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1
Gao, Y., L. Cheng, G. Su, Z. Wei, and G. Li. "55 STUDY ON THE INTERSPECIFIC NUCLEAR TRANSFER OF PRZEWALSKI'S GAZELLES AND BOVINES." Reproduction, Fertility and Development 25, no. 1 (2013): 175. http://dx.doi.org/10.1071/rdv25n1ab55.
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Przewalski’s gazelle (Procapra przewalskii), also known as Platts antelope, is an endangered species only found in China. It belongs to the Artiodactyla order, Bovidae family, antelope subfamily, and Gazella genus. In this study, 5 experiments were designed to examine the developmental potential of Przewalski’s gazelle somatic cells transplanted into bovine enucleated oocytes. Enucleation was conducted by Hoechst 33342 staining of the oocytes and guided by a fluorescent microscope to ensure the removal of the nuclei. The gazelle cells were then transferred to the enucleated oocytes and electrically fused to reconstructed embryos. The study resulted in 5 major findings. (1) When gazelle-bovine reconstructed embryos were treated with the deacetylase inhibitor valproic acid (VPA), at different concentrations and for different times, treatment of the cloned embryos with VPA at 0.5 mM for 24 h significantly increased the 8- to 16-cell-stage embryo development [61.9% (96/155) v. 33.8% (46/136) control]. However, the morula [1.3% (2/155) v. 1.5% (2/155); P > 0.05] and blastocyst (0.7% v. 1.5%; P > 0.05) development were similar to that of the control. In the intraspecific (bovine-bovine) control group, the cleavage, morula and blastocyst development of 3 cloned embryos were 72.6% (127/175), 28.0% (49/175), and 23.4% (41/175). (2) Octamer-binding transcription factor 4 (Oct-4), as a developmental potential and expression marker, was transfected to gazelle cells. When Oct-4-eGFP-confected cells were transferred, the cloned embryo development did not improve either with or without VPA treatment. (3) When the gazelle-bovine embryos were treated with the deacetylase inhibitor trichostatin A (TSA) for 24 h at 10 ng mL–1, blastocyst development was significantly higher than in the control group [3.6% (6/168) v. 0.8% (1/125); P < 0.05]. (4) When a reverse NT protocol, in which the oocyte nucleus was removed after the cell nucleus was fused to the oocyte, was used for NT, the cloned embryo development did not improve. (5) The gazelle-bovine and bovine-bovine cloned embryos at 8- to 16-cell stages, gazelle cells, bovine cells, and bovine oocytes transcriptomes were analyzed by Affymetrix microarray (Affymetrix Microarray Inc., Santa Clara, CA, USA) and repeated twice. A total of 643 genes were activated in gazelle-cattle embryos compared with oocytes, whereas 1527 genes were activated in bovine-bovine clones. A total of 1010 genes that were exclusively expressed in gazelle somatic cells were still expressed in the interspecies cloned embryos. In conclusion, TSA treatment of Przewalski’s gazelle somatic cells transferred into enucleated bovine oocytes improved development of cloned embryos to the blastocyst stage, although still with low efficiency. Data from microarray analyses of the gazelle-cattle embryos showed that over 1000 gazelle-specific genes were still expressed in the interspecific cloned embryos. This work was supported by the National Basic Research Program of China (no. 2012CB22306).
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Duszewska, Anna Maria, Magdalena Baraniewicz-Kołek, Jarosław Wojdan, Katarzyna Barłowska, Wojciech Bielecki, Paweł Gręda, Wojciech Niżański, and Wanda Olech. "Establishment of a Wisent (Bison bonasus) Germplasm Bank." Animals 12, no. 10 (May 11, 2022): 1239. http://dx.doi.org/10.3390/ani12101239.
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The wisent, or European bison (Bison bonasus), belongs to the same family (Bovidae) as the American bison and domestic cattle. The wisent is the largest mammal in Europe, and is called the “Forest Emperor”. The wisent is listed as “Vulnerable” on the IUCN Red List, and is protected by international law. Achievements in reproductive biotechnology have opened new possibilities for the cryoconservation of the wisent germplasm. Therefore, this research aimed to improve a strategy for the protection and preservation of the European bison through the creation of a wisent germplasm bank, based on the following procedures: isolation and in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) of matured oocytes, in vitro embryo culture (IVC), and embryo cryopreservation. Wisent ovaries were isolated from females outside the reproductive season, and eliminated from breeding for reasons other than infertility. Cumulus–oocyte complexes (COCs) were isolated from follicles greater than 2 mm in diameter and matured for 24 h and 30 h. After IVM, COCs were fertilized in vitro with wisent sperm. The obtained wisent zygotes, based on oocytes matured for 24 h and 30 h, were cultured for 216 h. Embryos at the morula and early blastocyst stages were vitrified and then warmed and transferred to interspecies recipients (Bos taurus). USG and biochemical tests were used to monitor pregnancies. This study obtained embryos in the morula and early blastocyst stages only after oocytes were fertilized and matured for 30 h. On average, per oocyte donor, 12.33 ± 0.5 COCs were isolated, and only 9.33 ± 0.61 COCs were qualified for in vitro maturation (75.68%), while 9.16 ± 0.48 COCs were matured (84.32%). On average, per donor, 5.5 ± 0.34 embryos were cleaved (59.96%) after 48 h post-fertilization (hpf), and 3.33 ± 0.21 achieved the eight-cell stage (36.52%) after 96 hpf, while 1 ± 0.21 morula and early blastocyst stages (10.71%) were achieved after 216 hpf. A total of six embryos (one morula and five early blastocysts) were obtained and vitrified; after warming, five of them were interspecies transferred to cattle (Bos taurus). On day 41 after fertilization, 3 out of 5 pregnancies were detected based on USG, P4, and PAG tests. However, no pregnancy was observed on day 86 after fertilization, indicating embryo resorption. This study shows that obtaining wisent embryos in vitro, and subsequent cryopreservation to create a wisent embryo bank, can be applied and implemented for the wisent protection program.
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Krisher, R., A. Auer, K. Clark, K. Emsweller, S. Rogers, K. Thomas, F. Chatiza, and P. Bartels. "246 IN VITRO PRODUCTION OF SPRINGBOK (ANTIDORCAS MARSUPIALIS) EMBRYOS." Reproduction, Fertility and Development 19, no. 1 (2007): 239. http://dx.doi.org/10.1071/rdv19n1ab246.
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The objective of this experiment was to develop in vitro embryo production (IVP) technologies in springbok (Antidorcas marsupialis), a southern African antelope. Springbok, a fairly common species on game farms in parts of South Africa, may be used as a model species for gamete rescue and IVP techniques to be applied to the conservation of other threatened antelope species. Springbok belong to the family bovidae, subfamily antilopinae, tribe antilopini, which comprises about twenty species in genera Gazella, Antilope, Procapra, Antidorcas, Litocranius, and Ammodorcas. In this tribe alone, there are 4 species or subspecies that are critically endangered, 3 that are endangered, and 10 that are considered vulnerable, demonstrating the need for antelope conservation efforts. In addition, our studies contributed to the South African biological resource bank, so that banked springbok semen and embryos might be used in the future for managed genetic contribution to isolated captive or wild populations via assisted reproductive technologies. Oocytes were recovered (3 replicates) from ovaries obtained at supervised culls for management purposes in South Africa, and cultured in defined Gmat or undefined TCM-199 with FCS maturation medium for 28-30 h (Brad et al. 2004 Reprod. Fertil. Dev. 16, 223). Oocytes were fertilized with frozen-thawed springbok epididymal spermatozoa in modified SOF fertilization medium with caffeine (Herrick et al. 2004 Biol. Reprod. 71, 948–958). Eighteen hours after insemination, a randomly selected subset of the zygotes were fixed to determine fertilization success. The remaining zygotes were cultured in G1/G2 media. On Day 7 of culture, embryos were analyzed for development to the morula or blastocyst stage. A total of 259 selected oocytes were collected from 50 females (5.2 selected oocytes/female on average). There was no difference in the percentage of oocytes normally fertilized (2 pronuclei, PN) between oocytes matured in Gmat (n= 43; 12%) and those matured in TCM-199 (n= 42; 10%). There were significantly (P < 0.05) more oocytes penetrated (e2 PN) when matured in TCM (50%) compared to Gmat (23%). There were no differences in embryonic cleavage or morula/blastocyst development (of total oocytes inseminated) between treatments (Gmat,n= 89, 54%, 9.0%; TCM-199, n= 85, 68%, 9.4%, respectively). In both treatments, the average blastocyst grade was 2.125 using the standard bovine grading system (Curtis, Cattle Embryo Transfer Procedure, 1991). In conclusion, in vitro oocyte maturation, fertilization, and embryo culture to the blastocyst stage is possible in springbok. Importantly, blastocysts can be produced in vitro under semi-defined conditions, demonstrating that oocyte maturation without serum does support developmental competence. This is important for the potential international movement of IVP embryos to be used for genetic management in the conservation of antelope species.
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Farrar, N. C., M. E. Staines, G. J. McCallum, P. Haggarty, J. J. Robinson, and T. G. McEvoy. "Effects of serum or fatty acid supplementation of synthetic oviduct fluid medium on development of bovine embryosin vitro." Proceedings of the British Society of Animal Science 1999 (1999): 62. http://dx.doi.org/10.1017/s1752756200002179.
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Serum, which is routinely included in many embryo culture media, can decrease the viability of bovine and ovine embryos produced in cultures employing synthetic oviduct fluid (SOF; Kuran et al., 1999) and represents a possible route for transmission of disease. Alternative approaches include the use of chemically defined culture media but results from studies which avoid sera and its derivatives (e.g., albumin) are generally less favourable due to a lack of knowledge regarding the embryo's response to specific nutrients, most notably fatty acids. As a preliminary step towards investigating fatty acid influences on bovine embryo developmentin vitro, the present study examined the effect of adding palmitic acid (C16:0) to SOF plus bovine serum albumin (BSA) on the performance of this semi-defined culture medium and contrasted it with embryo production in SOF supplemented with serum.
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Wang, Shujuan, Baoru Liu, Wenju Liu, Yao Xiao, Hualin Zhang, and Liguo Yang. "The effects of melatonin on bovine uniparental embryos developmentin vitroand the hormone secretion of COCs." PeerJ 5 (July 7, 2017): e3485. http://dx.doi.org/10.7717/peerj.3485.
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Melatonin is a unique multifunctional molecule that mediates reproductive functions in animals. In this study, we investigated the effects of melatonin on bovine parthenogenetic and androgenetic embryonic development, oocyte maturation, the reactive oxygen species (ROS) levels in parthenogenetic and androgenetic embryos and cumulus—oocyte complexes (COCs) hormone secretion with melatonin supplementation at four concentrations (0, 10, 20, and 30 pmol/mL), respectively. The results showed that melatonin significantly promoted the rates of bovine parthenogenetic and androgenetic embryonic cleavage and morula and blastocysts development (P < 0.05). The rate of cleavage was higher in the androgenetic embryo than that in the parthenogenetic embryo. Compared with the parthenogenetic embryos, the androgenetic embryos had a poor developmental competence from morula to blastocyst stage. Moreover, the levels of ROS were significantly lower in the parthenogenetic and androgenetic embryoes with melatonin-treated group than that of the control group (P < 0.05). Melatonin supplemented significantly increased the maturation rate of oocytein vitro(P < 0.05). More importantly, melatonin significantly promoted the secretion of progesterone and estradiol by COCs (P < 0.05). To reveal the regulatory mechanism of melatonin on steroids synthesis, we found that steroidogenic genes (CYP11A1, CYP19A1andStAR) were upregulated, suggesting that melatonin regulated estradiol and progesterone secretion through mediating the expression of steroidogenic genes (CYP11A1,CYP19A1andStAR). In addition, MT1 and MT2 were identified in bovine early parthenogenetic and androgenetic embryos using western blot. It could be concluded that melatonin had beneficial effects on bovine oocytein vitromaturation, COC hormone secretion, early development of subsequent parthenogenetic and androgenetic embryos. It is inferred that melatonin could be used to enhance the efficiency ofin vitrodeveloped embryos.
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Wells, Cara, Anders Wiik, John Hanks, Amir Zavareh, and Russell Killingsworth. "Embryo Morphokinetic Activity Evident in Short Videos of In Vitro Bovine Embryos." Dairy 3, no. 4 (November 23, 2022): 849–61. http://dx.doi.org/10.3390/dairy3040058.
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Embryo transfer (ET) and in vitro fertilization (IVF) are increasing in use by dairy producers as a means to breed their animals as these assisted reproductive techniques can optimize the genetics of the dairy breed or enable “beef on dairy” programs to increase the profitability of the dairy. Due to the advantages of ET and IVF, it is anticipated that their use will continue to increase despite the status of underwhelmingly low pregnancy outcomes. Pregnancy rates of bovine ET/IVF remain below 56%, with many dairy producers implementing beef on dairy programs reporting pregnancy to be lower than 23%. The inability to objectively evaluate embryo health prior to transfer into a recipient is a contributing factor to this problem as 20% of transferred embryos are inviable at the time of transfer and have little chance of establishing a pregnancy. The objective of this research was to evaluate bovine embryo real-time morphokinetic activity based on 30 s video recordings of day 7.5 morulas and correlate morphokinetic activity to developmental outcomes. Eighty-eight embryos were recorded in standard embryo culture conditions with an SMZ-1000 Stereo zoom microscope and TE-300 Nikon inverted microscope. The difference in the embryo’s morphokinetic activity was measured frame-by-frame and correlated to embryo hatching outcomes. It was found that embryos with lower morphokinetic activity demonstrated higher hatching rates and developmental outcomes, suggesting measurement of embryo morphokinetic activity is a noninvasive and non-subjective method to evaluate embryo competency prior to transfer and can be used to improve the reproductive efficiency and profitability of IVF/ET of dairy cattle.
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Yaacobi-Artzi, Shira, Dorit Kalo, and Zvi Roth. "Association between the morphokinetics of in-vitro-derived bovine embryos and the transcriptomic profile of the derived blastocysts." PLOS ONE 17, no. 10 (October 26, 2022): e0276642. http://dx.doi.org/10.1371/journal.pone.0276642.
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The time-lapse system is a non-invasive method that enables a continuous evaluation through embryo development. Here, we examined the association between the morphokinetics of the developing embryo and the transcriptomic profile of the formed blastocysts. Bovine oocytes were matured and fertilized in vitro; then, the putative zygotes were cultured in an incubator equipped with a time-lapse system. Based on the first-cleavage pattern, embryos were categorized as normal or abnormal (68.5±2.2 and 31.6±2.3%, respectively; P<0.001). A cleaved embryo was defined as normal when it first cleaved into two equal blastomeres; it was classified as synchronous or asynchronous according to its subsequent cleavages. An abnormal pattern was defined as direct, unequal, or reverse cleavage. Direct cleavage was classified as division from one cell directly into three or more blastomeres; unequal cleavage was classified as division that resulted in asymmetrically sized blastomeres; and reverse cleavage of the first division was classified as reduced number of blastomeres from two to one. Of the normally cleaving embryos, 60.2±3.1% underwent synchronous cleavage into 4, 8, and 16 blastomeres, and 39.7±3.1% cleaved asynchronously (P<0.001). The blastocyte formation rate was lower for the synchronously vs. the asynchronously cleaved embryos (P<0.03). The abnormally cleaved embryos showed low competence to develop to blastocysts, relative to the normally cleaved embryos (P<0.001). Microarray analysis revealed 895 and 643 differentially expressed genes in blastocysts that developed from synchronously and asynchronously cleaved embryos, respectively, relative to those that developed from directly cleaved embryos. The genes were related to the cell cycle, cell differentiation, metabolism, and apoptosis. About 180 differentially expressed genes were found between the synchronously vs. the asynchronously cleaved embryos, related to metabolism and the apoptosis mechanism. We provide the first evidence indicating that an embryo’s morphokinetics is associated with the transcriptome profile of the derived blastocyst, which might be practically relevant for the embryo transfer program.
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Choe, C. Y., S. R. Cho, J. K. Son, S. H. Choi, C. Y. Cho, J. B. Kim, S. J. Kim, D. Kang, and D. S. Son. "145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE." Reproduction, Fertility and Development 22, no. 1 (2010): 231. http://dx.doi.org/10.1071/rdv22n1ab145.
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Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.
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Suwik, Katarzyna, Emilia Sinderewicz, Dorota Boruszewska, Ilona Kowalczyk-Zięba, Joanna Staszkiewicz-Chodor, Krzysztof Łukaszuk, and Izabela Wocławek-Potocka. "mRNA Expression and Role of PPARγ and PPARδ in Bovine Preimplantation Embryos Depending on the Quality and Developmental Stage." Animals 10, no. 12 (December 10, 2020): 2358. http://dx.doi.org/10.3390/ani10122358.
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Peroxisome proliferator-activated receptors (PPARs), a nuclear receptors for prostacyclin (PGI2) have been recognized as being essential for early embryo development. The objectives of the present study were to determine if the bovine early- and late-cleaved embryos in different stages of early development express PPARγ and PPARδ. Since embryo developmental competence depends on numerous biological factors, we evaluated if the expression of PPARγ and PPARδ correlate with selected embryo quality markers (SOX2, OCT4, PLAC8, IGF1R) in the in vitro produced embryos at different stages of their development. Developmental rates and embryo quality for early- and late-cleaved embryos were provided according to International Embryo Transfer Society (IETS; developmental stages: 2-, 4-, 16-cell embryo, morula, blastocyst (1—early, 2—developing, 3—expanded, 4—hatched); quality stages: A—high quality, B—moderate quality, C—low quality). We found that bovine embryos expressed mRNA of PPARδ and PPARγ at all stages of early development, independently of their quality. In addition, the expression of PPARδ and PPARγ correlated with the expression of quality markers in bovine blastocysts. Positive correlations were stronger and more frequent in the group of early-cleaved embryos, whereas the negative correlations were typical for the group of late-cleaved embryos. Obtained results and available literature reports may indicate the participation of PGI2, via PPARδ and PPARγ, in the processes related to the early embryo development, through the participation of this factor in the modulation of blastocyst hatching, implantation, and post-implantation development.
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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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Gutierrez-Adan, Alfonso, Carlee R. White, Ann Van Soom, and Mellissa R. W. Mann. "Why we should not select the faster embryo: lessons from mice and cattle." Reproduction, Fertility and Development 27, no. 5 (2015): 765. http://dx.doi.org/10.1071/rd14216.
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Many studies have shown that in vitro culture can negatively impact preimplantation development. This necessitates some selection criteria for identifying the best-suited embryos for transfer. That said, embryo selection after in vitro culture remains a subjective process in most mammalian species, including cows, mice and humans. General consensus in the field is that embryos that develop in a timely manner have the highest developmental competence and viability after transfer. Herein lies the key question: what is a timely manner? With emerging data in bovine and mouse supporting increased developmental competency in embryos with moderate rates of development, it is time to question whether the fastest developing embryos are the best embryos for transfer in the human clinic. This is especially relevant to epigenetic gene regulation, including genomic imprinting, where faster developing embryos exhibit loss of imprinted methylation, as well as to sex selection bias, where faster developmental rates of male embryos may lead to biased embryo transfer and, in turn, biased sex ratios. In this review, we explore evidence surrounding the question of developmental timing as it relates to bovine embryo quality, mouse embryo quality and genomic imprint maintenance, and embryo sex.
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Herholdt, G. J. M., and D. M. Barry. "139 BOVINE AMNIOTIC FLUID FOR THE CULTURE OF TWO-CELL MURINE EMBRYOS." Reproduction, Fertility and Development 17, no. 2 (2005): 220. http://dx.doi.org/10.1071/rdv17n2ab139.
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The objective of the experiment was to evaluate bovine amniotic fluid as an alternative medium for culture of two-cell murine embryos. Variability in embryo development rate and high purchase prices of medium supplements such as fetal bovine serum have prompted the search for a serum alternative. Amniotic fluid was collected postmortem from first-trimester bovine fetuses, pooled, heat inactivated at 56°C for 30 min and stored at −20°C until used. Two-cell mouse embryos were collected from (Balb/C × C57BL6) F1 females superovulated with 10 IU PMSG and 10 IU hCG. The experiment was performed to evaluate the effect of commercial fetal bovine serum (F4135, Sigma, South Africa) supplementation to amniotic fluid. Treatments consisted of (1) bovine amniotic fluid, (2) bovine amniotic fluid supplemented with 10% fetal bovine serum, (3) M16 (M7292, Sigma) supplemented with 10% fetal bovine serum, and (4) Medium 199 (M4530, Sigma) supplemented with 10% fetal bovine serum. The latter two media were controls. Twenty-four hours before use the culture media were supplemented with 1% antibiotic antimycotic solution (A5955, Sigma). Media were equilibrated for 24 hours at 37°C and 5% CO2 before use. Embryos were cultured in 50-μL droplets with oil overlay at 37°C in 5% CO2. A minimum of 5 and maximum of 10 embryos per droplet were allowed. Six replications per treatment were done giving a total of 292 embryos in treatment (1), 318 in treatment (2), 304 in treatment (3), and 303 in treatment (4). The embryos were monitored under an inverted microscope (Olympus model IX70) at 24-h intervals for 72 h for blastocyst formation. The differences between embryo growth in the different culture media were assessed by one-way analysis of variance. All culture media supported the development of mouse embryos to the hatched blastocyst stage. A higher (P < 0.05) number of embryos hatched in M16 (64.6%) and Medium 199 (55.0%) supplemented with 10% fetal bovine serum than in frozen bovine amniotic fluid (12.2%) and frozen bovine amniotic fluid supplemented with 10% fetal bovine serum (17.8%). M16 was superior to all other treatments in supporting embryo development up to the morula stage. More than twice the number of embryos (94.9%) reached the morula stage in M16 than in frozen bovine amniotic fluid with (37.4%) or without (29.1%) serum supplementation. Bovine amniotic fluid obtained from postmortem first trimester fetuses supported the development of two-cell mouse embryos; however, embryo development in frozen fetal fluid was lower (P < 0.05) than that obtained in the control media. Fetal bovine serum, when added to amniotic fluid, did not increase the development rate. It seems likely that freezing the amniotic fluid had an adverse effect on in vitro embryo development.
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Novo, Sergi, Roser Morató, Oriol Penon, Sara Duran, Leonardo Barrios, Carme Nogués, José Antonio Plaza, Luisa Pérez-García, Teresa Mogas, and Elena Ibáñez. "Identification of bovine embryos cultured in groups by attachment of barcodes to the zona pellucida." Reproduction, Fertility and Development 26, no. 5 (2014): 645. http://dx.doi.org/10.1071/rd13066.
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The low number of oocytes collected from unstimulated donors by ovum pick-up means that embryos produced from each individual female have to be cultured individually or in very small groups. However, it has been demonstrated that single-embryo culture is less efficient than embryo culture in groups. To overcome this limitation, we developed a direct embryo-tagging system, which allows the collective culture of embryos from different origins whilst preserving their pedigree. Presumptive bovine zygotes were tagged with eight wheat-germ agglutinin biofunctionalised polysilicon barcodes attached to the outer surface of the zona pellucida (ZP). Four different barcodes were used to encode groups of 20–25 embryos, which were then cultured in the same drop. Cleavage, Day-7 and Day-8 blastocysts and barcode retention rates were assessed. In addition, Day-7 blastocysts were vitrified and warmed. Barcode attachment to the ZP of bovine embryos affected neither in vitro embryo development nor post-warming survival of the tagged embryos. All the embryos maintained barcodes attached until Day 8 of culture (3.63 ± 0.37 barcodes per embryo) and could be identified. In conclusion, identification of embryos by barcodes attached to the ZP is feasible and will allow the culture of embryos from different donors in the same drop.
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Melo-Baez, Bárbara, Yat S. Wong, Constanza J. Aguilera, Joel Cabezas, Ana C. F. Mançanares, Gonzalo Riadi, Fidel O. Castro, and Lleretny Rodriguez-Alvarez. "MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence." International Journal of Molecular Sciences 21, no. 23 (November 24, 2020): 8888. http://dx.doi.org/10.3390/ijms21238888.
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During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.
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Alonso, R. V., J. A. A. Hellú, S. H. V. Perri, J. A. Visintin, and J. F. Garcia. "258 FACTORS AFFECTING COMMERCIAL SEXING PROGRAM AND MULTIPLE GENETIC ANALYSIS PERSPECTIVES OF IN VITRO-PRODUCED BOVINE EMBRYOS." Reproduction, Fertility and Development 21, no. 1 (2009): 227. http://dx.doi.org/10.1071/rdv21n1ab258.
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The present study aimed to evaluate the interactions among different factors on the viability and sex ratio of in vitro-produced (IVP) bovine embryos, submitted to a large scale sexing program. Additionally, whole genome amplification (WGA) technology was used to amplify genomic DNA from IVP bovine embryo biopsies, in order to perform multiple genetic analyses. The survey was performed in a 4650 IVP bovine sexed embryo database. Embryos were biopsied by a microaspiration technique and sex was determined by PCR of DNA from the biopsy. Only female embryos were transferred to synchronized recipients. Pregnancy diagnosis and fetal sex determination were carried out by ultrasound. The variables were classified in accordance with embryo sex (male, female, and indeterminate), five laboratories (A, B, C, D, and E), six bovine breeds (Nellore, Brahman, Girolando, Simmental, Holstein, and Jersey), embryo stage (MO, EB, BL, XB, and HB), embryo quality (1, 2, and 3) and biopsy quality (“standard” and “nonstandard”). The statistical analysis was carried out by association chi-square test, chi-square for a 1:1 ratio, and logistic regression analysis (PROC LOGISTIC) of SAS. PCR showed 93.3% efficiency, 93.2% accuracy, and male and female rates of 52.9% and 47.1%, respectively. Mortality rate of biopsied embryos was 10.3% and pregnancy rate was 31.7%. Significant differences were not observed between male and female viability, although indeterminate embryos resulted in more death after micromanipulation. For quality 2 and 3 embryos, the mortality rate after biopsy was 3.19 and 11.37 fold higher, respectively, than for quality 1 embryos. For embryos whose biopsies were classified as nonstandard, the embryonic mortality rate was 3.6-fold higher than standard ones. Mortality rate was not affected by embryo stage at biopsy (P > 0.05). Although sex ratio was significantly skewed to male embryos, differences were not observed among laboratories (P > 0.05) and breeds (P > 0.05) on the sex ratio of IVP bovine embryos. To test the feasibility of using WGA method for multiple genetic analysis, biopsies from 28 IVP embryos were submitted to the GenomePlex Single Cell System (Sigma-Aldrich, St. Louis, MO, USA). Aliquots from each DNA sample were purified using column chromatography and submitted to PCR using sexing primers BRY4a, SRY, UMN0920, and S4B. PCR was successful and in agreement among tested DNA aliquots from each single biopsy. The WGA strategy used herein was a useful tool for applications involving restricted amounts of starting genetic material (DNA), such as in preimplantation genetic diagnosis using IVP bovine embryos. To FAPESP and UNESP.
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Lee, Jong Ho, Joong Hoon Park, Eun Joo Choi, Jong Taek Yoon, Chang Sik Park, Seong Ho Lee, Kyung Soon Im, and Dong Il Jin. "Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR." Zygote 11, no. 1 (February 2003): 87–93. http://dx.doi.org/10.1017/s0967199403001114.
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Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (>45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.
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Aparicio, I. M., M. Garcia-Herreros, T. Fair, and P. Lonergan. "Identification and regulation of glycogen synthase kinase-3 during bovine embryo development." REPRODUCTION 140, no. 1 (July 2010): 83–92. http://dx.doi.org/10.1530/rep-10-0040.
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The aim of this study was to examine the presence and regulation of glycogen synthase kinase-3α (GSK3A) and GSK-3β (GSK3B) in bovine embryos and their possible roles in embryo development. Our results show that GSK3A and GSK3B are present in bovine embryos at the two-cell stage to the hatched blastocyst stage. Bovine embryo development was associated with an increase in the phosphorylation of both isoforms, being statistically significant at blastocyst and hatched blastocyst stages, compared with earlier stages. Inhibition of GSK3 with CT99021 (3 μM) resulted in a significant increase in the percentage and quality of blastocysts, while inhibition of GSK3 with lithium chloride (LiCl; 20 mM) significantly reduced at the proportion of eight-cell embryos on day 3 and inhibited blastocyst formation. The use of LY294002 (10 μM), a specific inhibitor of phosphatidylinositol-3 kinase, also produced a significant decrease in embryo development. In addition, treatment with LiCl and LY294002 produced a significant decrease in the serine phosphorylation of both isoforms of GSK3. Finally, CT99021 and LiCl reduced the phosphorylation of β-catenin on Ser45 in two-cell embryos, while LY294002 increased it. Despite the fact that LiCl inhibited GSK3 activity, as demonstrated by β-catenin phosphorylation, its effects on the bovine embryo could be mediated through other signaling pathways leading finally to a decrease in the phosphorylation of GSK3 and a reduction in embryo development. Therefore, in conclusion, GSK3A/B serine phosphorylation was positively correlated with embryo development, indicating the importance of an accurate regulation of GSK3 activity during developmental stages to achieve normal bovine embryo development.
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Kim, B. K., H. J. Chung, B. C. Yang, D. H. Kim, J. H. Woo, J. H. Choi, H. H. Seong, J. K. Jung, and W. K. Chang. "153EXPRESSION OF TGF-BETA I AND TYPE I AND TYPE II OF TGF-BETA RECEPTORS IN BOVINE EMBRYOS." Reproduction, Fertility and Development 16, no. 2 (2004): 198. http://dx.doi.org/10.1071/rdv16n1ab153.
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Although the effects of TGFβ1, as an important factor in the mice embryo development have been reported, little information relevant to this subject is known in the bovine embryo. The objectives of this study were to investigate the presence and expression patterns of TGFβ1 and TGFβ1 receptors, types I and II, in unfertilized oocytes and fertilized bovine embryos in normal and NT embryo development. We postulated that TGFβ1 may have a beneficial effect on the preimplantation embryo and show different expression patterns at different stages of bovine embryo development. Immature bovine oocytes were aspirated from follicles of ovaries obtained from a local abattoir and they were cultured for up to 24h and fertilized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the presence of TGFβ1 and type I and type II of TGFβ1 receptors (the essential components of the TGFβ1 signaling pathway) in unfertilized oocytes and preimplantation embryos. Also, mRNA and protein expression patterns of TGFβ1 and their receptors at various stages of embryos were examined. It was found that both receptors, as well as TGFβ1, were present in the unfertilized bovine oocytes, indicating that TGFβ1 is a maternally expressed protein. Although the type I TGFβ1 receptor was present at the morulae and blastocyst stages, the type II TGFβ1 receptor was not present at both stages. It was also confirmed that the expression level of TGFβ1 was high at the 8-cell stage, and mRNA and protein expression patterns of TGFβ1 and their receptors were not coincident. Interestingly, TGFβ1 protein was not detected at blastocyst stage of embryos, whereas the mRNA expression level was high at this stage. The results of this experiment indicate that TGFβ1 protein may be needed by embryos after the blastocyst stage and may be expressed in hatched embryos for implantation. These findings support the hypothesis that there may be an interaction between the TGFβ1 and TGFβ1 receptors in the unfertilized oocytes and preimplantation embryos, and that TGFβ1 signaling may be important for the development of the oocytes and the preimplantation embryos.
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Vandaele, Leen, Bart Mateusen, Dominiek G. D. Maes, Aart de Kruif, and Ann Van Soom. "Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity." Reproduction 133, no. 4 (April 2007): 709–18. http://dx.doi.org/10.1530/rep-06-0109.
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Embryo quality is most frequently evaluated at the blastocyst stage, although quality parameters further back along the developmental axis, such as early developmental kinetics or oocyte quality, can be equally valuable. Despite the fact that previous studies in bovine have linked oocyte diameter and early developmental kinetics with blastocyst formation and viability, their relation with the incidence of apoptosis during embryo development remains relatively unexplored. Therefore, we related non-invasive parameters of oocyte and embryo quality, such as embryo kinetics, embryo morphology, and oocyte diameter, to the incidence of apoptosis throughout embryo development using fluorescent detection of active caspase-3 and -7. First, bovine in vitro embryos were selected according to developmental kinetics and morphology at four set times during culture and subjected to fluorescent detection of active caspase-3 and -7. Caspase activity was significantly higher in slow developing embryos in comparison with fast cleavers (P < 0.05), but was not related to embryo morphology. Second, bovine oocytes were divided into three groups on the basis of oocyte diameter and the resulting embryos were used for staining at the same four set times. Caspase activity was significantly higher in embryos derived from growing oocytes compared with those of fully grown oocytes at 45, 80, and 117 hours post-insemination (hpi; P < 0.05), but not at 168 hpi.
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Costa, L. F. S., M. S. N. Machado, J. F. C. Oliveira, J. C. Silva, R. S. Loguercio, and P. B. D. Gonçalves. "Profile and regulation of annexin II expression during early embryogenesis in cattle." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 59, no. 6 (December 2007): 1493–99. http://dx.doi.org/10.1590/s0102-09352007000600023.
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The presence of annexin II (Ann-II) during the initial stages of bovine embryo development and the regulation of Ann-II expression by retinol and insulin-like growth factor I (IGF-I) were studied. Bovine embryos at different stages of development were produced in vitro on Synthetic Oviductal Fluid (SOF) medium (control group), SOF supplemented with retinol (retinol group; 0.1ng/ml), or IGF-I (IGF-I group; 10ng/ml). The embryos were processed for mRNA extraction, cDNA production and polymerase chain reaction (PCR) using Ann-II-specific oligonucleotides. Ann-II was detected in all stages of early embryo development, except for the 16-cell stage. The blastocyst rates were significantly higher (P<0.05) in the group supplemented with retinol (37.8%, 45/119) during in vitro embryo culture (IVC) than in those cultured in SOF (20.5%, 24/117) or SOF with IGF-I (25.8%, 24/93). Semiquantitative analysis of Ann-II expression in embryos produced in medium supplemented with IGF-I or retinol revealed a lower expression of this gene when compared with embryos cultured in SOF (P<0.05). The Ann-II expression was not different in embryos cultured in the presence of retinol and IGF-I. The presence of retinol increased the production of embryos in vitro by decreasing the expression of Ann-II in early-stage of bovine embryo.
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Méndez, M. S., M. E. Soria, L. R. Galarza, F. P. Perea, and D. E. Argudo. "31 Effect of fetal calf serum on production and cryotolerance of in vitro bovine embryos from Ecuadorian Creole heifers." Reproduction, Fertility and Development 31, no. 1 (2019): 141. http://dx.doi.org/10.1071/rdv31n1ab31.
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In the Ecuadorian Andes there is a Creole bovine biotype whose population is disappearing. In vitro embryo production and cryopreservation is an important biotechnology that allows the conservation of animals threatened with extinction. The objective of this study was to determine the in vitro production and cryopreservation of embryos from creole heifers raised in the highlands of Ecuador. Immature cumulus-oocyte complexes were retrieved by ovum pickup from 10 Creole heifers (OPU) and from abattoir ovaries (control). The experiment was completed within 8 replicates. Cumulus-oocyte complexes were cultured in a maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 100µg mL−1 of sodium pyruvate, 0.75mg mL−1 of l-glutamine, 4µg mL−1 of FSH-p, 100µM cysteamine, and 250µg mL−1 of gentamicin) following IVF (SOF medium supplemented with 10µg mL−1 heparin) and in vitro culture (citrate SOF medium). After denudation (Day 1 after IVF), presumptive embryos from each oocyte source (OPU and control) were split into 2 groups: with (FCS+) and without (FCS−) FCS (2.5%), which was added on Day 5 after IVF. On Day 7, embryos were evaluated, and those with quality 1 were vitrified. After warming, embryo re-expansion at 2h and embryo re-expansion and hatching at 24 and 48h were evaluated. Data were analysed by logistic regression in SAS software (SAS Institute Inc., Cary, NC, USA). Results of embryo rate at Day 7 and rates of vitrified, re-expanded, and hatched embryos are shown in Table 1. Regardless of the oocyte source, the addition of 2.5% FCS decreased embryo re-expansion at 2h and reduced embryo hatching at 48h in the OPU group. In conclusion, FCS did not improve embryo production and adversely affected the cryotolerance of embryos produced in vitro from Ecuadorian creole heifers. Table 1.Production and cryotolerance of in vitro bovine embryos
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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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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.
Full textAbstract:
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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Liang, Shuang, Zheng-Wen Nie, Jing Guo, Ying-Jie Niu, Kyung-Tae Shin, Sun A. Ock, and Xiang-Shun Cui. "Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle." Microscopy and Microanalysis 24, no. 1 (February 2018): 29–37. http://dx.doi.org/10.1017/s1431927618000016.
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AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.
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Martinez, A. C., R. C. M. Tramontini, M. A. Saito Jr, C. O. Abreu, and C. R. Alcalde. "153 IN VITRO CULTIVATION OF CAPRINE EMBRYOS PRODUCED IN VIVO." Reproduction, Fertility and Development 22, no. 1 (2010): 235. http://dx.doi.org/10.1071/rdv22n1ab153.
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Independent of embryo stage, when grade III embryos are transferred, they usually provide poor pregnancy rates. The aim of this study was to compare the effect of different in vitro cultivation media on the development of in vivo derived grade III caprine embryos. Twelve adult crossbreed goats from the Animal Breeding and Reproduction Laboratory of Maringá State University were used for this experiment. The goats were not pregnant or lactating. After embryo collection, embryos were classified regarding their development and quality, according to the International Embryo Transfer Society manual. Morulae classified as grade III were used in this experiment. The embryos were cultivated in 2 different media: Holding Plus medium (Bioniche Animal Health, Belleville, Ontario, Canada) or PBS plus 10% of bovine fetal serum (Nutricell, Campinas, Brazil) in 38.5°C waterbath with water circulation for 24 hours. After 24 h, the embryos were morphologically assessed. The percentage of embryos that developed during the cultivation period was calculated to evaluate the effectiveness of culture media. The embryos that produced new cell divisions and changed from grade III (poor quality) to grade II or grade I (excellent or good) were considered developed embryos. Chi-square was used to determine statistical differences between media. In the present work, the rate of embryos that developed in Holding Plus medium (Bioniche Animal Health) was 75%, and with PBS plus 10% bovine fetal serum was 40% of the total cultivated embryos. Table 1.Embryo development assessment after in vitro cultivation process
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De Stefano, A., A. Gambini, and D. Salamone. "362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS." Reproduction, Fertility and Development 27, no. 1 (2015): 269. http://dx.doi.org/10.1071/rdv27n1ab362.
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Embryo aggregation has been shown to improve embryo development in several species. However, the effects seem to be different among species. Thus, the aim of this study was to compare the effect of embryo aggregation over in vitro development and blastocyst quality of bovine and feline parthenogenetic (PA) embryos. To this aim, bovine cumulus-oocyte complexes (COC) were collected from slaughterhouse ovaries, whereas cat ovaries were obtained from ovariectomized animals. The COC were in vitro matured in TCM199 supplemented following standard protocols for each species. After 24 h, cumulus cells and zona pellucidae were removed. Matured oocytes were selected and activated by 5 µM ionomycin treatment for 4 min followed by incubation in 1.9 mM 6-DMAP. Bovine and feline PA embryos were cultured in SOF medium in the well of well system in two different groups: only one PA embryo per microwell (1X); and three PA embryos per microwell (3X, aggregated embryos). Cleavage and blastocyst rates from all groups were assessed at Days 2 and 7, respectively. Size of blastocysts was measured at Day 7 using a millimetre eyepiece, and total cell number was determined by Hoechst 33342 staining. Blastocyst rates and embryo size were analysed by Fisher's test (P < 0.05) and total cell numbers by Kruskal–Wallis test with Dunn's correction (P < 0.05). Statistical differences were found in PA blastocyst rates between experimental groups (1X: 15/104, 24.6% v. 3X: 27/37, 62.2% for feline; and 1X: 21/113, 19.4% v. 3X: 20/32, 62.5% for bovine), but no differences were found between species. In addition, there was no statistical difference in the number of blastocysts obtained per oocyte used in any of the experimental groups. Bovine aggregated PA blastocysts were significantly larger than non-aggregated embryos (>200 microns, 1X: 2/20, 10% v. 3X: 9/19, 47.4%), but no differences were found in cell number. On the other hand, cat aggregated PA blastocysts had significantly higher cell numbers (1X: 122.4 ± 79.66 cells v. 3X: 259.8 ± 137.1 cells), but no differences were found in blastocyst size. This observation can contribute in the understanding of embryo physiology, suggesting that benefits of embryo aggregation in parthenogenic embryos vary among these species.
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Zhou, W. J., S. Liang, and X. S. Cui. "32 MicroRNA-29b Improves the Quality and Developmental Potential of Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle." Reproduction, Fertility and Development 30, no. 1 (2018): 155. http://dx.doi.org/10.1071/rdv30n1ab32.
Full textAbstract:
MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse cellular processes. miR-29b plays a crucial role during somatic cell reprogramming. However, studies of the function of miR-29b in embryogenesis are limited. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared with IVF embryos (P < 0.05). To determine the function of miR-29b in the bovine SCNT embryo, we microinjected a miR-29b mimic and inhibitor into bovine SCNT zygotes. The results showed that miR-29b significantly decreased the expression of Dnmts (Dnmt3a/3b and Dnmt1) in bovine SCNT embryos (P < 0.05). We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency (P > 0.05) but down-regulation inhibits developmental potency (P < 0.05). Although miR-29b overexpression does not improve the developmental potency of bovine SCNT embryos, the quality of bovine SCNT embryos at the blastocyst stage improved significantly (P < 0.05). The expression of pluripotency factors (OCT4 and SOX2) and cellular proliferation rate were significantly higher in blastocysts from the miR-29b overexpression group than the control and down-regulation groups (P < 0.05). In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and down-regulation groups (P < 0.05). Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.
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Schultz, GA, A. Hogan, AJ Watson, RM Smith, and S. Heyner. "Insulin, insulin-like growth factors and glucose transporters: temporal patterns of gene expression in early murine and bovine embryos." Reproduction, Fertility and Development 4, no. 4 (1992): 361. http://dx.doi.org/10.1071/rd9920361.
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mRNA phenotyping by the reverse transcription-polymerase chain reaction (RT-PCR) method was used to compare the patterns of expression of insulin and insulin-like growth factor (IGF) ligand and receptor genes in preimplantation bovine embryos with those established previously for preimplantation murine embryos. In the early bovine embryo, transcripts for IGF-I, IGF-II and mRNAs encoding receptors for insulin, IGF-I and IGF-II were all detectable at all embryo stages from the 1-cell zygote to the blastocyst. In the mouse, IGF-II ligand and receptor mRNAs were not expressed until the 2-cell stage, and the insulin and IGF-I receptor mRNAs were not detectable until the 8-cell stage. Since transcriptional activation of the embryonic genome occurs at the 8- to 16-cell stage in the bovine embryo and at the 2-cell stage in the murine embryo, it is suggested that these transcripts are products of both the maternal and embryonic genomes in the bovine embryo whereas in the mouse they are present only after activation of the embryonic genome. Transcripts for insulin were not detected in preimplantation embryos of either species. Colloidal-gold immunocytochemistry with antibodies directed against the insulin receptor, IGF-I receptor and IGF-I ligand has confirmed the presence of these molecules in bovine blastocysts. RT-PCR and indirect immunofluorescence procedures demonstrated that the glucose transporter (GLUT) isoform 1 is present in murine embryos from the oocyte to blastocyst stage whereas GLUT 2 expression begins at the 8-cell stage.
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Balboula, Ahmed Z., Mansour Aboelenain, Jianye Li, Hanako Bai, Manabu Kawahara, Mohammed A. Abdel-Ghani, and Masashi Takahashi. "Inverse relationship between autophagy and CTSK is related to bovine embryo quality." Reproduction 159, no. 6 (June 2020): 757–66. http://dx.doi.org/10.1530/rep-20-0036.
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Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.
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Hamdi, Meriem, María J. Sánchez-Calabuig, Beatriz Rodríguez-Alonso, Sandra Bagés Arnal, Kalliopi Roussi, Roger Sturmey, Alfonso Gutiérrez-Adán, Patrick Lonergan, and Dimitrios Rizos. "Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo." Reproduction 158, no. 1 (July 2019): 85–94. http://dx.doi.org/10.1530/rep-18-0561.
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During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.
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Pereira, A. F., L. M. Melo, V. J. F. Freitas, and D. F. Salamone. "Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos." Zygote 23, no. 4 (April 15, 2014): 485–93. http://dx.doi.org/10.1017/s0967199414000100.
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SummaryIn vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.
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Travassos Vieira, Joane Isis, José Carlos Ferreira-Silva, Fabiana Aparecida Cavalcante Silva, Elton Pedro Nunes Pena, Lucas Carvalho Freitas, Maiana Silva Chaves, João Gabriel Viana Grázia, et al. "Proteomic Profile of Vitrified in Vitro-Produced Bovine Embryos (Bos Taurus Indicus)." Cryoletters 43, no. 4 (July 1, 2022): 206–21. http://dx.doi.org/10.54680/fr22410110512.
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BACKGROUND: The proteomic profile of cryopreserved in vitro produced bovine embryos is little known but can provide insights on the successful application of cryo procedures in support of animal breeding. OBJECTIVE: To identify embryonic proteins and biomarkers related to improved cryotolerance of vitrified in vitro produced bovine embryos. MATERIALS AND METHODS: Proteins were isolated from embryo pools (n = 25 embryos per replicate) and analyzed using the nanoLC - MS/MS system. Further, the UniProtKB database (Uniprot – http://www.uniprot.org/) was used for protein identification. Proteins were classified based on their molecular mass, isoelectric point, and enzymatic activity. Post-translational modification predictions and functional gene ontology analysis were performed as well. Finally, a protein-protein interaction network was created to shed light on the embryo interactome. RESULTS: Based on the MS/MS approach, 66 proteins were identified from vitrified Bos taurus embryos. The retrieved proteins were presumably annotated, which allowed a description of the qualitative and functional aspects of the embryo proteome after the vitrification process. CONCLUSION: These findings allowed us to conclude that in vitro-produced vitrified embryos expressed proteins that underlie biological processes related to reproduction, stress and lipid metabolic process, which are essential to maintain embryo viability.
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Xu, Lianguang, Seok-Hwan Song, Muhammad Idrees, Ayman Mesalam, Myeong-Don Joo, Tabinda Sidrat, Yiran Wei, Kyeong-Lim Lee, Wenfa Lu, and Il-Keun Kong. "Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology." International Journal of Molecular Sciences 22, no. 11 (May 29, 2021): 5841. http://dx.doi.org/10.3390/ijms22115841.
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Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.
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Harvey, A., M. Lane, and J. Thompson. "162 IMPROVED EMBRYO SURVIVAL AND QUALITY WITH EMCARE II." Reproduction, Fertility and Development 18, no. 2 (2006): 189. http://dx.doi.org/10.1071/rdv18n2ab162.
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Collection of embryos exposes them to a number of stresses, including light, air, and changes in temperature. Improvement of holding media to reduce the impact of handling stresses on the embryo during in vivo collection and transfer is therefore beneficial to ensure maintenance of viability following transfer. The aim of this study was to compare the effect of holding IVP-derived blastocysts at 25°C in Emcare I (ECMI, Emcare, Dallas, TX, USA) with those held in Emcare II (ECMII), a proprietry formulation designed to reduce in vitro-induced stress. In vitro-produced bovine embryos were generated using standard protocols. Blastocysts were randomly allocated to either ECMI or ECMII (ICPBio, Aukland, New Zealand) on Day 7 and were held at 25°C for a period of 24 h, after which they were cultured in Cook Bovine Blast (Cook Australia, Brisbane, Australia) supplemented with 10% fetal calf serum for 48 h. At 24 and 48 h, embryos were scored for hatching, and a cohort removed for TUNEL staining at each time point. Differences were analyzed by Student's t-test. At both 24- and 48-h culture, hatching rates tended to be higher for embryos held in ECMII than in ECMI (Table 1). The level of apoptosis at 48 h was reduced in blastocysts held in ECMII (P = 0.06). Moreover, the total cell number of hatched blastocysts at 48 h was significantly increased (1.5-fold) in those held in ECMII (P = 0.01). Results suggest that the formulation of ECMII improves the ability of IVP bovine blastocysts to re-expand and hatch following an imposed stress (25°C for 24 h). Furthermore, ECMII improves overall embryo quality through a reduction in the percentage of cells undergoing apoptosis as well as through increased cell numbers, evident 48 h following cessation of the stress. We suggest that Emcare II reduces the impact of (or increases the embryo's tolerance to and recovery from) an imposed stress, which, although severe in the present study, may provide improved outcomes following embryo transfer in field situations. Table 1. Hatching and apoptosis of blastocysts held at 25°C for 24 h in Emcare I or Emcare II This work was supported with funding by ICPBio (NZ).
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Bridi, A., I. Motta, G. Andrade, M. Del Collado, A. Ávila, L. Silva, G. Pugliesi, F. Meirelles, J. Silveira, and F. Perecin. "91 Invivo- and invitro-produced bovine embryos have different microRNA profiles after invitro individual culture." Reproduction, Fertility and Development 32, no. 2 (2020): 171. http://dx.doi.org/10.1071/rdv32n2ab91.
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Invivo- and invitro-produced bovine embryos have different metabolic characteristics, embryonic development, and gene transcription. Additionally, pregnancy rates at 30 days (on average 51% and 34% when using fixed-time AI and invitro production, respectively) are different in beef cattle. Between Days 8 and 17 of the oestrous cycle, concurrent with embryo-maternal recognition, is when 40% of embryonic losses occur. These losses may occur due to altered embryo-maternal cross-talk. MicroRNA (miRNA) can be involved in this communication; however, its potentially regulated pathways in invivo and invitro embryos on Day 9 are unknown. Our hypothesis is that bovine embryos produced invivo and invitro contain different miRNA profiles, even after invivo bovine embryo were invitro cultured. Cows had the follicular wave synchronized and were superovulated to produce invivo or invitro bovine embryos. For the invitro group, on Day −8 of the protocol, the dominant follicles were recovered by ovum pickup, and invitro embryo production was performed to obtain embryos. For the invivo group, on Day −8, the cows were inseminated 12 and 24h after GnRH analogue application and on Day 7 after expected oestrus, uterine flushing was performed to obtain the embryos. Embryos from both groups were individually cultured for 48h. Three pools (of 5 embryos each) per group were used for reverse transcription of miRNAs from total RNA using miScript II RT Kit (Qiagen). Relative levels of 383 bovine miRNAs were determined using the geometric mean of miR-99b, RNU43 snoRNA, and Hm/Ms/Rt U1 snRNA by RT-qPCR. Differences in relative levels of miRNAs were determined by Student's t-test. A total of 210 miRNAs were detected in invivo and invitro embryos, and 13 out of 210 were differently identified between the groups. In invivo embryos, 6 miRNAs were up-regulated, whereas 7 miRNAs were up-regulated in invitro embryos. TARGETSCAN software was used to identify genes predicted as modulated by each miRNA. The top 100 genes predicted were used to identify enriched pathways according to DAVID Bioinformatics Resources. The miRNAs (miR-129, miR-132, miR-155, miR-192, miR-215, and miR-377) up-regulated in invivo embryos modulated pathways that include signaling pathways regulating pluripotency of stem cells (16 genes), TGF-β (11), hippo (10), oestrogen (8), and cell cycle (7). Moreover, miR-23a, miR-338, miR-34a, miR-491, miR-92b, miR-940, and miR-1271, which were increased in invitro embryos, regulate PI3K-Akt (17 genes), signaling pathways regulating pluripotency of stem cells (10), oestrogen (9), toll-like receptor (9), Wnt (9), and HIF-1 (7). The results demonstrate that even after 48h of invitro culture, bovine embryos produced invivo and invitro have different miRNA profiles that modulate pathways associated with embryonic development on Day 9. Furthermore, these results suggest that bioactive molecules, such as miRNAs, can modify embryo-maternal cross-talk, depending on the environment where the embryos are produced. Funding was provided by FAPESP 2017/19681-9, 2014/22887-0, and 2018/13155-6.
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Hasler, J. F. "37 EFFECT OF EMBRYO STAGE ON PREGNANCY RATE FOLLOWING DIRECT TRANSFER OF BOVINE EMBRYOS FROZEN IN ETHYLENE GLYCOL." Reproduction, Fertility and Development 24, no. 1 (2012): 131. http://dx.doi.org/10.1071/rdv24n1ab37.
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Annually, more than 400 000 in vivo-recovered bovine embryos are officially reported by members of the Canadian and American Embryo Transfer Associations. Between 65 and 70% of these embryos are cryopreserved and more than 95% are frozen in ethylene glycol (EG). Statistics on factors affecting embryo freezing are difficult to obtain because many cattle breeders/farmers no longer report pregnancy rates back to embryo transfer (ET) practitioners. Concerns are often expressed as to the optimal stage at which to freeze bovine in vivo-derived embryos. This is a retrospective analysis of results from 5 commercial ET programs (1 in the United States, 3 in Canada and 1 in the Netherlands) for which pregnancy data relative to embryo stage at freezing were made available. Embryos representing 4 stages of development, as defined by the IETS (4 = late morula, 5 = early blastocyst, 6 = mid blastocyst and 7 = expanded blastocyst) are included in the data. The number of embryos thawed and transferred ranged from 3954 to 24 827 for the 5 programs, with a total of 72 828. Embryos were frozen in either 1.5 M EG or 1.5 M EG + 0.1 M sucrose and exposure time to cryoprotectant before cooling ranged from 4 to 40 min. Pregnancy rates are shown in Table 1. Although the pregnancy rate for stage 6 embryos was only 2.6 and 3.2 percentage points lower than stages 4 and 5, respectively, these differences were highly significant and pregnancy rates for stage 6 embryos were lower than those for stages 4 and 5 in 4 of the 5 ET programs. The small decreased survival of stage 6 embryos is probably only moderately important in a commercial context. However, the pregnancy rate of stage 7 embryos was lower than all other stages for the combined dataset as well as in all 5 ET programs, with the difference between stages 5 and 7 ranging from 6.5 to 16.4 percentage points. Clearly, stage 7 embryos survive freezing at a significantly lower rate than stages 4, 5 and 6 and neither time of exposure to EG nor inclusion of sucrose in the freezing medium provided an obvious improvement. Although bovine ET practitioners routinely attempt to collect embryos on day 7 post-oestrus, recovery of stage 7 embryos cannot always be avoided. Further investigation into factors contributing to the decreased survival of stage 7 embryos is warranted. Table 1.Effect of embryo stage on pregnancy rate of bovine embryos frozen in EG
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Orsi, N. M., J. B. Reischl, and H. J. Leese. "Glucose metabolism of in vitro-produced bovine embryos in cell-free and co-culture systems." Proceedings of the British Society of Animal Science 2001 (2001): 216. http://dx.doi.org/10.1017/s1752756200005986.
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In vitro-produced (IVP) bovine embryos are used in a wide range of biotechnologies but develop less well than their in vivo counterparts and can give rise to foetal/neonatal anomalies after embryo transfer. The quality of bovine IVP embryos and the systems in which they are produced are traditionally assessed in terms of morphological and developmental criteria; namely, embryo grade and blastocyst formation rate. Lane and Gardner (1996) showed that mouse embryos selected for transfer on the basis of a low glycolytic activity (conversion of glucose to lactate), measured non-invasively, were 4 times more likely to implant than those selected randomly. Comparable data are not available for bovine embryos. The aim of this study was to assess linear glycolytic index of cattle blastocysts in vitro as a marker of viability. We have measured glucose consumption and lactate production by individual bovine IVP embryos grown in cell-free conditions and in a novel co-culture system (Orsi et al., 2000) involving confluent bovine oviduct epithelial cell monolayers on permeable supports. This preparation allows the epithelial cells to be fed by a nutritionally-rich medium via the physiological, basal, route, while the apical medium, containing the embryos, is more dilute, mimicking oviduct fluid.
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Velasquez, Alejandra E., Fidel O. Castro, Daniel Veraguas, Jose F. Cox, Evelyn Lara, Mario Briones, and Lleretny Rodriguez-Alvarez. "Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation." Zygote 24, no. 1 (December 11, 2014): 18–30. http://dx.doi.org/10.1017/s0967199414000677.
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SummaryEmbryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7–9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.
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Pavani, Krishna, An Hendrix, Wim Van Den Broeck, Liesbeth Couck, Katarzyna Szymanska, Xiaoyuan Lin, Jenne De Koster, Ann Van Soom, and Bart Leemans. "Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro." International Journal of Molecular Sciences 20, no. 1 (December 21, 2018): 38. http://dx.doi.org/10.3390/ijms20010038.
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Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.
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Gopichandran, Nadia, and Henry J. Leese. "The effect of paracrine/autocrine interactions on the in vitro culture of bovine preimplantation embryos." Reproduction 131, no. 2 (February 2006): 269–77. http://dx.doi.org/10.1530/rep.1.00677.
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Bovine preimplantation embryos develop more successfully when cultured in groups, proibably because of the increased production of, and exposure to, embryotrophic autocrine and paracrine factors. Using a novel embryo culture technique, this study had two aims: 1. to determine the distance over which potential paracrine interactions affect bovine embryo development in terms of blastocyst and hatching rates, cell counts and carbohydrate metabolism; 2. to investigate the effect of platelet-activating factor (PAF) supplementation on bovine embryo development and metabolism. Groups of 16 presumptive zygotes were attached to the bottom of a culture dish by the cell adhesive Cell-Tak in a 4 × 4 equidistant array. The distance between individual embryos in each group was 0–689 μm. Optimal blastocyst formation rate occurred when embryos were cultured 165 μm apart compared with control non-attached zygotes (Kruskal–Wallis followed by Mann–Whitney U test post-hoc; P < 0.05). Increasing the distance between embryos resulted in a further decline in blastocyst rate, which reached zero at 540 μm apart. Blastocyst cell number, pyruvate/glucose uptake and lactate production decreased as the interembryo distance increased from 240 to 465 μm (P < 0.05). Supplementation with PAF during conventional group culture enhanced blastocyst cell number, hatching rates and the oxidative metabolism of pyruvate and glucose. The data indicate that the distance between individual bovine embryos in culture influences preimplantation development, in particular blastocyst formation, cell number and metabolism. It is suggested that diffusible paracrine/autocrine factors, such as PAF, are in part responsible for the regulation of early embryo development.
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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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Landry, A. M., M. Murakami, R. S. Denniston, J. L. Williams, Y. Echelard, and R. A. Godke. "155 DEVELOPMENT OF BOVINE AGGREGATE EMBRYOS CONSTRUCTED FROM NUCLEAR TRANSFER EMBRYOS AND ELECTROFUSED IVF-DERIVED EMBRYOS." Reproduction, Fertility and Development 17, no. 2 (2005): 228. http://dx.doi.org/10.1071/rdv17n2ab155.
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The production of animals by nuclear transfer can be hindered by placental and developmental abnormalities in the fetus. Studies in mice have indicated that tetraploid embryo complementation can be used to rescue embryos with lethal placental deficiencies and produce live offspring. The objectives of this experiment were to produce bovine electrofused embryos by blastomere fusion and to utilize those embryos for aggregation with nuclear transfer (NT) embryos. Oocytes were obtained from a commercial source (BoMed, Madison, WI, USA) and were allocated for use in either NT or in vitro fertilization (IVF). NT embryos were produced using standard procedures. Bovine transgenic fibroblast cells maintained in active culture were used as the nuclear donor. Oocytes were fertilized with frozen semen using standard IVF procedures. Zygotes were observed for cleavage and selected at the 2-cell stage for electrofusion at 28, 30, 32, and 34 h post-insemination in 0.3 M mannitol fusion buffer. Embryos were aligned using a 5 s, 7.5 V AC pulse and were fused using a 1.4 kV cm−1 100 μs DC pulse. Treated embryos were observed after ∼1 h for fusion of cell membranes between the two blastomeres and were returned to culture (IVF-Fused). Good quality 8-cell embryos produced from the NT and IVF-Fused groups were selected for aggregation at 72 h post-insemination. Aggregate embryos were constructed by removing 3–4 blastomeres from an 8-cell NT embryo. The zona pellucida of an 8-cell IVF-Fused embryo was then removed by placing the embryo in a 0.25% pronase solution for approximately 1 min. After the zona pellucida was removed, 3–4 blastomeres were aspirated from the IVF-Fused embryo and were injected into the NT embryo using a glass pipette. Embryos were returned to culture in CR1aa media and were examined at 168 and 192 h post-insemination for blastocyst (BLST) development (Table 1). This study demonstrates that NT/IVF-Fused aggregate embryos can be constructed and develop at the same rate as controls. An attempt was made to determine the nuclear status of the electrofused embryos but technique limitations did not permit differentiation between tetraploid and multinucleate cells. Further research is needed to determine nuclear status of IVF-Fused embryos and the allocation of NT and IVF-Fused cell lineages within the developing embryo. Table 1. Blastocyst rates of NT/IVF-Fused aggregate embryos constructed at the 8-cell stage
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Brooks, K. E., B. L. Daughtry, S. S. Fei, M. Y. Yan, B. Davis, L. Carbone, and S. L. Chavez. "41 Delineating the molecular connections between mitotic aneuploidy, micronucleation, and cellular fragmentation in pre-implantation bovine embryos." Reproduction, Fertility and Development 31, no. 1 (2019): 146. http://dx.doi.org/10.1071/rdv31n1ab41.
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Whole chromosomal abnormalities (aneuploidy) that arise during early embryo development are a major contributor to in vitro fertilization failure. It is estimated that ~50 to 80% of human embryos contain aneuploid cells, which contribute to high levels of chromosomal mosaicism detected by pre-implantation genetic screening. Previous studies estimate that 32 to 88% of bovine embryos are aneuploid at the 2-cell stage, advocating cattle as a physiologically relevant model to study the mechanisms mediating meiotic and/or mitotic errors. In cleavage-stage human embryos, a process called cellular fragmentation is associated with aneuploidy, and when used in conjunction with assessment of early mitotic timing, can largely distinguish chromosomally normal and abnormal embryos. We recently demonstrated that some cellular fragments contain chromosomal material that likely began as mis-segregated chromosomes that were encapsulated into micronuclei. Given that bovine embryos exhibit cellular fragmentation, albeit to a lesser extent than human embryos, we hypothesise that cellular fragmentation is a response to micronucleation and represents a conserved mechanism to eliminate mis-segregated chromosomes from the pre-implantation embryo. Using a combination of live-cell imaging, single-cell DNA-sequencing, whole-embryo RNA-sequencing, quantitative RT-PCR, and multicolour confocal microscopy, we aim to further investigate the correlation between these phenomena using in vitro-produced bovine embryos. Similar to humans, the first three mitotic divisions are able to successfully predict progression to the blastocyst stage (N=84). Bovine embryos frequently contained multi-/micro-nuclei, and DNA-sequencing of individual bovine blastomeres up to 12 cells confirmed that ~58 to 87% of cleavage-stage bovine embryos are aneuploidy (N=38) and often detectable by abnormal cell divisions. Transcriptional profiling of fragmented versus non-fragmented bovine embryos via RNA-sequencing identified a small subset of differentially abundant genes at the 4-cell stage. Pathway analysis showed reduced abundance of genes associated with the cytoskeleton, microtubules, and spindle in 4-cell embryos with cellular fragmentation as well as enrichment of membrane targeting and vesicle fusion pathways. The potential role of these cellular components in micronucleation and cellular fragmentation is being assessed by microinjecting bovine zygotes with fluorescently labelled mRNA mCherry-H2B (chromatin marker) and mCitrine-LaminB1 (nuclear envelope marker), followed by overnight live-cell multicolour confocal imaging (Zeiss LSM 880 with AiryScan; Zeiss, Thornwood, NY, USA). Results from these studies contribute to our knowledge of early embryogenesis with translational application to help ameliorate embryonic loss in women and cattle.
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Ming, H., J. Sun, R. Pasquariello, J. R. Herrick, Y. Yuan, E. Gutierrez, L. Gatenby, K. R. Bondioli, R. L. Krisher, and Z. Jiang. "2 The landscape of accessible chromatin in bovine oocytes and early embryos." Reproduction, Fertility and Development 32, no. 2 (2020): 125. http://dx.doi.org/10.1071/rdv32n2ab2.
Full textAbstract:
Chromatin reorganization governs gene expression regulation during pre-implantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored in bovine. In this study, we constructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We analysed pools of 20 germinal vesicles or MII oocytes or 2-, 4-, 8-, 16-cell, morula, and blastocyst stage invitro-produced embryos. We conducted ATAC-seq on six pools for each stage and an additional four pools of invivo-derived morula and blastocysts and six replicates using individual Day 14 elongating embryos. We obtained ~110 million paired end reads uniquely mapped to the bovine reference genome for each stage. Hierarchical clustering, t-distributed stochastic neighbour embedding, and principal component analysis showed four distinct patterns for open chromatin status: (1) low accessibility in germinal vesicles and MII oocytes and in 2- and 4-cell embryos; (2) significantly elevated accessibility in 8-cell, 16-cell, and morula embryos; (3) less accessibility in blastocysts; and (4) extremely high accessibility in elongating embryos. This dynamic and sequential chromatin remodelling is consistent with transcription activation during the bovine minor embryonic genome activation from fertilization to 4-cell, major embryonic genome activation at 8-cell, first differentiation at blastocyst and drastic transcription initiation for embryo elongation. Genome-wide characteristics of accessible chromatin showed (1) accessible chromatin near the transcription start sites of active genes and CpG-rich promoters; (2) widespread accessible chromatin regions extensively overlapped with transposable elements; (3) distal peaks preferentially enriched for repeats including LINE, SINE, and LTR from 8-cell to morula embryos, especially for LTR, whereas enrichment in simple repeats were found from oocytes to 4-cell and in elongating embryos; and (4) highly stage-specific transcription factor motifs in distal peaks were unveiled. By integrating the maps of chromatin accessibility with bovine embryo transcriptomes and DNA methylomes, we found promoter accessibility and DNA methylation in bovine embryos correlated with both gene activities and CpG densities. Most importantly, we constructed the regulatory networks of stage-specific expressed genes and stage-specific activated genes with three omics datasets in bovine early embryos and revealed conserved and distinctive transcriptional regulatory networks between invivo- and invitro-derived embryos. This comprehensive analysis revealed critical features of the chromatin landscape and epigenetic reprogramming during bovine early embryo development.
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Torres, C. A. A., C. A. C. Fernandes, F. A. Oliveira, J. M. Penitente Filho, C. T. S. A. M. Oliveira, M. C. R. Santos, C. R. Jiménez, E. L. C. Triana, and M. M. N. F. Oliveira. "110 PREGNANCY RATES OF BOVINE EMBRYOS AND HEMI-EMBRYOS TRANSFERENCE." Reproduction, Fertility and Development 24, no. 1 (2012): 167. http://dx.doi.org/10.1071/rdv24n1ab110.
Full textAbstract:
Embryo transfer (ET) in cattle speeds up genetic gains, but its use is limited by cost-benefit analysis. In addition other techniques can be developed, such as sexing and bipartition of embryos. An alternative to improve the economic viability of ET in cattle would be to bipartition embryos in order to increase the yield of the technique. The aim of this study was to investigate the operational viability of embryo bipartition technique in an ET program in cattle and to study aspects related to the viability and development of bovine hemi-embryos (HE) compared with intact ones. The embryos were collected by nonsurgical technique 7 days after the onset of oestrus. Viable structures (130) from 49 embryo collections from 25 Aberdeen and Simmental donor cows and heifers were used. Only embryos in compact morula, early blastocyst and blastocyst stage, with a morphological range from excellent to good grade (IETS Grade 1), were split. The embryos, split (78) or not (52), were transferred into the uterine horn ipsilateral to the corpus luteum of the recipients 7 days after oestrus. The treatment groups evaluated were T1 (intact embryo, n = 52), T2 (1 HE, n = 27) and T3 (2 HE, n = 51). Crossbred heifers were used as recipients and pregnancy diagnosis was done at 60 to 80 days of gestation. The embryos of T1, T2 and T3 were classified morphologically as excellent or good and by developmental stage as morula, early blastocyst, or blastocyst, distributed as follows: T1: 30, 22 and 16, 17 and 19; T2: 15, 12 and 7, 9 and 11; and T3: 29, 22 and 15, 15 and 21, respectively. The birth rate per original embryos was greater for T2 than for T1 and T3 (Table 1). The pregnancy rates for excellent and good embryos and morulae, early blastocysts and blastocysts were not different (P > 0.05). The T1, T2 and T3 twin births were 0, 1 and 5, respectively. It is concluded that embryo bipartition technique applied in a commercial ET program is a viable operational technique. Table 1.Pregnancy at 60 to 80 days and birth rate per original embryos used Supported by CAPES, CNPq, FAPEMIG and BIOTRAN.
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Li, Geng, Karam Khateeb, Erin Schaeffer, Bao Zhang, and Hasan Khatib. "Genes of the transforming growth factor-beta signalling pathway are associated with pre-implantation embryonic development in cattle." Journal of Dairy Research 79, no. 3 (June 12, 2012): 310–17. http://dx.doi.org/10.1017/s0022029912000210.
Full textAbstract:
One of the main factors affecting cattle fertility is pre-implantation development of the bovine embryo, which is a complex process regulated by various signal-transduction pathways. The transforming growth factor-β (TGF-β) signalling system, which is responsible for many biological processes including cell proliferation, differentiation and apoptosis, also is involved in embryo development. We hypothesized that altered expression of TGF-β genes in pre-implantation bovine embryos is associated with morphological abnormalities of these embryos. To test this hypothesis, we produced embryos in vitro and classified them at the blastocyst stage as either normally developed blastocysts or degenerates (growth-arrested embryos). The expression patterns of 25 genes from the TGF-β pathway were assessed using quantitative real time PCR. Ten genes showed differential expression between the two embryo groups, four genes displayed similar expressional profiles, and 11 genes had no detectable expression. An altered expression profile was statistically significant for 10 of the 14 expressed genes, and all were up-regulated in degenerate embryos vs. blastocysts. Furthermore, genomic association analysis of the cows from which embryos were produced revealed a significant association of ID3 and BMP4 polymorphisms—two of the most significant differentially expressed genes—with fertilization rate and blastocyst rate, respectively. Taken together, we conclude that TGF-β pathway genes, especially BMP4 and ID3 play a vital function in the regulation of pre-implantation embryo development at both embryo and maternal levels. Hence, these genes may be suitable as genetic markers for embryo development and fertility in cattle.
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Taniyama, A., Y. Watanabe, Y. Nisino, and T. Inoue. "205 INCREASED PREGNANCY RATES OF POOR QUALITY BOVINE EMBRYOS BY ASSISTED HATCHING." Reproduction, Fertility and Development 19, no. 1 (2007): 219. http://dx.doi.org/10.1071/rdv19n1ab205.
Full textAbstract:
Embryo transfer after superovulation is commonly used for efficient embryo and animal production and for genetic improvement in cattle. However, the quality of collected embryos varies greatly, which affects pregnancy rate. Usually, poor quality embryos are related to low pregnancy rates after embryo transfer and low viability after cryopreservation. Therefore, it is important to improve chances for survival of poor quality embryos after embryo transfer. The objective of this experiment was to improve pregnancy rates by applying the assisted hatching technique to poor quality embryos. Embryos were collected from Japanese Black cows after superovulation on Day 7 post-insemination. After being washed, embryos were morphologically classified. Embryos having more than 30% degenerated cells were assigned as poor quality embryos. The assisted hatching of embryos (cutting the zona pellucida) was performed under a stereoscope or an inverted microscope by making a cutting slit on the zona pellucida for about 20% of its circumference using a micromanipulator equipped with a cutting needle and holding pipette. After cutting, single or two embryos were transferred fresh to one uterine horn of recipient cows on Day 7 of the estrous cycle. Pregnancy and calf production rates were compared between 2 embryo transfer groups composed of fresh zona-cut embryos (ZC group) or fresh embryos with non-cut zonae pellucidae (NZC group). Pregnancy rates were determined by rectal palpation on Day 45, and calf production rates were calculated by the following formula: number of calves born/number of pregnancies. Statistical analysis was carried out using the chi-square test. Pregnancy rates of poor quality embryos in the double ET ZC group (60.3%; 44 pregnancies/73 transfers) were significantly higher (P < 0.05) than those in the single ET NZC group (25.0%; 6 pregnancies/24 transfers) and in the single ET ZC group (44.0%; 37 pregnancies/84 transfers). Calf production rates were 67.3%, 45.5%, and 35.6% for the double ET ZC group, the double ET NZC group, and the single ET ZC group, respectively. Pregnancy rates of poor quality bovine embryos after double ET were remarkably improved by assisted hatching compared with those of single ET with non-assisted hatching. These results suggest that the combined methods of assisted hatching and double ET may be beneficial to produce calves from poor quality embryos.
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Hobbs, D., C. Holcomb, and J. Gibbons. "59 Incubation of frozen/thawed bovine embryos." Reproduction, Fertility and Development 33, no. 2 (2021): 136. http://dx.doi.org/10.1071/rdv33n2ab59.
Full textAbstract:
High-quality, properly developed bovine embryos are crucial to the embryo transfer industry and enable cryopreservation; however, ∼30% of invivo embryos do not develop to the appropriate stage and are discarded. Currently, frozen/thawed embryos are transferred directly into a recipient, and the quality of embryos post-thaw is seldom evaluated. The objective of this experiment was to evaluate frozen/thawed bovine embryos immediately post-thaw, and again after incubation in different environments. Frozen/thawed bovine embryos (n=30/treatment) processed for direct transfer were thawed (30°C water; 30s) and graded and staged in holding medium. Embryos were then placed into either holding medium, phosphate-buffered saline supplemented with 15% fetal bovine serum (v/v) and antibiotic/antimycotic (2μL mL−1) (PBS+FBS), or a commercially available invitro culture (IVC) medium for ∼18h. Embryos in holding medium and PBS+FBS were loaded into 0.25-mL straws with the appropriate medium, and the straws were sealed and submerged in 38.5°C water. The IVC embryos were placed individually into 25-μL culture drops on tissue-coated 60-mm plastic Petri dishes overlaid with mineral oil and incubated (18h) at 38.5°C in 5% CO2 and air at 100% humidity. Embryos were then collected, regraded, and staged, and comparisons among groups were analysed via the Kruskal–Wallis H-test. Quality score of all embryos decreased by at least one-third post-thaw; however, the developmental stage was unaffected by the freeze/thaw cycle. Following incubation, all embryos suffered a significant (P<0.05) decrease in embryo quality but the IVC group demonstrated less (P<0.05) of a decline in resulting quality (Table 1). The IVC group demonstrated significant development (P<0.05) during incubation compared to the Holding and PBS+FBS groups (Table 1) indicating that on average, viability was maintained during IVC. Regardless of group, the zona pellucida was damaged during the freeze/thaw process in 31.1% of embryos. These data illuminated embryo damage after cryopreservation, and demonstrated that short-term invitro incubation of frozen/thawed embryos (IVC) facilitated continued development and may be a practical mechanism to salvage poor quality or developmentally suppressed embryos. Future research will focus on salvaging fresh embryos that are classified as degenerate and may prove useful in the bovine embryo industry, and for cattle producers alike, by ultimately increasing the number of transferable embryos that would otherwise be discarded. Table 1. Descriptive statistics (mean±s.e.m.) of bovine embryos pre-freeze and post-thaw and incubation Item Media Pre-freeze Post-thaw Post-incubation Grade Holding 1.4±0.1 1.7±0.1a 3.3±0.1b,x PBS+FBS 1.2±0.1 1.6±0.1a 3.1±0.1b,x IVC1 1.2±0.1 1.6±0.1a 2.5±0.2b,y Stage Holding 4.0±0.1 4.0±0.1 3.9±0.1x PBS+FBS 4.7±0.2 4.4±0.2 4.5±0.2y IVC 4.3±0.1 4.3±0.1a 5.8±0.2b,z a,b, x–zDifferent superscripts within row (a, b) and column (x, y, z) indicate a significant difference (P<0.05, Kruskal-Wallis H-test). 1Invitro culture medium.
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Hobbs, D., C. Holcomb, and J. Gibbons. "59 Incubation of frozen/thawed bovine embryos." Reproduction, Fertility and Development 33, no. 2 (2021): 136. http://dx.doi.org/10.1071/rdv33n2ab59.
Full textAbstract:
High-quality, properly developed bovine embryos are crucial to the embryo transfer industry and enable cryopreservation; however, ∼30% of invivo embryos do not develop to the appropriate stage and are discarded. Currently, frozen/thawed embryos are transferred directly into a recipient, and the quality of embryos post-thaw is seldom evaluated. The objective of this experiment was to evaluate frozen/thawed bovine embryos immediately post-thaw, and again after incubation in different environments. Frozen/thawed bovine embryos (n=30/treatment) processed for direct transfer were thawed (30°C water; 30s) and graded and staged in holding medium. Embryos were then placed into either holding medium, phosphate-buffered saline supplemented with 15% fetal bovine serum (v/v) and antibiotic/antimycotic (2μL mL−1) (PBS+FBS), or a commercially available invitro culture (IVC) medium for ∼18h. Embryos in holding medium and PBS+FBS were loaded into 0.25-mL straws with the appropriate medium, and the straws were sealed and submerged in 38.5°C water. The IVC embryos were placed individually into 25-μL culture drops on tissue-coated 60-mm plastic Petri dishes overlaid with mineral oil and incubated (18h) at 38.5°C in 5% CO2 and air at 100% humidity. Embryos were then collected, regraded, and staged, and comparisons among groups were analysed via the Kruskal–Wallis H-test. Quality score of all embryos decreased by at least one-third post-thaw; however, the developmental stage was unaffected by the freeze/thaw cycle. Following incubation, all embryos suffered a significant (P<0.05) decrease in embryo quality but the IVC group demonstrated less (P<0.05) of a decline in resulting quality (Table 1). The IVC group demonstrated significant development (P<0.05) during incubation compared to the Holding and PBS+FBS groups (Table 1) indicating that on average, viability was maintained during IVC. Regardless of group, the zona pellucida was damaged during the freeze/thaw process in 31.1% of embryos. These data illuminated embryo damage after cryopreservation, and demonstrated that short-term invitro incubation of frozen/thawed embryos (IVC) facilitated continued development and may be a practical mechanism to salvage poor quality or developmentally suppressed embryos. Future research will focus on salvaging fresh embryos that are classified as degenerate and may prove useful in the bovine embryo industry, and for cattle producers alike, by ultimately increasing the number of transferable embryos that would otherwise be discarded. Table 1. Descriptive statistics (mean±s.e.m.) of bovine embryos pre-freeze and post-thaw and incubation Item Media Pre-freeze Post-thaw Post-incubation Grade Holding 1.4±0.1 1.7±0.1a 3.3±0.1b,x PBS+FBS 1.2±0.1 1.6±0.1a 3.1±0.1b,x IVC1 1.2±0.1 1.6±0.1a 2.5±0.2b,y Stage Holding 4.0±0.1 4.0±0.1 3.9±0.1x PBS+FBS 4.7±0.2 4.4±0.2 4.5±0.2y IVC 4.3±0.1 4.3±0.1a 5.8±0.2b,z a,b, x–zDifferent superscripts within row (a, b) and column (x, y, z) indicate a significant difference (P<0.05, Kruskal-Wallis H-test). 1Invitro culture medium.
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Tutt, D. A., R. E. Lyons, and M. K. Holland. "92 DOES BLASTOCENTESIS AFFECT CRYOPRESERVATION SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS?" Reproduction, Fertility and Development 29, no. 1 (2017): 153. http://dx.doi.org/10.1071/rdv29n1ab92.
Full textAbstract:
The cattle industry primarily employs embryo bisection in order to obtain genetic samples for pre-implantation screening and selection of embryos. Although practical and rapid, bisection is invasive and adversely affects embryo viability and cryopreservation. An alternative biopsy approach is to aspirate the blastocoele fluid (referred to as blastocentesis), which not only provides a genetic sample, but also has the potential to improve cryopreservation (Palini et al. 2013 Repro. Biomed. 26, 603–610). This study investigates blastocentesis as a low impact biopsy procedure to rapidly sample bovine blastocysts with limited effect on embryo cryopreservation survival. In vitro-produced embryos were selected at expanded blastocyst stage and placed in a 50-μL drop of holding media on an inverted microscope. The embryo was held using a glass holding pipette attached to a micromanipulator, oriented so that the inner cell mass was toward the bottom of the view. A 7-μm spiked intracytoplasmic sperm injection pipette attached to the other micro-manipulator was used to pierce the blastocoele cavity and aspirate the blastocoele fluid. Once removed, the aspirate was transferred into 4-μL TE buffer for later genetic analysis. Collapsed blastocysts were then vitrified in ~7 μL 16.5% ethylene glycol, 16.5% dimethyl sulfoxide in TCM-199 (Hanks salts) with 20% FCS and 0.5 M sucrose. Embryos were held for a minimum of 1 week and then thawed and assessed for survival. Post-cryopreservation embryo survival was measured as the proportion of embryos that re-expanded after 48 h in culture. One-way ANOVA was used for statistical testing. A total of 181 control (intact) and 182 blastocentesis embryos were vitrified over 6 replicates. In all but one replicate, non-biopsied control embryos had higher re-expansion rates. Overall, the re-expansion rate was significantly (P = 0.05) higher for control embryos (73.5%) than blastocentesis embryos (61.5%) (Table 1). Initial experiments would suggest embryo survival is affected by the biopsy procedure; however, because this was not the case with every replicate, this may be batch or technician/human error dependent. Further study is required to assess full effect of blastocoele fluid aspiration on embryo cryopreservation, particularly investigating effectiveness for in vivo-produced embryos and subsequent effect on pregnancy rates. Likewise, further investigation is required to assess whether the sample collected is sufficient to allow accuracy over a variety of genetic tests. More than 20 embryos can easily be sampled in an hour using this technique, making it a rapid and efficient process. Given the speed and compatibility with cryopreservation, this sampling procedure may offer an alternative to current techniques used for cattle embryo genetic assessment. Table 1. Post-thaw survival rates of in vitro-produced embryos vitrified after blastocentesis1