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)

Effects of perinatal period administration of clomiphene citrate in sexual behavior, organ weights and hormone concentration of Wistar male and female rats / Efeito da administração do citrato de clomifeno durante o período perinatal no comportamento sexual, peso dos órgãos e concentração hormonal de ratos Wistar machos e fêmeas

The effects of prenatal exposure to clomiphene citrate in sexual behavior, organ weight and hormone concentrations of male and female rats was evaluated. The animals received four doses of clomiphene citrate 2 mg/mL each during the prenatal period (21 days of gestation DG21) on days 1 (DN1), 2 (DN2) and 3 (DN3) after the birth of the puppies. The treatment led to the development of polycystic ovaries in 70% of the females, masculinization of female sexual behavior and changes in sexual behavior of males evidenced by the reduction in the number of ejaculations. In regards to hormone levels, a decrease in the FSH levels in male offspring was observed. It was concluded that clomiphene citrate interferes with the reproductive capacity of male and female rats and female sexual orientation when prenatally administered. (AU)

Foram investigados os efeitos da exposição perinatal ao citrato de clomifeno no comportamento sexual, peso dos órgãos e concentração hormonal de ratos machos e fêmeas. Os animais receberam quatro doses de 2 mg/mL de citrato de clomifeno, no período perinatal (21 dias de gestação DG21), nos dias 1 (DN1), 2 (DN2) e 3 (DN3) após o nascimento dos filhotes. O tratamento causou desenvolvimento de ovário policístico em 70% das fêmeas, masculinização do comportamento sexual das fêmeas e alteração do comportamento sexual dos machos evidenciado pela redução no número de ejaculações. Em relação aos níveis hormonais, observou-se diminuição de FSH na prole masculina. Concluiu-se que o citrato de clomifeno interfere na capacidade reprodutiva de ratos machos e fêmeas, e na orientação sexual de fêmeas, quando administrado perinatalmente. (AU)

CATETERIZAÇÃO URETRAL APÓS INDUÇÃO FARMACOLÓGICA PARA COLETA DE SÊMEN EM CÃES

Os agentes alfa adrenérgicos influenciam a ereção e a ejaculação, sendo o reflexo ejaculatório o principal evento mediado por receptores alfa adrenérgicos. Desta forma, o objetivo deste trabalho foi descrever a coleta farmacológica de sêmen pela técnica de cateterismo uretral em cães domésticos utilizando a associação dexmedetomidina (Dexdomitor, Zoetis, Finlandia) e cetamina (Ketalex, Vencofarma, Brasil). Para a determinação da dose, a coleta de sêmen foi realizada com doses crescentes de dexmedetomidina (5g / kg, n = 2; 10g / kg, n = 2 e 15g / kg, n = 2) associadas a 3mg / kg de cetamina em seis animais. Uma vez estabelecida a dose, foi realizada a coleta adicional de sêmen com 15g / kg de dexmedetomidina e 3mg / kg de cetamina em nove cães. Após o procedimento, volume espermático (0,087 ± 0,027 mL), motilidade total (60,5 ± 8,08%), vigor (1,9 ± 0,28), concentração espermática (2458 ±61303,44 x 10 espermatozoides / mL), integridade de membrana plásmatica (84,2 ± 13,97%), integridade acrossomal(75,2 ±18,83%) e morfologia normal dos espermatozóides (57,35 ± 6,49%) foram valiados. De acordo com os resultados apresentados, este estudo demonstra que a coleta de sêmen por cateterismo uretral após estimulo farmacológico utilizando a associação dexmedetomidina-cetamina pode ser realizada em cães, permitindo obter sêmen apto à aplicações em biotecnologias, apesar da contaminação por urina.

Erection and ejaculation are influenced for -adrenergic agents, and ejaculatory reflex is primarily an -adrenergically mediated event. This paper aims to describe the semen pharmacological collection by urethral catheterization technique in domestic dogs using dexmedetomidine (Dexdomitor, Zoetis, Finland) and ketamine (Ketalex,Vencofarma, Brazil) association. For dose determination, semen collection was performed using dexmedetomidine increasing doses (5g/kg, n=2; 10g/kg, n=2 and 15g/kg, n=2) associated to 3mg/kg ketamine in six animals. Once the dose was established, additional semen collection using 15g/kg dexmedetomidine and 3mg/kg ketamine was carried out in nine dogs. After the procedure, sperm volume (0.087 ± 0.027 mL), total motility (60.5 ±8.08%), progressive motility (1.9± 0.28), sperm 6 concentration (2458 ± 1303.44 x 10 sperm / mL), sperm viability (84.2 ± 13.97%), acrosomal integrity (75.2 ± 18.83 %) and normal sperm morphology ( 57.35 ± 6.49 %)were assessed. According the results, this study demonstrates that urethral catheterization for semen collection using dexmedetomidine-ketamine association can be performed in dogs and it may be possible to obtain semen for application in biotechnologies, despite urine contamination.

MÉTODOS DE COLETA, AVALIAÇÃO E PRESERVAÇÃO DE SÊMEN DE PACAS (Cuniculus paca Linnaeus, 1766) (CUNICULIDAE) EM DOIS DIFERENTES DILUENTES

ALVES, Augusto Luiz Faino. Universidade Federal do Acre, Março de 2018. Métodos de coleta, avaliação e preservação de sêmen de pacas (Cuniculus paca Linnaeus,1766) (Cuniculidae) em dois diferentes diluentes. Orientador:Vânia Maria França Ribeiro, Co-orientador: Fernando de Andrade Souza. Botusemen special Ao todo, sete animais foram submetidos a dois métodos para recuperação espermática: a eletroejaculação e a recuperação de espermatozoides diretamente da cauda do epidídimo por flutuação. Este ultimo, com diluição em dois meios: ACP123 e Botusemen special®. O protocolo anestésico foi feito com acepromazina 1% (0,1 mg/kg/IM) e associação de quetamina (20mg/kg) e xilazina (1,5mg/kg). As amostras coletadas pelo método de eletroejaculação obtiveram volume médio de 0,43ml (±0,33DP), concentração média de 45,5x106 espermatozoides/ml (±42,44DP) motilidade de 33,33% (±32,14DP) e vigor médio de 2,6 (±1,15DP). As amostras obtidas diretamente da cauda do epidídimo tiveram volume médio de 1,5ml, concentração média de 197,1x106 espermatozoides/ml (±84,9DP), sendo as médias da motilidade e vigor do diluente ACP 123, 63,8% (±34,2DP) e 4,2 (±1,7DP) respectivamente. Por outro lado, as amostras diluídas em Botusemen Special® apresentaram motilidade média de 29,8% (±34,2DP) e vigor médio de 2,4 (±1,9DP). A integridade de membrana dos espermatozoides quando diluídos com ACP 123 foi conservada em 84% (±0,07DP) e a viabilidade de membrana mantida em 53,9% (±3,78DP), enquanto o diluente Botusemen special® manteve 73% (±0,21DP) das células espermáticas integras e 39% (±17,9DP) de membranas viáveis. Os resultados foram comparados pelo teste de Wilcoxon-Mann-Whitney e descritos por médias e desvio padrão. Conclui-se, portanto que, a coleta epididimária obteve melhores resultados com relação aos parâmetros espermáticos em comparação ao protocolo de eletroejaculação. As avaliações dos diferentes diluidores indicaram que o diluente ACP-123 proporcionou melhor viabilidade espermática em comparação com o diluente comercial Botusemen special®, porém, ambos os compostos não conservaram as amostras espermáticas até o tempo de 24h.

ALVES, Augusto Luiz Faino. Universidade Federal do Acre, March 2018. Methods of collection, evaluation and preservation of paca(Cuniculus paca Linnaeus,1766) (Cuniculidae) semen in two different diluents. Advisor: Vânia Maria França Ribeiro,Co-advisor: Fernando de Andrade Souza. The objective of this study was to evaluate the efficiency of two methods of collecting semen from bales and to evaluate two diluents (ACP 123 and Botusemen special®) in the conservation of spermatozoa of this species. In all, seven animals were submitted to two methods of sperm retrieval: the electroejaculation and the recovery of the spermatozoa directly from the tail of the epididymis by flotation. The latter, with dilution in two media: ACP 123 and Botusemen special®. The anesthetic protocol was performed with acepromazine 1% (0.1mg/kg/IM) and combination of ketamine (20mg/kg) and xylazine (1.5 mg/kg). The samples collected by the electroejaculation method obtained an average volume of 0.43ml (±0.33DP), mean concentration of 45.5x106 spermatozoa/ml (±4.44DP), motility of 33.33% (±32.14DP) and mean vigor of 2.6 (±1.15DP). Samples obtained directly from the tail of the epididymis had a mean volume of 1.5ml, mean concentration of 197.1x106 spermatozoa/ml (±84.9DP). The mean motility and vigor of the ACP 123 diluent, 63.8% (±34.2DP) and 4.2 (±1.7DP), respectively. On the other hand, the samples diluted in Botusemen Special® presented mean motility of 29.8% (±34.2DP) and mean vigor of 2.4 (±1.9DP). Sperm membrane integrity when diluted with ACP 123 was conserved at 84% (±0.07DP) and membrane viability maintained at 53.9% (±3.78DP), while Botusemen special® Diluent maintained 73% (±0.21DP) of whole spermatozoa and 39% (±17.9DP) of viable membranes. The results were compared by the Wilcoxon-Mann-Whitney test and described by means and standard deviation. It was concluded, therefore, that the collection of the epididymis obtained better results in relation to the sperm parameters compared to the protocol of electro- ejaculation. The evaluations of the different diluents indicated that the ACP-123 diluent provided better sperm viability when compared to the commercial diluent Botusemen special®, however, both compounds did not retain the sperm samples for up to 24h.

Principais distúrbios reprodutivos observados em garanhões no Brasil / / Reproductive disturbs of stallions in Brazil

A presente publicação tem por objetivo compartilhar com colegas uma experiência clínica de 20 anos. Não diferente da literatura, em nossa experiência a grande maioria dos problemas que acometem a função reprodutiva do macho eqüino são decorrentes de disfunções da espermatogênese. Sendo estes distúrbios consequentes de alterações na termoregulação testicular. A babesiose por ser endêmica na maioria das regiões do Brasil é um fator importante na instalação do processo degenerativo testicular pelos piques febris que esta ocasiona. O aumento da temperatura testicular vai levar a um aumento do metabolismo celular com conseqüente aumento da necessidade de oxigênio tecidual. Como o suporte sanguíneo ao testículo é deficitário em não havendo oxigênio disponível ocorre morte celular e produção de radicais livres. A seqüência de aparecimento dos defeitos morfológicos dos espermatozóides vai depender da gravidade e tempo da injuria ao testículo. Acompanhar a evolução do quadro espermático através da avaliação da motilidade, concentração e morfologia espermática é importante não só para o diagnóstico diferencial como para determinarmos a severidade do dano testicular, contudo na maioria dos casos o prognóstico é favorável. A avaliação do perfil hormonal pode elucidar o dano testicular principalmente pela dosagem de estrogeno circulante...(AU)

Similar with the literature on our clinical experience problems related with spermatogenesis (testicle degeneration) and ejaculatory dysfunctions are the most frequent reproductive problems of stallions. Most of problems are related with disturbs on testicle thermoregulation. Equine Piroplasmosis (Babesiose) is endemic in most states from Brazil and is a important factor related with the testicle degeneration installation because of high fever induced by the disease. The increase on testicle temperature will induce a increase on cell metabolism with a consequent increase on oxygen needs. As the blood supply to testicle is poor and few oxygen is available the cell dies. The sequence of sperm abnormalities is related with the severity of the injury. The evolution of the sperm abnormalities and daily sperm production is important to determine a testicle degeneration prognosis that usually is favorable. The hormonal profile can also help to determine the degree of testicular damage being important to measure the estrogen levels...(AU)

Principais distúrbios reprodutivos observados em garanhões no Brasil / / Reproductive disturbs of stallions in Brazil

A presente publicação tem por objetivo compartilhar com colegas uma experiência clínica de 20 anos. Não diferente da literatura, em nossa experiência a grande maioria dos problemas que acometem a função reprodutiva do macho eqüino são decorrentes de disfunções da espermatogênese. Sendo estes distúrbios consequentes de alterações na termoregulação testicular. A babesiose por ser endêmica na maioria das regiões do Brasil é um fator importante na instalação do processo degenerativo testicular pelos piques febris que esta ocasiona. O aumento da temperatura testicular vai levar a um aumento do metabolismo celular com conseqüente aumento da necessidade de oxigênio tecidual. Como o suporte sanguíneo ao testículo é deficitário em não havendo oxigênio disponível ocorre morte celular e produção de radicais livres. A seqüência de aparecimento dos defeitos morfológicos dos espermatozóides vai depender da gravidade e tempo da injuria ao testículo. Acompanhar a evolução do quadro espermático através da avaliação da motilidade, concentração e morfologia espermática é importante não só para o diagnóstico diferencial como para determinarmos a severidade do dano testicular, contudo na maioria dos casos o prognóstico é favorável. A avaliação do perfil hormonal pode elucidar o dano testicular principalmente pela dosagem de estrogeno circulante...

Similar with the literature on our clinical experience problems related with spermatogenesis (testicle degeneration) and ejaculatory dysfunctions are the most frequent reproductive problems of stallions. Most of problems are related with disturbs on testicle thermoregulation. Equine Piroplasmosis (Babesiose) is endemic in most states from Brazil and is a important factor related with the testicle degeneration installation because of high fever induced by the disease. The increase on testicle temperature will induce a increase on cell metabolism with a consequent increase on oxygen needs. As the blood supply to testicle is poor and few oxygen is available the cell dies. The sequence of sperm abnormalities is related with the severity of the injury. The evolution of the sperm abnormalities and daily sperm production is important to determine a testicle degeneration prognosis that usually is favorable. The hormonal profile can also help to determine the degree of testicular damage being important to measure the estrogen levels...

Effects of perinatal period administration of clomiphene citrate in sexual behavior, organ weights and hormone concentration of Wistar male and female rats / Efeito da administração do citrato de clomifeno durante o período perinatal no comportamento sexual, peso dos órgãos e concentração hormonal de ratos Wistar machos e fêmeas

The effects of prenatal exposure to clomiphene citrate in sexual behavior, organ weight and hormone concentrations of male and female rats was evaluated. The animals received four doses of clomiphene citrate 2 mg/mL each during the prenatal period (21 days of gestation DG21) on days 1 (DN1), 2 (DN2) and 3 (DN3) after the birth of the puppies. The treatment led to the development of polycystic ovaries in 70% of the females, masculinization of female sexual behavior and changes in sexual behavior of males evidenced by the reduction in the number of ejaculations. In regards to hormone levels, a decrease in the FSH levels in male offspring was observed. It was concluded that clomiphene citrate interferes with the reproductive capacity of male and female rats and female sexual orientation when prenatally administered.

Foram investigados os efeitos da exposição perinatal ao citrato de clomifeno no comportamento sexual, peso dos órgãos e concentração hormonal de ratos machos e fêmeas. Os animais receberam quatro doses de 2 mg/mL de citrato de clomifeno, no período perinatal (21 dias de gestação DG21), nos dias 1 (DN1), 2 (DN2) e 3 (DN3) após o nascimento dos filhotes. O tratamento causou desenvolvimento de ovário policístico em 70% das fêmeas, masculinização do comportamento sexual das fêmeas e alteração do comportamento sexual dos machos evidenciado pela redução no número de ejaculações. Em relação aos níveis hormonais, observou-se diminuição de FSH na prole masculina. Concluiu-se que o citrato de clomifeno interfere na capacidade reprodutiva de ratos machos e fêmeas, e na orientação sexual de fêmeas, quando administrado perinatalmente.

Analysis of some normal parameters of the spermiogram of captive capuchin monkeys (Cebus apella Linnaeus, 1758 ) / Análise de alguns parâmetros normais do espermograma de macaco-prego (Cebus apella Linnaeus, 1758)

Nine adult males of capuchin monkey (Cebus apella) were electro-ejaculated with a rectal bipolar probe under general anaesthesia with tiletamine -zolazepan association. From the obtained ejaculate, we only analysed the liquid fraction in order to avoid any kind of chemical treatment to attain dissolution of the seminal coagulum. The mean obtained volume was 0,2ml with the sperm concentration of 56.169 x 10(6) sperm/ml and mean motility 68,4% and vigor 2,6. The mean percentage of morphologic normal spermatozoa was 39%. The technique was efficient for semen collection in Cebus apella and allowed seminal evaluation without possible sperm lesions caused by chemical treatment for coagulum dissolution.

Foram submetidos a eletro-ejaculação 9 macacos-prego, adultos, pertencentes à Fundação Parque Zoológico de São Paulo, com o uso de eletrodo retal bipolar, sob anestesia geral obtida pela aplicação da associação tiletamina-zolazepan. Do sêmen assim obtido foram analisadas apenas as frações líquidas resultantes após a coagulação do ejaculado, não havendo nenhum tratamento químico para sua dissolução, apenas a separação das frações após a ejaculação. O volume médio obtido foi de 0,2ml, a concentração média de 56.169 x 10(6) espermatozóides/ml, com motilidade média de 68,4% e vigor médio de 2,6.O percentual médio de espermatozóides morfológicamente normais foi de 39%. A técnica é eficiente em macacos-prego (Cebus apella) e estes resultados permitem a avaliação do sêmen evitando-se os danos causados pela dissolução química do coágulo seminal.