Morphology, sex ratio and gene expression of Day 14 in vivo and in vitro bovine embryos

The present study was designed to compare Day 14 bovine embryos that were produced entirely in vitro using the post-hatching development (PHD) system with in vivo-derived embryos without or with transient PHD culture from Day 7 to Day 14. Embryos on Day 14 were used for sex determination and gene expression analysis of PLAC8, KRT8, CD9, SLC2A1, SLC2A3, PGK1, HSF1, MNSOD, HSP70 and IFNT using real-time quantitative (q) polymerase chain reaction (PCR). First, Day 7 in vivo-and in vitro-produced embryos were subjected to the PHD system. A higher rate of survival was observed for in vitro embryos on Day 14. Comparing Day 14 embryos produced completely in vivo or completely in vitro revealed that the mean size of the former group was greater than that of the latter (10.29±1.83 vs 2.68±0.33mm, respectively). Expression of the HSP70 and SLC2A1 genes was down-and upregulated, respectively, in the in vitro embryos. The present study shows that in vitro embryos cultured in the PHD system are smaller than in vivo embryos and that of the 10 genes analysed, only two were differentially expressed between the two groups. These findings indicate that, owing to the poor survival rate, the PHD system is not reliable for evaluation of in vitro embryo quality. © 2013 CSIRO.

Influência do método de separação dos espermatozóides viáveis (swim-up) na eficiência de seleção do sexo de bovinos por gradiente descontínuo de densidade e o impacto no melhoramento genético animal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxicity assessment of TiO2 nanoparticles in zebrafish embryos under different exposure conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Assessment of the reproductive parameters, laparoscopic oocyte recovery and the first embryos produced in vitro from endangered Caninde goats (Capra hircus)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Pregnancy Monitoring of In Vitro Produced Embryos in Buffaloes

In the present study, pregnancies obtained from 115 in vitro produced embryos were monitored by ultrasonography on days 30 and 60 after embryo transfer (ET), and at calving. Additionally, the health of newborns and recipients were also evaluated. On day 30 after ET, positive pregnancy was diagnosed in 50 animals (43.5%). A total of 8 fetal mortalities (16.0%) were verified from 30 days until calving, in which 2 occurred from 30 to 60 days after ET (4.0%), and 6 occurred from 60 days until calving (12.0%). In this last period of pregnancy, 3 pregnancy losses were due to abortion, and the other 3 were stillbirth. One additional animal was eliminated from the study, remaining 41 pregnancies. From these 41 pregnancies, a total of 20 female calves (48.8%) and 21 male calves (51.2%) were born. Pregnancies from female and male calves had a mean length of 309.8 and 310.9 days, respectively (range 300 to 328 days, and 297 to 320 days, respectively). Weight at calving was a mean of 31.4 and 33.8 kg for female and male calves, respectively. All calving occurred without intervention and dystocia was not observed in any case. No large offspring syndrome, hydramnios, hydroallantois, or umbilical cord anomalies were observed in calves. Delivery was normal in all recipients, and no puerperal infections, or retained placenta occurred. Suckling assistance was not necessary in any newborn. All genetic pedigree was confirmed later by DNA tests.

Lethal effect of high concentrations of Parecoxib and Flunixin meglumine on the in vitro culture of bovine embryos

Since cyclooxygenase (COX) inhibitors have been pointed out as potential treatments to increase pregnancy rates after embryo transfer, the present experiment aimed to evaluate the effects of flunixin meglumine (FM) and parecoxib (P), a COX-1 and 2 or COX-2 specific inhibitor, respectively, on the development of bovine embryos until the hatched blastocyst stage. In vitro produced bovine embryos were cultured in media with different concentrations of FM (0.14; 1.4; 14; 140 or 1400 mu g/ml) or P (0.09; 0.9; 9; 90 or 900 mu g/ml) and the production rates were evaluated. Concentrations of FM <= 14 mu g/ml and P <= 90 mu g/ml did not impair embryo development, although compiled data from non-lethal FM concentrations (<= 14 mu g/ml) indicated a toxic effect enough to decrease the hatching rate of blastocysts. Concentrations of FM at 140 and 1400 mu g/ml and P at 900 mu g/ml were lethal as no cleavage was detected on presumptive zygotes.

State of the art on animal embryonic chimeras

Embryonic chimerism is generally used in basic research and in vivo diagnosis of undifferentiated embryonic stem cells (ESC), mostly using mice embryos, although there have been reports in the literature on using rat, rabbit, sheep, chicken, primate, bovine, goat and pig embryos. Several techniques can currently be used to produce chimeric embryos, including microinjection, co-culture with ESC, fusion and aggregation. Although microinjection is the most commonly used method in mice, the mere aggregation of embryos with ESC may result in viable chimeras and be as efficient as microinjection. In mice, this chimerism technique has been shown to have the advantage of aggregating embryos in different stages of development with different ploidy, in addition to using ESC in the tetraploid complementation assay. Compared to other techniques for producing chimeras, the aggregation technique is a cheaper, faster and easier methodology to be performed. Moreover, aggregation can be simplified by chemically removing the zona pellucida with pronase or acidic Tyrode’s solution and be enhanced by using the Well of the Well culture system in combination with adhesion molecules, such as phytohemagglutinin. The most commonly used stages for chimerism by aggregation are those that precede the full compaction of the morula. In these stages, embryos have low-tension adherent junctions at the tangential point between two blastomeres. During the embryonic development of mice, the inner cell mass differentiates into epiblast and hypoblast. These layers will originate the fetal tissues and a portion of the extraembryonic tissues (yolk sac, allantois and amnion), whereas the trophectoderm (TE) gives rise to the chorion. A functional TE is essential for the complex molecular communications that occur between the embryo and the uterus. Embryos produced by somatic cell nuclear transfer, such as commercial cattle clones or endangered species, are subject to large fetal and neonatal losses. Hence embryo complementation with heterologous TE could be of assistance to decrease these losses and might as well assist development of high-value embryos in other approaches.

Imprinted gene expression in in vivo- And in vi/ro-produced bovine embryos and chorio-allantoic membranes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro production of bovine embryos using Sigma antioxidant supplement®, α-tocopherol and L-ascorbic acid

This study tested the effect of Sigma antioxidant supplement®, α-tocopherol (vitamin E) and L-ascorbic acid (vitamin C) in the culture medium of bovine embryos. In experiment 1, in vitro produced bovine zygotes were cultured in Human Tubal Fluid (HTF): Eagle’s Basic Medium (BME) with: Group 1 – 50 µm vitamin C; Group 2 – 200 µm vitamin E; Group 3 – 25 µm vitamin C and 100 µm vitamin E; Group 4 – 1 µl/ml Sigma antioxidant supplement®; and the Control group – HTF:BME only. In experiment 2, embryos were cultured in high or low oxygen tension with HTF:BME + Sigma antioxidant supplement® or in HTF:BME alone (Control). The data were analyzed using ANOVA followed by Tukey’s test. The results of experiment 1 showed a negative effect (P < 0.05) of vitamin E on blastocyst production in Group 2 (19.7 ± 0.1%). This effect was reduced in Group 3 by the addition of vitamin C (26.1 ± 0.2%). The use of vitamin C alone (34.9 ± 0.3%) or the Sigma antioxidant supplement® (33.3 ± 0.7%) did not increase (P > 0.05) the number of blastocysts produced compared with the control group (30.1 ± 0.5%). During experiment 2, there was no effect (P > 0.05) from the culture medium or the O2 concentrations used, indicating that the reduction of the O2 concentration did not improve blastocyst production.

AÇÃO REGULATÓRIA DO GnRH NO DESENVOLVIMENTO EMBRIONÁRIO PRECOCE E VITRIFICAÇÃO DE EMBRIÕES SUÍNOS PELO MÉTODO DE MICROGOTA (ROLE OF GnRH DURING EARLY EMBRYONIC DEVELOPMENT AND DEVELOPMENT OF SWINE EMBRYOS FOLLOWING VITRIFICATION BY MICRODROPLET METHOD)

The objective of this study was to investigate the role of GnRH on the preimplantation development of mouse embryos in vitro. GnRH-I, GnRH-II, and GnRH agonists: Des-Gly, Des-Trp and histrelin did not improve embryo development. However, treatment with the specific GnRH antagonist SB-75 blocked embryo development at morula stage. The inhibition of embryo development by SB-75 could be rescued by the addition of histrelin. To determine which intracellular signaling cascade is involved following binding of GnRH to the GnRHR, embryos were cultured in the presence of specific PKC (GFX) or PKA (SQ22536) inhibitors. The PKC inhibitor blocked embryo development at a similar stage as SB-75, whereas SQ22536 had an inhibitory effect, diminishing blastocyst formation and hatched rates. There are evidences that GnRH has an essential autocrine effect on mouse embryonic development via GnRHR, probably by activating PKC signaling cascade while the inhibition of the GnRH signaling does not activate apoptotic mechanisms involving caspase-3. In another experiment, development in vitro of embryos from Chinese Meishan (M) and occidental white crossbred (WC) females were investigated after improving the vitrification protocol for pig embryos. Efficient cryopreservation of zona pellucida-intact porcine embryos and studies of the difference among breeds could greatly impact the swine industry. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than WC (44%). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than frozen embryos from both breeds at expanded blastocyst stage, but not at hatched blastocyst stage. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98 and 95%, respectively). With a new procedure to warm vitrified pig embryos, the survival rates may be improved. The optimal stages to vitrify pig embryos using the microdroplet method ranges from late compact morula to early expanded blastocyst. The results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos. O objetivo do presente estudo foi investigar a importância do GnRH no desenvolvimento embrionário precoce em camundongos. GnRH-I, GnRH-II e os GnRH agonistas: Des-Gly, Des-Trp e histrelina não incrementaram o desenvolvimento embrionário. Entretanto, o tratamento com SB-75, um antagonista específico do GnRH, bloqueou o desenvolvimento embrionário no estádio de mórula. A inibição do desenvolvimento embrionário pelo SB-75 pôde ser revertida com a adição de histrelina. Para determinar a cascata do sinal intracelular desencadeada pela ligação do GnRH com o seu receptor, embriões foram cultivados na presença de inibidores específicos da PKC (GFX) e da PKA (SQ22536). O inibidor da PKC bloqueou o desenvolvimento embrionário em estádio similar ao bloqueio mediado pelo SB- 75, enquanto o SQ22536 teve efeito inibitório diminuindo a formação de blastocisto e taxas de eclosão. Os resultados sugerem que o GnRH tem um efeito autócrino essencial no desenvolvimento embrionário através do GnRHR, provavelmente, ativando a cascata da PKC. Por outro lado, a inibição do sinal do GnRH não ativa mecanismos apoptóticos que involvam caspase-3. Em outro experimento, foi investigado o desenvolvimento in vitro de embriões da raça Meishan (M) e branco cruzado (WC) após vitrificação pelo método microgota. O desenvolvimento de protocolos eficientes para criopreservação de embriões suínos com a zona pelúcida intacta e a avaliação das diferenças entre raças pode ter um significativo impacto na suinocultura. A percentagem de embriões que sobreviveram à criopreservação depois de 24 h foi maior na M (72%) do que na WC (44%). No entanto, o desenvolvimento in vitro dos embriões que sobreviveram à criopreservação não foi diferente entre M e WC nos estádios de blastocisto expandido (64%) ou eclodido (22%). Os índices de desenvolvimento foram significativamente mais altos para os embriões controle do que para os embriões vitrificados nas duas raças no estádio de blastocisto expandido, porém não foram diferentes para o estádio de blastocisto eclodido. A formação de blastocisto expandido não diferiu entre os embriões controle M e WC (98 e 95%, respectivamente). Com o novo procedimento (“hot warm”) para descongelar embriões vitrificados pelo método de microgota, pode-se aumentar dos índices de sobrevivência. Os melhores estádios embrionários para a vitrificação de embriões suínos variam de mórula compacta tardia até blastocisto expandido inicial. Os resultados sugerem que embriões M têm mais capacidade de sobreviver ao processo de vitrificação do que embriões WC.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2 degrees C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows (R). Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 +/- 5.94% and 9.43 +/- 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 +/- 3.37 and 8.67 +/- 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2 degrees C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring a-fetoprotein, alpha-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-alpha-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-alpha-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.