84 PRODUCTION OF CLONED PIGLETS FROM NUCLEAR TRANSFER EMBRYOS AFTER VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 123
Author(s):  
N. Nakayama ◽  
R. Tomii ◽  
S. Ueno ◽  
H. Matsunari ◽  
H. Saito ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Nuclear Transfer ◽  
Lipid Droplets ◽  
Cytochalasin B ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Early Cleavage ◽  
C 23 ◽  
Morula Stage

Cryopreservation of cloned embryos is expected to be beneficial in improving the efficiency of somatic cell cloning in pigs. We have already demonstrated that normal piglets can be produced from in vitro-matured and fertilized (IVM/IVF) embryos vitrified at an early cleavage stage after delipation (Nagashima et al. 2007 Biol. Reprod. 76, 900–905). In this study we utilized this technique in an attempt to produce piglets from cloned embryos reconstructed with IVM oocytes. Nuclear transfer (NT) embryos were reconstructed using oocytes matured in vitro in NCSU23 and preadipocytes as nuclear donors. The embryos were cultured in PZM-5 for approximately 98 h, and those that had developed to the morula stage were delipated using a noninvasive method described previously (Esaki et al. 2004 Biol. Reprod. 71, 432–437). The embryos were treated with 4% trypsin at 38�C for 1 to 4 min to induce a slight swelling of the zona pellucida, and then centrifuged (12 000g, 38�C, 23 min) with 7.5 µg mL–1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. The embryos were cultured for 1 h and vitrified by the minimum volume cooling (MVC) method using a MVC plate (Cryotop�; Kitasato Supply Co., Tokyo, Japan) in the presence of 15% ethylene glycol, 15% DMSO, and 0.5 m sucrose as cryoprotectants. Vitrified embryos were rewarmed by immersing the MVC plate diretly into rewarming solution containing 1 m sucrose and 20% calf serum at 38�C for 1 min, followed by stepwise dilution of the cryoprotectants. The rewarmed embryos were cultured for 2 days to the blastocyst stage, and then treated with 0.5% pronase to remove the zona pellucida before transfer to the uterine horn of recipients. A total of 103 vitrified blastocysts were transferred to 2 recipient gilts. Both gilts became pregnant and farrowed 2 and 4 piglets, respectively (6/103, 5.8%). These results demonstrate that cloned piglets can be produced from NT embryos that have been cryopreserved at the morula stage using noninvasive delipation and vitrification procedures. This study was supported by PROBRAIN.

185 EMBRYONIC LOSS IN PIGS ASSOCIATED WITH OVIDUCT TRANSPLANTATION OF EARLY-STAGE EMBRYOS WITH DAMAGES IN THE ZONA PELLUCIDA

2006 ◽  
Vol 18 (2) ◽  
pp. 200
Author(s):  
S. Ueno ◽  
M. Kurome ◽  
R. Tomii ◽  
K. Hiruma ◽  
N. Maeda ◽  
...  
Keyword(s):  
Immune Response ◽  
Zona Pellucida ◽  
Nuclear Transfer ◽  
Early Stage ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Embryo Recovery ◽  
Embryonic Loss ◽  
The Impact

It is assumed that if porcine early-stage embryos with damages in their zonae pellucidae are transplanted to the recipient's oviduct, they may suffer from mechanical and immunological stresses by oviduct contraction and the recipient's immune response. This study aimed to examine the impact of zona pellucida damages, which might arise during nuclear transfer and intra cytoplasmic sperm injection (ICSI), on the development and survival of transplanted embryos. Cumulus-oocyte complexes were collected from ovaries obtained at a local slaughterhouse and matured in vitro in NCSU23 to prepare MII-stage oocytes. The zonae pellucidae of these oocytes were either penetrated with 8- to 10-�m square-ended microinjection pipettes or incised with 35- to 40-�m beveled enucleation pipettes. Intact oocytes were used as controls. The oocytes were electroactivated to induce parthenogenesis and transplanted to the oviducts of estrus-synchronized recipient gilts (estrus-synchronized with 1000 IU eCG and 1500 IU hCG). After 5 to 7 days, the recipient uteri were flushed with PBS supplemented with 1% fetal bovine serum (FBS) to collect embryos, and their development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-penetrated, 129 zona-incised, and 57 intact embryos were transplanted to four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (59.0 to 81.8%). However, the zona-penetrated and zona-incised embryos showed inconsistent development and survival compared with controls; the development and survival rate were 92.6% (25/27) to 96.7% (29/30) and 77.8% (21/27) to 96.7% (29/30) in control embryos, respectively, whereas those of zona-penetrated embryos were 57.1% (28/49) to 95.7% (22/23) and 8.2% (4/49) to 78.3% (18/30), and those of zona-incised embryos were 47.6% (30/63) to 92.3% (36/39) and 23.8% (15/63) to 92.3% (22/23), respectively. Large foci of cells that appeared to be macrophage giant cells were observed at the surface or inside of the degenerated zona-damaged embryos. These results indicate that the recipient's immune response may impair development after transplantation of the embryo to the oviduct, when there is damage in the zona pellucida. This may be one of the factors attributable to the reduced efficiency of live progeny production by ICSI and nuclear transfer. This work was supported by PROBRAIN.

The cyclin-dependent kinase inhibitor, JNJ-7706621, improves in vitro developmental competence of porcine parthenogenetic activation and somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (7) ◽  
pp. 1002 ◽  
Author(s):  
Qing Guo ◽  
Long Jin ◽  
Hai-Ying Zhu ◽  
Xiao-Xu Xing ◽  
Mei-Fu Xuan ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Kinase Inhibitor ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Cyclin Dependent Kinase ◽  
Parthenogenetic Activation ◽  
Cyclin Dependent Kinase Inhibitor

In this study we examined the effects of JNJ-7706621, a cyclin-dependent kinase inhibitor, on the in vitro growth of pig embryos that had been produced either by parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT). A significantly higher percentage of PA embryos reached the blastocyst stage by Day 7 after exposure to 10 µM JNJ-7706621 for 4 h compared with embryos exposed to 5 µg mL−1 cytochalasin B for 4 h (P < 0.05). Similarly, the rate of Tyr15 phosphorylation of the complex of cyclin and p34cdc2 (CDK1) was significantly elevated in the JNJ-7706621-treated embryos compared with embryos exposed to cytochalasin B or non-treated controls (P < 0.05). In contrast, Thr161 phosphorylation of CDK1 was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated as well as the non-treated group (P < 0.05). Similarly, the level of M-phase-promoting factor (MPF) in embryos was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated and non-treated groups (P < 0.05). In addition, more SCNT embryos reached the blastocyst stage after treatment with JNJ-7706621 than following exposure to cytochalasin B (P < 0.05). In conclusion, these results reveal that exposure to 10 µM JNJ-7706621 for 4 h improves early development of PA and SCNT porcine embryos by suppressing the activity of CDK1 and a concomitant reduction in the level of MPF.

236 PARTHENOGENETIC ACTIVATION OF IN VITRO-MATURED OVINE OOCYTES WITH STRONTIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 197
Author(s):  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Blastocyst Stage ◽  
Oocyte Activation ◽  
Parthenogenetic Development ◽  
Activation Rate ◽  
Electrical Pulses ◽  
Blastocyst Rate ◽  
Activation Rates

The objective of the present experiments was to examine whether strontium could activate in vitro-matured ovine oocytes. Oocytes were collected and matured as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). Briefly, selected cumulus–oocyte complexes were cultured in modified TCM-199 medium supplemented with 20% sheep serum and hormones for 22–23 h, at 39°C, 5% CO2 in air. Matured oocytes were randomly divided into four groups and treated as follows: (1) cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (2) electrically activated in Ca2+-containing medium, then cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (3) electrically activated in Ca2+-containing medium and then cultured in SOF medium containing 5 μg mL–1 cytochalasin B for 4–5 h; and (4) electrically activated in Ca2+-free medium and then transferred into SOF medium + 5 μg mL–1 cytochalasin B for 4–5 h. This experiment was repeated three times. Activation rates based on the number of pronuclear formations/the number of oocytes cultured were 96.7% (147/152), 95.9% (116/121), 75.9% (101/133), and 43.0% (56/107) in Groups 1–4, respectively. After 7 days of culture in SOF medium, 26.8%, 33.3%, 19.6%, and 0% of oocytes in Groups 1, 2, 3, and 4 developed to the blastocyst stage, respectively. Significant differences in blastocyst rate were observed across these groups except between groups 1 and 2 (P < 0.01). However, there were no significant differences in mean number of nuclei/blastocyst across Groups 1, 2, and 3 (P > 0.05). Our results demonstrated that in vitro-matured ovine oocytes can be effectively activated with strontium alone, resulting in an activation rate of 96.7% and a blastocyst rate of 26.8% (blastocysts/oocytes). Also, a combination of strontium and electrical pulses could benefit sheep oocyte activation and embryo development to the blastocyst stage (95.9% and 33.3%, respectively). We conclude that strontium is an effective activator for sheep oocyte activation and it could be used for sheep nuclear transfer. Table 1. Parthenogenetic development of oocytes activated by SrCl2+ and electrical pulses

Nuclear transplantation using bovine primordial germ cells from male fetuses

1995 ◽  
Vol 7 (5) ◽  
pp. 1217 ◽  
Author(s):  
F Delhaise ◽  
FJ Ectors ◽  
Roover R de ◽  
F Ectors ◽  
F Dessy
Keyword(s):  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
Nuclear Transplantation ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Developmental Potential ◽  
Morula Stage

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.

scholarly journals Parthenogenetic development of rabbit oocytes after electrical stimulation

2011 ◽  
Vol 51 (No. 9) ◽  
pp. 400-405
Author(s):  
A. Wierzchos
Keyword(s):  
Pulse Generator ◽  
Electric Pulse ◽  
Calf Serum ◽  
Functional Condition ◽  
Blastocyst Stage ◽  
White Female ◽  
Foetal Calf Serum ◽  
Electric Pulses ◽  
Morula Stage

The aim of this study was to determine the effect of electric pulses on the structural and functional condition of rabbit oocytes. The New Zealand White female rabbits at 3&ndash;5 months of age and at 3&ndash;4 kg body weight served as oocyte donors. Oocytes after flushing from the oviducts were placed between two electrodes in an electroporation chamber which was filled with a dielectric solution. Following a short incubation in B2 medium, oocytes were subjected to an electric pulse released by an electrical pulse generator. Oocytes were then incubated in 500 &micro;l of B2 medium supplemented with 20% foetal calf serum (FCS) at 38&deg;C in an atmosphere of 5% CO<sub>2 </sub>in air. Oocytes were cultured until the morula/blastocyst stage (approx. 72 h). The experiment was conducted using 430 oocytes obtained post mortem. In vitro cultured oocytes not subjected to an electric pulse were the control. Each group was subdivided into replications according to electric current intensity. The analysis of experimental variants shows that in the first variant all embryos developed to the morula stage but only 10% of them continued to develop to the blastocyst stage. In the second variant we observed that 5&ndash;10% of oocytes developed to the blastocyst stage after treatment with 2.0 and 2.5 kV/cm pulse but in the group of 1.0 kV/cm pulse 35% of oocytes developed only to the 2&ndash;12 b stage. In the third variant only 1 oocyte (5%) continued to develop to the blastocyst stage, but in the fourth variant oocyte development stopped at the morula stage. In the fifth variant, called an &ldquo;extreme&rdquo; one, oocytes stopped to develop at the stage of 2&ndash;12 b (about 25%) and the percentage of degenerated oocytes dramatically increased (about 60%). &nbsp;

28 EFFECT OF TRICHOSTATIN A ON IN VITRO EMBRYO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED FROM CAT DONOR NUCLEI AND BOVINE CYTOPLASM

2013 ◽  
Vol 25 (1) ◽  
pp. 161 ◽  
Author(s):  
M. Wittayarat ◽  
Z. Namula ◽  
V. V. Luu ◽  
L. T. K. Do ◽  
Y. Sato ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Cell Stage ◽  
Histone Deacetylase Inhibitor Trichostatin ◽  
Invaluable Tool ◽  
Morula Stage ◽  
Bovine Oocytes

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus-cytoplasm interactions and may provide an alternative for cloning endangered animals whose oocytes are difficult to obtain. The developmental ability of iSCNT embryos decreases with increases in taxonomic distance between the donor and recipient species. The development of cat-bovine iSCNT embryos is reportedly blocked at the 8-cell stage (Thongphakdee et al. 2008 J. Reprod. Dev. 54, 142–147). Abnormal epigenetic reprogramming, such as DNA methylation or histone modifications, may cause low iSCNT efficiencies. The present study was conducted to evaluate the effect of the histone deacetylase inhibitor trichostatin A (TSA), previously used to enhance nuclear reprogramming following SCNT, on the developmental ability of cat iSCNT embryos using bovine oocytes matured in vitro. The matured bovine oocyte was enucleated by the glass needle and the domestic cat fetal fibroblast used as the donor nuclei was then placed into the perivitelline space adjacent to the plasma membrane of the oocyte. Couplets with bovine ooplasm were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 µs, respectively, using an electro cell fusion generator followed by cycloheximide treatment. Reconstructed cat-bovine embryos were treated with 0, 25, 50, and 100 nM concentrations of TSA for 24 h following fusion. The percentages of embryos cleaved and embryos developed to the blastocyst stage were subjected to arc sin transformation before ANOVA. The TSA treatment at 50 nM contributed significantly higher rates of cleavage and blastocyst formation (n = 139; 84.3 and 4.6%, respectively) compared with untreated embryos (n = 187; 63.8 and 0%, respectively) and embryos treated with 100 nM TSA (n = 172; 71.4 and 0%, respectively; P < 0.05). Development to the morula stage of iSCNT embryos was observed in the TSA treatment groups, whereas no embryos developed beyond the 16-cell stage in the untreated group. In conclusion, our results indicate that TSA treatment for 24 h following fusion improves the development of iSCNT embryos. Specifically, 50 nM TSA treatment provides a beneficial effect on cleavage and development to the blastocyst stage of cat iSCNT embryos using bovine oocytes matured in vitro as recipients and domestic cat fibroblasts as donor nuclei.

126 CRYOPRESERVATION OF PORCINE EMBRYOS DERIVED FROM IVM OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 180
Author(s):  
N. Nakayama ◽  
K. Hiruma ◽  
M. Kurome ◽  
R. Tomii ◽  
S. Ueno ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Lipid Droplets ◽  
Cell Number ◽  
High Rate ◽  
Cell Stage ◽  
Cryoprotective Agents ◽  
Stage 4 ◽  
Morula Stage ◽  
Cytoplasmic Lipid Droplets

We have reported that a combination of delipation (removal of cytoplasmic lipid droplets from blastomeres) and vitrification by means of the minimum-volume cooling (MVC) method successfully cryopreserves porcine in vitro-matured/fertilized (IVM/IVF) embryos, and that normal piglets are produced from these embryos (Hiruma et al. 2006 Reprod. Fertil. Dev. 18, 157). We have also reported that IVM-derived embryos that undergo noninvasive delipation (i.e. micromanipulation is not required) and vitrification develop into blastocysts at a high rate (Esaki et al. 2004 Biol. Reprod. 71, 432–437). In this study, we examined whether fetuses can be produced from the IVM-derived embryos that have been delipated noninvasively and vitrified. Cumulus–oocyte complexes that had been collected from slaughterhouse ovaries were in vitro-matured in NCSU23 medium. The IVM oocytes were activated to produce parthenogenetic embryos. We used the embryos at the 4- to 8-cell (67 h after activation) and morula (98 h) stages in the following experiments. Embryos were treated with 4% trypsin (in PBS) at 38�C for 1 to 4 min to expand the zona pellucida. Next, the embryos were centrifuged (12 000g, 38�C, 23 min) in TL-HEPES-PVP containing 7.5 �g mL-1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. These embryos were cultured for 1 to 3 h and then vitrified. The post-thaw viability of the embryos was assessed based on their ability to develop into blastocysts and fetuses (21 to 23 days old). The embryos were vitrified using the MVC method with 15% ethylene glycol, 15% DMSO, and 0.5 M sucrose as cryoprotective agents. PZM-5 was used for culturing the embryos. In embryo transfer experiments, after thawing, the embryos were cultured for 36 or 72 h until they developed into morulae or 4- to 8-cell blastocysts, respectively; they were then treated with 0.5% pronase to remove the zona pellucida, and transferred to the uterine horns of estrus-synchronized recipients 6 days after onset of estrus. The proportion of vitrified embryos that developed into blastocysts and the mean cell number of the blastocysts were similar to those of non-vitrified control embryos, irrespective of the embryonic stage (4- to 8-cell stage: 42.1%, 22/51, 63.0 � 7.8 vs. 64.7%, 22/34, 74.2 � 7.1, respectively; morula stage: 77.6%, 38/49, 69.6 � 7.2 vs. 83.3%, 45/54, 66.2 � 5.9, respectively). Seventeen embryos that had been vitrified at the 4- to 8-cell stage gave rise to 3 fetuses after transfer into one recipient (17.6%). Fifty-three embryos that had been vitrified at the morula stage were transferred into 3 recipients. All recipients became pregnant and produced a total of 17 fetuses (32.1%). These results suggest that porcine IVM-derived embryos that have been cryopreserved by the combination of noninvasive delipation and vitrification by the MVC method are highly viable.

55 PRELIMINARY STUDIES ON THE DEVELOPMENT OF RECONSTRUCTED EMBRYOS AFTER NUCLEAR TRANSFER IN DROMEDARY CAMEL (CAMELUS DROMEDARIUS)

2009 ◽  
Vol 21 (1) ◽  
pp. 128 ◽  
Author(s):  
N. A. Wani ◽  
J. A. Skidmore ◽  
U. Wernery
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Light Exposure ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Reconstructed Embryos

Experiments were conducted to study the in vitro development of reconstructed dromedary camel embryos after nuclear transfer by a modified zona-free method. Cumulus oocyte complexes, collected from slaughterhouse ovaries were cultured in TCM199 at 38.5°C in an atmosphere of 5% CO2 in air for 32 to 36 h. Matured oocytes were denuded of cumulus cells by repeated pipetting and the zona pellucida was removed by brief incubation in 5 mg mL–1 pronase dissolved in Ca- and Mg-free PBS. Zona-free oocytes were stained with 5 mg mL–1 Hoechst 33342 in H199 supplemented with 7.5 μg mL–1 cytochalasin B and 10% FCS. They were enucleated under constant UV-light exposure in H199 supplemented with cytochalasin B and 10% FCS. The granulosa cells at passage numbers 4 to 15 were used as nuclear donors. The zona-free cytoplasts were individually washed for a few seconds in 300 μg mL–1 of Phytohemagglutinin in H199, then quickly dropped on a single donor cell settled to the bottom of a drop of H199 with 0.5% FCS and pushed together with the mouth pipette. Couplets were electrically fused, at room temperature, with two DC pulses of 100 V cm–1 for 15 μs. Reconstructs were activated 2 h post-fusion, with 5 μm ionomycin for 3 min followed by culture in 6-diethylaminopurine for 4 h. The reconstructs were then cultured individually in either 5 μL drops under oil, in agar wells or in wells of wells (WOW) in a well of 4-well culture plate. Embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 μg mL–1 gentamycine, 1% insulin-transferrin-selenium (ITS), and 15% estrous dromedary serum. The number of oocytes that had cleaved was recorded on day 2, whilst those developing to morulae and blastocysts were recorded on day 7 of culture. For cell count, the blastocysts were stained with Hoechst and cells counted under a fluorescent microscope at ×400. Data obtained was analysed by chi-square test. About 92% (349/380) of the oocytes were successfully enucleated and 76% (259/340) fused with the attached cells. The cleavage rate was significantly lower (P < 0.05) in reconstructed embryos cultured in droplets (10/72, 14%) as compared with those cultured in agar wells (37/87, 42%) or WOW system (42/96, 44%). The proportions of cleaved embryos reaching morula stage were 0, 83, and 89% in droplets, agar wells, and WOW, respectively. However, only 8% and 5% of the cleaved embryos developed to the blastocyst stage in the agar well and WOW culture systems, respectively. No difference was observed in the cell number of blastocysts produced in agar wells (77.3 ± 8.02) or WOW (78.0 ± 4.2) culture system. To the best of our knowledge, this is the first report of embryo production up to the blastocyst stage after NT in camelids and it shows that NT can be successfully applied for embryo production in camelids. Further studies are needed to optimize the parameters and to improve the efficiency for production of transferable blastocysts in this species. This study was kindly sponsored by H.H. General Sheikh Mohammed bin Rashid Al Maktoum, Ruler of Dubai.

scholarly journals 275FOLLICULAR ATRESIA IN SMALL, NON-FSH-DEPENDENT BOVINE FOLLICLES IS ASSOCIATED WITH INCREASED DEVELOPMENTAL POTENTIAL OF OOCYTES FOLLOWING IVP

2004 ◽  
Vol 16 (2) ◽  
pp. 258
Author(s):  
H. Irving-Rodgers ◽  
S. Morris ◽  
R. Collett ◽  
K. Catanzariti ◽  
T. Peura ◽  
...  
Keyword(s):  
Basal Lamina ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Early Cleavage ◽  
Blastocyst Development ◽  
Chi Square ◽  
Developmental Potential ◽  
Chi Square Analysis

Oocytes from small, non-FSH-dependent follicles are associated with reduced developmental competence following in vitro embryo production (IVP) compared to oocytes from larger follicles. It has been suggested that, for small follicles, oocytes derived from atretic follicles are more developmentally competent than those from healthy follicles (Blondin P and Sirard MA, 1995 Mol. Reprod. Dev. 41, 54–62). Little is known of the characteristics of small follicles that support developmentally competent oocytes. Here we examine the development to blastocyst stage of oocytes collected from histologically-assessed bovine 2–5mm follicles. Ovaries were obtained at a local abattoir;; 4 follicles were dissected from each ovary and oocytes were recovered. A section of each follicle wall was taken and fixed in 2.5% glutaraldehyde for histological assessment of the follicle and characterization of the morphology of the follicular basal lamina by electron microscopy (Irving-Rodgers HF and Rodgers RJ, 2000 J. Reprod. Fert. 118, 221–228). Oocytes recovered from follicles underwent IVP utilizing a novel single IVP system. Oocytes were matured for 24h (10μL per COC) in TCM199, supplemented with FSH, hCG, FCS, cysteamine and pyruvate. Mature oocytes were inseminated with 1×106 sperm mL−1 for an additional 24h using Bovine Fertilization Medium (10μL per COC;; Cook, Australia). Following insemination, putative zygotes were stripped of remaining cells and placed within individual micro-wells prepared in 1% agar in Bovine Early Cleavage Medium, Cook, Australia. The agar (350μL) was prepared within wells of a 4-well plate and small plugs of agar were removed to form micro-wells. The agar was over-laid with 450μL of Early Cleavage Medium and 250μL mineral oil, and equilibrated overnight before putative zygotes were placed individually within micro-wells. Culture was performed under 7% O2, 6% CO2, and 87% N2 at 39°C. On Day 5 following insemination, fetal calf serum (final concentration 10% v/v) was added to facilitate blastocyst development. Blastocyst formation was assessed on Day 8. A total of 211 oocytes were cultured and 69% were from healthy follicles;; 67 oocytes (32%) had developed to the blastocyst stage by Day 8. Forty-three percent of oocytes recovered from atretic follicles (28/65) had developed to the blastocyst stage by Day 8, as compared to only 27% (39/146) oocytes recovered from healthy follicles, this difference was significant (P&lt;0.05, chi-square analysis). Seventy-eight percent (14/18) of oocytes from healthy follicles with additional follicular basal lamina material (Irging-Rodgers HF and Rodgers RJ, 2000 J. Reprod. Fert. 118, 221–228) failed to develop, whereas only 44% (4/9) of oocytes from healthy follicles with a normal basal lamina failed to develop (P&gt;0.08). The present study finds a direct association between the follicle morphology and oocyte maturational potential within non-FSH dependent follicles, revealing that high levels of development (&gt;40%) can be obtained from atretic follicles. Furthermore, differences between healthy follicles may also contribute to developmental variation.

Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1061 ◽  
Author(s):  
RD Schramm ◽  
BD Bavister
Keyword(s):  
Growth Hormone ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Morula Stage

Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.

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