264 IDENTIFICATION OF THE PREDOMINANT TRANSCRIPT IN BOVINE OOCYTES, IN VITRO-DERIVED BLASTOCYSTS, AND IN NUCLEAR TRANSFER BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 239
Author(s):  
R. S. Prather ◽  
S. Korte ◽  
R. Woods ◽  
L. Spate ◽  
N. Bivens ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Expressed Sequence Tag ◽  
Sequence Data ◽  
Sequence Similarity ◽  
Blastocyst Stage ◽  
Mrna Abundance ◽  
Genomic Research ◽  
Cell Stage ◽  
Number Of Clusters

Identification of transcripts produced during bovine embryogenesis is the first step in describing the normal developmental program. To that end, mRNA was isolated from in vitro-matured metaphase II oocytes (MPII), in vitro-produced 2-cell-stage (2-Cell), in vitro-produced precompact morula-stage (PCM), in vitro-produced blastocyst-stage (BL), and in vitro-produced nuclear transfer blastocyst-stage (NTBL) embryos. The mRNA was isolated by using Dynabeads® (Dynal, Inc., Lake Success, NY, USA), and amplified by using the SMART system. PCR products were purified and ligated into pSPORT1 and electroporated into E. coli. Random clones were selected for DNA sequencing. Sequence data were evaluated for quality and clustered by sequence similarity with sequences generated from a larger expressed sequence tag (EST) project (http://genome.rnet.missouri.edu/Bovine/) by using the tlcluster program from the University of Iowa. Sequences over 100 bp in length with average Phred scores of over 20 for the entire sequence were submitted to GenBank (NIH genetic sequence database). Sequences were compared to the bovine TIGR (The Institute for Genomic Research) and human databases to gather annotation. The best comparison is listed below by using the HUGO Gene Nomenclature Committee standards (http://www.gene.ucl.ac.uk/nomenclature/) when possible. The number of unique clusters, i.e. no match in GenBank, was 53, 120, 109, 115, and 135, for MPII, 2-Cell, PCM, BL, and NTBL, respectively. The total number of clusters per tissue ranged from 224 to 992. The percent of clusters (number of clusters per total number of ESTs) per library was 12% (224/1762), 42% (746/1771), 48% (819/1715), 49% (900/1818) and 53% (992/1876) for MPII, 2-Cell, PCM, BL, and NTBL, respectively. Either the quality of the MPII library was lower or the complexity of the MPII mRNA was less than mRNA in the other tissues. Examples of mRNA that were in different abundance are shown in Table 1. Clearly, as in other species, there are significant changes in mRNA abundance during early embryogenesis. Furthermore, NTBL embryos, even though they are morphologically similar to BL, possess a population of mRNA that is distinct from that in BL. Table 1. Comparison of mRNA Abundance During Bovine Embryogenesis This work was funded by the USDA NRI 2003–35205–12812 and Food for the 21st Century.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

scholarly journals Birth of rats following nuclear exchange at the 2-cell stage

Zygote ◽  
2003 ◽  
Vol 11 (4) ◽  
pp. 317-321 ◽  
Author(s):  
Sangho Roh ◽  
Jitong Guo ◽  
Nakisa Malakooti ◽  
John R. Morrison ◽  
Alan O. Trounson ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Perivitelline Space ◽  
Sprague Dawley ◽  
Cell Stage ◽  
Rat Embryos ◽  
Sprague Dawley Rats ◽  
Nuclear Exchange

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Blastocyst Stage ◽  
Total Cell ◽  
Mrna Abundance ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

45 The role of TRIM28 in porcine somatic cell nuclear transfer embryo development

2019 ◽  
Vol 31 (1) ◽  
pp. 148
Author(s):  
Y. H. Zhai ◽  
X. L. An ◽  
Z. R. Zhang ◽  
S. Zhang ◽  
Z. Y. Li
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chromatin Modification ◽  
Blastocyst Stage ◽  
Imprinted Genes ◽  
Cell Stage ◽  
Genomic Methylation

During fertilization, the parental genome undergoes extensive demethylation. Global DNA demethylation is a hallmark of epigenetic reprogramming. Embryos engage non-canonical DNA methylation maintenance mechanisms to ensure inheritance of exceptional germline features. However, the mechanisms ensuring demethylation resistance in light of global reprogramming remain poorly understood. TRIM28 is a maternal-effect factor that controls genomic imprinting during early embryonic reprogramming. In this study, cytoplasmic injections of siRNA were performed into oocytes matured in vitro for 26h to interfere with the expression of TRIM28 in oocytes. The injected oocytes were continually matured in vitro until 42h and used to construct somatic cell nuclear transfer (SCNT) embryos. During 2-cell to blastocyst stages, the expression of development-related genes (NANOG, POU5F1, CDX2, BAX, and BCL2), maternal imprinting genes (IGF2, DIO3, PLAGL1, and DLK1), paternal imprinting genes (H19 and PEG3), TRIM28-recruitment complex-associated genes (ZFP57, PGC7, SETDB1, and DNMT), and epigenetic chromatin modification enzymes were detected by quantitative PCR in the constructed TRIM28-interfered SCNT embryos. The DNA methylation levels in the promoter regions of the imprinted genes (H19 and IGF2) and chromatin repeats (PRE-1 and SATELLITE) were analysed by sodium bisulfite genomic sequencing. The results showed that the TRIM28-interfered SCNT embryos had significantly lower cleavage and blastocyst rates (53.9±3.4% and 12.1±4.3%, respectively) than those in control SCNT embryos (64.8±2.7% and 18.8±1.9%, respectively). The expression levels of development-related genes (NANOG and POU5F1) and TRIM28-recruited transcriptional repression complex-associated genes (PGC7, ZFP57, and DNMT1) in the 4-cell stage were significantly reduced (P&lt;0.05). The imprinted genes were significantly up-regulated (P&lt;0.05) from the 2-cell to blastocyst stage in constructed TRIM28-interfered SCNT embryos, except H19 at the 2-cell and blastocyst stage decreased remarkably (P&lt;0.05). The DNA methylation levels of IGF2 decreased 2-fold from the 2-cell to blastocyst stage in TRIM28-interfered SCNT embryos. The PRE-1 and SATELLITE had a remarkably lower (P&lt;0.05) methylation levels in the TRIM28-interfered 2-cell embryos than in control SCNT embryos. The cluster analysis showed some of the chromatin modification enzymes had abnormal expression in the TRIM28-interfered SCNT embryos, especially in the 8-cell stage, where 48 enzymes were significantly decreased (P&lt;0.05). The down-regulation enzymes were mainly clustered in the histone H3K4 methyl transferase and histone acetylase. These results indicate that down-regulation of maternal TRIM28 breaks the steady-state of genomic methylation at a particular locus of the imprinted gene, disrupts the expression of imprinted gene and epigenetic modifications enzymes, and is detrimental to normal development of SCNT embryos. Maternal TRIM28 is needed in maintaining a stable state of genomic methylation and epigenetic modification state during SCNT embryo development.

Nuclear transplantation using bovine primordial germ cells from male fetuses

1995 ◽  
Vol 7 (5) ◽  
pp. 1217 ◽  
Author(s):  
F Delhaise ◽  
FJ Ectors ◽  
Roover R de ◽  
F Ectors ◽  
F Dessy
Keyword(s):  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
Nuclear Transplantation ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Developmental Potential ◽  
Morula Stage

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.

28 EFFECT OF TRICHOSTATIN A ON IN VITRO EMBRYO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED FROM CAT DONOR NUCLEI AND BOVINE CYTOPLASM

2013 ◽  
Vol 25 (1) ◽  
pp. 161 ◽  
Author(s):  
M. Wittayarat ◽  
Z. Namula ◽  
V. V. Luu ◽  
L. T. K. Do ◽  
Y. Sato ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Cell Stage ◽  
Histone Deacetylase Inhibitor Trichostatin ◽  
Invaluable Tool ◽  
Morula Stage ◽  
Bovine Oocytes

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus-cytoplasm interactions and may provide an alternative for cloning endangered animals whose oocytes are difficult to obtain. The developmental ability of iSCNT embryos decreases with increases in taxonomic distance between the donor and recipient species. The development of cat-bovine iSCNT embryos is reportedly blocked at the 8-cell stage (Thongphakdee et al. 2008 J. Reprod. Dev. 54, 142–147). Abnormal epigenetic reprogramming, such as DNA methylation or histone modifications, may cause low iSCNT efficiencies. The present study was conducted to evaluate the effect of the histone deacetylase inhibitor trichostatin A (TSA), previously used to enhance nuclear reprogramming following SCNT, on the developmental ability of cat iSCNT embryos using bovine oocytes matured in vitro. The matured bovine oocyte was enucleated by the glass needle and the domestic cat fetal fibroblast used as the donor nuclei was then placed into the perivitelline space adjacent to the plasma membrane of the oocyte. Couplets with bovine ooplasm were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 µs, respectively, using an electro cell fusion generator followed by cycloheximide treatment. Reconstructed cat-bovine embryos were treated with 0, 25, 50, and 100 nM concentrations of TSA for 24 h following fusion. The percentages of embryos cleaved and embryos developed to the blastocyst stage were subjected to arc sin transformation before ANOVA. The TSA treatment at 50 nM contributed significantly higher rates of cleavage and blastocyst formation (n = 139; 84.3 and 4.6%, respectively) compared with untreated embryos (n = 187; 63.8 and 0%, respectively) and embryos treated with 100 nM TSA (n = 172; 71.4 and 0%, respectively; P < 0.05). Development to the morula stage of iSCNT embryos was observed in the TSA treatment groups, whereas no embryos developed beyond the 16-cell stage in the untreated group. In conclusion, our results indicate that TSA treatment for 24 h following fusion improves the development of iSCNT embryos. Specifically, 50 nM TSA treatment provides a beneficial effect on cleavage and development to the blastocyst stage of cat iSCNT embryos using bovine oocytes matured in vitro as recipients and domestic cat fibroblasts as donor nuclei.

The development and expression of pluripotency genes in embryos derived from nuclear transfer and in vitro fertilization

Zygote ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 540-548 ◽  
Author(s):  
Li-Bing Ma ◽  
Xiao-Ying He ◽  
Feng-Mei Wang ◽  
Jun-Wei Cao ◽  
Teng Cheng
Keyword(s):  
Nuclear Transfer ◽  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Pluripotency Genes

SummarySomatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep–sheep intra-species cloned embryos, goat–sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.

Effect of embryonic cell cycle of nuclear donor embryos on the efficiency of nuclear transfer in Japanese black cattle

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 165-171
Author(s):  
M. Kishi ◽  
R. Takakura ◽  
Y. Nagao ◽  
K. Saeki ◽  
Y. Takahashi
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Embryonic Cell ◽  
Embryonic Cells ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
Cell Cycle Stage

SummaryIn the present study, the development in vitro and in vivo of nuclear transfer (NT) embryos reconstructed with embryonic cells (blastomeres) at the 32- to 63-cell (sixth cell cycle) and 64- to 127-cell (seventh cell cycle) stages was investigated to determine the optimum range of embryonic cell cycles for yielding the highest number of identical calves in Japanese black cattle. Rates of development to the blastocyst stage (overall efficiency) were higher in the sixth cell-cycle stage (45%) than in the seventh cell-cycle stage (12%). After the transfer of the blastocysts reconstructed with blastomeres of the sixth and seventh cell cycle-stage embryos to recipient heifers, there were no differences in the pregnancy (14/35: 40% versus 3/13: 23%, respectively) or calving rates (11/39: 28% versus 3/13: 23%, respectively). These results indicate that the highest number of identical calves would be obtained by using sixth cell cycle (32- to 63-cell)-stage embryos as nuclear donors.

scholarly journals 57EMBRYO DEVELOPMENT FOLLOWING INTERSPECIES NUCLEAR TRANSFER OF AFRICAN BUFFALO (SYNCERUS CAFFER), BONTEBOK (DAMALISCUS DORCUS DORCUS) AND ELAND (TAUROTRAGUS ORYX) SOMATIC CELLS INTO BOVINE CYTOPLASTS

2004 ◽  
Vol 16 (2) ◽  
pp. 150 ◽  
Author(s):  
M. Matshikiza ◽  
P. Bartels ◽  
G. Vajta ◽  
F. Olivier ◽  
T. Spies ◽  
...  
Keyword(s):  
South Africa ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Critically Endangered ◽  
African Buffalo ◽  
Syncerus Caffer ◽  
Cell Stage ◽  
Wildlife Species ◽  
Interspecies Nuclear Transfer

Wildlife conservation requires traditional as well as innovative conservation strategies in order to preserve gene and species diversity. Interspecies nuclear transfer has the potential to conserve genes from critically endangered wildlife species where few or no oocytes are available from the endangered species, and where representative cell lines have been established for the wildlife population while numbers were still abundant. The purpose of this study was to investigate the developmental ability of embryos reconstructed with transfer of somatic cells from the African buffalo (Syncerus caffer), bontebok (Damaliscus dorcus dorcus) and eland (Taurotragus oryx) to enucleated domestic cattle (Bos taurus) oocytes. Skin tissue from the three wildlife species were collected by surgically removing approx. 1.0×1.0cm ear skin notches from animals immobilized with a combination of etorphine hydrochloride (M99; South Africa) and azaperone (Stressnil, South Africa). The biopsies were placed into physiological saline and transported to the laboratory at 4°C within 2h, cleaned with chlorohexidine gluconate and sliced finely in Minimal Essential Medium supplemented with 10% fetal calf serum. The resultant tissue explants were treated as previously described (Baumgarten and Harley 1995 Comp. Biochem. Physiol. 110B, 37–46) and actively growing fibroblast cultures made available for the nuclear transfer process. Nuclear transfer was performed using the HMC technique (Vajta et al., 2003 Biol. Reprod. 68, 571–578) using slaughterhouse-derived bovine oocytes. Culture was performed in SOFaaci (Vajta et al., 2003 Biol. Reprod. 68, 571–578) medium supplemented with 5% cattle serum using WOWs (Vajta et al., Mol. Reprod. Dev. 50, 185–191). Two identical replicates were made with somatic cells of each species. After successful reconstruction, 57, 42 and 48 nuclear transferred and activated buffalo, bontebok and eland embryos were cultured, respectively. All except for 2 buffalo embryos cleaved; 22 (39%) developed to or over the 8-cell stage, and 2 (3.5%) of them to the blastocyst stage. All but 3 bontebok embryos cleaved, 17 (40%) developed to or over the 8-cell stage, but none of them reached the compacted morula or blastocyst stage. Sixteen (33%) of the eland embryos developed to or over the 8-cell stage with one (2%) reaching the blastocyst stage. In conclusion, buffalo, bontebok and eland embryos developed from reconstruction using their respective somatic cells combined with bovine cytoplasts, however, in vitro developmental ability to the blastocyst stage was limited. Additional basic research that establishes the regulative mechanisms involved with early preimplantation development together with optimising nuclear transfer techniques may have the potential to one day play a role in the conservation of critically endangered wildlife species.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

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