57 DEVELOPMENTAL CHARACTERISTICS OF LATER-STAGE PORCINE EMBRYOS PRODUCED IN VIVO OR IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 158 ◽  
Author(s):  
H. Callesen ◽  
P. Holm
Keyword(s):  
In Vitro Culture ◽  
Cell Mass ◽  
Morphological Characteristics ◽  
Viable Cell ◽  
Outer Diameter ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Cell Stage

Kinetics and morphological characteristics during pre-implantation embryo development are well established in most mammals. In porcine, such studies are few because of limited use of in vitro culture, but this has changed in recent years due to increasing use of in vitro production and cloning. Therefore, additional characterisation of especially later stage porcine embryos is needed. Here we studied in vitro development of porcine in vivo- and in vitro-derived zygotes collected from weaned, inseminated sows (slaughtered 2 days after first insemination; in vivo group, n = 112) or produced from immature oocytes from sow ovaries (matured and fertilized: Theriogenology 63, 2040–2052; in vitro group, n = 210). Both types were cultured for 7 days in vitro (Theriogenology 63, 2040–2052) in a time-lapse system (Theriogenology 50, 1285–1299) with images recorded every 0.5 h. Individual embryos were followed and characterised for stage and quality from first cleavage. The following was recorded: (i) zygote: inner (i.e. ooplasma) and outer diameter [i.e. including zona pellucida (ZP)], ZP thickness; (ii) compact morula: areas of compacted inner cells and of cellular debris; (iii) blastocyst: partial or total collapse of blastocoelic cavity and diameter at maximal expansion immediately before hatching. At the 1-cell stage, no difference was found between in vivo v. in vitro zygotes in ZP diameter (approximately 150 µm) and thickness (approximately 15.6 µm). In both groups, cleavage rate was around 65%, but more in vivo (85%) than in vitro (28%) zygotes developed beyond the morula stage. Embryos of both types that did not develop to this stage (n = 212) blocked predominantly at the 1st (50%) or 2nd (21%) cell cycle. Cell cycles were generally shorter in in vivo v. in vitro zygotes from compact morula until the hatched blastocyst stage (mean 128 v. 139 h from the 2-cell stage for in vivo v. in vitro; P < 0.05). Compacted in vivo morulae were 25% larger than in vitro, and the debris area was more than twice as large in in vitro. Hatching occurred after approximately two collapses in both in vivo and in vitro but at a larger ZP diameter in vitro. See Table 1 for further details. This study illustrates differences and similarities between morphology and developmental kinetics of in vivo- and in vitro-derived porcine zygotes, but also how various morphological characteristics indicate some of the possible causes of the reduced developmental ability of in vitro embryos. The in vitro period seems to result in more stressful conditions for the embryos, both during early development, but also during the later stages leading to the hatching process. Thus, further optimization of in vitro culture conditions is still needed in porcine. Table 1.Compact morula (CM) viable cell mass and debris, hatched blastocyst (HB) diameter, and early-hatched blastocyst (EB-XB) and expanded-hatched blastocyst (XB-HB) collapses for in vivo- and in vitro-derived zygotes

248 IN VITRO PRODUCTION OF CATTLE × BUFFALO HYBRID EMBRYOS USING BOVINE OOCYTES AND AFRICAN BUFFALO (SYNCERUS CAFFER CAFFER) EPIDIDYMAL SPERM

2007 ◽  
Vol 19 (1) ◽  
pp. 240
Author(s):  
O. D. Owiny ◽  
D. M. Barry ◽  
M. Agaba ◽  
R. A. Godke
Keyword(s):  
Bison Bison ◽  
Abnormal Development ◽  
African Buffalo ◽  
Syncerus Caffer ◽  
Epididymal Sperm ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Interspecies hybridization of bovids occurs between domestic cattle and at least 3 other species: the American bison (Bison bison), yak (Bos grunniens), and banteng (Bos banteng). Birth of a cattle � buffalo hybrid was reported in Russia, but the report was never authenticated. Such hybrids could be important in improving livestock production and managing diseases that impede production in tropical Africa. We investigated hybridization between cattle and their closest African wild relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce pre-implantation cattle � buffalo hybrid embryos in vitro, matured bovine oocytes were subjected to a standard IVF procedure with either homologous (n = 1166 oocytes) or heterologous (n = 1202 oocytes) buffalo epididymal sperm. After IVF, 67.2% of the oocytes inseminated with homologous sperm cleaved. In contrast, insemination with buffalo sperm resulted in a 4.6% cleavage rate. Cleavage was also slower in hybrids than in cattle embryos. Up to 52.2% of cleaved homologous embryos progressed to the morula stage compared with 12.7% for hybrids. No hybrid embryos developed beyond the 16-cell stage, whereas 40.1% of the cleaved bovine embryos developed to the blastocyst stage. Developmental anomalies such as polyspermy, uneven cleavage, vacuolization, and absence of nuclei in some blastomeres were common in the hybrid embryos. We conclude that interspecies fertilization of cattle oocytes with African buffalo sperm occurs in vitro and that the barrier to hybridization is in the early stages of embryonic development. Also, chromosomal disparity is the likely cause of fertilization abnormalities, abnormal development, and subsequent arrest, impairing the formation of pre-implantation hybrid embryos. Investigation into developmental abnormalities, including reciprocal hybridization and genetic studies of the hybrid embryos, are recommended.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
C. Pontes Godoi ◽  
P. D. Moço ◽  
B. Cazari ◽  
P. T. Mihara ◽  
P. V. Silva ◽  
...  
Keyword(s):  
Cell Mass ◽  
Farm Animals ◽  
Control Group ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Chi Square Test ◽  
Aggregation Technique

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

68 TRANSCRIPTIONAL GENOME ACTIVATION IN CANINE EMBRYOS COLLECTED IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 146 ◽  
Author(s):  
S. Chastant-Maillard ◽  
C. Viaris de Lesegno ◽  
S. Thoumire ◽  
M. Chebrout ◽  
K. Reynaud
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Laser Scanning ◽  
Transcriptional Activity ◽  
Staining Intensity ◽  
Laser Scanning Confocal Microscopy ◽  
Cell Stage ◽  
Morula Stage

Early embryonic stages are supported by maternal transcripts from the oocyte cytoplasm. Progressive transcription of embryonic genome is a key step for further embryonic development, especially during in vitro culture. To date, in vitro culture from fertilization to the blastocyst stage is inefficient in the canine species. The objective of this work was to identify minor and major activation in in vivo-produced dog embryos. Ovariectomies were performed in 31 Beagle bitches from 102 to 266 h after ovulation (post-ov), precisely timed by transabdominal ultrasonography. Embryos were collected by tubal flushing with M199-Hepes and immediately transferred into transcription buffer. Transcriptional activity was evaluated through 5-bromouridine 5′-triphosphate (BrUTP) incorporation in nascent RNA, without microinjection (Aoki et al. 1997). Oocytes from anoestrus ovaries were used as positive controls. 5-Bromouridine 5′-triphosphate incorporation was revealed by immunocytochemistry (anti-bromodeoxyuridine primary antibody) and embryonic DNA was stained by ethidium homodimer-2. Staining was quantified under laser scanning confocal microscopy. Transcriptional activity was calculated as (mean nuclear intensity – cytoplasmic mean intensity) × nuclear area and expressed in arbitrary units (AU). It was compared to 1 (similar intensity in nucleus and cytoplasm; i.e. no transcriptional activity) by t-test; levels of transcriptional activity were compared between stages by variance analysis. Seventy embryos (from 7 to 21 per stage) from 31 bitches were analysed, from 2 pronuclei to morula stage. Between 28 and 125 nuclei were quantified per stage. At each stage, transcriptional activity was calculated per embryo and per nucleus. A significant transcriptional activity was detected as early as the 2 pronuclei stage (102–132 h post-ov; 1.15 ± 0.05 AU). Transcriptional activity per embryo significantly increased between the 2- and the 4-cell stage and between the 8-cell and the morula stage. In early 8-cell embryos, staining intensity of the various nuclei was markedly heterogeneous within the same embryo, all nuclei being intensively stained from the late 8-cell stage onwards. Transcriptional activity per nucleus increased also from the 2- to the 4-cell stage (respectively, 120–161 h post-ov, 1.15 ± 0.02 AU and 133–154 h post-ov, 1.35 ± 0.04 AU) until the 8-cell stage (153–225 h post-ov, 5.12 ± 0.55 AU). Transcriptional levels at these 3 stages differed significantly. It decreased between the 8-cell and the morula stage (230–266 h post-ov, 3.06 ± 0.13 AU), probably reflecting the acquisition of a selectivity in gene expression at major activation, as in other species; Nothias et al. 1995). Addition of the transcriptional inhibitor α-amanitin during BrUTP incubation decreased the transcriptional activity by 60% (P < 0.05). Embryonic gene expression (minor activation) thus begins in the canine embryo as early as the 2 pronuclei stage, with major activation taking place during the 8-cell stage.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

In vitro production of cattle-water buffalo (Bos taurus - Bubalus bubalis) hybrid embryos

Zygote ◽  
2002 ◽  
Vol 10 (2) ◽  
pp. 155-162 ◽  
Author(s):  
H.P.S. Kochhar ◽  
K.B.C. Appa Rao ◽  
A.M. Luciano ◽  
S.M. Totey ◽  
F. Gandolfi ◽  
...  
Keyword(s):  
Water Buffalo ◽  
Bos Taurus ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Developmental Potential

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using interspecies gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.

199 IN VITRO EMBRYO PRODUCTION IN THE CLOUDED LEOPARD (NEOFELIS NEBULOSA)

2010 ◽  
Vol 22 (1) ◽  
pp. 258 ◽  
Author(s):  
C. E. Pope ◽  
M. C. Gómez ◽  
B. L. Dresser
Keyword(s):  
Oocyte Retrieval ◽  
Ovarian Response ◽  
Ovarian Activity ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Total N ◽  
Clouded Leopard ◽  
Vitro Production

The clouded leopard, a spotted, mid-sized cat native to southeast Asia, is classified as Endangered by the U.S. Fish and Wildlife Service. The Species Survival Plan (SSP) program has designated that clouded leopards in North American zoological institutions are a research population with the focus of enhancing management and developing assisted reproductive techniques. In this study, we examined (1) ovarian response to exogenous gonadotropin stimulation, (2) in vitro production of embryos by IVF and intracytoplasmic sperm injection (ICSI), and (3) in vivo developmental ability of in vitro-derived embryos. Eight females at Audubon Center for Research of Endangered Species (ACRES) in New Orleans, LA, were used as oocyte donors and embryo recipients. During non-estrual, but otherwise unknown, stages of the estrous cycle, females 4 to 10 years old at our first treatment were administered a total of 15 (n = 8) or 20 (n = 7) IU of porcine FSH (i.m; Sioux Biochemical Co., Sioux City, IA) daily for 4 days. On Day 5, 10 (n = 8) or 15 (n = 7) IU of porcine LH (i.m; Sioux Biochemical Co.) was given and laparoscopic oocyte retrieval (LOR) was done 24 h later. One, 2, or 3 LOR were performed on 3, 3, and 2 females, respectively (total n LOR = 15), 10 (67%) of which were done on females that were 8 to 12 years of age. A total of 176 pre-ovulatory oocytes (mean = 14.7; range = 3-31) were recovered from 12 LOR performed on 6 females. Two females, a 5-year-old and an 11-year-old, did not respond to gonadotropin treatment. Of 5 females with ≥2 LOR, an average of 16.2 and 14.6 oocytes were recovered at the first and second LOR, respectively. Luteal tissue was present on the ovaries at 3 of 4 LOR performed during January-April as compared with 0 of 11 LOR performed from June-December. Semen was obtained by electroejaculation of 3 males (6, 9, and 11 years of age) and used after cryopreservation [TEST-yolk (media prepared with egg yolk, TES, and Tris) + 6% glycerol] or after storage at 4°C for 24 h (cooled). In vitro production, cryopreservation, and transfer of embryos were done as described by Pope et al. (2006 Theriogenology 66, 1518-1524). Cleavage frequency was 43% overall, 55% (64/117) after IVF, and 20% after ICSI with frozen (10/43) or cooled (2/16) sperm. After IVF with frozen sperm, cleavage rate was 63% (48/76) v. 39% (16/41) with cooled sperm. On Days 5, 57, 26, and 17% of embryos were morula (> 16 cells), early morula (≈16 cells) and pre-morula (< 16 cells), respectively. Forty-four Day 5 embryos were cryopreserved at a slow, controlled rate, 24 of which were thawed and transferred by laparotomy to the uteri of 3 Day 5 gonadotropin-treated recipients (7-9 embryos/female). Similarly, 28 Day 5 fresh embryos were auto-transferred to 3 Day 5 recipients (5-15 embryos/female). Most embryos transferred were morulae (83%) produced by IVF using frozen sperm (71%). None of the recipients established pregnancies, a failure that, undoubtedly, was multifaceted. Age may have been a factor because most recipients were 8 to 11 years old. Nonetheless, we have (1) shown that clouded leopard females are responsive to exogenous gonadotropins, (2) produced embryos in vitro, both by IVF and ICSI, using both cryopreserved and cooled sperm, and (3) provided further evidence of seasonality in ovarian activity.

190 INFLUENCE OF IN VITRO CULTURE OF BOVINE EMBRYOS ON THE STRUCTURE OF THE ZONA PELLUCIDA

2007 ◽  
Vol 19 (1) ◽  
pp. 211 ◽  
Author(s):  
E. Mertens ◽  
U. Besenfelder ◽  
M. Gilles ◽  
M. Hölker ◽  
F. Rings ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Zona Pellucida ◽  
Outer Part ◽  
Negative Influence ◽  
Total Thickness ◽  
Microscopical Investigation ◽  
Cell Stage ◽  
Nutritional Factors

The zona pellucida is an extracellular structure at the direct interface between the maternal and embryonic sides, through which all signals of the embryo–maternal dialogue as well as nutritional factors have to pass. Up to now there has been no investigation as to whether in vitro culture influences the structure of the zona pellucida compared to that of in vivo embryos. Therefore, in vitro (oocyte, zygote, 2-, 4-, 8-, 16-cell, morula, and Day 7 blastocyst, using the protocol published by Nganvongpanit et al. 2006 Reproduction 131, 861–874) and in vivo (zygote, 4-cell, morula, and blastocyst) embryos were prepared for microscopical investigation. In total, 191 in vitro and 99 in vivo embryos of quality 1 and 2 from at least 2 replicates were used. Araldit-embedded embryos were semi-thin-sectioned and stained with hematoxilin. A morphometrical evaluation was performed to determine the percentage of the more reticular outer part compared to the total thickness of the zona. Furthermore, the total thickness of the zona pellucida was compared between in vitro and in vivo embryos. In parallel, embryos were analyzed by scanning electron microscopy. Up to the 16-cell stage, the zona of in vivo and in vitro embryos is similar, but in vivo morulae and blastocysts show significantly thicker zonae than the in vitro stages. The reticular part of the zona is thicker in in vivo embryos than in in vitro embryos (30.2 � 2.1% vs. 12.4 � 1.8%, respectively). Investigating the pores of the zona pellucida, in vivo morulae/blastocysts show smaller-sized pores than in those derived in vitro. Most of the in vivo morulae/blastocysts are totally covered by secreted granules, and therefore the pores could not be investigated. Furthermore, 30-50% of the in vitro embryos show partly degenerated outer layers of the zona pellucida. This investigation demonstrates that in vitro and in vivo zonae pellucidae are significantly different, reflecting a negative influence of the IVM and IVC.

scholarly journals Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

2005 ◽  
Vol 17 (8) ◽  
pp. 799 ◽  
Author(s):  
Natalie I. Alexopoulos ◽  
Gábor Vajta ◽  
Poul Maddox-Hyttel ◽  
Andrew J. French ◽  
Alan O. Trounson
Keyword(s):  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Collagen Gel ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Term Survival

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

142 CHIMERISM BY AGGREGATION OF BISECTED IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 175
Author(s):  
E. M. Razza ◽  
I. P. Emanuelli ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Aggregation Rate ◽  
Stage Of Development

Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

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