ABSTRACT
The aim of this study was to assess the effects of chemical restraint, general anesthesia and opioid treatment on hematological components in Cuniculus paca. Eight healthy, adult, captivity female animals , underwent three laparoscopic procedures with a 15-day interval were evaluated. After physical restraint, an association of ketamine (25mg/kg) and midazolam (0.5mg/kg) was administered intramuscularly for chemical restraint. Posteriorly, anesthesia was induced and maintained with isoflurane; and randomly administered methadone (0.5mg/kg), tramadol (5mg/kg) or saline-placebo (0,1mL/kg) intramuscularly. After pharmacological restraint and in the final laparoscopy stage, venous blood samples were obtained for complete blood count, total plasma protein (TP), creatinine, alanine aminotransferase (ALT), sodium, potassium, chloride and ionized calcium analysis. During general anesthesia, hemoglobin, TP concentration and lymphocytes decreased (P=0.029; <0.001; 0.022 respectively), whereas the potassium levels increased (P=0.034). In conclusion, chemical restraint with ketamine/midazolam association causes a slight decrease in blood cellular components. Isoflurane anesthesia for laparoscopic procedure lead to decrease in hemoglobin, lymphocytes and protein concentrations, while potassium increased, without any influence from the tramadol or methadone treatment. However, these alterations were transient, and its hematologic values can collaborate in carrying out epidemiological, pathophysiological or case studies in the Cuniculus paca.
Keywords:
isoflurane; ketamine; methadone; midazolam; rodents
RESUMO
O objetivo do presente estudo foi avaliar os efeitos de contenção química, anestesia geral e tratamento com opiáceos nos parâmetros hematológicos em Cuniculus paca. Foram avaliados oito animais saudáveis, fêmeas, adultas, de cativeiro, que foram submetidas a três procedimentos laparoscópicos, com intervalo de 15 dias. Após a contenção física, uma associação de cetamina (25mg/kg) e midazolam (0,5mg/kg) foi administrada por via intramuscular para contenção química. Posteriormente, a anestesia foi induzida e mantida com isoflurano, e administrou-se aleatoriamente metadona (0,5mg/kg), tramadol (5mg/kg) ou placebo salina por via intramuscular. Após a contenção farmacológica e em estágio final da laparoscopia, foram obtidas amostras de sangue venoso para contagem sanguínea completa, proteína de plasma total (TP), creatinina, alanina aminotransferase (ALT), cálcio, sódio, potássio e cloreto ionizado. Durante a anestesia geral, a concentração de hemoglobina, TP e linfócitos diminuiu (P= 0,029;< 0,001; 0,022, respectivamente), enquanto os níveis de potássio aumentaram (P= 0,034). Em conclusão, a contenção química com associação de cetamina/midazolam promove uma ligeira diminuição dos componentes celulares do sangue. A anestesia com isoflavano para o procedimento laparoscópico levou a uma diminuição das concentrações de hemoglobina, linfócitos e proteínas, enquanto o potássio aumentou, sem qualquer influência do tratamento com tramadol ou metadona. No entanto, essas alterações foram transitórias, e os seus valores hematológicos obtidos podem colaborar na realização de estudos epidemiológicos, fisiopatológicos ou casuísticas para Cuniculus paca.
Palavras-chave:
ecocardiografia; eletrocardiografia; roedores; neotropical; selvagem
INTRODUCTION
Cuniculus paca, previously identified as Agouti paca, is the second largest rodent in South America, naturally found from Mexico to Argentina (Emmons et al., 2016EMMONS, L. Cuniculus paca. In: Red list of threatened species.Gland: IUCN, 2016. ), and their habitat destruction and hunting threatens wildlife maintenance (Chiarello et al., 2008CHIARELLO, A.G.; AGUIAR, L.M.S.; CERQUEIRA, R. et al. Mamíferos Ameaçados de Extinção no Brasil. In: MACHADO, A.B.M.; DRUMMOND, G.M.; PAGLIA A.P. (Eds.). Livro vermelho da fauna brasileira ameaçada de extinção. Ministério do Meio Ambiente, Brasília, 2008. p.680-880.). Trying to preserve the species, commercial breeding has been assembled, and biological studies propitiated, particularly in regard to clinical and reproductive characteristics (Lourenço et al., 2008LOURENÇO, R.F.S.; DIAS, R.S.; GOMES, A.P. A criação de paca (Agouti paca) como alternativa de diversificação de produção e renda em Minas Gerais. In: CONGRESSO DA SOCIEDADE BRASILEIRA DE ECONOMIA, ADMINISTRAÇÃO E SOCIOLOGIA RURAL, 46., 2008, Rio Branco. Anais... Rio Branco: Sociedade Brasileira de Economia, Administração e Sociologia Rural, Rio Branco, 2008. p.1-15.). In this context, hematologic analyses are scarce in Cuniculus paca and only a few reference values have been published (Pachaly et al., 2001PACHALY, J.R.; ACCO, A.; LANGE, R.R. et al. Order Rodentia (Rodents), In: FOWLER, M.E.; CUBAS, Z.S. (Eds.). Biology, medicine, and surgery of South American wild animals. Iowa: State University Press, 2001. p.225-237.; Estrada et al., 2011ESTRADA, G.C.A.; ESTRADA, C.G.E.; POMPA, A. Indicadores morfo-fisiológicos de la especie Agouti paca en cautiverio. Moment. Cienc. v.8, p.102-105, 2011. ; Stradiotti et al., 2015STRADIOTTI, C.G.P.; LOPES, G.L.; STRADIOTTI JÚNIOR, D. et al. Estudo hematológico de pacas sob condições de cativeiro. Rev. Bras. Med. Vet., v.37, p.227-232, 2015. ).
Due to the behavioral characteristics and great susceptibility to stress, chemical restraint and/or anesthesia are necessary for clinical procedures (Pachaly and Werner 1998PACHALY, J.R.; WERNER, P.R. Restraint of the paca (Agouti paca) with ketamine hydrochloride, acetylpromazine maleate, and atropine sulfate. J. Zoo Wildl. Med., v.29, p.303-306, 1998.), and for this reason it is important to define and characterize if these procedures cause hematologic alterations as described in other species (Marini et al., 1997MARINI, R.P.; CALLAHAN, R.J.; JACKSON, L.R. et al. Distribution of technetium 99m-labeled red blood cells during isoflurane anesthesia in ferrets. Am. J. Vet. Res., v.58, p.781-785, 1997.; Kiliç, 2008KILIÇ, N. Cardiopulmonary, biochemical, and haematological changes after detomidine-midazolam-ketamine anaesthesia in calves. Bull. Vet. Inst. Pulawy, v.52, p.423-426, 2008. ). The aim of the study was to evaluate some hematological components in healthy female Spotted Pacas in captivity, that underwent laparoscopy under ketamine/midazolam chemical restraint, isoflurane anesthesia and tramadol or methadone treatment.
MATERIAL AND METHODS
The animals were cared for in accordance with guidelines such as the Brazilian Council for Control of Animal Experimentation guidelines (available from http://dx.doi.org/10.1590/S0102-86502009000100015 [accessed on July10, 2017]), and their use of animals was reviewed and approved by institutional Ethics Committee (027420/11) and our Department was registered in the Institute of Environment and Renewable Natural Resources (IBAMA), as Brazilian fauna specimens’ scientific purposes breeder (482508).
A prospective randomized cross-over blind clinical trial was conducted in eight(1±5 years) healthy female adults, adapted to captivity, in specialized confinements, identified by microchip and fed with rodents’ dry food, fruits, vegetables and water. Each animal was subjected to the experimental protocol 3 times, with a 15 day interval.
On the day of the experiment, the animals were captured and physically restrained with a proper sized net. Immediately, the midazolam 0.5mg/kg (Dormonid®, Roche, São Paulo, Brazil) and ketamine 25mg/kg (Ketalar®, Pfizer, Itapevi, Brazil) association was administered intramuscularly (IM) in the thigh, for chemical restraint (CR). Once having lost postural reaction, the animals were transferred to the research center, placed in supine position and the fore limbs were shaved and disinfected for catheterization (over-the-needle catheter 22G) of the cephalic vein. Venous blood was collected by dripping and stored in purple and red tubes (0.5mL) for immediate analysis on an automatic complete blood counter (ABC vet counter®, Horiba, Northampton, United Kingdom), microscope differential leukocytes count, and measurement (BIO-2000 IL®, Bioplus, Barueri, Brazil) of total plasmatic protein (TP), serum creatinine, alanine amino transferase (ALT), sodium (Na), potassium (K), chloride (Cl), calcium free ionized fraction (iCa) and osmolality (Osm). This moment was identified as CR.
Subsequently, Ringer's lactate was infused at 10mL/kg/h, anesthesia induction and maintenance was achieved by isoflurane vaporization (in oxygen 100%; 0.2L/kg/min) in the concentration necessary to maintain the plane of surgical anesthesia (eyeball rotation, medium arterial pressure > 65mm Hg and absence of sensitivity) via face mask using a partial rebreathing circuit with the animals breathing spontaneously. Once on surgical plane, the animals were randomly chosen to receive IM tramadol 5mg/kg (TRA), methadone 0.5mg/kg (MET) or saline placebo 0.1mL/kg (CON), with the evaluators blinded to the treatment. After 15 minutes of stable plane of anesthesia, laparoscopic ovum pick-up (LOPU) was performed as described by Barros et al. (2016BARROS, F.F.P.C.; TEIXEIRA, P.P.M.; USCATEGUI, R.A.R. et al. Laparoscopic ovum pick-up in spotted paca (Cuniculus pacas). Arq. Bras. Med. Vet. Zootec., v.68, p.858-864, 2016.).
Once the LOPU ended, the blood sampling and analysis were repeated and this moment was identified as AS. At this moment, the animals in the CON group received tramadol 5mg/kg intravenously (IV), and 10 minutes later isoflurane was suspended. Oxygen inhalation was maintained for 10 minutes and IM penicillin 20.000IU/Kg, dihydrostreptomycin 2.0mg/kg (Penfort®, Ourofino, Cravinhos, Brazil) and meloxicam 0.3mg/kg (Maxicam®, Ourofino, Cravinhos, Brazil) were administered. Subsequently, the animals were transferred to individual stalls until they recovered from anesthesia, and then were returned to collective enclosures and treated with IM meloxicam 0.2mg/kg every 24 hours for 3 days, using a pole syringe. Daily inspection was performed until complete recovery was deemed.
Statistical analysis was performed using the R software (R Foundation for Statistical Computing, Vienna, Austria), normality distribution of residuals (Shapiro-Wilk test) and homoscedasticity of variances (Barlett test) were tested. Real or transformed variables were compared between moments and analgesic treatments by repeated measures ANOVA and Tukey post-test (P< 0. 05).
RESULTS
Cephalic vein catheterization had not been described before in the species. Although, not visible or palpable due to low skin elasticity, this vessel is located on the cranial face of the forelimb in the middle of a distance between the carpus and the elbow, and it was considered a simple, practical, safe and feasible technique for blood collection.
Values from the hematological analysis in Cuniculus paca under chemical restraint, general anesthesia and treated with tramadol, methadone or placebo are summarized in Table 1 and 2. Under general anesthesia, hemoglobin, TP and lymphocytes decreased (P= 0.029;< 0.001 and 0.022 respectively), whereas potassium levels increased (P= 0.034) when compared to the values during chemical restraint. None of the other studied parameters suffered anesthesia/analgesia influences (P> 0.05). When comparing inter-evaluation periods (15 days), all variables were similar (P> 0.05).
DISCUSSION
There were no anesthetic or surgical complications and all animals recovered satisfactorily. The blood cell counts in Pacas under chemical restraint were slightly lower than those reported in conscious (Estrada et al., 2011ESTRADA, G.C.A.; ESTRADA, C.G.E.; POMPA, A. Indicadores morfo-fisiológicos de la especie Agouti paca en cautiverio. Moment. Cienc. v.8, p.102-105, 2011. ; Stradiotti et al., 2015STRADIOTTI, C.G.P.; LOPES, G.L.; STRADIOTTI JÚNIOR, D. et al. Estudo hematológico de pacas sob condições de cativeiro. Rev. Bras. Med. Vet., v.37, p.227-232, 2015. ) and sedated animals (Pachaly et al., 2001PACHALY, J.R.; ACCO, A.; LANGE, R.R. et al. Order Rodentia (Rodents), In: FOWLER, M.E.; CUBAS, Z.S. (Eds.). Biology, medicine, and surgery of South American wild animals. Iowa: State University Press, 2001. p.225-237.). This alteration may be related with ketamine and its associations since this causes reduction of the blood cells by sympathetic nervous system function modifications (Kiliç, 2008KILIÇ, N. Cardiopulmonary, biochemical, and haematological changes after detomidine-midazolam-ketamine anaesthesia in calves. Bull. Vet. Inst. Pulawy, v.52, p.423-426, 2008. , Ahmed et al., 2009AHMED, A.F.; AL-SOBAYIL, F.A.; AL-RAUJI, Y. An evaluation of acepromazine/ketamine for immobilization of white-tailed deer (Odocoileus virginianus). Res. J. Vet. Sci., v.2, p.1-9, 2009.; Khalaf et al., 2014KHALAF, F.H.; AL-ZUHAIRI, A.H.; AL-MUTHEFFER, E.A. Clinical and hematological effect of acepromazine, midazolam, ketamine as general anesthetic protocol in rabbits. Int. J. Sci. Nat., v.5, p.328-331, 2014.), or with the stress of blood sampling in conscious or sedated animals, which lead to splenic contraction (Thrall, 2012THRALL, M.A. Mammalian hematology: laboratory animals and miscellaneous species. In: THRALL, M.A.; WEISE, G.; ALLISON, R.W.; CAMPBELL, T.W. (Eds.). Veterinary hematology and clinical chemistry. 2.ed. Ames: Wiley-Blackwell, 2012. p.225-237.) and is related with a high lymphocytes count, reported by these authors (Table 3).
Cellular and biochemical histological components (Mean±SE) in Cuniculus paca undergoing ketamine/midazolam chemical restraint (n= 8, 3-times), compared with previous studies in conscious (Estrada et al., 2011ESTRADA, G.C.A.; ESTRADA, C.G.E.; POMPA, A. Indicadores morfo-fisiológicos de la especie Agouti paca en cautiverio. Moment. Cienc. v.8, p.102-105, 2011. , n= 20; Stradiotti et al., 2015STRADIOTTI, C.G.P.; LOPES, G.L.; STRADIOTTI JÚNIOR, D. et al. Estudo hematológico de pacas sob condições de cativeiro. Rev. Bras. Med. Vet., v.37, p.227-232, 2015. , n= 11) and tranquilized animals (Pachaly et al., 2001PACHALY, J.R.; ACCO, A.; LANGE, R.R. et al. Order Rodentia (Rodents), In: FOWLER, M.E.; CUBAS, Z.S. (Eds.). Biology, medicine, and surgery of South American wild animals. Iowa: State University Press, 2001. p.225-237., n= 12)
The Na, Cl, iCa and K concentrations were considered similar to those already reported in Cuniculus paca and other neotropical rodents (Pachaly et al., 2001PACHALY, J.R.; ACCO, A.; LANGE, R.R. et al. Order Rodentia (Rodents), In: FOWLER, M.E.; CUBAS, Z.S. (Eds.). Biology, medicine, and surgery of South American wild animals. Iowa: State University Press, 2001. p.225-237.; Corredor-Matus and Rodríguez-Pulido, 2012; Ribeiro et al., 2008RIBEIRO, E.E.A.; BATISTA, M.C.S.; CARVALHO, M.A.M. et al. Níveis iônicos e enzimáticos de cutias (Dasyprocta sp.) hígidas, criadas em cativeiro, influência do sexo e da idade. Arq. Bras. Med. Vet. Zootec., v.60, p.651-655, 2008.).). Note that in our study the free ionized calcium fraction assessed has not been reported in such species, which in normal conditions corresponds to approximately 50% of the total calcium, as has been evaluated by same authors. We consider it important to describe that even our maximum K values in the MET group (4.5±0.8mmol/L) does not reach the levels (6.41±1.28mmol/L) reported as normal in Pacas by Pachaly et al. (2001).
The anesthetic/laparoscopic procedure led to approximately 8% hemoglobin, TP and lymphocytes decrease. This effect may be explained by different mechanisms: 1) oxidative stress induced by isoflurane, leading to hemolysis and lymphopenia (Deckardt et al., 2007DECKARDT, K.; WEBER, I.; KASPERS, U. et al. The effects of inhalation anaesthetics on common clinical pathology parameters in laboratory rats. Food Chem. Toxicol., v.45, p.1709-1718, 2007.); 2) blood cell sequestration on hematopoietic organs, also attributed to isoflurane (Marini et al., 1997MARINI, R.P.; CALLAHAN, R.J.; JACKSON, L.R. et al. Distribution of technetium 99m-labeled red blood cells during isoflurane anesthesia in ferrets. Am. J. Vet. Res., v.58, p.781-785, 1997.); or 3) surgical hemorrhage, iatrogenic hemodilution or water movement between body compartments (Kiliç, 2008KILIÇ, N. Cardiopulmonary, biochemical, and haematological changes after detomidine-midazolam-ketamine anaesthesia in calves. Bull. Vet. Inst. Pulawy, v.52, p.423-426, 2008. ). We believed that blood cell sequestration is the most plausible explanation for these hematological alterations, since acute hemolysis would probably lead to an unobserved HCM and CMHC increase (Thrall, 2012THRALL, M.A. Mammalian hematology: laboratory animals and miscellaneous species. In: THRALL, M.A.; WEISE, G.; ALLISON, R.W.; CAMPBELL, T.W. (Eds.). Veterinary hematology and clinical chemistry. 2.ed. Ames: Wiley-Blackwell, 2012. p.225-237.) and during the surgical procedure there was no apparent bleeding episode or a potential fluid overload. Another alteration resulting from this procedure was a rise in blood K, probably associated to respiratory acidosis reported in anesthetized rodents submitted to laparoscopy (Fuentes et al., 2004FUENTES, J.M.; HANLY, E.J.; BACHMAN, S.L. et al. Videoendoscopic endotracheal intubation in the rat: a comprehensive rodent model of laparoscopic surgery. J. Surg. Res., v.122, p.240-248, 2004.).
It’s important to note that all of these hematological alterations appeared to be transient, since after the 15 day inter-experimental period, the evaluated parameters were similar to the baseline, as described by Inada et al. (2004INADA, T.; YAMANOUCHI, Y.; JOMURA, S. et al. Effect of propofol and isoflurane anaesthesia on the immune response to surgery. Anaesthesia, v.59, p.954-959, 2004.) due to autonomic nervous or immune systems function imbalances and with limited clinical relevance.
In conclusion, chemical restraint with ketamine/midazolam association causes a slight decrease in blood cellular components. Isoflurane anesthesia for laparoscopic procedure lead to decrease in hemoglobin, lymphocytes and protein concentrations, while potassium increased, without any influence from the analgesic treatment with tramadol or methadone. However, these alterations were transient, and these hematologic values can collaborate in carrying out epidemiological, pathophysiological or case studies in the Cuniculus paca.
ACKNOWLEDGEMENTS
The authors would like to thank the PEC-PG program of the Coordination for Improvement of Higher Education Personnel (CAPES Process 14835126), National Counsel of Technological and Scientific Development (CNPq) and the State of Sao Paulo Research Foundation (FAPESP) for the financial support provided; the Veterinary Hospital "Governador Laudo Natel" (UNESP) for lending the equipment, infrastructure, and staff; to Prof. Dra. Marcia Machado of the Morphology and Animal Physiology Department, for giving animals used in the experiment; and to Marina Brito, Leandro Coutinho, Luciana Padilha-Nakaghi, Ricardo Nociti, Mariana Rodriguez, Renata Mariano and Roberta Crivelaro, for their help with data collection.
REFERENCES
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- BARROS, F.F.P.C.; TEIXEIRA, P.P.M.; USCATEGUI, R.A.R. et al. Laparoscopic ovum pick-up in spotted paca (Cuniculus pacas). Arq. Bras. Med. Vet. Zootec., v.68, p.858-864, 2016.
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- ESTRADA, G.C.A.; ESTRADA, C.G.E.; POMPA, A. Indicadores morfo-fisiológicos de la especie Agouti paca en cautiverio. Moment. Cienc. v.8, p.102-105, 2011.
- FUENTES, J.M.; HANLY, E.J.; BACHMAN, S.L. et al. Videoendoscopic endotracheal intubation in the rat: a comprehensive rodent model of laparoscopic surgery. J. Surg. Res., v.122, p.240-248, 2004.
- INADA, T.; YAMANOUCHI, Y.; JOMURA, S. et al. Effect of propofol and isoflurane anaesthesia on the immune response to surgery. Anaesthesia, v.59, p.954-959, 2004.
- KHALAF, F.H.; AL-ZUHAIRI, A.H.; AL-MUTHEFFER, E.A. Clinical and hematological effect of acepromazine, midazolam, ketamine as general anesthetic protocol in rabbits. Int. J. Sci. Nat., v.5, p.328-331, 2014.
- KILIÇ, N. Cardiopulmonary, biochemical, and haematological changes after detomidine-midazolam-ketamine anaesthesia in calves. Bull. Vet. Inst. Pulawy, v.52, p.423-426, 2008.
- LOURENÇO, R.F.S.; DIAS, R.S.; GOMES, A.P. A criação de paca (Agouti paca) como alternativa de diversificação de produção e renda em Minas Gerais. In: CONGRESSO DA SOCIEDADE BRASILEIRA DE ECONOMIA, ADMINISTRAÇÃO E SOCIOLOGIA RURAL, 46., 2008, Rio Branco. Anais... Rio Branco: Sociedade Brasileira de Economia, Administração e Sociologia Rural, Rio Branco, 2008. p.1-15.
- MARINI, R.P.; CALLAHAN, R.J.; JACKSON, L.R. et al. Distribution of technetium 99m-labeled red blood cells during isoflurane anesthesia in ferrets. Am. J. Vet. Res., v.58, p.781-785, 1997.
- PACHALY, J.R.; ACCO, A.; LANGE, R.R. et al. Order Rodentia (Rodents), In: FOWLER, M.E.; CUBAS, Z.S. (Eds.). Biology, medicine, and surgery of South American wild animals. Iowa: State University Press, 2001. p.225-237.
- PACHALY, J.R.; WERNER, P.R. Restraint of the paca (Agouti paca) with ketamine hydrochloride, acetylpromazine maleate, and atropine sulfate. J. Zoo Wildl. Med., v.29, p.303-306, 1998.
- RIBEIRO, E.E.A.; BATISTA, M.C.S.; CARVALHO, M.A.M. et al. Níveis iônicos e enzimáticos de cutias (Dasyprocta sp.) hígidas, criadas em cativeiro, influência do sexo e da idade. Arq. Bras. Med. Vet. Zootec., v.60, p.651-655, 2008.
- STRADIOTTI, C.G.P.; LOPES, G.L.; STRADIOTTI JÚNIOR, D. et al. Estudo hematológico de pacas sob condições de cativeiro. Rev. Bras. Med. Vet., v.37, p.227-232, 2015.
- THRALL, M.A. Mammalian hematology: laboratory animals and miscellaneous species. In: THRALL, M.A.; WEISE, G.; ALLISON, R.W.; CAMPBELL, T.W. (Eds.). Veterinary hematology and clinical chemistry. 2.ed. Ames: Wiley-Blackwell, 2012. p.225-237.
Publication Dates
-
Publication in this collection
28 Oct 2019 -
Date of issue
Sep-Oct 2019
History
-
Received
28 Jan 2018 -
Accepted
13 Dec 2018