritation.
After drug injection, animals were placed in a dorsal recumbent position every minute or until the righting re ex was lost and the animals remained recumbent for at least 30 s. Then, animals were observed until they regained the righting re ex, i.e., animals returned to their normal prone position. Elapsed time from drug administration to loss and regaining of the righting re ex was recorded and considered as the onset and end of anesthesia. The induction time was the time from drug injection to loss of the righting re ex. The maintenance time was de ned as the time from complete loss of the righting re ex to the regaining of the righting re ex. The recovery time was the time from the regaining of the righting re ex to the time at which the animal could stand up and start walking. In addition, an adverse reaction was de ned as nausea and vomiting. After all the experiments, euthanasia were conducted that the animals were given 5 ml/kg pentobarbital by intravenously.

Physiological parameters
Throughout the experimental period, heart rate, respiration, mean arterial pressure, and body temperature were recorded pre-anesthesia (T0), after induction (T1), 30 min after operation (T2), 60 min after operation (T3), and at the end of anesthesia (resuscitation, T4). The mean arterial pressure, respiration and heart rates were measured by connecting an arterial catheter with a pressure transducer; body temperature was measured with a rectal thermo-probe. Both devices were connected to a monitor (model PM-2B; Honeywell, Minneapolis, MN).

Statistical analysis
Data analysis was performed with SPSS 19.0 software (SPSS Inc., Chicago, IL, USA), and the data were expressed as the mean ± SEM. Differences between groups were analyzed by Student's t test, and oneway ANOVA with Tukey's test was applied for analyses of more than two groups. A value of P < 0.05 indicated that the results were statistically signi cant.

Results
Body weight, body length, and experimental period Table 1 shows the basic measurements of the three miniature pig treatment groups. There were no signi cant differences in body weight, body length, or experimental period between the three groups (P > 0.05). Induction time, maintenance time, recovery time, and mortality rates Table 2 lists the anesthetic effects of the three treatments. The induction time of group A (sumianxin II + pentobarbital) was 7.4 ± 1.1 min. The induction times of group B (ketamine + pentobarbital) and C (sumianxin + ketamine) were 2-3 min, signi cantly shorter than that of group A. The maintenance time of group B was 136 ± 11.4 min, which was signi cantly different from those of groups A (234 ± 11.4 min) and C (232 ± 17.8 min). The recovery time of group C was 4 ± 1.0 min, compared with 9 ± 0.6 min in group A and 10 ± 1.5 min in group B. Five pigs in group A had adverse reactions, such as nausea and vomiting, and one pig died. In group B, all pigs had adverse reactions, and one pig died. The pigs in group C fared better and experienced no adverse reactions during the experimental period. Heart rate, respiration, body temperature, and mean arterial pressure Table 3 presents the heart rate, respiration, body temperature, and mean arterial pressure of the miniature pigs. The heart rates and respiration of the animals in group C were decreased at T2 (30 min after operation) and T3 (60 min after operation) compared with groups A and B; there were no signi cant differences in heart rates and respiration between groups A and B. In addition, we did not observe any differences in body temperature or mean arterial pressure between the three treatment groups.

Discussion
Usually, anesthetics are administered to large animals intraperitoneally, intravenously, or intramuscularly. Intraperitoneal injection has the disadvantage of a long induction time, which may cause visceral damage. Intravenous injection requires high and precise doses. Too great a dose may cause deep anesthesia, cardiovascular and respiratory depression, and death. Intramuscular injection offers convenience with accurate dosage control, light pain, fast absorption, and a short induction time; nonetheless, local muscle spasms can be a problem (9,10). In the present study, we delivered the experimental drugs intramuscularly, and all pigs became anesthetized within a few minutes of injection.
Pentobarbital, used in animal experiments, may result in dose-dependent toxicity, and overdose may cause perioperative cardiac and respiratory depression and a long postoperative recovery time that may lead to animal death (5,11). In this study, one pig died in groups A and B when we added pentobarbital for maintenance during experiment. Clearly, pentobarbital should be used with caution in large animal experiments. Sumianxin II has high analgesic, sedation, and muscle relaxation properties and a maintenance time of up to 1.5 h; however, a long induction time makes sumianxin II an inappropriate choice for large animal experiments (4,12). Ketamine produces strong analgesic and minimal circulation effects, but it has the disadvantage of a short maintenance time (13)(14)(15). Combination anesthesia offers balance and avoids the side effects of any one drug, but few studies have been conducted to compare the anesthesia effects of sumianxin II-pentobarbital, ketamine-pentobarbital, and ketamine-sumianxin II in miniature pigs. Thus, we selected the individual dosages of sumianxin II, ketamine, and pentobarbital on the basis of our experience and review of the literature (12,16,17). We also rst selected three important evaluating indexes, induction, maintenance, and recovery times, to evaluate the anesthetic effects. Ketamine yielded rapid induction, and sumianxin II produced long maintenance. Pentobarbital was associated with slow recovery; thus, injection of pigs with ketamine plus sumianxin II would be the best anesthetic because of short induction and recovery times and a long maintenance time.
Heart rates and respiration are related to anesthesia depth (18,19). The heart rates and respiration in group C (ketamine + sumianxin II) were greatly decreased at T2 and T3 compared with those of groups A and B. In addition, half of the pigs in group A experienced adverse reactions, and all the animals in group B had adverse reactions. Thus, we speculated that the anesthesia depth in group A and B was shallow enough to cause adverse reactions. The body temperature of most animals continuously decreased during the anesthesia process likely due to reduction in metabolic rate, inhibition of the temperature regulation center, and cessation of skeletal muscle activity (20). The body temperatures in all three groups declined, and we observed an upward trend in temperature at the end of anesthesia. We also observed that the three treatment groups had the same trend for inhibition of mean arterial pressure.
In conclusion, we demonstrated that the intramuscular injection of ketamine combined with sumianxin II is a highly e cacious anesthesia therapy in miniature pigs. Advantages of this treatment protocol include short induction and recovery times and long maintenance time. However, we did not investigate the speci c mechanisms of action of ketamine and sumianxin II; thus, further investigation is needed to expand on our ndings.
Minggang Huang (data acquisition and analysis, statistical analysis); Kai Lu (study conception and design, nal approval of the article, obtained funding, overall supervision). All authors read and approved the nal manuscript.