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Isermann, B., S. B. Hendrickson, K. Hutley, M. Wing, and H. Weiler. "Tissue-restricted expression of thrombomodulin in the placenta rescues thrombomodulin-deficient mice from early lethality and reveals a secondary developmental block." Development 128, no. 6 (March 15, 2001): 827–38. http://dx.doi.org/10.1242/dev.128.6.827.
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The endothelial cell surface receptor thrombomodulin (TM) inhibits blood coagulation by forming a complex with thrombin, which then converts protein C into the natural anticoagulant, activated protein C. In mice, a loss of TM function causes embryonic lethality at day 8.5 p.c. (post coitum) before establishment of a functional cardiovascular system. At this developmental stage, TM is expressed in the developing vasculature of the embryo proper, as well as in non-endothelial cells of the early placenta, giant trophoblast and parietal endoderm. Here, we show that reconstitution of TM expression in extraembryonic tissue by aggregation of tetraploid wild-type embryos with TM-null embryonic stem cells rescues TM-null embryos from early lethality. TM-null tetraploid embryos develop normally during midgestation, but encounter a secondary developmental block between days 12.5 and 16.5 p.c. Embryos lacking TM develop lethal consumptive coagulopathy during this period, and no live embryos are retrieved at term. Morphogenesis of embryonic blood vessels and other organs appears normal before E15. These findings demonstrate a dual role of TM in development, and that a loss of TM function disrupts mouse embryogenesis at two different stages. These two functions of TM are exerted in two distinct tissues: expression of TM in non-endothelial extraembryonic tissues is required for proper function of the early placenta, while the absence of TM from embryonic blood vessel endothelium causes lethal consumptive coagulopathy.
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Abecia, J. A., F. Forcada, I. Palacín, L. Sánchez-Prieto, C. Sosa, A. Fernández-Foren, and A. Meikle. "Undernutrition affects embryo quality of superovulated ewes." Zygote 23, no. 1 (October 9, 2013): 116–24. http://dx.doi.org/10.1017/s096719941300035x.
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SummaryTo determine the effect of undernutrition on embryo production and quality in superovulated sheep, 45 ewes were allocated into two groups to be fed diets that provided 1.5 (control, C; n = 20) or 0.5 (low nutrition, L; n = 25) times daily requirements for maintenance, from oestrous synchronization with intravaginal sponges to embryo collection. Embryos were collected 7 days after the onset of oestrus (day 0). Low nutrition resulted in lower live weight and body condition at embryo collection (P < 0.05). Diet (P < 0.01) and day of sampling (P < 0.001) significantly affected plasma non-esterified fatty acid (NEFA) and insulin concentrations. Plasma leptin concentrations decreased on day 7 only in L ewes. A significant effect of dietary treatment (P < 0.05) and day (P < 0.0001) was observed on plasma insulin-like growth factor (IGF)-I concentrations. The number of recovered oocytes and embryos did not differ between the groups (L: 15.4 ± 0.4; C: 12.4 ± 0.4). Recovery rate was lower (P < 0.05) in the L (60%) than in the C group (73%). The total number of embryos and number of viable-transferable embryos (5.0 ± 0.3 and 3.4 ± 0.3 embryos, respectively) of the L group were lower (P < 0.1) when compared with controls (8.4 ± 0.4 and 6.2 ± 0.4 embryos, respectively). Undernutrition during the period of superovulation and early embryonic development reduced total and viable number of embryos. These effects might be mediated by disruption of endocrine homeostasis, oviduct environment and/or oocyte quality.
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Tuska, Habib Syaiful Arif, Nursalsabila Khamalt, Muhammad Arfan Lesmana, Reza Yesica, Viski Fitri Hendrawan, Budiono, and Gretania Residiwati. "Effect of Artemisia vulgaris Supplementation on Zebrafish Embryo Under Heat Stress Condition during In Vitro Culture." Journal of Applied Veterinary Science And Technology 5, no. 1 (April 30, 2024): 20–25. http://dx.doi.org/10.20473/javest.v5.i1.2024.20-25.
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Background: Artemisia vulgaris contains flavonoids, which play a vital role in counteracting free radicals. Purpose: To determine the effect of Artemisia vulgaris extract supplementation on embryo development, heart rate and survival of zebrafish under heat stressed and non-heat stressed conditions. Methods: The research used a completely randomized design. Zebrafish embryos (n=240) were divided into heat stressed (36°C) and non-heat stressed (28°C) groups, while for each group were divided into three subgroups, namely T1/control (without Artemisia vulgaris supplementation); and supplemented group T1 and T2, with 2 µL and 4 µL of Artemisia vulgaris supplementation, respectively. The efficacy of Artemisia vulgaris supplementation was determined by observing the embryo development, heart rate, and survival rate of zebrafish up to 96 hours post fertilization (hpf). Results: The development of zebrafish embryos under heat stressed treated with Artemisia vulgaris extract gave the same quality as the control treatment without heat stressed exposure. Zebrafish embryos exposed to heat stressed with 4 µL Artemisia vulgaris supplementation gave the highest survival rate on the heat stressed group. Artemisia vulgaris supplementation improved the heart rate of zebrafish exposed to heat stressed as in the non-heat stressed group. Conclusion: Artemisia vulgaris extract can reduce the detrimental effects of heat stressed induction on zebrafish embryos, as evidenced by the improvement in embryonic development, heart rate, and survival rate of zebrafish embryos after supplementation.
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Fieni, F., M. Oseikria, K. Laroucau, F. Vorimore, D. Tainturier, S. Destrumelle, and J. L. Pellerin. "111 RISK OF CHLAMYDIA ABORTUS TRANSMISSION VIA EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS." Reproduction, Fertility and Development 28, no. 2 (2016): 186. http://dx.doi.org/10.1071/rdv28n2ab111.
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Chlamydia abortus (C. abortus) in cattle has been reported sporadically throughout the world and is implicated in respiratory, ocular, and reproductive disease as abortion, infertility, chronic mastitis, vaginal discharge, and endometritis. In addition, C. abortus presents a zoonotic risk exposure of pregnant women to infected animal and can lead to severe septicaemia in the mother, resulting in spontaneous abortion or stillbirth of the fetus. To investigate the risk of C. abortus transmission via bovine embryo transfer, our study aims to determine whether the embryonic ZP of in vitro-produced embryos protects early embryo cells against C. abortus infection and whether the bacteria adhere to or infect the cells of early bovine embryos (ZP-free) after in vitro infection. We also evaluated the efficacy of the washing procedure recommended by the IETS to decontaminate bovine embryos exposed to C. abortus in vitro. Ninety (8 to 16 cells) bovine embryos, produced in vitro, were randomly divided into 10 batches. Eight batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4.8 × 107 Chlamydia/mL of AB7 strain (ANSES, Maisons-Alfort, France). After incubation for 18 h at 37°C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a PBS and 5% FCS solution without trypsin nor antibiotics in accordance with IETS guidelines. In parallel, 2 batches of 5 embryos (1 ZP-intact and 1 ZP-free) were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13 000 × g. The embryos and wash pellets were tested using RT-PCR. Chlamydia abortus DNA was found in all ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the tenth wash fluid for 1 batch (1/4) of ZP-intact infected embryos and in 3 batches (3/4) of ZP-free infected embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. These results demonstrate that C. abortus adheres to or penetrates the ZP as well as the early embryonic cells of in vitro-produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients or their offspring. Nevertheless, the finding of C. abortus DNA by RT-PCR did not imply that the bacteria found is still infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. abortus would eliminate the bacteria from the ZP.
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Pellerin, J. L., A. Ashraf, M. Oseikria, K. Laroucau, F. Vorimore, C. Roux, M. Larrat, S. Michaud, and F. Fieni. "162 CAN CHLAMYDIA ABORTUS BE TRANSMITTED BY EMBRYO TRANSFER IN GOATS?" Reproduction, Fertility and Development 27, no. 1 (2015): 172. http://dx.doi.org/10.1071/rdv27n1ab162.
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Chlamydia abortus is a gram-negative obligate intracellular bacterium. Its lifecycle includes a resistant infectious form and a metabolically active non-infectious form. Chlamydia abortus infection results in abortion in goats; in nonpregnant animals the infection is usually subclinical. Chlamydia abortus presents a major zoonotic risk for pregnant women. The aim of this study was to investigate whether the embryonic zona pellucida (ZP) protects early embryo cells from infection and to test the efficacy of the washing protocol recommended by the IETS for bovine embryos. The study was performed in triple replicate: 14 donor goats, certified negative by ELISA and PCR to C. abortus, were synchronized, superovulated, and subsequently inseminated by males controlled negative for C. abortus. Fifty-two ZP-intact embryos (8–16 cells) were collected 4 days later, by laparotomy. The embryos were randomly divided into 12 batches. Nine batches of 5 embryos were incubated in a medium containing 4 × 107 Chlamydia mL–1, AB7 strain. After incubation for 18 h at 37°C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of PBS and 5% FCS solution in accordance with IETS guidelines for bovine embryos. In parallel, 3 batches of ZP-intact embryos (2, 2, and 3 embryos in the first, second, and third batches, respectively) were used as controls by being subjected to similar procedures, but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 h at 13 000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at –20°C before examination for evidence of C. abortus using RT-PCR. Chlamydia abortus DNA was found in all batches of infected ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the tenth wash fluid for 4 batches (4/9) of infected embryos. As expected, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goat to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of infected caprine embryos can eliminate C. abortus from the ZP.
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Gentles, R., and C. O'Neill. "340. ANALYSIS OF PROTEINS SECRETED BY THE PREIMPLANTATION MOUSE EMBRYO." Reproduction, Fertility and Development 22, no. 9 (2010): 140. http://dx.doi.org/10.1071/srb10abs340.
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Many embryos generated by assisted reproductive technologies do not have the capacity for full development. A non-destructive test that allows the most viable embryos to be identified would allow development of a range of strategies to improve these technologies. Analysis of the proteinaceous secretome of the preimplantation mouse embryo by electrospray mass spectrometry of tryptic digests of embryo-conditioned culture media identified 20 peptides.1 Lactate dehydrogenase beta (LDH) and protein disulfide isomerase (PDI) were consistently among the most abundant peptides identified. PDI has been found to be secreted and surface expressed in a number of other cell types suggesting a biologically significant role. LDH-beta is a marker of plasma membrane integrity and is widely used in medicine for assessment of cardiac health. The aim of this study was to assess the expression of these proteins in the early embryo to gain some understanding of the potential significance of their presence within the embryonic secretome. Zygotes (B6CBF2) were collected 20–22 h post hCG. They were cultured in groups of 10 embryos for 96 h in 10 mL of modHTF 30 µg BSA/mL media. Embryonic protein was extracted for western blot analysis or embryos subjected were to immunolocalization studies for PDI and LDH. Western blot analysis revealed presence of proteins of expected molecular mass. Staining for both proteins occurred at high levels at each stage of development throughout pre-implantation development. Staining was predominantly cytoplasmic and excluded from nuclei. Cultured blastocysts had less LDH than fresh but the level for PDI was similar for both. Staining of non-permeabilized embryos revealed extracellular staining of PDI. The results show high levels of cellular expression of two proteins reported to be released by the embryo in vitro. (1) Beardsley A, Li Y, O’Neill C (2010). Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro. Reproductive Biology and Endocrinology 8, 71.
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Alsaleh, A., J. L. Pellerin, C. Roux, M. Larrat, G. Chatagnon, and F. Fieni. "166 CAN COXIELLA BURNETII BE TRANSMITTED BY GOAT EMBRYO TRANSFER?" Reproduction, Fertility and Development 25, no. 1 (2013): 231. http://dx.doi.org/10.1071/rdv25n1ab166.
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Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Detection of significant bacterial loads in flushing media and tissue samples (oviducts and uterine horns) from the genital tracts of nonpregnant goats is a risk factor for in utero infection and transmission during embryo transfer (Alsaleh et al. 2011 CIMID 34, 355–360). The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the washing protocol recommend by the IETS for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and PCR, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9–9, 11–11, and 4–4 in replicates 1, 2, and 3, respectively) were placed in 1 mL of MEM containing 107 C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37°C and 5% CO2, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3–3, 5–5, and 2–2 in replicates 1, 2, and 3, respectively) were submitted to the same procedures but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 h at 13 000g. The washed embryos and pellets were tested by PCR. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first 5 washing fluids for ZP-free embryos and in the first 8 washing fluids for ZP-intact embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly demonstrate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to cling to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from the ZP.
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Martinez, Cristina A., Marie Rubér, Heriberto Rodriguez-Martinez, and Manuel Alvarez-Rodriguez. "Pig Pregnancies after Transfer of Allogeneic Embryos Show a Dysregulated Endometrial/Placental Cytokine Balance: A Novel Clue for Embryo Death?" Biomolecules 10, no. 4 (April 5, 2020): 554. http://dx.doi.org/10.3390/biom10040554.
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Pig embryo transfer (ET) is burdened by high embryo mortality, with cytokines playing a significant role in recruitment of immune cells during embryo attachment and placentation. We hereby tested if their levels in endometrium and placenta from sows carrying hemi-allogeneic (artificially inseminated sows; C+ positive control) or allogeneic embryos (sows subjected to ET; ET) during peri-implantation (D18) or post-implantation (D24) are suitable mirrors of embryo rejection or tolerance after ET. Non-pregnant sows (C−) were used as negative controls. A set of cytokines was assayed in the tissues through multiplexed microsphere-based flow cytometry (Luminex xMAP, Millipore. USA). Fewer (58.7%. p < 0.003) conceptuses were recovered at D24 after ET compared to C+ (80.9%); with more than 20% of the ET conceptuses being developmentally delayed. Cytokine levels shifted during implantation. Anti-inflammatory IL-10 levels were significantly (p < 0.05) lower in ET sows compared to C+ at D24 of pregnancy. The C+ controls (carrying hemi-allogeneic embryos) consistently showed higher levels of pro-inflammatory TNF-α, IFN-γ, and IL-2 cytokines at D18 and IL-1α at D24, compared to the ET group. This clear dysregulation of pro- and anti-inflammatory cytokine levels in sows subjected to ET could be associated with an impaired maternal immune tolerance, explaining the high embryonic mortality of ET programs.
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Abdul Rahman, Nor-Shahida, Nor-Ashikin Mohamed Noor Khan, Zolkapli Eshak, Mimi-Sophia Sarbandi, Aqila-Akmal Mohammad Kamal, Mastura Abd Malek, Fathiah Abdullah, Maizaton Atmadini Abdullah, and Fezah Othman. "Exogenous L-Glutathione Improves Vitrification Outcomes in Murine Preimplantation Embryos." Antioxidants 11, no. 11 (October 25, 2022): 2100. http://dx.doi.org/10.3390/antiox11112100.
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Vitrification is an important tool to store surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and results in decreased viability. Studies have reported that L-glutathione (GSH) supplementation improves the preimplantation development of murine embryos. Glutathione constitutes the major non-protein sulphydryl compound in mammalian cells, which confers protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has yet to be reported. This study aims to determine whether GSH supplementation in culture media improves in vitro culture and vitrification outcomes, as observed through embryo morphology and preimplantation development. Female BALB/c mice aged 6–8 weeks were superovulated through an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG), followed by 10 IU of human chorionic gonadotrophin (hCG) 48 h later. The mated mice were euthanized by cervical dislocation 48 h after hCG to harvest embryos. Two-cell embryos were randomly assigned to be cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH-supplemented medium), or Group 4 (0.01 mM GSH-supplemented medium with vitrification). Non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were observed for morphological quality and preimplantation development at 24, 48, 72, and 96 h. In the non-vitrified groups, there were significant increases in the number of Grade-1 blastocysts in GSH cultures (p < 0.05). Similarly, in the vitrified groups, GSH supplementation was also seen to significantly increase blastocyst formation. Exogenous GSH supplementation resulted in a significant increase in intracellular GSH, a release of cytochrome c from mitochondria, and a parallel decrease in intracellular reactive oxygen species (ROS) levels in vitrified eight-cell embryos (p < 0.05). GSH supplementation was shown to upregulate Bcl2 expression and downregulate Bax expression in the vitrified preimplantation embryo group. The action of exogenous GSH was concomitant with an increase in the relative abundance of Gpx1 and Sod1. In conclusion, our study demonstrated the novel use and practical applicability of GSH supplementation for improving embryonic cryotolerance via a decrease in ROS levels and the inhibition of apoptotic events by improvement in oxidative status.
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Coutinho, A. R. S., M. P. Milazzotto, M. A. Peres, M. G. Marques, A. C. Nicacio, J. A. Visintin, and M. E. O. A. Assumpção. "273 APOPTOSIS EVALUATION OF IN VITRO-PRODUCED PIG EMBRYOS (PARTIAL RESULT)." Reproduction, Fertility and Development 18, no. 2 (2006): 244. http://dx.doi.org/10.1071/rdv18n2ab273.
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Apoptosis is a physiological event involved with death and tissue replication, fulfilling an important function of tissue organizations during embryogenesis. This mechanism occurs in in vivo as well as in vitro pre-implantation embryos, but most frequently in the latter. The transcriptional activation of pig embryos occurs at the four-cell stage, which is the longer stage during the pre-implantation period. This stage is characterized by embryonic developmental blockage that decreases the production rates (embryos loss). The aim of this study was to evaluate a correlation between apoptosis mechanism and developmental blockage of IVP porcine embryos. Immature oocytes after IVM/IVF were submitted to IVC in PZM-1 medium containing BSA 3 mg/mL at 38.5�C, 5% CO2 in air and high humidified atmosphere. The embryo development was analyzed at 96 h of cultute (Day 4) in order to verify cleavage rate and blockage (4 cells) and non-blockage (e8 cells) embryo rates. Out of 625 grade I, II, and III oocytes submitted to IVP, 70.3 � 5.2% (430/625) cleaved from which 27.1 � 10.3% (166/625) were blocked and 43 � 10.8% (264/625) were non-blocked. Blocked and non-blocked embryos were assessed to evaluate apoptosis rates. Qualitative assays of embryo cells were achieved with two different DNA stains: YOPRO-1 (Molecular Probe�; Invitrogen Brasil, Ltd., Sao Paulo, Brazil), permeable though plasma membrane in the early stage of apoptosis, and TUNEL (Roche�; Amersham Biosciences, Sao Paulo, Brazil), which detects DNA fragmentation in the last stages of apoptosis. The embryos were stained with 0.1 �M YOPRO-1/mL PBS, incubated 15 min at 38�C, 5% CO2 in air and high humidified atmosphere, and immediately observed by means of confocal microscopy. For the TUNEL assay, embryos were fixed in 4% paraformaldehyde solution (w/v) in PBS for 1 h at room temperature, and incubated in permeabilization solution [0.5% (v/v) Triton X-100, 0.1% (w/v) sodium citrate in PBS] for 2 h. For positive control, samples were treated with DNase-I at 37�C for 1 h. The negative control and experimental samples were incubated with buffer solution under the same conditions. The positive control and experimental samples were incubated with enzymatic and stain solution (FITC) at 37�C for 1 h; the negative control was incubated with only enzymatic solution. The embryos were stained with Hoechst 33342 (5 �/mL) and observed by means of fluorescence microscopy. Blocked embryos showed more apoptosis (66% and 40% to YOPRO-1 and TUNEL, respectively) than non-blocked embryos (25% and 0% to YOPRO-1 and TUNEL, respectively). In conclusion, the developmentally blocked embryos suffered more apoptosis, although morphologic apoptosis assays (light and electronic microscopic) must be performed to confirm this finding. This work was supported by FAPESP 04/01252-4.
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Gäreskog, Mattias, and Parri Wentzel. "N-Acetylcysteine and α-cyano-4-hydroxycinnamic acid alter protein kinase C (PKC)-δ and PKC-ζ and diminish dysmorphogenesis in rat embryos cultured with high glucose in vitro." Journal of Endocrinology 192, no. 1 (January 2007): 207–14. http://dx.doi.org/10.1677/joe.1.06966.
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Malformations and growth disturbances are two- to threefold more common in infants of diabetic mothers than in offspring of non-diabetic pregnancy. Several suggestions have emerged to explain the reasons for diabetic embryopathy, including enhanced mitochondrial production of reactive oxygen species leading to altered activation of protein kinase C. This study aimed to evaluate the effect of α-cyano-4-hydroxycinnamic acid (CHC) and N-acetylcysteine (NAC) addition on morphology and activity of protein kinase C-δ and protein kinase C-ζ in rat embryos exposed to a high glucose concentration in vitro. Day 9 embryos from normal rats were cultured in 10 or 30 mM glucose concentrations with or without supplementation of CHC, NAC, or protein kinase C inhibitors specific for protein kinase C-δ and protein kinase C-ζ. Embryos were evaluated for malformations, crown rump length, and somite number. Protein kinase C-δ and protein kinase C-ζ activities were estimated by western blot by separating membranous and cytosolic fractions of the embryo. We found increased malformations and growth retardation in embryos cultured in high versus low glucose concentrations. These abnormalities were diminished when CHC and NAC or specific protein kinase C-inhibitors were added to the culture medium. The activities of embryonic protein kinase C-δ and protein kinase C-ζ were increased in the high glucose environment after 24-h culture, but were normalized by the addition of CHC and NAC as well as respective inhibitor to the culture medium. These findings suggest that mitochondrial overproduction of reactive oxygen species is involved in diabetic embryopathy. Furthermore, such overproduction may affect embryonic development, at least partly, by enhancing the activities of protein kinase C-δ and protein kinase C-ζ.
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Zhang, Lei, Yan Zhang, Zhuo Han, Jingshuai Fang, Huanhuan Chen, and Zekun Guo. "Transcriptome Analyses Reveal Effects of Vitamin C-Treated Donor Cells on Cloned Bovine Embryo Development." International Journal of Molecular Sciences 20, no. 11 (May 28, 2019): 2628. http://dx.doi.org/10.3390/ijms20112628.
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Somatic cell nuclear transfer (SCNT) is a very powerful technique used to produce genetically identical or modified animals. However, the cloning efficiency in mammals remains low. In this study, we aimed to explore the effects of vitamin C (Vc)-treated donor cells on cloned embryos. As a result, Vc treatment relaxed the chromatin of donor cells and improved cloned embryo development. RNA sequencing was adopted to investigate the changes in the transcriptional profiles in early embryos. We found that Vc treatment increased the expression of genes involved in the cell–substrate adherens junction. Gene ontology (GO) analysis revealed that Vc treatment facilitated the activation of autophagy, which was deficient in cloned two-cell embryos. Rapamycin, an effective autophagy activator, increased the formation of cloned blastocysts (36.0% vs. 25.6%, p < 0.05). Abnormal expression of some coding genes and long non-coding RNAs in cloned embryos was restored by Vc treatment, including the zinc-finger protein 641 (ZNF641). ZNF641 compensation by means of mRNA microinjection improved the developmental potential of cloned embryos. Moreover, Vc treatment rescued some deficient RNA-editing sites in cloned two-cell embryos. Collectively, Vc-treated donor cells improved the development of the cloned embryo by affecting embryonic transcription. This study provided useful resources for future work to promote the reprogramming process in SCNT embryos.
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JING, Ruyue, Peilan WANG, Zhen HUANG, and Zhihui LI. "Histocytological Study of Somatic Embryogenesis in the Tree Cinnamomum camphora L. (Lauraceae)." Notulae Botanicae Horti Agrobotanici Cluj-Napoca 47, no. 4 (December 23, 2019): 1348–58. http://dx.doi.org/10.15835/nbha47411655.
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Histocytological studies were conducted on primary, secondary, and malformed embryos produced during somatic embryogenesis of Cinnamomum camphora L. to better understand its development. Exploring its callus types and structures provided a theoretical basis for clarifying the mechanism of somatic embryogenesis, which may shed light on the mechanism of zygotic embryogenesis. We used immature zygotic embryos as explants to induce somatic embryos, forming many embryogenic calli that differentiated into mature somatic embryos. Our results showed that somatic embryogenesis of C. camphora was similar to that of zygotic embryos. We have been dedifferentiated four types of callus. Compared with non-embryogenic cells, embryogenic cells had a closer arrangement, larger nucleus, thicker cytoplasm, more starch granules and easier to stain into black. Somatic embryogenesis had two pathways: direct (predominate) and indirect (rare). Embryogenic cells of C. camphora could have either an internal or external origin, the latter being primary, for which occurrence sites include epidermis and near-epidermis (little internally). Mostly arising from single cells, C. camphora follows two developmental pathways: single-cell equal as opposed to unequal, wherein both divide to form multi-cell proembryos. However, multicellular origins can occasionally occur and feature physiological isolation during somatic embryo development. This development has four embryo stages: globular, heart-shaped, torpedo, and cotyledon, with procambium cells apparent in globular embryos and late cotyledons forming “Y-shaped” vascular bundles. Secondary embryos were present in all stages, directly occurring on primary embryo’s germ and radicle end surfaces. We conclude that secondary and primary embryos of C. camphora undergo similar developmental processes. At the same time, conjoined cotyledon embryos and morphological abnormal embryos were found, with an internal origin more likely to generate abnormal embryos.
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In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.
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Mallol, Anna, Laia Piqué, Josep Santaló, and Elena Ibáñez. "Morphokinetics of cloned mouse embryos treated with epigenetic drugs and blastocyst prediction." REPRODUCTION 151, no. 3 (March 2016): 203–14. http://dx.doi.org/10.1530/rep-15-0354.
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Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for non-treated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success.
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Muñoz, M., S. Carrocera, D. Martin, N. Peynot, C. Giraud-Delville, E. Correia, O. Sandra, V. Duranthon, and E. Gómez. "75 EXPRESSION OF GROWTH FACTOR GENES IN IN VITRO-PRODUCED BLASTOCYST CHANGES AFTER UTERINE PASSAGE, BUT ENDOMETRIAL EXPRESSION IS UNAFFECTED BY THE PRESENCE OF EMBRYOS." Reproduction, Fertility and Development 28, no. 2 (2016): 167. http://dx.doi.org/10.1071/rdv28n2ab75.
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Growth factors (GFs) exert recognised roles in mammalian reproduction. However, little is known about the role of GFs in very early development. We examined here endometrial and embryonic transcription of genes coding for GF proteins identified in bovine uterine fluid (UF): artemin, stanniocalcin-1 (STC-1); heparin-binding EGF-like growth factor (HBEGF); connective tissue growth factor (CTGF); and stem cell factor (SCF). Embryos were in vitro produced (IVP) including culture in SOF+BSA. On Day 6, embryos (n = 50) were transferred into the uteri of oestrus-synchronized heifers (n = 10). On Day 8, embryos were flushed, and groups (n = 6) of expanded blastocysts (n = 10) were snap frozen in LN2 and stored at –145°C. Day 8 embryos cultured in vitro from Day 6 were used as controls (n = 6 × 10 embryos). Transcript levels were also analysed in endometrial tissue collected from Day 8 slaughtered females that were embryo (n = 4) or sham transferred (n = 4). Samples were incubated overnight at 4°C in RNAlater and stored at –145°C in an ultrafreezer. Samples analysed (n = 3 caruncle; n = 3 intercaruncle) were taken from ipsilateral middle horn region. Embryonic and endometrial samples were analysed in duplicate for RT-qPCR analysis. Data were analysed by Wilcoxon Mann-Whitney test. In embryos, uterine passage compared with IVP down-regulated artemin (P < 0.01) and STC1 (P < 0.05) expression (0.61 ± 0.04 and 0.81 ± 0.06 v. 1.54 ± 0.10 and 1.25 ± 0.17, respectively). Conversely, abundance of CTGF (P < 0.05) and HBEGF (P < 0.01) decreased in IVP v. uterus-exposed embryos (0.79 ± 0.04 and 0.81 ± 0.03 v. 1.38 ± 0.18 and 1.37 ± 0.23, respectively). No changes were observed for SCF (0.96 ± 0.16 v. 1.09 ± 0.05 in IVP embryos). Endometrial gene expression for each GF did not change in response to embryos v. sham transfer (P > 0.10). Caruncular expression was higher (P < 0.05) for SCF compared with intercaruncles (1.32 ± 0.13 v. 0.88 ± 0.13). However, strong positive correlations (P < 0.006 to P < 0.0001) were seen for HBEGF with STC-1 (r = 0.67) and SCF (r = 0.72); for SCF with STC-1 (r = 0.61); and for CTGF with STC-1 (r = 0.59) and SCF (r = 0.50), suggesting a common transcription regulation among some GFs. Changes in embryonic gene expression reflect a regulatory response to the cognate GFs in the UF, which suggests a relevant role for GFs in early embryo-maternal interactions. Within the endometrium, our observations are consistent with studies postulating faster, non-transcriptional responses to early embryos (Gómez and Muñoz, Reproduction 2015 150, R35–R43). Alternatively, it is possible that very local endometrial responses for the analysed GFs were not detected despite the presence of multiple embryos. Research was supported by MICINN, project AGL2012–37772, and FEDER. The authors are members of the COST Action FA1201 (Epigenetics and Periconception environment).
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Alsaleh, A., J. L. Pellerin, D. M. Garcia, D. Tainturier, and F. Fieni. "101 RISK OF COXIELLA BURNETII TRANSMISSION BY EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS." Reproduction, Fertility and Development 26, no. 1 (2014): 165. http://dx.doi.org/10.1071/rdv26n1ab101.
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Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main sources of infection for humans. In cattle, infection is frequently asymptomatic, but it may cause abortion, reproductive failure (metritis, placentitis, and infertility), and economic losses. A previous study in goats showed that Coxiella burnetii had a strong tendency to cling to the zona pellucida (ZP) after in vitro infection and the washing procedure recommended by IETS for bovine embryos failed to remove it (Alsaleh et al. 2013 Theriogenology). The aims of this study were to determine (1) whether Coxiella burnetii would adhere to the intact ZP (ZP-intact) of early in vitro-produced bovine embryos, (2) whether the bacteria would adhere to or infect the embryo cells (ZP-free) after in vitro infection, and (3) the efficiency of the washing protocol recommended by the IETS. One hundred and sixty 8- to 16-cell bovine embryos produced in vitro were randomly divided into 16 batches of 10 embryos each. Twelve batches (8 ZP-intact and 4 ZP-free) were incubated in medium containing C. burnetii CbB1 (IASP, INRA Tours, France). After 18 h of incubation at 37°C and 5% CO2 in air, the embryos were washed in 10 successive baths of a phosphate buffer saline (PBS) and 5% FCS solution in accordance with the IETS guidelines. In parallel, 4 batches (2 ZP-intact and 2 ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. The 10 washing fluids for all batches were collected and centrifuged for 1 h at 13 000 × g. Embryo and pellet washing were tested by C-PCR. Coxiella burnetii DNA was found in all ZP-intact and ZP-free embryo batches after 10 successive washes. It was also detected in the first 4 washing fluids for ZP-intact embryos and in the 10th washing fluid for 2 of the 4 batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adhere and (or) penetrate the early embryonic cells as well as the ZP of in vitro bovine embryos after in vitro infection and the standard washing protocol recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients or their offspring, or both. Further studies are needed to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP.
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Sekirina, Galina G., and Irina E. Neganova. "The microenvironment created by non-blocking embryos in aggregates may rescue blocking embryos via cell–embryo adherent contacts." Zygote 3, no. 4 (November 1995): 313–24. http://dx.doi.org/10.1017/s0967199400002744.
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SummaryUnder our culture conditions, mouse embryos from the BALB/c inbred mouse strain develop successfully in culture only from the late 2-cell stage onwards (so-called 2-cell block), whether or not EDTA is added to the culture medium. (CBA × C57BL) F2 embryos do not exhibit a 2-cell block. Medium conditioned by culture of non-blocking embryos from the 2-cell to the 8-cell stage did not improve the development of blocking embryos, nor did co-culture of blocking and non-blocking embryos, with or without conditioned medium. On the other hand phytohaemagglutinin (PHA)-assisted aggregation of an early 2-cell BALB/c embryo with five surrounding non-blocking F2 embryos (2-cell or 8-cell) or five BALB/c 8-cell embryos allowed the early 2-cell BALB/c embryos to develop into blastocysts within 72 h. Aggregation of blocking BALB/c 2-cell embryos with each other had no ‘rescue’ effect. When blocking and non-blocking 2-cell embryos were aggregated together, an integrated blastocyst was formed; but when the early 2-cell BALB/c embryos were aggregated with non-blocking 8-cell embryos, the blocking embryos formed a separate small blastocyst, which nonetheless retained adherent contact with the non-blocking embryos throughout the culture period. Ultrastructural analysis showed that 2-cell embryos aggregated with the aid of PHA form close adherent cell contacts up to several micrometres in length.
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Díaz, R. Rodríguez, R. Blanes Zamora, R. Vaca Sánchez, J. González Pérez, and J. C. Alberto Bethencourt. "Embryo sHLA-G secretion is related to pregnancy rate." Zygote 27, no. 02 (March 15, 2019): 78–81. http://dx.doi.org/10.1017/s0967199419000054.
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SummaryHLA-G expression has been detected in early preimplantation embryos and it has been postulated that a relationship between embryonic expression of this factor and successful pregnancy may exist. Forty-six patients were prospectively selected from our centre ‘Unidad de Reproducción Humana, Hospital Universitario de Canarias’ for conducting this study. In all cases, metaphase II (MII) oocytes were fertilized using intracytoplasmic sperm injection 2–4 h after retrieval. Embryos were cultured individually in 20 µl droplets of G-1 medium (VitroLife) under oil at 37°C and a 6% CO2environment. Fertilization was assessed at 18 h postinsemination and all oocytes fertilized were passed into a new culture plaque individually in 300 µl culture medium until day 3 of culture. The culture medium was examined for the expression and secretion of sHLA-G with a sandwich enzyme-linked immunosorbent assay kit (BioVendor, Heidelberg, Germany) according to the manufacturer’s instructions. We found statistical significance between higher levels of sHLA-G secretion and pregnancy rate. When both groups were compared there was no difference in embryo quality of transferred embryos, but a significant difference in the number of oocytes and the embryo quality of the cohort existed that was greater in the pregnant group. A standardized sHLA-G assay with a specifically defined range and standard units provides a non-invasive method to identify the most competent embryos for transfer.
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Lopes, A. S., N. Ramsing, L. H. Larsen, M. Räty, J. Peippo, T. Greve, and H. Callesen. "2 CORRELATION BETWEEN OXYGEN RESPIRATION RATES AND MORPHOLOGY, SEX, DIAMETER AND DEVELOPMENTAL STAGE OF SINGLE BOVINE IVP-EMBRYOS." Reproduction, Fertility and Development 17, no. 2 (2005): 151. http://dx.doi.org/10.1071/rdv17n2ab2.
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A simple, non-invasive, rapid and sensitive oxygen microsensor system was developed to investigate correlations between oxygen respiration rates of individual bovine embryos and their morphology, sex, diameter and developmental stage. Bovine IVP-embryos (n = 78; Holm et al. Theriogenology 52, 683–700) were analysed around the 8-cell stage (Day 3; n = 18) and at various blastocyst stages (Day 7; n = 60). Each embryo was morphologically evaluated, its outer diameter measured and was then loaded into a glass tube (i.d. 0.68 mm, length 3 mm). After 1 h, oxygen concentration gradients generated by the embryo’s respiration were measured over app. 8 min with an oxygen microelectrode (www.unisense.com). Five embryos were measured in one round together with an empty tube as control. The procedure was repeated twice for each embryo with app. 1 h interval. Individual respiration rates in nL O2/embryo/h (nL/h) were calculated from these gradients. The measurements were performed at 38.5°C under constant flow of humidified 5% CO2 in air (app. 19% O2). After this, 64 embryos (14 Day 3; 50 Day 7) were lysed for sex diagnosis by PCR. Values are given as mean ± SD. The sensitivity of the oxygen measurement system was high (controls: 0.034 ± 0.035 nL/h, n = 15) and its repeatability from 1st to 2nd measurement was 99.7 ± 9.8% (n = 71). The average embryo respiration rate was 0.39 ± 0.05 nLl/h on Day 3 (n = 18) and 1.31 ± 0.52 nLl/h on Day 7 (n = 60). For Day 7 embryos, the respiration rates varied according to their morphological quality, being 1.87 ± 0.46a (n = 18), 1.17 ± 0.32b (n = 23), 0.95 ± 0.27b,c (n = 14) and 0.72 ± 0.24c (n = 4) nL/h for quality 1, 2, 3, and 4 embryos, respectively (Proc Mixed,a,b,c: P < 0.05; values with different superscripts differ significantly). The sex ratio (male:female) was 9:5 (Day 3) and 32:18 (Day 7), and on Day 7 this ratio varied between qualities: 11:2, 12:8, 8:4, and 1:3 for quality 1, 2, 3, and 4, respectively. The average respiration rate on day 3 was the same for males and females, as it was on day 7 (1.22 ± 0.43 nL/h (females) and 1.31 ± 0.58 nL/h (males), P > 0.05). There was a correlation between embryo diameter and respiration rate (r2 = 0.65, n = 74), which was even stronger for Day 7 male embryos (r2 = 0.72, n = 32). In conclusion, a highly reliable, repeatable and sensitive system was established for measuring respiration rates in single bovine embryos, even at early developmental stages. The respiration rate was lower on day 3 compared to Day 7 embryos, and it was correlated with the morphological embryo quality on Day 7. Oxygen consumption could be a valuable supplementary indicator of embryo viability, especially in difficult evaluations (e.g. quality 2 and 3 after IVP). It remains to be demonstrated if such measurements can also reveal quality differences already at Day 3, which would be of interest in, e.g. the human field. ASL is supported by FCT, Portugal.
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Scherzer, J., R. A. Fayrer-Hosken, L. Ray, and G. Heusner. "217 A NEW APPROACH TO CRYOPRESERVATION OF LARGE EQUINE EMBRYOS BY VITRIFICATION AFTER BLASTOCOEL MICROMANIPULATION." Reproduction, Fertility and Development 19, no. 1 (2007): 225. http://dx.doi.org/10.1071/rdv19n1ab217.
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Vitrified large equine embryos >800 �m recovered on Day 8 after ovulation have not been successfully transferred yet. In this study, we examined the effects of reduction of the blastocoelic fluid and microinfusion of a cryopreservative prior to vitrification on pregnancy outcome. In 2006, 33 embryos, recovered at the expanded blastocyst stage, were transferred fresh with an average pregnancy rate of 91% (30/33). However, suitable recipients are not always available. The sizes of embryos used for this vitrification project were 805 �m (embryo #1), 820 �m (#2), 1120 �m (#3), 1286 �m (#4), and 979 �m (#5). They were all morphologically graded excellent (according to IETS guidelines). These embryos were assigned to either no micromanipulation (embryos #1–#3) or microinfusion of VS1 (1.4 M glycerol in PBS; embryos #4 and #5) after additional aspiration of the blastocoelic fluid and before microinfusion for embryo #5. To facilitate aspiration and microinfusion, a laser system (XYclone; Hamilton Thorne Biosciences, Beverly, MA, USA) was applied. Approximately 20% of the total blastocoelic fluid was removed using a standard micromanipulator and microcapillary system (Eppendorf). All embryos were then vitrified as previously described (Eldridge-Panuska et al. 2005 Theriogenology 63, 1308–1319). In brief, embryos were exposed to VS1 and VS2 (1.4 M glycerol + 3.6 M ethylene glycol in PBS) for 5 min, and VS3 (3.4 M glycerol + 6.6 M ethylene glycol in PBS) for 1 min. Embryos in VS3 were then individually loaded into 0.25-mL straws, separated by 2 air bubbles from columns of 0.5 M galactose. Then straws were placed for 1 min into a cooled plastic goblet surrounded by liquid nitrogen. The goblet was finally plunged into liquid nitrogen. Digital images of all embryos were taken prior to and during the vitrification procedure, and also after thawing prior to embryo transfer. All expanded blastocysts initially decreased in size. After transfer to VS2 and VS3, they lost their spherical shape and blastocoels collapsed. Four vitrified embryos were transfered to recipients on Day 8 after ovulation. After thawing in air at room temperature for 5 s and then in water at 30�C for 15 s, straw contents were emptied into a Petri dish and mixed. After 5 min, single embryos were loaded into an AI pipette and nonsurgically transferred to recipients. The blastocoel of only one embryo re-expanded during the 5 min after thawing (#3) and one embryo was split into two halves (#4). One week after transfer of embryos, recipients were examined by ultrasonography. None of the control embryos nor the split embryo in the treatment group led to the formation of an embryonic vesicle. However, the blastocyst, which had undergone both aspiration of blastocoelic fluid and microinfusion of VS1 (#5), had formed an embryonic vesicle at Day 15 after ovulation. During a further exam on Day 28, the uterine tone in the recipient was still increased, but ultrasonography revealed resorption of the embryo, which was probably caused by heat stress. Nevertheless, we will test this protocol for the cryopreservation of large equine embryos on a larger scale during the next breeding season.
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Herranz, José M., Elena Copete, and Pablo Ferrandis. "Non-deep complex morphophysiological dormancy inNarcissus longispathus(Amaryllidaceae): implications for evolution of dormancy levels within sectionPseudonarcissi." Seed Science Research 23, no. 2 (March 1, 2013): 141–55. http://dx.doi.org/10.1017/s0960258513000056.
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AbstractNarcissus longispathus(Amaryllidaceae) is a perennial geophyte and a Mediterranean narrowly endemic species. At dispersal time,N. longispathusseeds are dormant and have underdeveloped embryos. This work aimed to determine requirements for dormancy break and germination and to compare dormancy traits with those of the two endemic Iberian congeners. Phenology of embryo growth and germination were studied by regularly exhuming seeds sown in near-natural conditions. Temperature and light requirements for embryo growth, breaking of dormancy and germination were determined by incubating seeds under controlled laboratory conditions. Mean embryo length in fresh seeds was 1.50 mm, and embryos had to grow to 3.80 mm before radicle emergence. Embryos grew to full size and seeds germinated when they were warm stratified for 2 months (optimum 1 month at 20/7°C+1 month at 15/4°C), then cold stratified at 5°C for 2 months, and finally incubated at cool temperatures (15/4°C) for 30 d. However, in seeds only subjected to either warm or cold stratification, the embryos hardly grew and did not germinate. In natural conditions, the embryos elongate in autumn–winter, and in late winter–early spring (March) almost all radicles and seedlings emerged. Velocity of embryo growth and germination percentages increased with seed storage duration. Seeds ofN. longispathushave non-deep complex morphophysiological dormancy (MPD). This is the first report of such a level of MPD inNarcissus. Our data suggest that non-deep complex MPD may have been derived from intermediate complex MPD in the sectionPseudonarcissi.
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de Sousa e Brito, Clara Sattler. "Biopatenting: “Angst” v. European Harmonization – The ECJ Decision on Stem Cell Patents." European Journal of Risk Regulation 3, no. 1 (March 2012): 130–34. http://dx.doi.org/10.1017/s1867299x00001938.
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Case C-34/10 Brüstle v. Greenpeace1.Article 6(2)(c) of Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions must be interpreted as meaning that:–any human ovum after fertilisation, any non-fertilised human ovum into which the cell nucleus from a mature human cell has been transplanted, and any non-fertilised human ovum whose division and further development have been stimulated by parthenogenesis constitute a ‘human embryo’;–it is for the referring court to ascertain, in the light of scientific developments, whether a stem cell obtained from a human embryo at the blastocyst stage constitutes a ‘human embryo’ within the meaning of Article 6(2)(c) of Directive 98/44.2.The exclusion from patentability concerning the use of human embryos for industrial or commercial purposes set out in Article 6(2)(c) of Directive 98/44 also covers the use of human embryos for purposes of scientific research, only use for therapeutic or diagnostic purposes which is applied to the human embryo and is useful to it being patentable.3.Article 6(2)(c) of Directive 98/44 excludes an invention from patentability where the technical teaching which is the subject-matter of the patent application requires the prior destruction of human embryos or their use as base material, whatever the stage at which that takes place and even if the description of the technical teaching claimed does not refer to the use of human embryos.
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Czerski, J., and K. Janowska. "Growth of apple-tree seedlings (Malus domesticd) Borkh cv. Antonówka obtained from non-stratified embryos." Acta Societatis Botanicorum Poloniae 46, no. 4 (2015): 647–68. http://dx.doi.org/10.5586/asbp.1977.054.
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The growth of seedlings (<i>Malus domestica</i> Borkh cv. Antonówka) obtained from embryos isolated from non-stratified and stratified (90 days at 4-5°C) seeds was compared. The lack of differences in the germination power of embryos isolated from non stratified and stratified seeds, and the observed normal growth of plants obtained from non-stratified seeds, point to the fact, that the seeds of the autumn apple 'Antonówka' do not pass the stage of embryonal dormancy.
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Mancini, M. P. M., B. C. S. Campanha, D. M. Souza, C. P. Godoi, F. Frei, and M. F. G. Nogueira. "162 EVALUATION OF THE DEMI-EMBRYOS AGGREGATION TECHNIQUE EFFICACY ON THE PRODUCTION OF CHIMERAS WITH IN VIVO PRODUCED MURINE EMBRYOS." Reproduction, Fertility and Development 22, no. 1 (2010): 239. http://dx.doi.org/10.1071/rdv22n1ab162.
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Mixing embryo cells coming from different fertilizations (i.e. embryonic chimera) have been used as a tool to understand embryogenesis, organo- genesis, and pluripotency, as well as a source to obtain transgenic mammals. The objectives of this work were to evaluate the potential of mice demi-embryos, in advanced stage of the development (morulae and blastocysts) to aggregate in chimeras; to compare the chimerism rate of those embryos with the rate of whole 8- to 16-cell-stage embryos; and to measure the genotype composition of the resultant chimera. One-month-old transgenic (C57/BL6/EGFP strain, GFP) or non-transgenic (Swiss Webster strain, SW) mice weighing approximately 35 g were superstimulated with 5 or 10IU of eCG (for GFP or SW mice, respectively) followed with hCG injection of 5 or 10IU (GFP or SW mice, respectively) 48 h later. Embryos were harvested at different stages of development and allocated in 3 groups for aggregation technique. Blastocysts and morulae were bisected (microblade mounted on TransferMan NK-2, Eppendorf), whereas 8- to 16-cell-stage embryos had their zona pellucida mechanically removed (23-gauge needle). Embryos were manipulated in M2 culture medium at room temperature, and aggregation groups consisted of G1 (2 demi-blastocysts, n = 28), G2 (demi-blastocyst and demi-morula, n = 20), and G3 (2 whole 8- to 16-cell-stage embryos, n = 25). All embryos were placed in wells (Embryo GPS dish, SunIVF) containing KSOMaa medium (EmbryoMax, Millipore) under oil (Sigma, St. Louis, MO, USA) and were incubated at 37°C, 5% CO2 in air saturated with humidity. After 24 h of incubation, the presence of chimera was verified, and the percentage of area (square pixel) occupied by each embryonic type (GFP or SW) from both G2 (n = 3) and G3 (n = 3) were measured by the ImageJ program (v. 1.42i, USA). General results of the chimerism rate were 3.6%, 15.0%, and 60.0% (G1, G2, and G3, respectively; P < 0.001, chi-square). The G3 group differed from others (G1, P < 0.001 and G2, P = 0.003), which appeared similar (P = 0.294; Fisher’s exact test). The mean percentage (±SD) of GFP cells in the resultant chimera were 51.3 ± 4.1% and 50.6 ± 10.0% (for G2 and G3, respectively; P = 0.91, t-test). Moreover, the percentages of GFP cells within the same group of G2 or G3 at 0 v. 24 h of culture were not statistically different (data not shown). It was concluded that in our conditions, the embryonic chimerism by aggregation of murine demi-embryos is a feasible procedure, even for embryos in an advanced stage of development (morulae and blastocysts). Nevertheless, the chimerism rate with whole pre-compaction embryos (G3) was higher than that of G1 and G2 groups. Furthermore, the phenotype of embryonic chimera was equally composed, with no effect of strain (GFP or SW cells) or culture (0 or 24 h) on its composition. Supported by FAPESP, Brazil: 2006/06491-2 and 2007/07705-9 (MFGN) and 2007/04291-9 (MPMM).
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Machado, G. M., C. R. Laender, M. M. Franco, L. O. Leme, R. Rumpf, and M. A. N. Dode. "132 POST-HATCHING BOVINE EMBRYO DEVELOPMENT IN VITRO: RELATIONSHIP BETWEEN SEX AND SIZE." Reproduction, Fertility and Development 20, no. 1 (2008): 146. http://dx.doi.org/10.1071/rdv20n1ab132.
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In vitro post-hatching embryos culture is a procedure that allows the establishment of more accurate tools for evaluating embryo developmental potential without the need of transferring them to recipient animals (Vejlsted et al. 2006 Theriogenology, 65, 153–165). It is well established that in the in vitro embryo production (IVP) technique, the sex ratio is imbalanced in favor of male embryos. The difference in sex ratio observed in the blastocyst stage at day 7 may be attributed to a variety of factors including developmental speed. However, whether or not this difference in sex ratio and speed of development continues after hatching is not known. The objective of this study was to evaluate post-hatching embryonic development until day 11 after in vitro fertilization (day 0) associating embryo size and gender. A total of 468 oocytes, obtained from abattoir-derived ovaries, were used. They were matured, fertilized, and cultured in vitro for 8 days in synthetic oviduct fluid medium (SOF Nutricell�) and incubated at 39�C in 5% CO2 in air. Degenerated embryos on day 8 and non-hatching embryos on day 9 were removed from culture droplets, and only hatched blastocysts were kept. Then, embryos were measured using a graduated ocular and post-hatching development (PHD) medium (Brand�o et al. 2005 Biol. Reprod. 71, 2048–2055) was added in each well, being the final medium 1:1 of SOF:PHD. On day 11, the embryos were evaluated under stereomicroscope and only morphologically normal blastocysts were measured and frozen at –80�C, for gender diagnosis. The DNA from frozen samples was extracted with trizol reagent, sodium citrate solution (0.1 m), and ethanol. Sex embryos determination was performed by PCR and visualized in 2% agarose gel. Data were analyzed using the Mann-Whitney test. The results show that the majority (69%) of the embryos that reached blastocyst stage at day 7 developed in the PHD system until day 11. From the initial oocytes, 144 embryos (30.1%) and 146 (31.1%) embryos had reached the blastocyst stage at days 7 and 8, respectively. At day 9, 89 (19%) embryos were hatched and 65 embryos (13.9%) developed until day 11, of which only 48 embryos (73.8%) had a clear trophoblast. No difference (P > 0.05) in the percentage of male and female embryos was observed when embryos were evaluated at day 11 of culture. In addition the mean size was similar (P > 0.05) for female (467.24 � µm, n = 19) and male (478.84 � 190.21 µm, n = 29) embryos. The results suggest that after post-hatching culture the differences in sex ration and in gender development in IVP bovine embryos are not evident. The development until day 11 showed that post-hatching in vitro culture of bovine blastocyst can be used for embryo evaluation in later phase of development However, several questions still remain to be investigated regarding post-hatching culture of bovine blastocysts before it can be used as a tool to evaluate in vitro embryos. Supported by Embrapa and UnB, Brazil.
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Czerski, Jerzy, and Krystyna Jankowska. "Germination of embryos from stratified and non-stratified seeds and growth of apple seedlings (Malus domestica Borkh cv. "Antonówka")." Acta Societatis Botanicorum Poloniae 50, no. 4 (2014): 595–605. http://dx.doi.org/10.5586/asbp.1981.081.
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The germination of whole seeds, the seeds without coat and isolated embryos of apple cv. "Antonówka Zwykła" after 90 days of cold-stratification was compared with the germination of embryos isolated from non-stratified seeds. They were germinated under 16hrs during a day at temperature 25°C and 20°C during the night. It has been found that after 2 weeks whole stratified seeds germinated in 5 per cent, seeds without coat in 25 per cent and isolated embryos in 98 per cent. Isolated embryos from nun-stratified seeds, after 2 weeks, germinated in the range from 75 to 88 per cent. The results indicate the similar germination ability of embryos isolated from nun-stratified seeds. The seedling populations obtained from embryo's stratified and non-stratified seeds were fully comparable and they evaluated: 1) a wide range of individual differences within population, 2) a similar number of seedlings in each class of shoot length, 3) a similar morphological habitus in each class of shoot length, 4) a similar fresh leaf weight and whole plant increment.
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Shaw, JM, L. Diotallevi, and AO Trounson. "A simple rapid 4.5 M dimethyl-sulfoxide freezing technique for the cryopreservation of one-cell to blastocyst stage preimplantation mouse embryos." Reproduction, Fertility and Development 3, no. 5 (1991): 621. http://dx.doi.org/10.1071/rd9910621.
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This study applies a 4.5 M dimethyl-sulfoxide freezing procedure, developed for 2-cell mouse embryos, to pronuclear to hatched blastocyst stage mouse embryos. The embryos were plunged into liquid nitrogen after 3 min equilibration at room temperature, or 3-60 min equilibration at 0 degrees C. Equilibration at 0 degrees C gave survival rates as high as or higher than rates after equilibration at room temperature. Optimal blastocyst formation, or re-expansion, rates for embryos frozen after equilibration at 0 degrees C were 76% for pronuclear stage embryos and 96-100% for 2-cell to mid-blastocyst stage embryos. The optimal rates of fetus formation, per embryo frozen, ranged from 62 to 88% for pronuclear to mid-blastocyst stage embryos. These results compared favourably with non-frozen control embryos (80-100% blastocyst formation, and 67-78% fetus formation).
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Du, F. L., A. Dinnyes, L. Y. Sung, J. Xu, S. Jiang, C. X. Tian, and X. Yang. "174EMBRYO TRANSFER OF VITRIFIED IVF EMBRYOS IN CATTLE: PREGNANCY COMPARISON AFTER SINGLE AND DOUBLE TRANSFER." Reproduction, Fertility and Development 16, no. 2 (2004): 209. http://dx.doi.org/10.1071/rdv16n1ab174.
Full textAbstract:
Advancement in vitrification of in vitro-produced bovine embryos will benifit the cattle breeding and production industry. The objective was to evaluate whether bilateral (double) embryo transfers (ET) can improve pregnancy rate compared to ipsilateral (single) transfers. Bovine cumulus-oocyte complexes collected from slaughterhouse ovaries were matured for 20–22h, and subsequently subjected to a standard Brackett and Oliphant in vitro fertilization (IVF). Six hours after IVF, embryos denuded of cumulus were cultured in defined CR1 medium supplemented with essential and non-essential amino acids (CR1aa), plus 6mgmL−1 BSA for 2 days at 39°C under 5% CO2, 5% O2 and 90% N2, and then cultured in CR1aa medium supplemented with 7.5% FBS for a further 5 days on bovine cumulus monolayers. Expanded blastocysts with tighter compaction of the inner cell mass (quality 1) were selected on Day 7 for cryopreservation via modified solid surface vitrification (Dinnyes et al., 2000 Biol. Reprod. 513–8). Vitrification solution contained HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol and dimethylsulphoxide. A droplet of 1–2μL vitrification solution containing 4–5 blastocysts was dropped directly onto a cooled surface within 30s after 3-min incubation in equilibration solution. Prior to ET, embryos were warmed and subsequently washed several times in 0.25M sucrose rehydration solution and M199+7.5% FBS medium. The warmed embryos from initial trials were cultured for 2 to 72h to evaluate their viability after vitrification. During ET trails, vitrified embryos were loaded into transfer straws (one embryo per straw) after warming. The treatments were as following, (1) single transfers, one embryo was transferred into the horn ipslateral to CL; (2) double transfers, one embryo was transferred by non-surgical means into each uterine horn of a synchronous recipient on Day 7. ET trails were conducted in both the USA (double transfers) and China (single v. double transfers). Pregnancy was determined by palpation per rectum around Day 70 after transfer. The data were compared by Student’s t-test. The survival rate of vitrified IVF embryos reached as high as 91.4% (n=256) 2h post-warming, and hatching rate was 70.7% (n=154) 72h after culture in vitro, respectively. The data (Table 1) show that double transfers resulted in a significantly higher pregnancy rate than did single transfers (P<0.05). With double transfers, a higher pregnancy rate was achieved in the USA than in China (76.2% v. 45.6%, P=0.079). This study confirms that double embryo transfers can improve the pregnancy outcome after ET, perhaps because bilateral placement of embryos may increase embryonic signals to the maternal environment. Further evaluation of gestation length, single/twin conception and calving difficulty is under investigation. Table 1 Pregnancy rate (Day 70) of vitrified bovine IVF embryos following single and double transfer
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Rodriguez, M. D., A. Gambini, A. Sestelo, O. Briski, R. Fernandez-Martin, and D. F. Salamone. "81 Generation of presumptive domestic cat tetraploid embryos and its application for asynchronic complementation with diploid blastomeres." Reproduction, Fertility and Development 31, no. 1 (2019): 166. http://dx.doi.org/10.1071/rdv31n1ab81.
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Tetraploid complementation has been extensively used to verify the pluripotency of stem cells and also for improving placenta formation when tetraploid embryos are aggregated synchronously or asynchronously with diploid (2n) embryos. Generation of tetraploid embryos can be achieved by the electric fusion of a 2-cell embryo. However, the optimal electric intensity pulse to generate tetraploid embryos has not been studied in the feline. The aims of this study were to (1) evaluate the optimal fusion conditions to achieve the highest fusion rate without affecting embryo developmental competence, (2) compare the in vitro development of synchronic and asynchronic aggregated domestic cat IVF embryos, and (3) assess pre-implantation development of embryos generated by asynchronic complementation of presumptive 1-cell tetraploid embryos with diploid blastomeres. Domestic cat cumulus-oocyte complexes were matured in vitro on 21% O2 in air at 38.5°C for 22h. The IVF embryos were generated by co-incubation of in vitro-matured oocytes with 2×106 motile spermatozoa mL−1 on 21% O2 in air at 38.5°C for 18 to 20h. After 24h of IVF, 2-cell embryos were selected. For Experiment 1, membrane fusion of 2-cell IVF embryos (n=164) was performed with two 30-ms DC pulses at different electric field (0.8, 2, 4, and 8 kV/cm) in fusion media (Mannitol, MgSO4, CaCl2, and polyvinyl alcohol). Presumptive fused embryos and nonfused were cultured in vitro in 50-µL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C (Pope et al. 2006 Methods in Molecular Biology 254, 227-244). Cleavage was determined 24h after pulse. For Experiment 2, zona pellucida-free IVF embryos (n=110) were synchronically (two 4-cell embryos) or asynchronically (one 4-cell embryo and one 2-cell embryo) aggregated in 1 microwell. For Experiment 3, 1-cell presumptive tetraploid embryo (2-cell fused embryo) was asynchronically complemented with a 4-cell embryo (n=38). For all experiments, blastocyst stage was evaluated at Day 8, and embryos presenting more than one structure per microwell were considered non-aggregated. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), and differences were considered significant at P<0.05. The highest fusion rates (30 and 46%) with the best developmental competence (31 and 46%) were observed with 4 and 8 Kv/cm electric pulses, respectively. Electric fusion did not affect the embryo developmental competence. We observed that synchronic and asynchronic complementation reached similar blastocysts rates (54 and 65%, respectively), indicating that both techniques are suitable for tetraploid embryo complementation. Finally, when presumptive tetraploid embryos were asynchronically complemented with diploid blastomeres, the high blastocyst rate (90%) was obtained from embryos that form only one structure (aggregated embryos). Further experiments will be performed to track the distribution of cells using mitotrackers after complementation using tetraploid IVF and diploid somatic cell nuclear transfer embryos.
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Uchikura, A., T. Wakayama, S. Wakayama, H. Matsunari, M. Maehara, Y. Matsumura, K. Nakano, et al. "49 PRACTICAL APPLICATION OF THE HOLLOW FIBER VITRIFICATION METHOD FOR CRYOPRESERVATION OF MAMMALIAN EMBRYOS." Reproduction, Fertility and Development 26, no. 1 (2014): 138. http://dx.doi.org/10.1071/rdv26n1ab49.
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We recently developed the hollow fibro vitrification (HFV) method, which is a novel, high-performance embryo cryopreservation method (Matsunari et al., 2012). In this study, we aimed to verify the applicability of the HFV method for cryopreserving various types of embryos; BDF1 mouse embryos at the 2-cell stage, porcine parthenogenetic morulae derived from in vitro-matured oocytes, bovine morulae produced by in vitro maturation/fertilization (LIAJ Animal Biotechnology Center, Tokyo, Japan), and in vivo-derived blastocysts of common marmosets were vitrified, and their survival was assessed by culture or transfer. The embryos were vitrified using 20 mM HEPES-buffered TCM-199 containing 20% calf serum as a base medium. Cellulose acetate hollow fibres (25 mm) containing 1 to 20 embryos were placed in an equilibration solution containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 5 to 7 min, followed by incubation for 1 min in vitrification solution containing 15% EG, 15% DMSO, and 0.5 M sucrose. The embryos were then vitrified by immersion in LN. The embryos were devitrified by immersing the hollow fibre in a 1 M sucrose solution at 38.5°C, which was followed by stepwise dilution of the cryoprotectants and washing. For a subset of the vitrified mouse embryos, rewarming in a non-ultra-rapid manner by melting a hollow fibre in air at room temperature for 5 s was tested. Embryo transfer was performed to assess the viability of the vitrified mouse embryos. For porcine embryos, vitrification in LN vapor (–150°C) was tested. Development of the vitrified mouse embryos to blastocysts was equal to that of the non-vitrified embryos [105/110 (95.5%) v. 109/110 (99.1%)]. Post-transfer development to fetuses was also equal between the vitrified and non-vitrified embryos [pregnancy rates: 4/4 v. 2/2; developmental rates: 55/80 (68.8%) v. 35/40 (87.5%)]. Non-ultra-rapid rewarming did not decrease the survival of the vitrified mouse embryos [blastocysts: 94/100 (94.0%); pregnancy: 4/4; fetuses: 55/80 (68.8%)]. Blastocyst formation was equivalent for vitrification of porcine embryos in LN vapor [27/34 (79.4%)], direct immersion into LN [28/35 (80.0%)], and the non-vitrified control [31/32 (96.9%)]. Vitrification of 191 bovine morulae resulted in 153 (80.1%) blastocysts. In preliminary experiments, survival of marmoset blastocysts was 100% (n = 6). These data demonstrate that the HFV method is (1) effective for embryos of various species and production methods; (2) effective even for porcine in vitro-derived morulae, which are highly cryosensitive; and (3) amenable to modifications such as non-ultra-rapid warming and cooling in LN vapor, increasing the potential applicability of the HFV method. For instance, vitrification in LN vapor may allow embryo cryopreservation with high hygienic standards. This study was supported by JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project.
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Beggs, Kerry, Jeanne Young, Arthur Georges, and Peter West. "Ageing the eggs and embryos of the pig-nosed turtle, Carettochelys insculpta (Chelonia: Carettochelydidae), from northern Australia." Canadian Journal of Zoology 78, no. 3 (April 1, 2000): 373–92. http://dx.doi.org/10.1139/z99-214.
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Standard series of embryonic stages are the primary basis for organising information in embryological studies and for ageing eggs and embryos in field studies. In this paper we calibrate the developmental series for the pig-nosed turtle, Carettochelys insculpta, from northern Australia against an established series for Chelydra serpentina, carefully noting unique attributes of C. insculpta. We also extend existing non-destructive approaches to staging embryos by identifying several additional specific embryological attributes visible externally or by candling. A chronological sequence of attributes visible by candling is established as a viable alternative to the destructive approaches requiring direct examination of embryos.
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Camargo, L. S. A., T. Aguirre-Lavin, P. Adenot, T. D. Araujo, E. D. Souza, and N. Beaujean. "77 EFFECT OF HEAT SHOCK DURING IN VITRO MATURATION ON HETEROCHROMATIN COMPACTION IN BOVINE EMBRYOS AT 4- AND 8-CELL STAGES: PRELIMINARY STUDY." Reproduction, Fertility and Development 27, no. 1 (2015): 132. http://dx.doi.org/10.1071/rdv27n1ab77.
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High temperatures cause several reproductive losses in cattle. Under in vitro conditions, heat shock decreases oocyte developmental competence and influences embryonic gene expression (Gendelman and Roth 2012 Anim. Reprod. Sci. 134, 125–134). This preliminary study aimed to evaluate whether heat shock during oocyte in vitro maturation (IVM) could have any further effect on chromatin remodelling of fertilized embryos at 4- and 8-cell stages, once such modifications are required for the gene activation in bovine embryos. We evaluated the distribution of heterochromatin 1 (HP1β) and of histone H3 trimethylated at lysine 9 (H3K9me3), both reportedly correlated with heterochromatin formation, in 4- and 8-cell stage embryos derived from control (C) and heat-shocked (HS) bovine oocytes. Immature cumulus-oocyte complexes (COC) collected from crossbred cows in Brazil were exposed for 12 h to 38.8°C (C group) or 41.0°C (HS group) followed by 12 h at 38.8°C, totalizing 24 h of IVM at 5% CO2 in air. Oocytes were in vitro fertilized (IVF) with non-sexed sperm and denuded zygotes were in vitro cultured in CR2aa medium at 38.8°C and 5% CO2, 5% O2 and 90% N2. Four- and 8-cell embryos at 44 h post-IVF were fixed in 4% paraformaldehyde and stained with anti-mouse HP1β and anti-rabbit H3K9me3 first antibodies. Immunofluorescence was evaluated by confocal microscopy (Zeiss LSM 700, MIMA platform, INRA) and 3D images processed by ZEN Lite software (Zeiss, Jena, Germany). Three different distribution patterns of fluorescence were identified based on morphological criteria: diffuse, little clusters, and big clusters. Proportions of embryos in every distribution pattern were compared between C and HS groups by Chi-squared test. No difference (P > 0.05) on cleavage rate was found between C and HS groups until 44 h post-fertilization. Embryos at the 4-cell stage from HS group displayed an increased (P < 0.01) proportion of nuclei with H3K9m3 big clusters (44%, n = 7/16 embryos), whereas embryos from C group displayed only few nuclei with this pattern (5%, n = 1/18). At the 8-cell stage, distribution of H3K9m3 was similar (P > 0.05) between C and HS groups. For HP1β, embryos at the 4-cell stage from HS group displayed an increased (P < 0.05) proportion of nuclei with little clusters (81%, n = 13/16 embryos), whereas embryos from C group had low proportion of nuclei with this same pattern (40%, n = 7/18). Mostly 4-cell stage embryos from C group presented the diffuse pattern (61%, n = 11/18 v.18%, n = 3/16 in the HS group; P < 0.05). At the 8-cell stage, some embryos from the C group (31%, n = 5/16) still showed nuclei with diffuse distribution of HP1β, whereas no nucleus with this pattern was found for the HS group. These preliminary data suggest that bovine embryos derived from heat-shocked oocytes can display precocious heterochromatin compaction, represented by the accumulation of H3K9me3 and HP1β at the 4-cell stage, compared with embryos derived from non-heat-shocked oocytes, which may affect embryonic genome activation with consequences for further gene expression.Research was supported by CNPq, FAPEMIG, FAPES and Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73).
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Blackman, Sheila A., and Eric E. Roos. "441 PB 244 CULTURE OF ISOLATED EMBRYOS OF DETERIORATED MAIZE SEED: A STRATEGY FOR RESCUING GERMPLASM." HortScience 29, no. 5 (May 1994): 494d—494. http://dx.doi.org/10.21273/hortsci.29.5.494d.
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The low quality of some seed lots received by germplasm repositories such as the National Seed Storage Laboratory can thwart efforts to regenerate seed for storage. This germplasm is in danger of irretrievable loss. The aim of this work is to promote the germination, and hence regeneration, of such low quality seeds through sterile culture of the isolated embryos. Hybrid (B73×LH51) maize seeds were aged 5 y at 32°C and 0.037 g H2O g-1 dry wt. Vigor - but not viability -declined under these conditions. The effects of four factors on growth and germination were systematically examined. These were: seed pretreatments; antibiotics and fungicides; nutrients; and growth substances. Amongst the pretreatments, none surpassed partial hydration of seeds for 24 hr to 0.55 g H2O g-1 dry wt at 25°C prior to embryo dissection. Thiram (2.4 mg mL-1) and kanamycin (50 ug ml1) effectively controlled bacterial and fungal growth with no deleterious effects on growth during culture of the isolated embryos. Exogenous sucrose (optimum 5 % wt/vol) significantly stimulated radicle growth in both deteriorated and non-deteriorated embryos. No other organic or inorganic nutrient stimulated growth. Naphthalene acetic acid did not affect growth while kinetin reduced radicle growth and stimulated coleoptile growth. Gibberellic acid (GA3 at 10-5M) significantly stimulated radicle growth in deteriorated embryos, whereas it promoted coleoptile growth in both deteriorated and non-deteriorated embryos. These data suggest GA or a GA-stimulated process may limit the growth of aged embryos.
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FREITAS, Rodrigo Therezan de, Renato PAIVA, Thais Silva SALES, Diogo Pedrosa Corrêa da SILVA, Michele ValquÃria dos REIS, Ana Cristina de SOUZA, and Sttela Dellyzete Veiga Franco da ROSA. "Cryopreservation of Coffea arabica L. Zygotic Embryos by Vitrification." Notulae Botanicae Horti Agrobotanici Cluj-Napoca 44, no. 2 (December 14, 2016): 445–51. http://dx.doi.org/10.15835/nbha44210553.
Full textAbstract:
As a consequence of the difficulty in conventional coffee seed storage, biotechnological alternatives such as cryopreservation have been investigated. The objective of this study was to develop a protocol for the cryopreservation of Coffea arabica L. (cv. ‘Catuaà Vermelho’ - IAC 144) zygotic embryos by vitrification. For the cryopreservation study, the embryos were immersed in Plant Vitrification Solution 2 at different times (0, 10, 25, 50, 100, and 250 min) and two temperatures (0 and 25 °C). Subsequently, the best thawing time was determined in a water bath (1, 3, 5 minutes or directly in Recovery Solution). An anatomical study was conducted on non-stored and stored embryos, with or without the use of Plant Vitrification Solution 2. The immersion in cryoprotectant solution for 100 min at 0 °C allows embryo cryopreservation. Embryos can be directly thawed in Recovery Solution after storage in liquid nitrogen. It was observed that Plant Vitrification Solution 2 reduced internal water content in the cells, allowing subsequent embryo growth resumption.
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Moriyasu, S., H. Hirayama, K. Sawai, S. Kageyama, S. Aoyagi, H. Shiku, T. Matsue, et al. "204 RELATIONSHIP BETWEEN RESPIRATORY ACTIVITY AND THE PREGNANCY RATE OF BISECTED BOVINE EMBRYOS IN VIVO." Reproduction, Fertility and Development 19, no. 1 (2007): 219. http://dx.doi.org/10.1071/rdv19n1ab204.
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Oxygen consumption is an important indicator of the metabolic activity of living cells, which may provide valuable information for evaluating embryo quality. We have found that the bovine embryos with high oxygen consumption possess stronger potential for further development. However, the relationship between respiratory activity and the pregnancy rate of embryos is still unclear. In this study, we investigated the respiration rates of bisected bovine embryos and the pregnancy rates of demi-embryos after embryo transfer. Compact morula-stage embryos were bisected evenly by micro glass needle. One hundred bisected embryos were incubated for 24 h in embryo culture medium (IVD101; Research Institute for the Functional Peptides, Yamagata, Japan) at 39�C under 5% CO2, 5% O2, 90% N2. After the incubation, demi-embryos were classified into 2 groups: blastocoel-formed (BC) and blastocoel-not-formed (CM) embryos. Oxygen consumption rates of demi-embryos were measured by scanning electrochemical microscopy (SECM; Hokuto Denko Corporation, Tokyo, Japan). Within 3 h after the measurement, 80 demi-embryos were transferred into recipient cows (one demi-embryo/one recipient) at 7–8 days after estrus. Recipient cows were diagnosed for pregnancy by ultrasonography approximately 40 days after estrus. Statistical difference was analyzed by Tukey's post-hoc test and chi-square test. A total of 27 recipient cows became pregnant; the pregnancy rates for cows with CM and BC demi-embryos were 40.6% (13/32) and 29.2% (14/48), respectively. Mean oxygen consumption rates (� 10-14 mol s-1) in pregnant and non-pregnant cows were 0.47 and 0.39 for CM demi-embryos and 0.63 and 0.52 for BC demi-embryos, respectively. Retrospective analysis showed that the respiratory activity of demi-embryos in the pregnant group was higher than those in the non-pregnant group. In particular, the pregnancy rates for demi-embryos with respiratory activity higher than 0.35 in CM and 0.40 in BC groups were 52.0% (13/25) and 35.9% (14/39), respectively. On the other hand, cows with demi-embryos having an oxygen consumption rate under 0.35 in CM (n = 7) and 0.40 in BC (n = 9) groups did not become pregnant. These results demonstrated that bovine demi-embryos with higher respiratory activity showed a high pregnancy rate after embryo transfer. It is generally known that the pregnancy rate after the transfer of bisected embryos is lower than that of whole embryos. The measurement of oxygen consumption by SECM procedures is a useful tool to assess the quality of pre-implantation embryos and may contribute to the improvement of the success rate for bisected embryo transfer.
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Kusakabe, Manabu, Kazuteru Hasegawa, Michito Hamada, Megumi Nakamura, Takayuki Ohsumi, Hirona Suzuki, Tran Thi Nhu Mai, et al. "c-Maf plays a crucial role for the definitive erythropoiesis that accompanies erythroblastic island formation in the fetal liver." Blood 118, no. 5 (August 4, 2011): 1374–85. http://dx.doi.org/10.1182/blood-2010-08-300400.
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Abstract c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the activated protein-1 super family of basic leucine zipper proteins. Despite its overexpression in hematologic malignancies, the physiologic roles c-Maf plays in normal hematopoiesis have been largely unexplored. On a C57BL/6J background, c-Maf−/− embryos succumbed from severe erythropenia between embryonic day (E) 15 and E18. Flow cytometric analysis of fetal liver cells showed that the mature erythroid compartments were significantly reduced in c-Maf−/− embryos compared with c-Maf+/+ littermates. Interestingly, the CFU assay indicated there was no significant difference between c-Maf+/+ and c-Maf−/− fetal liver cells in erythroid colony counts. This result indicated that impaired definitive erythropoiesis in c-Maf−/− embryos is because of a non–cell-autonomous effect, suggesting a defective erythropoietic microenvironment in the fetal liver. As expected, the number of erythroblasts surrounding the macrophages in erythroblastic islands was significantly reduced in c-Maf−/− embryos. Moreover, decreased expression of VCAM-1 was observed in c-Maf−/− fetal liver macrophages. In conclusion, these results strongly suggest that c-Maf is crucial for definitive erythropoiesis in fetal liver, playing an important role in macrophages that constitute erythroblastic islands.
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Buzzo, A. R., A. R. Pupulim, J. Mazucheli, F. V. Meirelles, and I. P. Emanuelli. "168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS." Reproduction, Fertility and Development 24, no. 1 (2012): 196. http://dx.doi.org/10.1071/rdv24n1ab168.
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Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O2, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.
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Copete, Elena, José M. Herranz, Miguel A. Copete, Jerry M. Baskin, and Carol C. Baskin. "Non-deep complex morphophysiological dormancy in seeds of the Iberian Peninsula endemic geophyteMerendera montana(Colchicaceae)." Seed Science Research 21, no. 4 (August 5, 2011): 267–81. http://dx.doi.org/10.1017/s096025851100016x.
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AbstractHeretofore, no detailed account was available on seed dormancy and germination of a member of the Colchicaceae (Liliales). Thus, the primary aim of this study was to do an in-depth investigation of the temperature requirements for dormancy break and germination in seeds ofMerendera montana(Colchicaceae) at the embryo and whole-seed levels under near-natural temperatures in a non-heated frame shade-house and under controlled conditions in the laboratory. Mean embryo length in fresh seeds wasc. 0.57 mm and embryos had to grow to at least 1.30 mm before radicle emergence. Embryos grew to full size and seeds completed germination (radicles emerged) when they were stratified at 28/14°C for 60 d followed by a cool temperature for 60 d and then incubated at a cool temperature for 30 d. The optimum cool stratification temperature for dormancy-break was 10°C. Thus, after the moist pretreatment at 28/14°C+10°C, radicle emergence was>93% at all incubation temperatures (5, 15/4 and 20/7°C). In its natural habitat,M. montanaseeds are dispersed in June, the embryo elongates to full size in autumn and radicles emerge from early November to early February. Although the shoot does not emerge until March and April, it is not physiologically dormant. The shoot emerged from 80% of the radicle-emerged seeds in 13 d at 20/7°C without a previous cold pretreatment. Seeds ofM. montanahave non-deep complex morphophysiological dormancy, C1b1aB-C1a. This is the first study on seeds with complex MPD to show a delay in shoot emergence following root emergence despite the shoot being physiologically non-dormant.
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Isom, S. Clay, Randall S. Prather, and Edmund B. Rucker III. "Enhanced developmental potential of heat-shocked porcine parthenogenetic embryos is related to accelerated mitogen-activated protein kinase dephosphorylation." Reproduction, Fertility and Development 21, no. 7 (2009): 892. http://dx.doi.org/10.1071/rd08268.
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Recently, we demonstrated that a 9-h heat shock of 42°C can have marked stimulatory effects on porcine parthenogenetic embryo development if applied immediately after oocyte activation. Developmental discrepancies between heat-shocked (HS) and non-HS embryos were manifest as early as 3 h after activation, suggesting involvement of maturation promoting factor (MPF) and/or mitogen-activated protein kinase (MAPK). Analysis of cdc2 kinase activity showed that MPF inactivation occurred at similar rates in HS and control embryos upon oocyte activation. However, MAPK dephosphorylation was accelerated in HS embryos compared with controls. Okadaic acid, a protein phosphatase inhibitor, maintained MAPK activity at high levels in both non-HS and HS embryos and sensitised HS embryos to the effects of elevated temperatures. No increase in heat shock proteins was observed in pronuclear-stage HS embryos. These data suggest that the acceleration of development observed in HS porcine parthenogenetic embryos is associated with a precocious inactivation of the MAPK signalling cascade. The faster cleavage divisions observed in HS embryos may be linked physiologically to their enhanced developmental potential in vitro.
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Romek, M., B. Gajda, M. Rolka, and Z. Smorag. "107 DIFFERENCES IN THE INNER MITOCHONDRIAL MEMBRANE POTENTIAL BETWEEN NON-CULTURED AND CULTURED PORCINE EMBRYOS." Reproduction, Fertility and Development 23, no. 1 (2011): 159. http://dx.doi.org/10.1071/rdv23n1ab107.
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In comparison to in vivo derived pig embryos, in vitro culture conditions produce embryos with altered metabolic rates of carbohydrates and fatty acids (Romek M et al. 2010 Theriogenology 74, 265–276), which may compromise embryo viability. Because various energy substrates are metabolized via several aerobic pathways leading to generation of the inner mitochondrial membrane potential (ΔΨm), value of ΔΨm is a key indicator of embryo metabolic activity, closely related to oxygen consumption and cellular energy needs. Therefore, the aim of this study was to compare ΔΨm between non-cultured and cultured pig embryos during early development. The non-cultured embryos were obtained from 6-month-old gilts, whereas those derived in vitro were cultured from zygotes to the appropriate stage in North Carolina State University 23 (NCSU-23) medium supplemented with 4 mg mL–1 of bovine serum albumin. The ΔΨm measurements were carried out on both non-cultured and cultured 4 to 8 cell embryos, morulae, blastocysts and late blastocysts. For this, embryos were labelled with 0.5 μM Mito Tracker Orange CMTMRos (MtOR) for 30 min at 39°C and then with 0.5 μM Mito Tracker Deep Red (MtDR) for 30 min at 10°C. Using a LSM 510 Meta Zeiss confocal microscope, we measured the amounts of fluorescence (IMtOR and IMtDR) emitted from embryos and values of ΔΨm were estimated as the IMtOR/IMtDR ratios. The results were analysed by ANOVA and Tukey's test. From the zygote to morula stages, ΔΨm remained unchanged and did not differ between developmentally matched non-cultured and cultured embryos (P < 0.001). The value of ΔΨm increased significantly (P < 0.05) from 0.90 ± 0.26 arbitrary units (a.u.) for morulae to 3.92 ± 0.63 and 2.06 ± 0.38 a.u. for non-cultured and cultured early blastocysts, respectively. Whereas the mean value of ΔΨm was almost 2 times higher in non-cultured than in cultured early blastocysts, the mitochondrial membrane potential was statistically similar (P < 0.05) in the in vivo derived (2.10 ± 0.37 a.u.) compared to cultured (1.87 ± 0.30 a.u.) blastocysts. The lower ΔΨm in cultured early blastocysts may be explained by several-fold higher glucose concentration in NCSU-23 medium than in the oviductal fluid. It was reported that high levels of glucose decreases the Krebs cycle metabolism of pyruvate, glutamine, and glucose, and reduces oxidation rates of fatty acids in cultured pig embryos in comparison with in vivo counterparts. Hence, this impaired metabolism reflected by decreased ΔΨm may be responsible for insufficient energy production and reduced developmental competence of cultured early blastocysts. Therefore, because embryo-cavitation is a critical event in pig development, further effort should be focused on proper blastocyst culture. Research was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.
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Srirattana, K., C. Laowtammathron, R. Devahudi, S. Imsoonthornruksa, A. Sangmalee, W. Tunwattana, C. Lorthongpanich, et al. "52 EFFECT OF TRICHOSTATIN A ON DEVELOPMENTAL POTENTIAL OF INTER-SPECIES CLONED GAUR (BOS GAURUS) EMBRYOS." Reproduction, Fertility and Development 21, no. 1 (2009): 126. http://dx.doi.org/10.1071/rdv21n1ab52.
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This study was carried out to investigate the effect of trichostatin A (TSA) treatment on interspecies cloned gaur (Bos gaurus) embryos development and implantation rate after transfer to bovine (Bos taurus) recipients. The bovine (Bos taurus) enucleated oocytes were used as recipient cytoplasm for male and female gaur fibroblasts. After electrical fusion, oocytes were separated into two groups, TSA treatment and control. For the TSA group, the oocytes were placed in EmCare (ICPbio, Ltd., Auckland, New Zealand) holding medium + 50 nm TSA for 1 h. The fused oocytes were activated by 7% ethanol + 50 nm TSA for 5 min at room temperature and 10 μg mL–1 cycloheximide + 1.25 μg mL–1 cytochalasin D + 50 nm TSA at 38.5°C under 5% CO2 in air for 5 h. Then the embryos were cultured in mSOFaa medium + 3 mg mL–1 bovine serum albumin (BSA) + 50 nm TSA up to 10 h. After 10 h, the reconstructed embryos were transferred to embryo culture medium without TSA and culture for 2 days at 38.5°C under 5% CO2, 5% O2, 90% N2. The control embryos were cultured with the same culture system without TSA supplementation. Eight-cell stage embryos were selected and co-cultured with bovine oviductal epithelial cells in culture medium at 38.5°C under 5% CO2 in air for 5 days. Half volume of the culture medium was replaced daily. Two blastocysts at days 7 or 8 derived from male fibroblasts of treated and non-treated TSA were non-surgically transferred to each synchronized estrous bovine recipients. The statistical analysis was done by ANOVA and the comparison of means by Duncan’s Multiple Range Test (DMRT). The development to blastocyst stage was not different among male and female, treated and non-treated TSA embryos which range between 34.8 to 39.3%. The pregnancy rate at 40 days after recipients received cloned embryos derived from male fibroblasts treated v. non-treated TSA was 11% (2/18) v. 10% (1/10) (Table 1). One recipient which received a non-treated embryo gave birth by C-section on March 4, 2008. The male gaur calf died from respiratory problem at 12 h after birth. Eight bovine microsatellite markers analysis confirmed that the newborn gaur was derived from the donor gaur fibroblast. In this study, TSA has no effect on pre-implantation cloned gaur embryos development either derived from male or female gaur fibroblasts. Cloned gaur calves could be produced by interspecies cloning using bovine oocytes as recipient cytoplasm. Table 1.Pregnancy and birth rates after transferred cloned gaur embryos derived from male fibroblasts to recipients This study was supported by National Center for Genetic Engineering and Biotechnology (BIOTEC) and Suranaree University of Technology.
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Martino, N. A., G. Marzano, A. Mastrorocco, G. M. Lacalandra, L. Vincenti, K. Hinrichs, and M. E. Dell'Aquila. "Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection." Reproduction, Fertility and Development 31, no. 12 (2019): 1862. http://dx.doi.org/10.1071/rd19223.
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Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.
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Hidayati, Siti N., David J. Merritt, Shane R. Turner, Kingsley W. Dixon, and Jeffrey L. Walck. "Temporal dynamics of seedling emergence among four fire ephemerals: the interplay of after-ripening and embryo growth with smoke." Seed Science Research 29, no. 2 (June 2019): 104–14. http://dx.doi.org/10.1017/s0960258519000084.
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AbstractThe flora of Mediterranean ecosystems contains families with species having fully and under-developed embryos in their seeds. After-ripening for physiological dormancy release and smoke influence germination in many species. We investigated how after-ripening and embryo growth interact with smoke to influence the temporal dynamics of seedling emergence among fire ephemerals. Seeds were placed in the field and under standardized (50% relative humidity, 30°C) laboratory conditions to test the effects of summer conditions on physiological dormancy loss. Germination was tested with water or smoke compounds (smoke water, KAR1) at a simulated autumn/winter temperature (18/7°C). The timing and amount of seedling emergence with smoke was observed for seeds exposed to near-natural conditions. During summer, physiological dormancy was broken in all species, enabling germination at autumn/winter but not summer temperatures; no embryo growth occurred in seeds with under-developed embryos. At the start of the wet season, seedling emergence from seeds with fully developed embryos occurred earlier than from seeds with under-developed embryos. In a non-consistent manner among our study species, smoke and smoke compounds influenced the rate of embryo growth and amount of germination. Effects of smoke were noticeable in terms of number of emergents in the first emergence season. Among ecologically similar species, we have shown (1) that both thermal and embryo traits exclude germination in the summer, (2) how embryo size influences the timing of seedling emergence in autumn–winter, and (3) a reduced requirement for smoke in the second emergence season after a fire with a shift to reliance on seasonal cues for emergence.
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Polak, Michal, Leigh W. Simmons, Joshua B. Benoit, Kari Ruohonen, Stephen J. Simpson, and Samantha M. Solon-Biet. "Nutritional geometry of paternal effects on embryo mortality." Proceedings of the Royal Society B: Biological Sciences 284, no. 1864 (October 11, 2017): 20171492. http://dx.doi.org/10.1098/rspb.2017.1492.
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Well-established causal links exist between maternal nutritional deficits and embryo health and viability. By contrast, environmental effects operating through the father that could influence embryo mortality have seldom been examined. Yet, ejaculates can require non-trivial resource allocation, and seminal plasma components are increasingly recognized to exert wide-ranging effects on females and offspring, so paternal dietary effects on the embryo should be expected. We test for effects of varying levels of protein (P), carbohydrate (C) and caloric load in adult male diet on embryo mortality in Drosophila melanogaster . We demonstrate that macronutrient balance and caloric restriction exert significant effects, and that nutritional effects are more impactful when a prior mating has occurred. Once-mated males produced embryos with marginally elevated mortality under high-caloric densities and a 1 : 8 P : C ratio. In contrast, embryos produced by twice-mated males were significantly more likely to die under male caloric restriction, an outcome that may have resulted from shifts in ejaculate quality and/or epigenetic paternal effects. Body nutrient reserves were strongly and predictably altered by diet, and body condition, in turn, was negatively related to embryo mortality. Thus, sire nutritional history and resultant shifts in metabolic state predict embryo viability and post-fertilization fitness outcomes.
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Behluli, B., M. Jahnke, J. K. West, and C. R. Youngs. "99 BIRTH OF THE FIRST BOVINE EMBRYO TRANSFER CALF IN THE REPUBLIC OF KOSOVA." Reproduction, Fertility and Development 29, no. 1 (2017): 157. http://dx.doi.org/10.1071/rdv29n1ab99.
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The objective of this applied field study was to assess the feasibility of successfully performing bovine embryo transfer in the Republic of Kosova—a feat that had not yet been accomplished in this newly independent (2008) eastern European country. Three Holstein heifers at the Iowa State University dairy farm were superovulated with a conventional descending dose regimen of FSH (Folltropin). Approximately 12 and 24 h after the observed onset of oestrus, heifers were inseminated with semen from a single Red Holstein bull. Embryos were non-surgically collected and washed in accordance with IETS procedures for sanitary handling of embryos. Embryos were cryopreserved for subsequent direct transfer. After obtaining an import permit from the Kosovo Food and Veterinary Agency, embryos were approved for export to the Republic of Kosova by the US Department of Agriculture, Animal Plant Health Inspection Service. Embryos were shipped via an express courier service. A total of 19 embryos were received in the Republic of Kosova. Recipients were monitored for signs of naturally occurring oestrus, and immediately before transfer, embryos were thawed by holding in air for 3 to 5 s followed by placement into a 37°C water bath for 25 to 30 s. The first-ever bovine embryo transfer calf in the Republic of Kosova was born July 6, 2015. A total of 9 calves were born from the 19 embryos transferred (47.4% embryo survival rate). Results of this applied field study show that bovine embryo transfer is feasible in the Republic of Kosova. Embryo transfer will be used to improve the quality of dairy cattle genetics in the Republic of Kosova and to subsequently increase the national supply of milk, decrease dependence on milk imports, and increase food security of the nation.
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Takagi, Y., M. Shimizu, M. Morimura, S. Yokomizo, K. Hara, and M. Sakamoto. "135 SHORT-TERM CRYOPRESERVATION OF VITRIFIED MOUSE MORULAE AT - 79°C." Reproduction, Fertility and Development 19, no. 1 (2007): 185. http://dx.doi.org/10.1071/rdv19n1ab135.
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Embryos of various species are successfully vitrified and cryopreserved in liquid nitrogen (<−150°C). Like the preservation of frozen somatic cells cooled by dry ice (−79°C), the cryopreservation of embryos at −79°C is useful for a reduction in the shipping costs. The purpose of this study was to evaluate the effect of the cryopreservation period at −79°C on the in vitro embryo viability of vitrified mouse morulae after thawing. Morula-stage mouse embryos were collected from superovulated ICR donors 70 h after hCG injection. The embryos were exposed first to 5% DMSO + 5% ethylene glycol (EG) in Dulbecco's PBS + 20% FCS (mPBS) for 2 min, and then equilibrated for 20–30 s in a vitrification solution composed of 10% DMSO + 10% EG + 0.6 M sucrose in mPBS. The embryos were loaded onto cryoloops (Lane et al. 1999 Nat. Biotech. 17, 1234–1236) and plunged directly into liquid nitrogen. The cryoloops were placed in 1.2-mL cryotubes and stored in a −79°C freezer for 1–7 days. The embryos were warmed by passing through 4 dilution media and rinsed with mWM culture medium. They were then cultured at 37°C in 5% CO2 for 44 h. Non-cryopreserved embryos and embryos cryopreserved in liquid nitrogen served as controls. Data were analyzed by the chi-square test and the Student's t-test. Results are shown in Table 1. There was no significant difference (P > 0.01) in the developmental abilities to the blastocyst stage of the vitrified embryos that were cryopreserved at −79°C for 1 day, 3 days, and 5 days, the embryos cryopreserved in liquid nitrogen, and the non-vitrified control. The blastocyst rate of embryos was significantly lower (P < 0.01) for the Day 7 group than for the control group. The cell numbers of blastocysts were significantly lower (P < 0.01) for the Day 1, Day 3, Day 5, and Day 7 groups than for the control group. This study suggests that vitrified mouse morulae can be successfully cryopreserved at −79°C for 5 days. Table 1. Effect of the cryopreservation period on the viability of vitrified mouse morulae preserved at −79°C
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Razza, E. M., I. P. Emanuelli, C. M. Barros, and M. F. G. Nogueira. "142 CHIMERISM BY AGGREGATION OF BISECTED IN VITRO PRODUCED BOVINE EMBRYOS." Reproduction, Fertility and Development 23, no. 1 (2011): 175. http://dx.doi.org/10.1071/rdv23n1ab142.
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Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).
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Liu, Zhuolin, Mingyue Li, Meiru Zhu, Rosana López, Roberto L. Salomón, and Peng Zhang. "Thermodormancy and Germination Response to Temperature of Pyrus ussuriensis Seeds." Agronomy 14, no. 3 (February 27, 2024): 475. http://dx.doi.org/10.3390/agronomy14030475.
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To determine the optimal germination temperature for Pyrus ussuriensis seeds and whether they experienced the phenomenon of thermodormancy and its inciting factors, several germination tests were conducted using non-dormant P. ussuriensis seeds for comparison. The results showed that the highest germination rate of P. ussuriensis seeds was reached at a constant temperature of 5 °C and variable temperature (night/day) of 5 °C/10 °C. Constant temperatures of 25 °C for three days induced thermodormancy, triggering significant drops in seeding emergence. Thermodormancy was related to the inhibitory effect of endogenous substances in the seed coat and an elevated abscisic acid concentration. The embryo, by contrast, remained non-dormant. Thermodormant and non-dormant seed embryos showed higher germination rates than dormant seed embryos when applied exogenous abscisic acid and gibberellic acid. We found that P. ussuriensis seeds showed thermodormancy; thus, during early spring sowing, high temperatures should be avoided to prevent low seed germination capacity. Additionally, applying exogenous gibberellic acid, shading and increasing soil moisture can be helpful to enhance the species seed germination.
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Wang, H., R. Q. Li, X. L. Zhang, Q. Z. Pan, J. L. Li, M. B. Zhao, Y. Z. Wang, and D. Y. Chen. "393 A STUDY OF THE USE OF SEXED SEMEN IN SUPERSTIMULATED HOLSTEIN COWS." Reproduction, Fertility and Development 19, no. 1 (2007): 312. http://dx.doi.org/10.1071/rdv19n1ab393.
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A study was designed to investigate the use of sexed semen in superstimulated Holstein cows. Non-lactating Holstein cows (n = 107; those that did not superovulate were excluded) were superstimulated and inseminated with frozen–thawed, sexed semen (at 1 insemination dose) or control semen (at 2 different insemination doses) in 3 groups. Cows received a Cue-Mate (Bioniche Animal Health, Beijing, China) and were injected with 5 mg of estradiol-17β plus 100 mg of progesterone on Day 0. Cows were treated from Days 4 to 7 with decreasing doses of FSH for a total of 300 mg Folltropin-V (Bioniche; 50, 50, 40, 40, 30, 30, 30, and 30 mg) at 12 h intervals. On Day 7, Cue-Mates were removed and cows were injected twice with 0.4 mg cloprostenol (PGF; Institute of Family Planning, Shanghai, China). Bilateral deep uterine horn AI was performed 12 and 24 h after the onset of estrus on Day 9 (Group A: 4 × 106 sexed, frozen–thawed sperm per insemination from 1 of 3 bulls; Group B: 4 × 106 non-sexed, frozen–thawed sperm, or Group C: 10 × 106 non-sexed, frozen–thawed sperm per insemination from 1 of 3 bulls). Cows were assigned to bulls and insemination groups at random. Embryos were collected and evaluated on Day 16. Group A had a significantly lower fertilization and usable embryo (IETS grades 1 and 2) rate than the other 2 groups (Table 1). Although the rate of usable embryos between Groups B and C did not differ, the fertilization rate in Group C was significantly higher, suggesting that 4 million sperm in Group B was an inadequate insemination dose for superstimulated cattle. The significantly lower number of usable embryos in Group A suggests that the use of sexed sperm in superovulation and embryo transfer may not be economically feasible. More research is required to optimize and standardize bovine superstimulation and AI with sexed semen. Table 1. Embryo production following insemination of superstimulated Holstein cows with frozen–thawed sexed (Group A) or unsexed (Groups B and C) semen
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Barceló-Fimbres, M., and G. E. Seidel Jr. "122 EFFECTS OF EMBRYO SEX AND GLUCOSE OR FRUCTOSE IN CULTURE MEDIA ON BOVINE EMBRYO DEVELOPMENT." Reproduction, Fertility and Development 20, no. 1 (2008): 141. http://dx.doi.org/10.1071/rdv20n1ab122.
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The objective of the present study was to determine if glucose, fructose, or both, in culture medium affects female embryos differently from male embryos. Embryos of predetermined sex were produced by use of sexed semen. Blastocysts were produced in vitro in a factorial design from 1851 oocytes fertilized with 3 sperm types [non-sexed control, X-bearing, and Y-bearing (~95% accuracy)]. Semen from 3 bulls (A, B, and C) was used, with 2 replicates for each bull. Zygotes were cultured with 4 energy substrates (no-hexose control, glucose, fructose, and glucose + fructose). Chemically defined medium (CDM) plus 0.5% fatty acid-free BSA (De La Torre-Sanchez et al. 2006 Mol. Reprod. Dev. 18, 585–596) was the culture medium to which the energy substrates were added. For the first 2.5 d of culture, the medium contained 0.5 mm hexose, and for the last 4.5 d, it contained 2.0 mm hexose. Data were analyzed by ANOVA. There was no effect of hexose treatments on cleavage rates (P > 0.1; Table 1). However, blastocyst rates per oocyte were higher for the fructose treatment than for the control, glucose, or glucose + fructose (P < 0.05) groups. Glucose treatment resulted in retarded development compared with no hexose or fructose (P < 0.05). Analysis of a subset of data with only the fructose and glucose treatments resulted in an interaction of hexose (glucose and fructose) and X-bearing andY-bearing sperm for blastocysts per oocyte: fructose X-sperm 20.4%a; fructoseY-sperm 17.7%ab; glucose X-sperm 10.6%bc, and glucoseY-sperm 14.7%c (means without common superscripts differ; P < 0.05). Glucose was detrimental to female embryos, and fructose improved development to the blastocyst stage for both sexes. Lower cleavage rates were found after fertilization with X-bearing sperm (Table 1); however, blastocyst rates per oocyte were not different after fertilization with X- or Y-bearing sperm or non-sexed sperm (P > 0.1). There was no bull effect on rates of cleavage or blastocysts per oocyte (P > 0.1). In conclusion, fructose improved embryonic development of embryos of both sexes; glucose retarded development of, and was toxic to, female embryos. Sexed sperm resulted in similar cleavage and blastocyst production as non-sexed sperm, and female embryos had retarded embryonic development relative to male embryos. Table 1. Main effect least-squares means for development of bovine embryos (±SE)