357 FERTILIZATION AND EMBRYONIC DEVELOPMENT OF PORCINE OOCYTES FOLLOWING INTRACYTOPLASMIC SPERM HEAD INJECTION ENHANCED BY ELECTRIC STIMULATION TO OOCYTES BUT NOT BY PRE-TREATMENT OF SPERMATOZOA WITH DITHIOTHREITOL

2006 ◽  
Vol 18 (2) ◽  
pp. 285
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
A. Takizawa ◽  
N. Maedomari ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Electric Stimulation ◽  
Morphological Changes ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
The Mean ◽  
Pronuclear Formation ◽  
Vitro Development ◽  
Nuclear Decondensation

Failure of sperm nuclear decondensation has been reported after injection into oocytes in pigs (Kren et al. 2003 J. Reprod. Dev. 49, 271-273). We examined the effects of pretreatment of spermatozoa with Triton X-100 (TX-100) and dithiothreitol (DTT) and electric stimulation of oocytes after injection on sperm decondensation, male pronuclear formation, and in vitro development to the blastocyst stage. We performed three replicates in each experimental group, with a total of about 70 oocytes per group. In Experiment 1, spermatozoa were pretreated with 1% TX-100 and 5 mM DTT (T+D), and injected into IVM oocytes that were collected from crossbred gilts. Electric stimulation (1.5 kV/cm, 20 �s; Nakai et al. 2003 Biol. Reprod. 68, 1003-1008) was applied 1 h to the oocytes after the injection (the stimulated group) or was not applied (the nonstimulated group). Some of the oocytes in each group were evaluated for morphological changes of sperm nuclei at hourly intervals until 10 h post-injection. Of nonstimulated oocytes, those injected with untreated spermatozoa showed a delayed peak in nuclear decondensation (39.4 to 44.1%, 3-6 h after the injection) compared to that of oocytes injected with T+D treated spermatozoa (57.0 to 52.6%, 1-1 h). The rate of male pronuclear formation increased after 4 h post-stimulation (by 40 to 60%) when the injected oocytes were stimulated, whether or not spermatozoa were pretreated. In nonstimulated oocytes, the rate of male pronuclear formation stayed at the basal level (less than 20%) throughout the culture period regardless of sperm treatments. Thus, the T+D treatment of spermatozoa did not affect decondensation and pronuclear formation. In Experiment 2, the effects of electric stimulation and sperm treatments with T+D on the rate of blastocyst formation and the mean numbers of cells per blastocyst were evaluated. Oocytes that were stimulated after injection of either T+D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1%, respectively) than did nonstimulated oocytes (1.1% and 4.1% for T+D-treated and untreated, respectively; P < 0.01). The rate of blastocyst formation was not different between the T+D-treated and the untreated groups. The mean number of cells per blastocyst was not different among all groups (14.0-29.4). In conclusion, the pretreatment of sperm with TX-100 and DTT shifted the timing of sperm nuclear decondensation forward. However, pronuclear formation and development to the blastocyst stage in vitro were not improved by the sperm treatment. Electric stimulation to the injected oocytes enhances in vitro development to the blastocyst stage in pigs.

294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 262
Author(s):  
W. Fujii ◽  
H. Funahashi
Keyword(s):  
Chemical Activation ◽  
Formation Rate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Female Rats ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Pronuclear Formation ◽  
Vitro Development

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.

scholarly journals 205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

2004 ◽  
Vol 16 (9) ◽  
pp. 205 ◽  
Author(s):  
K. M. Morton ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Oocyte Collection ◽  
Chi Squared ◽  
Development In Vitro ◽  
Stage 1 ◽  
Vitro Development

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

scholarly journals Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (3) ◽  
pp. 603-611 ◽  
Author(s):  
Michiko Nakai ◽  
Naomi Kashiwazaki ◽  
Akiko Takizawa ◽  
Naoki Maedomari ◽  
Manabu Ozawa ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
The Mean ◽  
Sperm Nuclei ◽  
Morphologic Changes ◽  
Number Of Cells ◽  
Stimulation Of ◽  
Nuclear Decondensation

In pigs, failure of sperm nuclear decondensation has been reported after injection into oocytes. We examined the effects of pretreating sperm heads with Triton X-100 (TX-100) and dithiothreitol (DTT) and of electrical stimulation of oocytes after sperm head injection on time-dependent morphologic changes in sperm nuclei andin vitrodevelopment to the blastocyst stage. In experiment 1, spermatozoa were pretreated with 1% TX-100 and 5 mM DTT (T + D) or not treated, and then injected intoin vitromatured oocytes. Electrical stimulation (1.5 kV/cm, 20 μs DC pulse) was applied to the oocytes 1 h after injection (stimulated group) or was not applied (unstimulated group). Some of the oocytes in each group were evaluated at hourly intervals until 10 h after injection for morphologic changes in the sperm nuclei. Unstimulated oocytes injected with untreated spermatozoa showed a delayed peak in the rate of nuclear decondensation (39.4–44.1%, 3–6 h after injection) compared with oocytes injected with T + D-treated spermatozoa (57.0% and 52.6%, 1 and 2 h, respectively). The rate of male pronucleus formation peaked 6 h after stimulation (by 40–60%) after injected oocytes had been stimulated with an electrical pulse, irrespective of whether or not the spermatozoa had been pretreated. In unstimulated oocytes, the rate of male pronucleus formation did not increase and stayed at the basal level (less than 20%) throughout the culture period, regardless of the sperm treatment. Thus, T + D treatment of spermatozoa did not affect completion of fertilization. In experiment 2, we evaluated the effects of electrical stimulation and sperm treatment with T + D on the rate of blastocyst formation and the mean number of cells per blastocyst. Oocytes stimulated after injection with either T + D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1% respectively) than did unstimulated oocytes (1.1% and 4.1% for T + D-treated and untreated respectively;P< 0.01 by Duncan’s multiple-range test). The rate of blastocyst formation did not differ between the T + D-treated and untreated groups. The mean number of cells per blastocyst did not differ among any of the groups (14.0–29.4 cells). These results suggest that pretreatment of sperm with TX-100 and DTT shifted the timing of sperm nuclear decondensation forward. However, pronucleus formation and development to the blastocyst stagein vitrowere not improved by sperm treatment. Thus, electrical stimulation of injected oocytes enhancesin vitrodevelopment to the blastocyst stage in pigs.

In vitro development of non-enucleated rat oocytes following microinjection of a cumulus nucleus and chemical activation

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 117-125 ◽  
Author(s):  
Wataru Fujii ◽  
Hiroaki Funahashi
Keyword(s):  
Wistar Rats ◽  
Chromatin Condensation ◽  
Chemical Activation ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Spindle Formation ◽  
Female Wistar Rats ◽  
Pronuclear Formation ◽  
Vitro Development

SummaryThe present study examined in vitro development and the cytological status of non-enucleated rat oocytes after microinjection of cumulus nuclei and chemical activation. Oocyte–cumulus complexes were collected from gonadotropin-treated prepubertal female Wistar rats 14 h after human chorionic gonadotropin (hCG) injection. Cumulus nuclei were injected into ovulated oocytes and then stimulated in the presence of 5 mM SrCl2 for 20 min at various time points (0–3.5 h) after injection. Some of the reconstituted eggs were cultured to observe the pronuclear formation, cleavage, and blastocyst formation. The incidences of eggs forming at least one pronucleus or containing two pronuclei were not significantly different among the periods (82.4–83.5% and 43.4–51.9%, respectively). Nor did the incidences of eggs cleaving (86.7–97.7%) and developing to the blastocyst stage (0–3.5%) differ depending on when, after injection, stimulation began. When some of the reconstituted eggs were observed for cytological morphology 1–1.5 h after injection, 71.7% of the eggs caused premature chromatin condensation, but only 46.2% of them formed two spindles around each of maternal and somatic chromatins. However, the morphology of the somatic spindles differed from that of the spindles, which formed around the oocyte chromatins. Only 7.5% of the eggs contained the normal chromosomal number. In many reconstituted oocytes, before activation, an abnormal spindle formation was observed in the somatic chromatins. In conclusion, these results show that non-enucleated rat oocytes injected with cumulus nuclei can form pronuclei and cleave following chemical activation, whereas blastocyst formation is very limited, probably caused by abnormalities in the spindle formation and distribution of somatic chromatids.

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

Development of porcine oocytes from preovulatory follicles of different sizes after maturation in media supplemented with follicular fluids

2000 ◽  
Vol 12 (4) ◽  
pp. 133 ◽  
Author(s):  
Ki-Won Yoon ◽  
Tae-Young Shin ◽  
Jong-Im Park ◽  
Sangho Roh ◽  
Jeong M. Lim ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
Developmental Potential ◽  
Large Follicle ◽  
In Vitro Development ◽  
Maturation Medium ◽  
Preovulatory Follicles ◽  
Vitro Development ◽  
Small Follicle ◽  
Follicular Fluids

The development of porcine oocytes from large (3.1–8.0 mm in diameter) or small (<3.1 mm) follicles was examined after maturation culture in medium containing porcine follicular fluid (pFF). Large follicles yielded larger (256 m v. 221 m; P<0.05) cumulus–oocyte complexes and more (22 v. 14%) morphologically normal oocytes than small follicles (Experiment 1). In Experiments 2–4, maturation media supplemented with mixed pFF (10%) from small and large follicles was used. More oocytes from large follicles matured (58% v. 91%), formed pronuclei (81% v. 90%) and developed to the blastocyst stage (2% v. 10%) than oocytes from small follicles. In Experiments 5–7, the effects of pFF collected from either small or large follicles on oocyte development were examined. Regardless of the source of oocytes, large-follicle-derived pFF more significantly enhanced preimplantation development than did small-follicle-derived pFF. The highest rate of blastocyst formation (16%) was found when oocytes from large follicles were cultured in maturation medium containing large-follicle-derived pFF. These results suggest that oocytes from large follicles have greater developmental potential than oocytes from small follicles, and that the origin of pFF, which is added to the maturation media, might be an important factor for improving in vitro development of porcine oocytes.

scholarly journals 43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH 6-DMAP FOLLOWING FUSION

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
G.-S. IM ◽  
L. Lai ◽  
Z. Liu ◽  
Y. Hao ◽  
C.M. Murphy ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Developmental Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Electric Pulses ◽  
Developmental Rates ◽  
Vitro Development

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P&lt;0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P&lt;0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.

267 TARGETED SUPPRESSION OF THE EXPRESSION OF MATERNAL AND EMBRYONIC GENES DURING IN VITRO DEVELOPMENT OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 241
Author(s):  
D. Tesfaye ◽  
K. Nganvongpanit ◽  
F. Rings ◽  
M. Gilles ◽  
D. Jennen ◽  
...  
Keyword(s):  
Free Water ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
Bovine Oocytes ◽  
E Cadherin

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39�C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P < 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P < 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

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