vetalar ( 10% ketamine ) and xylazine ( 2% xylazine ) were sourced from the designated veterinarian . fisher rats bred at rodent facility of university college cork ( originally sourced from charles river laboratories , uk ) were used in this study . rats used in experiments were 14 - 20 weeks of age weighing 250 - 380 g. the experimental procedures on animals were reviewed and approved by animal use ethics committee at university college cork . based on preliminary trials we decided to use 1/10 the recommended anesthetic dose of urethane ( 1.3 g / kg )   in our study . while ketamine ( 90 mg / kg ) and xylazine ( 10 mg / kg ) were used at the recommended dose . a combination of ketamine and xylazine was prepared as follows : 0.9 ml 10% ketamine solution + 0.5 ml of 2% xylazine solution mixed with 0.6 ml of sterile saline . this ketamine - xylazine mix was optimally vortexed and prepared fresh on the day of procedure . the volume of urethane ( 0.65 g / ml ) was kept constant at 0.1 ml / rat while the volume of ketamine - xylazine mix was varied to match the body weight . for administration 0.1 ml of urethane was drawn first into a 1 ml syringe , and the ketamine - xylazine mix was then drawn into the syringe to the required pre - calculated volume . following this the entire mixture was administered to the rat by the intraperitoneal route . the duration of anesthetic induction , surgical plane and recovery time were recorded . additionally the body temperature , heart rate and respiratory rate were also continuously monitored and recorded . these parameters were compared with rats anesthetized using combination of ketamine ( 90 mg / kg ) and xylazine ( 10 mg / kg ) without urethane for balloon injury of carotid artery procedure . the rats were observed up to 1 - 3 months post procedure following which detailed post mortem examination was performed to evaluate any anatomical adverse effects of urethane use . data from 57 rats used for abdominal aortic graft or sham procedures and 24 rats used for balloon injury of carotid artery procedure were analysed . the data are presented as meansd and were compared by un - paired t - test using graph pad prism version 5 ( graph - pad software inc , san diego , usa ) . fisher rats bred at rodent facility of university college cork ( originally sourced from charles river laboratories , uk ) were used in this study . rats used in experiments were 14 - 20 weeks of age weighing 250 - 380 g. the experimental procedures on animals were reviewed and approved by animal use ethics committee at university college cork . based on preliminary trials we decided to use 1/10 the recommended anesthetic dose of urethane ( 1.3 g / kg )   in our study . while ketamine ( 90 mg / kg ) and xylazine ( 10 mg / kg ) were used at the recommended dose . a combination of ketamine and xylazine was prepared as follows : 0.9 ml 10% ketamine solution + 0.5 ml of 2% xylazine solution mixed with 0.6 ml of sterile saline . this ketamine - xylazine mix was optimally vortexed and prepared fresh on the day of procedure . the volume of urethane ( 0.65 g / ml ) was kept constant at 0.1 ml / rat while the volume of ketamine - xylazine mix was varied to match the body weight . for administration 0.1 ml of urethane was drawn first into a 1 ml syringe , and the ketamine - xylazine mix was then drawn into the syringe to the required pre - calculated volume . following this the entire mixture was administered to the rat by the intraperitoneal route . the duration of anesthetic induction , surgical plane and recovery time were recorded . additionally the body temperature , heart rate and respiratory rate were also continuously monitored and recorded . these parameters were compared with rats anesthetized using combination of ketamine ( 90 mg / kg ) and xylazine ( 10 mg / kg ) without urethane for balloon injury of carotid artery procedure . the rats were observed up to 1 - 3 months post procedure following which detailed post mortem examination was performed to evaluate any anatomical adverse effects of urethane use . data from 57 rats used for abdominal aortic graft or sham procedures and 24 rats used for balloon injury of carotid artery procedure were analysed . the data are presented as meansd and were compared by un - paired t - test using graph pad prism version 5 ( graph - pad software inc , san diego , usa ) . use of urethane - ketamine - xylazine ( ukx ) combination resulted in smooth induction of anesthesia similar to that achieved with use of ketamine - xylazine ( kx ) combination . the time for induction of anesthesia in ukx group ( 4.250.87 minutes ) was significantly ( p<0.0001 ) lesser compared to that required with use of kx combination ( figure 1a ) . while the duration of surgical plane of anesthesia and recovery from anesthesia observed using ukx was significantly ( p<0.0001 ) longer compared to kx ( figure 1b , c ) . the duration of surgical anesthesia achieved by ukx combination ranged from 55 to 90 minutes with a mean duration of 73.939 minutes , which was almost 2 folds higher duration of surgical anesthesia compared to that achieved by kx combination ( 38.214.61 minutes ) ( figure 1b ) . the longer duration of surgical anesthesia concurrently resulted in a prolonged recovery period from anesthesia in rats administered ukx combination , which ranged from 180 - 280 minutes , with a mean recovery duration of 227.4632.6 minutes , which was 2.2 fold longer than that observed with the kx combination ( figure 1c ) . physiological parameters , such as rectal temperature ( figure 2a ) , heart rate ( figure 2b ) and respiratory rate ( figure 2b ) were within the normal range in either group . however the physiological parameters measured were statistically better in ukx group compared to kx group ( figure 2 ) . detailed postmortem examination did not reveal any gross abnormalities , indicating that urethane use in rats at the indicated dose is feasible and safe . we evaluated the potential of low dose urethane in combination with ketamine and xylazine to achieve extended duration of surgical anesthesia in rats . urethane is often used as an anesthetic for acute non - recovery procedures in rodents , specifically for procedures involving surgical intervention on cardiovascular system . the preference of urethane is due to its minimal adverse effects on cardiovascular and respiratory physiology in addition to its superior analgesic effects . however use of urethane for chronic recovery procedures has been limited due its carcinogenic and mutagenic effects evident on repeated use . however whether such carcinogenic and mutagenic effects can be observed on single or sub - anesthetic dose administration of anesthetic urethane ( ethyl urethane ) is not documented . a hugely positive feature of urethane anesthesia in laboratory animals is its ability to produce prolonged duration of surgical anesthesia ,   which is often vaguely termed as irreversible anesthesia . considering these facts on urethane , in the present study we hypothesized that use of urethane in combination with ketamine and xylazine ( which is the most widely used anesthetic approach in laboratory rodents ) can help to achieve extended duration of surgical anesthesia . based on a pilot study we limited the urethane dose to 1/10th of its recommended anesthetic dose ( 1.3 g / kg ) in rodents . it was feasible to achieve up to 90 minutes of surgical anesthesia using our combination of urethane , ketamine and xylazine ( ukx ) , which to best of our knowledge is not previously reported . the smooth and rapid induction of anesthesia with ukx with minimal effects on physiological parameters is a highly desirable anesthetic feature and we recommend this approach for surgical procedures in rats requiring 50 - 90 minutes of surgical anesthesia . which in our opinion is favorable and preferred over repeated administration of ketaminexylazine combination . although we did observe prolonged duration of recovery from anesthesia using ukx , which may not be desirable , however the fact that the recovery phase was smooth and without any adverse events is indeed promising for the wider use of the ukx anesthetic combination . however in surgical procedures involving major vascular structures in the abdomen , a prolonged recover period post surgery may be beneficial as this helps achieve optimal stabilization of the vascular anastomosis site . further at 3 months of follow up we did not observe any evidence of tumors or any other adverse events , supports our claim on utility of ukx combination to induce anesthesia for prolonged surgical recovery procedures in rats . hence use of urethane at sub - anesthetic dose in combination with xylazine and moreover the superior analgesic effects of urethane may be additionally beneficial during and post complex surgical procedures . although we did not evaluate a dose dependent effects of urethane , as our required duration of surgical anesthesia was met with this combination , nevertheless it will be interesting to assess in further studies , if altering the relative ratio of urethane in ukx mix will facilitate further extension of surgical plane of anesthesia . moreover such an approach may also help to reduce the dose of ketamine - xylazine , which in our opinion will be beneficial during surgical procedures on cardiovascular and/or respiratory system in rodents .
procedures involving complex surgical techniques in rats , such as placement of abdominal aortic graft require extended duration of surgical anesthesia , which often can be achieved by repeated administrations of xylazine - ketamine combination . 
 however such repeated anesthetic administration , in addition to being technically challenging , may be associated with potential adverse events due to cumulative effects of anesthesia . 
 we report here the feasibility of using urethane at low dose ( ~1/10 the recommended anesthetic dose ) in combination with a xylazine - ketamine mix to achieve an extended duration of surgical anesthesia in rats . 
 the anesthesia induction phase was quick and smooth with an optimal phase of surgical anesthesia achieved for up to 90 minutes , which was significantly higher compared to that achieved with use of only xylazine - ketamine combination . the rectal temperature , heart rate and respiratory rate were within the physiological range with an uneventful recovery phase . 
 post surgery the rats were followed up to 3 months without any evidence of tumor or any other adverse effects related to the use of the urethane anesthetic combination . 
 we conclude that low dose urethane can be effectively used in combination with xylazine and ketamine to achieve extended duration of surgical anesthesia up to 90 minutes in rats .