Individual assessment of bovine embryo development using a homemade chamber reveals kinetic patterns of success and failure to reach blastocyst stage.

Most early developmental data are lost in bovine embryo culture systems. We developed and validated a method for culture of bovine embryos in groups that allow individual assessment. An autoclavable low-cost multiembryo chamber (MEC) was prepared using a polyester mesh fixed to a glass coverslip. Embryonic development was not affected by MEC. Compared to conventional bovine culture system (oil-covered drops, control), cleavage (C, 71.2 ± 7.8%; MEC, 74.3 ± 6.0%), blastocyst rate (C, 29.9 ± 4.4%; MEC, 28.3 ± 5.0%) and blastocyst cell number (C, 94.1 ± 9.7; MEC, 92.9 ± 5.3) were similar. Caspase 3 positive cell index in blastocysts was increased in MEC group, but apoptosis rate was below 5% (C, 2.9 ± 0.5; MEC, 4.6 ± 0.6). Using MEC, we performed a retrospective analysis for 'failure' and 'success' embryos, based on their ability to reach the blastocyst stage. We detected the majority of 'success' embryos displayed 8 cells at 48 h post-insemination (hpi) (48.7%), but blastocysts derived from this pattern presented lower cell numbers (91.3 ± 4.2 vs. 107.9 ± 4.9) and higher apoptosis index (6.2 ± 0.6 vs. 4.4 ± 0.5) than blastocysts from 4-cell embryos at 48 hpi. Most (72.0%) embryos that were at morula stage 120 hpi reached blastocyst stage at 168 hpi. Those blastocysts presented more number of cells than blastocysts derived from embryos exhibiting 16 cells at 120 hpi (108.6 ± 4.1 vs. 83.9 ± 4.8). Combination of embryo kinetics data at 48 and 120 hpi revealed high chances of blastocyst formation for patterns: 8 cells/morula, 4 cells/morula, 8 cells/16 cells and 4 cells/16 cells. Blastocysts formed from 4-cell/morula and 8-cell/morula patterns represented 69% of all 168 hpi blastocysts. Blastocysts derived from 4 cells/16 cells displayed decreased apoptosis (3.1 ± 0.6). Our results suggest that MEC can be used for bovine embryo culture without detrimental effects on development and can help to predict blastocyst formation and quality of in vitro fertilization (IVF) embryos. Abbreviations: BSA: bovine serum albumine; COC: cumulus-oocyte complex; FERT-TALP: Tyrode's albumin lactate pyruvate fertilization; FBS: fetal bovine serum; IVF: in vitro fertilization; MEC: multiembryo chamber; PBS: phosphate buffered saline; SOF-AA: synthetic oviductal fluid with amino acids medium; TCM: Tissue Culture Medium.

Kinetics data from bovine sex-specific embryo development from three different bulls.

Here we present kinetics data from bovine sex-specific embryo development. Embryos were originated using sex-sorted semen from three different Nelore bulls, and semen from the same batch was used for X-and Y-chromosome spermatozoa sorting. Data was obtained for six time points (24, 48, 96, 120, and 144 h.p.i.). Analyses for each bull׳s embryos (1, 2 and 3) is presented for female and male groups separately. Also, grouped data analysis, considering bull and sex interaction, is shown. For further interpretation and discussion, see "Cell death is involved in sexual dimorphism during preimplantation development" (Oliveira et al., 2015 [1]).

Cell death is involved in sexual dimorphism during preimplantation development.

In bovine preimplantation development, female embryos progress at lower rates and originate smaller blastocysts than male counterparts. Although sex-specific gene expression patterns are reported, when and how sex dimorphism is established is not clear. Differences among female and male early development can be useful for human assisted reproductive medicine, when X-linked disorders risk is detected, and for genetic breeding programs, especially in dairy cattle, which requires female animals for milk production. The aim of this study was to characterize the development of female and male embryos, attempting to identify sex effects during preimplantation development and the role of cell death in this process. Using sex-sorted semen from three different bulls for fertilization, we compared kinetics of bovine sex-specific embryos in six time points, and cell death was assessed in viable embryos. For kinetics analysis, we detected an increased population of female embryos arrested at 48 and 120h.p.i., suggesting this time points as delicate stages of development for female embryos that should be considered for testing improvement strategies for assisted reproductive technologies. Assessing viable embryos quality, we found 144h.p.i. is the first time point when viable embryos are phenotypically distinct: cell number is decreased, and apoptosis and cell fragmentation are increased in female embryos at this stage. These new results lead us to propose that sex dimorphism in viable embryos is established during morula-blastocyst transition, and cell death is involved in this process.

Phenotypic correlations between ovum pick-up in vitro production traits and pregnancy rates in Zebu cows.

The growth of the Gyr breed in Brazil in terms of genetic gain for milk, along with conditions for market, has led to the use of ovum pick-up in vitro production (OPU-IVP) as a leader in biotechnology for the multiplication of genetic material. The aim of this study was to investigate phenotypic correlations between OPU-IVP-linked characteristics and pregnancy rates registered in an embryo transfer program using Gyr cows as oocyte donors. Data collected from 211 OPU sessions and 298 embryo transfers during the years 2012 and 2013 were analyzed and statistical analysis was performed. Estimates of simple Pearson correlations were calculated for NVcoc and PVcoc (number and proportion of viable cumulus-oocyte complexes, respectively); NcleavD4 and PcleavD4 (number and proportion of cleaved embryos on day 4 of culture, respectively); NTembD7 and PTembD7 (number and proportion of transferable embryos on day 7 of culture, respectively); NPrD30 and PPrD30 (number and proportion of pregnancies 30 days after transfer, respectively); and NPrD60 and PPrD60 (number and proportion of pregnancies 60 days after transfer, respectively). Moderate to moderately high correlations were found for all numerical characteristics, suggesting these as the most suitable parameters for selection of oocyte donors in Gyr programs. NVcoc is proposed as a selection trait due to positive correlations with percentage traits and pregnancy rates 30 and 60 days after transfer.

Comparison of conventional freezing and vitrification with dimethylformamide and ethylene glycol for cryopreservation of ovine embryos.

The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re-expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re-expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p < 0.05). The number of dead cells in vitrified embryos of Gr. 2 and Gr. 3 was higher (42.6 ± 26.2 and 63.2 ± 34.65, respectively) in comparison with Gr. 1 (conventional freezing, 10.1 ± 8.5, p < 0.05). Embryos vitrified with dimethylformamide included the same quality of apoptotic cells that Gr. 1 (conventional freezing) and fresh embryos. In conclusion, the dimethylformamide and ethylene glycol used as cryoprotectant to vitrify ovine embryos, in the concentrations and exposition time tested in this work, were not as efficient as the conventional freezing for cryopreservation of ovine embryos Thus, the conventional freezing with ethylene glycol was the most efficient method to cryopreserve ovine embryos in comparison with vitrification.

Quantificação de transcritos maternos em oócitos bovinos submetidos a diferentes condições de maturação / Quantification of maternal transcripts in bovine oocytes under different maturation systems

Comparou-se a quantidade relativa de transcritos de origem materna entre oócitos bovinos maturados in vivo e maturados em diferentes condições in vitro. Avaliou-se também o efeito dos sistemas de maturação in vitro sobre a viabilidade das células do cumulus. Para a maturação in vivo, os oócitos foram coletados 19-20h após aplicação de gonadorelina em doadoras superestimuladas com FSH e sincronizadas com implante de progesterona. Para a maturação in vitro, oócitos imaturos, obtidos de ovários coletados em matadouro, foram maturados sob diferentes tensões de oxigênio e suplementação proteica. Avaliou-se a abundância dos transcritos de Zar1, MATER e GDF9 por PCR em tempo real. A viabilidade das células do cumulus de oócitos maturados in vitro foi analisada pela coloração de Azul de Tripan. Observou-se sub-regulação (P<0,05) dos transcritos em oócitos submetidos às diferentes condições de maturação in vitro em relação aos maturados in vivo. Não houve diferença (P>0,05) na viabilidade das células do cumulus. Conclui-se que o sistema de maturação influencia a quantidade de transcritos de origem materna armazenados no citoplasma de oócitos bovinos.

The relative abundance of maternal transcripts among bovine oocytes in vivo matured or under different in vitro conditions was compared. Viability of cumulus cells of in vitro matured oocytes was also evaluated. For in vivo maturation, oocytes were recovered from 19 to 20h after gonadorelin injection in donor cows, which were previously superestimulated with FSH and synchronized with progesterone implant. For in vitro maturation, immature cumulus-oocyte complexes, obtained from ovaries collected at slaughterhouse, were matured under different oxygen tensions and protein supplementation. Relative amount of Zar1, MATER, and GDF9 transcrispts were analyzed by real time PCR. Cumulus cell viability was analyzed by trypan blue. The expression of maternal effect genes were down-regulated (P<0.05) in oocytes matured under different in vitro conditions when compared to those in vivo matured. There was no difference (P>0.05) on cumulus cell viability among different in vitro maturation conditions. In conclusion, different maturation conditions affect the relative abundance of maternal transcripts stored into oocyte cytoplasm.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules.

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P<0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless, there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. Similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis.

Developmental competence and expression of the Hsp 70.1 gene in oocytes obtained from Bos indicus and Bos taurus dairy cows in a tropical environment.

Bos indicus cows usually have better reproductive performance in tropical and subtropical regions than Bos taurus cows, presumably due to their better adaptation to tropical environments. The aim of this study was to evaluate the developmental competence and expression of the Hsp 70.1 gene in immature oocytes from B. taurus (Holstein) and B. indicus (Gyr) dairy cows raised in a tropical region. Cumulus-oocyte complexes were obtained by transvaginal ultrasound-guided follicle aspiration between spring and early autumn, and subjected to in vitro maturation and fertilization. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa media with 10% fetal calf serum; Grade 1 blastocysts were transferred to synchronized crossbred recipients. The total RNA was extracted from immature Holstein and Gyr oocytes (three pools for each breed) and relative quantification of the Hsp 70.1 transcripts was performed by real time PCR after reverse transcription. Cleavage and blastocyst rates were greater (P<0.05) for Gyr (n=390 oocytes) than Holstein (n=505) breed (66.7% versus 53.1% of cleavage and 19.6% versus 10.8% of blastocysts, respectively), but pregnancy rates were not significantly different following transfer to recipients (44.5% for 36 Gyr embryos; 60% for 10 Holstein embryos). Holstein immature oocytes had a higher level (P<0.05) of Hsp 70.1 relative expression (1.82+/-0.22; mean+/-S.E.M.) than Gyr oocytes (1.12+/-0.11). In conclusion, Gyr oocytes obtained in a tropical region were less subject to stress and more likely to develop (after IVF) than Holstein oocytes.

Secreção de interferon-tau em embriões bovinos produzidos in vitro frescos e congelados / Interferon tau secretion in cattle embryos in vitro fertilized before and after cryopreservation

Avaliou-se a interferência da criopreservação sobre a secreção de interferon-tau (IFN-t) por embriões bovinos produzidos in vitro. Usaram-se dois grupos de tratamentos: I) constituído por embriões não criopreservados (fresco) e II) embriões criopreservados. Os embriões, após atingirem a fase de blastocisto (fresco ou imediatamente após o descongelamento dos criopreservados), continuaram a ser cultivados individualmente por mais sete dias. Do meio de cultivo em que se mantiveram os blastocistos retiraram-se alíquotas com três e sete dias do início do cultivo, para a avaliação da secreção de IFN-t pelos embriões cultivados. Os embriões congelados secretaram menos IFN-t do que aqueles não criopreservados (P<0,05), e com sete dias houve maior secreção do interferon do que com três dias (P<0,05). A criopreservação prejudicou a produção de IFN-t pelo trofoblasto e pode comprometer o reconhecimento materno da gestação e o desenvolvimento do embrião pós-descongelamento.

The effect of cryopreservation in IFN-tau, from bovine embryos produced in vitro was evaluated. Two treated groups (G1= fresh bovine embryos, n=59 and G2= freezed embryos, n=84) were used to study the effect of cryopreservation on IFN-tau secretion. After reaching the blastocyst phase, the embryos were kept on individual culture for additional period of 7 days. On days 3 and 7 after the beginning of embryos cultivation, samples of the media culture were taken for IFN-tau secretion titration. Oocysts taken from follicles ranging from 3 to 5mm in diameter were obtained from ovaries of females at slaughterhouse. The embryos were frozen, after being dehydrated with ethylene glycol (1.8m), conditioned on 0.5ml palletes and frozen. Frozen embryos secreted lower IFN-tau than fresh embryos (P<0.05). At day 7 it was registered higher IFN-tau secretion from trophoblast than at day 3 (P<0.05). The increasing of IFN-tau secretion was observed when the blastocyst began to longed and it was directly related to the embryos development. The synthesis of IFN-tau is related to the capability of development of the blastocyst. Cryopreservation is a method that affects the maternal recognition of pregnancy and the post-freezing embryo development.

Secreção de interferon-tau em embriões bovinos produzidos in vitro frescos e congelados / Interferon tau secretion in cattle embryos in vitro fertilized before and after cryopreservation

Avaliou-se a interferência da criopreservação sobre a secreção de interferon-tau (IFN-t) por embriões bovinos produzidos in vitro. Usaram-se dois grupos de tratamentos: I) constituído por embriões não criopreservados (fresco) e II) embriões criopreservados. Os embriões, após atingirem a fase de blastocisto (fresco ou imediatamente após o descongelamento dos criopreservados), continuaram a ser cultivados individualmente por mais sete dias. Do meio de cultivo em que se mantiveram os blastocistos retiraram-se alíquotas com três e sete dias do início do cultivo, para a avaliação da secreção de IFN-t pelos embriões cultivados. Os embriões congelados secretaram menos IFN-t do que aqueles não criopreservados(P<0,05), e com sete dias houve maior secreção do interferon do que com três dias (P<0,05). A criopreservação prejudicou a produção de IFN-t pelo trofoblasto e pode comprometer o reconhecimento materno da gestação e o desenvolvimento do embrião pós-descongelamento.

Efeito de diferentes meios de cultivo no desenvolvimento e proporção do sexo de embriões bovinos produzidos in vitro / Effect of different culture media on development and sex ratio of bovine embryos fertilized in vitro

Avaliou-se o efeito da suplementação de meios de cultivo sobre o desenvolvimento e proporção do sexo de embriões bovinos fertilizados in vitro. Complexos cumulus-oócitos obtidos de ovários de matadouro foram maturados e fertilizados in vitro. Os zigotos (n= 484) foram distribuídos aleatoriamente em meio CR2aa, contendo soro fetal bovino (SFB) (T1), albumina sérica bovina (BSA) (T2) ou BSA mais insulina:transferrina:selênio e vitaminas (BSA+) (T3), no cultivo embrionário in vitro, a uma atmosfera de 5 por cento CO2 a 38,8ºC em ar. A taxa de clivagem foi observada 72-76 horas pós-fertilização (PF) e a taxa de blastocistos com sete e oito dias PF. Os blastocistos (n= 63) foram sexados pela técnica de reação em cadeia de polimerase. A taxa de clivagem em T2 foi maior (P<0,05) do que em T1 e T3. A taxa de blastocistos foi similar (P>0,05) entre T2 e T3, porém menor (P<0,01) do que em T1. A proporção do sexo dos embriões não diferiu (P>0,05) entre os tratamentos. O T1 influenciou o desenvolvimento de blastocistos, mas não teve efeito sobre a proporção do sexo.