Adequate Concentration and Duration of Vinblastine Treatment for Chromosome Preparation in Mouse Embryos

Matsukawa

Matsumoto

et al. 1998

Self Cite

Abstract. The required concentration and time of vinblastine sulfate (a mitotic inhibitor) treatment were determined for successful cytogenetic diagnoses, such as normality and sex in bovine blastocysts, derived from in vitro fertilization. Chromosome preparations were made from blastocysts treated for 17 h, in media containing vinblastine sulfate, with various concentrations of 0, 30, 100, 200 and 300 ng/ml. Vinblastine sulfate was effective at a concentration of 100-300 ng/ml, with respect to the average numbers of metaphase plates and the mitotic indexes (% of mitoses) in the embryos. To determine the sufficient treatment time, embryos were placed in a medium containing 100 ng/ml vinblastine sulfate for 10, 17 and 24 h. The required time of vinblastine sulfate treatment was 10 h. The average length of the no.1 chromosome shortened with the prolongation of treatment time but not with significant difference. Most of the embryos (approximately 98%) treated with vinblastine sulfate had metaphase plates, and approximately 75% of them were numerically analyzable. A high incidence of polyploidy and a sex ratio showing male advantage were found in the present study.

Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 97%

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Required Concentration and Time of Vinblastine Treatment for Chromosome Preparation in Bovine Blastocysts Derived from In Vitro Fertilization.

Matsukawa

Matsumoto

et al. 1998

Self Cite

“…In the present study, the low mitotic index (6.3) was similar to that reported in Day-6 porcine blastocysts produced by IVP (7.7) by McCauley et al [15]; this indicates that relatively few cells had undergone mitosis. A mitotic index of 20% has been reported for bovine blastocysts produced by IVP and treated with vinblastine for 17 h [33], while a higher mitotic index (92%) has been reported for mouse morulae derived from natural mating after vinblastine treatment for 10 h [39]. The low mitotic indices of porcine blastocysts produced by IVP compared with bovine and mouse embryos suggest that cell division occurs more slowly in porcine embryos than in bovine and mouse embryos.…”

Section: Discussionmentioning

confidence: 97%

The Blastocyst Production Rate and Incidence of Chromosomal Abnormalities by Developmental Stage in In Vitro Produced Porcine Embryos

Ullo

Komoriya

et al. 2008

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Abstract. The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 × 10 5 sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3-to 4-cell, 5-to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3-to 4-cell and 5-to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3-to 4-cell and 5-to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology. Key words: Blastocyst, Chromosomal abnormality, Embryo, In vitro production (IVP), Pig (J. Reprod. Dev. 54: [22][23][24][25][26][27][28][29] 2008) n mammals, the technology of in vitro production (IVP) of embryos, which involves in vitro maturation and fertilization of oocytes and in vitro culture of the resultant embryos, has become important for increasing the number of offspring from selected high-quality animals and for reducing the generation intervals for breed improvement in livestock [1,2]. Moreover, IVP technology has been used in the production of cloned and transgenic animals, which can be used in biomedical research [3][4][5]. In swine, although embryos have been successfully produced using the IVP system, the development rates to the blastocyst stage and to full term after embryo transfer are still low [6][7][8]. The viability of porcine embryos produced by IVP is low compared with that of in vivo-derived porcine embryos. For example, porcine embryos produced by IVP are of poor quality in terms of the number of cells compared with those produced in vivo [9].A...

“…Chromosome samples were produced following the method of Yoshizawa et al [20]. Embryos that developed to the blastocyst stage were treated in Whitten 514 medium with 30 ng/ml vinblastine sulfate for 10 h and individually fixed onto individual glass slides.…”

Section: Chromosome Preparationmentioning

confidence: 99%

Cytogenetic Analysis and Developmental Assessment of Mouse Embryos Derived from In Vitro Fertilization of Oocytes Reconstructed by Meiosis-II Chromosome Transplantation

Mitsui

2007

Self Cite

Abstract. An electrofusion methodology for transferring meiosis-II chromosomes (M-II-t) has not been completely established. The present study compared the use of two temperatures (fusion at 37 C for Group A and 25 C for Group B) during an electrofusion procedure for mouse oocyte M-II-t and investigated the cytogenetic normality and developmental competence of embryos derived from in vitro fertilization using oocytes reconstructed by M-II-t. The M-II-t oocytes were fertilized in vitro and cultured to the blastocyst stage; the resultant embryos were analyzed cytogenetically. Subsequently, chromosomal normality of the resultant embryos at the prometaphase stage of first cleavage division and the integrity of the meiosis-II spindles of the reconstructed oocytes were analyzed. The success rate of electrofusion in Group B was 92.1%, which was significantly different from that in Group A (49.2%) (P<0.05). The fertilization rates (A, 80.7%; B, 77.2%) and development rates (A, 70.9%; B, 65.5%) in the M-II-t groups were significantly lower than those in the control group (95.0 and 92.2%, respectively) (P<0.05). The incidence of chromosomal abnormalities in the Group A embryos (20.5%) at the blastocyst stage was significantly higher than that in the control group embryos (8.5%) (P<0.05), but the incidence of chromosomal abnormalities in Group B (12.5%) was not significantly different compared with the other groups. A temperature of 25 C during the electrofusion procedure for M-IIt resulted in a good fusion rate, good development rate, and efficient production of chromosomally normal blastocysts. Furthermore, the incidence of chromosomal abnormalities in the first cleavage embryos at the prometaphase stage in Group B (9.6%) did not differ significantly from that in the control group (6.6%). The spindle morphology of the M-II-t oocytes in Group B was normal.