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EFFECTS OF DETOMIDINE AND MIDAZOLAM ADMINISTERED ALONE OR COMBINED, ON INDUCTION DOSE AND QUALITY OF PROPOFOL AND KETAMINE ANESTHESIA IN GOATS.

Byline: M. Asif, M. A. Khan, H. Akbar and M. O. Omer

Keywords: Detomidine, midazolam, sedatives, propofol, ketamine, anesthetics, goat.

INTRODUCTION

Surgical management of animals needed safe pre-anesthetics and ideal anesthetic which produced deep sleep, analgesia, amnesia, and muscle relaxation (Zeedan et al., 2014). The concept of pre-anesthetic medication before the induction of anesthetic agents is well acknowledged in veterinary practice. A suitable pre-anesthetic treatment may help in cardiovascular stability, good sedation and analgesia and better recovery from anesthesia (Waelbers et al., 2009). Pre-anesthetic treatment directly affects dose of anesthetic agents that may result in least complication due to low anesthesia intake (Bednarski et al., 2011). Pre-anesthetic treatment have also been recommended in goats before administering the induction agents (Galatos, 2011). Different sedatives (xylazine, detomidine, dexmedetomidine, daizepam, midazolam and butorphenol) are being used now a days to restraint the animals as a pre-anesthetic agents (Brighton, 2013; Shah et al., 2013).

Detomidine produced dose dependent sedation and analgesia in ruminants (Tunio et al., 2016; EL-Kammar et al., 2014). Detomidine was found better option due to its good sedative, strong analgesic and less cardiorespiratory effects than xylazine (Hall et al., 2001). Pre-anesthetic treatment with midazolam have been found to cause reduction in the amount of inhalant or injectable anesthetic agents both in man and different animal species (Hendrickx et al., 2008; Dzikiti et al., 2011). Pre-anesthetic treatment with midazolam was found to be associated with minimal adverse cardiovascular effects (Dzikiti et al., 2011). Propofol (2, 6-di-isopropylphenol) is a phenolic compound commonly used in veterinary and human anesthetic practice that has capacity to induce smooth anesthesia and permit fast and uneventful recovery (Hall et al., 2001; Bodh et al., 2013).

Propofol alone or in combination with other anesthetic agents had been proved a good anesthetic agent for induction and maintenance of anesthesia (Mattos et al., 2013; Chui et al., 2014; Dahi et al., 2015). Ketamine is 2-(O-chlorophenyl)-2-(methylamine)-cyclohexane chloride that produced a dissociative state of anesthesia (Mama et al., 2005). Ketamine produced analgesic, anesthetic and cataleptic effects without any hypnosis (Clarke et al., 2014). The muscle relaxant limiting property of ketamine can be improved by mixing it with benzodiazepines (Barletta et al., 2011). To the knowledge of the author, currently, very little scientific findings have been reported regarding dose sparing effects of different sedatives on recently available induction agents as well as the impact of these agents on the quality of general anesthesia in goats.

Hence, six combinations of these pre-anesthetic and anesthetic agents were made keeping in view their pharmacokinetics, pharmacodynamics and their safety margin in different species. The five different combinations included in present study had already been trialed in canines, equines and bovines but very few in caprines. In addition to these already trialed combinations in different species including goats, a new combination (detomidine+midazolam+propofol+ketamine) is first ever being trialed in goats. The objective of this study was to investigate the effects of detomidine and midazolam administered alone or combined, on induction dose and quality of propofol and ketamine anesthesia in goats. This research was initiated to answer if detomidine and midazolam administered alone or combined as a co-induction agents, could reduce the dose of propofol and ketamine in goats.

Furthermore, if a reduction in dose was achieved, an additional aim was to determine if it would also impact on the quality of general anesthesia and ameliorate any potential negative cardio-pulmonary effects of these induction agents.

MATERIALS AND METHODS

The protocol for this study was approved by Ethical Review Committee, University of Veterinary and Animal Sciences (UVAS), Lahore (No. DR/551). The experiment was conducted on twelve (12) healthy female goats aged between 08-12 months with average body weight of 24-30 kg. The experimental animals were procured form local market. They were kept at Institute of Continuing Education and Extension (ICE and E, UVAS Ravi Campus, Pattoki, Pakistan) for twenty one (21) days as adaptation period. During this period, they were served with feed and water. They were dewormed by giving Albendazole @ 10 mg/kg two weeks before experiment. Clinico-physiological parameters were also noted regularly throughout the period of acclimatization. A day before the experimental trial, the animals were shifted to Surgery Section, A-block Ravi campus Pattoki. All the animals were deprived of feed and water for 12 hours before sedation or anesthesia (Adetunji et al., 2002).

The animals were weighed 30 minutes before the experiment using an electronic scale (JadeverA(r) Richter Scale, Jadever, Scale Co. Ltd, Taipei, Taiwan). The left jugular vein site was prepared aseptically for administration of drugs. The control dose of propofol used for induction of anesthesia was chosen based on previous study (Prassinos et al., 2005). The goats were randomly divided into six groups (A, B, C, D, E and F) comprising two animals each. Sedative, analgesic, dose sparing, anesthetic and clinico-physiological parameters were evaluated before sedation, after sedation and during anesthesia to total recovery period.

Parameter evaluated:

Sedation: The degree of sedation was noted by observing behavioral changes fifteen minutes after pre-anesthetic treatment and was graded as following (Kalhoro et al., 2000):

1 = no sedation (animal alert with all reflexes present)

2 = mild sedation (animal dull with minimum reduction in reflexes)

3 = moderate sedation (animal calm with weak reflexes and partially closure of eyelids)

4 = deep sedation (animal recumbent with very weak reflexes and complete closure of eyelids)

Anesthetic parameters: Anesthesia parameters (induction quality, muscle relaxation quality, recovery quality, induction time, induction dose,% age reduction in induction dose and duration of anesthesia) were recorded at 5 minutes interval post induction to throughout observation period. Quality of sedation, analgesia, induction, muscle relaxation and recovery were evaluated subjectively during anesthesia by using score card (Table 1).

Clinico-physiological parameters: Clinico-physiological parameters (heart rate, respiratory rate and rectal temperature AdegF) were recorded at 0 min before sedation, 15 min after sedation, 1 min after anesthetic induction and then every 5 minutes during anesthesia to total recovery period. The heart rate (beats/minute) was measured with Littman stethoscope (Littman Classic II, USA). The respiratory rate (breaths/min) was noted by observing the thoraco-abdominal movements. Rectal temperature (AdegF) was recorded using Medicare digital thermometer (distributed by MANA and Co, Pakistan).

Statistical Analysis: The collected data were arranged for dependent variables such as sedation and analgesia quality, induction quality, muscle relaxation quality and recovery quality. Treatment groups were considered as independent variables. Anesthetic and clinico-physiological parameters are much dependent on post-medication time interval. To rule out such effects, data were also arranged for time effects in a single treatment group. Further, analysis was performed using one-way analysis of variance (ANOVA) technique in Statistical Analysis Software (SAS) 9.1 version at (pa$?0.05). Significant differences among means were separated through Duncan's Multiple Range test.

Pre-anesthetic and Anesthetic Medication in Goats.

Groups###Pre-anesthetic medication###Induction

Group A###Normal Saline @ 10ml/whole body weight###Propofol @ 4.20+-0.10 mg/kg body weight

Group B###Detomidine @ 15ug/kg body weight###Different doses of Propofol

Group C###Detomidine @ 15ug/kg body weight###Different doses of Ketamine

Group D###Midazolam @ 0.25mg/kg body weight###Different doses of Propofol

Group E###Midazolam @ 0.25mg/kg body weight###Different doses of Ketamine

Group F###Combination of detomidine+ midazolam###Different doses of combination of Propofol+

###@7ug/kg+ 0.12mg/kg body weight###Ketamine

Table 1. A score card used for assessment of quality of different anesthetic parameters.

###Criteria of scoring induction quality

Score###Quality###Characteristic

###1###Smooth###Gradual falling to the ground with no paddling and no stiffness of limbs

###2###Fair###Gradual falling to the ground with mild paddling and no stiffness of limbs

###3###Rough###Gradual falling with vigorous paddling and strong stiffness of limbs

###(Ghurashi et al., 2009)

###Criteria of scoring analgesia quality

Score###Quality###Characteristic

###1###Excellent###Response is absent

###2###Fair###Mild response, with mild muscle fasciculation

###3###Poor###very responsive, with moderate to severe muscle fasciculation

###(Ghurashi et al., 2009)

###Criteria of scoring muscle relaxation quality

Score###Quality###Characteristic

###1###Excellent###Jaws, neck, abdomen and limbs muscles fully relaxed

###2###Good###Relaxation of neck, abdomen and limbs

###3###Poor###Jaws, neck, abdomen and limbs muscles fully rigid

###(Ghurashi et al., 2009)

###Criteria of scoring recovery quality

Score###Quality###Characteristic

###1###Smooth###Standing at first attempt -mild ataxia

###2###Fair###Needed two or three attempts to stand clear

###3###Poor###Animal remained calm but assisted to stand

###4###Very Poor###Showing signs of excitement during recovery

###(Ghurashi et al., 2009)

Table 2. Sedative, analgesic, dose sparing and anesthetic assessment of normal saline @ 10ml/whole b. wt., detomidine @ 15 ug/kg, midazolam @ 0.25 mg/kg and combination of detomidine and midazolam @ 7 ug/kg + 0.12 mg/kg, on induction doses of propofol and ketamine in goat.

Treatment groups###Sed. score Anal.###I.D (mg/kg)###(%) Red in###I.Q###M.R.Q###I.T###D. o. A###Rec. Q

###Score###I.D###(Sec)###(min)

A N.S + Prop###1.00+-0.00c 1.00+-0.00c###4.20+-0.10a###0.00+-0.00d###1.00+-0.00b###2.00+-0.00b###24.00+-1.00d###2.31+-0.24c###1.00+-0.00c

B Det + Prop###2.00+-0.00b 2.00+-0.00b###3.25+-0.05d###22.60+-0.65b###1.00+-0.00b###2.00+-0.00b###42.00+-4.00c###5.76+-0.64b###1.00+-0.00c

C Det + Ket###2.00+-0.00b 2.00+-0.00b###3.40+-0.00cd###19.00+-1.93bc###3.00+-0.00a###3.00+-0.00a###55.00+-4.00b###6.42+-0.90b###3.00+-0.00a

D Mid + Prop###2.00+-0.00b 2.00+-0.00b###3.50+-0.00bc###16.62+-1.99c###1.00+-0.00b###2.00+-0.00b###52.00+-2.00b###6.89+-0.33b###2.00+-0.00b

E Mid + Ket###2.00+-0.00b 2.00+-0.00b###3.60+-0.00b###14.24+-2.04c###1.00+-0.00b###2.00+-0.00b###74.00+-2.00a###5.77+-0.44b###1.00+-0.00c

F Det+Mid+Prop+Ket###4.00+-0.00a 4.00+-0.00a###2.70+-0.00e###35.68+-1.53a###1.00+-0.00b###1.00+-0.00c###19.00+-1.00d###9.22+-0.09a###1.00+-0.00c

###p-value###<.0001###<.0001###<.0001###<.0001###0.0001###<.0001###<.0001###0.0013###<.0001

Table 3. Mean value (+-S.E.) of heart rate (beats/minute) following premedication with normal saline @ 10ml/whole b. wt., deto midine @ 15ug/kg, midazolam @ 0.25 mg/kg and combination of detomidine and midazolam @ 7ug/kg+0.12 mg/kg and induction with different doses of propofol and ketamine in goat.

Treatment###Post-sedation###Post-induction###p-

groups###0 min###15 min###1 min###5 min###10 min###15 min###20 min###25 min###30 min###35 min###40 min###valu

###e

A N.S +###59.00+-1.###60.00+-1.###83.00+-3.###97.00+-1.###101.50+-0###107.00+-1.###103.00+-1###106.00+-1###<.00

###Prop###00eA###00eB###00dA###00cA###.50bcA###00aA###.00abA###.00abA###01

###Det +###63.00+-1.###55.00+-2.###56.50+-0.###57.50+-0.###53.50+-1.###55.00+-1.0###53.50+-0.###52.50+-1.###0.00

B Prop###00aAB###00bcC###50bcC###50bC###50bcC###0bcD###50bcD###50cD###46

C Det +###61.00+-1.###46.00+-1.###54.00+-1.###51.00+-1.###52.00+-1.###50.50+-2.5###51.00+-0.###52.00+-0.###49.00+-0.###48.50+-0.###48.00+-0.###<.00

###Ket###00aAB###00eD###00bC###00bcdD###00bcC###0bcdDE###00bcdD###00bcD###00cdeB###50cdeB###00deB###01

D Mid +###62.00+-2.###67.00+-1.###70.00+-1.###77.00+-1.###80.00+-1.###83.00+-1.0###87.00+-1.###87.00+-1.###93.00+-1.###92.00+-0.###92.00+-0.###<.00

###Prop###00fAB###00eA###00eB###00dB###00cdB###0cB###00bB###00bB###00aA###00aA###00aA###01

E Mid +###59.00+-1.###66.00+-2.###71.00+-1.###74.00+-1.###76.00+-2.###76.00###79.00+-1.###80.00+-1.###<.00

###Ket###00eB###00dA###00cB###00bcB###00abB###+-1.00aC###00aC###00aC###01

F Det+Mid+Pr###64.00+-0.###54.50+-0.###58.00+-0.###54.00+-1.###53.00+-1.###48.00+-0.0###46.00###46.00+-1.###<.00

###op+Ket###00aA###50cC###00bC###00cD###00cC###0dE###+-0.00dF###00dE###01

###p-value###0.0946###0.0003###<.0001###<.0001###<.0001###<.0001###<.0001###<.0001###0.0005###0.0001###<.0001

Table 4. Mean value (+-S.E.) of respiration rate (breaths/minute) following premedication with normal saline @ 10ml/whole b. w t., detomidine @ 15 ug/kg, midazolam @ 0.25 mg/kg and combination of detomidine and midazolam @ 7ug/kg+0.12 mg/kg and induction with different doses of propofol and ketamine in goat.

Treatment groups###Post-sedation###Post-induction###p-value

###0 min###15 min###1 min###5 min###10 min###15 min###20 min###25 min###30 min###35 min###40 min

A N.S + Prop###17.00+-1.00aA###16.00+-0.00aAB###13.50+-0.50bB###13.50 +-0.50bB###12.50+-0.50bB###12.50+-0.50bB###12.00+-0.00bB###12.00+-0.00bB###0.0006

B Det + Prop###17.00+-1.00aA###14.00+-0.00bC###13.00+-0.00bcB###12.50+-0.50cdBC###12.00+-0.00cdB###12.00+-0.00cdB###11.50+-0.50cdB###11.00+-0.00dB###0.0002

C Det + Ket###16.50+-0.50aA###13.50+-0.50bC###12.50+-0.50bcBC###12.50+-0.50bcBC###12.00+-0.00cdB###11.50+-0.50cdB###11.50+-0.50cdB###11.50+-0.50cdB###11.00+-0.00d###11.00+-0.00d###11.00+-0.00d###<.0001

D Mid +Prop###17.00+-1.00aA###14.50+-0.50bBC###13.50+-0.50bcB###12.50+-0.50cdBC###12.00+-0.00cdB###11.50+-0.50dB###11.50+-0.50dB###11.00+-0.00dB###11.50+-0.50d###11.00+-0.00d###11.00+-0.00d###<.0001

E Mid +Ket###15.00+-1.00dA###17.00+-1.00cA###18.00+-0.00bcA###19.50+-0.50abA###19.50+-0.50abA###20.00+-0.00 abA###20.50+-0.50aA###20.50+-0.50aA###0.0020

F Det+Mid+Prop+Ket###14.00+-0.00aA###11.00+-0.00cD###11.50+-0.50bcC###11.50+-0.50bcC###12.00+-0.00bcB###12.50+-0.50bB###11.50+-0.50bcB###12.00+-0.00bcB###0.0072

###p-value###0.1677###0.0016###0.0003###0.0002###<.0001###<.0001###<.0001###0.05) from each other.

Anesthetic parameters: The mean value of induction dose was found significantly (pa$?0.05) lower in group F (2.70 +-0.00 mg/kg) than control group A (4.20+-0.10 mg/kg) and other treatment groups. While, the mean value of induction dose required in group B and C were found (3.25+-0.05 mg/kg) and (3.40+-0.00 mg/kg) respectively that differed non-significantly (p>0.05) from each other. Similarly, the mean value of induction dose required in group D and E were found (3.50+-0.00 mg/kg) and (3.60 +-0.00 mg/kg) respectively that also differed non-significantly (p>0.05) from each other. The maximum% age reduction in induction dose was noted in group F (35.68+-1.53%) that differed significantly (pa$?0.05) than control group and other treatment groups. The% age reduction in induction dose in group B and C were found (22.60+-0.65%) and (19.00+-1.93%) respectively that differed non-significantly (p>0.05) from each other.

Similarly, the% age reduction in induction dose in group D and E were found (16.62+-1.99%) and (14.24+-2.04%) respectively that also differed non-significantly (p>0.05) from each other. The induction score in groups A, B, D, E and F was found (1.00+-0.00) indicating smooth induction that differed significantly (pa$?0.05) from group C (3.00+-0.00) indicating rough induction. The muscle relaxation score was found (1.00+-0.00) in group F indicating excellent, (2.00+-0.00) in groups A, B, D and E indicating good and (3.00+-0.00) in group C, indicating poor muscle relaxation quality. All treatment groups differed significantly (pa$?0.05) from each other. The recovery score in groups A, B, E and F was found (1.00+-0.00) indicating smooth, (2.00+-0.00) in group D, indicating fair and (3.00+-0.00) in group C, indicating poor recovery quality. All treatment groups differed significantly (pa$?0.05) from each other.

The maximum duration of anesthesia was produced in group F (9.22+-0.09 min) that differed significantly (pa$?0.05) than control group and other treatment groups. The average duration of anesthesia in group B and C was found (5.76+-0.64 min) and (6.42+-0.90 min) respectively that differed non-significantly (p>0.05) from each other. Similarly, the average duration of anesthesia in group D and E was found (6.89+-0.33 min) and (5.77+-0.44 min) respectively that also differed non-significantly (p>0.05) from each other.

Clinico-physiological parameters: Of the clinico-physiological parameters assessed, the statistical analysis showed significant (pa$?0.05) differences for heart rate, respiratory rate and non-significant (p>0.05) differences for rectal temperature among or within all groups (Table 3, 4, 5). These parameters returned to normal values after recovery. The heart rate increased significantly (pa$?0.05) within groups A, D and E from base value to onwards till recovery with peak increase at 15, 30 and 25 min respectively. While, the heart rate decreased significantly (pa$?0.05) within groups B, C and F from base value to onwards till recovery with a peak decrease at 25, 40 and 25 min respectively. The comparison among the treatment groups revealed that the heart rate differed non-significantly (p>0.05) at 0 minute before sedation then differed significantly (pa$?0.05) at 15 minute after sedation and 1 minute after induction to onwards till recovery in all groups (Table 3).

Similarly, the respiratory rate decreased significantly (pa$?0.05) within groups A, B, C, D and F from base value to onwards till recovery with peak decrease at 25, 25, 40, 40 and 15 min respectively. Whereas, the respiratory rate increased significantly (pa$?0.05) within group E with peak increase at 25 min. While the comparison among the treatment groups revealed that respiratory rate differed non-significantly (p>0.05) at 0 minute before sedation then differed significantly (pa$?0.05) at 15 minute after sedation and 1 minute after induction to onwards till recovery in all groups (Table 4). The rectal temperature also decreased non-significantly (p>0.05) within groups A, B, C, D, E and F from base value to onwards till recovery with a peak decrease at 20, 5, 5, 30, 15 and 10 min respectively.

The comparison among the treatment groups revealed that the rectal temperature differed non-significantly (p>0.05) at 0 minute before sedation, 15 minute after sedation and 1 minute after induction to onwards till recovery in all groups (Table 5).

Adverse effects: Mild and hyper-salivation was observed in group A and B respectively. While, mild salivation along-with snoring, regurgitation, ataxia and incoordination was observed in group C and D. Urination and mild ataxia was observed in group E. However, no adverse effects were noticed in group F.

DISCUSSION

In the present study, detomidine and midazolam when administered combined, caused deep sedation and strong analgesia in goats. A deep level of sedation and strong analgesia was obtained probably due to synergism between detomidine and midazolam. The synergistic interaction between dexmedetomidine and midazolam was also reported by (Bol et al., 2000). Midazolam as a single agent has a mild sedative effect but it shows additive or synergistic activity when administered with other sedatives (Cwiek et al., 2009). The combination of sedative drugs reinforced the sedative and analgesic actions of the single drug (Itamoto et al., 2000). The deep sedation and strong analgesia observed in the present study was also confirmed by earlier studies following the administration of medetomidine-midazolam (Kojima et al., 2002; Ahmad et al., 2011; Le-Chevallier et al., 2019).

Detomidine produced sedative effects by activating [alpha]-2 adrenergic receptor and analgesia due to stimulation of receptors within the brain and spinal cord (Sinclair, 2003). The premedication with detomidine-midazolam followed by induction with propofol-ketamine caused maximum reduction in induction dose and% age reduction in induction dose. The% age reduction in induction dose might be due to direct effects of premedication agents on induction agents. The maximum% age reduction in induction dose was obtained probably due to a synergistic action of detomidine-midazolam with propofol-ketamine. However, the synergic mechanism of action of detomidine-midazolam with propofol-ketamine is unknown. The combination of sedatives and anesthetic agents with different mechanisms of action has the benefit of using low doses of individual agents (Ukwueze et al., 2014).

Similar findings were also previously recorded by Kojima et al. (2002) who reported that sedation with combination of medetomidine-midazolam showed maximum dose sparing effects on induction dose of thiopental and propofol in dogs The combined therapy with detomidine-midazolam-propofol-ketamine resulted in significant (pa$?0.05) increase in duration of anesthesia. The increase in duration of anesthesia might be due to the combined sedative effects of detomidine-midazolam on propofol-ketamine. The maximum increase in duration of anesthesia along-with smooth induction, excellent muscle relaxation and smooth recovery may be probably attributable through a synergistic action of detomidine-midazolam with propofol-ketamine. The combinations of various pre-anesthetic had been used to prolong the duration of anesthesia along with shorter recovery time, thereby improving quality of anaesthesia (Potliya et al., 2015). Similar findings were also reported in calves (Kilic, 2008) and dogs (Sahoo et al., 2018).

The smooth induction recorded in most of the groups may be attributed to the combined muscle relaxing effect of detomidine, midazolam and propofol. The sedation with detomidine-midazolam followed by induction with propofol-ketamine caused significant (pa$?0.05) reduction in heart rate and respiratory rate and non-significant (p>0.05) reduction in rectal temperature among or within all groups. The reduction in heart rate, respiratory rate and rectal temperature was obtained probably due to direct depressant action of drugs on central nervous system in general and in particular respiratory centre and hypothalamic thermoregulatory center. Decrease in the values of physiological parameters was attributed due to decreased metabolic rate, muscle relaxation and direct depressant action on central nervous system (Kandpal et al., 2005). It could also have resulted from the synergistic effect of detomidine-midazolam with propofol-ketamine.

The detomidine-midazolam-ketamine when administered combined caused significant reduction in heart rate in calves (Kilic, 2008). The propofol-ketamine-xylazine when administered combined caused significant reduction in heart rate and respiratory rate in goat (Okwudili et al., 2014). Bradycardia is a common cardiovascular effect of detomidine (Tunio et al., 2016), midazolam (Mehlisch, 2002), propofol (Njoku, 2015). However, tachycardia induced by ketamine was masked by detomidine which was also reported by (Hopster et al., 2014). Respiratory depression has been reported as a classical complication of xylazine (Clarke and Trim, 2014), midazolam (Biboulet et al., 2010), propofol (Wiese et al., 2010) and ketamine (Clarke and Trim, 2014). Hypothermia is a common classical complication of detomidine (Maravi et al., 2018), midazolam (Bodh et al., 2013) and propofol (Bodh et al., 2013).

Of the adverse effects observed; mild and hyper-salivation was noticed in animals treated with normal saline-propofol and detomidine-propofol respectively. While, mild salivation along-with snoring, regurgitation, ataxia and incoordination were observed in animals treated with detomidine-ketamine and midazolam-propofol. Urination and mild ataxia were observed in animals treated with midazolam-ketamine. However, no adverse effects were observed in the animals treated with combination of detomidine-midazolam-propofol-ketamine. Salivation after induction with either propofol or ketamine in the present study might be due to delayed effects of detomidine.

Conclusion: It is concluded that the combined therapy with detomidine-midazolam-propofol-ketamine was found to be safe and economical for short surgical procedures in goats. The significant changes in heart rate, respiratory rate and non-significant changes in rectal temperature were noticed in all groups.

Acknowledgments: We fully acknowledge the efforts and support provided by the administration of Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore.

Conflict of Interest: There is no conflict of interest shown by any author.

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Author:M. Asif, M. A. Khan, H. Akbar and M. O. Omer
Publication:Journal of Animal and Plant Sciences
Date:Jan 5, 2021
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