Analysis of the influence factors of blastomere damage of frozen thawed embryos / 重庆医学

Objective To observe the factors that affect cryosurvival of frozen-thawed embryos .Methods A retrospective study was conducted on 98 patients undergoing FET with two embryos of which one was complete cryosurvival and the other not .1∶1 matched samples logistic regression analysis was employed to observe the influence of cytoplasm with granulation ,vacuolar cyto-plasm ,embryo fragments ,blastomere number and equality of size on the cryosurvival of frozen-thawed embryos .Results Presence of vacuolar cytoplasm(OR=13 .413) or embryo fragments(OR= 1 .101) significantly increased blasstomere damage ,but the in-creased blastomere number(OR=0 .569) decreased it(P<0 .05) .Conclusion Embryos with vacuolar cytoplasm ,or embryo frag-ments and blastomere number are very vital factors that affect the blastomere damage after cryopreservation .

Advantages and Applications of Cryopreservation in Fisheries Science

Cryopreservation is a long-term storage technique to preserve the biological material without deterioration for extended period of time at least several thousands of years. The ability to preserve and store both maternal and paternal gametes provides a reliable source of fish genetic material for scientific and aquaculture purposes as well as for conservation of biodiversity. Successful cryopreservation of fish sperm have been achieved for more than 200 fish species and many fish species have been adequated for the purpose of cryobanking. Cryopreservation of fish embryo is not viable, mainly because of the same limitations as in fish oocytes, i.e., high chilling sensitivity and low membrane permeability. However, cryopreservation of isolated embryonic cells is another option for preserving both maternal and paternal genome. In this paper, an overview of the current state of aquatic species is followed by a discussion on the sperm, embryos, oocytes and embryonic cells - blastomeres.

Desarrollo embrionario del capaz pimelodus grosskopfii (Steindachner, 1879) / Embryonic development of capaz pimelodus grosskopfii (Steindachner, 1879)

El conocimiento del desarrollo embrionario en los peces es especialmente importante en especies nativas con potencial para la piscicultura, en virtud que permite identificar eventos morfológicos y cronológicos, necesarios para establecer prácticas de manejo durante las fases de incubación y larvicultura. El capaz (Pimelodus grosskopfii) es una especie con potencial para cultivo comercial, por sus hábitos alimenticios omnívoros y aceptación de su carne en el mercado. Para estudiar el desarrollo embrionario de la especie, ejemplares adultos sexualmente maduros fueron inducidos a la reproducción con extracto de hipófisis de carpa (5,75 y 4,0 mg Kg-1, hembras y machos, respectivamente). Los óvulos seminados fueron incubados en un sistema de flujo ascendente de 30 L a 27 +/- 1 °C. Las muestras (n=30) fueron colectadas al momento de la extrusión, durante la fertilización y cada 15 minutos a partir de las 0 horas postfertilización (HPF) hasta las 2 horas y cada 30 minutos desde las 2 HPF hasta 5 HPF; finalmente, entre las 5 HPF y la eclosión, cada 60 minutos. Los óvulos fertilizados presentaron forma esférica, sin adherencias y con amplio espacio perivitelino. El desarrollo embrionario finalizó a las 12 HPF. La diferenciación del polo animal y vegetal ocurrió a las 0,2 HPF, el primer clivaje a las 0,3 HPF, el blastodisco alto y estratificado a las 1,8 HPF, el blastodisco achatado a las 3,3 HPF, la epibolia < a 50 por ciento se observó a las 4 HPF, el cierre del blastoporo a las 5,7 HPF, la diferenciación cráneo caudal e inicio de la neurolación a las 7 HPF, la diferenciación de las vesículas ópticas, óticas y vesícula de Kupffer a las 8,5 HPF, la liberación de la cola del vitelo a las 10 HPF, los primeros movimientos se observaron a las 10,5 HPF y finalmente la eclosión ocurrió a las 12 HPF. Las larvas al eclosionar presentaron una longitud total de 2987+/-67 um, sin pigmentación, tracto digestivo rudimentario, sin abertura bucal ni anal y presencia de cromatóforos...

The knowledge of embryonic development in fish is important in native species with potential for fish farming, by virtue of which it makes possible to identify morphological and chronological events to establish management practices during incubation periods and larviculture. The capaz (Pimelodus grosskopfii) is a species with potential for commercial crop, due to their omnivorous eating habits and acceptance of its meat in the market. To study the embryonic development of the species, sexually mature adult specimens were induced to reproduce with carp pituitary extract (5.75 and 4.0 mgKg-1, females and males, respectively). The inseminated oocytes were incubated in an upward flow system 30 a 27 +/- 1 ° C. The samples (n = 30) were collected at the same time of the extrusion, during fertilization, and every 15 minutes starting from 0 to 2 hours post fertilization (HPF) and every 30 minutes from 0 to 2 HPF, and every 30 minutes from 2 to 5 HPF; finally, between 5 HPF and hatching every 60 minutes. The fertilized oocytes had a spherical shape without adhesions and large perivitelline space. Embryonic development took 12 HPF. The differentiation in animal and vegetal pole occurred at 0.2 HPF, the first cleavage at 0.3 HPF, stratified and high blastodisc at 1.8 HPF, flattened blastodisc at 3.3 HPF, the epiboly <50 percent was observed at 4 HPF, the closure of the blastopore at 5.7 HPF, cranial-caudal differentiation and starting the neurolation at 7 HPF, the differentiation of the optic vesicles, otic and Kupffer's vesicle at 8.5 HPF, tail of the vitelum was released at 10 HPF, first movements were observed at 10.5 and finally hatching occurred at 12 HPF. When the larvae hatched, they showed a total length of 2987+/-67 µm, without depigmentation, rudimentary digestive system without oral and anal opening and the presence of chromatophores on the yolk sac.

Progeny of 2-cell embryo blastomeres distribute in mouse blastocyst randomly / 解剖学报

Objective Kunming strain(KM) mice were used as animal models. Nontoxic dextran conjugated with tetramethylrhodamine(TMR)and fluorecein isothiocyante(FITC)was microinjected to two of the 2-cell blastomere as molecular probe to trace the development fate of the blastomere ,in order to figure out the mechanisms of the formation of Em-Ab axis. Methods FITC- dextran was injected to zygote in order to make sure if it is noxious. Two blastomeres of 2-cell embryo were injected FITC- dextran and TMR- dextran respectively. Results When labeled embryo develeped to blastocyst, distribution of progeny of 2-cell embryo blastomeres can be detected.Conclusion The cells of blastomere randomly distributed either embryonic parts or extraembryonic parts of blastocyst.

Establishment of Embryonic Stem Cell Line from Isolated Blastomeres from Mouse Preimplantation Embryos / 대한불임학회지

OBJECTIVE: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. METHODS: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific gene expressions with immunocytochemistry and RT-PCR. RESULTS: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cells specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. CONCLUSION: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

The Effects of Free Radical Scavenger, Rutin, on the Development and the Cell Number of Blastocyst in Mouse Early Embryos / 대한산부인과학회잡지

It is well known that developmental delay or arrest occurs before implantation inmammals, which have undergone in vitro culture. Recently, these phenomenon are being attributedto oxygen free radicals, and successful cell culture are being obtained by lowering theoxygen environment of in vitro culture. This is due to the fact that the oxygen concentrationin the fallopian tube is around 5%, which is lower than the room air 20% concentrationfor in vitro culture.Rutin, which is a free radical scavenger, was added to early embryo mice culture andcompared the free radical level at blastocyst stage with that of different culture conditions,and found that free radical level was markedly decreased. Also, there was increased embryodevelopment with decreasing free radical levels in the experimental group, and there wassignificant increase in the blastulation rate and blastomere count.This study therefore suggests the possibility of improved in in-vitro embryo culture.

The Effects of Free Radical Scavenger, Rutin, on the Development and the Cell Number of Blastocyst in Mouse Early Embryos / 대한산부인과학회잡지

It is well known that developmental delay or arrest occurs before implantation inmammals, which have undergone in vitro culture. Recently, these phenomenon are being attributedto oxygen free radicals, and successful cell culture are being obtained by lowering theoxygen environment of in vitro culture. This is due to the fact that the oxygen concentrationin the fallopian tube is around 5%, which is lower than the room air 20% concentrationfor in vitro culture.Rutin, which is a free radical scavenger, was added to early embryo mice culture andcompared the free radical level at blastocyst stage with that of different culture conditions,and found that free radical level was markedly decreased. Also, there was increased embryodevelopment with decreasing free radical levels in the experimental group, and there wassignificant increase in the blastulation rate and blastomere count.This study therefore suggests the possibility of improved in in-vitro embryo culture.