Effects of laser-assisted hatching and exposure time to vitrification solution on mouse embryo development / 대한생식의학회지

OBJECTIVE: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. METHODS: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. RESULTS: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p < 0.05). In the control-8 group (22.1±4.6), the cell number of the inner cell mass was higher than in the LAH groups (p < 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group (92.8±8.9) than in the others (p < 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group (19.5±5.1, p < 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group (73.2±12.1) than in the other groups, but without statistical significance. CONCLUSION: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

Desarrollo embrionario del pargo colorado Lutjanus colorado (Jordan & Gilbert, 1882) / Embryonic development of pargo colorado Lutjanus colorado (Jordan & Gilbert, 1882)

El pargo colorado (Lutjanus colorado) es una especie con un alto valor comercial en el mercado mexicano, con potencial para su cultivo. Hasta la fecha no existen estudios sobre su reproducción, cultivo larvario y engorda en cautiverio. El presente trabajo es el primer reporte sobre la descripción a detalle del desarrollo embrionario de la especie bajo condiciones de cultivo. Los huevos fertilizados viables del pargo colorado son pelágicos, esféricos, transparentes y con una sola gota de aceite. Midieron 0,77±0,09 mm de diámetro y la gota de aceite 0,14±0,01 mm. La primera división ocurrió a las 0,05 horas post fertilización (HPF). La eclosión se llevó a cabo a las 17,22 HPF bajo las condiciones del presente estudio. Las larvas recién eclosionadas midieron 1,8±0,1 mm de longitud total (LT). El desarrollo embrionario de esta especie fue similar a la descrita para especies de la misma familia. Los resultados del presente estudio aportan información básica para iniciar el desarrollo de la biotecnología para la producción de semilla de esta especie a escala comercial.

The Colorado snapper (Lutjanus colorado) is one of the most commercially important fish species in México and it is considered a suitable candidate for culture. Until now, no research has been carried out on its reproduction, larviculture and fattening in captivity. This study is the first description of embryonic development of this species under controlled conditions. Fertilized eggs of Colorado snapper are pelagic, spherical and transparent and contain one drop of oil. Eggs measured 0.77±0.09 mm and the drop of oil 0.14±0.01 mm. First cell division occurred at 0.05 h post-fertilization (HPF), hatching at 17.22 HPF under the above described conditions. Larvae total length (LT) was 1.8±0.1 mm. Embryonic development of this species was similar to other lutjanidae species. These results provide basic information for developing the necessary biotechnology for commercial seed production of the Colorado snapper.

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.

Embryonic Developmental Capacity and Pregnancy Rates of Fertilized Oocytes in IVF, ICSI and TESE-ICSI Cycles / 대한불임학회지

OBJECTIVE: This study was performed to evaluate and compare the embryonic developmental capacity and pregnancy rates in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with ejaculated sperm or testicular sperm cycles. MATERIALS AND METHODS: Fertilization was examined in the following morning after IVF (group I), ICSI (group II) or TESE-ICSI cycles (group III). Fertilized oocytes were co-cultured with Vero cells until embryo transfer (ET). On day 2 and 5~7, grades of embryos ( or =4-cell) and blastocysts (BG1, 2, 3 or early) were evaluated. Clinical pregnancy rate was determined by detecting G-sac with transvaginal ultrasonogram. We analyzed the results bychi2 and Student's t-test and considered statistically significant when P value was less than 0.05. RESULTS: Fertilization rate was significantly higher (p<0.05) in group I (79.0+/-21.2%) than in group II and III (56.8+/-21.6% and 36.7+/-25.3%). Cleavage and blastulation rate of group I (95.8+/-13.8% and 59.5+/-25.3%) were significantly higher (p<0.05) than those of group III (83.4+/-18.6% and 40.4+/- 36.5%). Clinical pregnancy rate was significantly higher (p<0.05) in group I and II (40.7% and 41.7%) than that in group III (12.5%). No differences were found in the rates of multiple pregnancy and abortion among three groups. Embryonic implantation rate was higher in group I (15.1+/-20.2%, p<0.05) and II (14.7+/-20.6%, NS) than that in group III (5.1+/-15.6%). However, embryonic implantation rate was increased in ET with blastocyst(s) among three groups. CONCLUSIONS: Fertilized oocytes obtained from TESE-ICSI were harder to be successfully cultured to blastocyst stage for 5~7 days than that from IVF cycles. However, all blastocyst(s) ET increased the embryonic implantation rate equally in IVF, ICSI and TESE-ICSI cycles.

Effects of Medium on Blastocyst Formation, Cell Number and Percentage of ICM in Mice / 대한불임학회지

OBJECTIVE: The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. MATERIALS AND METHODS: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values<0.05 were accepted as statistically significant. RESULTS: The blastulation rate in MEM (64.9+/-4.95%) was significantly higher (p=0.0031) than that in TCM (57.2+/-5.22%). No differences were found in the number of ZiB and ZeB between MEM (31.9+/-2.62, 33.0+/-4.58%), and TCM (27.2+/-4.28, 30.1+/-4.58%). A total 314 blastocysts (MEM=166; TCM= 148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM (73.1+/-3.3) than in MEM (61.7+/-2.5). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM (20.9+/-1.3 vs. 17.1+/-1.2%, p=0.0281). The ICM:TE ratio was higher in TCM than in MEM (1 : 4.85+/-0.68 vs. 1 : 3.78+/-0.78, NS). CONCLUSION: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.

Effects of Medium on Blastocyst Formation, Cell Number and Percentage of ICM in Mice / 대한불임학회지

OBJECTIVE: The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. MATERIALS AND METHODS: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values<0.05 were accepted as statistically significant. RESULTS: The blastulation rate in MEM (64.9+/-4.95%) was significantly higher (p=0.0031) than that in TCM (57.2+/-5.22%). No differences were found in the number of ZiB and ZeB between MEM (31.9+/-2.62, 33.0+/-4.58%), and TCM (27.2+/-4.28, 30.1+/-4.58%). A total 314 blastocysts (MEM=166; TCM= 148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM (73.1+/-3.3) than in MEM (61.7+/-2.5). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM (20.9+/-1.3 vs. 17.1+/-1.2%, p=0.0281). The ICM:TE ratio was higher in TCM than in MEM (1 : 4.85+/-0.68 vs. 1 : 3.78+/-0.78, NS). CONCLUSION: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.

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.