Hatched Blastocyst Rates Research Articles

Relationship Between Differential Expression of Bax and Bcl-2 Genes and Developmental Differences of Porcine Parthenotes Cultured in PZM-3 and NCSU-23

Abstract The developmental competence of porcine parthenotes cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 (NCSU-23) media was investigated. After in vitro maturation oocytes were electro-activated, and the subsequent developmental competence, rates of apoptotic, fragmented and arrested embryos from the two media were evaluated. Also, the ratio of mRNA expression of Bcl-2 and Bax gene was determined. Results demonstrated that cleavage, blastocyst, hatched blastocyst rates, and blastocyst cell numbers were significantly higher in PZM-3 than in NCSU-23. The rate of fragmented embryos in PZM-3 was lower than in NCSU-23 on d 1 and 3 ( P<0.05) while the percentage of arrested embryo was lower in PZM-3 than in NCSU-23 on d 4 and 5 ( P<0.05). TUNEL positive signals were higher in NCSU-23 than in PZM-3 from d 3 to 7 ( P<0.05). The ratios of Bcl-2 and Bax mRNA expression in embryos were higher on d 5 than on d 3 and 1 in PZM-3 ( P<0.05). In contrast, the ratios of Bcl-2 and Bax mRNA expression in embryos on d 1 were higher than on d 3 and 5 in NCSU-23 ( P<0.05). Additionally, the ratios of Bcl-2 and Bax mRNA expression in embryos in PZM-3 were higher than in NCSU-23 on d 3 and 5 ( P<0.05). In conclusion, lower apoptotic embryo rates and down-regulating Bax together with up-regulating expression of Bcl-2 transcripts may be responsible for the better developmental competence of porcine parthenotes in PZM-3.

Effect of different culture systems on mRNA expression in developing rabbit embryos

The rate of zygotes in vitro developed to hatched blastocyst stage was evaluated between two different commercial media (TCM-199 and Ham's F10) and two different culture systems (renewal and non-renewal single medium) to determine the effects of culture conditions on rabbit embryo preimplantation development. The relative transcript abundances of OCT4, vascular endothelial growth factor (VEGF) and epidermal growth factor receptor 3 (erbB3) of resultant blastocysts were also analysed and compared with in vivo developed blastocysts. Results showed an important divergence in mRNA expression between embryos developed under in vivo and in vitro conditions despite there being no significant difference in hatching blastocyst rates between different culture systems and different media. For OCT4, transcript abundance of in vitro culture embryos differs from their in vivo chronological counterparts, but, when the medium is renewed, mRNA expression seemed similar to in vivo developed 4-day-old embryos. In addition, VEGF and erbB3 expression showed marked variation between different in vitro conditions. Therefore, the study of specific transcript abundance in rabbit blastocyst provides a more detailed description of which alterations in gene expression occur due in vitro conditions, and further studies should be carried out to reduce current limitations of long-term culture of rabbit pre-implantation embryos.

Scriptaid Improves In Vitro Development and Nuclear Reprogramming of Somatic Cell Nuclear Transfer Bovine Embryos

The present study evaluated the effect of Scriptaid, a novel histone deacetylase inhibitor (HDACi), on the in vitro development of somatic cell nuclear transfer (SCNT) bovine embryos. Average fluorescence intensity of two epigenetic markers (H3K9ac and H3K9m2) at two-cell, eight-cell, and blastocyst stages, and the expression levels of two developmental important genes (Oct4 and IFN-t) at the blastocyst stage were also examined to assess the influence of Scriptaid on the nuclear reprogramming of bovine SCNT embryos. The results showed that treatment with 500 nM Scriptaid for 14 h after activation significantly increased the cleavage rate, blastocyst formation rate, and blastocyst hatching rate of SCNT embryos compared with those of nontreated counterparts, but the total number of blastomeres per blastocyst did not differ. Scriptaid treatment also significantly increased the immunofluorescent signal for H3K9ac in SCNT embryos at two-cell, eight-cell, and blastocyst stages, and the fluorescent signal for H3K9m2 was decreased at two-cell, eight-cell, and blastocyst stages. The expression levels of Oct4 and IFN-t were significantly higher in Scriptaid-treated SCNT blastocysts than in Scriptaid nontreated SCNT blastocysts. The results indicated that Scriptaid treatment improved the in vitro developmental capacity and the nuclear reprogramming of bovine SCNT embryos.

Latrunculin a Enhances the Parthenogenetic Developmental Potentials of Electrically Activated Porcine Oocytes Matured &lt;i&gt;In Vitro&lt;/i&gt;

The present study was carried out to determine the effect of latrunculin A (Lat-A) and FBS on the developmental potentials of electrically activated porcine oocytes maturedin vitro. Electrically activated MII oocytes were incubated for 2 hr in mPZM-3 supplemented either with 0、20 nM、1 μM Lat-A or 2.2 μg/ml cytochalasin B (CB). They were then removed and placed in mPZM-3 with or without 10% FBS (addition) on Day 4 for further development. The nuclear activation rate was significantly improved when activated oocytes were cultured in mPZM-3 with Lat-A (P&lt;0.05). At higher dosage (1μM) of Lat-A, the extrusion of second polar body was inhibited while the cleavage rate increased significantly (P&lt;0.05). Significant interaction between Lat-A and FBS supplementation on Day 4 was observed duringin vitrodevelopment (P&lt;0.05). The hatched blastocyst rates and cell numbers per blastocyst were significantly enhanced with the supplementation of FBS (P&lt;0.05). The blastocyst formation, hatched blastocyst rates and mean cell numbers per blastocyst of 1 μM Lat-A treatment with FBS supplementation were significantly higher than those of 20 nM Lat-A and CB treatments with FBS supplementation (P&lt;0.05), but the individual effect of FBS supplementation was not observed on blastocyst formation in 20 nM and 1 μM Lat-A treatments. While significant difference was seen in blastocyst formation and cell numbers per blastocyst among all the treatments without FBS supplementation (P&lt;0.05). These results indicate that Lat-A especially at the dosage of 1 μM can significantly improve the developmental potential of electrically activated porcine oocytes, and the FBS supplementation on Day 4 of development have an additional effect on porcine embryo development.

Short-term urea feeding decreases in vitro hatching of bovine blastocysts

Cows fed high-protein diets may have impaired reproductive performance. Although the pathogenesis has not been completely elucidated, it appears that not only the uterus, but also the follicle and oocyte, are affected by excessive plasma urea nitrogen (PUN) concentrations. Thus, the objective was to determine the effects of short-term urea feeding on the competence of bovine oocytes. Forty crossbred heifers (Bos indicus vs Bos taurus) were allocated to two groups, namely CONTROL (maintenance diet) and UREA (maintenance diet supplemented with 75 g of urea/day), following a cross-over design. Heifers received their respective diets for 6 d (without adaptation). On the sixth day, blood samples were harvested both before and 3 h after feeding, and cumulus oocyte complexes (COCs) were collected by ovum pick-up. Although PUN concentrations were higher in UREA than CONTROL heifers (31.31 mg/dL ± 1.13 vs 22.12 mg/dL ± 0.86; mean ± SEM), neither the number of COCs recovered (8.8 ± 1.0 vs 9.2 ± 0.8, UREA vs CONTROL, respectively) nor their quality (based on morphology) differed significantly between groups. Next, oocytes were fertilized and cultured in vitro to assess developmental rates. There was an absence of significant differences between groups for rates of cleavage (Day 3) or blastocyst formation (Days 6, 7 and 9), but the hatched blastocyst rate on Day 11 after fertilization was lower (P < 0.05) in the UREA than the CONTROL groups (64.3 vs 83.5%). Therefore, we inferred that the effects of urea were only manifest later in development. In conclusion, high PUN concentrations decreased oocyte competence in heifers, reinforcing the hypothesis that poor reproductive performance in cows with high PUN was due, at least in part, to a deleterious effect on oocytes.

Vitrification of Biopsied Mouse Embryos At 8-cell, Morula and Blastocyst Stages Using A Modified Vitrification Medium

BACKGROUND: Vitrification of biopsied embryos plays an important role in preimplantation genetic diagnosis cases using CGH and array CGH METHODS which may require cryopreservation of the biopsied embryos for later transfer.OBJECTIVE(S): The objective of this study is to investigate the effect of vitrification on biopsied embryos at various stages using a modified vitrification medium.MATERIAL AND METHOD(S): The mouse embryos were biopsied and vitrified at 8-cell, morula and blastocyst stages using a modified vitrification medium with low concentration of DMSO to avoid potential toxicity. Non-biopsied mouse embryos at the same stages were vitrified as control. The vitrified embryos were thawed and analyzed by their survival rate, blastocyst formation and hatching rates.RESULT(S): At 8-cell stage, the overall survival rate of the non biopsied embryos was significantly higher than the biopsied embryos (94% versus 68%), P<0.05). The blastocyst formation rates of both groups after thawing are similar (87% versus 85%, P>0.05). At morula stage, both biopsied and non biopsied groups had similar survival rate (96% versus 92%, P>0.05). After thawing, the blastocyst formation rate of the biopsied group is comparable to that of the non biopsied group (93% versus 94%, P>0.05). At blastocyst stage, both biopsied and non biopsied embryos had similar survival rate (92% versus 91%, P>0.05). The hatching blastocyst rates in both groups are similar (86% verse 89%, P>0.05).CONCLUSION(S): The above data suggests that the survival rate and developmental potential of the biopsied mouse embryos at morula and blastocyst stages are similar to those of the non biopsied embryos using the modified vitrification medium. Moreover, vitrification of biopsied embryos at advanced stage shows better survival rate when compared to cleavage stage embryos using the modified vitrification medium.SUPPORT: Institutional funds BACKGROUND: Vitrification of biopsied embryos plays an important role in preimplantation genetic diagnosis cases using CGH and array CGH METHODS which may require cryopreservation of the biopsied embryos for later transfer. OBJECTIVE(S): The objective of this study is to investigate the effect of vitrification on biopsied embryos at various stages using a modified vitrification medium. MATERIAL AND METHOD(S): The mouse embryos were biopsied and vitrified at 8-cell, morula and blastocyst stages using a modified vitrification medium with low concentration of DMSO to avoid potential toxicity. Non-biopsied mouse embryos at the same stages were vitrified as control. The vitrified embryos were thawed and analyzed by their survival rate, blastocyst formation and hatching rates. RESULT(S): At 8-cell stage, the overall survival rate of the non biopsied embryos was significantly higher than the biopsied embryos (94% versus 68%), P<0.05). The blastocyst formation rates of both groups after thawing are similar (87% versus 85%, P>0.05). At morula stage, both biopsied and non biopsied groups had similar survival rate (96% versus 92%, P>0.05). After thawing, the blastocyst formation rate of the biopsied group is comparable to that of the non biopsied group (93% versus 94%, P>0.05). At blastocyst stage, both biopsied and non biopsied embryos had similar survival rate (92% versus 91%, P>0.05). The hatching blastocyst rates in both groups are similar (86% verse 89%, P>0.05). CONCLUSION(S): The above data suggests that the survival rate and developmental potential of the biopsied mouse embryos at morula and blastocyst stages are similar to those of the non biopsied embryos using the modified vitrification medium. Moreover, vitrification of biopsied embryos at advanced stage shows better survival rate when compared to cleavage stage embryos using the modified vitrification medium. SUPPORT: Institutional funds

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5–17.7%, 1.5–4.3% and 2.8–3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4–25.7 vs 13.7–26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8–3.7%) than in fresh controls (28.6–35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.

Targeted Suppression of Connexin 43 in Ovine Preimplantation Embryos by RNA Interference Using Long Double-stranded RNA

RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.

Effects of Melatonin on In Vitro Development of Mouse Two-Cell Embryos Cultured in HTF Medium

Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10–13 to 10–3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10–13 to 10–5 M and decreased by melatonin at 10–3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10–9 M. In comparison to control, 10–9 M melatonin increased blastocyst formation rate from 48.08 ± 5.25% to 82.08 ± 2.34% (p < 0.05), hatched blastocyst rate from 25.65 ± 11.79% to 66.47 ± 4.94% (p < 0.05), and cell number per blastocyst 62.71 ± 5.97 to 77.91 ± 10.63 (p < 0.05). Thus, our datas demonstrated firstly that melatonin has beneficial effects on the in vitro development of 2-cell mouse embryos cultured in HTF medium.

110 THE USE OF FORSKOLIN AND ITS EFFECT ON IN VITRO-PRODUCED BRAHMAN-SIRED EMBRYOS SUBMITTED TO SLOW COOL FREEZING OR VITRIFICATION

The objective of this study was to further test the lipolytic effect of 10 μM forskolin on developmental differences between bovine in vitro produced (IVP) embryos submitted to slow cool (SC) freezing or vitrification (VT). Previously reported (phase I) IVP embryo hatching rates for control embryos (62%) were no different than 10 μM forskolin (67%; Pryoretal et al. 2009 Reprod. Fertil. Dev. 21, 163). For phase II: on Day 6 post-fertilization (IVF = Day 0), 207 Brahman-sired viable embryos were evenly divided and cultured for 24 h in G2.5 medium (Vitrolife, Englewood, CO, USA) with or devoid of 10 μL forskolin (Sigma, St. Louis, MO, USA). On Day 7, compact morula (CM, n = 31), blastocyst (BL, n = 76), and expanded BL (XBL, n = 100) were washed in Vigro Holding Plus medium (Bioniche, Pullman, WA, USA) and randomly allocated to 4 treatment groups; control SC (CSC; no treatment, n = 52), 10 μM forskolin SC (FSC; n = 55), control VT (CVT; n = 49), and 10 μM forskolin VT (FVT; n = 51). All embryos were packaged in sterile 0.25-mL plastic straws. The SC embryos were submitted to Vigro Ethylene Glycol Freeze Plus medium (Bioniche) for 5 min before freezing at 0.5°C/min from -6°C to -32°C and plunging in LN2. Embryos were vitrified using a bovine VT kit (Bioniche): VS1, 3 min; VS2, 45 s in 15 μL; diluent, in straw with VS2 separated by air columns, vitrified in LN2 vapor 1 cm from liquid for 1 to 15 min before plunging. The SC embryos were air thawed 5 s and placed in 30°C H2O bath for 10 s. The VT straws were air warmed 10 s and then in 35°C H2O for 20 s prior to shaking them down to mix columns. All embryos were cultured in G2.5 for 24-h survival and 48-h hatching rates. All percentage data were transformed using arcsin square root function prior to analysis, and means were compared for statistical significance using Student’s t. For mean survival rates, FSC was different than CSC but showed no difference between FVT and CVT (81.7 ± 0.09, 42.6 ± 0.09, 59.4 ± 0.09, 49.0 ± 0.10, respectively (P &lt; 0.01). There were no statistical differences for hatching rates for combined embryo stages (58.2 ± 0.10, 37.8 ± 0.10, 34.4 ± 0.10, 28.1 ± 0.11 for FSC, FVT, CSC, and CVT, respectively; P &gt; 0.07). However, when comparing hatching rates of only the blastocyst stage embryos (n = 176), FSC was superior to CSC and CVT but not different than FVT (74.8 ± 0.11, 29.5 ± 0.11, 29.1 ± 0.11, 48.5 ± 0.11, respectively; P &lt; 0.01). In conclusion, FSC yielded significantly higher survival and blastocyst hatching rates than CSC, but there were no differences between CVT and FVT for survival and FVT for blastocyst hatching rates. These results indicate that the addition of 10 μM forskolin to culture 24 h prior to freezing 7 d IVP Brahman-sired embryos can increase survival and blastocyst hatching rates. The authors acknowledge support from the American Brahman Breeders Association.

291 INFLUENCE OF THE BREED OF BULL (BOS INDICUS × BOS TAURUS) IN TOLERANCE TO HEAT SHOCK IN BOVINE EMBRYOS PRODUCED IN VITRO

There is evidence that deleterious effects of heat shock (HS) on fertility are less pronounced in breeds tolerant to high temperatures, due mainly to differences in their thermoregulatory capacity. In vitro experiments have shown that Bos indicus embryos are more resistant to HS than Bos taurus. In order to better understand the differences related to HS resistance between Bos indicus and Bos taurus, the main objective of this study was to determine if tolerance to HS is caused by genetic contribution from the oocyte, spermatozoa, or both. Additionally, the influence of the time between collection of ovaries in the abattoir and oocyte aspiration in the laboratory on early embryo development was ascertained. In experiment 1, oocytes from Nellore and crossbreed Holstein cows (cHOL) were collected in a local abattoir, matured and fertilized using semen (n = 6 for each breed) from Nellore (NEL), Angus (ANG), Brahman (BRA,) and Gir (GIR) bulls. In experiment 2, oocytes from Nellore and Holstein (HOL) cows were collected in an abattoir and the oocytes were aspirated in the laboratory 4 (group 4 h) or 6.5 h (group 6.5 h) later, matured and fertilized using semen (n = 6 for each breed) from NEL, GIR, and HOL. In both experiments, 96 h post-insemination (hpi), embryos with &gt; 16 cells were separated in 2 groups: control and HS. In the control group the embryos were cultured at 39°C, whereas in the HS group the embryos were submitted to 41°C for 12 h, and then returned to 39°C. In experiments 1 and 2 the results were analyzed by ANOVA (Proc MIXED, SAS Institute, Cary, NC, USA). In experiment 1, there was no effect of HS on blastocyst and hatched blastocyst rates in all breeds studied. The percentage of oocytes that cleaved and reached the morula stage was significantly lower (P &lt; 0.05) in cHOL × GIR compared with the other breeds. Additionally, blastocyst rate was higher in cHOL × NEL than in cHOL × ANG and cHOL × GIR (P &lt; 0.05). In experiment 2, cleavage, morula, and blastocyst rates in group 4 h were higher (P &lt; 0.05) compared with group 6.5 h. The HS decreased blastocyst rates in all breeds (NEL × NEL, HOL × HOL, and HOL × GIR), and in both time intervals (4 and 6.5 h). The breed NEL × NEL had higher cleavage rate (P &lt; 0.05) for both time intervals compared with HOL × HOL and HOL × GIR. In addition, Nellore oocytes fertilized with Nellore semen (NEL × NEL) originated higher blastocyst rates (P &lt; 0.05) in control and HS group than the other breeds. We conclude that (a) embryos from Holstein are more susceptible to HS than embryos from crossbred Holstein; (b) the oocyte is more important than the spermatozoa for the development of thermotolerance, because the breed of the bull did not influence embryo development after HS; (c) in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the time interval between ovary collection and oocyte aspiration. Fellowships to T. Nabhan from CAPES and to R. A. Satrapa, R. A. L. Simoes, and E. M. Razza from FAPESP. Funding from FAPESP (Sao Paulo, Brazil).

98 EIGHT-CELL STAGE DELINEATES CRYOTOLERANCE OF VITRIFIED RABBIT EMBRYOS

A number of groups have successfully vitrified rabbit embryos at morula or blastocyst (BL) stage. For earlier stages (1- to 8-celled), however, there are very limited studies and the results are generally unsatisfactory. In this study, we examined the survival and developmental competence of rabbit embryos vitrified at different preimplantational stages. Sexually matured female New Zealand White (NZW) rabbits were superovulated with our standard protocols, followed by mating with NZW males. At 18h post-hCG treatment, viable fertilized embryos were collected and cultured in 2.5% FBS B2 medium (Laboratories CCD, Paris, France) in 5% CO2 with humidified air at 38.5°C. A total of 691 rabbit embryos at pronuclear, 2-celled, 4-celled, 8-celled, and morula/early blastocyst (BL; Day 3) stages were vitrified by the open pulled straw (OPS) method in HEPES buffered TCM-199 medium containing 20% fetal calf serum, ethylene glycol, and dimethyl sulfoxide. After stored in liquid nitrogen for at least 1 month, embryos were sequentially warmed, rehydrated, and washed before culture in B2 culture medium. Survival and developmental rates were analyzed by general linear model analysis (SPSS 11.0, SPSS Inc., Chicago, IL, USA). Embryos vitrified at 8-celled stage or beyond showed greater survival, and expanded BL and hatching rates than those at pronuclear, 2-celled and 4-celled embryos (Table 1). In particular, the expanded and hatched BL rates were significantly higher in the 8-celled group than those in 4-celled group, suggesting that the 8-celled is the threshold stage to tolerate the vitrification procedure in rabbit embryos. In addition, the rates of expanded and hatched blastocysts in morula/BL group were still significantly higher than those in the 8-celled group. Our results may provide the proper timing of cryopreserving fertilized, transgenic, and/or cloned rabbit embryos at early stages for biomedical research. Table 1.Survival and developmental competence of rabbit embryos vitrified at different stages after thawing This study was supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

Effects of IGF-I and EGF Supplemented to PZM3 Culture Medium on the Development of Porcine Embryos In vitro

This study investigated the effects of IGF-I and EGF on the development of blastocysts or hatched blastocysts during the in vitro culture of embryos from immature porcine oocytes. After the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs) and their culture in vitro in PZM3 medium, we examined the embryo development rate for 168 h. When different concentrations of IGF-I (0, 1, 10, 20 ng/ml) were supplemented to fertilized porcine embryos in vitro, there were no significant differences in cleavage rate, blastocyst development rate or blastocyst hatching rate among the treated groups. On the other hand, when different concentrations of EGF (0, 1, 10, 20 ng/ml) were supplemented to the in vitro culture medium, blastocyst development rate was highest in the group in which EGF was not supplemented and, specifically, it was higher than in the 20 ng/ml treatment group (p<0.05). When 10 ng/ml IGF-I and 1 ng/ml EGF were supplemented separately or simultaneously, there were no significant differences among the treated groups in blastocyst hatching rate and the number of cells in each condition. This study demonstrated that the addition of IGF-I and EGF into PZM3 medium did not enhance development of the blastocyst stage and total cell number in blastocysts. (Key Words : Embryo Development, Porcine Embryo, Growth Factor, PZM3)

Influence of Superovulatory Protocols onIn VitroProduction of Nellore (Bos indicus) Embryos

There are indications in the literature that delaying the period between ovarian superestimulation and ovum pick up (OPU) would induce follicles to a condition of initial atresia, which could be beneficial to oocyte development. In this work, we compared three protocols for OPU and in vitro production (IVP) of embryos, in Nellore cattle. Nellore cows (n = 18) were randomly allocated in three groups: Group 1 (OPU), Group 2 [Follicle stimulating hormone (FSH) and OPU] and Group 3 (FSH deprivation and OPU). Three OPUs were performed, and the animals were switched to a different group each time (crossover), in such a way that at the end of the experiment all cows received the 3 protocols. At random stage of the oestrous cycle (D-2), all follicles ≥ 6 mm were aspirated to induce a new follicular wave 2 days afterwards (D0). In Group 1, OPU was performed on D2 and oocytes were processed to IVP. In Group 2, starting on D0, cows were superstimulated (FSH, Folltropin(®), 30 mg administered daily, i.m., during three consecutive days, total dose = 180 mg), and 6 h after the last FSH dose, they received exogenous luteinizing hormone (LH) (12.5 mg, i.m., Lutropin(®), D3). The OPU was performed 6 h after LH administration, i.e. 12 h after the last dose of FSH. Animals in Group 3 received the same treatment as those in Group 2, except that LH was administered 42 h after the last dose of FSH, and OPU occurred 6 h later. Therefore, in this group, follicles were deprived of FSH at 48 h. Both cleavage and blastocyst rates were similar (p > 0.05, anova) among oocytes from Groups 1, 2 and 3, respectively: 77.4% (144/185) and 42.70% (79/185); 75.54% (105/139) and 31.65% (44/139); 63.52% (101/159) and 33.33% (53/159). However, hatched blastocyst rate was higher (p < 0.01) in Group 1 (30.27%, 56/185) when compared with Group 2 (11.51%, 16/139) or 3 (15.72%, 25/159). It is concluded that, contrary to previous work on European breeds (Bos taurus), ovarian superstimulation associated with deprivation of FSH and OPU (Group 3) did not increase IVP of Nellore embryos (Bos indicus). On the contrary, the highest hatched blastocyst rates were observed in oocytes from non-superstimulated cows.

127 LETHAL EFFECT OF HIGH CONCENTRATIONS OF PARECOXIB AND FLUNIXIN MEGLUMINE ON THE IN VITRO CULTURE OF BOVINE EMBRYOS

The aim of this experiment was to evaluate the effects of cycloxigenase inhibitor drugs, i.e. flunixin meglumine (FM) and parecoxib (P), on the development of bovine embryos cultured in vitro until the blastocyst and hatched blastocyst stages. Immature oocytes were aspirated from slaughterhouse ovaries and morphologically selected for IVM (Monteiro FM et al. 2007 Anim. Reprod. 4, 51–58). Twenty hours after maturation (39°C and 5% CO2 in air), matured oocytes were transferred to fertilization media, inseminated with frozen–thawed semen, and incubated for 10 to 12 h. Presumptive zygotes (PZ) were then transferred to TCM 199 HEPES medium, vortexed to remove cumulus cells and finally to drops of IVC media (SOFaaci plus 5% BFS [Gibco] with 13 mm sodium pyruvate). Each drop of IVC medium had appropriate concentrations of FM (0.14/n = 123; 1.4/n = 122; 14/n = 117; 140/n = 44 or 1400 μg mL–1/n = 44 PZ) or P (0.09/n = 134; 0.9/n = 109; 9/n = 118; 90/n = 113 or 900 μg mL–1/n = 45 PZ), besides extra drops as control groups (CFM; n = 124 and CP; n = 149 PZ). Based on published data from bovine (FM) and human (P) administered concentrations, it was calculated the blood concentration to a bovine weighing 450 kg (FM = 14 and P = 9 μg mL–1). Both drugs were used from available commercial preparations, and in a pilot test, there were no deleterious effects of the solvent itself on the blastocyst and hatched blastocyst rates. During culture, petri dishes containing PZ/embryos were kept into plastic bags, under controlled atmosphere of 5% O2, 5% CO2 and 90% N2 at 39°C. There were 11 replicates for each treatment. In all drops (both drug concentrations and control group) the blastocyst and hatched blastocyst rates (BR and HBR, respectively) were evaluated at 144 and 192 h after fertilization, respectively. Statistical analysis was performed with ANOVA on ranks (Dunn’s test a posteriori and significance being considered when P &lt; 0.05; BioEstat version 5.0). According to the results, FM (1400 and 140 μg mL–1) and P (900 μg mL–1) concentrations were toxic enough for a complete inhibition of in vitro bovine embryo development. There were no significant differences among the other drug concentrations and their respective control group, on the BR (27.7 ± 3.9; 29.6 ± 3.4 and 29.8% ± 4.8) and HBR (13.5 ± 4.4; 15.6 ± 3.8 and 22.1% ± 5.1), respectively to 0.14; 1.4 and 14 μg mL–1 for FM; on BR (26.0 ± 2.6; 18.2 ± 4.6; 25.8 ± 5.9 and 23.2% ± 4.8) and HBR (14.1 ± 3.3; 10.2 ± 3.3; 16.8 ± 3.8 and 12.0% ± 3.4), respectively to 0.09; 0.9; 9 and 90 μg mL–1 for P; and on BR (35.3 ± 5.2 and 36.5% ± 3.4) and HBR (26.6 ± 4.5 and 19.8% ± 3.6), respectively for CFM and CP. The results suggest that, during in vitro bovine embryo culture, there was no significant toxicity of either drug, with exception of the complete lethal concentrations of 140 and 1400 μg mL–1 (flunixin meglumine) and 900 μg mL–1 (parecoxib) on blastocyst production. Supported by FAPESP – Brazil (MFGN 06/06491-2 and 07/07705-9; EMR 07/04284-2; RAS; CFS and RALS) and CAPES – Brazil.

126 THE EFFECT OF FORSKOLIN ON IN VITRO PRODUCED BRAHMAN-SIRED BOVINE EMBRYOS

Forskolin (Colforsin) is a lipolytic agent that can be used to reduce intracellular lipids within in vitro produced (IVP) porcine embryos (Men et al. 2006 Theriogenology 66, 2008–2016). Lowering lipids within bovine embryos has shown to decrease sensitivity to cryopreservation. The objective of this preliminary experiment was to determine developmental differences and lipolytic effect on bovine IVP embryos exposed to 10 or 40 μm forskolin. Bovine oocytes (obtained from an abattoir) were fertilized with frozen/thawed Brahman semen (Day 0) and cultured in G1.5/G2.5 medium supplemented with 8 mg mL–1 BSA (Vitrolife, Englewood, CO, USA). On Day 6 post-fertilization, a total of 352 viable embryos were randomly submitted to 3 treatment groups; Control (no treatment, n = 116), 10 μm (n = 122) and 40 μm (n = 114) forskolin (Sigma, St. Louis, MO, USA) in G2.5 medium for 24 h. On Day 7, embryos were washed in holding medium (Vigro Holding Plus, Bioniche, Pullman, WA, USA). From each treatment group, a randomized half was cultured 72 h in G2.5 and assessed for hatched blastocyst rates (development), with the balance stained on Day 7 in Hoechst 33342 (2.5 μg mL–1) under UV light for total cell counts before being fixed/stained in Nile red (1 μg mL–1). Nile red stained embryos were mounted on glass slides with cover slips gently applied over each embryo. Embryos were photographed under UV light using the proper excitation filters. The area measurements of compact morula and blastocysts did not include the zona pellucida. Nile red units (NRU) were measured with computer software Image J (www.rbsweb.nih.gov) from 0 to 255 shades/pixel (0 = no lipids; 255 = highest lipid accrual). Each image was adjusted for variation in embryo size. Student’s t-test was used for statistical analysis. There was no difference (P &gt; 0.05) between development rates for control, 10 μm and 40 μm forskolin (62%, 67% and 65%, respectively). Mean NRU by total cell numbers were calculated per embryo for each treatment group: control, 10 μm and 40 μm with the resulting means being 72.4 ± 5.0, 63.1 ± 5.1 and 66.6 ± 4.8 respectively. With treatment groups combined by embryo stage, compact morula tended to exhibit a higher mean NRU than blastocysts (78.6 ± 6.8 to 64.7 ± 3.1, respectively, P = 0.07). In conclusion, forskolin did not significantly affect embryo development rates or lipid content as measured by Nile Red staining. Compact morula tended to yield a higher NRU mean than blastocysts indicating an elevation in intracellular lipids. Unlike pig IVP embryos exposed to forskolin, forskolin failed to show a significant decrease in intracellular lipids within bovine IVP embryos. However, further research is in progress to test forskolin’s lipolytic effect on cryopreservation of in vitro and in vivo produced bovine embryos. The authors acknowledge support from the American Brahman Breeders Association and Dr. Moisés Barceló Fimbres.

Comparative Study between Slow Freezing and Vitrification of Mouse Embryos Using Different Cryoprotectants

The objective of this study was to evaluate the effects of different cryoprotectants and different cryopreservation protocols on the development of mouse eight-cell embryos. Mouse eight-cell embryos were cryopreserved by using propylene glycerol (PROH), ethylene glycerol (EG), dimethyl sulfoxide (DMSO) or glycerol (G) as cryoprotectant with slow-freezing or Vit-Master vitrification protocol. After thawing, the survival rate, blastocyst formation rate and blastocyst hatching rate of the embryos were compared. When the mouse eight-cell embryos were cryopreserved by the slow-freezing, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with PROH were significantly higher than those of DMSO and G (p < 0.05, respectively), but not significantly different among those of DMSO, G and EG (p > 0.05, respectively), and not significantly different between those of PROH and EG (p > 0.05, respectively). When the mouse eight-cell embryos were cryopreserved by Vit-Master vitrification, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with EG were significantly higher than those of PROH, DMSO and G (p < 0.05, respectively). Yet, there were no significant differences among those of PROH, DMSO and G (p > 0.05, respectively). In conclusion, PROH was the optimal cryoprotectant for the cryopreservation of mouse eight-cell embryos by slow-freezing protocol. EG was the optimal cryoprotectant for the cryopresevation of mouse eight-cell embryos by Vit-Master vitrification protocol, which may be commonly used in clinical and laboratory practice.

Early apoptosis is associated with improved developmental potential in bovine oocytes

The poor quality of oocytes may be the main reason for the low efficiency of the current in vitro embryo production. However, efforts are required to understand the mechanisms of oocyte development, which is believed to be largely regulated by apoptosis in vivo. The aim of this study was to investigate the levels of apoptosis in bovine immature oocytes with different developmental potentials and to determine whether early apoptosis in bovine oocytes is correlated with their subsequent development. Cumulus–oocyte complexes (COCs) were selected and classified into four groups according to oocyte cytoplasm and cumulus status. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively. Developmental competence was evaluated by nuclear maturation (MII) after in vitro maturation and development rates in different stages following in vitro fertilization. Meanwhile, the transcripts of Bcl-2 and Bax genes were carried out in immature oocytes by real-time RT-PCR. Results indicated that Annexin-V-positive oocytes were detected in various groups at different percentages, and Group III showed the highest positive ratio. No TUNEL-positive oocytes were found in any immature COCs. Group III oocytes demonstrated the highest nuclear maturation, cleavage, blastocyst, and hatching blastocyst rates. Meanwhile, Group III oocytes exhibited the highest Bax (initiating apoptosis) transcriptional level and the lowest Bcl-2 (preventing apoptosis) transcriptional level. Taken together, Annexin-V and quantitative PCR results indicated that early apoptosis was beneficial for developmental competence, while TUNEL staining showed that none of the immature oocytes were undergoing late-stage apoptosis. This is the first time that Bax and Bcl-2 transcripts were characterized in the immature bovine oocyte, and results indicated that the genes are good markers of early apoptosis and embryo development. This research overthrows the traditional view that oocytes undergoing apoptosis have poor developmental competence, and the findings will facilitate oocyte selection and improvement of in vitro embryo production.

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

Cat blastocysts produced in vitro from oocytes vitrified using the cryoloop technique and cryopreserved electroejaculated semen

Oocyte preservation is still a challenge in the cat. The aim of this study was to evaluate the efficiency of oocyte vitrification in cryoloop in the domestic cat and to assess the embryonic development after IVF with cryopreserved semen. In vitro matured cat oocytes were vitrified in cryoloop after exposure to 10% ethylene glycol (EG, 0.9 M) in hepes synthetic oviductal fluid (HSOF) for 1 min, 20% EG (1.8M) in HSOF for 1 min, and 40% EG (3.6M), 10mg/ml Ficoll 70 and 0.3M sucrose in HSOF for 20s. Warmed oocytes were fertilized in vitro with frozen-thawed semen collected by electroejaculation and presumptive zygote were cultured in vitro for 10 days. Results showed that percentage of degenerated oocytes was higher (P<0.01), while cleavage rate and morulae blastocysts rate on day 6 were significantly lower (P<0.01) for vitrified oocytes than control. Blastocyst rate on day 8 was higher (P<0.01) for control oocytes than vitrified counterparts, and also developmental ability was higher (P<0.05) for non-vitrified oocytes, while the hatched blastocyst rate on day 10 was higher (P<0.05) for vitrified oocytes than control. In conclusion cat oocytes can be vitrified in cryoloop with a fairly good survival rate, cleavage rate and embryo development until pre-implantation stage.