scholarly journals 305DEVELOPING AN ACTIVATION PROTOCOL FOR SOMATIC CELL NUCLEAR TRANSFER (SCNT) IN THE DOMESTIC CAT

2004 ◽  
Vol 16 (2) ◽  
pp. 272 ◽  
Author(s):  
T. Shin ◽  
T. Otoi ◽  
D.C. Kraemer ◽  
M.E. Westhusin
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation ◽  
Maturation Rate

In order to establish an activation protocol for somatic cloning in the domestic cat, we evaluated the developmental competence of cat embryos derived from in-vitro matured ova after parthenogenetic activation treatment. The quality of parthenogenetic embryos was assessed by D3 cleavage rates, D8 rates of blastocyst formation and total nucleus numbers in expanded/hatching blastocysts. Parthenogenetic activation treatments were as follows;; Treatment I: 3.0kVcm−1 (25μs, twice) in 0.3M mannitol containing 0.1mM CaCl2· 2H2O and 0.1mM MgSO4, administered to matured cat oocytes and followed by 10μgmL−1 cycloheximide +5μgmL−1 cytochalasin B in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7h. Treatment II: The first electric stimulation was performed as described for treatment I except that the activation medium consisted of 0.3M mannitol containing Mg, but without Ca. Two hours later, pre-pulsed MII oocytes were electropulsed by applying 1.0kVcm−1 (50μs, twice, 5s apart) in 0.3M mannitol containing Ca and Mg for additional activation, followed by culture in 10μgmL−1 cycloheximide +5μgmL−1 cytochalasin B treatment in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7h. Immature cat oocytes were obtained from ovaries by mincing/dissection and matured in vitro for 26–30h as previously described (Gomez et al., 2001, Therigenology, 55, 472). Only MII oocytes with a 1st polar body were utilized for the activation procedure after removal of cumulus cells with 0.1% hyaluronidase by gentle pipetting. A total of 1120 oocytes were collected and the overall maturation rate was 49.8% (551/1120). After parthenogenetic activation of the MII oocytes, the embryos were cultured in vitro as described previously (Pope et al., 2000, Theriogenology, 53, 163–174). The results are shown in Table 1. Treatment II resulted in significantly higher (P<0.01) D3 cleavage rates;; however, there were no significant differences in D8 blastocyst formation and total nucleus numbers. These data suggest that an additional electric activation (Treatment II) may increase the in vitro cleavage rates compared to using a fusion and electrical stimulation simultaneously (Treatment I). In addition, we demonstrated the developmental competence of domestic cat embryos derived from in vitro maturation, activation, and culture for development to the pre-implantation stage. By using these procedures for SCNT, several pregnancies were established and a healthy cloned kitten resulted in our laboratory (Shin et al., 2002, Nature, 415, 859). Therefore, this protocol can be useful, not only for prediction of the developmental competence of domestic cat oocytes matured in vitro, but also when used with SCNT to produce cloned cats. Comparison of cleavage rates and developmental competence to blastocyst stage following parthenogenetic activation treatments in domestic cat oocytes matured in vitro

scholarly journals 301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 270
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Cytochalasin B ◽  
Average Rate ◽  
Polar Body ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Average Cell ◽  
Electric Impulse ◽  
Polar Body Extrusion

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
First Polar Body

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.

198 RESVERATROL SUPPLEMENTATION DURING IN VITRO MATURATION AND FERTILISATION ENHANCES DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 230
Author(s):  
P. Kordowitzki ◽  
S. M. Bernal ◽  
D. Herrmann ◽  
P. Aldag ◽  
H. Niemann
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Vehicle Control ◽  
Sirtuin 1 ◽  
Developmental Competence ◽  
Foxo Transcription Factors ◽  
High Concentrations

Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin identified in various plant species, particularly in grapevine peel. It is a strong antioxidant, induces mitochondrial biogenesis and enhances Sirtuin 1 (SIRT1) activity by inhibiting phosphodiesterase. SIRT1 belongs to the family of NAD+-dependent histone deacetylates and has been shown to regulate several key cellular processes, including transcriptional silencing, aging, chromatin remodeling, and genomic stability, via deacetylation of p53, FoxO transcription factors, and nuclear factor kappa B (NF-κB). The aim of this study was to determine whether supplementation of the maturation and fertilisation medium with resveratrol influences bovine oocyte maturation and subsequent embryonic development and whether these effects are mediated via SIRT1 pathway. Three different resveratrol concentrations were used during in vitro maturation (IVM) and IVF. Cumulus-oocyte complexes (n = 2878) were collected from slaughterhouse ovaries and subjected to IVM medium supplemented with 0.2 µM, 1 µM, or 20 µM resveratrol® (Sigma-Aldrich, Buchs, Switzerland) for 24 h followed by IVF with the same concentrations of resveratrol for 19 h. The IVM and IVF medium without resveratrol (controls) and dimethyl sulfoxide supplementation as vehicle control were also included. Presumptive zygotes were cultured in vitro until Day 8 to assess embryo development, and maturation rates, cleavage, and blastocyst formation were evaluated. Maturation rates as determined by polar body extrusion (0.2 µM: 64.2% ± 7; 1 µM: 82.3% ± 4; 20 µM: 68.8% ± 2; control: 74.6% ± 5 and vehicle control: 70.2% ± 6, respectively; P ≤ 0.05) did not differ dramatically. Oocytes in 1 µM resveratrol supplemented maturation medium showed distinct detachment of cumulus cells in comparison with those in the other treatment and control groups. Cleavage rates were reduced in the 0.2 µM and 20 µM group compared with controls (0.2 µM: 44.21% ± 2; 1 µM: 58.4% ± 3; 20 µM: 40.9% ± 5; control: 56.6% ± 2 and vehicle control: 55.2% ± 6, respectively; P ≤ 0.05). Blastocyst rates were impaired in the low and high resveratrol concentration groups compared to all other groups (0.2 µM: 11.3% ± 1; 1 µM: 33.4% ± 3; 20 µM: 8.2% ± 4; control: 26.7% ± 4 and vehicle control: 20.8% ± 2, respectively; P ≤ 0.05). Relative mRNA abundance of SIRT1 in matured oocytes from the 1 µM group did not differ significantly compared to the controls. Results so far indicate that very low and high concentrations of resveratrol impair development to the blastocyst stage. In conclusion, a 1 µM resveratrol supplementation during IVM and IVF seems to improve the developmental competence of oocytes, which is reflected not only in the elevated blastocyst rates but also in higher degree of expansion of cumulus cells after IVM and maturation rates.

scholarly journals 55 NUCLEUS CHANGES AND DEVELOPMENT OF PORCINE RECONSTRUCTED (NT) AND PARTHOGENETICALLY ACTIVATED (PA) EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 177
Author(s):  
N.R. Mtango ◽  
T. Kono
Keyword(s):  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Normal Embryo ◽  
Basic Medium ◽  
Cell Nuclei ◽  
First Polar Body ◽  
Porcine Oocyte

Nuclear reprogramming is characterized by functional modification(s) of the transferred nucleus that allows it to direct normal embryo development with the potential to grow to term. The aim of our study was to investigate the process of nuclear changes in reconstructed and activated embryos as well as their developmental competence. All chemicals used were from Sigma Chemicals (St. Louis, MO, USA). Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries of prepurbetal gilts and matured for 42 h in vitro. The cumulus cells were removed by adding in 1 mg mL −1 hyaluronidase in TLP-HEPES. For the NT experiment, oocytes with first polar body were cultured in 0.4 μg mL−1 demecolcine for 1 h. A protruding membrane was removed by micromanipulator and a single donor nucleus from fetal fibroblast was injected subzonally. Fusion was conducted immediately after transfer in 0.3 M mannitol, 0.5 mM HEPES, 0.1% PVA, and 0.1 mM MgCl2 in a fusion chamber with parallel electrodes set 1 mm apart using a singe DC pulse of 125 V mm−1 for 80 s. Activation was done 2–4 h after fusion in the same medium as fusion but with 0.1 mM CaCl2 added; embryos were cultured in 5 μg mL−1 cytochalasin B and 10 μg mL−1 cyclohexamide for 6 h. The embryos were cultured in glucose-free NCSU-37 containing 4 mg mL−1 BSA as basic medium supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2, and then in basic medium with 5.55 mM D-glucose from Days 2–6 (Kikuchi K et al. 2002 Biol. Reprod. 66, 1033–1041). Non-manipulated oocytes (PA) were electrically activated as stated above. For observing the changes of donor cells, some reconstructed oocytes were fixed 2 h after fusion, prior to activation, and some 12 h after activation in acetic acid:ethanol (1:3) and stained in 1% orcein. The activated oocytes were fixed at 12 h and stained as stated above. There were 47.5% (38/80) of reconstructed oocytes with premature chromosome condensation (PCC), and 23.7% (19/80) with nuclear swelling two hours after fusion. Pronuclear like formation 12 h after activation was 45% (27/60) and 83.3% (50/60) in NT and PA, respectively. The blastocyst rate was 8.3% (5/60) and 46% (69/150) for NT and PA, respectively. The results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of NT embryos to the blastocyst stage.

355 IMPROVEMENT OF CUMULUS-FREE IN VITRO MATURATION AND ITS APPLICATION TO MICROINSEMINATION WITH PRIMARY SPERMATOCYTES IN MICE

2006 ◽  
Vol 18 (2) ◽  
pp. 284
Author(s):  
H. Miki ◽  
N. Ogonuki ◽  
K. Inoue ◽  
T. Baba ◽  
A. Ogura
Keyword(s):  
Embryo Transfer ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Parthenogenetic Activation ◽  
Maturation Rate ◽  
Mitochondrial Distribution

Manipulation of immature oocytes requires removal of cumulus cells, which may compromise ooplasmic maturation. This problem can be overcome by cytoplasm exchange at the MII stage, but this requires a second micromanipulative step. This study was undertaken to determine whether cumulus-free IVM can be improved by modifying IVM medium. We assessed the quality of oocytes by several cytochemical parameters, and by the subsequent development of embryos constructed by IVF, parthenogenetic activation, or microinsemination using primary spermatocytes. GV-stage oocytes were collected from superovulated B6D2F1 females. After freeing the oocytes from cumulus cells, they were subjected to IVM in either �MEM or TYH media, or a 1:1 mixture of the two (termed TaM). IVM was performed under chemically semidefined conditions by using BSA as the only protein source. Oocytes that reached the MII stage were analyzed for distribution of mitochondria, activity of the catalytic p34cdc2 kinase subunit of the maturation promoting factor (MPF), and chromosomal alignment within the MII spindle. Microinsemination with primary spermatocytes was performed in oocytes at the MI stage as described previously (Ogura et al. 1998 PNAS 95, 5611-5615), but without MII chromosome exchange. TYH and �MEM media had opposing effects on several parameters. TYH resulted in a better maturation rate (181/196, 92.3%) than �MEM (184/257, 71.6%). In contrast, �MEM supported better embryo development to the morula/blastocyst stage than TYH following IVF (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse in �MEM, but aggregated in TYH. MPF activity in MII oocytes was significantly higher in TYH than in �MEM (P < 0.05). Oocytes derived from TaM had intermediate characteristics between TYH- and �MEM-oocytes and essentially resembled in vivo-matured oocytes, with the mitochondrial distribution pattern being most typically intermediate. By using TaM, 23.8% of GV oocytes developed into full-term fetuses following IVF and embryo transfer (compared to TYH: 6.3%, and �MEM: 18.2%). This IVM system was then applied to oocytes to be injected with spermatocytes when in the MI stage. Approximately 60% of the oocytes survived injection. After 127 two-cell embryos were transfered, two normal-looking offspring were obtained. This is the first successful birth of pups derived from fertilization with spermatocytes by single injection, without serial nuclear transfer. Chromosomal analysis at MII revealed that the low developmental efficiency after embryo transfer could be attributed mainly to premature sister chromatid separation, as reported previously (Ogura et al. 1998). These results demonstrate that optimization of IVM media can improve the quality of cumulus-free IVM oocytes in mice, although it cannot overcome defects in the kinetics of injected spermatocyte chromosomes.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p &gt; 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

249 FOLLICLE-STIMULATING HORMONE AND DIBUTYRYL cAMP TREATMENTS DURING IN VITRO MATURATION IMPROVE THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
R. Oishi ◽  
Y. Isaji ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
In Vitro Maturation ◽  
Basal Medium ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Medium ◽  
Dibutyryl Camp ◽  
Mouse Oocytes ◽  
Junctional Communication

The high level of cyclic adenosine monophosphate (cAMP), which is provided to the oocytes from cumulus cells via gap junctional complexes in cumulus-enclosed oocytes (CEOs), is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of CEOs. However, whether intraoocyte cAMP during the period of in vitro maturation (IVM) affects postfertilization developmental competence of mouse oocytes still remains unclear. The aim of this study was to examine the effects of FSH or dibutyryl cAMP (dbcAMP) treatment during IVM on in vitro development of mouse oocytes after in vitro fertilization (IVF). Whether a junctional association between cumulus cells and the oocyte would be essential for a cytoplasmic maturation-promoting effect was also examined. CEOs were isolated from and eCG-primed 3-week-old ICR mouse by rupturing preovulatory follicles with needles in M16 medium with 5% FCS and essential and nonessential amino acids (basal medium). IVM media used were basal medium without (control) or with 100 µm dbcAMP or 1 IU mL–1 FSH. Carbenoxolone (100 µm, CBX), an inhibitor of gap junction, was used to inhibit a junctional association between cumulus cells and the oocyte. Denuded oocytes (DOs) were prepared by repeatedly pipetting in basal medium with 0.2% hyaluronidase. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h, and then transferred to TYH medium (a modified Krebs-Ringer bicarbonate medium) containing 0.4% BSA, followed by insemination with capacitated sperm. After 6 h of IVF, inseminated oocytes were cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was estimated at 24 h and 120 h after IVF, respectively. All experiments were done in 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's protected least-squares difference (PLSD) test. When CEOs were matured in IVM media, the rates of postfertilization development to the 2-cell and blastocyst stages of oocytes matured in the control medium were very low(29% and 13%, respectively), whereas those of oocytes matured with FSH or dbcAMP significantly increased (FSH: 61% and 52%, dbcAMP: 63 and 57%, respectively, v. control; P < 0.05). Next, when CEOs were matured in basal medium with 1 IU mL–1 FSH and 100 µm CBX, the developmental rate to the 2-cell stage (56%) was similar to that in medium with FSH alone (61%) but the rate to the blastocyst stage (40%) was little lower compared with that in medium with FSH alone (52%), although not significantly different (P > 0.05). Furthermore, when DOs were matured in IVM media, the developmental rates to the blastocyst stage after IVF of the oocytes matured with FSH or dbcAMP significantly increased (FSH: 25%, dbcAMP: 15%; P < 0.05) compared with those in control medium (7%). Taken together, it is suggested that increasing the concentration of intraoocyte cAMP during the IVM period is important to improve the developmental competence after IVF of mouse oocytes, and that the competence is acquired in part in a cumulus-oocyte junctional communication-independent manner.

74 IN VIVO SURVIVAL OF DOMESTIC CAT OOCYTES AFTER VITRIFICATION, INTRACYTOPLASMIC SPERM INJECTION, AND TRANSFER TO RECIPIENTS

2008 ◽  
Vol 20 (1) ◽  
pp. 118 ◽  
Author(s):  
M. C. Gómez ◽  
N. Kagawa ◽  
C. E. Pope ◽  
M. Kuwayama ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Oocyte Retrieval ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
Minimal Amount ◽  
Vitro Fertilization ◽  
First Polar Body

The ability to cryopreserve female gametes efficiently holds immense economic and genetic implications. The purpose of the present project was to determine if domestic cat oocytes could be cryopreserved successfully by use of the Cryotop method. We evaluated (a) cleavage frequency after in vitro fertilization (IVF) v. intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification, and (b) fetal development after transfer of resultant embryos into recipients. In vivo-matured cumulus–oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment, denuded of cumulus cells, and examined for the presence of the first polar body (PB). In vitro-matured COCs were obtained from ovaries donated by local clinics and placed into maturation medium for 24 h before cumulus cells were removed and PB status was determined. Oocytes were cryopreserved by the Cryotop method (Kuwayama et al. 2005 Reprod. Biomed. Online 11, 608–614) in a vitrification solution consisting of 15% DMSO, 15% ethylene glycol, and 18% sucrose. For IVF, oocytes were co-incubated with 1 � 106 motile spermatozoa mL–1 in droplets of modified Tyrode's medium in 5% CO2/air at 38�C (Pope et al. 2006 Theriogenology 66, 59–71). For ICSI, an immobilized spermatozoon was loaded into the injection pipette, which was then pushed through the zona pellucida into the ooplasm. After a minimal amount of ooplasm was aspirated into the pipette, the spermatozoon was carefully expelled, along with the aspirated ooplasm. After ICSI, or at 5 or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006). As assessed by normal morphological appearance after liquefaction, the survival rate of both in vivo- and in vitro-matured oocytes was >90% (93–97%). For in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 73% (16/22) and 53% (30/57), respectively, as compared to 68% (19/28) after ICSI of vitrified oocytes (P > 0.05). For in vivo-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 55% (18/33) and 35% (6/17), respectively, compared to 50% (10/20) after ICSI of vitrified oocytes (P > 0.05). At 18–20 h after ICSI, 18 presumptive zygotes and four 2-cell embryos derived from vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo-matured and 12 in vitro-matured vitrified oocytes were transferred by laparoscopy into the oviducts of two recipients at 24–26 h after oocyte retrieval. The two recipients were 9-month-old IVF/ET-derived females produced with X-sperm sorted by flow cytometry. At ultrasonography on Day 22, both recipients were pregnant, with three live fetuses observed in one recipient and one live fetus seen in the second recipient. On Day 63 and Day 66 of gestation, four live kittens were born, without assistance, to the two recipients. The one male and three female kittens weighed an average of 131 g. In summary, in vivo viability of zygotes/embryos produced by ICSI of cat oocytes vitrified by the Cryotop method was demonstrated by the birth of live kittens following transfer to recipients.

17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

2019 ◽  
Vol 31 (1) ◽  
pp. 134
Author(s):  
D. Veraguas ◽  
C. Aguilera ◽  
D. Echeverry ◽  
D. Saez-Ruiz ◽  
F. O. Castro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Relative Expression ◽  
Standard Curve ◽  
Morula Stage ◽  
Leopardus Guigna

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P&lt;0.05. The number of replicates was IVF=10, Ca1x=8, Ca2x=6, K1x=3, and K2x=8. The morulae rate was lower when clone embryos were cultured individually (IVF=97/153, 63.4%; Ca2x=28/51, 54.9%; K2x=63/110, 57.3%; Ca1x=48/126, 38.1%; K1x=22/87, 25.3%; P&lt;0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P&lt;0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P&gt;0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P&gt;0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P&gt;0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.

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