Causas e Tratamentos de Distúrbios Ejaculatórios em Garanhões / Causes and Treatments of Ejaculatory Disorders in Stallions

A ejaculação é um complexo processo de eventos neurofisiológicos sincronizados em coordenação com vários sistemas e órgãos. Antes do advento da injeção intracitoplasmática de espermatozoides (ICSI), a ejaculação era um passo absolutamente essencial na reprodução. Os distúrbios ejaculatórios por sua vez são caracterizados pela não ocorrência do processo ejaculatório, mesmo que todos os demais parâmetros relacionados ao comportamento sexual apresentem-se normais. As principais causas dessas falhas ou alterações na ejaculação estão relacionadas à sensibilidade dolorosa durante a cópula, ejaculação retrógrada, disfunção psicogênica, obstruções do aparelho reprodutor masculino, urospermia, oligospermia ou azoospermia, além de falhas na contração da musculatura lisa do trato genital ou alterações musculoesqueléticas e neurológicas. O tratamento é dependente da causa primária de cada alteração. O controle da dor, ajustes no manejo, aumento do estímulo antes da coleta associados a tratamentos farmacológicos que atuam nas sinapses neuromotoras penianas tendem a resolver os principais distúrbios ejaculatórios, levando-se em consideração que as respostas a estes tratamentos são variadas de acordo com a individualidade do garanhão com relação a doses, protocolos, vias de administração e combinações de fármacos.(AU)

Ejaculation is a complex process of neurophysiological events synchronized in coordination with various systems and organs. Before the advent of intracytoplasmic sperm injection (ICSI), ejaculation was an absolutely essential step in reproduction. Ejaculatory disorders, in turn, are characterized by the nonoccurrence of the ejaculatory process, even if all other parameters related to sexual behavior are normal. The main causes of these failures or changes in ejaculation are related to painful sensitivity during copulation, retrograde ejaculation, psychogenic dysfunction, obstructions of the male reproductive system, urospermia, oligospermia, azoospermia, in addition to failures in the contraction of the smooth muscles of the genital tract or musculoskeletal disorders and neurological. Treatment is dependent on the primary cause of each change. Pain control, management adjustments, increased stimulus before collection associated with pharmacological treatments that act on penile neuromotor synapses tend to resolve the main ejaculatory disorders, taking into account that the responses to these treatments vary according to the individuality of the stallion with regard to dosages, protocols, routes of administration and drug combinations.(AU)

Evaluation of fertilization capability of frozen-thawed completely immotile spermatozoa collected from a white bengal tiger after interspecific ICSI with bovine oocytes

The objective of this study was to evaluate the fertilization capability of White Bengal Tiger frozen-thawed completely immotile spermatozoa after interspecific intracytoplasmic sperm injection (ICSI) with bovine oocytes. The fertilization status of presumptive zygotes was assessed 18 h after ICSI by immunofluorescence staining and confocal microscopy. The fertilization rate was 34.8% (8/23), as confirmed by the extrusion of two polar bodies, or male and female pronuclei formation. For unfertilized oocytes (65.2%, 15/23), one activated oocyte had an activated spermatozoon but most were unactivated oocytes with unactivated spermatozoa (1/15, 6.7% vs 10/15, 66.7%, respectively, p < 0.05). These results showed that White Bengal Tiger frozen-thawed completely immotile spermatozoa retained the capacity to fertilize bovine oocytes after interspecific ICSI. This is the first report of in vitro produced zygotes using tiger immotile sperm with bovine oocytes by interspecific ICSI technique, which provides an efficient and feasible method for preservation and utilization of endangered feline animals.(AU)

Action of swim-up and caffeine on equine frozen sperm

Cryopreservation of equine semen is crucial to semen commercialization. However, it reduces sperm motility and longevity. Thus, sperm selection methods and addition of motility-activating substances to sperm, such as caffeine, may improve sperm quality of equine frozen semen. The objective of the current work was to evaluate the effects of caffeine on recovery and quality parameters of frozen-thawed sperm subjected to swim-up selection to be used in intracytoplasmic sperm injection (ICSI) in assisted reproductive techniques. Stallion semen were frozen and after thawing different caffeine concentrations were added to the samples performing four treatments control (no caffeine), 3, 5, and 7.5 mM caffeine. Sperm kinematic and motility were assessed by computer-assisted sperm analysis (CASA). Then, the four treated samples were submitted to the swim-up sperm selection, and the number of recovered sperm and morphology were evaluated at four times 20, 40, 60, and 80 min. The swim-up increased the recovery proportion of normal morphology sperm without (80.1±1%) or with caffeine addition (3mM: 81.2±1%, 5mM: 79.9±1% and 7.5 mM 78.9±1%) compared to the thawed semen (70±2%). However, the addition of 5 mM caffeine induced an increase in sperm motility (38.9±2.8 vs. 32.6±3.4%, P<0.05), and sperm recovery after swim-up (7.9x106 vs. 3.4x106 sperm/ml, P<0.05) compared to the control. The addition of 5 mM caffeine to frozen-thawed equine semen before swim-up selection improved sperm motility and increased the sperm recovery rate while not decreasing the percentage of morphologically normal sperm. Thus, caffeine addition to frozen-thawed equine semen before swim-up selection has potential clinical application in improving sperm quality for use in ICSI.(AU)

A interface entre a saúde humana e a reprodução assistida em primatas não humanos / The interface between human health and assisted reproductive technologies in non-human primates

Os primatas não-humanos (PNHs) são tidos como importantes modelos para estudos biomédicos devido à sua grande similaridade biológica com os seres humanos. As técnicas de reprodução assistida (TRAs) constituem uma importante ferramenta para realização de estudos que envolvam infertilidade, desenvolvimento de contraceptivos, gestação e desenvolvimento fetal, preservação da fertilidade em pacientes com câncer, geração de modelos experimentais por meio de técnicas de edição gênica, entre outros. O objetivo dessa revisão é discutir as principais TRAs utilizadas em PNHs e suas aplicações. Dentre as técnicas mais utilizadas em PNHs, podem-se citar colheita e criopreservação de sêmen, monitoramento do ciclo ovariano, estimulação ovariana controlada e aspiração de folículos ovarianos, fecundação in vitro, injeção intracitoplasmática de espermatozoides, inseminação artificial, microinjeção para injeção gênica, biopsia embrionária, criopreservação de embriões, transferência de embriões, diagnóstico de gestação, enxerto de gônadas e clonagem. Os estudos apresentados nesta revisão mostram a evolução das TRAs e suas diferentes aplicações. Particularmente, os estudos de edição gênica e clonagem representam um grande avanço na utilização combinada de diversas TRAs para geração de modelos biomédicos para doenças humanas, demostrando o papel dessas técnicas para avanços científicos no que diz respeito à saúde humana.

Nonhuman Primates (NHPs) are considered important models for biomedical studies due to their high biological proximity with humans. The Assisted Reproductive Technologies (ARTs) represent an important tool to perform studies related to infertility, development of contraceptives, gestation and fetal development, fertility preservation in cancer patients, generation of experimental models through gene editing techniques, among others. The objective of this review is to discuss the main ARTs used in NHPs and their application. Among the most used techniques in NHPs, we can include collection and cryopreservation of semen, ovarian cycle monitoring, controlled ovarian stimulation and follicle aspiration, in vitro fertilization, intracytoplasmic sperm injection, artificial insemination, microinjections for gene editing, embryo biopsy, embryo cryopreservation, embryo transfer, pregnancy diagnosis, grafting of gonadal tissue, and cloning. The studies cited in this review illustrate the evolution of the ARTs and their applications. The gene editing and cloning studies, in particular, represent the great advancements on the combined use of several different ARTs for the generation of biomedical models for human diseases, demonstrating the role of these techniques in the scientific advancement with regards to human health.

Thirty years of assisted reproductive technology in the domestic cat: A selected summary / Trinta anos de tecnologia de reprodução assistida no gato doméstico: Um resumo selecionado

The first kittens to be born after embryo transfer (ET) in the cat (Schriver and Kraemer, 1978) were reported four decades ago. The births of kittens after in vitro fertilization (IVF)/ET (Goodrowe et al., 1988) and after embryo cryopreservation/ET (Dresser et al., 1988) were described 10 years later. In an early report it was established that embryo donors had the ability ot exhibit repeated ovarian stimulatory response to multiple gonadotropin treatments (porcine FSH; Dresser et al., 1987). After studies on gonadotropin-induced hyperstimulation of follicular development for recovery and ET of fresh and cryopreserved in vivo derived uterine stage embryos from mated donors (Dresser et al., 1988; Pope et al., 1989), we transitioned into developing methods for in vitro production of domestic cat embryos for the purpose of applying the technology to support conservation of threatened and endangered felid species (Pope et al., 1993). Since then, techniques for the in vitro production of cat embryos have been developed sufficiently to allow births of kittens after transfer of embryos derived by an assortment of in vitro techniques, including cryopreservation (Pope et al., 1994; Gómez et al., 2003a; Pope et al., 2012a, b; Galiguis et al., 2014), intracytoplasmic sperm injection (ICSI; Pope et al., 1998; Gómez et al., 2000; Pope et al., 2012a), gender selection (Pope et al., 2009) and somatic cell nuclear transfer (SCNT) (Gómez et al., 2004; 2008; 2009).(AU)

Thirty years of assisted reproductive technology in the domestic cat: A selected summary / Trinta anos de tecnologia de reprodução assistida no gato doméstico: Um resumo selecionado

The first kittens to be born after embryo transfer (ET) in the cat (Schriver and Kraemer, 1978) were reported four decades ago. The births of kittens after in vitro fertilization (IVF)/ET (Goodrowe et al., 1988) and after embryo cryopreservation/ET (Dresser et al., 1988) were described 10 years later. In an early report it was established that embryo donors had the ability ot exhibit repeated ovarian stimulatory response to multiple gonadotropin treatments (porcine FSH; Dresser et al., 1987). After studies on gonadotropin-induced hyperstimulation of follicular development for recovery and ET of fresh and cryopreserved in vivo derived uterine stage embryos from mated donors (Dresser et al., 1988; Pope et al., 1989), we transitioned into developing methods for in vitro production of domestic cat embryos for the purpose of applying the technology to support conservation of threatened and endangered felid species (Pope et al., 1993). Since then, techniques for the in vitro production of cat embryos have been developed sufficiently to allow births of kittens after transfer of embryos derived by an assortment of in vitro techniques, including cryopreservation (Pope et al., 1994; Gómez et al., 2003a; Pope et al., 2012a, b; Galiguis et al., 2014), intracytoplasmic sperm injection (ICSI; Pope et al., 1998; Gómez et al., 2000; Pope et al., 2012a), gender selection (Pope et al., 2009) and somatic cell nuclear transfer (SCNT) (Gómez et al., 2004; 2008; 2009).

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...]

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...](AU)

Practical applications of sperm selection techniques for improving reproductive efficiency

Several selection techniques are available for processing spermatozoa. Apart from sperm washing to remove seminal plasma, only “swim-up” and colloid centrifugation have been used to any extent to prepare spermatozoa for in vitro fertilization, and only colloid centrifugation has been used to prepare sperm samples for artificial insemination. Single-layer centrifugation (SLC) through a species-specific colloid has been shown to be effective in selecting spermatozoa with good motility, normal morphology and intact chromatin in a range of species. This method is less timeconsuming than swim-up, and has been scaled-up to allow whole ejaculates to be processed in a practical manner. The applications of SLC are as follows: to improve sperm quality in insemination doses or in samples for in vitro fertilization, to increase the shelf life of normal sperm doses, to remove pathogens (viruses, bacteria), to improve cryosurvival by removing dead and dying spermatozoa before freezing or after thawing, to select spermatozoa for intracytoplasmic sperm injection, and to aid conservation breeding.

Practical applications of sperm selection techniques for improving reproductive efficiency

Several selection techniques are available for processing spermatozoa. Apart from sperm washing to remove seminal plasma, only “swim-up” and colloid centrifugation have been used to any extent to prepare spermatozoa for in vitro fertilization, and only colloid centrifugation has been used to prepare sperm samples for artificial insemination. Single-layer centrifugation (SLC) through a species-specific colloid has been shown to be effective in selecting spermatozoa with good motility, normal morphology and intact chromatin in a range of species. This method is less timeconsuming than swim-up, and has been scaled-up to allow whole ejaculates to be processed in a practical manner. The applications of SLC are as follows: to improve sperm quality in insemination doses or in samples for in vitro fertilization, to increase the shelf life of normal sperm doses, to remove pathogens (viruses, bacteria), to improve cryosurvival by removing dead and dying spermatozoa before freezing or after thawing, to select spermatozoa for intracytoplasmic sperm injection, and to aid conservation breeding.(AU)

Produção in vivo e in vitro de embriões em equinos / In vivo and in vitro production of equine embryos

A produção de embriões equinos in vivo ou in vitro tem como objetivo principal uma maximização daeficiência reprodutiva, principalmente por que a espécie equina é reconhecida por ter uma capacidadereprodutiva menor que as demais espécies. O objetivo desse trabalho foi fazer uma revisão nas principaistécnicas de produção embrionária in vivo e in vitro na espécie equina. A produção in vivo a partir datransferência de embriões (TE) tornou-se uma técnica rotineira e o Brasil se destaca entre os países de maiorprodução de embrião in vivo por essa técnica no mundo. Já com relação à produção in vitro apesar do grandeavanço adquirido nos últimos anos, vários aspectos ainda necessitam de esclarecimento. Desde a coleta deoócitos por aspiração folicular no animal vivo ou post-mortem, passando pela maturação oocitária in vitro, indopelas técnicas de transferência de oócitos (TO) ou de transferência intra falopiana dos gametas (GIFT) e aabordagem da técnica de Fertilização in vitro (FIV) ainda sem sucesso na espécie equina e a transposição dosobstáculos encontrados a partir da técnica de microinjeção intracitoplasmática de espermatozoide (ICSI).Abordamos ainda os aspectos mais recentes da clonagem na espécie equina auxiliando em uma maiorcompreensão dos diversos aspectos envolvidos nessa biotecnologia. Concluímos que maiores estudos serãonecessários para compreendermos alguns dos mecanismos propostos e esclarecermos essas dúvidas.(AU)

The production of equine embryos in vivo or in vitro has as main objective to reach a maximumreproductive efficiency, especially because the equine is recognized for having a less reproductive capacityamong other species. The objective of this work was to review main techniques of embryo production in vivo andin vitro in the equine species. In vivo production from embryo transfer (TE) has become a routine technique andBrazil stands out among the countries with the highest production of in vivo embryos by this technique in theworld. With regard to in vitro production despite the breakthrough acquired in recent years, many aspects stillrequire clarification. From the oocyte recovery by follicular aspiration in living animal or in post-mortem,oocyte in vitro maturation, going to oocyte transfer techniques or Gamete Intra Fallopian Transfer and theapproach of the technique of in vitro fertilization (IVF) yet without success and the transposition of obstaclesencountered using Intracytoplasmic Sperm Injection (ICSI) technique. We discuss the latest aspects of cloning inthe equine species aiding in a better understanding of the various aspects involved in this biotechnology. Weconclude that further studies will be necessary to understand some of the proposed mechanisms and clear thesedoubts.(AU)

Produção in vivo e in vitro de embriões em equinos / In vivo and in vitro production of equine embryos

A produção de embriões equinos in vivo ou in vitro tem como objetivo principal uma maximização daeficiência reprodutiva, principalmente por que a espécie equina é reconhecida por ter uma capacidadereprodutiva menor que as demais espécies. O objetivo desse trabalho foi fazer uma revisão nas principaistécnicas de produção embrionária in vivo e in vitro na espécie equina. A produção in vivo a partir datransferência de embriões (TE) tornou-se uma técnica rotineira e o Brasil se destaca entre os países de maiorprodução de embrião in vivo por essa técnica no mundo. Já com relação à produção in vitro apesar do grandeavanço adquirido nos últimos anos, vários aspectos ainda necessitam de esclarecimento. Desde a coleta deoócitos por aspiração folicular no animal vivo ou post-mortem, passando pela maturação oocitária in vitro, indopelas técnicas de transferência de oócitos (TO) ou de transferência intra falopiana dos gametas (GIFT) e aabordagem da técnica de Fertilização in vitro (FIV) ainda sem sucesso na espécie equina e a transposição dosobstáculos encontrados a partir da técnica de microinjeção intracitoplasmática de espermatozoide (ICSI).Abordamos ainda os aspectos mais recentes da clonagem na espécie equina auxiliando em uma maiorcompreensão dos diversos aspectos envolvidos nessa biotecnologia. Concluímos que maiores estudos serãonecessários para compreendermos alguns dos mecanismos propostos e esclarecermos essas dúvidas.

The production of equine embryos in vivo or in vitro has as main objective to reach a maximumreproductive efficiency, especially because the equine is recognized for having a less reproductive capacityamong other species. The objective of this work was to review main techniques of embryo production in vivo andin vitro in the equine species. In vivo production from embryo transfer (TE) has become a routine technique andBrazil stands out among the countries with the highest production of in vivo embryos by this technique in theworld. With regard to in vitro production despite the breakthrough acquired in recent years, many aspects stillrequire clarification. From the oocyte recovery by follicular aspiration in living animal or in post-mortem,oocyte in vitro maturation, going to oocyte transfer techniques or Gamete Intra Fallopian Transfer and theapproach of the technique of in vitro fertilization (IVF) yet without success and the transposition of obstaclesencountered using Intracytoplasmic Sperm Injection (ICSI) technique. We discuss the latest aspects of cloning inthe equine species aiding in a better understanding of the various aspects involved in this biotechnology. Weconclude that further studies will be necessary to understand some of the proposed mechanisms and clear thesedoubts.

CARACTERÍSTICAS DO SÊMEN CAPRINO DESCONGELADO APÓS A ADIÇÃO DE RINGER LACTATO, CITRATO DE SÓDIO E SOLUÇÃO TRIS

Cryopreservation of semen is of great importance for various reproductive biotechnologies such as artificial insemination (IA), In Vitro Embryo Production (PIV) and Intracytoplasmic Sperm Injection (ICSI). The objective of this work was to evaluate the stability and persistence of motility and strength of sperm, as well as changes in the plasma membrane after the addition of Ringer Lactate, sodium citrate 2.92% or TRIS solution in thawed goat semen. Semen was collected from two Alpine Brown goats, and standard procedures for cryopreservation and seminal analysis were performed. After thawing the semen, the extenders Ringer Lactate, sodium citrate 2.92% and TRIS solution were added and Thermoresistance (TTR), Supravital and Morphology Tests were carried out. In TTR, only the group received TRIS solution presented motility and strength for a longer period (90 minutes; P 0,05). No influence of the addition of the solutions was found in the morphological analysis, as well as in the Supravital Test, which showed values close to those found for motility (P> 0.05). We concluded that the addition of the solutions does not allow a large persistence of motility and strength of thawed sperm, but TRIS solution could be used for expansion of seminal doses used in vitro reproductive biotechnology.

A criopreservação do sêmen é de grande importância para diversas biotecnologias da reprodução, como Inseminação Artificial (IA), Produção de Embriões In Vitro (PIV) e Injeção Intracitoplasmática de Espermatozoides (ICSI). Avaliou-se a estabilidade e persistência da motilidade e vigor dos espermatozoides, assim como alterações da membrana plasmática, após a adição de Ringer com Lactato, citrato de sódio 2,92% ou solução TRIS ao sêmen caprino descongelado. O sêmen foi coletado de dois bodes da raça Parda Alpina, realizando-se os procedimentos padrões de análise e criopreservação seminal. Após a descongelação do sêmen, foram adicionados os diluentes Ringer Lactato, citrato de sódio 2,92% ou solução TRIS, realizando-se os Testes de Termorresistência (TTR), Supravital e Morfológico. No TTR, somente o grupo a que foi adicionada a Solução TRIS obteve motilidade e vigor por maior período (90 minutos; P 0,05). Não foi encontrada influência da adição das soluções na análise morfológica, assim como no teste Supravital, o qual apresentou valores próximos aos encontrados para motilidade (P> 0,05). Concluiu-se que a adição das soluções não permite uma grande persistência da motilidade e vigor dos espermatozoides descongelados, porém a solução TRIS poderia ser utilizada para expansão de doses seminais utilizadas em biotecnologias reprodutivas in vitro.

Características do sêmen caprino descongelado após a adição de Ringer lactato, citrato de sódio e solução Tris / Characteristics of thawed goat semen after addition of Ringer lactate, sodium citrate and Tris solution

Cryopreservation of semen is of great importance for various reproductive biotechnologies such as artificial insemination (IA), In Vitro Embryo Production (PIV) and Intracytoplasmic Sperm Injection (ICSI). The objective of this work was to evaluate the stability and persistence of motility and strength of sperm, as well as changes in the plasma membrane after the addition of Ringer Lactate, sodium citrate 2.92% or TRIS solution in thawed goat semen. Semen was collected from two Alpine Brown goats, and standard procedures for cryopreservation and seminal analysis were performed. After thawing the semen, the extenders Ringer Lactate, sodium citrate 2.92% and TRIS solution were added and Thermoresistance (TTR), Supravital and Morphology Tests were carried out. In TTR, only the group received TRIS solution presented motility and strength for a longer period (90 minutes; P 0,05). No influence of the addition of the solutions was found in the morphological analysis, as well as in the Supravital Test, which showed values ??close to those found for motility (P> 0.05). We concluded that the addition of the solutions does not allow a large persistence of motility and strength of thawed sperm, but TRIS solution could be used for expansion of seminal doses used in vitro reproductive biotechnology.

A criopreservação do sêmen é de grande importância para diversas biotecnologias da reprodução, como Inseminação Artificial (IA), Produção de Embriões In Vitro (PIV) e Injeção Intracitoplasmática de Espermatozoides (ICSI). Avaliou-se a estabilidade e persistência da motilidade e vigor dos espermatozoides, assim como alterações da membrana plasmática, após a adição de Ringer com Lactato, citrato de sódio 2,92% ou solução TRIS ao sêmen caprino descongelado. O sêmen foi coletado de dois bodes da raça Parda Alpina, realizando-se os procedimentos padrões de análise e criopreservação seminal. Após a descongelação do sêmen, foram adicionados os diluentes Ringer Lactato, citrato de sódio 2,92% ou solução TRIS, realizando-se os Testes de Termorresistência (TTR), Supravital e Morfológico. No TTR, somente o grupo a que foi adicionada a Solução TRIS obteve motilidade e vigor por maior período (90 minutos; P 0,05). Não foi encontrada influência da adição das soluções na análise morfológica, assim como no teste Supravital, o qual apresentou valores próximos aos encontrados para motilidade (P> 0,05). Concluiu-se que a adição das soluções não permite uma grande persistência da motilidade e vigor dos espermatozoides descongelados, porém a solução TRIS poderia ser utilizada para expansão de doses seminais utilizadas em biotecnologias reprodutivas in vitro.

Características do sêmen caprino descongelado após a adição de Ringer lactato, citrato de sódio e solução Tris / Characteristics of thawed goat semen after addition of Ringer lactate, sodium citrate and Tris solution

Cryopreservation of semen is of great importance for various reproductive biotechnologies such as artificial insemination (IA), In Vitro Embryo Production (PIV) and Intracytoplasmic Sperm Injection (ICSI). The objective of this work was to evaluate the stability and persistence of motility and strength of sperm, as well as changes in the plasma membrane after the addition of Ringer Lactate, sodium citrate 2.92% or TRIS solution in thawed goat semen. Semen was collected from two Alpine Brown goats, and standard procedures for cryopreservation and seminal analysis were performed. After thawing the semen, the extenders Ringer Lactate, sodium citrate 2.92% and TRIS solution were added and Thermoresistance (TTR), Supravital and Morphology Tests were carried out. In TTR, only the group received TRIS solution presented motility and strength for a longer period (90 minutes; P 0,05). No influence of the addition of the solutions was found in the morphological analysis, as well as in the Supravital Test, which showed values ??close to those found for motility (P> 0.05). We concluded that the addition of the solutions does not allow a large persistence of motility and strength of thawed sperm, but TRIS solution could be used for expansion of seminal doses used in vitro reproductive biotechnology.(AU)

A criopreservação do sêmen é de grande importância para diversas biotecnologias da reprodução, como Inseminação Artificial (IA), Produção de Embriões In Vitro (PIV) e Injeção Intracitoplasmática de Espermatozoides (ICSI). Avaliou-se a estabilidade e persistência da motilidade e vigor dos espermatozoides, assim como alterações da membrana plasmática, após a adição de Ringer com Lactato, citrato de sódio 2,92% ou solução TRIS ao sêmen caprino descongelado. O sêmen foi coletado de dois bodes da raça Parda Alpina, realizando-se os procedimentos padrões de análise e criopreservação seminal. Após a descongelação do sêmen, foram adicionados os diluentes Ringer Lactato, citrato de sódio 2,92% ou solução TRIS, realizando-se os Testes de Termorresistência (TTR), Supravital e Morfológico. No TTR, somente o grupo a que foi adicionada a Solução TRIS obteve motilidade e vigor por maior período (90 minutos; P 0,05). Não foi encontrada influência da adição das soluções na análise morfológica, assim como no teste Supravital, o qual apresentou valores próximos aos encontrados para motilidade (P> 0,05). Concluiu-se que a adição das soluções não permite uma grande persistência da motilidade e vigor dos espermatozoides descongelados, porém a solução TRIS poderia ser utilizada para expansão de doses seminais utilizadas em biotecnologias reprodutivas in vitro.(AU)

USO DO PEPTÍDEO Ctn[1-9], DERIVADO DA TOXINA CROTALICIDINA, NA ATIVAÇÃO DE OÓCITOS BOVINOS

Em mamíferos, o influxo do cálcio é fator primordial para a ativação oocitária após ovulação de um oócito maturado e com competência ao desenvolvimento. Biotecnologias da reprodução, como a injeção intracitoplasmática de espermatozoide e tranferência nuclear de células somáticas necessitam da ativação oocitária artificial para dar continuidade ao desenvolvimento embrionário. Os estudos com as catelicidinas, presentes no veneno de cascavéis, mostraram propriedades antimicrobianas, antitumorais e antifúngicas. Além disso, o peptídeo sintético de crotalicidina (Ctn1-9), uma vipericidina relacionado à família das catelicidinas, possui capacidade de promover influxo de cálcio em células cancerígenas. Portanto, este trabalho teve como objetivo avaliar o efeito do (Ctn1-9), ligado covalentemente com a Rodamina B (RhoB-Ctn1-9), na ativação oocitária e no posterior desenvolvimento de embriões bovinos produzidos por partenogênese. Ovários bovinos foram colhidos em abatedouro local e foram puncionados os folículos medindo entre 2-8 mm. Os complexos cumulus-oócito obtidos (n = 1599) foram submetidos à maturação in vitro por 26 h e então divididos (n= 1178) nos diferentes grupos experimentais: Ctn1-9 (0, 1, 10 e 40 M) e grupo controle (ionomicina 5 M), com tempo de exposição de 4 min em todos os grupos. Posteriormente, os oócitos foram incubados por 4 h com 6-DMAP em meio SOF, seguido de cultivo in vitro em meio SOF por sete dias. A análise estatística foi realizada utilizando o software Minitab. Os dados são apresentados como porcentagem e comparados através do teste exato de Fisher. Os resultados foram considerados significativos quando P < 0,05. No dia dois de cultivo, em relação à taxa de clivagem, não houve diferença estatística (P > 0,05) entre as concentrações 1 M (19,6%), 10 M (22,4%) e 40 M (14,7%), entretanto o grupo 1 M e 10 M foram significativamente maiores (P < 0,05) do que o grupo 0 M (8,4%). Para a taxa de formação de blastocisto, grupos 0 M (0,6%), 10 M (1,8%) e 40 M (2,2%) não demonstraram diferença significativa (P > 0,05) entre eles, porém o grupo 40 M foi significativamente maior (P < 0,05) do que o grupo 1 M (0%). Em conclusão, o RhoB-Ctn[1-9] demonstrou ser capaz de induzir a clivagem em oócitos bovinos. Além disso, a ativação e o desenvolvimento até o estádio de blastocisto mostram pouca ou nenhuma toxicidade deste peptídeo como agente ativador de oócitos bovinos.

In mammals, calcium influx is critical to oocyte activation post-ovulation of a matured oocyte with competence to development. Reproductive biotechnologies such as intracytoplasmic sperm injection and somatic cell nuclear transfer necessarily use artificial oocyte activation to continue embryo development. The studies on cathelicidins, present in pit rattlesnake venom, showed antimicrobial, antitumor and antifungal properties. In addition, the synthetic peptide from crotalicidin (Ctn1-9), a vipericidin related to the cathelicidin family, has the capacity to promote calcium influx in cancer cells. Thus, the aim of this study was to evaluate the effect of Ctn[1-9], covalently conjugated with Rhodamine B (RhoB-Ctn1-9), on oocyte activation and development of bovine embryos produced by parthenogenesis. Bovine ovaries were collected in local abattoir and follicles measuring 2-8 mm were punctured. Cumulus-oocyte complexes (n = 1599) were subjected to in vitro maturation for 26 h and then divided (n= 1178) to different experimental groups: Ctn1-9 (0; 1; 10 and 40 M) and control group (ionomycin 5 M), with 4 min exposure time for all groups. Then, oocytes were incubated for 4 h in 6-DMAP in SOF medium and followed for in vitro culture in SOF medium for seven days. Statistical analysis were performed using Minitab software. Data are shown as percentage and compared by Fishers exact test. Results were significant when P < 0.05. In day two of culture, concerning cleavage rate, there was no statistical difference (P > 0.05) between concentration 1 M (19.6%), 10 M (22.4%) and 40 M (14.7%), however group 1 M and 10 M were significantly higher (P < 0.05) than group 0 M (8.4%). For the blastocyst formation rate, groups with 0 M (0.6%), 10 M (1.8%) and 40 M (2.2%) showed no significant difference (P > 0.05) among them, yet group 40 M was significantly higher (P < 0.05) than group 1 M (0%). In conclusion, RhoB-Ctn[1-9] showed to be able to induced cleavage in bovine oocytes. In addition, the activation and development to the blastocyst stage show little or no toxicity of this peptide as an activating agent for bovine oocytes.

Vantagens e desafios das biotécnicas avançadas utilizadas na reprodução equina assistida / Advantages and challenges of advanced biotech used in assisted equine reproduction

Relatos apontam que a espécie equina foi a primeira a ser submetida à inseminação artificial. Apesar deste fato, o desenvolvimento da reprodução assistida nos equinos foi lento em comparação às demais espécies voltadas diretamente para a produção animal. A liberação do uso da transferência de embriões pela maioria das associações de raças estimulou o desenvolvimento e a expansão de algumas técnicas de multiplicação animal para uso em equinos. A presente revisão teve por objetivo abordar as biotécnicas avançadas atualmente disponíveis para a espécie, como a superovulação, a transferência de ovócitos, a transferência intratubárica de gametas, a injeção intracitoplasmática de gametas, a produção in vitro de embriões e a clonagem, enfatizando suas aplicações prática e clínica, bem como determinados desafios que ainda precisam ser superados em cada biotécnica.

Some reports suggest the equine species as the first one to be subjected to the artificial insemination. Despite this fact, the development of assisted reproduction in this species has been slow when compared to other production species. The authorization of the use of embryo transfer by some breed associations stimulated the development and expansion of several reproductive techniques for use in horses. The present study aimed to address the advanced biotechnologies currently available for this species, such as superovulation, transfer of oocytes, gamete intrafallopian transfer, in vitro production of embryos and cloning, emphasizing their practical and clinical applications, as well as certain challenges that still need to be overcome in each biotech.

Vantagens e desafios das biotécnicas avançadas utilizadas na reprodução equina assistida / Advantages and challenges of advanced biotech used in assisted equine reproduction

Relatos apontam que a espécie equina foi a primeira a ser submetida à inseminação artificial. Apesar deste fato, o desenvolvimento da reprodução assistida nos equinos foi lento em comparação às demais espécies voltadas diretamente para a produção animal. A liberação do uso da transferência de embriões pela maioria das associações de raças estimulou o desenvolvimento e a expansão de algumas técnicas de multiplicação animal para uso em equinos. A presente revisão teve por objetivo abordar as biotécnicas avançadas atualmente disponíveis para a espécie, como a superovulação, a transferência de ovócitos, a transferência intratubárica de gametas, a injeção intracitoplasmática de gametas, a produção in vitro de embriões e a clonagem, enfatizando suas aplicações prática e clínica, bem como determinados desafios que ainda precisam ser superados em cada biotécnica.(AU)

Some reports suggest the equine species as the first one to be subjected to the artificial insemination. Despite this fact, the development of assisted reproduction in this species has been slow when compared to other production species. The authorization of the use of embryo transfer by some breed associations stimulated the development and expansion of several reproductive techniques for use in horses. The present study aimed to address the advanced biotechnologies currently available for this species, such as superovulation, transfer of oocytes, gamete intrafallopian transfer, in vitro production of embryos and cloning, emphasizing their practical and clinical applications, as well as certain challenges that still need to be overcome in each biotech.(AU)

Injeção Intracitoplasmática de Espermatozoide: Métodos de ativação oocitária e desestabilização da membrana espermática

O desenvolvimento de embriões bovinos produzidos por injeção intracitoplasmática de espermatozóides (ICSI) é baixo em relação aos embriões fertilizados. Deficiência na ativação de oócitos, capacitação inadequada de espermatozóides e falta de descondensação de espermatozóides são os principais transtornos que afetam o sucesso de ICSI em bovinos. No presente trabalho, foram testados métodos de ativação com o intuito de estabelecer um protocolo efetivo para a ativação de oócitos bovinos após a ICSI. Para isso, um inibidor específico de CDK1 (RO-3306) e um ativador específico de PKC (OAG) foram utilizados após a incubação com Ionomicina (ION) para avaliar a retomada da meiose em oócitos bovinos. Além disso, a incubação em meio Fert durante 6 h e a eletroporação (El) de espermatozoides previamente a ICSI foram testadas para verificar o efeito sobre a descondensação do espermatozoide e formação do pró-núcleo (PN) masculino. Oócitos bovinos foram incubados, em diferentes concentrações e períodos de exposição aos tratamentos. Foram avaliados conforme a taxa de ativação oocitária (formação de PN), clivagem, desenvolvimento a blastocisto e número de células nos embriões que se desenvolveram a blastocisto. As taxas de PN foram maiores (P0,01) nos grupos ativados com ION+RO (48.5 %) e ION+RO+OAG (65.6 %) comparado com ION (12.3 %) e ION+OAG (9.2 %). Não houve efeito significativo entre as concentrações 5.0, 7.5 e 10.0 M de RO sobre a taxa de ativação. A taxa de ativação foi significativamente maior (P0,01) em oócitos tratados por 240 min (84.6 %) comparado a 60 (53.6 %) e 120 min (60.0%). No entanto, não houve diferença significativa na taxa de ativação quando o tratamento com RO foi iniciado a 0, 30 ou 60 minutos após a incubação com ION. A taxa de clivagem foi inferior nos grupos ION (11.8 %) e ION+OAG (22.8 %) comparada aos grupos ION+RO (70.2 %) e ION+RO+OAG (62.4 %). A taxa de blastocistos também foi maior no grupo ION+RO+OAG (24.1 %), mas não houve diferença estatística entre os grupos ION+RO (19.7 %) e ION+OAG (9.5 %). Não houve desenvolvimento a blastocisto quando os oócitos foram tratamento somente com ION. Não foi detectada diferença estatística entre os tratamentos sobre o número médio de células por blastocistos. No segundo experimento, espermatozoides não tratados (ICSI-Cont) ou tratados por electroporação (ICSI-El) foram microinjetados em oócitos, os quais foram ativados com ION+RO por 240 min e fixados cerca de 15 h após a injeção para determinar a taxa de formação de PNs masculino e feminino (2PN). A maioria dos oócitos apresentaram o PN feminino bem desenvolvido (66.4 %). A formação de 2PN (masculino e feminino) foi maior no grupo ICSI-El (33.3 %) comparado ao grupo ICSI-Cont (9.4 %). Em conclusão, esse estudo demonstrou que a inibição específica da CDK1 após o tratamento com ION promove a ativação de oócitos bovinos. O tratamento de espermatozoides com eletroporação melhora a formação do PN masculino após ICSI, mas a taxa é inferior a formação do PN feminino.

Development of bovine embryos produced by intracytoplasmic sperm injection (ICSI) is low compared to fertilized embryos. Deficient oocyte activation, inappropriate sperm capacitation, and lack of sperm decondensation are thought to be the main constrains affecting ICSI success in cattle. In the present study, activation compounds were tested to establish an effective protocol for activation of bovine oocytes, and then used for oocyte activation after ICSI. The activation approach consisted of exposing in vitro matured oocytes to Ionomycin (ION) following by a specific CDK1 inhibitor (RO-3306), a specific PKC activator (OAG) or both RO+OAG. In the first experiment, the rate of activation (pronuclear (PN) formation), cleavage, development to the blastocyst stage, and number of cells per blastocyst were evaluated after oocyte treatment. The PN rates were higher (P0.01) in the groups activated with ION+RO (48.5 %) and ION+RO+OAG (65.6 %) compared to ION (12.3 %) and ION+OAG (9.2 %). There was no significant effect between the RO concentrations tested (5, 7.5 and 10 M) on oocyte activation. The PN rate was significantly higher (P0.01) when oocytes were exposed to RO for 240 min (84.6 %) compared to 60 (53.6 %) and 120 min (60.0 %). However, there was no difference between groups when treatment with RO started at 0, 30 or 60 min after on ION exposure. Cleavage rate was higher in ION+RO (70.2 %) and ION+RO+OAG (62.4 %) groups compared to ION (11.8 %) and ION+OAG (22.8 %). Blastocyst rate was also higher in the ION+RO+OAG (24.1 %) group, but not statistically different between ION+RO (19.7 %) and ION+OAG (9.5 %) groups. There was no development to the blastocyst stage after treatment with ION alone. The average cell number in blastocysts was not statistically different among treatments. In the second experiment, the effect of activation with ION+RO (10 M for 240 min) was tested after ICSI using control (ICSI-Cont) or treated by electroporation (ICSI-El) sperm. Most oocytes presented a well-developed female PN (66.4%). Male PN formation was higher (P0.05) in the ICSI-El (33.3%) compared to the ICSI-Cont (9.4%) group. In conclusion, this study revealed that the specific inhibition of CDK1 after ION treatment is an effective approach to activate bovine oocytes. Male pronuclear formation after ICSI is increased by sperm electroporation, but is lower than female pronuclear formation. This indicates that deficient sperm decondensation and male PN formation rather than deficient oocyte activation is likely the main problem to develop an effective protocol for bovine ICSI.