Investigating a method for pharmacologic semen collection in alpacas

Abstract While semen evaluation is standard practice prior to a sale or when infertility is suspected in other species, it is rarely done in camelids due to the difficulties involved in collecting a sample. The reproductive physiology of alpacas differs to that of other domestic animals and is still poorly understood. In the stallion, a technique was developed for semen collection that pharmacologically induces ejaculation without copulation (ex copula). This study investigates whether semen could be reliably collected by ex copula ejaculation in male alpacas. Eleven male Huacaya alpacas were used in this study, and six ex copula treatment protocols were evaluated: (1) saline (control); (2) xylazine only (0.1 mg/kg); (3) xylazine only (0.2 mg/kg); (4) imipramine only (1.0 mg/kg); (5) imipramine (1.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg); and (6) imipramine (2.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg). Each treatment protocol was repeated two to five times. Azoospermic samples obtained from ex copula ejaculation contained numerous epithelial cells but no sperm. A reliable treatment for pharmacologically inducing ejaculation in alpacas remains to be found.

Investigating a method for pharmacologic semen collection in alpacas

While semen evaluation is standard practice prior to a sale or when infertility is suspected in other species, it is rarely done in camelids due to the difficulties involved in collecting a sample. The reproductive physiology of alpacas differs to that of other domestic animals and is still poorly understood. In the stallion, a technique was developed for semen collection that pharmacologically induces ejaculation without copulation (ex copula). This study investigates whether semen could be reliably collected by ex copula ejaculation in male alpacas. Eleven male Huacaya alpacas were used in this study, and six ex copula treatment protocols were evaluated: (1) saline (control); (2) xylazine only (0.1 mg/kg); (3) xylazine only (0.2 mg/kg); (4) imipramine only (1.0 mg/kg); (5) imipramine (1.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg); and (6) imipramine (2.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg). Each treatment protocol was repeated two to five times. Azoospermic samples obtained from ex copula ejaculation contained numerous epithelial cells but no sperm. A reliable treatment for pharmacologically inducing ejaculation in alpacas remains to be found.(AU)

Novo protocolo utilizando ocitocina para induzir a ejaculação em garanhão penectomizado / New protocol using oxytocin to induce ejaculation in penectomized stallion

Background: Several reproductive diseases can prevent ejaculation by the traditional method of collection. Neoplasias as squamous cell carcinoma is the most common tumor of the external genitalia of horses and its lesions usually prevent copulation. The pharmacological induction of ejaculation is an important alternative technique to obtain and preserve the genetic material of stallions incapable of ejaculating by traditional methods of semen collection. However, the protocols currently used have shown questionable results and new protocols are needed in order to increase the success rates. The aim of this study is to report the success of a new protocol in inducing ejaculation when oral imipramine and intravenous oxytocin and detomidine were administrated in a Crioulo stallion.Case: A 9-year-old Crioulo stallion was admitted at the Veterinary Hospital of the São Paulo State University, FMVZUNESP, Botucatu, Brazil, with a history of a mass located on the glans and body of the penis. The histopathological exam confirmed the diagnostic of Squamous cell carcinoma and penectomy was performed. After 10 days of surgery the stallion was submitted to 5 different protocols with 3 days interval between the follow protocols: Imipramine+Xylazine; Imipramine+ Xylazine+Oxytocin; Imipramine+Detomidine and Imipramine+Detomidine+Oxytocin.Discussion: The traditional protocol of pharmacologically-induced ejaculation with imipramine hydrochloride (3 mg/kg/v.o) and xylazine hydrochloride (0.66 mg/kg/iv) was not successful even when oxytocin (20 UI/iv) was added to this protocol. Administration of imipramine hydrochloride (3 mg/kg/v.o) two hours prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) also did not result in ejaculation. However, administration of imipramine hydrochloride (3 mg/kg/v.o) 2 h prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) associated with oxytocin (20 U.I/i.v) resulted in ejaculation.[...]

Novo protocolo utilizando ocitocina para induzir a ejaculação em garanhão penectomizado / New protocol using oxytocin to induce ejaculation in penectomized stallion

Background: Several reproductive diseases can prevent ejaculation by the traditional method of collection. Neoplasias as squamous cell carcinoma is the most common tumor of the external genitalia of horses and its lesions usually prevent copulation. The pharmacological induction of ejaculation is an important alternative technique to obtain and preserve the genetic material of stallions incapable of ejaculating by traditional methods of semen collection. However, the protocols currently used have shown questionable results and new protocols are needed in order to increase the success rates. The aim of this study is to report the success of a new protocol in inducing ejaculation when oral imipramine and intravenous oxytocin and detomidine were administrated in a Crioulo stallion.Case: A 9-year-old Crioulo stallion was admitted at the Veterinary Hospital of the São Paulo State University, FMVZUNESP, Botucatu, Brazil, with a history of a mass located on the glans and body of the penis. The histopathological exam confirmed the diagnostic of Squamous cell carcinoma and penectomy was performed. After 10 days of surgery the stallion was submitted to 5 different protocols with 3 days interval between the follow protocols: Imipramine+Xylazine; Imipramine+ Xylazine+Oxytocin; Imipramine+Detomidine and Imipramine+Detomidine+Oxytocin.Discussion: The traditional protocol of pharmacologically-induced ejaculation with imipramine hydrochloride (3 mg/kg/v.o) and xylazine hydrochloride (0.66 mg/kg/iv) was not successful even when oxytocin (20 UI/iv) was added to this protocol. Administration of imipramine hydrochloride (3 mg/kg/v.o) two hours prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) also did not result in ejaculation. However, administration of imipramine hydrochloride (3 mg/kg/v.o) 2 h prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) associated with oxytocin (20 U.I/i.v) resulted in ejaculation.[...](AU)

NOVOS PROTOCOLOS UTILIZANDO ASSOCIAÇÕES COM OCITOCINA NA INDUÇÃO FARMACOLÓGICA DA EJACULAÇÃO EM GARANHÕES

A indução farmacológica da ejaculação é uma alternativa utilizada para aumentar a função ejaculatória de garanhões incapazes de ejacularem pelos métodos tradicionais de coleta de sêmen. No entanto, os protocolos desenvolvidos até o presente momento apresentam baixas taxas de sucesso na indução da ejaculação, alta variabilidade de doses, vias de administração e efeitos adversos. A ocitocina é um hormônio que participa ativamente no desencadeamento da ejaculação, no entanto, não existem estudos avaliando sua atuação em protocolos de indução farmacológica da ejaculação. Nesse sentido, o presente estudo teve por objetivos: 1) Comparar a eficiência de diferentes protocolos na indução da ejaculação; 2) Avaliar a eficiência da ocitocina quando adicionada aos protocolos; 3) Comparar os parâmetros seminais de ejaculados coletados em vagina artificial e por indução farmacológica da ejaculação. Foram avaliados os protocolos X - Xilazina (0,66/mg/kg/i.v); XO - xilazina (0,66/mg/kg/i.v) + ocitocina (20UI/i.v); IX - Imipramina (3/mg/kg/v.o) + xilazina (0,66/mg/kg/i.v); IXO - Imipramina (3/mg/kg/v.o) + xilazina (0,66/mg/kg/i.v) + ocitocina (20UI/i.v); D- detomidina (0,02/mg/kg/i.v); DO - detomidina (0,02/mg/kg/i.v) + ocitocina (20 UI/i.v); ID-Imipramina (3mg/kg/v.o) + detomidina (0,02mg/kg/i.v); IDO-Imipramina (3mg/kg/v.o) + detomidina (0,02mg/kg/i.v) + ocitocina (20 UI/i.v); IO- Imipramina (3mg/kg/v.o) + ocitocina (20 UI/i.v). Nenhum dos 4 garanhões jovens ejacularam e 9 dos 12 (75%) garanhões adultos responderam a, pelo menos, 1 protocolo. Nenhum dos garanhões que responderam aos tratamentos com xilazina respondeu aos tratamentos com detomidina. Dois garanhões responderam ao X e XO (16,6%) em todas as tentativas, 4 garanhões responderam ao IX e IXO (33,33%) em 75% das tentativas. Um garanhão respondeu ao DO (8,33%) em todas as tentativas, enquanto 5 garanhões responderam ao IDO (41,6%) em 70% dos tentativas. A ereção ocorreu em 5 garanhões (31,25%), enquanto a masturbação ocorreu em apenas 2 garanhões (16,6%). Ereção e masturbação não foram observados nos protocolos sem a administração de imipramina (X, XO, D e DO). Os ejaculados obtidos em DO e IDO tiveram menor volume total, menor volume de gel livre e maior concentração (P <0,05), com um número total de espermatozóides, cinética e morfologia espermática semelhantes (P> 0,05) aos protocolos com xilazina (X, XO, IX e IXO) e ejaculados coletados por meio de vagina artificial. As características do sêmen sugerem diminuição dos fluidos das glândulas acessórias nos protocolos DO e IDO, provavelmente devido a não pré-estimulação sexual e também pelo uso de ocitocina, que aumenta contrações em cauda do epidídimo. Assim, a ocitocina parece desempenhar um papel na indução da ejaculação quando associada à detomidina, mas nenhuma quando associada à xilazina.

The pharmacological induction of ejaculation has been an alternative used to increase ejaculatory function of stallions incapable of ejaculating by the traditional methods of semen collection. However, the protocols developed to date are available in high rate of dose variation, routes of administration, adverse effects and ejaculation rates. In general, pharmacological protocols have shown low success rates in inducing ejaculation. Thus, the aims of the study were: 1) Compare the efficiency of different protocols to induce ex copula ejaculation when detomidine and oxytocin was added to the protocols; 2) Compare seminal parameters between in copula and ex copula ejaculates; We evaluated the nine protocols to ex copula ejaculation: X - xylazine (0.66mg/kg/i.v); XO - xylazine + oxytocin (20UI/i.v); IX - imipramine (3mg/kg/v.o) + xylazine (0.66mg/kg/i.v); IXO - imipramine (3mg/kg/v.o) + xylazine (0.66 mg/kg/i.v) + oxytocin (20UI); D - detomidine (0.02mg/kg/i.v); DO - detomidine (0.02mg/kg/i.v) + oxytocin (20UI/i.v); ID - imipramine (3mg/kg/v.o) + detomidine (0.02mg/kg/i.v); IDO- imipramine (3mg/kg/v.o) + detomidine (0.02mg/kg/i.v) + oxytocin (20 UI/i.v); IO- imipramine (3mg/kg/v.o) + oxytocin (20 UI/i.v). Four young stallions (2-3 y old) and 12 sexually mature stallions (6 to 26 y old) were each submitted to 2 treatment trials conducted at 3-day intervals. Induced ejaculates were collected into a plastic bag and compared with in copula ejaculates. None of the 4 young stallions ejaculated and 9 of the 12 mature stallions responded. Stallions that responded to xylazine did not respond to detomidine treatments. Two stallions responded to X and XO while 4 mature stallions responded to IX and IXO. One stallion respond to DO while 5 stallions responded to IDO. Erection occurred in 5 stallions and no erection and masturbation were observed in protocols without imipramine. The induced ejaculates obtained in DO and IDO had significantly (P<0.05) lower total volume, lower free-gel volume and higher concentration, with similar (P>0.05) total number of spermatozoa, sperm kinetics and morphology compared to in copula ejaculates and xylazine protocols. The semen characteristics suggests decreased accessory glands fluids in protocols DO and IDO, probably resulting from the use of oxytocin, which increase the epididymal tail contraction. Thus, oxytocin appears to play a role to induce ejaculation when associated with detomidine, but not when associated with xylazine.

Genotoxic and cytotoxic evaluation of venlafaxine in an acute and a subchronic assay in mouse

Abstract The present research was made to determine the micronuclei and cytotoxic capacity of the antidepressant venlafaxine in an in vivo acute and subchronic assays in mouse. In the first study, we administered once 5, 50, and 250 mg/kg of the drug, and included a negative and a daunorubicin treated group. Observations were daily made during four days. The subchronic assay lasted 5 weeks with daily administration of venlafaxine (1, 5, and 10 mg/kg) plus a negative and an imipramine administered groups. Observations were made each week. In the first assay results showed no micronucleated polychromatic erythrocytes (MNPE) increase, except with the high dose at 72 h. The strongest cytotoxic effect was found with 250 mg/kg at 72 h (a 51% cytotoxic effect in comparison with the mean control level). In the subchronic assay no MNPE increase was found; however, with the highest dose a significant increase of micronucleated normochromatic erythrocytes was observed in the last three weeks (a mean of 51% respect to the mean control value). A cytotoxic effect with the two high doses in the last two weeks was observed (a polychromatic erythrocyte mean decrease of 52% respect to the mean control value). Results suggest caution with venlafaxine.

Resumo A presente pesquisa foi feita para determinar a capacidade micronuclei e citotóxica do antidepressivo venlafaxina em ensaios agudos e subcrônicos in vivo em camundongos. No primeiro estudo, administramos uma vez 5, 50 e 250 mg/kg do medicamento e incluímos um grupo negativo e um grupo tratado com daunorubicina. As observações foram feitas diariamente durante quatro dias. O ensaio subcrônico durou cinco semanas com administração diária de venlafaxina (1, 5, e 10 mg/kg) mais um grupo negativo e um grupo administrado de imipramina. As observações foram feitas a cada semana. No primeiro ensaio, os resultados não mostraram aumento de eritrócitos policromáticos micronucleados (MNPE), exceto com a dose elevada a 72 h. O efeito citotóxico mais forte foi encontrado com 250 mg/kg a 72 h (um efeito citotóxico de 51% em comparação com o nível médio de controle). No ensaio subcrônico não foi encontrado aumento de MNPE; entretanto, com a dose mais alta, um aumento significativo de eritrócitos normocromáticos micronucleados foi observado nas últimas três semanas (média de 51% em relação ao valor médio de controle). Foi observado um efeito citotóxico com as duas altas doses nas últimas duas semanas (uma diminuição média de 52% em relação ao valor médio de controle dos eritrócitos policromáticos). Os resultados sugerem cautela com a venlafaxina.

Genotoxic and cytotoxic evaluation of venlafaxine in an acute and a subchronic assay in mouse / Avaliação genotóxica e citotóxica da venlafaxina em ensaio agudo e subcrônico em camundongos

Abstract The present research was made to determine the micronuclei and cytotoxic capacity of the antidepressant venlafaxine in an in vivo acute and subchronic assays in mouse. In the first study, we administered once 5, 50, and 250 mg/kg of the drug, and included a negative and a daunorubicin treated group. Observations were daily made during four days. The subchronic assay lasted 5 weeks with daily administration of venlafaxine (1, 5, and 10 mg/kg) plus a negative and an imipramine administered groups. Observations were made each week. In the first assay results showed no micronucleated polychromatic erythrocytes (MNPE) increase, except with the high dose at 72 h. The strongest cytotoxic effect was found with 250 mg/kg at 72 h (a 51% cytotoxic effect in comparison with the mean control level). In the subchronic assay no MNPE increase was found; however, with the highest dose a significant increase of micronucleated normochromatic erythrocytes was observed in the last three weeks (a mean of 51% respect to the mean control value). A cytotoxic effect with the two high doses in the last two weeks was observed (a polychromatic erythrocyte mean decrease of 52% respect to the mean control value). Results suggest caution with venlafaxine.

Resumo A presente pesquisa foi feita para determinar a capacidade micronuclei e citotóxica do antidepressivo venlafaxina em ensaios agudos e subcrônicos in vivo em camundongos. No primeiro estudo, administramos uma vez 5, 50 e 250 mg/kg do medicamento e incluímos um grupo negativo e um grupo tratado com daunorubicina. As observações foram feitas diariamente durante quatro dias. O ensaio subcrônico durou cinco semanas com administração diária de venlafaxina (1, 5, e 10 mg/kg) mais um grupo negativo e um grupo administrado de imipramina. As observações foram feitas a cada semana. No primeiro ensaio, os resultados não mostraram aumento de eritrócitos policromáticos micronucleados (MNPE), exceto com a dose elevada a 72 h. O efeito citotóxico mais forte foi encontrado com 250 mg/kg a 72 h (um efeito citotóxico de 51% em comparação com o nível médio de controle). No ensaio subcrônico não foi encontrado aumento de MNPE; entretanto, com a dose mais alta, um aumento significativo de eritrócitos normocromáticos micronucleados foi observado nas últimas três semanas (média de 51% em relação ao valor médio de controle). Foi observado um efeito citotóxico com as duas altas doses nas últimas duas semanas (uma diminuição média de 52% em relação ao valor médio de controle dos eritrócitos policromáticos). Os resultados sugerem cautela com a venlafaxina.

GC-MS analysis of organic fractions of Chrozophora tinctoria (L.) A.Juss. and their prokinetic propensity in animal models / Análise por GC-MS de frações orgânicas de Chrozophora tinctoria (L.) A.Juss. e sua propensão procinética em modelos animais

Chrozophora tinctoria (L.) A.Juss. is herbaceous, monecious annual plant used traditionally to cure gastrointestinal disorders. The present study was carried out to find the bioactive compounds by Gas Chromatography-Mass Spectrometry, the acetylcholinesterase inhibitory potential acute toxicity, and emetic activity present in the ethyl acetate fraction of Chrozophora tinctoria (EAFCT) and dichloromethane fraction of Chrozophora tinctoria (DCMFCT). The compounds detected in both fractions were mostly fatty acids, with about seven compounds in EAFCT and 10 in DCMFCT. These included pharmacologically active compounds such as imipramine, used to treat depression, or hexadecanoic acid methyl ester, an antioxidant, nematicide, pesticide, hypocholesterolemic, 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- is used as a cancer preventive, antiarthritic, antihistaminic, hepatoprotective, insectifuge, nematicide, Pentadecanoic acid, 14-methyl-, methyl ester have antifungal, antimicrobial and antioxidant activities, 10-Octadecanoic acid, methyl ester have the property to decrease blood cholesterol, Antioxidant and antimicrobial, 1-Eicosanol is used as an antibacterial, 1-Hexadecene has antibacterial, antioxidant, and antifungal activities. Both DCMFCT and EAFCT fractions inhibited acetylcholinesterase (AChE) activity with IC50 values of 10 µg and 130 µg, respectively. Both the fractions were found to be toxic in a dose-dependent manner, inducing emesis at 0.5g onward and lethargy and mortality from 3­5 g upwards. Both the fractions combined with distilled water showed highly emetic activity. The significant increase in the number of vomits was shown by EAFCT plus distilled water which are 7.50±1.29, 7.25±3.10, and 11.75±2.22 number of vomits at 1g, 2g, and 3g/kg concentration respectively, while DCMFCT plus distilled water showed 5.25±2.22, 7.50±2.52 and 10.25±2.22 number of vomits at 1g, 2, and 3g/kg correspondingly. The antiemetic standard drug metoclopramide has a higher impact against the emesis induced by both the fractions than dimenhydrinate. Metoclopramide decreases the number of vomits caused by EAFCT to 1.00±0.00, 2.00±0.00, 4.00±1.00 at 1g, 2, and 3g/kg sequentially, while dimenhydrinate decreases the number of vomits to 1.33±0.58, 2.33±1.15, 4.33±0.58 at 1g, 2, and 3g respectively. In the same way, Metochloprimide decreases the number of emesis caused by DcmCt from 5.25±2.22, 7.50±2.52, 10.25±2.22 to 1.33±0.58, 2.33±1.1, 4.33±0.58 at 1g, 2, and 3g/kg concentrations. The present study is the first documented report that scientifically validates the folkloric use of Chrozophora tinctoria as an emetic agent.

Chrozophora tinctoria (L.) A.Juss. é uma planta anual herbácea, monoica, usada tradicionalmente para curar distúrbios gastrointestinais. O presente estudo foi realizado para encontrar os compostos bioativos por Cromatografia Gasosa-Espectrometria de Massa (GC-MS), a toxicidade aguda do potencial inibitório da acetilcolinesterase e a atividade emética presente na fração acetato de etila de Chrozophora tinctoria (EAFCT) e fração diclorometano de Chrozophora tinctoria (DCMFCT). Os compostos detectados em ambas as frações foram principalmente ácidos graxos, com cerca de sete compostos em EAFCT e 10 em DCMFCT. Estes incluíam compostos farmacologicamente ativos, como a imipramina, usada para tratar a depressão, ou éster metílico do ácido hexadecanoico, um antioxidante, nematicida, pesticida, hipocolesterolêmico, ácido 9,12,15-octadecatrienoico, éster etílico, (Z,Z,Z)- é usado como preventivo do câncer, antiartrítico, anti-histamínico, hepatoprotetor, inseticida, nematicida, ácido pentadecanoico, 14-metil-, éster metílico tem atividades antifúngicas, antimicrobianas e antioxidantes, ácido 10-octadecanoico, éster metílico tem a propriedade de diminuir o colesterol no sangue, antioxidante e antimicrobiano, o 1-Eicosanol é usado como antibacteriano, o 1-Hexadeceno possui atividades antibacteriana, antioxidante e antifúngica. Ambas as frações DCMFCT e EAFCT inibiram a atividade da acetilcolinesterase (AChE) com valores de IC50 de 10µg e 130µg, respectivamente. Ambas as frações foram consideradas tóxicas de maneira dose-dependente, induzindo vômitos a partir de 0,5g e letargia e mortalidade de 3g a 5g para cima. Ambas as frações combinadas com água destilada apresentaram atividade altamente emética. O aumento significativo no número de vômitos foi demonstrado pelo EAFCT mais água destilada, que são 7,50±1,29, 7,25±3,10 e 11,75±2,22 número de vômitos nas concentrações de 1g, 2g e 3g/kg, respectivamente, enquanto DCMFCT mais água destilada mostrou 5,25±2,22, 7,50±2,52 e 10,25±2,22 número de vômitos em 1g, 2g e 3g/kg, respectivamente. A droga padrão antiemética metoclopramida tem um impacto maior contra a emese induzida por ambas as frações do que o dimenidrinato. A metoclopramida diminui o número de vômitos causados ​​por EAFCT para 1,00±0,00, 2,00±0,00, 4,00±1,00 a 1g, 2g e 3g/kg, sequencialmente, enquanto o dimenidrinato diminui o número de vômitos para 1,33±0,58, 2,33±1,15, 4,33± 0,58 a 1g, 2g e 3g, respectivamente. Da mesma forma, a metocloprimida diminui o número de vômitos causados ​​por DcmCt de 5,25±2,22, 7,50±2,52, 10,25±2,22 para 1,33±0,58, 2,33±1,1, 4,33±0,58 nas concentrações de 1g, 2g e 3g/kg. O presente estudo é o primeiro relato documentado que valida cientificamente o uso folclórico de Chrozophora tinctoria como agente emético.