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.

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

39 BOVINE SOMATIC CELL NUCLEAR TRANSFER USING CUMULUS - OOCYTE COMPLEXES COLLECTED FROM THE IDENTICAL INDIVIDUAL BY OVUM PICKUP

2007 ◽  
Vol 19 (1) ◽  
pp. 138 ◽  
Author(s):  
K. Hasegawa ◽  
S. Takahashi ◽  
S. Akagi ◽  
K. Takeda ◽  
K. Imai ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Single Strand Conformation Polymorphism ◽  
Blastocyst Stage ◽  
Polymorphism Analysis ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Developmental Rates ◽  
Vitro Development

We previously produced a cloned calf by nuclear transfer (NT) using cumulus cells removed from cumulus–oocyte complexes (COCs) after IVM. If both cumulus cells and oocytes are obtained identically and individually, and can be used simultaneously for NT, the production of cloned cows will be more expedient. And the cloned offspring produced from them will not exhibit the heteroplasmic mixed mtDNAs of donor cells and recipient oocytes. In this study, we examined the developmental potential of NT embryos using cumulus–oocyte complexes (COCs) collected from cows individually by ovum pickup (OPU). The cumulus cells were removed from COCs after IVM. The cumulus cells and cumulus-free MII oocytes derived from the same cow were used as donor nuclei and recipient oocytes, respectively. NT was performed as previously described (Akagi et al. 2003 Clon Stem Cells 5, 101–108). In Experiment 1, we examined the in vitro development of NT embryos using COCs collected by OPU. The aspiration of the follicles was performed once a week consecutively for 6 weeks in 6 cows (Cows A, B, C, D, E, and F) without hormone stimulation. In Experiment 2, we examined fetal development after the transfer of NT embryos. A Japanese black cow (Cow G) was used for OPU. On Day 7, 13 NT blastocysts were transferred to 7 recipient cows. The mtDNA genotypes of the donor cow and the cloned calf were analyzed by PCR-mediated single-strand conformation polymorphism analysis as previously described (Takeda et al. 2003 Mol. Reprod. Dev. 64, 429–437). The results of Experiment 1 are summarized in Table 1. Fusion rates did not differ among individual cows. However, the developmental rates of NT embryos at the blastocyst stage varied widely among individual cows, with a range of 19 to 64%. In Experiment 2, 2 of 7 recipient cows became pregnant on Day 30. One pregnant cow aborted on Day 60, and another cow calved a healthy calf. The mtDNA genotype of the cloned calf was confirmed to be identical with that of the donor cow. These results indicate that COCs from an identical individual can be used as donor nuclei and recipient oocytes for NT in order to produce female clones with the same mtDNA as that of the donor cow. Table 1.In vitro development of NT embryos using COCs collected by OPU

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

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.)

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

In vitro development of ovine oocytes cultured in the presence of GDF9 at chemically defined or undefined maturation medium

2014 ◽  
Vol 120 (1) ◽  
pp. 78-83 ◽  
Author(s):  
Sh. Rouhollahi Varnosfaderani ◽  
S. Ostadhosseini ◽  
S.M. Hosseini ◽  
M. Hajian ◽  
M.H. Nasr-Esfahani
Keyword(s):  
In Vitro Development ◽  
Maturation Medium ◽  
Vitro Development

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.

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.

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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