A Clinico-Biochemical Study of Diazepam as a Preanesthetic in Combination with Various Anesthetics During Orchidectomy in Dogs

| Dog has been a companion animal for humans since years. Being kept in a variety of environments dogs are prone to injuries, wounds and trauma that might require the use of anesthesia to repair and treat the animals. Injectable anesthetics are preferred over inhalant anesthetics in animals as the later require a complicated technique and mechanical instruments. So, the injectable anesthetics are preferred in dogs. Diazepam with Propofol and Diazepam with Ketamine HCl are important anesthetic combinations for induction. Ketamine HCl is a type of dissociative anesthesia has been used for years. Propofol unaided is used as general anesthesia and causes rapid induction provide sedation during anesthesia. To overcome the apnea caused by alone Propofol can be overcome by using combination of Diazepam with Propofol. At present, Diazepam with Ketamine HCl combination can also be used for better results. A total of 12 clinically healthy mongrel dogs were arranged and kept in experimental shed at Surgery Teaching Hospital University of Veterinary and Animal Sciences (UVAS), Ravi Campus, Pattoki. The dogs were placed in two group i-e. Group A and B. the animals were housed under normal conditions for duration of one week. Each dog of group A was administered Diazepam (Valium 2ml; Martin Dow Pharmaceuticals (Pak) LTD) with Propofol (Propofol 20ml; Sandoz Limited, Germany) combination (dose: Diazepam 0.2-0.5 mg/kg of b. wt. and Propofol 5 mg/kg of b. wt.) while each dog of group B was administered Diazepam (Valium 2ml; Martin Dow Pharmaceuticals (Pak) LTD) with Ketamine HCl (Ketamine Hydrochloride 10ml; West ward Pharmaceuticals, USA) combination (dose: Diazepam 0.2-0.5 mg/kg of b. wt. and Ketamine HCl 10-15 mg/kg of b. wt.). Physical parameters like temperature, pulse and respiration, Clinical Parameters like Palpebral reflexes, Pedal withdrawal reflex, degree of analgesia, degree of sedation, induction of anesthesia and ease of recovery was evaluated at different time intervals. LFTs (ALP, AST, ALT and TB) and RFTs (Creatinine and BUN) were performed to find out the effect of anesthesia on liver and kidney. Orchidectomy in dogs was performed after the anesthesia has been achieved and then the post-operative care was done for 5 days after surgery. It is concluded from this research that Diazepam with Ketamine HCl shows an ideal, safe, cost effective and easily available anesthetic with minimal hazards to perform minor and major surgical exercises on small animals.

times till now dogs are of utmost importance in human life and were used as creature for hunting and animal for burden and now as a pet animal also ( Jafari and Emam-Djomeh, 2007). Being kept in a verity of environment dogs are prone to injuries, wound and trauma which might require the use of anaesthetic for the repair and treatment of the conditions. Sometime highly painful procedures are required for injuries and operations. Anesthetics are used for both inhalational and parenteral routes for the surgery in dogs. Moreover, anesthetics can be classified into intramuscular and intravenous depending upon their route and ease of administration. People used ice for analgesia in ancient times and with the passage of time Ketamine Hydrogen Chloride (HCl) was introduced as a first injectable anesthetic in 1962 and accepted for usage in the United States in 1970. Ketamine HCl performs as specific antagonist of the receptor N-Methyl-D-aspartic acid NMDA, an inotropic glutamate base receptor (Anis et al., 1983). It induces analgesia, sedation, and amnesia (memory loss). Effects characteristically begin in five minutes when administered by injection with the main effects durable up to 25 minutes (Mair et al., 2009). Propofol is sole ultra-short acting intravenous, non-steroid and non-barbiturate anesthetic agent (Hofmeister et al., 2006). It is linked with a fast-smooth induction and a quick retrieval after the surgical intervention has been performed (VanNatta and Rex, 2006). It might be specified in assured clinical cases with cardiovascular depression (Haskins et al., 1986;Kolata, 1986;Hellyer et al., 1991;White et al., 2001;Boutureira et al., 2007;Hazra et al., 2008;Beteg et al., 2010). Diazepam and the Ketamine HCl induction of anesthesia and recovery features related with the Propofol have not been stated before (Seliskar et al., 2007). The goal of this potential clinical study is to relate Diazepam with Propofol combination verses Diazepam with Ketamine HCl for the induction of anesthesia and recovery. The mixture of Diazepam with Propofol at a dose rate of 0.2 -0.5 mg/kg and 10 -15 mg/kg correspondingly, is normally related with eagerness free induction of anesthesia in dogs.
Orchidectomy is usually performed at the age of 6 months, to avoid breeding in unwanted dogs. The foundation of this viewpoint is typically in the attention to control pets' population (Hart et al., 2016). Besides this, this intervention is also advised to prevent male dog from testicular cancer, reduction of prostate problems including prostate cancer and modification of behaviors of dog. This procedure as a major surgery requires use of ideal anesthesia to reduce the intra-operative pain. To check the effects of anesthesia on the body of animal by biochemical analysis like renal function test and liver function tests was carried out. The liver function tests (Serum alkaline phosphatase ALP, serum alanine transaminase ALT, and serum aspartate transaminase AST) and renal function tests (Total bilirubin level, BUN and Albumin concentrations) was performed (Rand et al., 1991) before and after the administration of anesthesia to figure out the effects of anesthesia on liver and kidney.

MAtErIALS And MEtHodS
The current study was performed on twelve dogs ranging between 1-3 years of age and weighing 25-40 Kg and kept under normal conditions for about a week before start of research at Surgery Teaching Hospital, University of Veterinary and Animal Sciences, Ravi Campus Pattoki. Before surgical interventions, dogs were evaluated physically for age, weight and physical health as well as responses. Finally, pre-anesthetic evaluation was carried out to evaluate the animal fit for anesthetic administration and further surgical interventions.

experiMeNtal desigN
A total of twelve (12) dogs were selected and divided into following two groups; Group A: 1A, 2A, 3A, 4A, 5A, 6A while Group B: 1B, 2B, 3B, 4B, 5B, 6B The present project was designed to evaluate the two combinations of anesthesia as an inducing agent which influences the superiority of induction, recovery in clinically healthy dogs experiencing anesthesia during orchidectomy and effect on liver and kidneys performing LFTs and RFTs as well before and after the administration of drugs.

preoperative coNsideratioNs
All dogs were completely examined physically and clinically to rule out any pre-operative problem and all the surgical protocols were adopted prior to surgery.

experiMeNtal desigN
Six dogs were allocated for this group. Animals were restrained and were administered with pre-anesthetic diazepam at the dose rate of 0.1-0.2 mg/kg intramuscular (Slatter, 2003) and catheterized intravenously to administer combination of Diazepam with Ketamine HCl for the induction of anesthesia. Each dog of group A was administered with Diazepam and Ketamine HCl combination dose: Diazepam (Valium 2ml; Martin Dow Pharmaceuticals (Pak) LTD) at 0.2-0.5 mg/kg of b. wt. (Van der Kleijn et al., 1971;Frey et al., 1984;Ferreira et al., 2015) and Ketamine HCl (Ketamine Hydrochloride 10ml; West ward Pharmaceuticals, USA) 10-15 mg/kg of b. wt. (Van der Kleijn et al., 1971;Frey et al., 1984;Ferreira et al., 2015). After sedation animal was placed on opreation table for surgery. Six dogs were allocated for this group. Animals were restrained and were administered with pre-anesthetic diazepam at the dose rate of 0.1-0.2 mg/kg intramuscular (Slatter, 2003) , 1971;Frey et al., 1984;Ferreira et al., 2015) and Propofol (Propofol 20ml; Sandoz Limited, Germany) 5 mg/kg of body weight (Ferreira et al., 2015;Slatter, 2003). After sedation animal was placed on table for surgery.

paraMeters evaluated
Observations were recorded for different parameters like;

physical paraMeters
Temperature has been taken from anus of every animal of both groups in degree Celsius at different time intervals of 0, 15, 30, 45 and 60 minutes (Hansen, 2003). Pulse has been taken from femoral artery of every animal of both groups in beats per minute at different time intervals of 0, 15, 30, 45 and 60 minutes (Hansen, 2003). Respiration rate has been taken from lungs by using stethoscope of every animal of both groups in beats per minutes at different time intervals of 0, 15, 30, 45 and 60 minutes (Hansen, 2003).

cliNical paraMeters
Dogs were observed for body responses according to the score card defined by (Ibrahim, 2017). Pedal withdrawal reflex of dogs was tested by score card assigned by (Ibrahim, 2017). This parameter was checked by assigning scores to dogs' reflexes of Toe pinching and Tail Response as defined by (Breivik et al., 2008). Four scores were assigned to test the degree of sedation in dogs using the references like palpebral response set by (Mondello et al., 2002). Four scores were assigned to this parameter depending upon various responses and reflexes of animal as defined by (White and Yates, 2017). Ease of recovery from anesthetic was checked using the parameters/scores defined by (White and Yates, 2017) for reappearance of consciousness or reflexes.

blood collectioN
The blood samples were collected at two different intervals; before and after surgery, directly from cephalic vein that was located in the front leg of dog. Blood samples were kept in gel containing vacutainer (yellow Lid) for hematological parameters and biochemical evaluation.
The anesthetized dogs were applied with Povidone iodine surgical scrub at surgical site for complete sterilization. Scrubbing was done in the circular manner from the center towards periphery. Two drape layers were used; first layer contained four drapes and they were placed over the animal. The second layer contained one big drape with central slit place over first layer. Pre-scrotal approach was used for performing neutering in all dogs. A skin and subcuticular tissue incision was made in front of the scrotum (pre-scrotal) over the shaft of the penis to expose the testicles. The scrotum was on the left under the hand. We used, "open procedure" and a tie or ligation was placed around internal cremaster, pampiniform plexus and vas deferens. When both testicles were removed then the skin incision was closed by using absorbable suture material (Vicryl#1) to close the subcutaneous tissue by using simple continuous suture pattern and skin was closed with simple interrupted pattern using non-absorbable (Glysilk#1) suture material. The same surgical procedure was done in other dogs of both groups by using above designed anesthetic combination and the effects of these pre-anesthetic mixtures were checked on hematological liver and kidney functions.

Figure4
: Skin tissues using absorbable and non-absorbable suture material respectively.

post-operative coNsideratioNs
After surgical procedure, the dogs were kept in cages of Surgery Teaching Hospital under complete observation and care. Postoperative care was provided to each dog accordingly in the cages. Analgesics were given for five days inj. Prefen (Ketoprofen-Fynk Pharmaceuticals) at the dose rate of 0.2-1 mg/Kg body weight up to 5 th day of operation. Antibiotic coverage was given with inj. Velosef (Cephradine -GlaxoSmithKline) at the dose rate of 25mg/ Kg body weight up to 5 th day of operation.

physical paraMeters
The effects of anesthetic combinations were evaluated focusing variations in physiological parameters and the results of clinical parameters including temperature, pulse and respiration are presented in Table 1.  Table 3 • Score 1: No limb extraction at the lock of 3 rd ratchet of hemostat braced on web • Score 2: Limb extraction at the lock of 3 rd ratchet of hemostat braced on web • Score 3: Limb extraction at the lock of 2 nd ratchet of hemostat braced on web • Score 4: Limb extraction at the lock of 1 st ratchet of hemostat braced on web

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Advances in Animal and Veterinary Sciences 2020 | Volume 8 | Issue 9 | Page 986      Table 8. There is a statistical non significance between the groups for ALP, AST and total Bilirubin pre-operatively (P>0.05), but they

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Advances in Animal and Veterinary Sciences 2020 | Volume 8 | Issue 9 | Page 987 are statistically significant post operatively (P<0.05). When two groups were compared for ALT, they showed statistical significance between groups (P<0.05) both pre and post operatively.    Table 9 shows the pre and post-operative comparison of different parameters of RFT (Creatinine and Blood Urea Nitrogen) analyzed through Paired T test which indicated a statistical non-significance between the groups for all the parameters for Creatinine (P>0.05) while shows significant results between groups for BUN (P<0.05).

dIScuSSIon
Anesthesia is a crucial requirement for the surgical interferences in animals as well as in human and that's why the surgeon can do surgical interference with accuracy and wisdom. Without the miracle of anesthesia, the surgical manipulations would have been impossible. The synergistic outcome of two or more drugs can lessen the dose of both primary to price efficiency of the practice which is of inordinate apprehension in animals (Pypendop and Ilkiw, 2005). Inhalant anesthetics have cardiovascular depressant effect in small animals so balanced anesthesia is used mostly to reduce the requirement of inhalant anesthetics. Therefore, a concept of balanced anesthesia is important that includes a combination of various sedative, anesthetic agents and analgesics. Diazepam with Ketamine HCl and Diazepam with Propofol are important anesthetics for induction. Propofol alone is used as general anesthesia and causes rapid induction provide sedation during anesthesia.
To overcome the apnea caused by alone Propofol can be overcome by using combination of Diazepam with Propofol. At present, Diazepam with Ketamine HCl combination can also be used. Orchidectomy in dogs was performed in dogs after the anesthesia has been achieved. The present project was designed to relate the two blends of anesthesia as an inducing agent which influences the superiority of induction, recovery in clinically healthy dogs experiencing anesthesia during orchidectomy and effect on liver and kidney performing LFTs and RFTs before and

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Advances in Animal and Veterinary Sciences 2020 | Volume 8 | Issue 9 | Page 988 after the administration of drugs. For this purpose, twelve dogs ranging between 1-3 years of age and weighing 25-40 Kg and were kept under normal conditions for about a week before start of research at University of Veterinary and Animal Sciences, Ravi Campus Pattoki. A total of twelve (12) dogs were selected and were divided into following two groups; Group A and Group B having 6 animals each. Efficacy of Diazepam with Ketamine and Propofol was assessed on the basis of physiological, clinical and biochemical parameters in dog. Slight reduction in rectal temperature of treated dogs was observed with all of the selected doses of diazepam Ketamine group and same observation was observed in other treatment group of diazepam Propofol. On statistical analysis of both groups by using Paired T test the results were non-significant at 0 mints, 15 mints, 30 mints, 45 mints and 60 mints having P value 0.65, 0.69, 1.00, 0.94 and 0.95 respectively (P>0.05) providing the information that there is no effect on temperature of the treatments similar observation was observed by Hansen and Bernie (2003). Diazepam induces hypothermia and allows better maintenance of body temperature due to peripheral vasoconstriction by central distribution of blood. By administering intravenously the anesthesia there was a drop in pulse rate associated with peripheral vasoconstriction. This vasoconstriction was followed by vasodilation which is in line with the pattern observed by (Hansen, 2003). On analysis by using paired T test for both groups the result was significant (P<0.05) at 15 mints, 30 mints, 45 mints and 60 mints but non-significant having P value 0.04, 0.03, 0.02 and 0.01 respectively while 0.06 (P>0.05) at 0 mint. By using Propofol as an anesthetic agent either in combination or alone results into induction apnea which shows that there will be decrease in respiration rate on induction and same observation was observed during respiration analysis in group B i.e. Diazepam and Propofol. On statistical analysis of both groups results were significant at 0 mints, 15 mints, 30 mints, 45 mints and 60 mints having P value 0.02, 0.01, 0.00, 0.00 and 0.00 respectively (P>0.05) statistically ensuing that the Diazepam Ketamine provides better results than Diazepam Propofol.
Anatomic extent of anesthesia was evaluated by checking palpebral and pedal reflex after 20 minutes of the administration of anesthesia. At 20 minutes of post drug administration, maximum dogs of Group B show absence of response while other shows lethargic response on checking of palpebral reflex while there was lethargic to moderate response in dogs of group A. statistical Analysis by using paired T test for Palpebral reflex between both groups reveals that the result was non-significant P value 0.07 (P>0.05) meaning that there is no difference on evaluating palpebral reflex between both groups. Pedal reflex was checked after 20 minutes in both groups which reveals that group Diazepam with Ketamine shows reflex of limb extraction at the lock of 2 nd ratchet of hemostat braced on web and a few shows reflex of limb extraction at the lock of 1 st ratchet of hemostat braced on web while diazepam Propofol shows limb extraction at the lock of 3 rd ratchet of hemostat braced on web. When both groups were statistically analyzed by using Paired T test the results shows that there was non-significance P value 0.07 (P>0.05) result between the groups hence showing that no difference in pedal reflex of both the animals as depicted by (Hansen, 2003).  Renal Function test is used to determine the effect of anesthetics over the kidneys by taking test like Creatinine and BUN before and after surgery. The Serum was collected for RFTs which described the values for Creatinine normal range is 0.3-0.9 mg/dl and Blood Urea Nitrogen Bun normal range is about 6-24mg/dl. On analysis before and after surgery has been performed, the statistical analysis by Paired T test describes P value preoperatively were 0.17 and 0.04 respectively showing nonsignificant results for Creatinine while significant result in BUN and have same values and same effects before surgery while post-operatively P values for Creatinine and BUN were 0.45 and 0.00 respectively described that the effect of Creatinine and Bilirubin were same post-operatively and have non-significant (P>0.05) results for Creatinine and Significance (P<0.05) for BUN and same results was similar discussed by Ferreira et al., 2015. concLuSIon Present research revealed that the anesthetic combination of Diazepam with Ketamine showed significant results for Physical Parameter (Pulse and Respiration), Clinical Parameters (Degree of analgesia, Degree of sedation, Induction of Anesthesia and Ease of recovery), Biochemical Parameters of Liver Function (ALP, ALT and AST) and Biochemical Parameters of Renal Function (BUN) when compared with Diazepam with Propofol Combination but showed non-significant results for Physical Parameter (Temperature), Clinical Parameters (Palpebral and Pedal Reflexes), Biochemical Parameters of Liver Function (Total Bilirubin) and Biochemical Parameters of Renal Function (Creatinine). From the above statistical facts it can be concluded that anesthetic combination of Diazepam and Ketamine provides safest injectable anesthetic cocktail for induction of anesthesia in dogs and is cost effective with least undesired effects on liver and Kidneys when compared with other Diazepam and Propofol.

rEcoMMEndAtIon
It is suggested from this research that Diazepam with Ketamine HCl shows an ideal, safe, cost effective and easily available anesthetic with minimal hazards to perform minor and major surgical exercises on small animals.