Criopreservação e cultivo de tecido testicular em mamíferos - estado da arte, desafios e perspectivas / Cryopreservation and culture of testicular tissue in mammals - state of the art, challenges and perspectives

O tecido testicular contém células germinativas, que possuem potencial para se desenvolver em espermatozoides viáveis por meio do cultivo in vitro ou xenotransplante, sendo uma alternativa interessante a ser utilizada na formação de biobancos. Esta revisão compila as atualizações, desafios e perspectivas relacionadas às técnicas de criopreservação e cultivo de tecido testicular como estratégia para a conservação de espécies mamíferas. O tecido testicular pode ser obtido tanto de indivíduos adultos como pré púberes, seja após orquiectomia ou até mesmo após a sua morte. O tecido fragmentado pode ser criopreservado por congelação lenta ou rápida e por métodos de vitrificação. Os crioprotetores são indispensáveis durante a criopreservação e podem variar o tipo e concentração de acordo com a espécie. Com os avanços da criopreservação deste material, espermatozoides podem ser obtidos por transplante de fragmentos testiculares ou células germinativas isoladas em camundongos imunodeficientes. No entanto, a obtenção de espermatozoides no cultivo in vitro ainda é um desafio.(AU)

The testicular tissue contains germ cells, which have the potential to develop into viable spermatozoa through in vitro culture or xenotransplantation, being an interesting alternative to be used in the formation of biobanks. This review compiles updates, challenges and perspectives related to cryopreservation techniques and testicular tissue culture as a strategy for the conservation of mammalian species. Testicular tissue can be obtained from both adult and pre-pubertal individuals, either after orchiectomy or even after their death. Fragmented tissue can be cryopreserved by slow or fast freezing and by vitrification methods. Cryoprotectants are indispensable during cryopreservation and may vary in type and concentration according to the species. With advances in cryopreservation of this material, spermatozoa can be obtained by transplanting testicular fragments or isolated germ cells into immunodeficient mice. However, obtaining spermatozoa in in vitro culture is still a challenge.(AU)

Advances and Challenges in Understanding and Assisting Reproduction of Wild Animal Species / Avanços e desafíos na compreensão e reprodução assistida de espécies selvagens

Studying reproduction in wild animals is complex as there are as many biological traits as there are species. What we currently know is minimal compared to the large number of species that remain unstudied. In addition to the impressive diversity of natural mechanisms, other complexities limit the progress of wildlife reproductive science - little interest in animal reproduction, difficult access to animals, lack of expertise, hard working conditions, and insufficient funding. Despite those challenges, some species are being saved from extinction with the help of a precise understanding of reproduction, development of assisted reproductive technologies, and creations of cryo-banks. Those advances originate from huge progresses in non-invasive measurements of steroid metabolites in urine or fecal samples to study and monitor reproductive functions and pregnancies. Progresses in cryobiology also have been impactful in animal conservation. Importantly, emerging technologies (transcriptomics, microfluidics) and additional research areas (reproductive aging, microbiomes) could lead to more successes and address current challenges in the reproduction of rare and endangered species. However, while some emerging approaches like stem cell technologies may sound promising, it is necessary to design holistic strategies considering all available tools to optimize investments, time, and efforts in conservation.

Influência de polissacarídeos sulfatados sobre a motilidade e morfologia espermática pós descongelação de Prochilodus brevis / (Influence of sulfated polysaccharides on post-thawing sperm motility and morphology of Prochilodus brevis

This research aims to verify the influence of sulfated polysaccharides, extracted from the skin of tilapia, on the after thawing motility and morphology of P. brevis sperm. For this, 17 males were hormonally induced to reproduce, through the application of two doses of pituitary carp extract, 0.4 and 4.0mg kg-1. After the seminal collection, objective analyzes were performed and samples with motility greater than 80% were selected to form the pools. Then, the pools were frozen in solution supplemented, or not, with different concentrations of glycosaminoglycans (GAGs): 0 (control); 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5 or 5.0mg mL-¹ (total of 10 treatments). The samples were placed in 0.25 mL French straws and submitted to an equilibrium time of 10 minutes at 4 ºC. Then, they were kept in the dry shipper for 15 minutes and finally stored in liquid nitrogen. After 15 days, samples were thawed in a water bath at 30 ºC for 16 seconds and evaluated for sperm motility and morphology. Thus, it was observed that the increase in GAGs caused a decrease in sperm motility, however the control and the concentration of 0.5mg mL-¹ presented very similar data. On the other hand, no decrease in normal sperm was observed with an increase in the concentration of GAGs. Therefore, it is concluded that the lowest concentration of GAGs is the most adequate to supplement the sperm freezing medium.

Influência de polissacarídeos sulfatados sobre a motilidade e morfologia espermática pós descongelação de Prochilodus brevis / (Influence of sulfated polysaccharides on post-thawing sperm motility and morphology of Prochilodus brevis

This research aims to verify the influence of sulfated polysaccharides, extracted from the skin of tilapia, on the after thawing motility and morphology of P. brevis sperm. For this, 17 males were hormonally induced to reproduce, through the application of two doses of pituitary carp extract, 0.4 and 4.0mg kg-1. After the seminal collection, objective analyzes were performed and samples with motility greater than 80% were selected to form the pools. Then, the pools were frozen in solution supplemented, or not, with different concentrations of glycosaminoglycans (GAGs): 0 (control); 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5 or 5.0mg mL-¹ (total of 10 treatments). The samples were placed in 0.25 mL French straws and submitted to an equilibrium time of 10 minutes at 4 ºC. Then, they were kept in the dry shipper for 15 minutes and finally stored in liquid nitrogen. After 15 days, samples were thawed in a water bath at 30 ºC for 16 seconds and evaluated for sperm motility and morphology. Thus, it was observed that the increase in GAGs caused a decrease in sperm motility, however the control and the concentration of 0.5mg mL-¹ presented very similar data. On the other hand, no decrease in normal sperm was observed with an increase in the concentration of GAGs. Therefore, it is concluded that the lowest concentration of GAGs is the most adequate to supplement the sperm freezing medium.(AU)

Preservation of female fertility in humans and animal species

A detailed understanding of the cryobiology of gametes and complex tissues has led to the development of methods that facilitate the successful low temperature banking of isolated mature human oocytes, or immature oocytes in situ within fragments of human ovarian cortex. Although many outstanding research challenges remain to be addressed, the successful development of new treatments to preserve female fertility for a range of clinical indications has largely been underpinned by the conduct of extensive, fundamental research on oocytes and ovarian tissues from a number of laboratory and commercially important farm species. Indeed, the most recent evidence from large animals suggests that it is also possible to cryopreserve intact whole ovaries along with their supporting vasculature for later autotransplantation and restoration of natural fertility. This review will explore how the methods developed to preserve human oocytes and ovarian tissues can now be used strategically to support the development of conservation strategies aimed at safeguarding the genetic diversity of commercially important domestic animals and also of preserving the female germplasm for wild animals and endangered species.

Preservation of female fertility in humans and animal species

A detailed understanding of the cryobiology of gametes and complex tissues has led to the development of methods that facilitate the successful low temperature banking of isolated mature human oocytes, or immature oocytes in situ within fragments of human ovarian cortex. Although many outstanding research challenges remain to be addressed, the successful development of new treatments to preserve female fertility for a range of clinical indications has largely been underpinned by the conduct of extensive, fundamental research on oocytes and ovarian tissues from a number of laboratory and commercially important farm species. Indeed, the most recent evidence from large animals suggests that it is also possible to cryopreserve intact whole ovaries along with their supporting vasculature for later autotransplantation and restoration of natural fertility. This review will explore how the methods developed to preserve human oocytes and ovarian tissues can now be used strategically to support the development of conservation strategies aimed at safeguarding the genetic diversity of commercially important domestic animals and also of preserving the female germplasm for wild animals and endangered species.(AU)

Viability of in vitro produced embryos of gyr cattle (Bos indicus) after cryopreservation by vitrification under field conditions / Viabilidade de embriões gir (Bos indicus) produzidos in vitro após criopreservação em condições de campo pela vitrificação

The aim of this study was to evaluate the pregnancy rate of in vitro produced embryos of Gyr cattle (Bos indicus) after cryopreservation by the vitrification method under field conditions. Blastocysts in different developmental stages were transferred to recipient cows either fresh (n = 140) or warmed after vitrification (n = 138). The pregnancy rates obtained for fresh embryos were 46.15% (initial blastocyst), 46.93% (blastocyst) and 50.0% (expanded blastocyst) at 35 days post-fertilization, and 43.58% (initial blastocyst), 46.93% (blastocyst) and 50.0% (expanded blastocyst) at 60 days. The pregnancy rates after embryo vitrification were 35.0% (initial blastocyst), 42.30% (blastocyst) and 43.47% (expanded blastocyst) at 35 days post-fertilization, and 32.50% (initial blastocyst), 38.46% (blastocyst) and 43.47% (expanded blastocyst) at 60 days. Embryo vitrification or blastocyst developmental stage did not affect pregnancy rates or incidence of embryonic death. In conclusion, vitrification of Gyr (Bos indicus) embryos under field conditions is an efficient method that can be implemented to use surplus in vitro produced embryos without affecting pregnancy rates...

Objetivou-se com o trabalho avaliar a taxa de prenhez a partir de embriões de bovinos da raça Gir (Bos indicus) produzidos in vitro após criopreservação em condições de campo pelo método de vitrificação. Blastocistos em diferentes estádios de desenvolvimento foram transferidos a fresco (n = 140) ou aquecidos após a vitrificação (n = 138). As taxas de prenhez de embriões frescos foram 46,15% (blastocisto inicial), 46,93% (blastocisto) e 50,00% (blastocisto expandido) aos 35 dias e 43,58% (blastocisto inicial), 46,93% (blastocisto) e 50,00% (blastocisto expandido) aos 60 dias pós-fertilização, respectivamente. As taxas de prenhez após a vitrificação foram 35,00% (blastocisto inicial), 42,30% (blastocisto) e 43,47% (blastocisto expandido) aos 35 dias e 32,50% (blastocisto inicial), 38,46% (blastocisto) e 43,47% (blastocisto expandido) aos 60 dias pós-fertilização, respectivamente. A vitrificação do embrião ou estádio do desenvolvimento não afetaram as taxas de prenhez ou incidência de morte embrionária. Em conclusão, a vitrificação de embriões de bovinos da raça Gir (Bos indicus) sob condições de campo é um método eficiente que pode ser implementado para aproveitar os embriões produzidos in vitro excedentes sem afetar a taxa de prenhez...

Viability of in vitro produced embryos of gyr cattle (Bos indicus) after cryopreservation by vitrification under field conditions / Viabilidade de embriões gir (Bos indicus) produzidos in vitro após criopreservação em condições de campo pela vitrificação

The aim of this study was to evaluate the pregnancy rate of in vitro produced embryos of Gyr cattle (Bos indicus) after cryopreservation by the vitrification method under field conditions. Blastocysts in different developmental stages were transferred to recipient cows either fresh (n = 140) or warmed after vitrification (n = 138). The pregnancy rates obtained for fresh embryos were 46.15% (initial blastocyst), 46.93% (blastocyst) and 50.0% (expanded blastocyst) at 35 days post-fertilization, and 43.58% (initial blastocyst), 46.93% (blastocyst) and 50.0% (expanded blastocyst) at 60 days. The pregnancy rates after embryo vitrification were 35.0% (initial blastocyst), 42.30% (blastocyst) and 43.47% (expanded blastocyst) at 35 days post-fertilization, and 32.50% (initial blastocyst), 38.46% (blastocyst) and 43.47% (expanded blastocyst) at 60 days. Embryo vitrification or blastocyst developmental stage did not affect pregnancy rates or incidence of embryonic death. In conclusion, vitrification of Gyr (Bos indicus) embryos under field conditions is an efficient method that can be implemented to use surplus in vitro produced embryos without affecting pregnancy rates...(AU)

Objetivou-se com o trabalho avaliar a taxa de prenhez a partir de embriões de bovinos da raça Gir (Bos indicus) produzidos in vitro após criopreservação em condições de campo pelo método de vitrificação. Blastocistos em diferentes estádios de desenvolvimento foram transferidos a fresco (n = 140) ou aquecidos após a vitrificação (n = 138). As taxas de prenhez de embriões frescos foram 46,15% (blastocisto inicial), 46,93% (blastocisto) e 50,00% (blastocisto expandido) aos 35 dias e 43,58% (blastocisto inicial), 46,93% (blastocisto) e 50,00% (blastocisto expandido) aos 60 dias pós-fertilização, respectivamente. As taxas de prenhez após a vitrificação foram 35,00% (blastocisto inicial), 42,30% (blastocisto) e 43,47% (blastocisto expandido) aos 35 dias e 32,50% (blastocisto inicial), 38,46% (blastocisto) e 43,47% (blastocisto expandido) aos 60 dias pós-fertilização, respectivamente. A vitrificação do embrião ou estádio do desenvolvimento não afetaram as taxas de prenhez ou incidência de morte embrionária. Em conclusão, a vitrificação de embriões de bovinos da raça Gir (Bos indicus) sob condições de campo é um método eficiente que pode ser implementado para aproveitar os embriões produzidos in vitro excedentes sem afetar a taxa de prenhez...(AU)

Criopreservação de tecido testicular / Criopreservation of testicular tissue

O uso da criobiologia tem sido importante ferramenta para a preservação dos gametas. Atualmente, um dos desafios da área consiste na preservação do tecido ovariano e testicular por tempo prolongado e a criobiologia tem se mostrado como parte inerente no processo. Embora existam relatos sobre a criopreservação de tecido ovariano e obtenção de nascimentos em diferentes espécies, a criopreservação de tecido testicular ainda encontra-se limitada ao nível da experimentação. O desafio é promover um protocolo de criopreservação capaz de manter o potencial do tecido de completar a espermatogênese. A criopreservação de tecido testicular pode vir a ser um método utilizado quando técnicas como a congelação do ejaculado não é possível.(AU)

The use of cryobiology has been extremely important for gametes preservation. Nowadays, the challenge is the study of long-term preservation of ovarian and testicular tissue and the use of cryobiology is an important tool in this process. Although ovarian cryopreservation has successfully produced live births in different species, cryopreservation of testicular tissue is still restricted to a limited experimental level. The challenge is to provide a successful cryopreservation protocol for testis tissue capable of maintaining the potential of the tissue for completion spermatogenesis. Cryopreservation of testicular tissues theoretically offers a practical method when other techniques such as cryopreservation of ejaculated sperm are not available or applicable.(AU)

Criopreservação de tecido testicular / Criopreservation of testicular tissue

O uso da criobiologia tem sido importante ferramenta para a preservação dos gametas. Atualmente, um dos desafios da área consiste na preservação do tecido ovariano e testicular por tempo prolongado e a criobiologia tem se mostrado como parte inerente no processo. Embora existam relatos sobre a criopreservação de tecido ovariano e obtenção de nascimentos em diferentes espécies, a criopreservação de tecido testicular ainda encontra-se limitada ao nível da experimentação. O desafio é promover um protocolo de criopreservação capaz de manter o potencial do tecido de completar a espermatogênese. A criopreservação de tecido testicular pode vir a ser um método utilizado quando técnicas como a congelação do ejaculado não é possível.

The use of cryobiology has been extremely important for gametes preservation. Nowadays, the challenge is the study of long-term preservation of ovarian and testicular tissue and the use of cryobiology is an important tool in this process. Although ovarian cryopreservation has successfully produced live births in different species, cryopreservation of testicular tissue is still restricted to a limited experimental level. The challenge is to provide a successful cryopreservation protocol for testis tissue capable of maintaining the potential of the tissue for completion spermatogenesis. Cryopreservation of testicular tissues theoretically offers a practical method when other techniques such as cryopreservation of ejaculated sperm are not available or applicable.

O POTENCIAL CRIOPROTETOR DO ALGINATO DE SÓDIO NA CRIOPRESERVAÇÃO DE TECIDO OVARIANO

A criopreservação de gametas é uma das ferramentas mais promissoras para a preservação de material genético, útil tanto na conservação de espécies ameaçadas de extinção quanto de interesse econômico e agropecuário. Embora a criopreservação de sêmen de peixes já esteja consolidada, são restritos os relativos sucessos na criopreservação de gametas femininos de peixes. Muitos são os problemas acarretados pela não perda de material genético materno tais como, redução na variabilidade genética, perda do material genético mitocondrial e RNAs mensageiros indispensáveis para o desenvolvimento inicial do embrião. Há muitas décadas, pesquisadores tentam encontrar o equilíbrio necessário entre as substâncias utilizadas como crioprotetores e a técnica adequada de resfriamento e aquecimento. No entanto, mesmo com as diversas variações experimentadas, os problemas ainda parecem ser os mesmos: injúrias celulares causadas pela variação de temperatura, estresse osmótico e o alto grau de citotoxicidade dos crioprotetores, além de problemas reportados mais recentemente como a mutagênese, fragmentação de DNA e cromossomos. Alguns criobiologistas e cientistas de áreas afins pesquisam novas alternativas e diferentes conceitos e etapas da criopreservação que vão desde a intensificação das curvas de resfriamento e aquecimento, quanto a busca por novas soluções crioprotetoras que de fato protejam as células. Nesse contexto, o objetivo desse estudo foi avaliar a capacidade crioprotetora do Alginato de Sódio. Trata-se de um polissacarídeo natural extraído de algas marrons, e foi escolhido com agente crioprotetor não permeável por possuir a capacidade de absorver e reter líquidos além de ser uma substância atóxica. Pela primeira vez utilizado na criopreservação de tecido ovariano de peixes, utilizamos o zebrafish como modelo animal. Além disso, avaliamos o potencial comportamento crioprotetor do Alginato de Sódio tanto isoladamente como crioprotetor, quanto em combinações com outros agentes crioprotetores convencionas como o Tese de Doutorado em Zootecnia Produção animal, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil. (89p.) Dezembro, 2020. C2H6OS (DMSO), propilenoglicol, metanol e sacarose. E os resultados obtidos com a utilização do Alginato de Sódio foram surpreendentes, dado que houve resultados positivos para a criopreservação de folículos em estágios I e II, além de ocorrências, mesmo que isoladas, de oócitos em elevado grau de maturação, sem alterações morfológicas. Embora haja a necessidade de estudos massivos acerca dessa biomolécula na criobiologia, o conteúdo deste estudo sugere que o uso do Alginato de Sódio pode ser uma escolha saudável de agente crioprotetor não permeável, além de torná-lo objeto de pesquisa na área.

Gamete cryopreservation is one of the most promising tools for preserving genetic material, useful both for the conservation of endangered species and for economic and agricultural interest. Although the cryopreservation of fish semen is already consolidated, relative successes in the cryopreservation of female fish gametes are restricted. There are many problems caused by the non-loss of maternal genetic material such as, reduction in genetic variability, loss of mitochondrial genetic material and mRNAs indispensable for the initial development of the embryo. For many decades, researchers have tried to find the necessary balance between the substances used as cryoprotectants and the appropriate cooling and heating technique. However, even with several variations experienced, the problems still seem to be the same, cell injuries caused by temperature variation, osmotic stress and the high level of cryoprotectants cytotoxicity, in addition to other problems that new studies discover with each new result, such as example mutagen, fragmentation of DNA and chromosomes. Some cryobiologists and scientists from related fields are researching new alternatives and different concepts and stages of cryopreservation process, ranging from intensifying the cooling and heating curves, to the search for new cryoprotective solutions that actually protect cells. In this context, the aim of this study was to evaluate the cryoprotective capacity of sodium alginate. Because it is one natural polysaccharide extracted from brown algae, and was chosen as a non-permeable cryoprotective agent because it has the ability to absorb and retain liquids in addition to being a non-toxic substance. For the first time used in the cryopreservation in fish ovarian tissue, we used zebrafish as an animal model. In addition, we evaluated the potential cryoprotective behavior of sodium alginate both alone and with cryoprotectant, and in combinations with other conventional cryoprotectants such as C2H6OS (DMSO), propylene glycol, methanol and sucrose. And the results obtained with the use of sodium alginate were surprising, given that there were positive results for the cryopreservation of follicles in stages I and II, in Doctoral thesis in Animal Science, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. (89 p.) Dezember, 2020. addition to occurrences even if isolated from oocytes in a high degree of maturation, without morphological changes. Although there is a need for massive studies about this biomolecule in cryobiology, the content of this study suggests that the use of sodium alginate may be a healthy choice of non-permeable cryoprotective agent, in addition to making it an object of research in this area.

Cryopreservation of Prochilodus lineatus semen: effect of cryoprotectants combination / Criopreservação do sêmen do Prochilodus lineatus: efeito da combinação de crioprotetores

The study was conducted with the objective of comparing the toxicity and the effect of the combination of intra and extracellular cryoprotectants in curimba Prochilodus lineatus sperm cells subjected to cryopreservation. Semen from 19 males were analyzed and diluted in four solutions comprise of intra and extracellular cryoprotectants in the following combinations: methanol+lactose, methanol+egg yolk, DMSO+lactose and DMSO+egg yolk. A portion of the diluted semen was frozen while the remaining fraction was kept in repose and evaluated after 10 min. For freezing, the diluted samples were packaged in 0.50 mL straws and placed into liquid nitrogen vapor for 24 h and, after this time, submerged into liquid nitrogen for 10 days. The combination of DMSO+lactose was less toxic to the diluted semen, resulting in higher motility rates and durations when compared to the other treatments. After thawing, the highest motility rate and duration were obtained using lactose as extracellular cryoprotectant, regardless of its combination. There was no significant difference between treatments when analyzing sperm morphology after thawing. Considering the effects of the tested treatments, the use of lactose as an extracellularcryoprotectant added with DMSO or methanol is the most suitable, since these combinations presented the highest motility rates and durations and low morphological change rate after thawing.(AU)

O estudo foi realizado com o objetivo de comparar a toxicidade e o efeito da combinação de crioprotetoresintra e extracelulares em espermatozóides de curimba Prochilodus lineatus submetidos à criopreservação. O sêmen de 19 machos foi analisado e diluído em quatro soluções com crioprotetores intra e extracelulares nas seguintes combinações: metanol+lactose, metanol+gema de ovo, DMSO+lactose e DMSO+gema de ovo. Uma porção do sêmen diluído foi congelada enquanto que a fração remanescente foi mantida em repouso e avaliada após 10 min. Para o congelamento, as amostras diluídas foram envasadas em palhetas de 0,50 mL e colocadas em vapor de nitrogênio líquido durante 24 h e, após este tempo, submersas em nitrogênio líquido durante 10 dias. A combinação de DMSO+lactose se mostrou menos tóxica para o sêmen diluído, resultando em taxas mais elevadas de motilidade e duração espermática quando comparadas com os outros tratamentos. Após descongelamento, as maiores taxas de motilidade e duração foram obtidas usando lactose como crioprotector extracelular, independentemente da sua combinação. Não houve diferença significativa entre os tratamentos ao analisar a morfologia espermática após a descongemento. Considerando-se os efeitos dos tratamentos utilizados, o uso de lactose como crioprotector extracelular, adicionada ao DMSO ou metanol, é o mais adequado, uma vez que estascombinações apresentaram maiores taxas de motilidade e duração espermática, e baixa taxa de alteração morfológica após o descongelamento.(AU)

Cryopreservation of Prochilodus lineatus semen: effect of cryoprotectants combination / Criopreservação do sêmen do Prochilodus lineatus: efeito da combinação de crioprotetores

The study was conducted with the objective of comparing the toxicity and the effect of the combination of intra and extracellular cryoprotectants in curimba Prochilodus lineatus sperm cells subjected to cryopreservation. Semen from 19 males were analyzed and diluted in four solutions comprise of intra and extracellular cryoprotectants in the following combinations: methanol+lactose, methanol+egg yolk, DMSO+lactose and DMSO+egg yolk. A portion of the diluted semen was frozen while the remaining fraction was kept in repose and evaluated after 10 min. For freezing, the diluted samples were packaged in 0.50 mL straws and placed into liquid nitrogen vapor for 24 h and, after this time, submerged into liquid nitrogen for 10 days. The combination of DMSO+lactose was less toxic to the diluted semen, resulting in higher motility rates and durations when compared to the other treatments. After thawing, the highest motility rate and duration were obtained using lactose as extracellular cryoprotectant, regardless of its combination. There was no significant difference between treatments when analyzing sperm morphology after thawing. Considering the effects of the tested treatments, the use of lactose as an extracellularcryoprotectant added with DMSO or methanol is the most suitable, since these combinations presented the highest motility rates and durations and low morphological change rate after thawing.

O estudo foi realizado com o objetivo de comparar a toxicidade e o efeito da combinação de crioprotetoresintra e extracelulares em espermatozóides de curimba Prochilodus lineatus submetidos à criopreservação. O sêmen de 19 machos foi analisado e diluído em quatro soluções com crioprotetores intra e extracelulares nas seguintes combinações: metanol+lactose, metanol+gema de ovo, DMSO+lactose e DMSO+gema de ovo. Uma porção do sêmen diluído foi congelada enquanto que a fração remanescente foi mantida em repouso e avaliada após 10 min. Para o congelamento, as amostras diluídas foram envasadas em palhetas de 0,50 mL e colocadas em vapor de nitrogênio líquido durante 24 h e, após este tempo, submersas em nitrogênio líquido durante 10 dias. A combinação de DMSO+lactose se mostrou menos tóxica para o sêmen diluído, resultando em taxas mais elevadas de motilidade e duração espermática quando comparadas com os outros tratamentos. Após descongelamento, as maiores taxas de motilidade e duração foram obtidas usando lactose como crioprotector extracelular, independentemente da sua combinação. Não houve diferença significativa entre os tratamentos ao analisar a morfologia espermática após a descongemento. Considerando-se os efeitos dos tratamentos utilizados, o uso de lactose como crioprotector extracelular, adicionada ao DMSO ou metanol, é o mais adequado, uma vez que estascombinações apresentaram maiores taxas de motilidade e duração espermática, e baixa taxa de alteração morfológica após o descongelamento.

Sperm biotechnologies in domestic species: state of the art

Modern livestock breeding is basically dependent on the proper use of semen for artificial insemination of femeles and of other reproductive biotechnologies such as the production of embryos in vitro for embryo transfer (IVP). Both these techniques have made possible not only the wide dissemmation of genetic material onto breeding populations but also anhanced the selection of best sires, owing to the development of better diagnostic techniques for sperm function and of preservation of seminal material over time. Although use ofliquid semen cooled to room temperature, to intermediate temperatures (+16-20ºC) or chilled (+5ºC) dominates in different species cryopreservation is preferred in bovine A1 and it is advancing in other species by the design of new containers, freezing methods and the use of better insemination strategies. Techniques to separate the aliquot of most robust spermatozoa from an ejaculate have shown a renascent particularly for sires with low sperm quality, and technological advances in separating spermatozoa for c hromosomal sex make the technique suitable for commercial use, following applicat ion of novel findings in sperm and seminal plasma (SP) diagnostics and function. Alongside, knowledge of the epigenome and signalling c apabilities of the semen (sperm and SP) call s for further studies regarding transgene production via ICSI for IVP or AI.

Sperm biotechnologies in domestic species: state of the art

Modern livestock breeding is basically dependent on the proper use of semen for artificial insemination (AU) of femeles and of other reproductive biotechnologies such as the production of embryos in vitro for embryo transfer (IVP). Both these techniques have made possible not only the wide dissemmation of genetic material onto breeding populations but also anhanced the selection of best sires, owing to the development of better diagnostic techniques for sperm function and of preservation of seminal material over time. Although use ofliquid semen cooled to room temperature, to intermediate temperatures (+16-20ºC) or chilled (+5ºC) dominates in different species cryopreservation is preferred in bovine A1 and it is advancing in other species by the design of new containers, freezing methods and the use of better insemination strategies. Techniques to separate the aliquot of most robust spermatozoa from an ejaculate have shown a renascent particularly for sires with low sperm quality, and technological advances in separating spermatozoa for c hromosomal sex make the technique suitable for commercial use, following applicat ion of novel findings in sperm and seminal plasma (SP) diagnostics and function. Alongside, knowledge of the epigenome and signalling c apabilities of the semen (sperm and SP) call s for further studies regarding transgene production via ICSI for IVP or AI.(AU)

Princípios básicos da criomicrobiologia: enfoque nos tipos de micro-organismos e nos principais agentes crioprotetores / Basic principles of criomicrobiologia: focus in kind of microorganisms and principals cryoprotectants agents

O reconhecimento da grande relevância da biodiversidade microbiana para o desenvolvimento humano tem conduzido ao aprimoramento de técnicas destinadas à conservação dos mais diversos espécimes microbiológicos. Contudo, ainda não existe uma fórmula singular, ideal ou universal que possibilite a eficiência da estocagem e preservação microbiana a longo prazo. Consequentemente, diversos micro-organismos para os quais há uma crescente demanda, ainda esperam pelo desenvolvimento de metodologias adequadas à sua conservação, fazendo da criobiologia um campo de vasto potencial para o desenvolvimento de pesquisas. Nesse contexto, a proposta dessa revisão é discutir os princípios básicos da criomicrobiologia, com enfoque na preservação dos variados tipos de micro-organismos e nos principais agentes crioprotetores, tomando como base estudos disponíveis na literatura científica.

The recognizing the great importance of microbial biodiversity for human development has led to the improvement of techniques for the conservation of diverse microbiological specimens. However, there isn't a singular, ideal or universal method to provice the efficiency of the long-term microorganisms stock and preservation. Many microorganisms for which there is a growing demand still wait for the development of satisfactory methodologies to their conservation, making the cryobiology a field of vast potential for the development of researches. Then, the proposal of this review is to discuss the basic principles of cryomicrobiology, focusing the preservation of kinds microorganisms and principals agents cryoprotectants, using the available studies in the comtemporary scientific literature as base.

Isolamento de células derivadas da pele de macacos Sapajus apella (Linnaeus, 1758) e de bovinos para fins de aplicação em biotécnicas da reprodução

Células-tronco presentes nos compartimentos da pele são indispensáveis para a manutenção da homeostase, regeneração, reparação tecidual e reconstrução da integridade e funcionalidade após lesões. Em 2001 um protocolo de isolamento de precursores neurais foi adaptado para o isolamento de células-tronco da pele, referidas como precursores derivados da pele (SKPs), as quais foram diferenciadas, posteriormente, em células da linhagem germinativa, abrindo, assim, uma via de exploração para aplicações em biotecnologias da reprodução. Outro relevante aspecto sobre a pele, são as metodologias desenvolvidas para sua conservação e manutenção por longos períodos, o que permite a estocagem de suprimento biológico para inúmeras pesquisas, inclusive em biologia da conservação e criobiologia. Nesse contexto, considerando-se a necessidade de novas técnicas que contribuam para a preservação de espécies de primatas neotropicais e ungulados silvestres, este trabalho teve como objetivo o isolamento de células derivadas da pele de macacos Sapajus apella e de bovinos, buscando-se, em acréscimo, o estabelecimento de um protocolo de criopreservação das amostras, para posterior utilização em biotécnicas da reprodução. Inicialmente, implementou-se um protocolo otimizado de isolamento enzimático de precursores derivados da pele, com o qual foi possível obter SKPs de fetos bovinos, as quais foram positivas para a atividade da enzima fosfatase alcalina, marcadora de pluripotência. Em Sapajus apella foi possível isolar e cultivar células por essa metodologia até a formação de aglomerados. Em seguida, células resistentes a estresse conhecidas como células Muse, foram isoladas de fibroblastos de macaco Sapajus apella e de bovino adulto e em idade fetal. As células resistentes a estresse formaram aglomerados semelhantes a SKPs em amostras dos três animais, indicando a técnica como um método com potencial para a obtenção de esferas multipotentes. Por fim, estabeleceu-se um protocolo para vitrificação de biópsias de pele de macacos Sapajus apella, o qual foi efetivo para manter a integridade de até 88% das células epidermais, preservar boa densidade de fibroblastos e viabilizar a obtenção de células através do cultivo primário por explante. O protocolo de vitrificação proposto nesse estudo, associado às técnicas de isolamento de células derivadas da pele executadas, constituem em valiosa metodologia para a formação de bancos de germoplasma que viabilizem a realização de biotécnicas da reprodução como a clonagem e a geração de gametas in vitro e contribuam para a preservação de espécies silvestres em risco de extinção.

Stem cells present in the skin compartments are indispensable for the maintenance of homeostasis, regeneration, tissue integrity repair and functionality recovering after injury. In 2001 a protocol for the isolation of neural precursors was adapted for the isolation of skin stem cells, referred as "skin derived precursors" (SKPs), which were able to differentiate into germline cells, thus opening up a line of research in the field of reproduction biotechnology.Another relevant aspect about the skin are the methodologies developed for its conservation and maintenance for long periods, which allows the storage of biological supplies for numerous researches, including conservation biology and cryobiology. In this context, considering the need for new techniques that contribute to the preservation of neotropical primates and wild ungulates species, this work aimed to isolate skin derived cells of Sapajus apella monkeys and bovines. In addition, it was stablished a cryopreservation protocol for the skin samples. Initially, an optimized protocol of enzymatic isolation of skin derived precursors was implemented. It was possible to obtain alkaline phosphatase positive SKPs from bovine fetuses and to isolate and cultures cells from Sapajus apella until cluster formation. Next, stress-tolerant cells also known as Muse cells, were isolated from fibroblastsof Sapajus apella, adult bovine and bovine fetus. The stress-tolerant cells isolated from all three animals generated SKP-like spheres, indicating the technique as a potential method for multipotent spheres obtention. Finally, a protocol for Sapajus apella skin biopsies vitrification was established, which was effective to maintain the integrity of up to 88% of epidermal cells, preserve good fibroblast density and to be established a primary culture of skin explants.The vitrification protocol proposed in this study, associated with the techniques of skin derived cells isolation, constitute a valuable methodology for the formation of germplasm banks that enable the performance of reproductive biotechniques such as cloning and in vitro gametes generation and contribute to the preservation of endangered or threatened wild species.

DNA fragmentation and membrane damage of bocachico Prochilodus magdalenae (Ostariophysi: Prochilodontidae) sperm following cryopreservation with dimethylsulfoxide and glucose

The endangered bocachico Prochilodus magdalenae is a native freshwater fish of Colombia, the most captured species locally and one of the most important species for ex-situ conservation (germplasm banks). The aim of this study was to examine the effect of three concentrations of Dimethylsulfoxide (DMSO) (5%, 10%, 15%) and three of glucose (305, 333, 361 mM) in the extender on spermatic DNA fragmentation (F-DNA) (by Halomax®, Chromatin dispersion) and membrane damage (D-Me) (by eosin-nigrosin staining). After assessment of sperm quality by computer analysis of motility, one part of semen from males was diluted separately with three parts of extender and filled into 0.5 ml straws. Freezing was carried out in liquid nitrogen vapor dry shipper for 30 minutes and thawed at 60ºC for 8 seconds in a water bath and evaluated for the percentage of cells found with F-DNA and D-Me. The results demonstrated that cryopreservation causes greater F-DNA (13.62 ± 1.6% to 28.91 ± 3.25) and D-Me (24.27 ± 1.1% to 58.33 ± 2.81%) when compared with pre-freezing semen (PFS) (6.71 ± 1.54% and 2.34 ± 0.5%, respectively for F-DNA and D-Me). A significant interaction was found between DMSO and glucose concentration in this experiment. Use of extender: 10% DMSO + 305 mM glucose + 12% chicken egg yolk and, 10% DMSO + 333 mM glucose + 12% chicken egg yolk, allow for lower F-DNA and D-Me during cryopreservation of bocachico semen. A high correlation between F-DNA and D-Me was found (r = 0.771).(AU)

O curimba Prochilodus magdalenae, é uma espécie nativa de água doce da Colômbia ameaçada de extinção, sendo a mais capturada localmente e uma das mais importantes para a conservação ex-situ (bancos de germoplasma). O objetivo deste estudo foi avaliar o efeito de três concentrações de dimetilsulfóxido (DMSO) (5%, 10%, 15%) e três de glicose (305, 333, 361 mM) no diluente sobre a fragmentação do ADN espermático (F-DNA) (através de Halomax®, dispersão da cromatina) e danos em membrana (D-Me) (através da coloração eosina-nigrosina). Depois de avaliar a qualidade espermática por análise computacional da mobilidade, uma parte do sêmen dos machos foi diluída separadamente com três partes do diluente e colocado dentro de canudos de 0.5 ml. O congelamento foi feito em tanque de vapores secos de nitrogênio líquido por 30 minutos e descongelado a 60ºC por 8 segundos em banho de água e avaliada a percentagem de células com F-DNA e D-Me. Os resultados demonstraram que a criopreservação causa grande F-DNA (13.62 ± 1.6% até 28.91 ± 3.25) e D-Me (24.27 ± 1.1% até 58.33 ± 2.81%) quando comparada com o sêmen pré-congelamento (PFS) (6.71 ± 1.54% e 2.34 ± 0.5%, respectivamente para F-DNA e D-Me). Uma interação significativa foi encontrada entre a concentração de DMSO e glicose neste experimento. O uso dos diluentes 10% DMSO + 305 mM glicose + 12% gema de ovo de galinha e 10% DMSO + 333 mM glicose + 12% gema de ovo de galinha permitem obter a menor F-DNA e D-Me durante a criopreservação do sêmen de curimba. Uma alta correlação entre F-DNA e D-Me foi encontrada (r = 0.771).(AU)

Papel de agentes antioxidantes na criopreservação de células germinativas e embriões / Role of Antioxidants Agents in Germ Cells and Embryos Cryopreservation

Background: Antioxidants are molecules or substances able to convert the reactive oxygen species (ROS) in water, preventing its overproduction. In an attempt to reduce or eliminate oxidative stress during cryopreservation, antioxidants, especially catalase and trolox®, have been added to the freezing medium to maximize cell survival after the process of freezing / thawing. These substances have been used mainly in the cryopreservation of semen, embryos and oocytes. Given the importance of adding these agents in cryopreservation of mammal germ cells, this review aims to describe issues related to the addition of catalase and trolox® for maintaining the viability of these cells in the cryopreservation process. Review: Numerous protocols for germ cells cryopreservation have achieved satisfactory results in different species, although some points of these protocols still require adjustments in order to succeed in the repeatability of results. Cryopreservation can affect negativelly cell and / or tissues viability by several factors, including the formation of intracellular ice crystals, solution effect and toxicity caused by the inappropriate use of cryoprotective agents. Currently, several studies have emphasized the damage caused by the formation of ROS during cryopreservation processes, leading to oxidative stress. ROS formed during the cryopreservation process can degrade essential molecules to cells, including the polyunsaturated lipids present in the cell membrane (lipid peroxidation), leading them to death. In order to prevent oxidative stress that occurs during cryopreservation, some researchers have tested the addition of antioxidants to the freezing medium in order to achieve higher rates of cell survival after the thawing process. Antioxidants are substances that can neutralize ROS, thus reducing its power of chemical reaction.[...]

Papel de agentes antioxidantes na criopreservação de células germinativas e embriões / Role of Antioxidants Agents in Germ Cells and Embryos Cryopreservation

Background: Antioxidants are molecules or substances able to convert the reactive oxygen species (ROS) in water, preventing its overproduction. In an attempt to reduce or eliminate oxidative stress during cryopreservation, antioxidants, especially catalase and trolox®, have been added to the freezing medium to maximize cell survival after the process of freezing / thawing. These substances have been used mainly in the cryopreservation of semen, embryos and oocytes. Given the importance of adding these agents in cryopreservation of mammal germ cells, this review aims to describe issues related to the addition of catalase and trolox® for maintaining the viability of these cells in the cryopreservation process. Review: Numerous protocols for germ cells cryopreservation have achieved satisfactory results in different species, although some points of these protocols still require adjustments in order to succeed in the repeatability of results. Cryopreservation can affect negativelly cell and / or tissues viability by several factors, including the formation of intracellular ice crystals, solution effect and toxicity caused by the inappropriate use of cryoprotective agents. Currently, several studies have emphasized the damage caused by the formation of ROS during cryopreservation processes, leading to oxidative stress. ROS formed during the cryopreservation process can degrade essential molecules to cells, including the polyunsaturated lipids present in the cell membrane (lipid peroxidation), leading them to death. In order to prevent oxidative stress that occurs during cryopreservation, some researchers have tested the addition of antioxidants to the freezing medium in order to achieve higher rates of cell survival after the thawing process. Antioxidants are substances that can neutralize ROS, thus reducing its power of chemical reaction.[...](AU)