scholarly journals 290EFFECTS OF HYALURONAN ON IN VITRO DEVELOPMENT OF PORCINE EMBRYOS CULTURED IN A CHEMICALLY DEFINED MEDIUM

2004 ◽  
Vol 16 (2) ◽  
pp. 264
Author(s):  
K. Yoshioka ◽  
H. Ekwall ◽  
H. Rodriguez-Martinez
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
Transmission Electron Microscopy Data ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Microscopy Data

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization and embryo development. We have previously developed an in vitro-production (IVP) system of porcine embryos, where porcine blastocysts can be produced by IVF and IVC in chemically defined media and can develop to full-term by transfer to recipients. The application of a chemically defined medium to IVP in pigs allows the analysis of the physical action of substances on the development of pre-implantation embryos. In the present study, the effects of HA on the development of porcine embryos in a chemically defined medium were investigated. Porcine presumptive zygotes were produced by IVM and IVF of COC from pre-pubertal gilts and frozen-thawed ejaculated boar semen. The zygotes were cultured in Porcine Zygote Medium (PZM)-5 containing different concentrations of HA (0 [control], 1, 2, 5, 10, 20 and 50μgmL−1) until 6 days after IVF, and representative specimens were fixed for cell counting and transmission electron microscopy. Data of percentages and cell numbers were statistically analyzed by one-way ANOVA and Fisher’s PLSD test. The percentage of embryos that developed to the blastocyst stage (15.8% [23/144] to 19.5% [27/139]) did not differ among treatments. However, addition of 5 or 10μgmL−1 HA increased (P<0.05) the total number of cells in blastocysts (56.1 and 58.3 cells [n=22 and 23], respectively) compared to control (no HA, 42.0 cells [n=23]). To evaluate proliferation rates of inner cell mass (ICM) and trophectoderm (TE), embryos were cultured in PZM-5 for various periods of exposure to 10μgmL−1 HA. The numbers of ICM and TE cells in Day-6 blastocysts cultured in the presence of exogenous HA from Day 0 to Day 3 (18.3 and 34.4 cells, respectively [n=38]) or Day 6 (17.9 and 35.9 cells, respectively [n=36]) were significantly (P<0.05) higher than those cultured without HA through the culture period (13.5 and 24.2 cells, respectively [n=26]). In the presence of HA from Day 3 to 6, only the number of TE cells (37.1 cells [n=33]) increased (P<0.05), compared to PZM-5 alone. Differences in ultrastructure were noticed among blastocysts cultured with or without 10mgmL−1 HA. Blastocysts cultured with HA had mainly mature mitochondria while many mitochondria appeared morphologically immature in the blastocysts cultured without HA. Lipid droplets in the blastocysts cultured with HA seemed to be more homogeneous in comparison with those in the blastocysts cultured in PZM-5 alone. Further differences were seen in the numbers of lysosome-like structures, which were greater in blastocysts cultured with HA. This study demonstrates that exogenous HA improves cell proliferation and normality of ICM and TE in porcine embryos cultured in a chemically defined medium, depending on the exposure periods to HA. (Supported by MAFF, Japan and STINT, Sweden.)

scholarly journals In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

1996 ◽  
Vol 5 (5) ◽  
pp. 509-514 ◽  
Author(s):  
Kristiina Bredbacka ◽  
Peter Bredbacka
Keyword(s):  
Bovine Serum Albumin ◽  
Culture Medium ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Cell Numbers ◽  
Vitro Production ◽  
Bovine Oocytes

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
Y. U. Kim ◽  
D. P. Bhandari ◽  
M. S. Hossein ◽  
S. M. Park ◽  
E. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Differential Staining ◽  
In Vitro Production ◽  
Fetal Fibroblasts

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.

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.

Development in vitro of mouse embryos from the two-cell egg stage to the early somite stage

Development ◽  
1974 ◽  
Vol 31 (1) ◽  
pp. 235-245
Author(s):  
Yu-Chih Hsu ◽  
John Baskar ◽  
Leroy C. Stevens ◽  
John E. Rash
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Cell Stage ◽  
Cord Serum ◽  
Somite Stage ◽  
Development In Vitro

About 1–3% of mouse blastocysts, which had initially been cultured from the two-cell stage in chemically defined medium or about 3–5% of blastocysts which were explanted from the uterus, developed to the early somite stage when cultured in vitro on collagen. Two-cell eggs were initially cultivated in chemically defined medium to the blastocyst stage. Blastocysts were then transferred to Eagle's minimal essential medium (MEM) plus 10% heat-inactivated calf serum. Two barriers to further development were overcome. First, the formation of endoderm and ectoderm from the inner cell mass immediately after attachment to collagen. Second, formation of the embryo proper from the embryonic region. Both barriers were overcome by using heat-inactivated human cord serum after the blastocysts hatched from the zona pellucida and attached to collagen. After attachment, embryos were cultured in MEM plus 20% heat-inactivated human cord serum which was changed daily until early somite stages. Apparently normal healthy development in vitro occurred, as judged by light and electron microscopic examination.

Maternal influence on oocyte collection and embryo development after transvaginal puncture and in vitro fertilisation in dairy cows

2001 ◽  
Vol 26 (2) ◽  
pp. 433-435
Author(s):  
S. Chastant-Maillard ◽  
H. Quinton ◽  
C. Douar ◽  
J. Marchal ◽  
C. Richard ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Formation Rate ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
In Vitro Fertilisation ◽  
Oocyte Collection

AbstractThe aim of the study was to evaluate differences between cows in the “quality” of their oocytes defined as their ability to support embryonic development. Ten cows from the same herd, all primiparous and non-pregnant were submitted for oocyte collection by Ovum-Pick Up (OPU). The oocytes were matured in vitro and fertilised with semen from the same bull. In vitro embryo development, both quantitatively (percentages showing cleavage and forming blastocysts) and qualitatively (differential cell counting in blastocysts) was determined at the blastocyst stage (Day 7). The number of oocytes collected, the number of blastocysts obtained and the blastocyst formation rate varied between cows (P<0.001). The mean percentage of inner cell mass cells tended to higher for embryos derived from one cow. These results provide evidence that the quality of the oocytes was influenced by their maternal origin. Follicular growth also varied between cows.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

104 BIRTH OF FIRST BUFFALO (BUBALUS BUBALS) CALF FOLLOWING EMBRYO SPLITTING AND POLYMERASE CHAIN REACTION SEXING AT BLASTOCYST STAGE

2012 ◽  
Vol 24 (1) ◽  
pp. 164 ◽  
Author(s):  
M. Zhang ◽  
H. H. Chen ◽  
J. W. Tang ◽  
X. W. Liang ◽  
M. T. Chen ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Buffalo Calf ◽  
Practical Procedure ◽  
Blastocyst Quality ◽  
Grade 3 ◽  
Vitro Development

Embryo-splitting technology provides an effective procedure for increasing the number of transferable embryos per donor, producing genetically identical offspring and facilitating embryo sexing. The ability to identify the sex of embryos before transfer will offer a reliable, economical and practical procedure for buffalo breeding. In this study, we have assessed the feasibility of production of offspring with controlled sex in buffalo by first comparing the effect of blastocyst quality on the viability of demi-embryos and then identifying the sex of a demi-embryo by multiplex-nested PCR before transfer into the recipient. In vitro-matured buffalo oocytes were fertilized by IVF and cultured to the blastocyst stage for 6 to 7 days as described by Lu et al. (2007 Anim. Reprod. Sci. 100, 192–196). These blastocysts were classified in terms of their developmental pattern and morphology on a scale of 1 to 3 grades as described by McEvoy et al. (1990 Theriogenology 33, 1245–1253). Blastocysts were split into 2 equal parts by a micromanipulation system. Viability of the resulting demi-embryos was confirmed by formation of a blastocoel cavity and definite inner cell mass after culture for 24 h. One of the zone-free demi-embryos derived from a grade-1 blastocyst was cultured in TCM 199 supplemented with 10% fetal bovine serum for another 2 h, then was transplanted to a spontaneous oestrous recipient. The other demi-embryo was used for sexing by multiplex-nested PCR (Fu et al. 2007 Theriogenology 68, 1211–1218). The results showed that grade-1 blastocysts yielded more viable demi-embryos than grade-2 and grade-3 blastocysts [P < 0.01; 73/92 (79.67%) vs 32/76 (47.05%) vs 26/94 (26.53%), respectively]. Transplantation of the presumed-Y demi-embryo derived from grade-1 blastocyst into a recipient resulted in the birth of a male buffalo calf. To the best of our knowledge, this is the first buffalo calf produced following embryo splitting and PCR sexing of the embryo at the blastocyst stage. Successful birth of the desired-sex offspring in the present study indicates the feasibility of using embryo splitting in combination with multiplex-nested PCR sexing to produce offspring of controlled sex in swamp buffalo. However, the quality of embryos before splitting was an important factor governing the in vitro development of viable demi-embryos. This study was supported by the Guangxi Science and Technology R&D Program (0626001-3-1, 0815008-2-4).

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation

1995 ◽  
Vol 29 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Th. Rülicke ◽  
P. Autenried
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Freeze Thaw ◽  
Foster Mothers ◽  
Vital Stains ◽  
Partial Damage

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

scholarly journals 45PRODUCTION OF CHIMERIC TRANSCHROMOSOMIC CALVES

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
P. Kasinathan ◽  
M.F. Nichols ◽  
J.E. Griffin ◽  
J.M. Robl
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Peripheral Blood Lymphocytes ◽  
Blastocyst Stage ◽  
Human Artificial Chromosome ◽  
Fish Analysis ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Fetal Fibroblasts

Chimeras have been used for investigating fundamental aspects of early embryonic development, and differentiation, and for introducing foreign genes into mammals (Robertson et al., 1986 Nature 323, 445–448; Cibelli et al., 1998 Science 280, 1256–1258). The main objective of this study was to determine if the transfer of blastomeres from in vitro-produced (IVP) embryos into cloned, transchromosomic embryos improved the efficiency of producing transchromosomic calves. Cloned embryos were produced using in vitro-matured bovine oocytes and bovine fetal fibroblasts containing a human artificial chromosome (HAC) (Kuroiwa et al., 2002 Nat Biotechnol 20, 889–894). IVP embryos were produced using standard procedures and blastomeres were harvested at the 8–16 cell stage by removing the zona pellucida with protease. Cloned embryos were randomly divided on Day 4 into two groups. One group received 3–4 IVP blastomeres while a second group served as a control (nonmanipulated cloned embryos). After transferring the blastomeres, the chimeric and cloned embryos were placed in culture (Kasinathan et al., 2001 Biol. Reprod. 64, 1487–1493) and on Day 7 development to the blastocyst stage was evaluated. Grades 1 and 2 embryos were transferred; two each per synchronized recipient. Pregnancy maintenance, calving, and calf survival were evaluated in both groups. Presence of a HAC in live calves was evaluated in both fibroblasts and peripheral blood lymphocytes (PBLs) using FISH analysis. Embryo development to the blastocyst stage, maintenance of pregnancy and number of calves born were analyzed using Chi-square. There were no differences in the rate of blastocyst development at day 7 or establishment of pregnancy at 40d (P&gt;0.05). However, pregnancy rate at 120d, and number of calves that developed to term and were alive at birth (chimera 14/54 and clone 4/90), and at 1 month of age (chimera 13/54 and clone 1/90) were lower (P&lt;0.01) for cloned embryos. The proportion of cells containing an HAC in PBLs, was higher in cloned calves (100%) compared to chimeric calves (26%). The HAC retension rates in PBLs in HAC-positive chimeric and cloned calves were 84% and 95%, respectively. These data indicate that, although the proportion of calves retaining an HAC was lower in chimeras compared to clones, more HAC-positive calves were produced in the chimeric treatment from fewer cloned embryos. We speculate that higher rates of development in the chimeras may be related to the normality of the placenta. Future studies will be required to determine the contribution of the IVP blastomeres to both the inner cell mass and trophectoderm. Therefore, a chimeric approach may be useful for improving the efficiency of producing cloned transchromosomic calves.

scholarly journals MCRS1 is essential for epiblast development during early mouse embryogenesis

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Wei Cui ◽  
Agnes Cheong ◽  
Yongsheng Wang ◽  
Yuran Tsuchida ◽  
Yong Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Early Mouse

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

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