Thesis Submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy in VETERINARY SURGERY AND RADIOLOGY

Transcription

1 COMPARISON OF MEDETOMIDINE AND DEXMEDETOMIDINE WITH AND WITHOUT BUTORPHANOL AND MIDAZOLAM AS PREANAESTHETICS TO PROPOFOL ANAESTHESIA IN SHEEP Thesis Submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy in VETERINARY SURGERY AND RADIOLOGY By Dr. Shongsir Warson Monsang Roll No To DEEMED UNIVERSITY INDIAN VETERINARY RESEARCH INSTITUTE Izatnagar (U.P.) 2011

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4 Acknowledgements The fear of the Lord is the beginning of knowledge Proverbs 1:7 I felt the drought of words to acknowledge the hands of all those people which had led me through to finally bring out a small ocean of knowledge in the journey of research field. I consider myself lucky and greatly privileged person to work under Dr. Abhijit M. Pawde, Principal Scientist, Division of Surgery and Radiology, IVRI, Izatnagar, Chairman Advisory Committee. His fatherly love, unwavering encouraging words, benevolent guidance, meticulous supervision, constructive criticism, tireless guidance and keen interest for the betterment of my carreer were my perennial source of inspiration throughout the course of the study and research work for which I will be indebted throughout my life. I feel proud of being associated with him. Members of my advisory committee Dr. Amarpal, Senior Scientist and Dr. Prakash Kinjavdekar, Principal Scientist, Division of Surgery and Radiology are highly appreciated for their love and affections, approachable and omni-available in times of needs and difficulties. Their tireless sacrifice and patience for srutinizing the manuscript proved instrumental in completion of this study. I also consider it a great privilege to place on record my respect and indebtedness to Dr S. Dey, Principal Scientist, Dr. Reena Mukherjee, Senior Scientist, Division of Veterinary Medicine, and Dr A. G. Telang, Senior Scientist, Division of Pharmacology and Toxicology, the members of my advisory committee, for their constructive suggestions, benevolent criticism, keen interest and encouragement during the course of my research. Dr. M. M. S. Zama, Professor and Head, Division of Surgery, IVRI, Izatnagar, will be remembered always for his valuable suggestions and providing all the necessary facilities for successful completion of this work. I would like to thank the Director, the Joint Director (Acad) and the Scientific Coordinator, IVRI, for providing necessary facilities and resources required for this piece of work. I also greatly acknowledge the financial assistance provided by ICAR in the form of Senior Research Fellowship (SRF).

5 I extend my gratitude to other Scientists of the Division of Surgery and Radiology, Dr. A. K. Sharma, Dr. M. Hoque, Dr. Naveen Kumar, Dr. H. P. Aithal, Dr. S. K. Maiti, Dr. A. C. Saxena, Dr. Rekha Pathak and Dr. A. Gopinathan for their concern and encouragement during the entire period of my course and research work. I owe a lot to Mina Rinkel, for issuing Dexmedetomidine and Medetomidine from her ends; without which my research would not have been completed. It is also a great privilege to place on record my respect and gratitude to Dr. N. P. Kurade, Senior Scientist, Referral Veterinary Polyclinic, Dr. S. Dey, Pr Scientist, Division of Medicine, and Dr. K. N. Bhilegaonkar, Principal Scientist, Division of Public Health, IVRI, for providing laboratory facilities in the lab and clinics. My sincere thanks to Dr. V.K. Chaturvedi, Head, Biological Products, for his generous help in giving me sheep for the entire period of my experimental study. The precious help rendered by Dr. Shreya, Mr. M. Pathak (Technical Officer, NRL), Dr. Himani (RA), Rajendra (Lab Technician) and Kundan, are highly appreciated too. I would regret if I failed to acknowledge the untiring help, support and endless love received from Dr. Sapunii Stephen Hanah, Dr. Surbhi, Dr. Madhu, D. N., Dr. Irawati P. Sarode, Dr. Jasmeet Singh and Dr. Raja Aijaz Ahmad throughout my stay here. They will remain in the centre core of my friendship. My thanks are also due to Mr Shekhar Saxena and Mr Jeevan Pandey, Clerks, in the Division for their help. I am also grateful to Mr Tara Singh, Mr Chauhan, Mr Dorilal, Mr Nisar, Mr Afroz, Mr Arya, Mr Gurbachan, Mr Pradeep, Mr Jai Swarup, Mr Sherbahadur Nabi, Mr Tika Ram, Mr Devi Singh, Mr Malkhan and Ms Jasoda for their cooperation and precious assistance in one way or the other. It is my pleasure to thank whole heartedly to my seniors and classmate, Dr G. D. Singh, Dr Rahul, Dr Ramesh Tiwary, Dr Dileep Kr K. M., Dr A. K. Gangwar, Dr Moin Ansari, Dr Suvendu Behera, Dr Mritunjay, Dr B. D. Sahu, Dr Ranjith, Dr Kalyan Sharma, Dr Santosh Marandi, Dr Thilakar, Dr Genesis Inigo and Dr Tumyak Loyi for their help and support. My special thanks are due for my divisional juniors, (Dr) Raouff, Amit Kumar, Santhosh, Devarathnam, Aloknath, Vineet Kumar, Dayamon Matthew, Ninu, Shahnawaz, Mudasir, Shivakumar, Karthick, Reihii John, Rohit, Siddharth, Giriraj, Sivanarayan, Arundeep and Remya who provided a colorful company during the entire course of study at IVRI.

6 The delightful company provided by my friends Deepak Sinha, Doni, Blessa, Joy Lamunapuiia, Jonathan, Soumen Choudhury, Norjit Singh, Romie Singh, Lalrinkima, Vikuolie, Bangkeng perme, Vikshe Sumi, Tilling Tayo, Rajib Deb, Yhuntilo Kent, Lokesh J. V., Zupenii, Donna, Beaulah, Guikinglung Pamei and Vikramjit Sharma are thankfully acknowledged. The hidden affections, love, care, and prayers I got from my friends and cousins Sinyorita, Reena, Lalmuanpuii, Dimdim, Gaicham, Wormichon, Umeshwori, Victoria Chanu, Sanjukta, Karuna, Namita, Navneet, Mandakini, Neeta, Delfina, Shylvia, Edward, Augustine and Khambi cannot be expressed in words. My sincere thanks and appreciation goes to the tireless efforts and skilled typing of the manuscript by Mr. Dharmendra (chacha), Aamil, Javed and his team in the early completion of this manuscript. Let me not forget the unconditional love, affection, financial help and support that I have been getting from the very beginning of my educational career from my uncle Manglem, Kothar and aunt Phamdung and Rebecca. I owe a lot to them. I would like to utilize this opportunity to acknowledge the care and support freely received from my brothers-, Mo Ginet, Ko Joyson, Be Bening, Jimmy Paul, Nicodim, Joyphangam, Wartung, Mosaingam and only sweet loving sister Bormila. The prayers of good health and strength pronounced everyday by my sister-in-laws, Bina, Ruth, Shamila and Regie will never be let forgotten. The deepest innocent love of my dearly niece, nephews and Bala (Sanyatjit Pawde) will always be cherished and remembered. My mere words will never match the quantum of unconditional love, affection and inspiration, which I received from my beloved parents, who sacrificed their comfort to see me happy and comfortable. I shall never be able to repay their sacrifices. I also thank the prayer families of Laymen Evangelical Fellowship (LEF, Ghy), VCSF (Ghy), Grace Fellowship (Bareilly), Br Sajii and Anup Jacob for their continuous prayers, encouragement and spiritual support. Family members of Br Sam Israel, Dr. R. K. Agarwal, Br Reji, Br William, Br Hatzaw, Br Sim and Dr. A. M. Pawde are duly acknowledged. At last, I remember the Almighty who gave me patience, strength, wisdom and knowledge to overcome the difficulties, which crossed my way in accomplishment of this endeavour. All may not be mentioned but none is forgotten. Date: Place: (S.W. Monsang)

7 Abbreviations µ : Micron α : Alpha ACTH : Adrenocorticotropic hormone β : Beta BUN : Blood urea nitrogen 0 C : Degree Celsius CRI : Continuous rate infusion CRT : Complete recovery time CNS : Central nervous system DLC : Differential leukocyte count DBP : Diastolic blood pressure et al. : and others g/l : Gram per litre Hb : Haemoglobin HR : Heart rate IM : Intramuscular IV : Intravenous MAP : Mean arterial pressure MAC : Minimum alveolar concentration MCVP : Mean central venous pressure mg/kg : Milligram per kilogram mmol/l : Millimole per litre NIBP : Non invasive blood pressure (monitor) nmol : Nanomole PCV : Packed cell volume PUN : Plasma urea nitrogen RR : Respiratory rate RT : Rectal temperature SBP : Systolic blood pressure SVR : Systemic vascular resistance SVRI : Systemic vascular resistance index SE : Standard error SpO2 : Oxygen saturation of haemoglobin SRT : Standing recovery time TIVA : Total intravenous anaesthesia TLC : Total leukocyte count µiu : Micro international units µmol : Micromole

8 List of Figures Fig 1 : Fig. : Fig. 3 : Fig. 4 : Fig. 5 : Fig. 6 : Fig. 7a : Fig. 7b : Fig. 7c : Fig. 8 : Fig. 9 : Fig. 10 : Fig. 11 : Fig. 12 : Fig. 13 : Recording of blood pressure, SpO 2 and ECG during general anaesthesia Recording of central venous pressure during general anaesthesia Mean±SE score of jaw relaxation in the animals of different subgroups Mean±SE score of palpebral reflex in the animals of different subgroups Mean±SE score of pedal reflex in the animals of different subgroups Mean±SE score of salivation in the animals of different subgroups Mean±SE of induction dose (mg/kg) in the animals of different subgroups Mean±SE of maintenance dose (mg/kg/min) in the animals of different subgroups Mean±SE of duration of anaesthesia (min) in the animals of different subgroups Mean±SE of recovery time (min) in the animals of different subgroups Mean±SE of standing recovery time (min) in the animals of different subgroups Mean±SE of complete recovery time (min) in the animals of different subgroups Mean±SE of heart rate (beats/min) in the animals of different subgroups Mean±SE of respiration rate (breaths/min) in the animals of different subgroups Mean±SE of rectal temperature ( o C) in the animals of different subgroups

9 Fig. 14 : Fig. 15 : Fig. 16 : Fig. 17 : Fig. 18 : Fig. 19 : Fig. 20 : Fig. 21 : Fig. 22 : Fig. 23 : Fig. 24 : Fig. 25 : Fig. 26 : Fig. 27 : Fig. 28 : Fig. 29 : Fig. 30 : Mean±SE of haemoglobin (g/l) in the animals of different subgroups Mean±SE of packed cell volume (L/L) in the animals of different subgroups Mean±SE of total leukocyte count (x10 9 /L) in the animals of different subgroups Mean±SE of lymphocyte count (%) in the animals of different subgroups Mean±SE of neutrophil count (%) in the animals of different subgroups Mean±SE of basophil count (%) in the animals of different subgroups Mean±SE of monocyte count (%) in the animals of different subgroups Mean±SE of eosinophil count (%) in the animals of different subgroups Mean±SE of plasma urea nitrogen (mmol/l) in the animals of different subgroups Mean±SE of plasma glucose (mmol/l) in the animals of different subgroups Mean±SE of plasma creatinine (µmol/l) in the animals of different subgroups Mean±SE of plasma cortisol count (nmol/l) in the animals of different subgroups Mean±SE of plasma insulin (µlu/ml) in the animals of different subgroups Mean±SE of systolic blood pressure (mmhg) in the animals of different subgroups Mean±SE of diastolic blood pressure (mmhg) in the animals of different subgroups Mean±SE of mean arterial pressure (mmhg) in the animals of different subgroups Mean±SE of central venous pressure (cm H 2 O) in the animals of different subgroups

10 Fig. 31 : Fig. 32 : Fig. 33 : Fig. 34 : Fig. 35 : Fig. 36 : Fig. 37 : Fig. 38 : Fig. 39 : Fig. 40 : Fig. 41 : Fig. 42 : Fig. 43 : Fig. 44 : Fig. 45 : Mean±SE of haemoglobin oxygen saturation (%) in the animals of different subgroups Electrocardiographic changes in A 1 subgroups Electrocardiographic changes in A 2 subgroups Electrocardiographic changes in A 3 subgroups Electrocardiographic changes in B 1 subgroups Electrocardiographic changes in B 2 subgroups Electrocardiographic changes in B 3 subgroups Mean±SE of P-wave duration (sec) in the animals of different subgroups Mean±SE of P-wave amplitude (mv) in the animals of different subgroups Mean±SE of PR interval (sec) in the animals of different subgroups Mean±SE of QRS complex duration (sec) in the animals of different subgroups Mean±SE of QRS complex amplitude (mv) in the animals of different subgroups Mean±SE of T-wave duration (sec) in the animals of different subgroups Mean±SE of T-wave amplitude (mv) in the animals of different subgroups Mean±SE of QT interval (sec) in the animals of different subgroups

11 List of Tables Table 1 : Table 2: Table 3 : Table 4 : Table 5 : Table 6 : Table 7 : Table 8 : Table 9 : Table 10: Table 11: Table 12: Table 13: Table 14: System of recording of various reflexes and responses Different drugs administered in the animals of various groups for sedation, induction and maintenance of anaesthesia Mean±SE score of jaw relaxation in the animals of different subgroups Mean±SE score of palpebral reflex in the animals of different subgroups Mean±SE score of pedal reflex in the animals of different subgroups Mean±SE score of salivation in the animals of different subgroups Mean±SE of induction, maintenance doses and duration of anaesthesia in the animals of different subgroups Mean±SE of recovery time in the animals of different subgroups Mean±SE of standing recovery time in the animals of different subgroups Mean±SE of complete recovery time in the animals of different subgroups Mean±SE of heart rate (beats/min) in the animals of different subgroups Mean±SE of respiration rate (breaths/min) in the animals of different subgroups Mean±SE of rectal temperature ( o C) in the animals of different subgroups Mean±SE of haemoglobin (g/l) in the animals of different subgroups

12 Table 15: Table 16: Table 17: Table 18: Table 19: Table 20: Table 21 : Table 22 : Table 23 : Table 24 : Table 25 : Table 26 : Table 27 : Table 28 : Table 29 : Mean±SE of packed cell volume (L/L) in the animals of different subgroups Mean±SE of total leukocyte count (x10 9 /L) in the animals of different subgroups Mean±SE of lymphocyte count (%) in the animals of different subgroups Mean±SE of neutrophil count (%) in the animals of different subgroups Mean±SE of basophil count (%) in the animals of different subgroups Mean±SE of monocyte count (%) in the animals of different subgroups Mean±SE of eosinophil count (%) in the animals of different subgroups Mean±SE of plasma urea nitrogen (mmol/l) in the animals of different subgroups Mean±SE values of plasma glucose (mmol/l) in the animals different subgroups Mean±SE of plasma creatinine (µmol/l) in the animals of different subgroups Mean±SE of plasma cortisol count (nmol/l) in the animals of different subgroups Mean±SE of plasma insulin (µiu/ml) in the animals of different subgroups Mean±SE of systolic blood pressure (mmhg) in the animals of different subgroups Mean±SE of diastolic blood pressure (mmhg) in the animals of different subgroups Mean±SE of mean arterial pressure (mmhg) in the animals of different subgroups -12-

13 Table 30 : Table 31 : Table 32 : Table 33 : Table 34 : Table 35 : Table 36 : Table 37 : Table 38 : Table 39 : Mean±SE of central venous pressure (cm H 2 O) in the animals of different subgroups Mean±SE of haemoglobin oxygen saturation (%) in the animals of different subgroups Mean±SE of P-wave duration (sec) in the animals of different subgroups Mean±SE of P-wave amplitude (mv) in the animals of different subgroups Mean±SE of PR interval (sec) in the animals of different subgroups Mean±SE of QRS complex duration (sec) in the animals of different subgroups Mean±SE of QRS complex amplitude (mv) in the animals of different subgroups Mean±SE of T-wave duration (sec) in the animals of different subgroups Mean±SE of T-wave amplitude (mv) in the animals of different subgroups Mean±SE of QT interval (sec) in the animals of different subgroups

14 Contents Sl. CHAPTER PAGE No. No. 1. INTRODUCTION REVIEW OF LITERATURE MATERIALS AND METHODS RESULTS DISCUSSION SUMMARY AND CONCLUSIONS MINI ABSTRACT HINDI ABSTRACT REFERENCES APPENDIX -14-

15 1 Introduction The concept of administration of sedatives before an injectable anaesthetic induction agent is well accepted and considered as an active practice in veterinary anaesthesia. Sedatives are used pre-operatively to induce sedation, improve the quality of induction of anaesthesia and more importantly, may result in lesser drug-related adverse effects by reducing the amount of injectable or inhalation anaesthetics required to induce and maintain general anaesthesia. General anaesthesia is regarded mandatory for any surgical intervention (Thurmon et al., 1996) as it provides complete unconsciousness, better insensitivity to pain, good muscle relaxation, and freedom from reflex responses and loss of motor ability (Lumb and Jones, 1984). Though most surgical procedures in ruminants are carried out under local or regional analgesia, general anaesthesia may be preferred in certain complicated procedures like diaphragmatic herniorrhaphy, thoracopericardiotomy, repair of ruptured suspensory ligaments, orthopaedic surgery, keratoplasty and repair of ventral hernia etc. (Riazuddin et al., 2004). In clinical anaesthetic practice, adequate general anaesthesia requires a minimum of two different classes of anaesthetic drugs. Use of a single drug for achieving anaesthesia may be unsuitable for any surgical patient and thus evolves a balanced anaesthetic technique, which utilizes a combination of drugs from different pharmacological groups. An ideal balanced anaesthetic technique

16 Introduction... helps in rapid and smooth induction, adequate hypnosis and analgesia for surgical interventions, minimal suppression of vital organ functions and rapid and uncomplicated complete recovery (Thurmon et al., 1996). Small ruminants are not the good candidates for general anaesthesia because their intestinal physiology is such that they are susceptible to bloat and regurgitation of gut contents when recumbent, with potentially fatal consequences. These hazards are minimized initially by inducing anaesthesia with an intravenous agent and securing the airway with a cuffed tube while the animal is still in sternal recumbency. However, the dangers return during the recovery period, until the animal regains good control of its pharyngeal and laryngeal reflexes and maintains sternal recumbency (Hall et al., 2001). Anaesthetic protocols that results in a rapid and complete recovery should therefore, reduce these risks. Anticholinergics are not usually administered to the domestic ruminants prior to induction of anaesthesia. They do not consistently decrease salivary secretions unless frequent higher doses are given. Anticholinergics, while decreasing the volume of secretions, make them more viscid and difficult to clear from the trachea. Alpha 2 -adrenoceptor agonists are frequently used as sedatives and preanaesthetic analgesics. When these agents are given in combination with opioids or dissociative agents, the sedative and analgesic effects of alpha 2 - adrenoceptor agonists may be enhanced. Xylazine is the first alpha 2 -agonist which has been used in veterinary practice for sedation and analgesia (Thurmon et al., 1996). The drug is often used to sedate or, in higher doses, restrain ruminants and is a more potent sedative in ruminants than in horses (Greene, 1999). In dogs and cats, xylazine has been used alone or in combination with opioids to provide sedation and analgesia for diagnostic and minor surgical -16-

17 Introduction... procedures. However, vomiting, bradycardia and arterial hypotension are some of the known side effects of xylazine in dogs. Medetomidine, is a potent and selective alpha 2 -adrenoceptor agonist that contains equal parts of two optical enantiomers, dexmedetomidine and levomedetomidine. It has been used as a sedative, analgesic and anaesthetic premedication in animals, primarily in dogs and cats (Cullen, 1996) as it rapidly produces dose-dependent and reliable sedation and analgesia with good muscle relaxation. Because a lower dose of medetomidine is associated with a reduction in the amount of sedation and analgesia, two opioids commonly used in veterinary practice (i.e, hydromorphone or butorphanol) were administered in combination with medetomidine to improve sedation and analgesia while maintaining cardiovascular stability (Wei-Chen Kuo et al., 2004). As compared to other alpha 2 -agonists, medetomidine is more lipophilic, selective, potent and efficacious, and eliminated faster (Scheinin and MacDonald, 1989). Sedative and analgesic effects of medetomidine have been studied in sheep (Mohammad et al., 1993; Muge et al., 1994; Kastner et al., 2001a), goats (Hugar, 1993; Pawde et al., 1996), dogs and cats (Aithal et al., 1998; Greene, 1999; Ahmad, 2010), horses (Bryant et al., 1991), pigs (Sakaguchi et al., 1995), dairy cows (Ranheim et al., 1999) and buffaloes (Kinjavdekar et al., 2003; Malik, 2008; Ahmad, 2009; Singh, 2011). Studies carried out with medetomidine in some animal species indicate that the pharmacological activity of the drug resides predominantly in its dextrorotatory optical isomer (dexmedetomidine) (Segal et al., 1988; Virtanen et al., 1988; MacDonald et al., 1991; Savola and Virtanen, 1991 and Kuusela et al., 2000). Thus dexmedetomidine was later developed, which is the most potent alpha 2 -agonist developed so far. Dexmedetomidine has been shown to be as safe and as effective as medetomidine when administered at equipotent dose (half the medetomidine dose) (Kuusela et al., 2001). Dexmedetomidine, a selective alpha 2 -adrenergic agonist has been studied for its potential use in anaesthetic practice because of its combined analgesic, -17-

18 Introduction... sedative, hypnotic, and anxiolytic effects (Peden and Prys-Roberts, 1992; Mizobe and Maze, 1995). Dexmedetomidine reduces the dose requirements of opioids and anaesthetic agents and attenuates the haemodynamic responses to tracheal intubation and surgical stimuli. In intensive care patients, dexmedetomidine has been used to achieve sedation without respiratory depression, and cardiac patients may benefit from the perioperative cardiovascular stability it induces (Mantz et al., 1999; Hall et al., 2000 and Venn et al., 2000). Dexmedetomidine has also been studied in dogs, and its clinical effects are presumed to be comparable with those of racemic medetomidine (Hayashi et al., 1991). Dexmedetomidine shows the highest affinity for alpha 2 -adrenergic receptors compared with other similar compounds such as xylazine and medetomidine and has gained interest in veterinary anaesthesiology over medetomidine (Kuusela et al., 2000). Opioids are widely used as analgesics to supplement anaesthesia for tolerance of surgical procedures. Butorphanol is an opioid agonist-antagonist with sedative and analgesic properties known to induce mild sedation accompanied by small decreases in arterial blood pressure, heart rate, and arterial oxygen tension in dogs (Trim, 1983). Combinations of butorphanol and alpha 2 -adrenoceptor agonists provide reliable and uniform sedation in dogs and cats, although significant decreases in heart and respiratory rates are observed (Batram et al., 1994; Ko et al., 1996). Synergistic interactions have been reported between alpha 2 -agonists and opioids and benzodiazepines in earlier studies (Gross et al., 1993; Amarpal et al., 1998b; Kojma et al., 2002). Combinations of alpha 2 -agonists and butorphanol have been used in horses, camelids and domestic ruminants to provide neuroleptanalgesia (Lumb and Jones, 1996). Midazolam is a water soluble imidazole benzodiazepine derivative with sedative, hypnotic, anticonvulsant and muscle relaxant properties (Marjorie, 2001). It has been used alone or in combination with opioids and alpha 2 -agonists to induce sedative and analgesic effects (Kojima et al., 2002; -18-

19 Introduction... Malik and Singh, 2008). Midazolam has minimal effects on cardiopulmonary system (Butola and Singh, 2007) and thus may be preferred for combination anaesthesia in animals. Midazolam has been reported to produce adequate sedation when used as premedicant with ketamine in goats (Stegmann, 1999) and horses (Malik and Singh, 2008), and with ketamine and halothane in ponies (Luna et al., 1993), experimental calves (Bishnoi, 2001) and buffaloes (Cheema, 2002; Malik, 2008). Propofol, is a substituted isopropylphenol, non-barbiturate, nonsteroidal, sedative-hypnotic drug having only minimal analgesic action at subanaesthetic dose. The drug can be used for induction, as well as maintenance of general anaesthesia. Preanaesthetic medication considerably reduces the induction dose of the drug. Propofol has been investigated as intravenous anaesthetic in horses (Oku et al., 2006), sheep (Lin et al., 1997), goats (Prassinos et al., 2005), swine (Graham et al., 1998) and buffaloes (Malik, 2008). Propofol provides rapid induction of anaesthesia, as well as smooth rapid recovery after its administration by both intermittent bolus and continuous intravenous infusion technique which entails as a safe and an ideal anaesthetic (Adetunji et al., 2002). Administration of a single dose of propofol transiently decreased arterial pressure, accompanied by a stable heart rate (Cullen and Reynoldson, 1993). Ovine species are immensely delicate and sensitive subjects to pain among the group of ruminants. In sheep, most of the surgical interventions can be carried out under local or regional analgesia. However, general anaesthesia is preferred by majority of the surgeons or clinicians as it provides a better way of unconsciousness, insensitivity to pain, loss of motor power, muscle relaxation and absence of reflex response, thereby enabling the surgeons to perform any surgical procedure successfully. There is paucity of literature regarding the use of medetomidine and dexmedetomidine and their combinations with other sedative drugs with special reference to sheep which entails the necessity to -19-

20 Introduction... explore the action of these drugs on various body systems of the animal model. Keeping this fact in mind, the present study was undertaken with the following objectives. Objectives: 1. To investigate the sedative, analgesic and clinical effects of medetomidine and dexmedetomidine in sheep. 2. To evaluate the efficacy of butorphanol with and without midazolam for augmentation of sedation and analgesia produced by medetomidine and dexmedetomidine in sheep. 3. To investigate the cardiorespiratory, haemodynamic, haematobiochemical profile and anaesthetic effects of propofol in sheep premedicated with medetomidine and dexmedetomidine with and without butorphanol and midazolam. -20-

21 2 Review of Literature In ruminants, sedation and anaesthesia are solely required for any type of surgical or diagnostic procedures as in other species of animals. Physical restrain in conjunction with local or regional anaesthesia, is often sufficient to allow completion of many procedures without any complications. However, general anaesthesia may be required for any complex diagnostic and surgical procedures. Domestic ruminants have a multi-compartment stomach with a large rumen which requires fasting thereby decreasing the likelihood of complications associated with lateral recumbency and anaesthesia. Preanaesthetic medications Anticholinergics are not usually administered to ruminants as a preanaesthetic medicant prior to induction of anaesthesia, as they do not consistently decrease salivary secretion unless used in very high doses and repeated frequently. Alpha 2 -adrenoreceptor agonists have been used in veterinary practice to induce sedation and analgesia. They are frequently combined with opioids and other dissociative agents to enhance the degree of sedation and analgesia. These agents attenuate the stress response to anaesthesia and surgery thereby reducing the anaesthetic and opioids requirements and produce good sedation and analgesia (Gertler et al., 2001).

22 Review of Literature... Xylazine, detomidine, romifidine and medetomidine induce sedation by stimulating the central alpha 2 -adrenoceptors (Lumb and Jones, 2007). Xylazine is often used to sedate or in higher doses, restrain ruminants. Detomidine, medetomidine and romifidine have been used to a lesser extent in ruminants. The alpha 2 -adrenoreceptor is a distinct sub-classification of á-adrenergic receptors, which are located in the central nervous system (CNS) and virtually every peripheral tissue (Vainio, 1997). Alpha 2 -adrenoreceptors are comprised of numerous subtypes, α 2 A, α 2 B, α 2 C and α 2 D, based on classical pharmacologic and molecular biologic studies (Ruffolo et al., 1994; Vainio, 1997) and these subtypes are distributed throughout the CNS (Scheinin et al., 1994; MacDonald and Scheinin, 1995). Species differences exist, based on the proportion of these subtypes centrally at the level of the brainstem. For example, α 2 A subtypes predominate in canine and rat brainstems (Schwartz et al., 1999), while the α 2 D subtype appears to predominate in the sheep brainstem (Schwartz and Clark, 1998). Alpha 2 -agonists exert their effects through their action on alpha 2 - adrenergic receptors. Alpha 2 -receptors exist presynaptically and postsynaptically in neuronal as well as nonneuronal tissues and extrasynaptically in the vasculature. Alpha 2 -receptors located on noradrenergic neurons are called autoreceptors and those on non-adrenergic neurons are called heteroreceptors. The sedative and anxiolytic effects of alpha 2 -agonists are mediated by activation of supraspinal auto receptors located in the pons (Locus coeruleus) whereas analgesic effects are mediated by activation of heteroreceptors located in the dorsal horn of the spinal cord (Lemke, 2004). Most currently available alpha 2 -agonists can activate the α 1 - adrenoreceptors; therefore, these receptors play some part in the effect of these agents, especially non specific agents such as xylazine. Activation of α 1 - adrenoreceptors induces arousal, restlessness, increased locomotor activity, and increased vigilance (Puumala et al., 1997). These effects may be noted when -22-

23 Review of Literature... high doses (4 and 8 mg/kg BW) of xylazine are used (Ambrisko and Hikashi, 2002a). Studies have demonstrated that central alpha 1 -adrenoreceptor stimulation antagonizes the hypnotic response to even potent alpha 2 -agonists, such as dexmedetomidine (Guo et al., 1991), and that the alpha 1 -adrenoreceptor effects will predominate with increased or toxic doses of alpha 2 -agonists (Ansah et al., 2000; Doze et al., 1989). Another study revealed that dexmedetomidine may offer sedative and analgesic benefits over medetomidine. In dogs, dexmedetomidine produces dose dependent sedation and analgesia and the intensity of these effects is similar to that produced by twice the dose of medetomidine (Kuusela et al., 2000). In recent years, a trend of combining an opioid with a tranquilizer has become common. Butorphanol has been combined with xylazine and other more selective alpha 2 -adrenergic agonists to improve analgesia and sedation in the horses, elephants, ruminants, dogs, cats and laboratory animals (Tranquilli et al., 1988; Faulkner et al., 1992; Marini et al., 1992; Amarpal et al., 1998a; Aithal et al., 1998). A detomidine-butorphanol combination has also been used in horses for sedation (Taylor et al., 1988) and buffaloes (Malik, 2008 and Ahmad, 2009). It has been known that the alpha 2 -agonist xylazine decreases partial pressure of oxygen in arterial blood (PaO 2 ) in cattle (DeMoor and Desmet, 1971), goats (Kumar and Thurmon, 1979), and sheep (Nolan et al., 1986). The degree of hypoxaemia in sheep after sedation with clonidine (Eisenach, 1988) is quite severe. Newer alpha 2 -agonists (detomidine, medetomidine and romifidine) also produce severe hypoxaemia when administered IV at equipotent sedative doses in conscious sheep (Celly et al., 1997a, b) The interaction between midazolam and dexmedetomidine has been found to be synergistic ensuing in deeper levels of sedation. In dogs medetomidine (20 µg/kg)-midazolam (0.3 mg/kg) and medetomidine (20 µg/ kg)-butorphanol (0.1 mg/kg) combinations were reported to produce smooth and rapid induction and more profound and longer sedation than those by medetomidine alone (20, 40, 80 µg/kg). Especially, medetomidine- midazolam -23-

24 Review of Literature... combination produced desirable sedation with moderate reflex depression, analgesia, excellent muscle relaxation and immobilization without further side effects (Hayashi et al., 1994). The literature regarding the drugs used as preanaesthetics in the present study is reviewed. Medetomidine Medetomidine is a very potent, efficacious and selective agonist for alpha 2 - adrenoceptors in the central and peripheral nervous system (Virtanen et al., 1988). The chemical name for the drug is (±)-4-(1-[2, 3-dimethylphenyl] ethyl)-1h-imidazole monohydrochloride. Medetomidine is a lipophilic compound that is rapidly and completely absorbed after IM injection. Following IV administration, it assumes action within 2 minutes; analgesia lasts for 45 minutes and sedation lasts for minutes. The absorption half-life is approximately 7 min with peak serum levels at 30 min in the dog (Salonen, 1989). The drug is not licensed for SC use, due to potentially less reliable and incomplete sedation compared with IM administration (England and Clarke, 1989). It is supplied as a 0.1% or 1 mg/ml (1000 µg/ml) solution and is marketed as a racemic mixture of 2 stereoisomers, dextro-medetomidine and levo-medetomidine. The dextro-isomer, dexmedetomidine, is the active isomer of the medetomidine formulation and when administered at half the dose induces similar effects to medetomidine (Savola and Virtanen, 1991; Kuusela et al., 2000). The levo-isomer lacks pharmacological activity and only shows mild sedative and analgesic activity at higher doses (Kuusela et al., 2000). Medetomidine has been used in combination with propofol infusion (Vainio, 1991; Ahmad, 2009) and propofol bolus (Hammond and England, 1994) and with anticholinergic medication (Vainio et al., 1989; Surbhi, 2008) in dogs. -24-

25 Review of Literature... The pharmacologic effects of medetomidine include: depression of CNS (sedation), decreased gastrointestinal secretions, varying effects on intestinal muscle tone and endocrine functions, peripheral and cardiac vasoconstriction, bradycardia, respiratory depression, diuresis, hypothermia, analgesia, muscle relaxation, blanched or cyanotic mucous membranes and anxiolytic effects. Effects on blood pressure are variable. It has been used to improve haemodynamic stability, alleviate stress, prevent tachyarrhythmias and reduce shivering in humans (Vainio, 1997). Decreased doses of medetomidine ranging from 2 to 10 µg/kg have been combined with various preanaesthetics (butorphanol, oxymorphone, hydromorphone, buprenorphine, meperidine, midazolam) to enhance sedation and analgesia, while potentially reducing the duration of the adverse cardiovascular effects associated with its use (Pypendop and Verstegen, 1998). With medetomidine sedation, vomiting was observed in 8% to 20% of dogs (Vainio et al., 1986, 1989; England and Clarke, 1989; Clarke and England, 1989; Vaha-Vahe, 1989; Nilsfors et al., 1989; Young et al., 1990; Pettifer and Dyson, 1993) and up to 90% of cats (Vainio et al., 1986; Vaha-Vahe, 1989b). When medetomidine is administered alone at a dose of 40 µg / kg IV to healthy beagle dogs, there is a dramatic increase in mean arterial blood pressure (MAP) (average of 175 mmhg within 3 min) (Pypendop and Verstegen, 1999). This hypertension induces a reflex baroreceptor-mediated physiologic bradycardia, associated bradyarrhythmias, and dramatic reduction in cardiac output (CO), which is perpetuated by the central effects of sedation and reduced sympathetic tone. Various research articles have demonstrated that the drop in CO is not due to a direct negative action of the alpha 2 -agonist on myocardial contractility, but is secondary to the increased systemic vascular resistance (SVR) and reduced heart rate (HR) (Schmeling et al., 1991; Autran et al., 1995). On respiratory system it has bradypneic effect. Values of blood pressure however, remain the same or are slightly elevated (Pypendop and Verstegen, 1998). Like xylazine, medetomidine also reduces the dose of propofol for induction and also prolongs propofol anaesthesia (Cullen and Reynoldson, 1993). -25-

26 Review of Literature... The beneficial effects of medetomidine are the same as those of other alpha 2 -agonists and include reliable sedation, analgesia, muscle relaxation, and anxiolysis, as well as a decrease in the anaesthetic requirements of injectable and inhalant agents (anaesthetic sparing). It is not a controlled substance and, therefore, does not require extensive record keeping. These qualities make medetomidine a viable option in small animal anaesthesia. Unfortunately, the negative cardiovascular effects of earlier alpha 2 -agonists (xylazine), including bradycardia and associated arrhythmias, hypertension or hypotension, and reduced cardiac output, are still observed with medetomidine and cause concern among clinicians with respect to their use as premedication or sedative agents. Medetomidine is the most potent alpha 2 -agonist available for use in veterinary anaesthesia, since it induces a longer duration of sedation and analgesia compared with xylazine. These characteristics likely make medetomidine the overall best choice for small animal clinical use. Medetomidine is marketed only as a sedative-analgesic agent to facilitate clinical examinations; clinical procedures; minor surgical procedures, with the exception of those requiring muscle relaxation; and minor dental procedures, where intubation is not required in healthy exercise tolerant dogs. It is contraindicated in dogs that are debilitated; in shock; or stressed due to extreme heat, cold, or fatigue; as well as in dogs with cardiovascular, respiratory, liver, or renal dysfunction. Currently, the cardiovascular alterations induced by medetomidine are the most problematic effects produced, and they usually preclude its use in critical and cardiovascular compromised patients in veterinary medicine. Despite the obvious negative cardiovascular effects, alpha 2 - agonists are increasingly being utilized in human anaesthesia to improve hemodynamic stability, alleviate stress, prevent tachyarrhythmias, and reduce shivering (Maze, 1992; Vainio, 1997). Premedication with medetomidine dramatically reduces the amount of all other anaesthetic agents that must be administered to maintain anaesthesia. -26-

27 Review of Literature... This is important both during the induction and maintenance phase of anaesthesia and relates equally to intravenous and volatile anaesthetic agents. Most anaesthetic drugs, such as propofol, thiopental, isoflurane and halothane have cardiovascular and respiratory side effects that are dose dependent. Therefore, a reduction in the dose of these agents can lead to improved cardiovascular stability and contributes to the provision of balanced anaesthesia. This drug sparing action results from both intrinsic potency of medetomidine and a reduction in the rate of hepatic metabolism of other drugs. Administration of medetomidine decreases injectable and inhalant anaesthetic requirements in several species and it has been reported that propofol requirements for induction and maintenance of anaesthesia are greatly reduced by premedication with medetomidine (Vainio, 1991). Similarly, intramuscular administration of medetomidine at a dose of 20 µg/kg in dogs decreased the amount of propofol required for intubation to 1.8 mg/kg (Redondo et al., 1999). In sheep, intramuscular medetomidine (5 and 10 µg/kg) reduced the propofol and isoflurane requirements for induction and maintenance of anaesthesia, respectively, and cardiovascular variables and blood gas measurements remained stable over the course of anaesthesia but hypoxaemia developed in one of 16 sheep (Kastner et al., 2006). Characterisation of the cardiovascular pharmacology of medetomidine in the horse and sheep was studied by Bryant et al. (1998). Medetomidine administered to sheep and horses at a dose rate of 5 µg/ kg (IV) induced bradycardia and a biphasic blood pressure response consisting of a transient hypertension followed by hypotension. Compared to other alpha 2 -agonists, medetomidine is more lipophilic, selective, potent, and efficacious, and is eliminated faster (Scheinin and McDonald, 1989). Sedative and analgesic effects of medetomidine have been studied in sheep (Mohammad et al., 1993; Muge et al., 1994; Kastner et al., 2001a); goats (Hugar, 1993; Raekallio et al., 1994; Pawde et al., 1996); calves (Raekallio et al., 1997); dog and cats (Amarpal et al., 1998a; Greene, 1999); horses (Bryant et al., 1991); pigs (Sakaguchi et al., 1995); elephant (Sarma et al., 2002); dairy cows -27-

28 Review of Literature... (Ranheim, 1999) and buffaloes (Kinjavdekar et al., 2003; Malik, 2008; Ahmad, 2009; Singh, 2011). At lower doses the sedative and analgesic properties of this drug make it attractive for premedication use and it has been reported that propofol requirements for induction and maintenance of anaesthesia are greatly reduced by premedication of medetomidine (Vainio, 1991). Dogs undergoing ovariohysterectomy by use of thiopental induction and halothane anaesthesia benefited from analgesia induced by medetomidine administered prior to anaesthesia induction (Ko et al., 2000b). The analgesic effects of intravenously administered medetomidine and its combination with ketamine in buffalo calves were evaluated by Kinjavdekar et al. (2003). The animals treated with 20 µg/kg medetomidine showed excellent sedation up to 45 minutes followed by mild sedation. 20 µg / kg medetomidine + 3 mg / kg ketamine IV produced significantly (P<0.05) longer sedation and analgesia than medetomidine alone. Complete muscle relaxation was observed in both groups. Kinjavdekar et al. (2003) recorded that medetomidine (20 µg / kg) in buffaloes could induce deep sedation and analgesia allowing minor surgical interventions for a period of 30 minutes and a surgical anaesthesia of min could be achieved after its supplementation with ketamine (3 mg/ kg). The extent and duration of muscle relaxation induced by medetomidine and ketamine were approximately twice that achieved by medetomidine alone. Similar type of synergism on sedation and analgesia has been reported on detomidine and ketamine administration in buffalo calves (Pawde et al., 2000) and medetomidine and ketamine in goats (Hugar et al., 1998). Medetomidine (2.5 µg / kg)-butorphanol (0.05 mg / kg) combination was reported to provide better quality sedation, analgesia and muscle relaxation than midazolam (0.25 mg/kg)-butorphanol (0.05 mg/kg) combination when used as preanaesthetics to thiopental sodium in buffaloes (Malik, 2008). Ahmad (2009) also reported better quality of sedation, analgesia and muscle relaxation when medetomidinebutorphanol was used as preanaesthetics as compared to butorphanol alone or acepromazine-butorphanol combination in buffalo calves. In similar study, -28-

29 Review of Literature... dexmedetomidine (5 µg/kg) and fentanyl (5 µg/kg) was found better preanaesthetic combination followed by medetomidine (2.5 µg/kg) and fentanyl as it provides better sedation and analgesia in buffaloes (Singh, 2011). Medetomidine (40 to 160 µg/kg), when used in dogs (Vainio et al., 1989) transiently increased blood pressure (18 to 26%) and within 30 min blood pressure returned to the base value or slightly below. However, respiratory rate decreased after medetomidine injection. Increasing the heart rate by the routine administration of atropine concurrent with medetomidine has been observed to have severe deleterious consequences for the cardiovascular system by causing tachycardia and hypertension (Alibhai et al., 1996). Medetomidine caused a significant (P<0.05) increase in SBP, DBP and MBP, when it was administered in dogs at the dose rate of 0.22 mg/kg following premedication with atropine (0.022 mg/kg) and butorphanol (0.22 mg/kg). Atropine prevented the medetomidine induced bradycardia (Grimm et al., 1998). In other study it had depressive cardiovascular effects with bradycardia, drop in cardiac output and rise in systemic vascular resistance. Values of blood pressure, however, remained the same or were slightly elevated (Pypendop and Verstegen, 1998). Like xylazine, medetomidine also reduced the dose of propofol for induction and also prolonged propofol anaesthesia (Cullen, 1996). Bradycardia of longer duration was recorded after medetomidine administration as compared to the medetomidine-ketamine combination (Kinjavdekar et al., 2003) and a significant depression of respiratory rate was observed in both groups. Cardiovascular effects of deep sedation by medetomidine (0.2 mg/kg) and ketamine (10 mg/kg) combination was reported in Yucatan mini swine. Hypertension (MAP from 116±12 mmhg to 142±18 mmhg) followed by bradycardia (from 107±22 bpm to 71±9 bpm), concomitantly, decrease in both rate of increase in ventricular pressure (48%) and ventricular wall thickening fraction (37%), increase in respiratory frequency and increase in PaCO 2, decrease in PaO 2 and rectal temperature were recorded (38.4±0.9 to 36.0±0.8ºC (Vainio et al., 1992). -29-

30 Review of Literature... On respiratory system, medetomidine has bradypneic effect (Pypendop and Verstegen, 1998). Sheep have been reported to develop adverse hypoxaemic effects after intravenous administration of alpha- 2 agonists (Kastner et al., 2001). Cardiac output (CO) has been reported to be reduced following administration of medetomidine in dogs (Pypendop and Verstegen, 1998), principally as a consequence of the peripheral vasoconstriction and therefore, increased afterload. Interestingly the cardiovascular effects of medetomidine, including changes in cardiac output, did not appear to be dose dependent, unlike most other anaesthetic agents. Pypendop and Verstegen (2000) compared renal, intestinal and muscle microvascular blood flow in dogs anaesthetised with either isoflurane alone or a combination of isoflurane with medetomidine, midazolam and butorphanol. The study also demonstrated that the medetomidine combination decreased intestinal and skeletal blood flow, while renal blood flow was similar to the dogs given isoflurane alone. Medetomidine is reported to inhibit the electrical activity of the small intestine and dramatically inhibit the motility of the colon in dogs (Maugeri et al., 1994). Effects of high doses of medetomidine on endocrine glands were evaluated in eight calves, eight lactating dairy cows and eight adult female sheep (Ranheim et al., 2000). All the animals were injected intravenously with medetomidine (40 µg/kg), followed 60 min later by atipamezole (200 µg/kg, IV). In every animal, medetomidine induced marked hyperglycaemia, which was reversed by atipamezole. Cortisol levels increased significantly in cows and sheep, reaching levels 4-8-fold higher than the baseline levels min after injection of medetomidine. Atipamezole did not affect the cortisol levels, except in sheep where an increase was observed. Plasma levels of noradrenaline decreased in cows and sheep after medetomidine injection, reflecting the inhibition of sympathetic activity by the drug. After injection of the antagonist, there was a large increase in noradrenaline levels. Ranheim et al. (2000) concluded that a high dose of medetomidine does not seem to reduce the overall endocrine stress response in cattle and sheep. Concentrations of epinephrine, -30-

31 Review of Literature... norepinephrine, and cortisol were significantly lower for dogs administered medetomidine-butorphanol in comparison to dogs administered with acepromazine-butorphanol. However, heart rates was significantly lower and mean arterial blood pressure was significantly higher in dogs administered with medetomidine, compared with values for dogs administered acepromazine (Vaisanen et al., 2002). It was concluded that preanaesthetic medications with medetomidine offered some advantages over acepromazine with respect to the ability to decrease perioperative concentrations of stress-related hormones. In particular, the ability to provide stable plasma catecholamine concentrations may help to attenuate perioperative activation of the sympathetic nervous system. The use of anticholinergic drugs aiming to reduce the bradycardia caused by medetomidine is usually not advised, as it leads to prolonged, severe hypertension leading to increased myocardial oxygen demand (Vainio and Palmu, 1989; Alibhai et al., 1996). In dogs apart from bradycardia, accentuated sinus arrhythmia and sinoatrial and atrioventricular (AV) heart blocks are the most commonly described arrhythmogenic effects of alpha 2 -adrenergic agonist drugs (Kuusela et al., 2003). Systemic administration of a 2 -adrenergic agonists has resulted in a decrease in rectal temperature (Ponder and Clarke, 1980), although higher doses of detomidine have been shown to produce a late onset of hyperthermia (Bueno et al., 1999). Hypothermia was reported in sheep after the use of xylazine or medetomidine (Ponder and Clarke, 1980; Aminkov and Pascalev, 1998; Kinjavdekar, 1998) and has been attributed to generalized sedation, a decreased metabolic rate, muscular relaxation and depression of hypothalamus. By contrast an increase in RT has been reported in cattle following epidural administration of xylazine and possible causes for the hyperthermia are unclear (Skarda and Muir, 1992). Hypotension is attributed to bradycardia and vasodilation, the stimulation of central alpha 2 -adrenoceptors, peripheral sympatholytic action and enhanced parasympathetic outflow (Ossipov et al., 1989; Tibirica et al., 1991). -31-

32 Review of Literature... Hypotension has also been reported following the systemic administration of romifidine in goats (Saxena, 1996). Bryant et al. (1996) studied the cardiopulmonary effects of medetomidine in sheep and in ponies and observed that medetomidine resulted in significant decreases in heart rate and cardiac output and, initially, in an increase in arterial blood pressure in both species. In the ponies this increase in blood pressure was followed by a significant and prolonged decrease, but in the sheep the secondary decrease in blood pressure was not statistically significant. In the sheep, the three doses 5 µg/kg, 10 µg/kg and 20 µg/kg of medetomidine resulted in profound and significant decreases in arterial oxygen tensions, which were significantly dose related, but in the ponies the arterial blood oxygen tensions were not significantly decreased. In both species medetomidine caused a small but significant increase in arterial blood carbon dioxide tensions. Raekallio et al. (1998) reported that medetomidine (15 µg/kg) with midazolam (0.1 mg/kg) administered intravenously alone or in combination in sheep to produce sedation causes a marked decrease in heart rate, PaO 2 and Hb oxygen saturation along with marked hypoxaemia as compared to medetomidine alone. Systolic and mean arterial pressures decreased after medetomidine-midazolam. The results indicate that a medetomidinemidazolam combination is unsafe for sheep at the doses studied. Kinjavdekar et al. (2005) reported that subarachnoid administration of romifidine (50 µg/kg) and lidocaine (1 mg/kg) in goats causes reduction in MAP for longer period. Similar effects on MAP have been observed after the subarachnoid administration of lidocaine and xylazine/medetomidine, and romifidine and ketamine in goats (Kinjavdekar, 1998). Kinjavdekar et al. (2005) also reported that subarachnoid injection of both romifidine or romifidine and lidocaine produced a significant increase in the mean central venous pressure (MCVP). The increase in MCVP was probably due to pooling of blood in the venous circulation as a result of low heart rate and decrease in cardiac function (Venugopalan et al., 1994). Klein and Sherman (1977) also reported, the central -32-

33 Review of Literature... shift of blood to the venous compartment might be associated with an increase in MCVP. Venugopalan et al. (1994), however, ascribed the increase in MCVP, after medetomidine administration, due to vasoconstriction as a result of stimulation of peripheral post synaptic alpha 2 -adrenoceptors. Increased MCVP has also been reported following the subarachnoid administration of xylazine or medetomidine alone or in combination with lidocaine or romifidine and ketamine in goats (Kinjavdekar, 1998; Kinjavdekar et al., 1999). Romifidine also increased the MCVP after its systemic administration (Saxena, 1996). Kinjavdekar et al. (2005) reported no significant changes in PCV and Hb following subarachnoid administration of romifidine alone or romifidine and lidocaine. Reduction in Hb, PCV and TLC has been reported following epidural or subarachnoid administration of xylazine or medetomidine in goats and other species of animals (Kinjavdekar, 1998; Jean et al., 1990; Malik, 2008; Ahmad, 2009, Singh, 2011). Kinjavdekar et al. (2005) reported significant increase in plasma glucose levels following subarachnoid administration romifidine alone and romifidine and lidocaine in goats. The hyperglycaemic effect has been reported following the administration of alpha 2 -agonists in different species (Eichner et al., 1979; Hsu and Hummal, 1981; Brikas et al., 1987; Cullen, 1996; Kinjavdekar, 1998; Malik, 2008; Ahmad, 2009; Singh, 2011). Serum insulin levels decrease significantly following medetomidine administration in dogs and cats, which return to base values after several hours. Although glucose concentration increases after medetomidine administration but it remains within physiological limits (Burton et al., 1997; Ambrisko and Hikasa, 2002b). Xylazine produces a dose dependent increase in plasma glucose in contrast to medetomidine, which does not produce the same effect after increasing the dose (Ambrisko and Hikasa, 2002). Kinjavdekar et al. (2005) reported that following the administration of alpha 2 -agonist changes in plasma urea nitrogen and creatinine were only slight -33-

34 Review of Literature... and non-significant. This observation is in agreement with the findings of Gasthuys et al. (1996) following intravenous administration of romifidine in horses, xylazine in goats (Kumar and Thurmon, 1979), subarachnoid administration of xylazine and medetomidine in goat (Kinjavdekar, 1998). Kaster et al. (2006) reported that intramuscular medetomidine at 30 µg/ kg in sheep resulted in deep sedation within 30 min with mean peak plasma concentration of 4.98 mg/ml and plasma concentrations of medetomidine correlated well with the behavioural effects with litter or no hysteresis and it was cleared very rapidly. The risk of adverse hypoxaemia is still present with intramuscular medetomidine at this dose. Surbhi et al. (2010) studied the effects of medetomidine-butorphanolpropofol anaesthesia in dogs undergoing orthopaedic surgery. Administration of Medetomidine (10 µg/kg, IV) and butorphanol (0.02 mg/kg, IV) as preanaesthetics to propofol caused a non-significant (P>0.05) decreased in heart and respiratory rate whereas propofol caused tachycardia, depression in respiratory rate and SpO 2. MAP increases significantly (P>0.05) after the administration of preanaesthetics, whereas after the administration of propofol hypotension was observed. Significantly higher base values of glucose and cortisol were also observed that further increased significantly (P<0.05) after the administration of drugs. Plasma urea nitrogen level was found to be decreased whereas creatinine values increased non-significantly (P>0.05). Singh et al. (2010) reported that complete muscle relaxations with depressed swallowing reflexes were observed up to 45 minutes interval when medetomidine (0.015 mg/kg BW) and ketamine (10 mg/kg BW) were administered intramuscularly after atropinisation in buffalo calves. There was no significant change in rectal temperature and heart rate at different intervals. However, increase in respiratory rate was significant at 5, 15 and 30 minutes intervals. All the haematological parameters remained within the normal range -34-

35 Review of Literature... with significant hyperglycaemia observed at 15 minutes onwards until 75 minutes. A non-significant increased in BUN was also noticed up to 75 minutes. Dexmedetomidine Dexmedetomidine, (+) -4-[1-(2, 3-dimethylphenyl) ethyl]-1h-imidazole, is a selective and potent alpha 2 -adrenergic agonist. Its molecular structure is given below: Dexmedetomidine is a potent alpha 2 -adreneceptor agonist and the active enantiomer of medetomidine (Aantaa et al., 1989). Dexmedetomidine contains an imidazole in its chemical structure and has an affinity for imidazoline receptors, despite weaker than that for alpha 2 -adrenoceptors (Wikberg et al., 1991; Khan et al., 1999b). The α 2 /α 1 selectivity of dexmedetomidine is several times higher than that of clonidine, detomidine or xylazine (Virtanen et al., 1988). Dexmedetomidine is therefore, considered a full agonist of the alpha 2 - adrenoceptor, and clonidine a partial agonist. More potent sedation and analgesia with minor cardiovascular depression from alpha 1 -adrenoceptor activation can be expected as a result of full agonism. Dexmedetomidine, as the active ingredient of the racemic mixture, has gained more interest than the racemate medetomidine in human anaesthesiology. In intensive care patients, dexmedetomidine is used to achieve sedation without respiratory depression, and cardiac patients may benefit from the perioperative cardiovascular stability it induces. In humans it is used for sedation in intensive care patients and as a premedicant before surgery (Aantaa et al., 1997; Lawrence and De Lange, 1997). Dexmedetomidine has also been studied in dogs, and its clinical effects are presumed to be comparable with those of racemic medetomidine. -35-

36 Review of Literature... Dexmedetomidine has minimum alveolar anaesthetic concentration (MAC)-sparing properties, but its use as an anaesthetic adjuvant has been complicated by persistent hypotension that has mandated IV fluid administration and vasopressor administration. In addition, its use in large doses is complicated by hypertension from alpha 2 -receptor- mediated vascular constriction. The mechanism of action of dexmedetomidine is unique and it is by activation of the alpha 2 - receptors in the brain and spinal cord and inhibiting neuronal firing, causing hypotension, bradycardia, sedation, and analgesia (Gertler et al., 2001). The activation of alpha 2 -receptors in other areas will cause respiratory depression, decreased salivation, decreased secretion, and decreased bowel motility in the gastrointestinal tract; contraction of vascular and other smooth muscles; inhibition of renin release, increased glomerular filtration, and increased secretion of sodium and water in the kidney; decreased intraocular pressure; and decreased insulin release from the pancreas (Metz et al., 1978). The presynaptically located alpha 2 - adrenoceptors are believed to inhibit the release of catecholamine. Therefore, this drug has anti-adrenergic effect (Starke, 1981). Dexmedetomidine has been shown to cause a marked reduction in the anaesthetic requirements of general anaesthetics. Compared to midazolam, dexmedetomidine decreases the need for intraoperative ketamine and is more effective in reducing ketamine-induced adverse central nervous system effects (Levanen et al., 1995). Kuusela et al. (2000) compared the clinical and pharmacokinetic effects of medetomidine and dexmedetomidine and found that overall sedative effect of medetomidine did not differ from that of the dexmedetomidine and analgesic effect of dexmedetomidine was of longer duration than racemate medetomidine. It was also observed that 10 µg /kg of dexmedetomidine intravenously induced maximum sedation; increasing the dose only increased duration of sedation not intensity (Kuusela et al., 2000). The degree of sedation induced by dexmedetomidine is not affected by exercise -36-

37 Review of Literature... or pre-treatment with dexamethasone (Raekallio et al., 2005). In animal models, dexmedetomidine is mainly metabolized in liver, mainly by hepatic hydroxylation in dogs (Salonen, 1992); while in human s hepatic glucuronidation of dexmedetomidine is more efficient than in dogs. Dexmedetomidine administration will cause an initial pressor response produced via vasoconstriction, followed by a secondary baroreflex-mediated decrease in heart rate. Systemic vascular resistance is increased and cardiac output is decreased. Bradycardia is produced mainly via diminished sympathetic (Schmeling et al., 1991; Xu et al., 1998; Hogue et al., 2002) and/or augmented parasympathetic tone (Bloor et al., 1992b), and blood pressure declines gradually to or beyond baseline level (Bloor et al., 1992a, 1992b). Dexmedetomidine has been used as a sedative and as a premedicant in dogs, cats, horses (Ansah et al., 1998; Kuusela et al., 2001; Bettschart-Wolfensberger et al., 2005; Ahmad, 2010) and buffaloes (Singh, 2011). It has been demonstrated to be at least as safe and effective as medetomidine prior to propofol-isoflurane anaesthesia (Kuusela et al., 2001) and propofol-halothane anaesthesia in dogs (Hayashi et al., 1991). However, no comparative studies could be found on the influence of dexmedetomidine and medetomidine during general anaesthesia using propofol-desflurane in dogs. The effect of dexmedetomidine on respiration and hypoxic reflexes are relatively mild but species specific variation may exist (Nguyen et al., 1992). In sheep, marked hypoxia may develop after IV and IM medetomidine or dexmedetomidine (Raekallio et al., 1998; Kastner et al., 2001a), probably due to pulmonary venospasm-induced alveolar oedema (Bacon et al., 1998). After dexmedetomidine or medetomidine administration IV or IM in dogs respiratory rate usually decreases with minimal effects on blood gas values (Vainio, 1989; Schmeling et al., 1991; Kramer et al., 1996). Dexmedetomidine has been found to depress cardiac function in dogs even after autonomic denervation (Flacke et al., 1990). Postsynaptic alpha

38 Review of Literature... adrenoceptors are considered non-existent in the mammalian heart (Dukes and Vaughan Williams, 1984; Hayashi et al., 1991), and dexmedetomidine has shown no direct depressant effect on the canine myocardium (Flacke et al., 1992). Schmeling et al. (1991) interpreted the depression in heart function caused by dexmedetomidine to be the result of increased arterial pressure, but in isolated canine hearts, dexmedetomidine-induced increased afterload did not impair cardiac function (Flacke et al., 1992). In a later study, Flacke et al. (1993) reported diastolic dysfunction and a decline in systolic contractility in conjunction with decreasing catecholamine levels after dexmedetomidine administration. When haemodynamic changes were antagonized by a calcium channel blocker, cardiac function was nevertheless reduced after dexmedetomidine administration in dogs, probably due to sympatholysis (Roekaerts et al., 1996). The decrease in cardiac output after dexmedetomidine is caused mainly by a decrease in heart rate. While the increase in blood pressure and the decrease in cardiac output occur simultaneously, the latter seems not to be completely mediated reflexly (Flacke et al., 1990). Reduced stroke volume, increased afterload, reduced metabolic demands (Bloor et al., 1992b); low catecholamine levels and coronary vasoconstriction (Flacke et al., 1990; Bloor et al., 1992b) have been suspected to contribute to the reduction in cardiac output. The pressor response to IV dexmedetomidine was enhanced after autonomic denervation (Flacke et al., 1990; Schmeling et al., 1991), but cardiac output decreased without a change in heart rate (Flacke et al., 1990). Likewise, when bradycardia was blocked by glycopyrrolate pre-treatment, the decrease in cardiac output was only partially reversed, but arterial pressure increased further (Bloor et al., 1992b). IV dexmedetomidine (Lawrence et al.,1996) and IM medetomidine (Pypendop and Verstegen, 2000) have been shown to considerably redistribute cardiac output in dogs, reducing blood flow to less vital organs and preserving it in vital organs to levels above those known to induce hypo-perfusion. The cardiovascular effects of IV racemic medetomidine were fully established with doses as low as 5 µg/kg, and increasing the dose prolonged -38-

39 Review of Literature... the effects (Pypendop and Verstegen, 1998). Lombard et al. (1989) studied the effects of IM medetomidine in dogs with compensated mitral regurgitation and considered the drug to be safe but recommended the use of an antagonist to reverse cardiovascular effects. The use of anticholinergic drugs aiming to reduce the bradycardia caused by medetomidine is usually not advised, as it may result in prolonged, severe hypertension leading to increased myocardial oxygen demand (Vainio and Palmu, 1989; Short, 1991; Alibhai et al., 1996). Besides bradycardia, accentuated sinus arrhythmia and sinoatrial and atrioventricular (AV) heart blocks are the most commonly described arrhythmogenic effects of alpha 2 -adrenergic agonist drugs in dogs. AV-blocks (first and second degree) develop as disturbances in the conductivity of the cardiac excitatory impulse. The appearance of AV-blocks after dexmedetomidine administration has not been reported. IV and IM medetomidine has been shown to induce occasional second-degree AV-blocks in dogs (Lombard et al., 1989; Vainio and Palmu, 1989; Kramer et al., 1996). The analgesic action of dexmedetomidine is mediated spinally (Hayashi et al., 1995). A direct action on locus coeruleus has been implicated, which results in activation of alpha 2 -receptors in the spinal cord through which antinociceptive effect is mediated (Guo et al., 1996). Dexmedetomidine has been shown to act on dorsal horn of spinal cord by interrupting the nociceptive pathway to the ventral root, thus reducing the spinal reflex (Savola and Virtanen, 1991). Species specific haemodynamic response occurs following administration of dexmedetomidine (Flackle et al., 1990). Talke et al. (1997) studied the postoperative sympatholytic effects of dexmedetomidine and found that plasma norepinephrine and epinephrine concentration decreased on an average by 72% which attenuates postoperative increase in heart rate and blood pressure but does not entirely abolish sympathetic tone, so as to produce significant hypotension and bradycardia. Medetomidine alone causes a small rise in mean arterial pressure which is followed by decrease in the MAP but hypotension has not been recorded (Alibhai et al., 1996). -39-

40 Review of Literature... Administration of dexmedetomidine results in decrease in respiratory rate with minimal effects on blood gases in dogs (Vainio, 1989). Isoflurane anaesthesia in dogs has been shown to cause more respiratory depression when used alone than when used along with medetomidine or dexmedetomidine (Bloor et al., 1989; Nguyen et al., 1992). Systemic administration of dexmedetomidine resulted in a dose dependent depression in the rate and slope of CO 2 response curve whereas intrathecal or epidural dexmedetomidine caused little change in the rate and or slope of CO 2 function suggesting that these actions are mediated supraspinally (Sabbe et al., 1994). Alpha 2 -agonists directly or indirectly obtund the stress response when administered systemically. Reduction in the perception of stressors is achieved indirectly through sedation and analgesia while directly by inhibiting neuroendocrine response. Delayed ACTH and cortisol response was observed in dogs undergoing ovariohysterectomy in which medetomidine was administered preoperatively (Benson et al., 2000). In dogs undergoing ovariohysterectomy plasma cortisol did not change significantly from baseline at 60 min after extubation when medetomidine was given preoperatively. Dexmedetomidine resulted in a decrease in blood glucose concentration at 30 min of its administration, which returned to baseline at 90 min in beagle dogs (Raekallio et al., 2005). In the same experiment plasma cortisol concentration decreased significantly in the dogs, which received dexamethasone with or without exercise. Kuusela et al. (2000) compared the clinical and pharmacokinetic effects of medetomidine and dexmedetomidine and found that overall sedative effects of medetomidine did not differ from that of dexmedetomidine and analgesic effect of dexmedetomidine was of longer duration than racemate medetomidine. It was also observed that 10 µg/kg of dexmedetomidine intravenously induced maximum sedation; increasing the dose only increased duration of sedation not intensity (Kuusela et al., 2000). The degree of sedation induced by -40-

41 Review of Literature... dexmedetomidine is not affected by exercise or pre-treatment with dexamethasone (Raekallio et al., 2005). Kuusela et al. (2001) studied various premedicant doses of dexmedetomidine administrated intravenously in dogs under propofol and isoflurane anaesthesia and found that dose level of 20 µg/kg preserved blood pressure but profound bradycardia occurred. Dose level of 2 µg/kg resulted in more stable cardiovascular effects but the effect was short term and a stable plane of anaesthesia was difficult to maintain, such that isoflurane concentration had to be increased. In equines sedated with dexmedetomidine, heart rate and central venous pressure did not differ significantly from presedation values but decrease in stroke volume and blood pressure occurred (Bettschart et al., 2005). Kastner et al. (2005) found that dexmedetomidine induced a transient decrease in heart rate and cardiac output in sheep anaesthetized with sevoflurane. A short-lived increase in mean arterial pressure (MAP) and systemic vascular resistance (SVR) was followed by a significant decrease in MAP and SVR. Kutter et al. (2006) found that arterial, pulmonary arterial, pulmonary capillary wedge and central venous pressures increased and heart rate and cardiac output decreased significantly after dexmedetomidine administration in sheep and goat anaesthetized with sevoflurane,. In a study carried out by Sabine et al. (2005) on cardiopulmonary effects of dexmedetomidine in sevoflurane-anaesthetized sheep with and without nitric oxide inhalation reported that dexmedetomidine induced a transient decrease in heart rate and cardiac output. Mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance increased transiently after dexmedetomidine injection. In sheep, alpha 2 - agonists can induce severe hypoxaemia. In goats, reports on changes in oxygenation are inconsistent. Alpha 2 -agonist induced pulmonary oedema in sheep which might be related to alterations in pulmonary Haemodynamic and/or activation of inflammatory processes. Intravenous dexmedetomidine (2 µg /kg) in sevoflurane-anaesthetized sheep causes -41-

42 Review of Literature... significant increased in mean pulmonary artery pressure, pulmonary arterial occlusion pressure and estimated capillary pressure to 34.5 mmhg, 22.2 mmhg and 27.1 mmhg, respectively (Kastner et al., 2007). Ahmad (2010) reported that the combination of dexmedetomidine, midazolam, fentanyl and ketamine administered intramuscularly, induces surgical anaesthesia lasting for 15 min, which was sufficient for minor surgical procedures. The induction was recorded to be smooth with rapid and recovery uneventful, although complete recovery was found to be prolonged. Ketamine along with dexmedetomidine, midazolam and fentanyl provides greater haemodynamic stability as compared to dexmedetomidine alone or its combinations with midazolam and fentanyl in dogs. Singh (2011) reported that drug combinations (fentanyl-medetomidinethiopental- isoflurane and (fentanyl-dexmedetomidine-thiopental-isoflurane) provided adequate sedation, analgesia and muscle relaxation in experimental buffaloes calves. However, the maintenance dose of isoflurane was reported to be lesser as compared to halothane due to the dose sparing effect of dexmedetomidine and fentanyl. Dexmedetomidine-fentanyl-thiopental and isoflurane combination provides better clinical, physiological and haemodynamic stability as compared to medetomidine-fentanyl-thiopental and halothane in buffaloes. Butorphanol Butorphanol is a centrally acting analgesic with both agonist and antagonist properties. It is a morphinan derivative and is chemically l-ncyclobutylmethyl-6, 10 aβ-dihydroxy-1, 2, 3, 9, 10, 10a-hexahydro-(4H)10, 4aiminoethanophenanthrene tartrate. The molecular formula is C21 H29 NO2 C4H6O6. It is white, crystalline powder, sparingly soluble in water and insoluble in alcohol. It has a bitter taste and a pka value of 8.6. The commercial injection has a ph of Butorphanol is an agonist at µ-opioid receptors and an -42-

43 Review of Literature... antagonist at κ-opioid receptors (Carroll et al., 1997). There are conflicting statements in the literature on effects of butorphanol on µ-opioid receptors as some authors claim it is a partial agonist (Doherty et al., 2002). Stimulation of these receptors on central nervous system neurons causes an intracellular inhibition of adenylate cyclase, closing of influx membrane calcium channels and opening of membrane potassium channels. This leads to hyperpolarization of the cell membrane potential and suppression of action potential transmission of ascending pain pathways. Because of its k-agonist activity, at analgesic doses, butorphanol increases pulmonary arterial pressure and cardiac work. Opioids are traditionally included in balanced anaesthesia protocols for their analgesic effects, but they also have sedative effects (Lemke, 2007). Butorphanol has analgesic properties in ruminants; however, it can also induce excitatory behavioural changes (Carroll et al., 2001). Other recognized uses include obstetric analgesia during labour and relief of moderate postpartum pain. In addition butorphanol has been used effectively for conscious sedation. Butorphanol has also been used for epidural analgesia or for intravenous patient controlled analgesia when allergies to opiates exist (Vogelsang and Hayes, 1991). Butorphanol at a dose of mg/kg, administered IM or IV, increases sedation from acepromazine or benzodiazepines (Hall et al., 2001), while at the same time the sedatives (acepromazine and benzodiazepines) would help diminish the behavioural effects of butorphanol (Carroll et al., 2001). Butorphanol produces good to excellent analgesia after parenteral administration in moderate to severe pain. Butorphanol is a potent analgesic agent with favourable side effects like sedation, nausea, elevated pulmonary vascular pressure and rarely CNS excitation with limited respiratory depression (Pachter et al., 1985). Butorphanol induces mild sedation and has minimal -43-

44 Review of Literature... cardiovascular effects. It may cause mild lowering of heart rate and arterial pressure or respiratory depression (Trim, 1983; Greene et al., 1990). Onset of effect after IV administration of butorphanol is within minutes and lasts for 2 to 4 hours (Hosgood, 1990). Butorphanol do not affect the halothane MAC in ponies (Matthews and Lindsay, 1990) but decreases the isoflurane MAC in dogs (Ko et al., 2000a). Opioids like butorphanol have been incorporated into balanced anaesthetic regimens to reduce the amount of inhalation anaesthetic required and potentially decrease cardiovascular depression (Short, 1987). The combination of butorphanol and romifidine was found to provide better sedation when compared with morphine as a preanaesthetic agent for field castration in ponies (Corletto et al., 2005), whereas Rauser and Lexmaulova (2002) reported that intravenous administration of medetomidine (10 µg/kg) and butorphanol (0.1 mg/kg) gives high quality sedation with quick onset. In another study, Ko et al. (2000c) suggested that a combination of medetomidine (30 µg/kg) with butorphanol (0.2 mg/kg) or ketamine (3 mg/kg) resulted in more reliable and uniform sedation in dogs than did medetomidine alone (30 µg/kg). Whereas, Ko et al. (1996) observed that the low dose medetomidine combined with butorphanol did not result in improved cardiovascular variables and no advantage would be gained from use of a dosage of 10 µg/kg of medetomidine with 0.2 mg/kg of butorphanol compared with 40 µg/kg of medetomidine given alone when administered in dogs. Adequate anaesthesia with adequate analgesic effect and side effects like bradycardia, hypertension and slight respiratory acidosis has been achieved by addition of 0.1 mg/kg butorphanol to medetomidine-midazolam anaesthesia in dogs (Itamoto et al., 2000). Butorphanol (0.1 mg/kg, IM) with reduced doses of medetomidine (10 µg/kg, IM) has generated profound sedation in dogs (Bartram et al., 1994). Butorphanol has been used extensively in a wide variety of animal species. As an analgesic, it is considered to be 4-7 times more potent than morphine, 20 times greater than pentazocine and 40 times greater than -44-

45 Review of Literature... meperidine (Pircio et al., 1976; Vandam, 1980). Adequate anaesthesia with adequate analgesic effect and side effects like bradycardia, hypertension and slight respiratory acidosis has been achieved by addition of 0.1 mg/kg butorphanol to medetomidine-midazolam anaesthesia in dogs (Itamoto et al., 2000). Butorphanol (0.1 mg/kg, IM) with reduced doses of medetomidine (10 µg/kg, IM) has generated profound sedation in dogs (Bartram et al., 1994). In another study, Rauser and Lexmaulova (2002) reported that IV administration of butorphanol (0.1 mg/kg) and medetomidine (10 µg/kg) provided high quality sedation in dogs with quick onset. Additionally, it has also been demonstrated that a xylazine-ketamine-butorphanol combination is better for surgical procedures than a xylazine-ketamine combination, even if the dose of ketamine was reduced to one third (Nishimura et al., 1992) and that butorphanol can enhance medetomidine induced sedation. The sedative effect induced by administering detomidine hydrochloride (0.01 mg/kg, IV) with or without butorphanol tartrate (0.05 mg/kg, IV) to standing dairy cattle has been studied in Holstein cows (Lin and Riddell, 2003). Cows in each treatment group showed significant decrease in heart rate, respiratory rate, ptosis, slow horizontal nystagmus and salivation following administration of detomidine and butorphanol. Duration of sedation was 47.0±8.1 minutes in detomidine group and 43.0± 14.0 minutes in detomidine with butorphanol group. Malik (2008) studied the different combinations of preanaesthetic drugs to thiopental general anaesthesia in buffaloes and observed that medetomidine (2.5 µg/kg)-butorphanol (0.05 mg/kg) combination provides better quality sedation, analgesia, muscular relaxation and dose sparing effects with transient but slightly more cardiac depression than midazolam (0.25 mg/kg) - butorphanol (0.05 mg/kg) combination when used as preanaesthetics to thiopental sodium in buffaloes. Ahmad (2009) also reported better sedation, analgesia and dose sparing effects of medetomidine - butorphanol combination as compared to acepromazine - butorphanol combination when used as preanaesthetic to thiopental and propofol anaesthesia in buffaloes. -45-

46 Review of Literature... Midazolam Midazolam is a new water-soluble benzodiazepine. It has a short duration of action, with a rapid elimination half-life and a total body clearance. Midazolam utilized for sedation, muscle relaxation and as an adjunct to the anaesthesia has a molecular weight of 362 and is 8-chloro-6-(2-fluorophenyl)-1- methyl-4h-imidazo (1, 5-α) (1, 4) benzodiazepine. Probably this is the most underutilized sedative in veterinary practice. The pka of midazolam is 6.15, which permits preparation of salts that are water soluble and in clinical practice it is buffered to an acidic ph (3.5) (Gerecke, 1983). Even though the ph of parenteral formulation is 3.5, after injection it changes its chemical configuration and becomes lipid soluble at physiological ph (Lumb and Jones, 2007). Like other benzodiazepines, midazolam possesses antianxiety, sedative, amnestic, anti-convulscent and skeletal muscle relaxant effects. Midazolam is 2 to 3 times more potent than diazepam (Mohler and Okada, 1977). The drug is prepared either as a hydrochloride or maleate salt and is available in the concentration of 1mg and 5mg per ml injection. Midazolam has a fused imidazole ring in its structure which makes it water soluble. Unlike diazepam, there is very little or no irritation after intravenous or intramuscular injection of midazolam. On intramuscular injection to dogs, midazolam is rapidly and completely absorbed (Court and Greenblatt, 1992). Peak levels reached within 15 minutes with systemic availability values greater than 90% in dogs. At physiological ph, midazolam has high lipophilicity leading to rapid onset of activity. The drug has very high metabolic clearance and rapid rate of elimination (Reves et al., 1985). The high lipophilicity has a number of clinical consequences, including rapid absorption of midazolam from the gastrointestinal tract and rapid entry of midazolam into brain tissue after intravenous administration. In dogs systemic elimination of midazolam is more rapid than man (Court and Greenblatt, 1992). In healthy dogs and cats, IV or IM administration does not induce anaesthesia. Most often it is best utilized as an adjunctive agent to provide -46-

47 Review of Literature... muscle relaxation in combination with ketamine, thiobarbiturates, or opioids such as oxymorphone. The usual dose is 0.1 to 0.2 mg/kg given either IV or IM. Midazolam is rapidly absorbed following IM injection and is relatively nonpainful when compared to ketamine or diazepam IM injections. Cats will often exhibit abnormal behaviour following midazolam injection. These actions can vary from restlessness to belligerence and vocalization. It has also been associated with increased food consumption in this species. Midazolam is a good adjunctive agent for use with oxymorphone or fentanyl to maintain anaesthesia in dogs. Midazolam has sedative, hypnotic and muscle relaxant properties. The hypnotic effect of midazolam is probably due to accumulation of GABA and its occupation of benzodiazepine receptors. These receptors are found in highest density in cerebral cortex and in the hypothalamus, cerebellum, midbrain, hippocampus, striatum, medulla oblongata-pons, and spinal cord in descending order (Mantegazza et al., 1982; Whitwam, 1983). The benzodiazepine and GABA receptors are coupled to a common chloride channel (Gallager, 1982). Occupation of the receptors leads to the hyperpolarization of membrane and neural inhibition. Accumulation of GABA is due to inhibition of its reuptake by midazolam (Cheng and Brunner, 1981). The anticonvulsant effects of midazolam are mediated through enhanced action of GABA on motor circuits in the brain (Richter, 1981).The study in mice shown that midazolam is more effective anticonvulsant than diazepam or lorazepam (DeJong and Bonin, 1981). The anxiolytic effects of midazolam are exerted presumably by increasing the glycine inhibitory neurotransmitters and the hypnotic effect of midazolam is related to GABA accumulation and occupation of the benzodiazepine receptors. Midazolam has a relatively high affinity for the benzodiazepine receptors, roughly two times that of diazepam (Mohler and Okada, 1977). Midazolam is commonly used with ketamine to enhance muscle relaxation and facilitate intubation in dogs and cats (Hellyer et al., 1991; Ilkiw et -47-

48 Review of Literature... al., 1996). Behavioral changes such as marked weakness and ataxia are demonstrated within 5 minutes of IV and IM administration of midazolam although a lag period of 3-4 min is observed when IM route is used (Court and Greenblatt, 1992). The signs are abated within 30 min and dogs can rise and walk (although unsteadily). Midazolam has been reported to cause a reduction in cerebral blood flow in a dose dependent manner and it parallels the behavioral and electroencephalographic effects in dogs (Nugent et al., 1982). These observations suggest that midazolam can be useful for patients who have impaired intracranial compliance or increased intracranial pressure (ICP). Thus midazolam can be an acceptable alternative to barbiturates for induction of anaesthesia in patients who have intracranial pathology. In dogs, cardiac functions are minimally altered. There is insignificant increase in heart rate along with cardiac output. A significant decrease in arterial pressure and minor fluctuations in CVP are observed (Butola and Singh, 2007). Midazolam alone or in combination with opioids and alpha 2 -agonists can be used for sedation of older dogs, small mammals, swine and birds. It is administered along with injectable anaesthetics to improve muscle relaxation and reduce the dose of anaesthetic. Because of its limited effects on cardiopulmonary system it is an ideal sedative for older and compromised animals. Midazolam alone is not a reliable sedatives in dogs and cats (Lemke, 2007), and has been combined with xylazine (Balicki et al., 2007), fentanyl (Natasa et al., 2007), medetomidine and with butorphanol (Leonardi et al., 2007) for premedication in small animals and buffaloes (Malik, 2008). Midazolam has been reported in combination with alpha 2 - agonists to improve the quality of sedation, analgesia and muscular relaxation with lesser cardiopulmonary depression. Midazolam-medetomidine combination has produced desirable sedation with moderate reflex depression, analgesia, excellent muscle relaxation and immobilization without further side effects (Hayashi et al., 1994). Similarly addition of midazolam to medetomidine- -48-

49 Review of Literature... propofol regimen in New Zealand white rabbits significantly prolonged the duration of ear- pinch analgesia, the time of extubation to sternal recumbency and the time from extubation to standing without inducing significant changes in heart rate, respiratory rate, arterial blood pressure and end tidal alveolar CO 2 (Ko et al., 1992). Marques et al. (1995) did not observe any significant decrease in heart rate after midazolam-fentanyl administration in pigs. Intravenous administration of midazolam-butorphanol combination induced significant decrease in heart rate and mean arterial pressure in isoflurane anaesthetized cat (Gross et al., 1993). Whereas, heart rate has been observed to decrease after intramuscular administration of medetomidine-butorphanol-ketamine and medetomidinebutorphanol-midazolam combinations in patas monkeys (Kalema-Zikusoka et al., 2003). It is clear that preservation of haemodynamic functions occurred with midazolam. This involves an intact sympathetic reflex as demonstrated by release of endogenous catecholamine (Glisson et al., 1982). Kalema- Zikusoka et al. (2003) observed an increase in mean arterial pressure after medetomidinebutorphanol-midazolam administration. It, however, remained with in normal physiological limit. Grecu et al. (2007) studied the use of midazolam (0.60 mg/kg) along with xylazine (0.5 mg/kg) and ketamine (10 mg/kg) in geriatric dogs found that the combination provides a good cardiovascular stability and recovery was easy and fast without excitatory trembling. Natasa et al. (2007) found that a combination of midazolam with fentanyl in geriatric dogs can be used for preanaesthesia and as postoperative analgesic with minimal ill effects on vital parameters. The combination had disadvantages like slow recovery and behavioral disturbances. Another study reported that mask induction in healthy dogs with sevoflurane resulted in shorter and smoother induction and smaller and smoother cardiopulmonary changes after premedication with fentanyl and midazolam (Mutoh, 2007). -49-

50 Review of Literature... Dzikiti et al. (2009) studied the sedative and cardiopulmonary effects of acepromazine, midazolam, butorphanol, acepromazine-butorphanol and midazolam-butorphanol on propofol anaesthesia in goats. They observed that sedation with midazolam alone, or midazolam combined with either acepromazine or butorphanol significantly reduces the induction dose of propofol with minimal cardiopulmonary effects in goats. Ahmad (2010) in his study in dogs also reported that addition of midazolam with or without fentanyl enhances sedation, analgesia and muscle relaxation produced by dexmedetomidine, although slightly more depression of cardio-respiratory parameters occurs, particularly when midazolam alone was used with dexmedetomidine. Induction of general anaesthesia A good basal anaesthesia is the foremost requirement in ruminants for smooth induction and a pleasant surgery under general anaesthesia. Any surgical intervention demands unconsciousness, freedom from pain and reflex responses, good muscle relaxation and loss of motor ability mandatorily, which are met with balanced anaesthesia. It is a multiple drug approach in which drugs are targeted to attenuate the individual components of the anaesthetic state. Several anaesthetic protocols have been described for induction and endotracheal intubation in ruminants using injectable drugs (Thurmon, 1986; Carroll and Hartesfield, 1996), but a few are acceptable for maintenance of anaesthesia. The use of propofol as the sole agent for TIVA is generally unsatisfactory, since the concentration levels required to eliminate responses to surgery, induce cardiovascular and respiratory depression. The propofol induction dose requirement should be appropriately decreased by 20% to 80% when propofol is administered in combination with sedative or analgesic agents as part of a balanced technique as well as in elderly and debilitated patients. As a general recommendation, the dose of propofol should always be carefully titrated -50-

51 Review of Literature... against the needs and responses of the individual patient, as there is considerable variability in anaesthetic requirements among patients (Short and Bufalari, 1999). The drug used for induction of anaesthesia in the present study is reviewed in the following text. Propofol Propofol is an alkyl phenol hypnotic and is used as an intravenous anaesthetic in dogs and cats (2, 6 di isopropylphenol) (Hall and Chambers, 1987; Watkins et al., 1987; Brearley et al., 1988; Morgan and Legge, 1989). It was first used in animals in 1977 (Bufalari et al., 1996). Propofol provided rapid induction of anaesthesia, as well as smooth rapid recovery after its administration by both intermittent bolus and continuous intravenous infusion techniques (Adetunji et al., 2002). Propofol is only slightly soluble in water and is marketed as an aqueous emulsion containing 10 mg of propofol, 100 mg of soyabean oil, 22.5 mg of glycerol, and 12 mg of egg lecithin /ml. Sodium hydroxide is added to adjust the ph. The emulsion has a ph of (Tranquilli et al., 2007). Propofol supplied as a 1% emulsion, is short-acting, rapidly metabolized agent, characterized by a virtual lack of any cumulative effect and by rapid recovery after its administration in bolus doses or by continuous infusion (Campbell, 2005). Propofol emulsion can support the microbial growth and endotoxin production. The rapid onset of action is caused by rapid uptake of drug into the CNS. The short action and rapid smooth emergence result from rapid redistribution from the brain to other tissues and efficient elimination from plasma by metabolism (Langley and Heel, 1988). As with other hypnotics, even -51-

52 Review of Literature... when the animal is rendered unconscious with propofol, it will respond to painful stimuli unless analgesic drugs such as the opioids or alpha 2 -agonists are administered concurrently. As a hypnotic, propofol has no reported intrinsic analgesic potency (Hall and Clarke, 1991). In spite of the milk-like appearance of propofol s emulsion (Glen and Hunter, 1984), it is free-flowing and could be injected easily through even a 21- gauge needle. Its aqueous emulsion formulation does not produce histamine release in the dog and no anaphylactoid response. The rate of administration must be sufficiently rapid to compensate for the rapid redistribution from the brain to other non-nervous tissues. The drug is administered by first injecting a loading (induction) dose followed by repeat bolus injection (RBI) of one-half the induction dose as needed or by continuous intravenous infusion (CII) at a rate up to 0.4mg/kg/min (Hall and Chambers, 1987). Propofol is as rapid acting as thiamylal. Inductions are smooth and excitement free. Recoveries are very smooth and rapid. Recovery is more rapid than from isoflurane anaesthesia. Rapidity of recovery is due to propofol s rapid metabolism. In cats, a smaller total dose/ duration have been reported and may be due to differences in metabolism of the phenol in this species (Glenn, 1980). Recoveries in cats can be prolonged following infusions in excess of 30 minutes. Propofol can be used with single infusion technique in cats for short procedure such as castrations, ear flushes, ultrasound biopsies, and laceration repairs. The rapid smooth recovery makes propofol an ideal, although expensive, outpatient anaesthetic. In cats CRI of propofol was seen to be clinically useful over propofol-ketamine combination (Ilkiw and Pascoe, 2003). Seliskar et al. (2007) used total intravenous anaesthesia using propofol and propofol/ketamine and reported smoother and uncomplicated recovery in propofol group. Propofol compatibility with a large variety of preanaesthetics may increase its use as a safe and reliable IV anaesthetic for the induction and maintenance of general anaesthesia and sedation in small animal veterinary -52-

53 Review of Literature... practice (Short and Bufalari, 1999). Drugs used with propofol include the opioid analgesics, fentanyl (Hughes and Nolan, 1999; Yamashita, 2004; Sartas et al., 2006); alfentanil (Flecknell et al.,1990); more recently remifentanil (Mertens et al., 2003) and butorphanol (Surbhi, 2008), while infusions of propofol with other agents such as ketamine, and the alpha 2 -agonists, like xylazine (Cullen and Reynoldson, 1993; Lim et al., 2000; Surbhi, 2008) and medetomidine have been described (Cullen and Reynoldson, 1993; Correia et al., 1996; Nolan et al., 1996; Hughes and Nolan, 1999, Malik, 2008; Ahmad, 2009). In dogs it has been given alone or after premedication with acepromazine, pethidine, papaveratum, diazepam or atropine (Hall and Chambers, 1987; Watkins et al., 1987; Morgan and Legge, 1989; Weaver and Raptopoulos, 1990; Surbhi, 2008), with midazolam (Dzikiti et al., 2006), dexmedetomidine (Gomez et al., 2006), medetomidine (Ko et al., 2006). The potential adverse effects of propofol are respiratory depression and apnoea after IV administration of propofol to dogs, particularly when administered at rapid rates of infusion (Muir and Gadawski, 1998), increased cost and marginal differences in recovery times compared with those of standard inhalant or balanced anaesthetic techniques (Short and Bufalari, 1999) and occasionally excitation (Davies and Hall, 1991). Apnoea has frequently been reported when using propofol (Keegan and Greene, 1993; Bufalari et al., 1997; Muir and Gadawski, 1998; Redondo-Garcia et al., 1999). Rapid rate of administration of propofol may account for the higher incidence of apnoea. Propofol can cause respiratory depression at anaesthesia dosages, the use of respiratory monitors (capnography and pulse oxymeter) and supplemental oxygen administration significantly improves safety and function even in cardiopulmonary compromised dogs (Short et al., 1997). Propofol can induce significant depression of respiratory function, characterized by a reduction in the rate of respiration. Potent alpha 2 -sedative/analgesics (e.g., xylazine, medetomidine) or opioids (e.g., oxymorphone, butorphanol) increase the probability of respiratory depression during anaesthesia. Cardiovascular -53-

54 Review of Literature... changes induced by propofol administration consist of a slight decrease in arterial blood pressures (systolic, mean, and diastolic) without a compensatory increase in heart rate. Selective premedicants markedly modify this characteristic response (Short and Bufalari, 1999). Appropriate consideration of dose reduction and speed of administration of propofol reduces the degree of depression (Short and Bufalari, 1999; Murison, 2001). Mathews et al. (2004) reported smooth inductions and recoveries in dogs and cats. Apnoea did not develop in any dogs, but it occurred in 30% of cats. Heinz bodies increased in 60% of the cats, but the increase was not considered clinically significant. No cardiopulmonary differences were detected, and no apparent cumulative effects were observed when metabisulfite propofol was administered on three consecutive days at an IV dose of 6 mg/kg in dogs and 10 mg/kg in cats. Paddling has been reported on recovery with propofol, but the incidence varies widely (Davies and Hall, 1991; Zoran et al., 1993) and is more prevalent when pre-anaesthetic medications are not used. When used as an induction agent, the calculated dose of propofol is approximately 6mg/kg. In healthy patients, 25% of the calculated dose is given every 30 seconds until intubation is possible. Following induction, the anaesthetic period ranges from 3 to 9 minutes, depending on the total dose required to achieve intubation. Anaesthesia can be maintained with propofol by rapid bolus administration or by constant infusion. A dose of approximately 0.4 mg/kg/min delivered via a constant infusion maintains surgical anaesthesia. If anaesthesia is inadequate, a small bolus dose (1mg/kg) can be given IV and the infusion rate increased 25%. If the patient is too deep, the infusion can be stopped until the patient lightens and the infusion is restarted at a lower rate (25% reduction). Propofol induces depression by enhancing the effects of the inhibitory neurotransmitter GABA and decreasing the brain s metabolic activity (Concas et al., 1991). Propofol decreases intracranial and cerebral perfusion pressure. It transiently depresses arterial pressure and myocardial contractility similar to -54-

55 Review of Literature... the ultrashort-acting thiobarbiturates. Hypotension is primarily the result of arterial and venous vasodilation (Ilkiw et al., 1992). Propofol is primarily metabolized in the liver by conjugation pathway to form inactive metabolites, which are then excreted through urine and, to a much lesser extent, in bile (Simons et al., 1991). Rapid onset of action and fast metabolism make propofol suitable induction agent for short-term anaesthesia or for maintenance for longer procedures when an inhalant anaesthetic is not available. Maintenance can be achieved by continuous intravenous infusion or intermittent bolus injections. Studies have shown that clinical effectiveness of propofol is similar to thiopental, methohexital or etomidate. The incidence of an excitatory response is lower with propofol than with methohexital, but apnoea after induction occurs more often with propofol than with other anaesthetics (Langley and Heel, 1988). The underlying mechanisms of propofol s muscle relaxing properties are not completely clear. These could be peripheral and/or central in origin and could affect any part of the motor pathway, from cortical motor neuron down to the muscle cells. A significant decrease in masseter muscle tone after succinylcholine administration was reported with propofol but not with thiopental, suggesting the involvement of a peripheral effect of propofolinduced muscle relaxation (Ummenhofer et al., 1998). Other studies recently demonstrated a central (spinal) effect of propofol with use of electromyographic (EMG) criteria (Kakinohana et al., 2002). However, myoclonus in response to administration of propofol has been reported in clinical studies, but is an infrequent event generally associated with recovery from anaesthesia (Smith et al., 1993). Cardiovascular depression with propofol is similar to the ultrashortacting barbiturates. Direct myocardial depression, peripheral vasodilation, and ventilation have been reported which results in minimum hypotension. Caution should be used in dogs and cats with known hypotension and myocardial dysfunction. Apnoea is common with bolus administration but can be managed as with the ultrashort- acting barbiturates (IIkiw, 1997). -55-

56 Review of Literature... Respiratory depression and apnoea were determined to be the serious adverse effects induced by intravenous administration of propofol to dogs (Muir and Gadawski, 1998). Duration of apnoea varied between the dogs, but increased in a dose dependent manner at doses more than 14 mg/kg. Propofol produces dose dependent respiratory depression with variable effects on heart rate (Cullen and Reynoldson, 1993). Schumacher et al. (1997) studied infusion of propofol for induction (5 mg/kg over 20 sec) and maintenance of anaesthesia (0.5 mg/kg/min) in adult wild turkeys. They reported apnoea for 10 to 30 seconds after propofol administration and non-significant decrease in heart rate, systolic, mean, and diastolic pressures and significant decrease in respiratory rate at 4 min after administration of propofol. Similarly, respiratory depression and apnoea have been reported to be the most common adverse effects associated with intravenous administration of propofol and when used alone, blood propofol levels in excess of 6 mg/ml were associated with apnoea in dogs (Beths et al., 2001). The use of propofol as the sole agent for TIVA is generally unsatisfactory, since the concentration levels required to eliminate responses to surgery induce cardiovascular and respiratory depression. Infusion of propofol with other agents such as ketamine and the alpha 2 -agonists like medetomidine has been described (Correlia et al., 1996; Nolan et al., 1996; Hughes and Nolan, 1999). However, Mathews et al. (2004) reported smooth induction and recoveries in dogs and cats. Apnoea did not develop in any dog but it occurred in 30% cats. No cardiopulmonary differences were detected and no cumulative effects were observed when metabisulfite propofol was administered on three consecutive days at an IV dose of 6 mg/kg in dogs and 10 mg/kg in cats. Paddling has been reported on recovery with propofol but the incidence varied widely (Davies, 1991; Zoran et al., 1993) and was more prevalent when preanaesthetic medications were not used. Apnea has also frequently been reported with propofol (Keegan and Greene, 1993; Bufalari et al., 1997; Redondo-Garcia et al., 1999). Propofol (2, 6-diisopropyl-phenol) is one of the induction agents commonly used in goats. It has a rapid and smooth onset of action and is cleared -56-

57 Review of Literature... rapidly from the tissues. Besides metabolism by the liver, extra-hepatic sites of metabolism, most prominently the lung, have been claimed for propofol (Grossherr et al., 2006). Propofol causes a dose-related decrease in blood pressure due to peripheral vasodilation and myocardial depression, bradycardia, epileptic form seizures and true convulsions (Bettschart-Wolfensberger et al., 2000). When administered at a dose of 4 7 mg/kg IV in unpremedicated goats and sheep, propofol will induce sufficient anaesthesia for endotracheal intubation (Pablo et al., 1997), while 3 mg/kg was shown to be sufficient for endotracheal intubation in premedicated goats (Bertens et al., 1993). One pharmacokinetic study of propofol in goats showed the half-life of propofol to be 15.5 minutes (Reid et al., 1993). A regional pharmacokinetic study in sheep showed that extra hepatic metabolism of propofol may contribute to a large percentage of the total propofol metabolism. Therefore, propofol may be advantageous in patients with hepatic insufficiency (Mather et al., 1989). Earlier it was reported that propofol has no reported intrinsic analgesic activity (Hall and Clarke, 1991), but later it was suggested that propofol may have some analgesic properties (Zancy et al., 1996), however, it is necessary to supplement the use of propofol with an analgesic drug. Langley and Heel, (1988) stated that the rate of propofol infusion would depend on any other adjunct drugs administered and the degree of surgical stimulation but is usually from 0.15 to 0.4 mg/kg/min in dogs. In spite of the milk like appearance of propofol emulsion (Glen and Hunter, 1984), it is free flowing and could be injected easily through even a 21 G needle. Its aqueous emulsion formulation does not produce histamine release or anaphylactic reaction in the dog. The rate of administration must be sufficiently rapid to compensate for the rapid redistribution from the brain to other nonnervous tissues. The elimination half life of propofol is up to 6 hr in dogs and 24 hr in humans. Propofol is a short-acting hypnotic that has been used in rabbits for shortterm anaesthesia as well as for the induction or maintenance of anaesthesia, but -57-

58 Review of Literature... it does not provide a substantial degree of analgesia (Erhardt et al., 2004; Haberstroh et al., 2004). Propofol reduces both cardiac index and MAP (Mayer et al. 1990). In one study a comparison of thiopental, propofol, and diazepamketamine anaesthesia for evaluation of laryngeal function was made in dogs. Jaw tone was significantly greater with diazepam-ketamine while exposure of the larynx was excellent in dogs receiving thiopental or propofol than with diazepam-ketamine (Gross et al., 2002). Haemodynamic and analgesic effects of propofol (2 mg/kg, IV as a bolus, and 165 µg / kg/ min, IV for 60 minutes, as an infusion) in medetomidine (30 µg / kg, IM) premedicated dogs have been studied by Thurmon et al. (1994). Sinoatrial and atrioventricular blockade developed in all the dogs within 3 minutes of administration of medetomidine, but disappeared within 10 minutes. No apnoea and strong and consistent analgesia was observed throughout 60 minutes of propofol infusion. In this study medetomidine significantly increased systemic vascular resistance and decreased cardiac output. Propofol appeared to alleviate medetomidine induced vasoconstriction. Ilkiw et al. (1992) reported increased mean pulmonary arterial pressure, pulmonary vascular resistance, oxygen utilization ratio, venous admixture, and arterial and mixed venous CO 2 tensions after propofol administration in hypovolemic dogs, whereas mean arterial pressure, arterial oxygen tension, mixed venous oxygen concentration and mixed venous ph decreased. However, by 30 min after propofol administration, all measurements had returned to values similar to those prior to propofol administration. In a study to compare the cardiopulmonary effects of anaesthesia maintained by continuous infusion of ketamine (0.03 mg/kg/min) and propofol (0.3 mg/kg/min) with anaesthesia maintained by inhalation of sevoflurane in 100% oxygen in goats undergoing magnetic resonance imaging, it was found that the mean and diastolic blood pressure values in the sevoflurane group were significantly (P<0.05) lower at most or all time points after induction of anaesthesia when compared to ketamine and propofol groups (Larenja et al., 2005). -58-

59 Review of Literature... When compared with xylazine-ketamine-halothane anaesthesia, propofol induced comparable anaesthetic effects in healthy sheep (Lin et al., 1997). Changes in the cardiopulmonary system were similar in both anaesthetic regimens and these changes were within acceptable ranges. The time taken for the sheep to stand was reported to be significantly shorter for propofol in comparison to xylazine-ketamine and halothane anaesthesia. In the same study, the average dose of propofol used to induce and maintain anaesthesia was 6.63±2.06 mg/kg and 29±11.7 mg/kg/hr (0.49±0.20 mg/kg/min), respectively. The median effective dosage (ED 50 ) of propofol for induction of anaesthesia was determined in 25 dogs premedicated with acepromazine (0.05 mg/kg) and in 35 unpremedicated dogs (Watney and Pablo, 1992). The ED 50 was found to be 2.2 mg/kg in premedicated dogs and 3.8 mg/kg in unpremedicated dogs. The mean ± SD total dosage of propofol required to induce anaesthesia in premedicated animals was 2.8±0.5 mg/kg and in unpremedicated animals was 4.7 ± 1.3 mg/kg. Five unpremedicated dogs showed signs of excitement, however, no animal after premedication showed any excitement. Among small ruminants propofol has shorter half life in the goat than in sheep. Caesarean sections were performed on sheep and goats using propofol as anaesthetic with no preanaesthetic medication. The lambs but not the kids were depressed by the anaesthetic (Vesce and Lucisano, 1991). Also in adult lions propofol was found to be a suitable and safe drug for maintenance of anaesthesia (Epstein et al., 2002). Amarpal et al. (1999) reported a reduction in induction and maintenance dose of propofol in goats using xylazine and medetomidine as premedicants. In cats premedicated with intramuscular acepromazine at 0.2 mg/kg and induced with 6.0 mg/kg of propofol IV, when compared, continuous infusion of 0.4 mg/kg and repeated doses of 3.0 mg/kg for maintenance, continuous infusion promoted a more stable anaesthesia ( Souza et al., 2004) as -59-

60 Review of Literature... seen in dogs (Yoo et al., 2002). Continuous infusion of propofol mg/kg/ min in dogs premedicated with methotrimeprazine produced a dose-dependent respiratory depression. Muscle relaxation was satisfactory, but analgesia was inadequate as seen by the presence of a pedal withdrawal reflex and marked cardiovascular responses to this noxious stimulus, suggests that anaesthesia may not be of sufficient depth for surgery to be carried out suggesting the determination of an adequate propofol infusion rate for the routine use during major surgical procedures in dogs (Aguiar et al., 2001). Alone propofol anaesthesia in dogs without premedication in a single dose of 6.5 mg/kg induced a general anaesthesia lasting for about 5 min and in continuous infusion at 0.5 mg/kg/min general anaesthesia was easy to control in depth and time. Continuous infusion of propofol caused a transient respiratory acidosis and a decrease of blood oxygenation and, a short-lived statistically significant increased pulmonary shunt (Komar and Balicki, 2000). The mean induction doses of propofol for unpremedicated dogs and cats were, 6.55 mg/kg and 8.03 mg/kg respectively. The mean induction doses after premedication with a tranquilizer were 4.5 mg/kg and 5.97 mg/kg for dogs and cats, respectively (Morgan and Legge, 1989). Combining propofol with alpha 2 -agonists offer many advantages and a number of studies are there in which alpha 2 -agonists and propofol combination has been evaluated. In one study propofol alone, propofol with acepromazine and propofol with medetomidine were compared in which the dogs in the medetomidine/propofol group had a significantly higher blood pressure and longer duration of anaesthesia. Both preanaesthetic agents reduced the dose of propofol required for induction of anaesthesia. Medetomidine significantly reduced the dose of propofol required for the maintenance of anaesthesia for a 30-minute period (Bufalari et al., 1995) and in beagle breed of the dogs (Hall et al., 1997). Medetomidine 10 µg/kg, IM, significantly reduced propofol dosage requirements from 6.6 mg/kg IV without medetomidine to induction dose of 2.2 mg/kg IV (Bufalari et al., 1996). Medetomidine when given at 20 µg/kg, as -60-

61 Review of Literature... premedication, followed by propofol, at 2 mg/kg after 10 minutes showed only slight depression of the respiratory and circulatory systems and the method was seen to be suitable for parenteral anaesthesia in dogs (Virtanen et al.,1998). The duration of action and cardiopulmonary effects of propofol (6.55 mg/kg IV), xylazine (0.8 mg/kg IM), medetomidine (30 µg/kg, IM), xylazine plus propofol (3 mg/kg IV) and medetomidine plus propofol (3 mg/iv) were compared in dogs. Xylazine and medetomidine premedication prolonged propofol anaesthesia in dogs. Propofol alone reduced blood pressure and transiently raised heart rate. The apnoea and hypoxaemia induced by propofol alone also occurred in the premedicated groups with hypoxaemia being most evident in the medetomidine/propofol group. Bradycardia was a common feature in all the dogs given xylazine or medetomidine, but hypertension was consistently recorded in all the dogs given medetomidine (Cullen and Reynoldson, 1993). Nolan and Hall (1985) described the use of TIVA with propofol in ponies and reported satisfactory anaesthesia, and good quality recovery. Since then the use of propofol by infusion to maintain anaesthesia has been reported in many species (Nolan and Hall, 1985; Hall and Chambers, 1987; Nolan and Reid, 1993; Correia et al., 1996; Bettschart- Wolfensberger et al., 2001). Total intravenous anaesthesia with propofol or ketamine-medetomidine-propofol combination in horses has shown that the ketamine-medetomidine infusion provided a sparing effect on propofol requirement for maintaining anaesthesia (Umar et al., 2006). TIVA with medetomidine-propofol infusion produces adequate conditions for a range of surgical procedures in horses with adequate cardiovascular function (Bettschart et al., 2005). The minimum infusion rate (MIR) of propofol for total intravenous anaesthesia after premedication with xylazine in horse was estimated at 0.14 mg/kg/min (Oku et al., 2005). Total intravenous anaesthesia with propofol was suggested to be suitable for long-term anaesthesia in horses (Oku et al., 2006). A six-compartment physiological model of the kinetics and dynamics of induction of anaesthesia with propofol in sheep was studied by Upton and -61-

62 Review of Literature... Ludbrook, (1997). Variables for the model were estimated from an extensive in vivo data set using hybrid modelling. Propofol was characterized by rapid transit through the lungs, but a slower transit time though the brain, leading to significant delay between arterial blood concentrations and cerebral effects. The effects of propofol on haemodynamics and renal blood flow in healthy and in septic sheep, and combined with fentanyl in septic sheep was studied by Booke et al. (1996). Anaesthesia was induced in all animals with propofol alone at the rate of 10mg/kg and maintained with 10mg/kg/hr for 30 mins. When propofol is used along with fentanyl, 0.05mg/kg fentanyl was administered first followed by 2mg/kg propofol given 5 mins later. Anaesthesia was maintained at for 30 minutes with a continuous infusion of propofol at the rate of 2mg/kg/hr. Results showed that anaesthesia with propofol in this septic state caused haemodynamic deterioration. Cardiac output decreased to baseline levels. Simultaneously, MAP, which was already lowered during the course of sepsis, decreased even further. The SVRI remained unchanged. The decrease in oxygen delivery was not accompanied by an increase in oxygen extraction, leading to a marked reduction in oxygen consumption. When propofol was combined with fentanyl, these haemodynamic variables were significantly less affected. Cardiac output remained above baseline level. MAP and SVRI, as well as oxygen consumption, remained unchanged. Ludbrook and Uptron (1997) studied a physiological model of induction of anaesthesia with propofol in sheep and found that the depth of anaesthesia occurred in 2-3 mins after cessation of injection. Injection over 2 mins minimized the induction dose. More rapid injection (<1 min) did not significantly hasten induction, but increase dose requirements and produced large peak arterial concentrations, potentially risking hypotension. Zheng et al. (1998) reported the influence of bolus injection of propofol on its cardiovascular effects and peak blood concentrations in sheep. Propofol was injected at 200 mg over 2 mins (slow injection) and 0.5 min (rapid injection) on separate occasions in random order in seven chronically instrumented sheep. -62-

63 Review of Literature... The rapid injection was associated with more profound decreases in arterial mean blood pressure which is dangerous to patient as compared to slow injection (35.7% vs 23.7% maximal reductions from baseline, respectively; P<0.02). Surbhi (2008) reported that the used of propofol after administration of atropine, xylazine and butorphanol in dogs caused tachycardia and decrease in RR. Decreased in the SpO 2 and rectal temperature was also recorded whereas the haemodynamic parameter (MAP) was found to increase after premedication after which it decrease after propofol administration. Both xylazine-butorphanolpropofol and medetomidine-butorphanol-propofol can be employed safely for anaesthetic-management of canine orthopaedic patients. Ahmad (2009) recorded a significant lower dose of propofol (1.64±0.03 and 2.04±0.87 mg/kg) for induction of anaesthesia in buffaloes premedicated with medetomidine-butorphanol and acepromazine-butorphanol as compared to butorphanol alone. A significant increased in the heart rate was recorded after propofol administration whereas; the respiratory rate was recorded to decrease even after propofol administration in all the groups. The haematological parameters like Hb and PCV was also found to be decreased in all the groups. In similar study, mg/kg/min was reported to be safe for constant rate infusion anaesthesia of 2 hr duration in buffaloes and halothane was considered better maintenance agent in terms of clinicophysiological and haematobiochemical stability as compared to propofol and ketamine in buffaloes (Malik, 2008). Upton et al. (2009) studied the effect of a range of doses of CNS 7056 (esterase-metabolized benzodiazepine) midazolam, and propofol on depth of sedation, the respiratory system, and the cardiovascular system in chronically instrumented sheep (n=5). The low, medium, and high doses of CNS 7056, midazolam, and propofol were 0.37, 0.74, and 1.47 mg/ kg; 0.05, 0.1, and 0.2 mg/ kg; and 1, 2, and 4 mg/ kg, respectively. Results suggested all three drugs produced dose-dependent respiratory (e.g. reductions in arterial oxygen tension) -63-

64 Review of Literature... and cardiovascular depression (e.g. reductions in mean arterial pressure). For CNS 7056, midazolam, and propofol, the magnitude of the cardiovascular and respiratory depression was proportional to the depth of sedation achieved for any given drug or dose. For all three drugs, the respiratory and cardiovascular depression was not of sufficient magnitude to endanger the animals. Sedation from propofol was comparable with that of CNS 7056 for medium and high doses only. The high doses produced 20 min of sedation. Sharma and Bhardwaj (2010) studied the effects of propofol-xylaxinemidazolam anaesthesia in eighteen healthy dogs to evaluate the quality of anaesthesia produced by propofol alone or its combinations with xylazine and midazolam as preanaesthetics. They reported that combinations of propofolxylazine produced maximum duration of anaesthesia (35.62± 2.84 min) and recumbency (92.72± 5.95 min), whereas the recovery time was 32.07± 2.99 min. Haemato-biochemical parameters showed non-significant changes in their values during the entire observation period. Common side effects observed were transient apnoea, vomiting, urination, paddling movements, opisthotonus and salivation, although none of these was clinically significant. The used of atropine-medetomidine-ketamine as balanced anaesthesia in neonatal calves was reported by Singh et al. (2010). The initial rise of blood pressure after the administration of atropine and a significant hypotension following medetomidine-ketamine administration were recorded. The overall non-significant increased in voltage parameters of ECG at different time intervals along with ST-elevation and biphasic T-wave were also recorded. -64-

65 3 Materials and Methods Place of work The study was conducted in the Division of Surgery, Indian Veterinary Research Institute, Izatnagar, Bareilly (U.P). Experimental Animals Six sheep of either sex between 1 and 2 years of age were used for the study. All the animals were dewormed with fenbendazole 1 (5 mg/kg) administered orally. All the animals were stall-fed and had free access to feed, water and were maintained in uniform managemental conditions throughout the period of the study. The clinical status of the animals was assessed by recording heart rate, respiratory rate and rectal temperature and by conducting haematological examination. The animals were kept off feed for 24 hours and water was withheld for 12 hours prior to the start of the experiment. The animals were secured in right lateral recumbency and their left ventro-lateral aspect of the neck was prepared for administration of drugs. Experimental design The animals were divided into two major groups viz: A and B, based on the alpha 2- agonist used. Further, six subgroups of animals (A 1, A 2, A 3, B 1, B 2 and B 3 ), each having four animals, were sub-divided randomly, on the basis of other preanaesthetics used. The animals of subgroups A 1 were administered 1 Panacure : Intervet (India) Ltd.

66 Materials and Methods... with medetomidine 2 (30µg/kg, IM); A 2 medetomidine (30µg/kg, IM) + butorphanol 3 (0.1mg/kg, IM) and A 3 medetomidine (30µg/kg, IM) + butorphanol (0.1mg/kg, IM) + midazolam 4 (0.3 mg/kg, IM). Animals of subgroups B 1 were administered with dexmedetomidine 5 (15µg/kg, IM); B 2 dexmedetomidine (15µg/kg, IM) + butorphanol (0.1mg/kg, IM); and B 3 with dexmedetomidine (15µg/kg, IM) + butorphanol (0.1mg/kg, IM) + midazolam (0.3 mg/kg, IM). All the preanaesthetics in each animal were administered simultaneously. Propofol 6 (1%) was used for induction of anaesthesia as well as maintenance agent in both groups (A and B). The dose of propofol was standardized and selected after conducting pilot trials in a few sheep before the start of the study. The drug combinations used in different subgroups have been shown in Table 2. Table 2: Different drugs administered in the animals of various groups for sedation, induction and maintenance of anaesthesia S.N. Sub No. of Preanaesthetic Induction and groups animals agents maintenance agent 1 A1 4 Medetomidine 1 % Propofol 2 A2 4 Medetomidine+ Butorphanol 1% Propofol 3 A3 4 Medetomidine + Butorphanol 1% Propofol + Midazolam 4 B1 4 Dexmedetomidine 1% Propofol 5 B2 4 Dexmedetomidine+ 1% Propofol Butorphanol 6 B3 4 Dexmedetomidine + 1% Propofol Butorphanol + Midazolam 2 Domitor : Orion Corporation, Formos Group Ltd., Turku, Finland 3 Butrum: Aristo Pharmaceuticals Pvt. Ltd. Mumbai Mezolam (5 mg/ml). Neon Laboratories Ltd., Mumbai- India 5 Dexdomitor : Orion Corporation, Formos Group Ltd., Turku, Finland 6 Neorof: Neon Laboratories Ltd., Mumbai- India -66-

67 Fig. 1 : Recording of Blood pressure, SpO 2 and ECG during general anaesthesia Fig. 2 : Recording of Central venous pressure during general anaesthesia

68 Materials and Methods... Technique of drug administration All the animals were restrained in right lateral recumbency and the proposed drugs were administered intramuscularly using disposable syringes. After 10 minutes of premedication, the animals were induced by administering half of the calculated induction dose of 1% propofol intravenously to the effect. The anaesthesia was maintained with 1% propofol for 60 minutes. The different treatments were evaluated on the basis of the following parameters: A. Clinical observations The following clinical parameters were recorded in all the groups during the period of anaesthesia at 0 (base value), 5, 10, 15, 20, 30, 45 and 60 minutes interval after induction. 1. Jaw relaxation Relaxation of the jaw was taken as a measure of muscle relaxation during the study. It was evaluated by observing the resistance to opening of the jaw on applying the pressure on lower and upper jaws. The subjective observation was graded on a 0 to 3 scoring scale as follows: 0 : Not allowing opening the jaws 1 : Resistant to open the jaws and closed quickly 2 : Less resistance to open the jaws and closed slowly 3 : No resistance and jaws remained open Jaw relaxation was recorded at 0, 5, 10 (just before propofol), 15, 20, 30, 45 and 60 min intervals after induction. At each interval mean value for jaw relaxation score was calculated and the muscle relaxation was graded as nil on a score of 0, very mild when the score was > 0 but < 1, mild when the score was = 1 but < 2, moderate when the score was = 2 but < 3 and excellent when the score was

69 Table 1 : System of recording of various reflexes and responses (adopted and modified after Amarpal et al. (1996) Sl. Parameters Score No Relaxation of jaw Not allowing to Resistant to opening Less resistance to No resistance open the jaws the jaws and closed opening the jaws and jaws remain quickly and closed quickly open 2. Palpebral reflex Intact and strong Intact but weak Very weak (very Abolished (quick blink) (slow response) slow and occasional (no response) response) 3. Pedal reflex Intact and strong Intact but weak Intact but very light Abolished completely (strong withdrawal) (animal responding (slow and occasional (no response) slowly) response) 4. Response to Not permitting entry Allow entry but Allowing deeper entry Difficult intubation Easy intubation intubation of tube in the mouth chewing but chewing with coughing without coughing 5. Salivation No salivation Mild salivation Moderate salivation Excessive salivation -69-

70 Materials and Methods Palpebral reflex Status of palpebral reflex was recorded as a measure of depth of sedation. It was assessed by observing the blink of eye lids on touching the medial canthus of the eyes with index finger. The status of palpebral reflex was graded on a 0 to 3 scoring scale as follows: 0 : Intact and strong reflex (quick blink) 1 : Intact but weak reflex (slow response) 2 : Very weak reflex (very slow and occasional response) 3 : Abolished reflex (no response) Palpebral reflex was recorded at 0, 5, 10 (just before propofol), 15, 20, 30, 45 and 60 min intervals after induction. At each interval mean value for palpebral reflex score was calculated and the sedation was graded as absent on a score of 0, mild when the score was > 0 but < 1, moderate when the score was = 1 but < 2, deep when the score was = 2 but < 3 and very deep when the score was Pedal reflex The status of pedal reflex was recorded as a measure of depth of analgesia. It was assessed by recording the response of the animal after a deep pin prick on the periosteum of the rib and at the coronary band with a 22G hypodermic needle. The response of the animal was graded on a 0 to 3 scoring scale as follows: 0 : Intact and strong reflex (strong withdrawal) 1 : Intact but weak reflex (slow response) 2 : Intact but very light reflex (slow and occasional response) 3 : Reflex abolished completely Response of animal was recorded at 0, 5, 10 (just before propofol), 15, 20, 30 and 60 min intervals after induction. -70-

71 Materials and Methods... At each interval mean value for pedal reflex score was calculated and the analgesia was graded as no analgesia on a score of 0, very mild analgesia when the score was > 0 but < 1, mild analgesia when the score was = 1 but < 2, moderate analgesia when the score was = 2 but < 3 and complete analgesia when the score was Salivation Extent of salivation was observed at different intervals as for various others reflexes and was graded on a 0 to 3 scoring scale as follows: 0 : No salivation 1 : Mild salivation 2 : Moderate salivation 3 : Excessive salivation 5. Recovery time Recovery time was recorded as the time elapsed from injection of the drug to the appearance of pedal reflex. 6. Standing recovery time Standing recovery time was recorded as the time elapsed from the time of injection of the drugs until the animal attained standing position and was able to walk. 7. Complete recovery Complete recovery was recorded as the time elapsed from the reappearance of pedal reflex to the time when the animal stood and walked unassisted. 8. Duration of anaesthesia Duration of anaesthesia was recorded as the time elapsed from the time of abolition of pedal reflex to the time of reappearance of the pedal reflex. -71-

72 Materials and Methods Any other observation Urination was also recorded during the period of anaesthesia. B. Physiological observations The following physiological parameters were recorded before and after administration of the drug(s) at 0 minute (base value) and at 5, 10, 15, 20, 30, 45, 60, 75, 90, 105 and 120 after administration of the drugs. 1. Heart rate (beats/min) Heart rate was recorded by non-invasive blood pressure (NIBP) monitor (Surgivet, Smith s medical PM, Inc. Waukesha, U.S.A). 2. Respiratory rate (breaths/min) Respiratory rate was recorded by counting and recording the costal excursion or movement of the thorax. 3. Rectal temperature ( 0 C) Rectal temperature was recorded with the help of a digital thermometer. C. Haematological observations One millilitre blood samples were collected from the jugular vein of each animal in heparinized (1:1000) disposable plastic syringes at 0 min (baseline), 30, 60 and 120 minutes after the administration of the drugs and were subjected to the estimation of the following parameters: 1. Haemoglobin (Hb) Haemoglobin was estimated using 0.1 N HCl with the help of Sahli s haemoglobinometer (Schalm et al., 1975). The values were expressed in g/l. 2. Packed cell volume (PCV) Haematocrit was estimated by microhematocrit method and using haematocrit tube reader (Schalm et al., 1975). The values were expressed in L/ L. -72-

73 Materials and Methods Total leukocyte count (TLC) Total leukocyte count was estimated by standard technique using Neubauer s cell counting chamber (Schalm et al., 1975). The values were expressed in x10 9 /L. 4. Differential leukocyte count (DLC) Blood smears were prepared, fixed in methanol and stained with Giemsa s stain and cells were counted by the method described by Schalm et al., The values were expressed in percent. D. Biochemical observations The blood samples (5 ml) were collected from the jugular vein in heparinized (1:1000) disposable syringes (sodium fluoride for glucose) at 0 min (base line) and at 30, 60 and 120 min after injection of the drug(s). The blood samples were centrifuged at 3000 rpm for 5 min and plasma was separated and stored at C until assayed. The plasma samples were subjected to estimation of the following parameters: 1. Plasma urea nitrogen estimation The plasma urea nitrogen was estimated by the procedure of Wybenga et al. (1971) using diacetyl monoxide (DAM) method. The values were expressed in mmol/l. 2. Plasma glucose The plasma glucose was estimated by GOD/POD method as described by Trinder (1969). The values were expressed in mmol/l. 3. Plasma creatinine Plasma creatinine was estimated by alkaline picrate method (Toro and Ackerman, 1975). The values were expressed in ìmol/l. 4. Cortisol Cortisol was estimated by RIA method using RIA kit. The values were expressed in nmol/l. -73-

74 Materials and Methods Insulin Insulin was estimated by RIA method using RIA kit. The values were expressed in ìiu/ml. E. Haemodynamic study The following parameters were recorded after the stabilization period: 1. Systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) The cuff of the non invasive blood pressure (NIBP) monitor 7 was applied around the metacarpal region or base of the tail for monitoring systolic, diastolic and mean arterial pressures. Systolic, diastolic and mean arterial blood pressures (mmhg) were recorded at 0 min (baseline) and at 5, 10, 15, 20, 30, 45 and 60 minutes after administration of the drug (s). 2. Central venous pressure (CVP) The CVP (cm of water) was recorded by polyethylene catheter passed through a 12 gauze hypodermic needle, anchored in the jugular vein, and was advanced up to anterior vena cava or right atrium. The catheter was connected to a saline manometer containing heparinized saline through a three way stop cock. The position of the catheter was confirmed by observing the pressure changes in the saline manometer due to respiration. The zero of the column was adjusted at the level of the sternal manubrium. A syringe containing heparinised saline was connected to the third end of the stop cock to flush the system. 3. Oxygen saturation of haemoglobin (SpO 2 ) SpO 2 was measured by means of a pulse oxymeter 8. The base value of haemoglobin oxygen saturation was recorded by applying the sensor of the pulse oxymeter on the anal fold or the tongue after anaesthesia at the same 7 Surgivet, Smith Medical PM, Inc, Waukeshya, WI, Pulse oxymeter : Nonin Medical Inc., Minneapolis, USA -74-

75 Materials and Methods... time intervals as in blood pressure. The values were expressed in percent. 4. Electrocardiographic study (ECG) The animal was kept in right lateral recumbency with the fore and hind limbs perpendicular to the long axis of the body on a nonconductive surface to eliminate electrical interference. Subcutaneous needle electrodes were placed at the posterior border of the scapula and at the 5th costochondral junction (base apex lead) for electrocardiography. Electrocardiographic recordings at 1 mv and 50 mm/s paper speed were made at 0 (base value), 5, 10, 15, 20, 30, 45 and 60 min intervals using ECG Machine 9, Cardioart 6108T, (BPL Limited, India). The electrocardiogram was analyzed for the duration and amplitude of P wave, QRS complex, T wave, P-R and Q-T intervals. Statistical Analysis ANOVA (Analysis of variance) and Duncan s multiple range test (DMRT) were used to compare the means at different time intervals among different groups. Paired t test was used to compare the mean values at different intervals with their base values in each group (Snedecor and Cochran, 1994). The subjective data generated from the scoring was analysed using Kruskal Wallis test (Snedecor and Cochran, 1994). 9 Cardiart : BPL Ltd. -75-

76 4 Results Clinical observations Jaw relaxation Mean±SE of jaw relaxation score in the animals of different subgroups have been presented in table 3 and fig. 3. Jaw relaxation score Time (min) A1 A2 A3 B1 B2 B3 Fig. 3 : Mean±SE score of jaw relaxation in the animals of different subgroups In all the subgroups, resistance to opening of jaws was recorded after the animals were premedicated at 5 and 10 min. However, animals of all the subgroups showed excellent muscle relaxation after induction with propofol until the end of the observation period.

77 Results... Comparison among different subgroups did not reveal any significant difference at different time intervals. Palpebral reflex Mean±SE of palpebral reflex score in the animals of different subgroups have been presented in table 4 and fig Palpebral reflex score Time (min) A1 A2 A3 B1 B2 B3 Fig. 4 : Mean±SE score of palpebral reflex in the animals of different subgroups In all the subgroups, the palpebral reflex remained intact during the premedication period at 5 and 10 min. In subgroups A 1, A 2, A 3, and B 1, a very weak palpebral reflex / moderate sedation was recorded immediately after induction with propofol starting from 15 until 60 min of maintenance period. In subgroups B 1 and B 2, a very weak palpebral reflex / moderate sedation was recorded after induction at 15 and 20 min followed by complete lost of palpebral reflex from 30 to 60 min of the observation period. Comparison among different subgroups did not reveal any significant difference however; better sedation was recorded in subgroup B 2 and B

78 Results... Pedal reflex Mean±SE of pedal reflex score in the animals of different subgroups have been presented in table 5 and fig Pedal reflex score Time (min) A1 A2 A3 B1 B2 B3 Fig. 5: Mean±SE score of pedal reflex in the animals of different subgroups In all the subgroups, signs of loss of pain reflex were not observed during premedication at 5 and 10 min. The pedal reflex was intact when the coronary band was pricked with the hypodermic needle. However, animals of all the subgroups recorded complete analgesia after induction with propofol until the end of the observation period. There was no significant difference (P<0.05) among the subgroups at different time intervals. Salivation Mean±SE of salivation score in the animals of different subgroups have been presented in table 6 and fig

79 Results... Salivation score Time (min) A1 A2 A3 B1 B2 B3 Fig. 6 : Mean±SE score of salivation in the animals of different subgroups No salivation was recorded in all the subgroups after administration of preanaesthetics at 5 and 10 min. A very mild degree of salivation was recorded in all the subgroups after induction with propofol from 15 min until the end of the observation period. Comparison among different subgroups did not reveal any significant change at any time interval. Dose of propofol for induction and maintenance Mean±SE of the induction and maintenance doses of propofol in the animals of different subgroups have been presented in table 7 and fig. 7. Induction dose Mean±SE doses of propofol required for induction of anaesthesia in the animals of different subgroups A 1, A 2, A 3, B 1, B 2 and B 3 were 2.90±0.49 mg/kg, 2.93±0.52 mg/kg, 1.55±0.10 mg/kg, 2.10±0.32 mg/kg, 1.48±0.73 mg/kg and 0.68±0.09 mg/kg respectively. Comparison among different subgroups revealed that subgroups A 3, B 2 and B 3 required significantly (P<0.05) lesser dose of propofol for induction of anaesthesia as compared to the subgroups A 1, A 2 and B

80 Results Induction Dose (mg/kg) Fig. 7a : Maintenance dose A1 A2 A3 B1 B2 B3 Subgroups Mean±SE of induction dose (mg/kg) in the animals of different subgroups Mean±SE infusion rates of propofol to maintain the adequate depth of anaesthesia in the animals of different subgroups A 1, A 2, A 3, B 1, B 2 and B 3 were 0.19±0.02 mg/kg/min, 0.18±0.04 mg/kg/min, 0.12±0.03 mg/kg/min, 0.15±0.04 mg/kg/min, 0.13±0.03 mg/kg/min and 0.07±0.02 mg/kg/min respectively Maintenance dose (mg/kg/min) Fig. 7b : 0 A1 A2 A3 B1 B2 B3 Subgroups Mean±SE of maintenance dose (mg/kg/min) in the animals of different subgroups -80-

81 Results... When comparison was made among different subgroups, it was recorded that animals of subgroup B 3 required significantly (P<0.05) lesser dose of propofol to maintain the depth of anaesthesia as compared to subgroups A 1 and A 2. Recovery time Mean±SE of recovery time (min) in the animals of different subgroups have been presented in table 8 and fig. 8. The mean values of recovery time recorded in the animals of different subgroups A 1, A 2, A 3, B 1, B 2 and B 3 were 68.00±5.32 min, 74.50±7.17 min, 77.25±3.90 min, 69.25±1.91 min, 68.25±5.15 min and 77.50±5.32 min respectively Recovery time (min) Fig. 8: 0 A1 A2 A3 B1 B2 B3 Subgroups Mean±SE of recovery time (min) in the animals of different subgroups Comparison among different subgroups did not reveal any significant difference although the recovery time was recorded to be decreased in subgroups A 1, B 1 and B 2 as compared to other subgroups. Standing recovery time Mean±SE of standing recovery time (min) in the animals of different subgroups have been presented in table 9 and fig

82 Results... Standing recovery time (min) Fig. 9 : 0 A1 A2 A3 B1 B2 B3 Subgroups Mean±SE of standing recovery time (min) in the animals of different subgroups The means values of standing recovery time recorded in the animals of different subgroups A 1, A 2, A 3, B 1, B 2 and B 3 were 60.75±18.71 min, 24.75±12.46 min, 24.25±4.61 min, 29.75±2.66 min, 21.00±5.34 min and 27.50±3.38 min respectively. Comparison among different subgroups revealed that standing recovery time in subgroup A 1 was significantly (P<0.05) longer as compared to the remaining subgroups. Complete recovery time Mean±SE of complete recovery time (min) in the animals of different subgroups have been presented in table 10 and fig. 10. The mean values of complete recovery time recorded in the animals of different subgroups A 1, A 2, A 3, B 1, B 2 and B 3 were 71.50±19.26 min, 37.00±11.71 min, 39.50±5.39 min, 40.75±8.51 min, 36.25±4.48 min and 46.00±5.34 min respectively. Comparison among different subgroups revealed that complete recovery time was significantly (P<0.05) longer in subgroup A 1 as compared to A 2 and B

83 Results... Complete recovery time (min) Fig. 10 : Mean±SE of complete recovery time (min) in the animals of different subgroups Duration of anaesthesia A1 A2 A3 B1 B2 B3 Subgroups Mean±SE of the duration of anaesthesia (min) in the animals of different subgroups have been presented in table 7 and fig Duration of anaesthesia (min) A1 A2 A3 B1 B2 B3 Subgroups Fig. 7c : Mean±SE of duration of anaesthesia (min) in the animals of different subgroups -83-

84 Results... Mean duration of anaesthesia in the animals of different subgroups A 1, A 2, A 3, B 1, B 2 and B 3 were 66.50±5.14 min, 72.75±7.31 min, 76.00±4.14 min, 67.50±2.02 min, 66.75±4.96 min and 75.75±5.45 min, respectively. Comparison among different subgroups did not reveal any significant difference although duration of anaesthesia was shorter in subgroup A 1 and longest in subgroup A 3. Other observations Voluntary urination was recorded at the end of the observation period and even after that in one animal each in subgroups A 1, A 2 and B 1 and two animals each in subgroups A 3, B 2 and B 3. Physiological observations Heart rate Mean±SE of heart rate (beats per min) in the animals of different subgroups have been presented in table 11 and fig Heart rate (beats/min) Time (min) A1 A2 A3 B1 B2 B3 Fig. 11 : Mean±SE of heart rate (beats/min) in the animals of different subgroups In subgroup A 1, the HR decreased after premedication and after induction throughout the observation period and the decrease was significant (P<0.05) at -84-

85 Results min after premedication and at 120 min during the observation period. In subgroup A 3, the HR decreased after premedication till 45 min of induction and thereafter, it increase non-significantly (P>0.05) till the end of the observation period. However, the decrease in HR was significant (P<0.05) at 5 and 10 min after premedication and at 15 min after induction. In subgroup B 2, the HR decreased after premedication and then increased until the end. However, the increase in the HR was significant (P<0.05) only at 90 min of the observation period. No significant difference in the HR was recorded in the animals of subgroups A 2, B 1, and B 3 at any time interval when it was compared with their respective base values. The HR decreased non-significantly (P>0.05) after premedication till the end of the observation period in subgroups B 1 and B 2. However, in subgroup A 2, the HR decreased at 5 and 10 min after premedication and thereafter, increased non-significantly (P>0.05) till 90 min of the observation period. Heart rate recorded at 0, 20 and 30 min after induction did not reveal significant difference among different subgroups (P<0.05). At 5 and 10 min after premedication, the HR was significantly (P<0.05) higher in subgroup A 2 as compared to subgroup B 1. Subgroup A 2 also recorded a significantly (P<0.05) higher heart rate at 15 min as compared to other subgroups. At 45 and 60 min, heart rate was significantly (P<0.05) higher in subgroup A2 as compared to subgroup B 3. Respiratory rate Mean±SE of respiratory rate (breaths per min) in the animals of different subgroups have been presented in table 12 and fig. 12. In subgroup A 2, the RR was variable after premedication and decreased after induction till the end of the observation period. However, the decrease in the RR was significant (P<0.05) at 75 min during the observation period. Subgroup B 1 also recorded a decrease in the RR after premedication and -85-

86 Respiration rate (beats/min) Results Time (min) A1 A2 A3 B1 B2 B3 Fig. 12 : Mean±SE of respiration rate (breaths/min) in the animals of different subgroups continued throughout the observation period. The decrease in RR was significant (P<0.05) at 90 min of the observation period. No significant difference in the RR was recorded in the animals of subgroups A 1, A 3, B 2 and B 3 at any time interval when it was compared with their respective base values. In subgroup A 1, the RR was variable after premedication and thereafter, decreased till the end of the observation period without any significant change. Subgroup A 3 also recorded a decrease in the RR after premedication till 20 min after induction and thereafter, RR increased non-significantly (P>0.05) till 120 min. However, in subgroups B 2 and B 3, the RR decreased non-significantly (P>0.05) after 5 min of premedication till the end of the observation period. When comparison was made among different subgroups at different time interval, a significant (P<0.05) difference in RR was recorded at 30, 90 and 120 min of the observation period. At 30 min, RR was significantly (P<0.05) higher in subgroup A 3 as compared to subgroup B 3. RR was also significantly (P<0.05) higher in subgroup A 3 as compared to subgroups B 1, B 2 and B 3 at 90 min. Subgroup A 2 recorded a significantly (P<0.05) higher RR at 120 min as compared to subgroup B

87 Results... Rectal temperature Mean±SE of rectal temperature ( o C) in the animals of different subgroups have been presented in table 13 and fig. 13. In subgroup A 1, the RT decreased after 5 min of premedication and continued till 120 min. However, the decrease was significant (P<0.05) from 20 to 75 min of the observation period. Subgroup A 2 also recorded a trend similar to that of subgroup A 1, however, the decrease was significant (P<0.05) at 20 and 60 min after induction. In subgroup A 3, the RT decreased from 5 min after premedication and continued till the end of the observation period. The decrease was significant (P<0.05) from 20 to 75 min which became highly significant (P<0.01) at 90 and 120 min of the observation period Rectal temperature ( o C) Time (min) A1 A2 A3 B1 B2 B3 Fig. 13 : Mean±SE of rectal temperature ( o C) in the animals of different subgroups In subgroup B 1, the decrease in RT was recorded after 5 min of premedication till the end of the observation period. However, the decrease was significant (P<0.05) at 5 and 10 min after premedication and at 15 and 30 min after induction which later became highly significant (P<0.01) from 45 to 120 min of the observation period. Subgroup B 3 recorded a trend similar to that -87-

88 Results... of subgroup B 1, however, the decrease in RT was significant (P<0.05) after induction at 30, 45, 90, 105 and 120 min of the observation period. No significant difference was recorded in the animals of subgroup B 2 at any time interval although the RT decreased after 5 min of premedication till the end of the observation period. Comparison of rectal temperature among different subgroups did not reveal any significant difference. Haematological observations Haemoglobin (Hb) Mean±SE of haemoglobin (g/l) in the animals of different subgroups have been presented in table 14 and fig. 14. Significant difference was recorded in subgroups A 1 and B 2 at 30 and 60 min respectively Haemoglobin (g/l) Time (min) A1 A2 A3 B1 B2 B3 Fig. 14: Mean±SE of haemoglobin (g/l) in the animals of different subgroups In subgroup A 1, the Hb decreased after the induction and it was significant (P<0.05) at 30 min as compared to the base value. Similarly, in subgroup B 2, the Hb decreased significantly (P<0.05) at 30 and 60 min. -88-

89 Results... No significant difference was recorded in subgroups A 2, A 3, B 1 and B 3 although the Hb decreased throughout the observation period. Comparison among different subgroups revealed that Hb was significantly (P<0.05) higher at 60 min in subgroup A 3 as compared to subgroup B 1. At 120 min, the Hb increased significantly (P<0.05) in subgroup A 2 as compared to subgroup B 2. However, no significant difference was recorded in any of the subgroups at 0 and 30 min. Packed cell volume (PCV) Mean±SE of PCV (L/L) in the animals of different subgroups have been presented in table 15 and fig. 15. Significant difference was recorded in subgroups B 2 and B 3 at 30 and 60 min respectively. In both subgroups, the PCV decreased significantly (P<0.05) at 30 and 60 min respectively when compared with their respective base values Packed cell volume (L/L) Time (min) A1 A2 A3 B1 B2 B3 Fig. 15 : Mean±SE of packed cell volume (L/L) in the animals of different subgroups No significant difference was recorded in subgroups A 1, A 2, A 3 and B 1 although the PCV increased throughout the observation period in subgroups A 1, A 2 and A 3 and decreased in subgroup B

90 Results... Comparison among different subgroups revealed that the PCV was significantly (P<0.05) higher in subgroup B 3 at 0 and 120 min as compared to other subgroups. A significant (P<0.05) increase was also recorded in subgroup B 3 at 30 min as compared to subgroups A 3, B 1 and B 2. The PCV increased significantly (P<0.05) at 60 min in subgroup B 3 as compared to subgroups B 1 and B 2. Total leukocyte count (TLC) Mean±SE of TLC (x10 9 /L) in the animals of different subgroups have been presented in table 16 and fig. 16. Significant difference was recorded in subgroups A 1, A 2 and B 3 at 30, 60 and 120 min respectively. 100 Total leukocyte count (x10 9 /L) Time (min) A1 A2 A3 B1 B2 B3 Fig. 16 : Mean±SE of total leukocyte count (x10 9 /L) in the animals of different subgroups In subgroup A 1, the TLC decreased throughout the observation period but the decrease was significant (P<0.05) at 30 min which became highly significant (P<0.01) at 60 min when compared with the base value. Similar trend was recorded in subgroup A 2, but the decrease in TLC was significant (P<0.05) -90-

91 Results... at 30 min. Subgroup B 3 also recorded the similar pattern, but the decrease was significant (P<0.05) at 60 and 120 min of the observation period. No significant differences were recorded in the subgroups A 3, B 1 and B 2 although the PCV decreased throughout the observation period. When comparison was made among different subgroups, no significant change was recorded in all the subgroups at 60 min. At 0 min, the TLC increased significantly (P<0.05) in subgroup A 1 as compared to subgroup B 2. Subgroup A 3 also recorded a significant (P<0.05) increase at 30 min as compared to subgroups B 1 and B 2. At 120 min, the TLC increased significantly (P<0.05) in subgroup A 2 as compared to subgroup B 2. Differential leukocyte count Lymphocytes Mean±SE of lymphocyte count (%) in the animals of different subgroups have been presented in table 17 and fig Lymphocyte count (%) Time (min) A1 A2 A3 B1 B2 B3 Fig. 17 : Mean±SE of lymphocyte count (%) in the animals of different subgroups -91-

92 Results... In all the subgroups, the lymphocyte count decreased till the end of the observation period. However, significant difference was recorded in subgroups A 3, B 1 and B 2 at 30 and 60 min interval when it was compared with their respective base values. In subgroups A 3 and B 2, the lymphocyte count decreased significantly (P<0.05) at 30 min. However, in subgroup B 1, a highly significant (P<0.01) decrease in the lymphocyte count was recorded at 60 min when it was compared with its base value. No significant differences were recorded in the subgroups A 1, A 2 and B 3 although the lymphocyte count decreased throughout the observation period. Comparison among different subgroups revealed no significant change in the lymphocyte count at 0 min. At 30 min, a significantly (P<0.05) higher lymphocyte count was recorded in subgroup B 1 when compared to subgroups A 1 and A 3. A significant (P<0.05) decrease in the count was also recorded in subgroup A 2 when compared to subgroups B 2 and B 3 at 60 min of the observation period. At 120 min, the lymphocyte count was significantly (P<0.05) less in subgroup A 1 when compared to subgroups A 3 and B 2. Neutrophils Mean±SE of neutrophil count (%) in the animals of different subgroups have been presented in table 18 and fig. 18. The neutrophil count increased in almost all the subgroups throughout the observation period when it was compared with their respective base values. However, significant increase was recorded only in subgroups B 1 and B 2. In subgroup B 1, the increase in neutrophil count was highly significant (P<0.01) at 60 min of the observation period. In subgroup B 2, the increase was significant (P<0.05) at 30 min when it was compared with the base value. No significant change was however, recorded in the subgroups A 1, A 2, A 3 and B 3 at any time interval. -92-

93 Results Neutrophil count (%) Time (min) A1 A2 A3 B1 B2 B3 Fig. 18 : Mean±SE of neutrophil count (%) in the animals of different subgroups Comparison among different subgroups revealed no significant change in the neutrophil count at 0 min. At 30 min, the neutrophil count decreased significantly (P<0.05) in subgroup B 1 as compared to subgroups A 1 and A 3. Subgroup A 2 recorded a significantly (P<0.05) higher count at 60 min as compared to subgroups B 2 and B 3. At 120 min, the count was significantly (P<0.05) higher in subgroup A 1 as compared to subgroup B 2. Basophils Mean±SE of basophil count (%) in the animals of different subgroups have been presented in table 19 and fig. 19. The basophil count decreased in almost all the subgroups except in subgroup B 3. However, no significant difference was recorded in all the subgroups at any time interval. Comparison among different subgroups also revealed no significant (P>0.05) difference among the subgroups at different time interval. -93-

94 Results Basophil count (%) Time (min) A1 A2 A3 B1 B2 B3 Fig. 19 : Mean±SE of basophil count (%) in the animals of different subgroups Monocytes Mean±SE of monocytes (%) in the animals of different subgroups have been presented in table 20 and fig Monocyte count (%) Time (min) A1 A2 A3 B1 B2 B3 Fig. 20 : Mean±SE of monocyte count (%) in the animals of different subgroups -94-

95 Results... The monocyte count increased in subgroups A 1, B 1 and B 3 at all the intervals. However, it decreased in subgroups A 2 and B 2 with negligible count in subgroup A 3 when it was compared with their respective base values. In subgroup B 2, the monocyte count decreased significantly (P<0.05) at 30 min and no significant change was recorded in other subgroups at any time interval. When comparison was made among different subgroups, the monocyte count was significantly (P<0.05) higher in subgroup B 2 when compared to subgroups A 1 and A 3 at 0 min. A significantly (P<0.05) increased count was recorded in subgroup B 3 at 30 min when compared to subgroups A 1 and A 3. At 60 min, the count significantly (P<0.05) increased in subgroup B 1 when compared to subgroups A 2 and A 3. Significant (P<0.05) increase in monocyte count was recorded at 120 min in subgroup B 2 when compared to subgroup A 3. Eosinophils Mean±SE of eosinophil count (%) in the animals of different groups have been presented in table 21 and fig Eosinophil count (%) Time (min) A1 A2 A3 B1 B2 B3 Fig. 21 : Mean±SE of eosinophil count (%) in the animals of different subgroups -95-

96 Results... In all the subgroups, eosinophil count did not vary considerably at any time interval and the count at different time intervals in different subgroups did not differ significantly (P>0.05) from their respective base values. Comparison of the eosinophil count revealed no significant (P>0.05) difference among the subgroups at different time interval. Biochemical observations Plasma urea nitrogen Mean±SE of plasma urea nitrogen (mmol/l) in the animals of different subgroups have been presented in table 22 and fig. 22. Plasma urea nitrogen (mmol/l) Time (min) A1 A2 A3 B1 B2 B3 Fig. 22 : Mean±SE of plasma urea nitrogen (mmol/l) in the animals of different subgroups The plasma urea nitrogen decreased non-significantly (P>0.05) throughout the observation period in subgroups A 1, A 3, B 1 and B 2. However, it increased in subgroups A 2 and B 3 without any significant difference. When comparison was made among different subgroups, a significantly (P<0.05) higher plasma urea nitrogen was recorded at 0 min in subgroup B 2 as compared to subgroups A 1, A 2 and B 1. At 30 min, a significantly (P<0.05) higher level was recorded in subgroup B 2 than subgroup B 1. The plasma urea nitrogen -96-

97 Results... was significantly (P<0.05) higher in subgroup B 3 when compared to subgroup B 1 at 60 min and no significant change was recorded at 120 min in any of the subgroups. Plasma glucose Mean±SE of plasma glucose (mmol/l) in the animals of different subgroups have been presented in table 23 and fig. 23. In all the subgroups, the plasma glucose increased after induction till the end of the observation period when it was compared with their respective base values except in subgroup B 2. Plasma glucose (mmol/l) Time (min) A1 A2 A3 B1 B2 B3 Fig. 23 : Mean±SE of plasma glucose (mmol/l) in the animals of different subgroups In subgroups B 1, a highly significant (P<0.01) increase in the plasma glucose was recorded at 30, 60 and 120 min of anaesthesia. However, no significant difference was recorded in the animals of subgroups A 1, A 2, A 3, B 2 and B 3 at any time interval. Comparison among different subgroups revealed a significantly (P<0.05) decreased level of plasma glucose at 0 min in subgroup A 2 as compared to subgroups B 2 and B 3. A significant (P<0.05) decrease was recorded in subgroups -97-

98 Results... A 1 and A 2 at 30 min when compared to subgroups A 3, B 1 and B 3. At 60 min, a significantly (P<0.05) increased plasma glucose was recorded in subgroups B 1 as compared to subgroups A 1 and A 2. A significantly (P<0.05) increased plasma glucose was also recorded in subgroups B 1 and B 3 at 120 min when compared to subgroups A 1 and A 2. Plasma creatinine Mean±SE of plasma creatinine (µmol/l) in the animals of different subgroups have been presented in table 24 and fig Plasma creatinine (µmol/l) Time (min) A1 A2 A3 B1 B2 B3 Fig. 24 : Mean±SE of plasma creatinine (µmol/l) in the animals of different subgroups In subgroups A 1 and A 2, the plasma creatinine decreased nonsignificantly (P>0.05) till 60 min and thereafter, increased at 120 min as compared to their respective base values. However, it decreased throughout the observation period in subgroup A 3. In subgroups B 1, B 2 and B 3, the plasma creatinine increased nonsignificantly (P>0.05) throughout the observation period but was significant (P<0.05) only at 120 min in subgroup B 2 as compared to their respective base values. -98-

99 Results... When comparison was made among different subgroups at different time intervals, the plasma creatinine was significantly (P<0.05) higher at 0 min in subgroup B 2 when it was compared to subgroup A 3. At 60 min, it was significantly (P<0.05) higher in subgroup B 2 when compared to subgroups A 1, A 2 and A 3. The plasma creatinine was significantly (P<0.05) higher in subgroup B 2 when it was compared to the other subgroups at 120 min and no significant change was recorded in any of the subgroup at 30 min. Plasma cortisol Mean±SE of plasma cortisol (nmol/l) in the animals of different subgroups have been presented in table 25 and fig. 25. In all the subgroups, the plasma cortisol decreased after induction till the end of the observation period when it was compared with their respective base values. 225 Plasma cortisol (mmol/l) Time (min) A1 A2 A3 B1 B2 B3 Fig. 25 : Mean±SE of plasma cortisol count (nmol/l) in the animals of different subgroups In subgroups A 1 and A 3, the decrease in plasma cortisol was highly significant (P<0.01) throughout the observation period at 30, 60 and 120 min when compared with their respective base values. In subgroup A 2, the decrease was significant (P<0.05) at 30 and 60 min. -99-

100 Results... In subgroup B 3, a significant (P<0.05) decrease was recorded at 30 min which became highly significant (P<0.01) at 60 min. No significant difference in the plasma cortisol was however, recorded in subgroups B 1 and B 2 at any time interval. Comparison among different subgroups revealed no significant difference at 0 and 60 min interval. At 30 min, the plasma cortisol was significantly (P<0.05) higher in subgroup B 3 when compared to subgroups A 1, A 2 and A 3. A significant (P<0.05) decrease was recorded at 120 min in subgroup B 3 when compared to subgroups A 1 and A 3. Plasma insulin Mean±SE of plasma insulin (µiu/l) in the animals of different subgroups have been presented in table 26 and fig. 26. In all the subgroups, the plasma insulin decreased after induction till the end of the observation period when it was compared with their respective base values except in subgroup B 2. Plasma insulin (µlu/ml) Time (min) A1 A2 A3 B1 B2 B3 Fig. 26 : Mean±SE of plasma insulin (µlu/ml) in the animals of different subgroups -100-

101 Results... In subgroup A 1, the decrease in plasma insulin was significant (P<0.05) at 30 and 60 min when it was compared with the base value and subgroup A 2 recorded a significant (P<0.05) decrease in plasma insulin at 60 min. Subgroup B 1 also recorded a significant (P<0.05) decrease in the plasma insulin at 120 min. No significant difference was recorded in the animals of subgroups A 3, B 2 and B 3 at any time interval. Comparison among different subgroups at different intervals revealed a significant (P<0.05) increase in plasma insulin at 0 min in subgroup A 1 when compared to subgroup B 2. However, the changes in plasma insulin among different groups at different intervals were non-significant (P>0.05). Haemodynamic studies Systolic blood pressure (SBP) Mean±SE of systolic blood pressure (mmhg) in the animals of different subgroups have been presented in table 27 and fig. 27. Systolic blood pressure (mmhg) Time (min) A1 A2 A3 B1 B2 B3 Fig. 27 : Mean±SE of systolic blood pressure (mmhg) in the animals of different subgroups -101-

102 Results... In subgroup A 1, the SBP increased after 5 min of premedication till the end of the observation period and the increase was significant (P<0.05) at 5 min after premedication and at 15, 20 and 30 min after induction as compared to the base value. In subgroup A 2, the SBP decreased non-significantly after premedication at 5 and 10 min and thereafter, increased till the end of the observation period. The increase in SBP was significant (P<0.05) at 15 min after induction. In subgroup A 3, the fall in SBP was significant (P<0.05) after premedication at 5 and 10 min and the subsequent increase in SBP after induction was significant (P<0.05) only at 20 min. In subgroup B 3, the increase in SBP was significant (P<0.05) at 5 min after premedication and no significant change was recorded thereafter, at any interval when compared with the base value. No significant difference in the SBP was recorded in the animals of subgroups B 1 and B 2 at any time interval. In subgroup B 1, the SBP decreased after premedication and then increased throughout the observation period without any significant change. However, in subgroup B 2, the SBP nonsignificantly (P>0.05) increased after premedication and continued till the end of the observation period. When comparison was made among different subgroups, the SBP was significantly (P<0.05) higher in subgroup A 3 than A 1, A 2, B 1, B 2 and B 3 at 0 min and at 20 min. At 5, 10 and 30 min, SBP was significantly (P<0.05) higher in subgroup A 3 as compared to subgroup A 2. The SBP was significantly (P<0.05) higher in subgroup A 3 as compared to subgroups A 2, B 1, B 2 and B 3 at 15 min. At 45 min, SBP was significantly (P<0.05) higher in subgroup A 3 as compared to subgroup B 3 and no significant change was recorded in all the subgroups at 60 min. Diastolic blood pressure (DBP) Mean±SE of diastolic blood pressure (mmhg) in the animals of different subgroups have been presented in table 28 and fig

103 Results... Diastolic blood pressure (mmhg) Time (min) A1 A2 A3 B1 B2 B3 Fig. 28 : Mean±SE of diastolic blood pressure (mmhg) in the animals of different subgroups Subgroup A 3 recorded a significant (P<0.05) fall in DBP at 5 min after premedication and subsequent increase after induction was non- significant (P>0.05). In subgroup B 1, the fall in DBP was significant (P<0.05) at 5 min after premedication and also at 60 min of the observation period and the increase was significant (P<0.05) at 15 min after induction. In subgroup B 2, the DBP increased from 5 min after premedication till the end of the observation period and the increase was significant (P<0.05) at 20 min after induction. No significant difference was recorded in the animals of subgroups A 1, A 2 and B 3 at any time interval when it was compared with their respective base values. In subgroups A 1 and B 3, non-significant (P>0.05) increase in DBP after premedication and induction was recorded whereas in subgroup A 2, the decrease in DBP recorded after premedication till the end of the observation period was non-significant (P>0.05) at all the intervals. Comparison among different subgroups revealed that significant (P<0.05) increase in DBP was recorded in the animals of subgroup A 3 at 0 min when compared to other subgroups. At 5, 10 and 60 min, significant (P<0.05) increase in DBP was also recorded in subgroup A 3 as compared to subgroup A

104 Results... Subgroup A 3 recorded a significant (P<0.05) increase in DBP at 15 min as compared to other subgroups. At 20 min, the DBP was significantly (P<0.05) higher in subgroup A 3 as compared to subgroups A 2 and B 2 and significantly (P<0.05) higher than subgroups A 2 and B 3 at 30 and 45 min of the observation period. Mean arterial pressure (MAP) Mean±SE of mean arterial pressure (mmhg) in the animals of different subgroups have been presented in table 29 and fig. 29. Mean arterial pressure (mmhg) Time (min) A1 A2 A3 B1 B2 B3 Fig. 29 : Mean±SE of mean arterial pressure (mmhg) in the animals of different subgroups In subgroup A 1, the MAP increased after premedication till the end of the observation period and the increase was significantly (P<0.05) higher at 5 min after premedication and also at 20 and 30 min after induction. In subgroups A 3 and B 1, the MAP non-significantly (P>0.05) decrease after premedication and the increase in the MAP after induction was significantly (P<0.05) higher at 20 and 15 min respectively during the observation period. No significant difference was recorded in the animals of subgroups A 2, B 2 and B 3 at any time intervals when it was compared with their respective base -104-

105 Results... value although the MAP increased after premedication till the end of the observation period. When comparison was made among different subgroups, MAP was significantly (P<0.05) higher in subgroup A 3 as compared to other subgroups at 0 and 20 min. At 5 and 10 min, MAP in subgroup A 3 was significantly (P<0.05) higher as compared to subgroup A 2. Subgroup A 3 also recorded a significantly (P<0.05) higher MAP at 15 min as compared to A 2, B 1, B 2 and B 3. At 30 min, MAP was significantly (P<0.05) higher in subgroup A 3 than subgroups A 2 and B 3 and at 45 min the MAP was significantly (P<0.05) higher in subgroup A 3 than subgroup B 3. No significant difference was recorded in the animals of all the subgroups at 60 min during the observation period. Central venous pressure (CVP) Mean±SE of central venous pressure (cmh 2 O) in the animals of different subgroups have been presented in table 30 and fig. 30. In all the subgroups, the CVP increased after premedication as well as after induction throughout the observation period. Central venous pressure (cm H 2 O) Time (min) A1 A2 A3 B1 B2 B3 Fig. 30 : Mean±SE of central venous pressure (cm H 2 O) in the animals of different subgroups -105-

106 Results... In subgroup A 1, the CVP was significantly (P<0.05) higher at 30 and 45 min after induction as compared to the base value. In subgroup A 2, the CVP was significantly (P<0.05) higher at 45 min after induction and at 15 min in subgroup A 3. No significant difference was recorded in the animals of subgroups B 1, B 2 and B 3 at any time intervals when it was compared with their respective base value. Comparison made among different subgroups did not reveal any significant difference between the groups at any interval of time. Haemoglobin oxygen saturation (SpO 2 ) Mean±SE of haemoglobin oxygen saturation (%) in the animals of different subgroups have been presented in table 31 and fig. 31. In all the subgroups, the SpO 2 decreased after premedication as well as after induction throughout the observation period. In subgroup A 2, SpO 2 significantly (P<0.05) decreased at 30 min of maintenance period when it was compared with the base value. In subgroup Haemoglobin oxygen saturation (%) Time (min) A1 A2 A3 B1 B2 B3 Fig. 31 : Mean±SE of haemoglobin oxygen saturation (%) in the animals of different subgroups -106-

107 Results... A 3, SpO 2 significantly (P<0.05) decreased below the base value at 30 to 60 min of anaesthesia. Subgroup B 1 recorded a significant (P<0.05) decrease in SpO 2 at 5 min after premedication. Subgroup B 2 animals recorded a significant (P<0.05) decrease in SpO 2 at 5 and 10 min after premedication. No significant difference was recorded in the animals of subgroups A 1 and B 3 at any time intervals when it was compared with their respective base values. When comparison was made among different subgroups, no significant (P>0.05) difference were recorded at all the intervals except at 45 and 60 min of anaesthesia. A significantly (P<0.05) higher SpO 2 was recorded in subgroup B 2 when it was compared to subgroups A 2 and A 3 at 45 min. At 60 min, SpO 2 was significantly (P<0.05) less in subgroup A3 as compared to subgroups A 1 and B 2. Electrocardiographic study In the subgroups A 1, A 2, A 3, B 1, B and B 2, a normal sinus rhythm was 3 recorded before the administration of preanaesthetics. In subgroup A 1, sinus bradycardia was recorded during the premedication period at 5 and 10 min. After induction, the heart rate improved and remained stable till the end of the observation period. In subgroup A 2, sinus bradycardia was recorded at 5 and 10 min after premedication. Thereafter, the heart rate improved at a steady level followed by sinus tachycardia at 45 and 60 min during the end of the maintenance period. Tachycardia persisted till the end of the observation period. In subgroup A 3, a similar trend of sinus bradycardia was recorded after premedication. The heart rate then began to improve at a considerable rate followed by sinus tachycardia at 60 min during the end of the maintenance period. In subgroup B 1, sinus bradycardia was recorded at 5 and 10 min after premedication. Thereafter, the heart rate increased from 5 min of induction and remained steady till 60 min of the maintenance period

108 Results... In subgroup B 2, sinus bradycardia was recorded at 5 and 10 min after premedication. Sinus tachycardia was a consistent finding after 5 min of induction and the heart rate remained stable throughout the duration of the maintenance period. In subgroup B 3, sinus bradycardia was recorded at 5 and 10 min after premedication and continued even after 5 min of induction. Thereafter, a similar pattern of sinus tachycardia was recorded as in subgroups B 1 and B 2 although tachycardia was recorded in one of the animal till 60 min during the induction period. P wave duration Mean±SE of P wave duration (sec) in the animals of different subgroups have been presented in table 32 and fig. 38. In all the subgroups A 1, A 2, A 3, B 1, B 2 and B 3, the P-wave duration did not reveal any significant change at different time intervals although it fluctuated around the base value P-wave duration (sec) Time (min) A1 A2 A3 B1 B2 B3 Fig. 38 : Mean±SE of P-wave duration (sec) in the animals of different subgroups -108-

109 (a) Normal sinus rhythum before the administration of any drug (b) Sinus bradycardia at 5 min after the administration of medetomidine (c) Biphasic T-wave at 15 min after the administration of medetomidine and propofol (d) Normal sinus rhytum at 45 min after the administration of medetomidine and propofol Fig. 40 : Electrocardiographic changes in A 1 subgroup

110 (a) Normal sinus rhythum before the administration of any drug (b) Sinus bradycardia at 10 min after the administration of medetomidine and butorphanol (c) Improvement in the heart rate at 45 min after the administration of medetomidine, butorphanol and propofol (d) Normal sinus rhytum at 60 min after the administration of medetomidine, butorphanol and propofol Fig. 41 : Electrocardiographic changes in A 2 subgroup

111 (a) Normal sinus rhythum before the administration of any drug (b) Sinus bradycardia at 5 min after the administration of medetomidine, butorphanol and midazolam (c) Improvement in the heart rate at 30 min after the administration of medetomidine, butorphanol, midazolam and propofol (d) Normal sinus rhythm at 60 min after the administration of medetomidine, butorphanol, midazolam and propofol Fig. 42 : Electrocardiographic changes in A 3 subgroup

112 (a) Normal sinus rhythum before the administration of any drug (b) Sinus bradycardia at 5 min after the administration of dexmedetomidine (c) Sinus arrhythmia at 45 min after the administration of dexmedetomidine and propofol (d) Normal sinus rhythm at 60 min after the administration of dexmedetomidine and propofol Fig. 43 : Electrocardiographic changes in B 1 subgroup

113 (a) Normal sinus rhythum before the administration of any drug (b) Sinus arrhythmia at 10 min after the administration of dexmedetomidine and butorphanol (c) Sinus bradycardia at 30 min after the administration of dexmedetomidine, butorphanol and propofol (d) Normal sinus rhythm at 45 min after the administration of dexmedetomidine, butorphanol and propofol Fig. 44 : Electrocardiographic changes in B 2 subgroup

114 (a) Normal sinus rhythum before the administration of any drug (b) Sinus bradycardia at 5 min after the administration of dexmedetomidine, butorphanol and midazolam (c) Improvement in the heart rate at 30 min after the administration of dexmedetomidine, butorphanol, midazolam and propofol (d) Normal sinus rhythm at 60 min after the administration of dexmedetomidine, butorphanol, midazolam and propofol Fig. 45 : Electrocardiographic changes in B 3 subgroup