Sperm membrane integrity and stability after selection of cryopreserved ovine semen on colloidal solutions.

The aim of this study was to evaluate the effect of four methods of sperm selection, on the integrity and stability of the plasma membrane, integrity of the acrosomal membrane and spermatic morphology in frozen/thawed ovine semen. Two types of colloidal silica: colloidal silica-silane and colloidal silica-polyvinylpyrrolidone (PVP), and two aliquots: 1 and 4 ml, were used for sperm selection. Probes FITC-PSA and PI were used to measure the integrity of the plasma and acrosomal membranes. Plasma membrane stability was measured, using fluorescent probes M540 and YOPRO1. Effective reduction in the incidence of spermatozoa with acrosomal pathologies was only achieved using 1 ml colloidal silica-silane. All methods were efficient in select viable and unreacted spermatozoa. Only methods using 1 ml of silica were efficient in decrease spermatozoa stained by PI (death). Methods using silica colloidal-silane were more efficient to decrease apoptotic cells after selection when compared to silica colloidal-PVP. In conclusion, sperm selection in colloidal silica-silane and colloidal silica-PVP improved sperm quality when compared to the controls. The method using 1 ml of colloidal silica-silane is the preferred method because its effectiveness and lower cost.

Viability of ovine spermatozoa collected from epididymides stored at 18°-25°C for 48 hours post mortem / Viabilidade dos espermatozoides ovinos coletados de epidídimos armazenados a 18°-25°C até 48 horas após a morte

The objectives of this study were to verify the time during which viable ovine spermatozoa could be recovered from the cauda epididymis kept at ambient temperature (18-25°C). Sperm collected in an artificial vagina (AV) were used as control. Spermatozoa samples were collected with an AV and from epididymis at 0 (G0), 6 (G6), 12 (G12), 24 (G24), and 48 (G48) hours post mortem. Total motility (TM), progressive motility (PM), hypo-osmotic membrane integrity test (HOST) and morphological changes were assessed. TM decreased (P<0.05) from 24 hours post mortem (70.0±1.9%) compared to AV (86.4±1.0%). PM decreased (P<0.05) from 12 hours after death (31.3±4.0%) compared to AV group (73.2±1.4%). The percentage of viable cells in HOST decreased (P<0.05) in the G48 (60.0±8.9%). Spermatozoa recovery was lower (P<0.05) 48 hours after death (2064.2±230.7 x 106 spermatozoa) compared to G0(2623.6±288.4 x 106 spermatozoa). In conclusion, under the conditions of this study, it would be possible to use epididymal spermatozoa recovered up to 24 hours after death for artificial insemination or in vitro fertilization; however, fertility trials are necessary to prove this hypothesis.(AU)

Os objetivos deste estudo foram avaliar o período pelo qual era possível recuperar espermatozoides ovinos viáveis da cauda de epidídimos mantidos em temperatura ambiente (18-25°C). O sêmen coletado em vagina artificial (AV) foi utilizado como controle. Os espermatozoides foram coletados dos epidídimos à zero hora (G0), às seis (G6), 12 (G12), 24 (G24) e 48 (G48) horas post mortem. A motilidade total (TM), a motilidade progressiva (PM), a integridade de membrana plasmática em solução hiposmótica (HOST) e a morfologia espermática foram avaliadas. A TM diminuiu (P<0,05) a partir de 24 horas após a morte (70,0±1,9%) comparado ao sêmen coletado em AV (86,4±1,0%). A PM diminuiu (P<0,05) a partir de 12 horas após a morte (31,3±4,0%) comparado ao grupo AV (73,2±1,4%). A porcentagem de espermatozoides viáveis no HOST diminuiu (P<0,05) no G48 (60,0±8,9%). A recuperação espermática foi menor (P<0,05) 48 horas após a morte (2064,2±498,1 x 106 espermatozoides) comparado ao G0 (2298,4±288,4 x 106 espermatozoides). Em conclusão, nas condições deste estudo, é possível utilizar espermatozoides epididimários recuperados até 24 horas após a morte para inseminação artificial ou fertilização in vitro, porém testes de fertilidade são necessários para comprovar essa hipótese.(AU)

Viability of ovine spermatozoa collected from epididymides stored at 18°-25°C for 48 hours post mortem / Viabilidade dos espermatozoides ovinos coletados de epidídimos armazenados a 18°-25°C até 48 horas após a morte

The objectives of this study were to verify the time during which viable ovine spermatozoa could be recovered from the cauda epididymis kept at ambient temperature (18-25°C). Sperm collected in an artificial vagina (AV) were used as control. Spermatozoa samples were collected with an AV and from epididymis at 0 (G0), 6 (G6), 12 (G12), 24 (G24), and 48 (G48) hours post mortem. Total motility (TM), progressive motility (PM), hypo-osmotic membrane integrity test (HOST) and morphological changes were assessed. TM decreased (P<0.05) from 24 hours post mortem (70.0±1.9%) compared to AV (86.4±1.0%). PM decreased (P<0.05) from 12 hours after death (31.3±4.0%) compared to AV group (73.2±1.4%). The percentage of viable cells in HOST decreased (P<0.05) in the G48 (60.0±8.9%). Spermatozoa recovery was lower (P<0.05) 48 hours after death (2064.2±230.7 x 106 spermatozoa) compared to G0(2623.6±288.4 x 106 spermatozoa). In conclusion, under the conditions of this study, it would be possible to use epididymal spermatozoa recovered up to 24 hours after death for artificial insemination or in vitro fertilization; however, fertility trials are necessary to prove this hypothesis.(AU)

Os objetivos deste estudo foram avaliar o período pelo qual era possível recuperar espermatozoides ovinos viáveis da cauda de epidídimos mantidos em temperatura ambiente (18-25°C). O sêmen coletado em vagina artificial (AV) foi utilizado como controle. Os espermatozoides foram coletados dos epidídimos à zero hora (G0), às seis (G6), 12 (G12), 24 (G24) e 48 (G48) horas post mortem. A motilidade total (TM), a motilidade progressiva (PM), a integridade de membrana plasmática em solução hiposmótica (HOST) e a morfologia espermática foram avaliadas. A TM diminuiu (P<0,05) a partir de 24 horas após a morte (70,0±1,9%) comparado ao sêmen coletado em AV (86,4±1,0%). A PM diminuiu (P<0,05) a partir de 12 horas após a morte (31,3±4,0%) comparado ao grupo AV (73,2±1,4%). A porcentagem de espermatozoides viáveis no HOST diminuiu (P<0,05) no G48 (60,0±8,9%). A recuperação espermática foi menor (P<0,05) 48 horas após a morte (2064,2±498,1 x 106 espermatozoides) comparado ao G0 (2298,4±288,4 x 106 espermatozoides). Em conclusão, nas condições deste estudo, é possível utilizar espermatozoides epididimários recuperados até 24 horas após a morte para inseminação artificial ou fertilização in vitro, porém testes de fertilidade são necessários para comprovar essa hipótese.(AU)

Kinematic and spermatic recovery after selection by centrifugation in colloid solutions of ovine cryopreserved semen / Cinética e recuperação espermática após seleção por centrifugação em soluções coloidais de sêmen ovino criopreservado

Frozen and thawed ovine semen undergo morphological and functional changes that prevent or decrease the efficiency of fertilization. Sperm selection methods seek to improve the quality and viability of the fertilizing materials. Four sperm selection methods were employed, using two silica colloidal solutions coated with silane (silica colloidal-silane) or by polyvinylpyrrolidone (silica colloidal-PVP), and varying the volume of colloidal solution. Sperm kinematic and sperm recovery were evaluated by means of CASA. The protocols using silica colloidal-silane showed higher total motility (TM), progressive motility (PM) and percentage of rapid sperm (%RAP) compared to the methods employing silica colloidal-PVP and to the samples prior to sperm selection. The silica colloidal-PVP had greater sperm recovery compared to the silica colloidal-silane. Only the method using 4mL of silica colloidal-PVP was not efficient in selecting samples with better quality compared to the samples analyzed prior to sperm selection. The methods using lower volumes of colloidal solution did not differ from those using higher volumes and the best results were shown by the method with 1mL silica colloidal-silane. The results found in the study indicated greater efficiency of the silica colloidal-silane solution for sperm selection of thawed ovine semen when compared to selection using silica colloidal-PVP. The method using 1mL of silica colloidal-silane was equally efficient to the method with higher volume, presenting itself as an alternative to process samples with lower sperm concentration.(AU)

O sêmen ovino congelado e descongelado sofre alterações morfofuncionais que impossibilitam ou diminuem a eficiência na fecundação. Os métodos de seleção espermática visam melhorar a qualidade e a viabilidade do material fecundante. Foram utilizados quatro métodos de seleção espermática utilizando duas soluções de sílica coloidal revestida por silano (sílica coloidal-silano) ou por polivinilpirrolidona (sílica coloidal-PVP), variando o volume de solução coloidal. Foram testadas a cinética espermática no CASA e a recuperação espermática. Os protocolos utilizando sílica coloidal-silano apresentaram maior motilidade total (MT), motilidade progressiva (MP) e porcentagem de espermatozoides rápidos (% RAP) quando comparados aos métodos utilizando a sílica coloidal-PVP e às amostras antes da seleção espermática. A sílica coloidal-PVP teve maior recuperação espermática quando comparada à sílica coloidal-silano. Somente o método utilizando 4mL de sílica coloidal-PVP não foi eficiente na seleção de amostras com melhor qualidade quando comparado às amostras analisadas antes da seleção espermática. Os métodos utilizando menores volumes de solução coloidal não diferiram dos métodos de maior volume, sendo a sílica coloidal-silano com 1mL o método que apresentou os melhores resultados. Como conclusão, os resultados encontrados no trabalho apontaram a maior eficiência da sílica coloidal-silano em selecionar sêmen ovino congelado e descongelado quando comparado à seleção em sílica coloidal-PVP. O método utilizando 1mL de sílica coloidal-silano foi igualmente eficiente ao método com maior volume, sendo uma alternativa para processar amostras com baixa concentração espermática.(AU)

Kinematic and spermatic recovery after selection by centrifugation in colloid solutions of ovine cryopreserved semen / Cinética e recuperação espermática após seleção por centrifugação em soluções coloidais de sêmen ovino criopreservado

Frozen and thawed ovine semen undergo morphological and functional changes that prevent or decrease the efficiency of fertilization. Sperm selection methods seek to improve the quality and viability of the fertilizing materials. Four sperm selection methods were employed, using two silica colloidal solutions coated with silane (silica colloidal-silane) or by polyvinylpyrrolidone (silica colloidal-PVP), and varying the volume of colloidal solution. Sperm kinematic and sperm recovery were evaluated by means of CASA. The protocols using silica colloidal-silane showed higher total motility (TM), progressive motility (PM) and percentage of rapid sperm (%RAP) compared to the methods employing silica colloidal-PVP and to the samples prior to sperm selection. The silica colloidal-PVP had greater sperm recovery compared to the silica colloidal-silane. Only the method using 4mL of silica colloidal-PVP was not efficient in selecting samples with better quality compared to the samples analyzed prior to sperm selection. The methods using lower volumes of colloidal solution did not differ from those using higher volumes and the best results were shown by the method with 1mL silica colloidal-silane. The results found in the study indicated greater efficiency of the silica colloidal-silane solution for sperm selection of thawed ovine semen when compared to selection using silica colloidal-PVP. The method using 1mL of silica colloidal-silane was equally efficient to the method with higher volume, presenting itself as an alternative to process samples with lower sperm concentration.(AU)

O sêmen ovino congelado e descongelado sofre alterações morfofuncionais que impossibilitam ou diminuem a eficiência na fecundação. Os métodos de seleção espermática visam melhorar a qualidade e a viabilidade do material fecundante. Foram utilizados quatro métodos de seleção espermática utilizando duas soluções de sílica coloidal revestida por silano (sílica coloidal-silano) ou por polivinilpirrolidona (sílica coloidal-PVP), variando o volume de solução coloidal. Foram testadas a cinética espermática no CASA e a recuperação espermática. Os protocolos utilizando sílica coloidal-silano apresentaram maior motilidade total (MT), motilidade progressiva (MP) e porcentagem de espermatozoides rápidos (% RAP) quando comparados aos métodos utilizando a sílica coloidal-PVP e às amostras antes da seleção espermática. A sílica coloidal-PVP teve maior recuperação espermática quando comparada à sílica coloidal-silano. Somente o método utilizando 4mL de sílica coloidal-PVP não foi eficiente na seleção de amostras com melhor qualidade quando comparado às amostras analisadas antes da seleção espermática. Os métodos utilizando menores volumes de solução coloidal não diferiram dos métodos de maior volume, sendo a sílica coloidal-silano com 1mL o método que apresentou os melhores resultados. Como conclusão, os resultados encontrados no trabalho apontaram a maior eficiência da sílica coloidal-silano em selecionar sêmen ovino congelado e descongelado quando comparado à seleção em sílica coloidal-PVP. O método utilizando 1mL de sílica coloidal-silano foi igualmente eficiente ao método com maior volume, sendo uma alternativa para processar amostras com baixa concentração espermática.(AU)

Detecting animal by-product intake using stable isotope ratio mass spectrometry (IRMS).

Sheep are used in many countries as food and for manufacturing bioproducts. However, when these animals consume animal by-products (ABP), which is widely prohibited, there is a risk of transmitting scrapie - a fatal prion disease in human beings. Therefore, it is essential to develop sensitive methods to detect previous ABP intake to select safe animals for producing biopharmaceuticals. We used stable isotope ratio mass spectrometry (IRMS) for 13C and 15N to trace animal proteins in the serum of three groups of sheep: 1 - received only vegetable protein (VP) for 89 days; 2 - received animal and vegetable protein (AVP); and 3 - received animal and vegetable protein with animal protein subsequently removed (AVPR). Groups 2 and 3 received diets with 30% bovine meat and bone meal (MBM) added to a vegetable diet (from days 16-89 in the AVP group and until day 49 in the AVPR group, when MBM was removed). The AVPR group showed 15N equilibrium 5 days after MBM removal (54th day). Conversely, 15N equilibrium in the AVP group occurred 22 days later (76th day). The half-life differed between these groups by 3.55 days. In the AVPR group, 15N elimination required 53 days, which was similar to this isotope's incorporation time. Turnover was determined based on natural 15N signatures. IRMS followed by turnover calculations was used to evaluate the time period for the incorporation and elimination of animal protein in sheep serum. The δ13C and δ15N values were used to track animal protein in the diet. This method is biologically and economically relevant for the veterinary field because it can track protein over time or make a point assessment of animal feed with high sensitivity and resolution, providing a low-cost analysis coupled with fast detection. Isotopic profiles could be measured throughout the experimental period, demonstrating the potential to use the method for traceability and certification assessments.

In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine.

The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 µm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.

Effect of oil overlay on inhibition potential of roscovitine in sheep cumulus-oocyte complexes.

Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 µm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/-). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.

Técnicas de análise de sêmen / Techniques in semen analysis

A análise de sêmen in vitro é importante para evitar prejuízos em programas de inseminação artificial eauxiliar o técnico na escolha de reprodutores para estações de monta. Ainda que as técnicas mais avançadas deavaliação de sêmen ovino estejam restritas a centros de pesquisa, avanços nessa área aumentam a acurácia e arepetibilidade das análises. Foram desenvolvidas tecnologias objetivas de avaliação do sêmen que utilizamsistemas computadorizados e softwares específicos para examinar imagens dos espermatozoides. A análise demotilidade assistida por computador utiliza fotografias sequenciais em que é possível desenhar o trajeto dascélulas espermáticas.Essa tecnologia tem sido aplicada na pesquisa de novos diluidores e crioprotetoresseminais. A morfometria computadorizada diminui a subjetividade da tradicional análise de morfologiaespermática realizada manualmente. A aplicação de sondas fluorescentes com leitura em microscopia defluorescência ou citometria de fluxo permite a avaliação estrutural e funcional dos espermatozoides de formamais ampla, criteriosa e rápida. A pesquisa da capacidade de degradação das espécies reativas de oxigênioauxilia na preservação do sêmen tanto congelado quanto descongelado, e vem sendo utilizada na melhoria demeios para criopreservação. Os testes biológicos, ainda que onerosos na pesquisa em ovinos, geram informaçõesimportantes sobre a sobrevida dos espermatozoides no trato reprodutivo feminino e sua capacidade fecundante.

In vitro semen analysis is important to prevent errors in artificial insemination programs and assist thetechnician in choosing rams for breeding seasons. While the most advanced assessment techniques ram semenare restricted to research centers, advances in this area increase the accuracy and repeatability of analyzes.Objective semen evaluation technologies using specific computer systems and software to examine images ofsperm were developed. The analysis of computer-assisted motility uses sequential shots that can draw the path ofthe sperm cells, this technology has been applied in search of new extenders and seminal cryoprotectants. Thecomputerized morphometry reduces the subjectivity of traditional sperm morphology analysis performedmanually. The use of fluorescent probes reading fluorescence microscopy or flow cytometry allows structuraland functional assessment of sperm in a more extensive and faster analysis. The research capacity ofdegradation of reactive oxygen species assists in the preservation of frozen and thawed semen, and has beenused in improving the means for cryopreservation. The biological tests, albeit costly research in sheep generateimportant information on the survival of spermatozoa in the female reproductive tract and their fertilizingcapacity.

Técnicas de análise de sêmen / Techniques in semen analysis

A análise de sêmen in vitro é importante para evitar prejuízos em programas de inseminação artificial eauxiliar o técnico na escolha de reprodutores para estações de monta. Ainda que as técnicas mais avançadas deavaliação de sêmen ovino estejam restritas a centros de pesquisa, avanços nessa área aumentam a acurácia e arepetibilidade das análises. Foram desenvolvidas tecnologias objetivas de avaliação do sêmen que utilizamsistemas computadorizados e softwares específicos para examinar imagens dos espermatozoides. A análise demotilidade assistida por computador utiliza fotografias sequenciais em que é possível desenhar o trajeto dascélulas espermáticas.Essa tecnologia tem sido aplicada na pesquisa de novos diluidores e crioprotetoresseminais. A morfometria computadorizada diminui a subjetividade da tradicional análise de morfologiaespermática realizada manualmente. A aplicação de sondas fluorescentes com leitura em microscopia defluorescência ou citometria de fluxo permite a avaliação estrutural e funcional dos espermatozoides de formamais ampla, criteriosa e rápida. A pesquisa da capacidade de degradação das espécies reativas de oxigênioauxilia na preservação do sêmen tanto congelado quanto descongelado, e vem sendo utilizada na melhoria demeios para criopreservação. Os testes biológicos, ainda que onerosos na pesquisa em ovinos, geram informaçõesimportantes sobre a sobrevida dos espermatozoides no trato reprodutivo feminino e sua capacidade fecundante.(AU)

In vitro semen analysis is important to prevent errors in artificial insemination programs and assist thetechnician in choosing rams for breeding seasons. While the most advanced assessment techniques ram semenare restricted to research centers, advances in this area increase the accuracy and repeatability of analyzes.Objective semen evaluation technologies using specific computer systems and software to examine images ofsperm were developed. The analysis of computer-assisted motility uses sequential shots that can draw the path ofthe sperm cells, this technology has been applied in search of new extenders and seminal cryoprotectants. Thecomputerized morphometry reduces the subjectivity of traditional sperm morphology analysis performedmanually. The use of fluorescent probes reading fluorescence microscopy or flow cytometry allows structuraland functional assessment of sperm in a more extensive and faster analysis. The research capacity ofdegradation of reactive oxygen species assists in the preservation of frozen and thawed semen, and has beenused in improving the means for cryopreservation. The biological tests, albeit costly research in sheep generateimportant information on the survival of spermatozoa in the female reproductive tract and their fertilizingcapacity.(AU)

Controle molecular do acúmulo, tradução e degradação de mRNA em oócitos e embriões / Molecular control of accumulation, translation and degradation of mRNA in oocytes and embryos

A competência oocitária para suportar os posteriores estágios do desenvolvimento depende não somenteda correta segregação cromossômica e de transformações citoesqueléticas, mas, principalmente, da adequadatranscrição e estoque de mRNAs cruciais ao desenvolvimento e à viabilidade celular. Com a retomada dameiose, no entanto, embora o oócito mantenha a capacidade de tradução gênica e de síntese proteica, suaatividade transcricional é interrompida, sendo restabelecida apenas com a ativação do genoma embrionário.Deste modo, todo mRNA materno mobilizado durante maturação, expansão do cumulus, fertilização eembriogênese inicial deve ser sintetizado e estocado, em sua forma traducionalmente inativa, nos oócitosmantidos no estágio diplóteno da prófase I. Complexos mecanismos regulatórios, os quais envolvem apoliadenilação e a desadenilação do mRNA, estão implicados no processo de ativação e silenciamento tantotranscricional quanto traducional. Assim, dada à relevância do tema, esta revisão se propõe a abordar osprincipais eventos moleculares envolvidos no controle da expressão, do estoque, da tradução e da degradação detranscritos maternos imprescindíveis ao desenvolvimento oocitário e embrionário.

The oocyte competence to support the later stages of development depends not only on correctchromosome segregation and cytoskeletal changes, but mainly, the proper transcription and storage of mRNAscritical to the cellular development and viability. However, with the resumption of meiosis, although the oocytekept the ability of gene translation and protein synthesis, its transcriptional activity is interrupted and restoredonly with embryonic genome activation. Thus, all maternal mRNA mobilized during maturation, cumulusexpansion, fertilization and early embryogenesis must be synthesized and stored, in its translationally inactiveform, in oocytes kept in the diplotene stage of prophase I. Complex regulatory mechanisms which involvedeadenylation and polyadenylation of mRNA are involved in this process of activation and silencing astranscriptional as translational. So, due to the importance of the topic, this review proposes to discuss the mainmolecular events involved in the control of expression, storage, translation and degradation of maternaltranscript essential to the oocyte and embryo development.

Avaliação da heterogeneidade espermática de carneiros por meio da análise morfométrica computadorizada não automatizada utilizando-se preparação úmida e coloração de Karras / Evaluation of spermatic heterogeneity in ram with computerized non automatized morphometric analysis using humid preparation and Karras stain

A avaliação espermática é fundamental para resultados satisfatórios na biotecnologia da reprodução emovinos. A heterogeneidade das células no ejaculado pode afetar o congelamento de sêmen e morfologiasalteradas podem formar embriões de má qualidade. A análise do sêmen de 10 ovinos da raça Somalis demonstraque existe, entre as células espermáticas, variação na morfometria de nove dos 11 parâmetros avaliados namensuração computadorizada não automatizada, ao comparar as técnicas utilizadas no trabalho (coloração deKarras sob microscopia óptica e preparação úmida sob microscopia de contraste de fase). As diferençasestatísticas entre a maioria dos parâmetros medidos tornam importante a busca por uma justificativa que possaesclarecer tal fenômeno com o objetivo de aprimorar e melhorar a qualidade da avaliação seminal, já que ela estádiretamente relacionada com os índices de fertilidade nos ovinos.

Sperm evaluation is critical for satisfactory results in the biotechnology of reproduction in sheepbreeding. The heterogeneity of cells in the ejaculate can affect semen freezing, and altered morphologies canproduce bad quality embryos. The semen analysis of 10 Somalis ram found that there is variation betweenmorphometric analysis of two techniques (staining Karras and wet preparation), finding statistical differencesamong most of the parameters measured. The semen analysis of 10 Somali sheep breed shows that there isvariation among sperm cells in 9 of the 11 morphometric parameters evaluated in non-automated computedmeasurement comparing the techniques used (Karras staining under optical microscopy and wet preparationunder phase contrast microscopy). Statistical differences between measured parameters is important to searchfor a justification that can clarify this phenomenon with the aim to enhance and improve quality of seminalreview, since it is directly related to fertility rates in sheep.

Avaliação da heterogeneidade espermática de carneiros por meio da análise morfométrica computadorizada não automatizada utilizando-se preparação úmida e coloração de Karras / Evaluation of spermatic heterogeneity in ram with computerized non automatized morphometric analysis using humid preparation and Karras stain

A avaliação espermática é fundamental para resultados satisfatórios na biotecnologia da reprodução emovinos. A heterogeneidade das células no ejaculado pode afetar o congelamento de sêmen e morfologiasalteradas podem formar embriões de má qualidade. A análise do sêmen de 10 ovinos da raça Somalis demonstraque existe, entre as células espermáticas, variação na morfometria de nove dos 11 parâmetros avaliados namensuração computadorizada não automatizada, ao comparar as técnicas utilizadas no trabalho (coloração deKarras sob microscopia óptica e preparação úmida sob microscopia de contraste de fase). As diferençasestatísticas entre a maioria dos parâmetros medidos tornam importante a busca por uma justificativa que possaesclarecer tal fenômeno com o objetivo de aprimorar e melhorar a qualidade da avaliação seminal, já que ela estádiretamente relacionada com os índices de fertilidade nos ovinos.(AU)

Sperm evaluation is critical for satisfactory results in the biotechnology of reproduction in sheepbreeding. The heterogeneity of cells in the ejaculate can affect semen freezing, and altered morphologies canproduce bad quality embryos. The semen analysis of 10 Somalis ram found that there is variation betweenmorphometric analysis of two techniques (staining Karras and wet preparation), finding statistical differencesamong most of the parameters measured. The semen analysis of 10 Somali sheep breed shows that there isvariation among sperm cells in 9 of the 11 morphometric parameters evaluated in non-automated computedmeasurement comparing the techniques used (Karras staining under optical microscopy and wet preparationunder phase contrast microscopy). Statistical differences between measured parameters is important to searchfor a justification that can clarify this phenomenon with the aim to enhance and improve quality of seminalreview, since it is directly related to fertility rates in sheep.(AU)

Controle molecular do acúmulo, tradução e degradação de mRNA em oócitos e embriões / Molecular control of accumulation, translation and degradation of mRNA in oocytes and embryos

A competência oocitária para suportar os posteriores estágios do desenvolvimento depende não somenteda correta segregação cromossômica e de transformações citoesqueléticas, mas, principalmente, da adequadatranscrição e estoque de mRNAs cruciais ao desenvolvimento e à viabilidade celular. Com a retomada dameiose, no entanto, embora o oócito mantenha a capacidade de tradução gênica e de síntese proteica, suaatividade transcricional é interrompida, sendo restabelecida apenas com a ativação do genoma embrionário.Deste modo, todo mRNA materno mobilizado durante maturação, expansão do cumulus, fertilização eembriogênese inicial deve ser sintetizado e estocado, em sua forma traducionalmente inativa, nos oócitosmantidos no estágio diplóteno da prófase I. Complexos mecanismos regulatórios, os quais envolvem apoliadenilação e a desadenilação do mRNA, estão implicados no processo de ativação e silenciamento tantotranscricional quanto traducional. Assim, dada à relevância do tema, esta revisão se propõe a abordar osprincipais eventos moleculares envolvidos no controle da expressão, do estoque, da tradução e da degradação detranscritos maternos imprescindíveis ao desenvolvimento oocitário e embrionário.(AU)

The oocyte competence to support the later stages of development depends not only on correctchromosome segregation and cytoskeletal changes, but mainly, the proper transcription and storage of mRNAscritical to the cellular development and viability. However, with the resumption of meiosis, although the oocytekept the ability of gene translation and protein synthesis, its transcriptional activity is interrupted and restoredonly with embryonic genome activation. Thus, all maternal mRNA mobilized during maturation, cumulusexpansion, fertilization and early embryogenesis must be synthesized and stored, in its translationally inactiveform, in oocytes kept in the diplotene stage of prophase I. Complex regulatory mechanisms which involvedeadenylation and polyadenylation of mRNA are involved in this process of activation and silencing astranscriptional as translational. So, due to the importance of the topic, this review proposes to discuss the mainmolecular events involved in the control of expression, storage, translation and degradation of maternaltranscript essential to the oocyte and embryo development.(AU)

Effect of hydrogen peroxide on thawed ovine sperm motility

Oxidative stress, resulting from excessive levels of ROS in semen, has a negative impact on functional parameters and sperm fertility. In this study we examined the influence of the oxidative stress induced by hydrogen peroxide (H 2 O 2 ) in ovine sperm motility after thawing and catalase (CAT) ability to preserve sperm motility. Semen was incubated at 37 °C with 100 μM H 2 O 2 , 1 U catalase + 100 μM H 2 O 2 or no treatment, for 30 min. Immediately after adding treatments, sperm motility was determined by computer - assisted semen analysis (CASA). Incubation with H 2 O 2 led to a significant (P < 0.05) decrease in motility parameters whereas catalase prevented a decline in motility secondary to oxidative stress. After 30 min of incubation with H 2 O 2 , total motility (12.0% v s . control 73.0%, H 2 O 2 +CAT 70.0%), progressive motility (0.0% vs . control 19.0%, H 2 O 2 +CAT 19.0%) and rapid motility (1.0% v s . control 43.0%, H 2 O 2 +CAT 40.0%) decreased significantly ( P < 0. 05 ), whereas percentage of static cells increased (84.0% v s . control 18.0%, H 2 O 2 +CAT 20.0%). We conclude that H 2 O 2 causes damage to ovine sperm motility and that catalase is able to avoid detrimental effect of H 2 O 2 on sperm motility. (AU)

Effect of hydrogen peroxide on thawed ovine sperm motility

Oxidative stress, resulting from excessive levels of ROS in semen, has a negative impact on functional parameters and sperm fertility. In this study we examined the influence of the oxidative stress induced by hydrogen peroxide (H 2 O 2 ) in ovine sperm motility after thawing and catalase (CAT) ability to preserve sperm motility. Semen was incubated at 37 °C with 100 μM H 2 O 2 , 1 U catalase + 100 μM H 2 O 2 or no treatment, for 30 min. Immediately after adding treatments, sperm motility was determined by computer - assisted semen analysis (CASA). Incubation with H 2 O 2 led to a significant (P < 0.05) decrease in motility parameters whereas catalase prevented a decline in motility secondary to oxidative stress. After 30 min of incubation with H 2 O 2 , total motility (12.0% v s . control 73.0%, H 2 O 2 +CAT 70.0%), progressive motility (0.0% vs . control 19.0%, H 2 O 2 +CAT 19.0%) and rapid motility (1.0% v s . control 43.0%, H 2 O 2 +CAT 40.0%) decreased significantly ( P < 0. 05 ), whereas percentage of static cells increased (84.0% v s . control 18.0%, H 2 O 2 +CAT 20.0%). We conclude that H 2 O 2 causes damage to ovine sperm motility and that catalase is able to avoid detrimental effect of H 2 O 2 on sperm motility.

Oocyte maturation in bitches

The canine species has been used as an experimental model for preservation of endangered species. Biotechnologies of reproduction, such as in vitro maturation (IVM), have been used to meet this objective. Several protocols for in vitro embryo production (IVEP) in swine and bovine species have been adapted for canids. Ho wever, the highest rate reported for in vitro maturation in canids is only 39%, which is still lower than those in other species. Therefore, current research on assisted reproduction in canids have focused on several IVM protocols, including the addition of proteins, hormones, meiosis inhibitors, growth factors and antioxidants to the maturation media and the determination of suitable timing for culture, so that variables involved in the process can be fine-tuned. This review has the main objective of describing major developments and limitations in the process of oocyte maturation in bitches.(AU)

Peculiaridades da coleta de oócitos para produção in vitro de embriões ovinos / Peculiarities of oocyte harvesting for in vitro production of sheep embryos

A expansão da ovinocultura brasileira impulsionou o desenvolvimento de novas biotécnicas de reprodução assistida visando ao aumento dos índices produtivos em concomitância com o acelerado progresso genético. Neste contexto, a produção in vitro de embriões (PIV) ovinos se intensificou, devido à possibilidade de otimização das fêmeas doadoras de oócitos e de incremento da quantidade de embriões produzidos. Entre os vários fatores implicados no sucesso dessa biotecnologia, a qualidade dos complexos cumulus-oophorus (COCs), caracterizada, principalmente, pela presença das células do cumulus, é considerada crucial. Deste modo, é imprescindível a ampliação do conhecimento referente aos diferentes métodos de coleta dos COCs e suas peculiaridades, visando a futuros aperfeiçoamentos biotecnológicos.(AU)

The expansion of Brazilian sheep rearing promoted the development of new biotechnologies of assisted reproduction in order to increase the production indices concomitantly with accelerated genetic progress. In this context, the in vitro production (IVP) of sheep embryos has intensified due to the possibility of optimization of the female oocytes donor and increase the number of embryos produced. Among the various factors involved in the success of this biotechnology, the cumulus-oophorus complexes (COCs) quality, characterized, mainly, by the presence of cumulus cells, is considered crucial. Thus, it is essential to increase the knowledge on the different methods of collection of COCs and their peculiarities, aiming for future biotechnological improvements. (AU)

Peculiaridades da coleta de oócitos para produção in vitro de embriões ovinos / Peculiarities of oocyte harvesting for in vitro production of sheep embryos

A expansão da ovinocultura brasileira impulsionou o desenvolvimento de novas biotécnicas de reprodução assistida visando ao aumento dos índices produtivos em concomitância com o acelerado progresso genético. Neste contexto, a produção in vitro de embriões (PIV) ovinos se intensificou, devido à possibilidade de otimização das fêmeas doadoras de oócitos e de incremento da quantidade de embriões produzidos. Entre os vários fatores implicados no sucesso dessa biotecnologia, a qualidade dos complexos cumulus-oophorus (COCs), caracterizada, principalmente, pela presença das células do cumulus, é considerada crucial. Deste modo, é imprescindível a ampliação do conhecimento referente aos diferentes métodos de coleta dos COCs e suas peculiaridades, visando a futuros aperfeiçoamentos biotecnológicos.

The expansion of Brazilian sheep rearing promoted the development of new biotechnologies of assisted reproduction in order to increase the production indices concomitantly with accelerated genetic progress. In this context, the in vitro production (IVP) of sheep embryos has intensified due to the possibility of optimization of the female oocytes donor and increase the number of embryos produced. Among the various factors involved in the success of this biotechnology, the cumulus-oophorus complexes (COCs) quality, characterized, mainly, by the presence of cumulus cells, is considered crucial. Thus, it is essential to increase the knowledge on the different methods of collection of COCs and their peculiarities, aiming for future biotechnological improvements.

Oocyte maturation in bitches

The canine species has been used as an experimental model for preservation of endangered species. Biotechnologies of reproduction, such as in vitro maturation (IVM), have been used to meet this objective. Several protocols for in vitro embryo production (IVEP) in swine and bovine species have been adapted for canids. Ho wever, the highest rate reported for in vitro maturation in canids is only 39%, which is still lower than those in other species. Therefore, current research on assisted reproduction in canids have focused on several IVM protocols, including the addition of proteins, hormones, meiosis inhibitors, growth factors and antioxidants to the maturation media and the determination of suitable timing for culture, so that variables involved in the process can be fine-tuned. This review has the main objective of describing major developments and limitations in the process of oocyte maturation in bitches.