Aspiração folicular em vacas Bos taurus e Bos indicus e vitrificação dos oócitos em condições de campo

The aim of this study was to evaluate the viability of cryopreservation of bovine oocytes from Bos taurus and Bos indicus cows, at field conditions. The average rate of oocyte recovered by Ovum Pick up (OPU) from Bos taurus taurus cows (Devon) or Bos taurus indicus cows (Nelore), and the embryo development after vitrification were determined. After 60 sessions, an average rate of 4.6 oocytes/session was obtained from Devon cows. This number was significantly lower than the 16.3 oocytes obtained from Nelore cows, in 12 sessions. Selected recovered oocytes were vitrified in 20% EG + 20% DMSO + 0.5 M sucrose, in TCM 199 medium, with open pulled straws (OPS) technology, and stored in a cryogenic container. Just after warmed, oocytes were submitted to standard in vitro maturation, fertilization and culture in the laboratory. There were no differences in cleavage rates obtained from Devon cows oocytes (17.6%) and Nelore cows oocytes (29.1%). Evaluation of embryo development resulted in only one blastocyst obtained from Devon cows. Data from this study showed that Bos taurus indicus cows had higher oocytes recovery rates when compared with Bos taurus taurus, and that association of OPU with oocytes vitrification at farm conditions resulted in unsatisfactory indexes of embryo development, regardless of cow breed.Com o objetivo de avaliar a viabilidade da criopreservação de oócitos de fêmeas taurinas e zebuínas em condição de campo, este estudo determinou a taxa média de oócitos obtidos por punção folicular in vivo (OPU) de fêmeas bovinas Bos taurus taurus (Devon) e Bos taurus indicus (Nelore), bem como o desenvolvimento embrionário após a vitrificação destes oócitos. Em 60 sessões, obteve-se média de 4,6 oócitos por sessão de OPU nas vacas Devon, número significativamente inferior aos 16,3 oócitos obtidos nas vacas Nelore, em 12 sessões. Os oócitos selecionados foram vitrificados em solução de 20% de EG + 20% de DMSO + 0,5 M de sacarose, em meio TCM 199, utilizando-se palhetas estiradas (OPS). O reaquecimento foi efetuado em laboratório, seguindo-se a maturação, fecundação e cultivo in vitro. As taxas de clivagem obtidas foram 17,6% no grupo de vacas Devon e 29,1% no grupo Nelore, não havendo diferença significativa entre as mesmas. Na avaliação do desenvolvimento embrionário, obteve-se apenas um blastocisto no grupo Devon. Concluiu-se que fêmeas zebuínas (Nelore) proporcionam maior taxa de oócitos recuperados por sessão em relação às fêmeas taurinas (Devon), e que a associação da OPU com a vitrificação dos oócitos na própria fazenda não proporciona taxas aceitáveis de desenvolvimento embrionário, independente da origem dos animais

Conjugated linoleic acid in cryotolerance of bovine in vitro - produced embryos

Embryo cryopreservation is an important tool that allows the storage of genetic material for long time, without loss of functional activity or genetic damage. Cryopreservation of in vivo-produced embryos is well established and provides similar results than those obtained after fresh embryos transfer. However, in vitro-produced (IVP) bovine embryos are easily damaged by conventional methods of cryopreservation, resulting in low survival rates after re-warming. Such sensitivity seems to be related to an excessive amount of lipid in the embryos cytoplasm during in vitro development. This might occur due to serum containing medium cultured embryos. Mechanical removal of lipid droplets can improve survival of early developmental stages embryos after cryopreservation. Nevertheless, delipidation method are laborious and time-consuming, besides altering embryo development after transfer to the recipients. Therefore, studies have searched for non invasive and less laborious techniques to reduce the amount of cytoplasmic lipids of bovine IVP embryos, thus improving their cryotolerance. Trans-10, cis-12 CLA, a conjugated isomer of linoleic can inhibit the fatty acid synthesis, thus decreasing the amount of lipid in several human and animal cells. It is known that lipid synthesis reduction involve down-regulation of mRNA expression of lipogenic enzymes associated with fat synthesis. Studies showed that addition of t10, c12 CLA to culture media, can reduce lipid content of bovine IVP embryos, improving their cryotolerance. Therefore, this study evaluated two CLA isomers on cryotolerance of IVP bovine embryos, as well as the enzymes acetyl-CoA carboxylase (ACCa), stearoyl-CoA desaturase (SCD1) and fatty acid synthase (FASN) mRNA expression of these embryos. In Experiment 1, embryos were cultured in media with increasing concentrations of trans-10, cis-12 CLA isomer: control group (0 μM), 50CLA group (50 μM), 100CLA group (100 μM) and 200CLA group (200 μM), looking for the higher concentration that does not negatively affect embryo development. In Experiment 2, zygotes were cultured in medium containing 100 μM (determined in Experiment 1) of different CLA isomers: control (CLA free) group, t10, c12 CLA group, c9, t11 CLA group and Mixture (50% c9, t11 CLA + 50% t10, c12) group. In Experiment 3, zygotes were allocated in 2 groups: CLA group, containing 100 μM of t10, c12 CLA isomer (determined in Experiment 2) and control (CLA free) group. Blastocysts were separated according to their stage (Bx or Bl) and subjected to vitrification or freezing. As viability criteria, cleavage and blastocyst rates were assessed after IVC and reexpanding and hatching rates were assessed after re-warming. Cell counting and mRNA expression of the ACCa, SCD1 and FASN enzymes were also assessed. The highest concentration of t10, c12 CLA that did not impair blastocyst rates was 100μM.In Experiment 2, the highest hatching rate after re-warming was obtained in c9, t11 group, vitrified at Bx stage (68.6%), not differing from the other treatments vitrified at this stage. The lowest hatching rate was obtained in Mixture group, vitrified at Bl stage (8.0%), not differing from the groups t10, c12 and c9, t11, vitrified at the same stage. In all treatments, Bx stage embryos showed higher viability than Bl stage embryos, except for the Control group, in whose the viability was similar. In Experiment 3, the highest hatching rate was obtained in Bx stage Control vitrified group (67.4%), which was similar to the Bx stage vitrified CLA group. The xv lowest hatching rate was observed in Bl stage frozen CLA group (10.3%), not differing from the other cryopreserved treatments at the same stage. In all frozen groups, despite CLA addition, hatching rates were all similar. There was also no difference in cell counting or mRNA expression of the enzymes ACCa, SCD1 and FASN, among embryos.Under the conditions of this study, the addition of CLA isomers t10, c12 and c9, t11 to culture media does not improve cryotolerance of IVP bovine embryos, and the isomer t10, c12 CLA does not influences the cell number and mRNA expression of enzymes ACCa, SCD1 and FASNA criopreservação de embriões é uma importante ferramenta que permite o armazenamento de material genético por período prolongado, sem causar perda de atividade funcional ou alterações genéticas nessas estruturas. A criopreservação de embriões gerados in vivo está bem estabelecida e proporciona resultados muito próximos aos obtidos após a transferência de embriões frescos. Já os embriões bovinos produzidos in vitro (PIV) são extremamente sensíveis aos métodos convencionais de criopreservação, apresentando reduzidas taxas de sobrevivência após o reaquecimento. Essa sensibilidade parece estar relacionada ao acúmulo excessivo de lipídeos no citoplasma dos embriões durante o desenvolvimento in vitro, principalmente quando o cultivo é realizado em meios adicionados de soro. Estudos realizados com embriões em estágios iniciais de desenvolvimento evidenciaram que a remoção física das gotas lipídicas é capaz de aumentar a tolerância destes embriões à criopreservação. Contudo, o método de delipidação é demasiadamente trabalhoso e demorado, além de alterar o potencial de desenvolvimento dos embriões após a transferência para as receptoras. Dessa forma, os estudos têm buscado métodos não invasivos e menos trabalhosos, que reduzam a quantidade de lipídeos no citoplasma dos embriões bovinos PIV, melhorando assim a sua criotolerância. O trans-10, cis-12 CLA, um isômero conjugado do ácido linoleico, é capaz de inibir a síntese de ácidos graxos, diminuindo o teor lipídico em diversos tecidos de animais e humanos. Sabe-se que um dos mecanismos pelos quais este isômero exerce esse efeito é através da diminuição da expressão de RNAm de enzimas responsáveis pela síntese de lipídeos. Estudos mostraram que a adição do t10, c12 CLA ao meio de cultivo pode reduzir o acúmulo lipídico de embriões bovinos PIV, aumentando a sua criotolerância. Assim, este estudo teve como objetivo avaliar o efeito dos dois principais isômeros do CLA na criotolerância de embriões bovinos PIV e adicionalmente a expressão de RNAm das enzimas acetil-CoA carboxilase (ACCa), estearoil-CoA dessaturase (SCD1) e ácido graxo sintase (FASN) desses embriões. No Experimento 1, embriões foram cultivados em meio com concentrações crescentes do isômero t10, c12 CLA: grupo controle (0 μM), grupo 50CLA (50 μM), grupo 100CLA (100 μM) e grupo 200CLA (200 μM), buscando a maior concentração que não afete o posterior desenvolvimento embrionário. No Experimento 2, os zigotos foram cultivados em meio com 100 μM, (concentração apontada no Experimento 1) de diferentes isômeros de CLA, sendo: grupo controle, sem adição de CLA, grupo t10, c12 CLA, grupo c9, t11 CLA e grupo Mistura, com 50% de cada isômero. No Experimento 3, os zigotos foram distribuídos em 2 grupos: grupo CLA, contendo 100 μM do isômero t10, c12 CLA (determinado no Experimento 2) e grupo controle, sem CLA. Os blastocistos obtidos foram separados em função do estágio (Bx ou Bl) e submetidos ao processo de vitrificação ou congelamento. Como critérios de viabilidade foram avaliados as taxas de clivagem e de blastocistos após o cultivo, e de re-expansão e eclosão após o reaquecimento. Ainda, foi determinada a densidade celular e a expressão de RNAm das enzimas ACCa, SCD1 e FASN destes embriões. A maior concentração de t10, c12 CLA que não reduziu a taxa de blastocistos foi a de 100μM. No Experimento 2, a maior taxa de eclosão após o reaquecimento foi a do grupo c9, t11 vitrificado no estágio de Bx xiii (68,6%), que não diferiu dos demais tratamentos vitrificados neste estágio. A menor taxa de eclosão foi a do grupo Mistura (c9, t11 + t10, c12, no estágio de Bl (8,0%), que não diferiu dos grupos t10, c12 e c9, t11, vitrificados no mesmo estágio. Em todos os tratamentos, embriões em estágio de Bx apresentaram taxas superiores aos Bl, com exceção do grupo Controle, cujas taxas foram similares. No Experimento 3, a maior taxa de eclosão foi observada no grupo Controle com Bx vitrificados (67,4%), que não diferiu do grupo CLA, com Bx vitrificados. A menor taxa de eclosão foi a do grupo CLA com Bl congelados (10,3%), que não diferiu dos demais tratamentos criopreservados neste estágio. Nos grupos submetidos ao congelamento, as taxas de desenvolvimento foram semelhantes, independente da exposição ao CLA. Não houve diferença na densidade celular entre os embriões expostos ou não ao t10, c12 CLA, e nem na expressão de RNAm das enzimas ACCa, SCD1 e FASN. Conclui-se que nas condições deste estudo a adição dos isômeros do CLA t10, c12 e c9, t11 ao meio de cultivo não melhora a criotolerância de embriões bovinos PIV, bem como que o isômero t10, c12 CLA, não interfere na densidade celular e na expressão de RNAm das enzimas ACCa, SCD1 e FASN destes embriõesCoordenação de Aperfeiçoamento de Pessoal de Nível Superio

Efeito do diâmetro folicular, momento da primeira clivagem e metilação da H3K4 na produção embrionária de vacas Bos indicus

This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus’ slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P 28 h e ? 34 h pós-FIV) e tardia (> 34 h e ? 54 h pós-FIV). Os blastocistos foram submetidos à imunofluorescência para investigação de H3K4me. A taxa de blastocisto para embriões provenientes de folículos grandes (36,3%) foi maior que de folículos pequenos (22,9%; p < 0,05). Ainda, as taxas de blastocisto para os grupos de clivagem precoce e intermediária (45,3% e 33,8%, respectivamente) foram maiores que para o grupo de clivagem tardia (13,5%; p < 0,05). Blastocistos de todos os grupos mostraram marcação para H3K4me à imunofluorescência, particularmente intensa no que pareciam ser células do trofectoderma e fraca ou ausente em células semelhantes às da massa celular interna. Pela primeira vez em embriões indicus, os dados deste estudo demonstram que maiores taxas de blastocisto são obtidas de embriões que clivam em até 34 h pós-fertilização e dos oriundos de folículos de 4 a 8 mm de diâmetro, indicando uma maior habilidade desses embriões de se desenvolverem até o estágio de pré-implantação embrionária. Este é o primeiro artigo demonstrando a ocorrência de H3K4me em embriões bovinos; sua presença em todos os blastocistos avaliados sugere que essa modificação de histona exerce função-chave na manutenção da viabilidade embrionária no estágio pré-implantação

Developmental block and programmed cell death in Bos indicus embryos: effects of protein supplementation source and developmental kinetics.

The aims of this study were to determine if the protein source of the medium influences zebu embryo development and if developmental kinetics, developmental block and programmed cell death are related. The culture medium was supplemented with either fetal calf serum or bovine serum albumin. The embryos were classified as Fast (n = 1,235) or Slow (n = 485) based on the time required to reach the fourth cell cycle (48 h and 90 h post insemination - hpi -, respectively). The Slow group was further separated into two groups: those presenting exactly 4 cells at 48 hpi (Slow/4 cells) and those that reached the fourth cell cycle at 90 hpi (Slow). Blastocyst quality, DNA fragmentation, mitochondrial membrane potential and signs of apoptosis or necrosis were evaluated. The Slow group had higher incidence of developmental block than the Fast group. The embryos supplemented with fetal calf serum had lower quality. DNA fragmentation and mitochondrial membrane potential were absent in embryos at 48 hpi but present at 90 hpi. Early signs of apoptosis were more frequent in the Slow and Slow/4 cell groups than in the Fast group. We concluded that fetal calf serum reduces blastocyst development and quality, but the mechanism appears to be independent of DNA fragmentation. The apoptotic cells detected at 48 hpi reveal a possible mechanism of programmed cell death activation prior to genome activation. The apoptotic cells observed in the slow-developing embryos suggested a relationship between programmed cell death and embryonic developmental kinetics in zebu in vitro-produced embryos

Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos

Conjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA. In addition, in Experiment 3, the hatching rate was higher at the EB stage in vitrified groups, while the rates of BL and EB were similar in frozen groups, thus proving that vitrification was more efficient than freezing for IVP bovine embryos. In Experiment 3, CLA isomer t10, C12 did not influence the embryonic cell number or mRNA expression of ACC1 and SCD1 enzymes, but decreased the mRNA expression of FASN. In conclusion, 100 ?M CLA did not affect subsequent embryonic development. However, neither CLA isomer improved the cryotolerance of IVP bovine embryos.</p

TUNEL assay.

<p>Epifluorescence photomicrography of embryos at 48 and 90 hpi classified according to the speed of development as either Fast (reached the fourth cell cycle at 48 hpi) or Slow/4 cells (exactly 4 cells and prior to the fourth cell cycle at 48 hpi). A) Fast embryo at 48 h of culture; B) Slow/4 cell embryo at 48 h of culture; C) embryo at 48 h of culture submitted to DNA fragmentation via exposure to DNase (technique positive control); D) Fast embryo at 90 h of culture; E) Slow/4 cell embryo at 90 h of culture; and F) embryo at 90 h of culture submitted to DNA fragmentation via exposure to DNase. The blue fluorescent nuclei indicate total cell number and red fluorescent staining indicates cells with fragmented DNA.</p

Differential staining by fluorochrome.

<p>Epifluorescence photomicrography of the differential staining by fluorochrome of hatched blastocysts cultured with two sources of protein and classified according to the speed of development: A) Fast developing embryo cultured with BSA; B) Slow developing embryo cultured with BSA; C) Fast developing embryo cultured with FCS; and D) Slow developing embryo cultured with FCS.</p

Nuclei number in the ICM and TE from hatched blastocysts 9 days after IVF (Be—D9), supplemented with FCS, classified according to the speed of development (Fast or Slow).

<p>*—There were no significant differences between treatments (P ≤ 0.05).</p><p>Nuclei number in the ICM and TE from hatched blastocysts 9 days after IVF (Be—D9), supplemented with FCS, classified according to the speed of development (Fast or Slow).</p