Survival Rate Of Embryos Research Articles

Early embryonic exposure of ionizing radiations disrupts zebrafish pigmentation.

Studies have demonstrated that zebrafish are powerful tools for monitoring environmental toxicity, including radiation hazard. Here we investigated the developmental toxicity of ionizing radiation (IR) in an in vivo embryonic zebrafish model. The effects of heavy ion (12 C6+ ), proton, and X-ray radiation on early zebrafish embryos were determined. A similar dose-dependent decrease in the hatch and survival rate of zebrafish embryos was observed after exposure to these irradiations. Exposure of zebrafish embryos to 1-4 Gy IR caused significant loss of pigmentation. Quantitative real-time reverse transcription polymerase chain reaction, western blot analysis, and in situ hybridization (ISH) experiment revealed that atp5α1 was markedly upregulated in irradiated zebrafish embryos. In addition, IR resulted in a rapid decrease in total adenosine triphosphate (ATP) generation. With dual functions of synthesizing or hydrolyzing ATP, ATP synthase regulated H+ transport crossing the mitochondrial inner. Administration of the mitochondrial ATP synthase inhibitor, oligomycin, partially restored pigmentation in irradiated zebrafish embryos, but the ATPase inhibitor, BTB06584, had no effect. Taken together, these results showed that IR exposure downregulated zebrafish pigmentation through regulation of H+ ion transport in mitochondria.

THE EFFECTS OF ILLUMINATION ON THE EARLY DEVELOPMENT OF AMPHIBIANS (AMPHIBIA: ANURA AND CAUDATA)

The effects of illumination on the early development of four amphibian species — Lissotriton vulgaris (Linnaeus, 1758), Triturus cristatus (Laurenti, 1768), Rana arvalis (Nilsson, 1842), and R. temporaria (Linnaeus, 1758) — have been studied. In general, the rate of their early development is rather independent of illumination. The eggs of tailless amphibians develop almost similarly under any illumination conditions, whereas the eggs of tailed amphibians better develop at an illumination of 700 lx. Illumination mainly influences the survival rate of embryos and prolarvae, which increases at low light intensities and decreases in the dark. Possible mechanisms and causes underlying the observed facts are discussed.

Minimum volume Spatula MVD vitrification method improves embryo survival compared to traditional slow freezing, both for in vivo and in vitro produced mice embryos

This study was conducted to compare the effect of minimum volume Spatula MVD vitrification (VIT) versus traditional slow freezing (SLF) of mouse embryos. A total of 2,617 8-cell in vivo derived and 2-cell in vitro produced B6D2 mouse embryos were subjected to freezing/thawing or vitrification/warming, while fresh embryos were used as control group. Embryo recovery, survival and development rate, pregnancy rate and offspring production were analyzed. In Experiment 1, 8-cell in vivo derived embryos were subjected to in vitro culture, resulting in greater survival and development rates at 3.5 days post coitum stage in VIT than in SLF group (P < 0.05). Although both methods reached an acceptable hatching rate (41.0% and 49.7% for VIT and SLF, respectively; P=NS), it was significantly lower respect to the control group (67.8%, P < 0.01). In Experiment 2, 2-cell in vitro produced mouse embryos showed a similar recovery rate from the device after freezing/thawing or vitrification/warming (∼84%), however survival rate was significantly higher for vitrified/warmed (94.7%) than frozen/thawed embryos (85.1%; P < 0.01). Vitrified/warmed and control fresh embryos were transferred to surrogate mothers, revealing no differences both in pregnancy and offspring production rates. Our data demonstrate that minimum volume Spatula MVD method is a simple home-made useful technique for vitrification of 2-cell and 8-cell mouse embryos produced either in vitro or in vivo.

ESTIMATION OF CADMIUM FROM DRINKING WATER SOURCES AND ITS EFFECT ON CHICK EMBRYO DEVELOPMENT

The aim of the study was to screen Cd contamination from the River Mutha and to investigate its effects on chick embryo development. The study was correlated with the toxicity of fetal development in humans, as chick embryo provides an excellent model system for studying the development of higher vertebrates including humans. The estimated average concentration of Cd in water column of River Mutha was 0.0986 mg/l, exceeded the safe limit of drinking water (0.0003 mg/l). To study the effects of Cd on chick embryo development, analogous concentration observed in River water was introduced in fertilized chicken eggs. Aqueous solutions of Cd ranging from 0.05 to 1.00 mg/l were injected into fertilized chicken eggs at 72 hours of incubation and metal injected eggs were further incubated for 10 days to see the toxic effects if any. Control eggs were treated in the similar way with sterile distilled water. Results showed abnormal development of blood vessels and decrease in the embryo survival rate in the Cd treated eggs.

A new, simple, automatic vitrification device: preliminary results withmurine and bovine oocytes and embryos.

This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos. Mice oocytes (n = 40) and embryos (8 cells, n = 35 and blastocysts, n = 165), bovine embryos (2PN, n = 35), and MII oocytes (n = 84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates. Ninety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos' survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts' rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts' rates. This novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.

Vitrification for bovine embryos with low-quality grade.

This study was conducted to examine the utility of vitrification for bovine embryos with low-quality grade, and simple cryoprotectants dilution method for practitioners. In Experiment 1, survival of frozen embryos was compared with that of vitrified embryos using minimum volume cooling (MVC). Then, vitrified embryos were used to confirm the optimum sucrose concentration in Experiment 2. The survival rates of embryos that had been vitrified following diluted cryoprotectants with the one-step in-straw method were compared with those of fresh control embryos in Experiment 3. Frozen-thawed or vitrified-warmed blastocysts were cultured with TCM-199 supplemented with 100μmol/L beta-mercaptoethanol +5% fetal bovine serum at 38.5°C in an atmosphere of 5% CO2 in air, their survival after 24hr were compared. The development to term of fair quality invivo embryos after vitrification was examined in Experiment 4. Results show that survival rates of frozen-thawed embryos were lower (p<.05) than that of vitrified-warmed ones. When vitrified embryos were warmed in 0.3mol/L sucrose in straws, their survival rate was 100%. The total cell numbers of vitrified-warmed embryos were comparable to those of fresh control embryos. The six calves from 13 vitrified embryos were delivered in Experiment 4. These results indicate that MVC vitrification following one-step cryoprotectants dilution is utilized to preserve low-quality bovine embryos.

Mapping of quantitative trait loci for microspore embryogenesis-related traits in the oilseed rape doubled haploid population DH4069 × Express 617

Oilseed rape (Brassica napus L.) is among dicotyledonous plants, a model species for microspore embryogenesis. Tremendous differences exist among oilseed rape genotypes in their embryogenic response and direct embryo to plant conversion. Despite some attempts to identify relevant genes, the genetic basis of these traits remains largely unknown. The objective of this work was to develop and to provide to the scientific community a doubled haploid (DH) population derived from a cross of the reported highly embryogenic genotype DH4079 and the low embryogenic inbred line Express 617. A population of 198 DH-lines was generated and genotyped with the Brassica 60 K Illumina Infinium™ SNP array. The parental and the F1 genotypes as well as between 81 and 107 DH-lines were characterized for their number of microspores, number of microspore-derived embryos, embryo survival rate, direct embryo to shoot conversion, and related traits. The results obtained for the F1 genotype were mostly in between the two parents. SNP markers in the DH population showed to 49% distorted segregation and of those 63% were in favor of DH4079. Significant genotypic differences were found for all traits and heritabilities ranged from 66 to 88%. Together, 13 quantitative trait loci (QTL) for the different traits were identified on linkage groups A01, A02, A05, A10, C04, and C06, and candidate genes were identified within their QTL confidence intervals.

Mouse Embryo Cryopreservation by Slow Freezing.

This embryo cryopreservation protocol is used by The Jackson Laboratory and several other repositories around the world. This protocol has clearly withstood the test of time; >2 million embryos from thousands of strains have been cryopreserved using this method. It requires a controlled-rate freezer, but is simple, effective, and can be applied to cryopreserve embryos from the two-cell to the blastocyst stage. Thawing requires neither special media nor equipment because the insemination straw contains all of the needed elements, making this very useful for distributing cryopreserved embryos. Embryo survival rates of greater than 90% can be expected when this method is used, making it reliable and enabling staff with prior embryo-handling experience to become proficient quickly.

Egg oxidation status, antioxidant enzyme activities, lipid classes, fatty acid composition profile and embryo survival rates during in vitro oocyte ageing in tenchTinca tinca(Linnaeus, 1758)

The effect of in vitro storage of oocytes on embryos survival rate, egg oxidation status, antioxidant enzyme activities, lipid classes and fatty acid composition profile was investigated in common tench Tinca tinca. In order to identify the role of oxidative stress in the progress of oocyte ageing the levels of thiobarbituric acid reactive substances (TBARS) and carbonyls as indicators of lipid and protein oxidation were measured and the activity of antioxidant enzymes were examined. Stripped ova from six females were stored in cell culture plates at 20°C for up to 10 hr post-stripping (HPS). The stored ova were fertilized and the embryo survival rates were assessed. The results indicated that tench eggs could be successfully stored in vitro for 4 hr after stripping at 20°C. Superoxide dismutase enzyme activity increased at 6 HPS, whereas glutathione peroxidase and glutathione reductase activities decreased in oocytes during in vitro storage (p < .05) at 4 and 8 HPSs, respectively. The level of malondialdehyde did not show any significant changes during the progress of oocyte ageing. Carbonyls increased up to 2 HPS and thereafter decreased significantly. However, ova ageing did not affect the main lipid class composition and the fatty acid composition of the eggs. Lower quality eggs exhibited lower levels of cholesterol but higher levels of triacylglycerol.

Detection of Genes Associated with Follicle Development Through Transcriptome Analysis of Bovine Ovarian Follicles GCs

Background: The ovarian follicle is an essential component of the reproductive process. It plays an important role in controlling the estrous cycle, determining estrous behaviour, ensuring oocyte competency and subsequent embryo survival rate, and determining both post ovulation corpus luteum function and progesterone synthesis. Traditionally, gene expression studies in the field of follicular development focus on the study of expression of candidate genes of interest. With the development of next-generation sequencing technologies, transcriptome profiling has become a powerful approach for identification of genes globally expressed in various tissues including ovarian follicles. Objective: To investigate potential differentially expressed genes associated with follicular development and gene expression profiles of bovine ovarian follicles at onset of deviation stages of a follicular wave. Method: Ovaries were removed, greatest (ODF1) and second greatest (ODF2) diameter follicles were isolated, and GCs were isolated from the two types of follicles (n=4). RNA samples of follicles were pooled within ODF1 and ODF2. The two cDNA libraries were sequenced, and mapped to the bovine RefSeq database. Gene ontology (GO) functional enrichment analysis was subjected and differentially expressed genes were analysed using visualization plug BiNGO of Cytoscape 3.0.0 software. KEGG pathways were performed and all the pathways and protein equivalent convert information of UniProt database of cattle. Twelve genes were selected randomly to verify their expression by QRT-PCR technology. Results: A total of 43,708,132 and 43,826,914 clean reads were obtained for ODF1 and ODF2, respectively. After being mapped to the bovine RefSeq database, a total of 15,519 genes were expressed (cut-off RPKM>0.5), of which 761 were highly expressed (cut-off RPKM>100) in both types of follicles. GO functional classification of these highly expressed genes revealed that many of them are involved in metabolic processes, multicellular organismal processes, and binding. Furthermore, 831 genes were identified as differentially expressed in ODF1 versus ODF2, in which 384 genes were up-regulated, and 447 genes were down-regulated. KEGG pathway analysis revealed eight differentially expressed genes in ODF1 vs. ODF2, QRT-PCR results showed that 9 of the 12 selected genes mRNA amounts were significantly greater in ODF1 than that ODF2 (P<0.05 or 0.01), which was consistent with the deep sequencing data. Conclusion: This study represents the first transcriptome investigation of bovine follicles at onset of deviation of the largest vs. second largest follicles. It provides a foundation for future investigation of the regulatory mechanisms involved in follicular development in cattle. Keywords: Follicular development, transcriptome, gene expression, GO analysis, onset of deviation.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos

BackgroundUnderstanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this “mid-embryonic lethality hypothesis” by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations.ResultsBy subjecting zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and chicken (Gallus gallus) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species.ConclusionIn opposition to the “mid-embryonic lethality hypothesis,” our results consistently showed that the stage with the highest lethality was not around the conserved pharyngula period, but rather around the early period in all the vertebrate species tested. These results suggest that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as the diversifying effect of positive selections against earlier and later stages, and developmental constraints which lead to conservation of mid-embryonic stages.

Efficiency of assisted hatching of the cryopreserved–melted embryos

Purpose . To measure outcomes of clinical research of efficiency of assisted hatching of cryopreserved embryos. Materials and methods. Patients who had un successful cycles IVF/ICSI with transfer of fresh embryos have been selected for participation in the research between 2014 and 2016 years. Patients were distributed in a random way for participation in the experiment and control groups. Results of embryos transfer of one or two cryopreserved and melted embryos were considered only. Embryos were cryopreserved at a stage of blastocyst, 5 days after extraction of oocytes by method of vitrification. Melting procedure was conducted in the morning of a day of embryos transfer following the instructions of the vitrification medium producer Cryotech (Japan). Assisted hatching was conducted with use of micropipettes of Holding Pipette Cook Medical (Australia) and Assisted Hatching/Zona Drilling Pipette Cook Medical (Australia). The treated embryos were cultivated up to a repeated estimation of morphology of embryos before transfer. Transfer of embryos has been conducted by a standard method with the use of catheter for non-invasive transfer of embryo Sydney IVF Cook Medical (Australia). The quantity of the transferred embryos varied from one to two. Results . 100 cryopreserved embryos were transferred which have been distributed in a random way either to the group with the assisted hatching or to the control group (without assisted hatching). A number of parameters of patients from both groups was analyzed, i.e. age of the patient at the time of melting of embryos, duration of infertility, causes of infertility, quantity of previous unsuccessful cycles IVF/ICSI. Any essential differences between patients within two groups based on the aforementioned parameters were not revealed. Also, there were no essential differences in number of the melted embryos, survival rate of embryos, quantity of the embryos transferred to patients. However, at the same time, parameters of positive results of tests on HCG (human chorionic gonadotropin) (42 % against 27 %) and quantity of clinical pregnancy (35 % against 24 %) were statistically higher in the group with assisted hatching comparatively to the control group (P < 0.004; P < 0.001 accordingly). Conclusions . Implantation of the transfered embryos and number of clinical pregnancies were statistically improved due to selection of the optimum freezing conditions and subsequent cultivation and conducting of procedure of mechanical incision of ZP.

Establishment of a technological system via zygote injection of CRISPR/Cas9 system for the generation of gene modified ziwuling black goats

Zygote injection of CRISPR/Cas9 system is an efficient method for generation of gene modified animals. Efficiency of zygote collection, embryo survival rate after micro-injection and pregnancy rate of surrogate are the key influence factors for the generation of gene modified animals using this strategy. Here, we investigated the influence of CIDR treatment at different time during the estrus cycle to the superovulation, and analyzed the influence of the estrus terminal time between embryo donor and embryo receptor to the pregnancy rate in Ziwuling black goats. We designed three sgRNAs targeting myostatin ( MSTN ) and also optimized the micro-injected parameter of the CRISPR/Cas9 system. The results showed that embryo donor goats effectively increase the corpus luteum amount (15.25±3.69) and zygote amount (13.875±4.015) when they were treated with CIDR at the fourth day of estrus cycle. The mixture including Cas9 mRNA (60 ng/μL) and sgRNAs (60 ng/μL) targeting MSTN was injected into zygote cytoplasm using a micromanipulator at the optimized injection parameters (Pi, Ti and Pc was 300, 0.5 and 60, respectively). Embryo cleavage rate was 76.79 % after 24 h of embryo injection. Pregnancy rate of embryo receptors whose terminal estrus time was delayed 12–16 h compared with that of embryo donor was the highest (38.46%). A total of 19 living lambs were obtained, and indels were detected in all the lambs using TA cloning sequencing. Overall, we established the technological system based on the zygote injection of CRISPR/Cas9 in Ziwuling black goats. The established technological system is the basic for generation of gene modified goats.

Pre-clinical validation of a closed surface system (Cryotop SC) for the vitrification of oocytes and embryos in the mouse model

Vitrification is currently a well-established technique for the cryopreservation of oocytes and embryos. It can be achieved either by direct (open systems) or indirect (closed systems) contact with liquid nitrogen. While there is not a direct evidence of disease transmission by transferred cryopreserved embryos, it was experimentally demonstrated that cross-contamination between liquid nitrogen and embryos may occur, and thus, the use of closed devices has been recommended to avoid the risk of contamination. Unfortunately, closed systems may result in lower cooling rates compared to open systems, due to the thermal insulation of the samples, which may cause ice crystal formation resulting in impaired results. In our study, we aimed to validate a newly developed vitrification device (Cryotop SC) that has been specifically designed for being used as a closed system. The cooling and warming rates calculated for the closed system were 5.254 °C/min and 43.522 °C/min, respectively. Results obtained with the closed system were equivalent to those with the classic Cryotop (open system), with survival rates in oocytes close to 100%. Similarly, the potential of the survived oocytes to develop up to good quality blastocysts after parthenogenetic activation between both groups was statistically equivalent. Assessment of the meiotic spindle and chromosome distribution by fluorescence microscopy in vitrified oocytes showed alike morphologies between the open and closed system. No differences were found either between the both systems in terms of survival rates of one-cell stage embryos or blastocysts, as well as, in the potential of the vitrified/warmed blastocysts to develop to full-term after transferred to surrogate females.

Single-injection ex ovo transplantation method for broad spinal cord engraftment of human pluripotent stem cell-derived motor neurons

BackgroundTransplantation of human pluripotent stem cell (hPSC)-derived neurons into chick embryos is an established preliminary assay to evaluate engraftment potential. Yet, with recent advances in deriving diverse human neuronal subtypes, optimizing and standardizing such transplantation methodology for specific subtypes at their correlated anatomical sites is still required. New methodWe determined the optimal stage of hPSC-derived motor neuron (hMN) differentiation for ex ovo transplantation, and developed a single injection protocol that implants hMNs throughout the spinal cord enabling broad regional engraftment possibilities. ResultsA single injection into the neural tube lumen yielded a 100% chick embryo survival and successful transplantation rate with MN engraftment observed from the rostral cervical through caudal lumbar spinal cord. Transplantation of HB9+/ChAT− hMN precursors yielded the greatest amount of engraftment compared to Pax6+/Nkx6.1+/Olig2+ progenitors or mature HB9+/ChAT+ hMNs. Comparison with existing method(s)Our single injection hMN transplant method is the first to standardize the optimal hMN phenotype for chick embryo transplantation, provide a rubric for engraftment quantification, and enable broad engraftment throughout the spinal cord with a single surgical intervention. ConclusionTransplantation of HB9+/ChAT− hMN precursors into chick embryos of Hamburger Hamilton (HH) stages 15–18 using a single luminal injection confers a high probability of embryo survival and cell engraftment in diverse regions throughout the spinal cord.

Post-cooling survival, growth and deformity rates in zebrafish embryos (Danio rerio).

This study investigated and analysed survival, growth and macro- and microscopic damage during the development of zebrafish embryos up to the adult stage after undergoing cooling. The embryos at 50% epiboly stage were selected, submerged in cryoprotectant solution of methanol and sucrose, cooled gradually to 0 ± 2°C temperature, and divided into two groups with different storage times (6 and 18 h). Subsequently, the embryos were reheated, rehydrated and incubated normally. The experiment lasted 5 months and, from hatching onward, the larvae were examined, collected and processed at pre-established time intervals. The hatching rate was significantly higher for the larvae stored for 18 h compared with the 6-h group. However, embryos from this group gave rise to a larger number of malformations, and these were much more severe compared with those in the 6 h group, which led to a higher mortality in the long term. Regarding larval length, the animals of the 6 h group had higher mean total length compared with the 18 h group, but both treatments were inferior to the control. Numerous macro- and microscopic malformations were observed and, in both treatments, only the morphologically normal individuals were able to develop to the adult stage, with organ development similar to the control, except for the gonads that were still undifferentiated in treated animals.

REPRODUCTIVE PERFORMANCE OF DOMESTICATED BROODSTOCK OF SILVER PERCH, Bidyanus bidyanus (MITCHELL 1838) AND THE RELATIONSHIP BETWEEN OIL GLOBULE FRAGMENTATION AND EGG QUALITY

The experiments investigated the reproductive performance of the domesticated broodstock of the silver perch and the relationship between various degrees of oil globule fragmentation and egg quality. Six years old of second generation broodstock (n=3) were evaluated based on the fecundity, fertilisation rate, hatching rate, the degree of oil fragmentation of egg, and the quality of embryos and larvae produced. The fragmentation were grouped into three categories: un-fragmented (cat-1), moderately fragmented (cat-2), and highly fragmented (cat-3). The results showed that the broodstock had a relatively high fecundity (132,400 ± 7,22), fertilization rate (94.27 ± 1.28%), and hatching rates (87.94 ± 1.23%). The survival rate of larvae at 12 days post hatching (dph) in cat-1 (71.3 ± 0.9%) was higher than cat-2 (66.7 ± 0.9%) whereas cat-2 was higher than cat-3 (61.3 ± 0.3%). The eggs was dominated by cat-1 (78.11 ± 2.44%) which was significantly higher than cat-2 (21.26 ± 2.45%) and cat-3 ones (0.40 ± 0.21%). The survival rate of embryo at 20 hours post spawning (hps) and hatching rate of cat-1 (95.33 ± 0.00% and 93.33 ± 0.00%) and cat-2 (90.00 ± 0.00% and 85.00 ± 0.00%) were significantly higher than cat-3 (72.33 ± 1.76% and 60.33 ± 0.00%). The total length (TL) of the larvae of cat-1 and cat-2 (8.44 ± 0.21 mm and 8.35 ± 0.23 mm respectively) were significantly higher than larvae of cat-3 (7.09 ± 0.14 mm). No significant difference was found in the larval deformities among any categories. In conclusion, the reproductive performance of six year-old broodstock silver perch showing acceptable performance and egg categorisation based on oil globule fragmentation can be used as a useful tool to indicate eggs quality of silver perch.

47 A New Device and Method for Successful Vitrification of In Vitro-Produced Ovine Embryos

To advance the use of embryo vitrification technology in veterinary practice, we developed a system in which embryo vitrification, warming, and dilution can be performed within a straw. An in-straw embryo cryopreservation method reduces the need for equipment and technical skills and can facilitate direct embryo transfer to the uterus. This study proposes the use of a new device named “Sarah” that is designed to permit all in-straw embryo cryopreservation procedures. Ovine in vitro-produced (IVP) embryos were vitrified at either early blastocyst stage (EB, n = 65, 6 days post-IVF) or fully expanded blastocyst stage (FB, n = 168, 7 days post-IVF). The vitrification procedure using Sarah constituted a 0.25-mL straw with a capsule having 50-µm pores inserted at one end. Embryos at each stage (EB and FB) were divided into 2 subgroups and vitrified by 1 of 2 methods: (1) multi-step (MS) group-a straw containing 2 embryos was sequentially loaded vertically into 1.5-mL tubes containing 6 different vitrification solutions: 10, 20, 40, 60, 80, or 100% ES (with 100% ES being 7.5% DMSO +7.5% EG + 20% FCS in TCM-199; 90 s each step) followed by 30 s each in 75 and 100% VS (100% VS being 18% DMSO +18% EG + 0.5 M trehalose + BSA in TCM-199); and (2) two-step (TS) group-the straw (2 embryos/straw) was loaded with 100% of ES (5 min), followed by 100% VS solution for 30 s. For both methods, at the end of the preparation steps, the straws were plunged directly into liquid N2. Non-vitrified embryos were maintained in in vitro culture as a control group (n = 102). The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, 25% WS (WS = 1 M sucrose in TCM-199+ 20% FCS) at 38.6°C (for first solution) and at room temperature for all the rest (5 min each), before being placed into the holding medium. Embryos were recovered from the straws, incubated at 38.6 C in 5% CO2 in air in TCM 199 + 5% FCS, and evaluated for blastocoel re-expansion, embryo survival, and hatching rate at 2, 14, 48 h post-warming. Blastocyst re-expansion (2 h) after warming increased as the developmental stage progressed and was not affected by the vitrification method. In fact, it was significantly (P &lt; 0.05) higher for FB vitrified in the MS and TS methods (77.90% and 71.25%, respectively) compared with the EB method (62.5% and 48.50%, respectively). At 24 h, survival rate of vitrified FB was significantly higher (P &lt; 0.05) in the MS system (95.35%) compared with those in TS (86.25%). Survival rates of FB embryos for both methods (MS and TS) were significantly higher (P &lt; 0.001) than EB embryos vitrified in MS (56.25%) and TS (56.55) methods. After 48 h of culture, the hatching rate for FB vitrified in the MS system (87.21%) was comparable with TS (77.5%) and control (85.3%) groups but significantly higher (P &lt; 0.001) than vitrified EB in MS (43.75%) and TS (36.36%). In conclusion, we showed that a high survival rate of IVP embryos can be achieved by this new in-straw vitrification and warming device (“Sarah”), with hatching rates in vitro comparable with that of control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

208 Cryotolerance of In Vitro-Produced Cattle Embryos Produced with Different Maturation and Culture Media

In vitro-produced (IVP) bovine embryos suffer from damage due to suboptimal culture conditions involving altered metabolism, reactive oxygen species generation and high lipid accumulation, which could be impacted by oocyte maturation × embryo culture interaction. We hypothesised that optimizing oocyte and embryo culture conditions would lead to a higher post-thaw embryo survival rate. The experiment was replicated 9 times in a factorial design with 3 oocyte maturation media: TCM-199 plus 10% serum and gonadotropins (control), BO-HEPES-IVM (BO, IVF Bioscience UK), and HMM (HEPES-buffered maturation medium) and 3 embryo culture media: BO-IVC (BOC), BBH7 (Cooley Biotech, Gainesville, FL, USA), and SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod Fertil Dev. 29, 129-130). Bovine oocytes (n = 2907) were aspirated from 2- to 8-mm follicles of abattoir ovaries, matured at 38.5°C for 22 to 24 h in 5% CO2 (control) or in BO and HMM in air, fertilized with semen from 1 of 3 bulls, and cultured in BOC, BBH7, and SCF1 at 38.5°C in 5.5% CO2, 5% O2, and 90% N2. Cleavage and blastocyst rates were evaluated at Day 3 and 7, respectively, after IVF. Selected stage 7 blastocysts were slow frozen using 1.5 M ethylene glycol supplemented with 1 mm l-ascorbic acid for 20 min (equilibration). The embryos were cooled to and seeded at –6°C, and then cooled to −32°C at 0.5°C/min. Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data (Table 1) was arcsin transformed and analysed by ANOVA, and means separated by l.s.d. Results indicate that oocytes matured in BO and embryos cultured in SCF1 had a higher blastocyst rate than all oocytes matured in control medium and all embryos cultured in BBH7 (P &lt; 0.05). However, oocytes matured in control medium and embryos cultured in BOC had lower post-thaw re-expansion rates than other treatment groups (P &lt; 0.05). These results suggest that a combination of oocyte maturation and embryo culture media plays an important role in post-thaw embryo survival, al though interactions of present treatments need to be further evaluated. Table 1.Effect of maturation and culture media on blastocyst rate and cryotolerance ± SEM)

Cathepsin B & D and the Survival of Early Embryos in Red Spotted Grouper, Ephinephelus akaara.

ABSTRACTSurvival of embryos largely depends on yolk processing during early development. Proteolytic enzymes, cathepsin B & D (ctsb & ctsd) are known to have some important roles in yolk processing of various fish species. Mature female red spotted groupers were injected with human chorionic gonadotropin (HCG) to induce ovulation. The fertilized eggs and embryos were sampled at 0, 4 and 24 HPF (hours post fertilization). Survivals of each groups of embryos were checked at 24 and 48 HPH (hours post hatching). Transcripts of ctsb & ctsd showed the highest level at 0 HPF and relatively high at 4 HPF, but greatly decreased at 24 HPF. In bad egg quality group (BE, embryos survived until 24 HPH), transcript level of ctsb at 4 HPF were significantly lower than the transcript level at the same stage in good egg quality group (GE, embryos survived until 48 HPH) while no significant change of ctsb transcript level was observed at 0 or 24 HPF between BE and GE. Transcript level of ctsd was decreased at 24 HPF, but the difference was not as strong as the case of ctsb transcript. These results suggest that maternal ctsb transcript rather than ctsd transcript is likely to be involved in egg quality resulting in the difference of survival rate of embryos at early developmental period in this species.