Higher Blastocyst Rate Research Articles

254 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF OOCYTES SCREENED FOR THEIR DEVELOPMENTAL COMPETENCE BASED ON G6PDH ACTIVITY

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes (Alm 2005 Theriogenology 63, 2194–2205). However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here we aim to compare the developmental, molecular, and subcellular characteristics of oocytes selected using brilliant cresyl blue (BCB) staining based on G6PDH activity. Immature compact cumulus–oocyte complexes (COCs) were stained with 26 µm BCB (B-5388, Sigma-Alderich, Taufenkirchen, Germany) for 90 min. Based on their coloration, oocytes were divided into BCB– (colorless cytoplasm, high G6PDH activity) and BCB+ (colored cytoplasm, low G6PDH activity). The chromatin configuration and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement (n = 337). The abundance and phosphorylation pattern of protein kinases Akt and MAP kinase were estimated by western blot analysis (n = 500). A bovine cDNA microarray with 2000 clones was used to analyze the gene expression profiles of BCB+ and BCB– oocytes (n = 580). BCB+ oocytes were found to result in a higher blastocyst rate (33.1 � 3.1%) until Day 8 of in vitro culture compared to BCB– ones (12.1 � 1.5%). Moreover, BCB+ oocytes showed higher phosphorylation levels of Akt and MAP kinase compared to the BCB– oocytes. After array data analysis, BCB+ oocytes were found to be enriched with genes regulating transcription (SMARCA5), cell cycle (NASP), and protein biosynthesis (RPS274A and EEF1A1), while the BCB– oocytes had a higher level of genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10), and growth factor activity (BMP-15). Independent real-time quantitative PCR validated 90% (9/10) of the genes investigated to be in agreement with the array expression profile. The study has shown evidence of differences in molecular and subcellular organization of oocytes with different G6PDH activity.

200 EFFECT OF OOCYTE SIZE AND MICROMANIPULATION TECHNIQUES (ICSI AND EMBRYO BIOPSY) ON GOAT EMBRYO QUALITY

The aim of this study was to evaluate embryo quality of prepubertal goat oocytes after micromanipulation techniques: ICSI and embryo biopsy. Prepubertal goat oocytes were recovered from a slaughterhouse and matured in IVM medium (TCM-199 with serum, hormones, and cystamine) for 27 h. Spermatozoa were selected by swim-up and capacitated with heparin (10 µg mL−1) plus ionomycin (200 nM) for 15 min. A total of 930 IVM oocytes were divided into 4 groups depending on size (Group I: 110–125 µm, and Group II: >125 µm) and fertilization procedure: ICSI or IVF. Zygotes were cultured in TALP fertilization medium. At 24 h post-insemination (hpi), zygotes were cultured in SOF. At 48 hpi, oocyte cleavage was recorded. Four days after insemination, a sample of 8-cell embryos was biopsied by removal of one blastomere. After biopsy, embryos were cultured in SOF for a further 4 days. Blastocysts were stained by TUNEL to assess apoptotic cell percentage. Results are shown in Table 1. Biopsied and non-biopsied embryos from Group II had higher blastocyst rates than those in Group I both in ICSI and IVF (P ≤ 0.02). Group II had also larger blastocyst cell numbers than Group I both in ICSI and in IVF (P ≤ 0.001). Moreover, blastocysts from Group II non-biopsied embryos had statistically larger cell numbers than those from the biopsied ones (P ≤ 0.05). Blastocysts from Group II had a lower apoptotic rate than those in Group I both in ICSI and IVF (P ≤ 0.05). In conclusion, oocytes larger than 125 µm were more competent to achieve embryo development than smaller oocytes. ICSI did not reduce embryo quality, either in blastocyst cell number or apoptotic index, when compared with IVF. Furthermore, embryo biopsy did not reduce blastocyst rates or apoptotic index. However, embryo biopsy reduced blastocyst cell number in Group II-derived blastocysts. Table 1.Effect of oocyte size and micromanipulation techniques on goat embryo quality

180 DEVELOPMENTAL CAPACITY OF SINGLE CULTIVATED EMBRYOS IN SMALL AND BIG DROPLETS

An in vitro production system where a single oocyte can be followed from the ovary to the blastocyst stage would be a useful tool for studies concerning developmental competence or follicular environment. Unfortunately, until now, only low blastocyst rates could be obtained after single embryo production, and there is still discussion about the ideal droplet size. The objective of the present experiment was to compare the developmental competence of single cultivated zygotes in 20- and 500-µL droplets. Cumulus–oocyte complexes were obtained from slaughterhouse ovaries and were matured and fertilized in groups of 100 for 22 h; the presumptive zygotes were divided into 4 groups. In treatment 1, 25 zygotes were transferred into 50 µL of SOF medium supplemented with 5% serum under oil, whereas in treatment 2, 25 zygotes were transferred into 500 µL of medium. Zygotes were cultivated separately in treatments 3 and 4: in treatment 3 in 20 µL of medium under oil and in treatment 4 in 500 µL of medium. Cleavage rates and division stages were assessed after 3 days of cultivation (5% CO2, 5% O2, 90% N2); blastocyst rates were determined after 7 days. Statistical analysis was performed by logistic regression using SAS (PROC LOGISTIC). There was no difference in cleavage rates between the 2 group treatments or between the 2 single treatments. Also, the division stages were not different between the 2 single treatments (16-cell: 2.0 vs. 1.3%; 8-cell: 25.8 vs. 31.6%; 4-cell: 41.2 vs. 38.0%; and 2-cell: 31.0 vs. 29.1% for the 20 µL and the 500 µL droplet sizes, respectively). Group cultivation after 7 days in 50 µL was significantly better than in 500 µL; however, both treatments resulted in significantly higher blastocyst rates compared with the individual cultures in 20 or 500 µL, between which no significant difference could be found. Noteworthy, only 4-cell and 8-cell stages on Day 3 resulted in blastocysts on Day 7 of cultivation. In conclusion, these results indicate that cultivation in groups gives higher blastocyst rates, although the same embryo density is used as in individual cultivation (1 embryo 20 µL in treatments 2 and 3). Moreover, no significant difference could be found between single cultivation in small and big droplets. This is confirmed by the cleavage stages on Day 3, which indicate no difference in timing of cleaving between small and big droplets; time of cleaving is indicative of further developmental capacity. Table 1.Cleavage and blastocyst rates after single and group cultivation

304 SHORT-PERIOD SPERM - OOCYTE INCUBATION AND FERTILIZATION MEDIA EFFECTS ON THE IN VITRO PRODUCTION OF SWINE EMBRYOS

The aim of this study was to evaluate the effect of 2 sperm–oocyte incubation periods (30 min and 6 h) and 3 IVF media (TCM-199, TCM-199 with Ca-lactate, or mTBM). Cumulus–oocyte complexes (COCs) from abattoir-derived swine ovaries were matured in TCM-199 supplemented with 3.05 mM glucose, 50µg mL−1 gentamycin, 0.91 mM sodium pyruvate, 10% swine follicular fluid, 0.57 mM cysteine, 10 ng mL−1 of epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 of hCG for 22 h, followed by a 22-h incubation without hormones. After in vitro maturation, oocyteswere allocated into 3 IVF media: TCM-199 (supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 50µg mL−1 gentamycin, 1 mg mL−1 BSA, and 3.06 mM mL−1 caffeine-standard medium);TCM-199 Ca (standard medium with 2.92 mM Ca lactate); or mTBM (supplemented with 5 mM mL−1 sodium pyruvate, 0.57 mM cysteine, 50µg mL−1 entamycin, 1 mg mL−1 BSA, and 2 mM mL−1 caffeine). The refrigerated semen at 15°C was centrifuged and capacitated for 2 h at 38.5°C in an atmosphere of 5% CO2 in air and under high humidity conditions in the respective IVF media. Oocytes were denuded, washed with the respective IVF media, and inseminated with the capacitated spermatozoa at a concentration of 6 × 105 sperm mL−1 in 90-µL microdroplets. After 30 min, a group of oocytes from each fertilization medium was gently washed to remove nonbound spermatozoa and returned to a new medium droplet for another 5.5 h (30-min group). The other group of oocytes remained in the same droplet with spermatozoa for 6 h (6-h group). After IVF, oocytes were cultured in porcine zygote medium-3 for 7 days. Data were analyzed by chi-squared test (P < 0.05).With regard to cleavage rates on the third day of embryo development (Day 3), no significant difference was observed among sperm–oocyte incubation periods; however, there was a difference in the TCM-199 group, compared with the mTBM and TCM-199 Ca groups, at the 30-min incubation period. With 6 h of incubation, no differences were observed among groups. When blastocyst (Day 7) rates were evaluated, no significant differences were observed between the 2 TCM-199 media groups; however, the mTBM groups showed higher blastocyst rates. The sperm–oocyte incubation period of 30 min or 6 h of IVF did not interfere with the blastocyst rate. In conclusion, the IVF medium mTBM was more efficient than the TCM-199 media when considering embryo production. Moreover, 30 min of IVF was sufficient for the spermatozoa responsible for oocyte fertilization to bind to the zona pellucida. Table 1.Cleavage (Day 3) and blastocyst (Day 7) rates of embryos incubated in two sperm–oocyte periods (30 min or 6 h) and cultivated in three IVF media This work was supported by the FAPESP 05/01420-7.

Evaluation of laser-assisted lentiviral transgenesis in bovine

Lentiviral transduction of oocytes or early embryos is an efficient strategy to generate transgenic rodents and livestock. We evaluated laser-based microdrilling (MD) of the zona pellucida, which is a physical barrier for viral infection, and subsequent incubation in virus suspension as a new route for lentiviral transgenesis in bovine. Lentiviral vectors carrying an eGFP expression cassette were used to transduce oocytes or zygotes after MD as compared to the established subzonal virus injection technique (MI). The type of manipulation (MD vs. MI) did not affect cleavage rates, but had a significant effect on blastocyst rates (P < 0.001). MI of virus or sham-MI (buffer) resulted in higher blastocyst rates as compared to MD, both in the oocyte and zygote treatment groups. The latter exhibited higher rates of early cleavage (P < 0.05) and blastocyst rates (P < 0.01). The proportion of eGFP expressing blastocysts was higher after infection of oocytes (MD: 44 +/- 9%; MI: 67 +/- 8%) than after infection of zygotes (MD: 26 +/- 8%; MI: 26 +/- 9%). Overall efficacy (eGFP-positive blastocysts per treated oocytes or zygotes) was highest after MI of oocytes (18 +/- 2%). Our study demonstrates the feasibility of laser-assisted lentiviral gene transfer into bovine oocytes and zygotes. However, further optimization of the procedure is required, mainly to reduce the incidence of polyspermy after MD of oocytes and to eliminate negative effects of MD on early embryonic development.

Reduction of body-weight gain enhances in vitro embryo production in overfed superovulated dairy heifers

The aim of our study was to test whether a reduction in dietary intake could improve in vitro embryo production in superovulated overfed dairy heifers. Cumulus-oocyte complexes of 16 Prim' Holstein heifers (14 +/- 1 months old) were collected by ovum pick-up (OPU), every 2 weeks following superovulation treatment with 250 microg FSH, before being matured and fertilized in vitro. Embryos were cultured in Synthetic Oviduct Fluid medium for 7 days. Heifers were fed with hay, soybean meal, barley, minerals and vitamins. From OPU 1 to 4 (period 1), all heifers received individually for 8 weeks a diet formulated for a 1000 g/day live-weight gain. From OPU 5 to 8 (period 2), the heifers were allocated to one of two diets (1000 or 600 g/day) for 8 weeks. Heifers' growth rates were monitored and plasma concentrations of metabolites, metabolic and reproductive hormones were measured each week. Mean live-weight gain observed during period 1 was 950 +/- 80 g/day (n = 16). In period 2 it was 730 +/- 70 (n = 8) and 1300 +/- 70 g/day (n = 8) for restricted and overfed groups respectively. When comparing period 1 and period 2 within groups, significant differences were found. In the restricted group, a higher blastocyst rate, greater proportions of grade 1-3 and grade 1 embryos, associated with higher estradiol at OPU and lower glucose and beta-hydroxybutyrate, were observed in period 2 compared with period 1. Moreover, after 6 weeks of dietary restriction (OPU 7), numbers of day 7 total embryos, blastocysts and grade 1-3 embryos had significantly increased. On the contrary, in the overfed group, we observed more <8 mm follicles 2 days before superovulation treatment, higher insulin and IGF-I and lower nonesterified fatty acids in period 2 compared with period 1 (no significant difference between periods for embryo production). After 6 weeks of 1300 g/day live-weight gain (OPU 7), embryo production began to decrease. Whatever the group, oocyte collection did not differ between period 1 and 2. These data suggest that following a period of overfeeding, a short-term dietary intake restriction (6 weeks in our study) may improve blastocyst production and embryo quality when they are low. However, nutritional recommendations aiming to optimize both follicular growth and embryonic development may be different.

Microfertilization of oocytes and culture of embryos in goat

AbstractIn vitro development of goat embryos obtained by fertilizing oocytes using intracytoplasmic sperm injection (ICSI) was investigated. The results showed that the blastocyst rate was significantly higher (P&lt;0.05) when the embryos were co-cultured with granular cells (GCs) in media CR1aa supplemented with 10% bovine follicular fluid (BFF) (22.2%) than with 5% (13.1%) and 15% BFF (2.7%). When embryos were co-cultured with GCs in SOFaa media, the highest blastocyst rate was obtained from supplementation with 10% BFF (25.1%), which was significantly different from 5% (12.9%) and 15% BFF (3.0%) groups. When 10% BFF and fetus bovine serum (FBS) were added into CR1aa or SOFaa media, the goat ICSI blastocyst rates were 22.6 and 26.9% or 5.8 and 6.1% respectively. These results suggest that both CR1aa and SOFaa could be used as culture media for goat ICSI embryos, 10% BFF could significantly increase the blastocyst rate and BFF was more efficient than FBS for the early in vitro development of goat ICSI embryos.

Metabolic regulation of in-vitro-produced bovine embryos. I. Effects of metabolic regulators at different glucose concentrations with embryos produced by semen from different bulls

The toxic and/or beneficial effects of four metabolic regulators on embryo development were evaluated. In-vitro-produced compact morulae were cultured for 3 days in a chemically defined medium + bovine serum albumin (BSA; CDM-2) plus regulators (4991 total embryos). Phenazine ethosulfate (PES), phloretin (PL), pyrroline-5-carboxylate (P5C), and sodium azide (NaN3) were evaluated at four doses each in factorial combinations with four concentrations of glucose: 0, 0.5, 2, and 8 mm. Phenazine ethosulfate at 0.9 microm resulted in poorer development than lower or no PES. Phloretin was, in general, detrimental for embryo development, but most markedly at the highest dose (270 microm). Pyrroline-5-carboxylate had little effect on post-compaction embryos at the doses studied, 9 to 81 microm. Sodium azide at the concentrations used (3, 9, and 27 microm) had little effect on embryo development compared with controls. Concentrations of glucose had little effect on development of embryos. A fifth metabolic regulator, 2,4-dinitrophenol (DNP), was studied at various doses at pre-morula or morula-blastocyst stages cultured in 2 mm glucose. Embryos (2189 total) cultured in 90 microm DNP developed more slowly and were darker than embryos cultured at lower doses. Embryos cultured in 30 microm DNP had a higher blastocyst rate (48.3%) than controls (34.9%). In the last experiment using G1.2/G2.2 media, DNP (30 microm) resulted in a marked decrease in embryonic development when embryos were exposed at the zygote to 8- to 16-cell stages but had little effect when morulae were exposed for 2 days. The dose-response information for these metabolic regulators is crucial for designing future experiments.

300 BLASTOCYST PRODUCTION BY IN VITRO MATURATION AND DEVELOPMENT OF PORCINE OOCYTES IN DEFINED MEDIA FOLLOWING INTRACYTOPLASMIC SPERM INJECTION

Most culture media for in vitro production (IVP) of porcine embryos contain serum or bovine serum albumin (BSA); especially in the case of in vitro maturation (IVM) porcine follicular fluid is added. The present study was carried out to establish porcine-defined IVP. In Experiment 1, we investigated the efficacy of additional 0.6 mM cystine, 100 �M cysteamine (Cys), or cystine + Cys to a defined TCM-199 maturation medium with regard to the developmental competence of in vitro-matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM-199 containing 0.05% (w/v) polyvinyl alcohol (PVA). In Experiment 2, the effect of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development following ICSI. As positive and negative controls, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the basic medium. Five or 10 ng mL-1 of EGF was supplemented in the negative control medium (mPZM-4). All percentage data on cleavage and blastocyst development were subjected to arcsine transformation before statistical analysis using the STATVIEW program (Abacus Concepts, Berkeley, CA). The mean cell numbers per blastocyst (assessed by Giemsa staining method) were analyzed by one-way ANOVA, and the differences among the groups were also analyzed using Tukey-Kramer multiple comparison tests. In Experiment 1, there were no significant differences in the rates of cleavage (31.4 to 64.3%) and blastocyst formation (6.5 to 22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups, without significant differences. In Experiment 2, the rates of cleavage (43.0 to 58.0%) and blastocyst formation (14.0 to 23.6%), and the mean cell numbers per blastocyst (30 to 37) were not significantly different among the groups. However, the addition of 5 ng mL-1 of EGF to the mPZM-4 resulted in a significantly (P d 0.05) higher blastocyst rate (48.6%) to cleaved embryos than the other two defined groups (mPZM-4 and mPZM-4 with 10 ng mL-1 EGF: 23.4% and 23.1%, respectively), but not significantly different with mPZN-3 (40.1%). The present results indicate that TCM-199 with added cysteamine (100 �M) can be used as a defined IVM medium for porcine oocytes, and further addition of cystine into the IVM medium is not necessary. The addition of 5 ng mL-1 of EGF to a defined IVC medium enhanced subsequent development after ICSI. These results show that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI, and IVC). This study was partly supported by a grant-in-aid for Scientific Research (17580242) from the Japan Society for the Promotion of Science.

259 DNA HYPOMETHYLATION OF FETAL FIBROBLASTS IMPROVES DEVELOPMENTAL COMPETENCY AND GENE EXPRESSION PATTERN IN PORCINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

The inhibition of methyl groups in the DNA of donor cells has been hypothesized to improve the potential reprogramming by the enucleated ooplasm after nuclear transfer (NT). Previously, we reported that treatment of porcine fetal fibroblasts (PFF) with an inhibitor of methylation, 5-azacytidine (5-azaC) at 0.5 �m, results in the retention of desirable characteristics with a relative reduction in methylation, making cells more conducive for reprogramming (Mohana Kumar et al. 2006 Cell Tissue Res. 325, 445-454). To understand these observations further, the present study investigated the developmental competence and expression pattern of gene transcripts in porcine NT embryos from PFF (control) and 0.5 �m 5-azaC-treated PFF (PFF + 5-azaC) at 4-cell, 8-cell, morula, and blastocyst stages, and compared these with those of IVF and in vivo embryos. Cleavage rate was significantly (P &amp;lt; 0.05) higher in IVF than in NT embryos from PFF and PFF + 5-azaC (86.7 � 5.2% vs. 65.8 � 5.3% and 69.3 � 4.4%, respectively). Similarly, significantly (P &amp;lt; 0.05) higher blastocyst rates were observed in IVF embryos (27.2 � 2.1%). However, NT embryos from PFF + 5-azaC showed enhanced developmental potential with significantly (P &amp;lt; 0.05) higher rates of blastocysts (21.3 � 2.2%) than NT embryos from PFF (14.8 � 1.9%). NT embryos from PFF + 5-azaC (33.8 � 4.1) had significantly (P &amp;lt; 0.05) higher total cell numbers than from PFF (24.6 � 3.5), but did not differ in the proportion of apoptotic cells (6.9 � 1.8% and 7.2 � 2.1%, respectively). However, the high total cell number and lower incidence of apoptosis were observed in IVF and in vivo embryos (45.3 � 3.8, 2.7 � 0.8%, and 53.9 � 3.5, 1.2 � 0.9%, respectively). Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4 and Stat3), DNA methylation (DNA methyltransferases: Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b), histone acetylation (histone acetyltransferase 1-HAT1), and histone deacetylation (histone deacetylases-Hdac1, Hdac2, and Hdac3) were observed in NT embryos from PFF and PFF + 5-azaC compared with that in IVF and in vivo counterparts. However, the expression of genes in PFF + 5-azaC-NT embryos closely followed those of in vivo-derived embryos compared with PFF-NT embryos, and, interestingly, there was lower variability in the expression of genes related to DNA methylation. Our findings demonstrate that remodeling of the epigenetic status by partial reduction of somatic DNA methylation from donor cells is beneficial in improving the developmental competency of porcine NT embryos. Further, hypomethylated donors may be more efficiently reprogrammed to re-activate the expression of early embryonic genes. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

262 ASSESSMENT OF HSP70-1 TRANSCRIPTION LEVELS IN IMMATURE OOCYTES FROM BOS TAURUS AND BOS INDICUS COWS RAISED IN A TROPICAL CLIMATE

Heat stress is one of the main causes of low conception rate in Bos taurus cows in a tropical climate. On the other hand, in this environment, oocytes from Bos indicus show greater developmental capacity after in vitro fertilization than those from Bos taurus, suggesting an adaptation to the hot climate. Heat shock proteins (HSP) are chaperones that promote protection against heat damage, and their transcription is associated to stress. The aim of this study was to evaluate the expression of HSP70-1 gene (Genbank NM174550), a member of HSP family, in oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) cows raised in the tropical climate located at 21�35′′S latitude, 43�51′′W longitude, and 435 m altitude. Cumulus–oocyte complexes were recovered by oocyte pickup from mature non-lactating Holstein (n = 4) and Gyr (n = 4) donor cows during the hot season. Cumulus cells of viable oocytes were removed by vortexing in TALP-HEPES plus BSA, and pools (3 for each breed) with 12 immature oocytes were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction. Total RNA extraction was performed using Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and first strands were synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000; Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) and cDNA equivalent to 1.2 oocytes and gene specific primers. Expression of the GAPDH gene was used as endogenous reference. Calculations of relative quantification were performed by the comparative Ct method, using the lowest value found in Bos indicus oocytes as calibrator; values (mean � SE) are shown as n-fold difference relative to the calibrator. Statistical comparison between breeds was performed by analysis of variance. Oocytes from Holstein cows showed a higher level (P &amp;lt; 0.05) of HSP70-1 expression (1.82 � 0.22) than oocytes recovered from Gyr cows (1.12 � 0.11). Previous study reported that oocytes from Gyr cows in a tropical climate showed a higher blastocyst rate after in vitro fertilization than Holstein oocytes (Camargo et al. 2006 Reprod. Fertil. Dev. 18, 243 abst). The lower level of HSP70-1 in Gyr oocytes suggests that they were less subject to stress than the Holstein ones, which may reflect their capacity to develop after fertilization. This effect may be, at least in part, due to the ability of Bos indicus cows to regulate body temperature in a hot environment, causing less stress on oocytes. Financial support was provided by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogen�tica, Vi�osa, Brazil, for the real-time PCR machine.

367 INFLUENCE OF SUPERESTIMULATION AND HORMONAL DEPRIVATION PROTOCOLS ON IN VITRO PRODUCTION OF NELORE EMBRYOS (BOS TAURUS INDICUS)

There are indications in the literature that delaying the period between ovarian superestimulation and ovum pickup (OPU) will induce follicles to a condition of initial atresia, which could be beneficial to oocyte development. Blondin et al. (2002 Biol. Reprod. 66, 38–43) superstimulated (FSH) Holstein heifers and delayed OPU for 48 h, i.e. they induced initial atresia in these follicles deprived of FSH (starvation) for 48 h. Additionally, 6 h before OPU, LH was administered to accelerate follicular maturation. This protocol yielded a surprisingly high blastocyst rate (80.4 ± 9.4%). In the present work, Blondin’s protocol was simultaneously compared to other protocols used for OPU and in vitro production of embryos (IVP), in Nelore cattle. Nelore cows (n = 18) were randomly allocated in three groups: Group 1 (just OPU), Group 2 (superestimulation and OPU), and Group 3 (superestimulation associated with FSH deprivation and OPU). Three OPU were performed, and the animals were switched to a different group each time in such a way that at the end of the experiment all cows received the three protocols. At a random stage of the estrous cycle (D2), follicles ≥6 mm were aspirated to induce a new follicular wave 2 days afterward (D0). In Group 1, OPU was performed on D2 and oocytes were processed to IVP. In Group 2, starting on D0, cows were superestimulated (pFSH, Folltropin®, 30 mg administered daily, IM, during 3 consecutive days; Bioniche Animal Health, Belleville, Ontario, Canada), and 6 h after the last FSH dose they received exogenous LH (12.5 mg, Im, Lutropin®, D3; Bioniche Animal Health). 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 in Group 2, except that LH was administered 36 h after the last dose of FSH, and OPU occurred 6 h later. Therefore, in this Group, follicles were deprived of FSH during 48 h. Both cleavage and blastocyst rates were similar (P &gt; 0.05, logistic regression) 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 &lt; 0.01) in Group 1 (30.27%, 56/185) when compared to Group 2 (11.51%, 16/139) or 3 (15.72%, 25/159). It is concluded that, contrary to previous work on European breeds (taurus), ovarian superestimulation associated with deprivation of FSH and OPU (Group 3) do not increase IVP of Nelore embryos (indicus). Additionally, the highest hatched blastocyst rates were observed in oocytes from non superstimulated cows (Group 1).

40 EFFECT OF CHEMICAL ACTIVATION ON DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION PORCINE EMBRYOS DERIVED FROM NUCLEAR TRANSFER

Activation is one of key factors for improving developmental ability of pre-implantation nuclear transfer (NT) embryos. This study investigated the effect of chemical activation following fusion/activation on the development and apoptosis of pre-implantation porcine embryos derived from NT. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 42 to 44 h. Donor cells were prepared from a 35-day-old porcine fetus. Matured oocytes were enucleated and donor cells were introduced into the perivitelline space. Fusion/activation was conducted with two electric pulse of 1.2 kV/cm for 30 �s. Fused embryos were divided into four groups. The first one was the control without chemical activation; the other three groups were treated with thimerosal (0.2 mM for 10 min; T) and then with dithiothreitol (8 mM for 30 min; DTT), 6-dimethylaminopurine (2 mM for 3 h; 6-DMAP), or cycloheximide (10 �g/mL for 6 h; CH). Treated embryos were cultured in porcine zygote medium-3 (PZM-3) at 38.5�C under 5% CO2 in air for 6 days. Cleavage and blastocyst rate were determined on Days 3 and 6, respectively. Apoptosis was analyzed with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay from day 1 to 7. Embryos treated with chemicals following fusion/activation showed significantly higher blastocyst rates compared to control embryos fused/activated by electric pulse alone (12.6% for control vs. 21.1% for DTT, 20.8% for 6-DMAP, 20.6% for CH; P &lt; 0.05). Although total cell number of blastocysts showed no significant difference, the ratio of inner cell mass to trophectoderm was significantly higher (P &lt; 0.05) in embryos with chemical activation than in those without it (11.9 vs. 19.4, 18.1, and 24.1%; P &lt; 0.05). Occurrence of apoptosis was first observed on Day 3, but there was no significant difference among treatments until Day 6. It was significantly increased in embryos with chemical activation on Day 7 compared to control embryos (5.1 vs. 7.1, 7.8, and 7.8%; P &lt; 0.05). These results indicate that chemical activation following fusion/activation could support significantly a higher blastocyst rate for pre-implantation porcine embryos derived from nuclear transfer; however, it can increase occurrence of apoptotic cells at blastocyst stage.

Developmental potential of embryos after 1 to 3 biopsy procedures for preimplantation genetic diagnosis (PGD)

To study the potential impact of multiple micromanipulation procedures for PGD on embryo development. Retrospective analysis of 701 PGD cycles (7737 oocytes) over a 15-month period with regard to the type and number of biopsy procedures in relation to fertilization and embryo development as compared to 1695 control cycles (17346 oocytes) during that same period. Intracytoplasmic sperm injection (ICSI) was performed on all oocytes in both control (non-tested) and PGD cycles. Cell sampling for PGD was accomplished by creating an opening in the zona pellucida using the mechanical method of partial zona dissection. The first polar body (PB1) was removed prior to ICSI for maternally derived disorders followed by second polar body (PB2) removal through the same opening after fertilization assessment. Both PB1 and PB2 were simultaneously removed following fertilization assessment for aneuploidy testing. Embryo biopsy was performed on day 3 of embryo development after an additional intersecting slit was made forming a flap type opening in the zona pellucida in order to remove a single blastomere. When PGD was performed for mutation analysis in conjunction with aneuploidy testing, as many as three micromanipulation procedures, in addition to ICSI, were performed on the same oocyte/embryo. Fertilization rates, for cases requiring PB1 removal prior to ICSI, and embryo development to the blastocyst stage for all micromanipulation groups were compared with the control group. Both PGD and control groups were further divided by age, patients ≤ to 34 and ≥ to 35 years. Chi-square analysis was used to determine statistical significance. For the group of patients ≤ 34 years, the fertilization rate for oocytes in which PB1 was removed prior to ICSI was 82.2% (499/607) compared to 79.4% (8413/10613) in the control group. No significant difference was found. Fertilization rates for the group of patients ≥ 35 years were as follows: control group 78.3% (5270/6733) vs. 86.5% (499/607) in the group in which PB1 was removed prior to ICSI. A significant difference was seen (p=0.001) between the control group and test group with a higher fertilization rate achieved in the group of oocytes in which PB1 had been removed prior to ICSI. Comparison of blastocysts rates between 1, 2 and 3 biopsy procedures compared to the control group in patients ages ≤ 34 years were 52.9 % (9/17), 55.2%, (374/677), 56.3% (198/352) and 54.3% (4216/7766) respectively. No significant difference was found. Comparison of blastocysts rates between 1, 2 and 3 biopsy procedures and the control group for patients ages ≥ 35 years were 41.0% (16/39), 47.8% (795/1664), 62.1%, (121/195) and 45.7% (1922/4204) respectively. No significant difference was seen with the exception of the group in which three micromanipulations were performed resulting in a higher blastocyst rate when compared to the control group (p=0.001). Multiple micromanipulations, to include ICSI, sequential PB1 and PB2 removal and embryo biopsy on day 3 can be performed on oocytes and subsequent embryos without detriment to embryo development.

2POSTHATCHING DEVELOPMENT SYSTEM: A NOVEL IN VITRO CULTURE OF BOVINE EMBRYOS

Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683–700). On Day 8, degenerated embryos were removed from each well and 400L of modified culture medium (SOFaaci plus 0.5% glucose and 10% fetal bovine serum) were added. At Day 11, hatched blastocysts were selected by scoring them as Quality 1 (Q1: &amp;gt;1.0mm, clear trophoblast, compact inner cell mass), Quality 2 (Q2: 0.5mm, dark spots in the trophoblast, less compact inner cell mass), or Quality 3 (Q3: &amp;lt;0.5mm, many dark spots in the trophoblast, spread inner cell mass). The resulting 304 blastocysts in 12 replicates were then loaded into 15mm×1.2 gel tunnels of 2.4% agarose in PBS, supplemented with either 5% (Agar5) or 10% (Agar10) fetal bovine serum, covered with the modified culture medium, and then incubated at 38.5°C in 5% CO2, 5% O2, 90% N2. Embryo morphology and length were evaluated using a stereomicroscope on Days 12, 13, 14 and 15. On Day 14, 75 embryos were removed, biopsed (1mm) for sex determination of each embryo, and processed for light and transmission electron microscopy. Qualitative and quantitative data were analyzed by χ2 test and GLM procedure of SAS, respectively, with P level of 0.05. A total of 170 embryos (56% of total) initiated elongation. This percentage was higher (LSmeansSD, n=12; P&amp;lt;0.05) in Agar10 v. Agar5 in both Q1 (889 v. 637), Q2 (667 v. 485) and Q3 embryos (529 v. 278). Mean embryo length (mm; LSmeansSEM) on Day 13 was higher (P&amp;lt;0.05) in Q1 (2.10.2, n=49) and Q2 (1.71.4, n=98) than Q3 (1.20.3, n=23). On Day 14, Q1 embryos (3.50.2) were longer (P&amp;lt;0.01) than Q2 and Q3 embryos (2.70.1 and 2.00.3). On Day 15, Q1, Q2 and Q3 embryos (4.40.5, n=24, 4.00.3, n=45 and 2.90.6, n=14, respectively) had similar length, probably influenced by the low number of Q3 embryos. The percentage of males was higher (P&amp;lt;0.001) in Q1 (95%; n=40), but similar in Q2 (39%; n=26) and Q3 (71%; n=7). Light microscopy confirmed hypoblast and epiblast formation. Ultrastructural analysis revealed that the latter had penetrated the trophoblast (Rauber’s layer), forming an embryonic disc including many degenerative cells. In conclusion, this culture system represents the first model for rapid growth, elongation, and initial differentiation of bovine posthatching embryos.

Onset of the First S-Phase Is Determined by a Paternal Effect During the G1-Phase in Bovine Zygotes1

The aim of this study was to characterize the respective influences of the paternal and the maternal components on the timing of the first S-phase in the bovine zygote. In vitro-matured oocytes were fertilized in vitro with sperm conferring a high blastocyst rate (embryos of group 1) or a low blastocyst rate (embryos of group 2). Resulting zygotes were either allowed to develop in vitro to the blastocyst stage or exposed to 5'-bromo-2'-deoxyuridine in order to characterize the timing of their first S-phases. Timing of pronuclear formation was similar in the two groups, but the onset of S-phase and the first cleavage occurred earlier in group 1 than in group 2. We also showed that the length of the S-phase represented 30% of the first cell cycle in group 1 and 20% in group 2. Differences in times of onset of the first S-phase observed between embryo groups concerned both male and female pronuclei in a similar manner and were not dependent on the maternal component of the zygote. Our data demonstrated that the precocity of the onset of the first S-phase stemmed from a paternal control exerted during a transient period of the G1-phase.

The effect of IVM and IVC media on in vitro development of bovine embryos.

The purpose of this study was to examine the effect of medium combination of IVM and IVC on the in vitro development of bovine embryos. The study involved 4 groups in a 2 (IVM medium) x 2 (IVC medium) factorial in a randomized block design. Each group was replicated for 5 times. The treatments were as follows: TCM-199/CR1aa (T1); TCM-199/SOF (T2); B- 199/CR1aa (T3) and B-199/SOF (T4). Oocytes were aspirated from ovaries collected at local abattoirs using aspiration medium of PBS supplemented with 3% FCS and 0.1% Penicillin and Streptomycin. The oocytes were matured in medium of TCM-199 or B-199 supplemented with 10% FCS, hormones: 10μg/ml FSH+ 10μg/ml hCG+ 1μg/ml Estradiol. Maturation was maintained at 37oC for 22 hours in 5% CO2 incubator with high humidity. A method of BRACKETT & Oliphant (BO) was used to fertilize the matured oocytes. The fertilization was incubated for 7 hours in the 5% CO2 incubator. Two culture media of CR1aa or SOF/AA/BSA were used to develop the fertilized oocytes undergo to morula and blastocyst embryos. The findings showed that the proportion of oocytes cleaved and formation of blastocysts were affected significantly by a combination of IVM and IVC media (P<0.05). A combination of B-199/SOF (T4) resulted in a higher blastocyst rate (32%) than others (T3= 29%; T2=T1= 23%). This study suggests that either SOF/AA/BSA or CR1aa has similar competence in development of bovine embryos in vitro . Key words: Bovine embryo, IVF, maturation medium, culture medium

Meiotic and developmental competence of bovine oocytes after induction of different levels of intracellular cAMP during IVM

Porcine IVF faces various problems such as incomplete cytoplasmic maturation of the oocyte and polyspermy. Previous studies proved the importance of cAMP in regulating nuclear and cytoplasmic maturation of oocytes. This study investigated the effect of the cAMP-modulating agents 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cAMP sodium salt (dbcAMP) on several parameters during in vitro production of porcine embryos. First, we wanted to see if oocyte collection in IBMX could meiotically arrest oocytes and, as such, improve synchronization of nuclear and cytoplasmic maturation. To this end, cumulus–oocyte complexes (COCs) were collected from gilts in HEPES-buffered Tyrode balanced salt solution medium with 0.5-mM IBMX or without IBMX. At the end of oocyte collection, the effect of IBMX on chromatin configuration was evaluated. However, no differences could be observed in nuclear configuration between IBMX− and IBMX+ oocytes (P > 0.05). Second, we added dbcAMP during IVM to improve cytoplasmic maturation and evaluated cumulus expansion (lack of adhesion), a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS-1) levels in cumulus cells, fertilization, and blastocyst rates. Cumulus–oocyte complexes were matured in modified North Carolina State University medium 37 with or without 1-mM dbcAMP. Frozen-thawed, epididymal, boar spermatozoa were used for IVF. After IVF, presumed zygotes were cultured for 7 days in North Carolina State University medium 23. Penetration rate decreased in dbcAMP+ (57.3%) compared with dbcAMP− (67.8%), but the polyspermy rate also decreased (43.3% vs. 53.4%, respectively) leading to an increased normal fertilization rate (56.7% vs. 46.6%, respectively; P < 0.05). Only 7.2% of the COCs showed adhesion in dbcAMP+ which was lower than 15.7% in dbcAMP− (P < 0.05) probably because of an upregulation of the ADAMTS-1 protein by dbcAMP. When the adherent oocytes were removed during maturation, no difference could be detected between the blastocyst rate of dbcAMP− and dbcAMP+ (17.1% and 21.0% on Day 7, respectively; P > 0.05). In conclusion, the use of IBMX during collection did not cause a meiotic arrest. Using dbcAMP during IVM caused a greater normal fertilization rate, a lower rate of adherent COCs during IVM, higher levels of ADAMTS-1 in cumulus cells, and an equal blastocyst rate after screening out adherent COCs. These findings contribute to a better understanding of cAMP involvement in porcine oocyte maturation and provide a basis to develop an improved system with less polyspermy and higher blastocyst rates.

Support for the development of bovine embryos in vitro by secretions of bovine trophoblastic vesicles derived in vitro.

This study evaluated whether trophoblastic tissue derived in vitro secretes factors that support bovine embryonic development in vitro. The embryotrophic activity of these secretions was analysed in three different culture conditions based on TCM-199: (1) in a routine culture system using cumulus cells and 10% oestrous cow serum; (2) without cells but with 10% oestrous cow serum; and (3) under serum-free conditions. Rates of development to the 5-8-cell and blastocyst stages, as well as numbers of inner cell mass and trophectoderm cells of blastocysts were determined. In the absence of cumulus cells, cleavage rates of 5-8-cell embryos were significantly (P < 0.05) higher in trophoblastic vesicle-conditioned medium than in TCM-199 in both the presence (71% versus 49%) and absence (70% versus 49%) of serum. Trophoblastic vesicle-conditioned medium had a significant (P < 0.05) positive effect on the rate of development to the blastocyst stage when compared with TCM-199 in the presence of cumulus cells and serum (39% versus 33%), only serum (26% versus 19%), or in the absence of cells and serum (21% versus 5%). The numbers of inner cell mass and trophectoderm cells, and total number of cells in blastocysts produced in the cumulus cell coculture system in serum-free trophoblastic vesicle-conditioned medium or TCM-199 supplemented with serum were greater than those of blastocysts produced without cumulus cells or serum. Fractionation of serum-free trophoblastic vesicle-conditioned medium by ultrafiltration (10 kDa cut off) confined the embryotrophic activity mainly to the low molecular mass fraction. This study shows that serum-free trophoblastic vesicle-conditioned medium contains potent embryotrophic factors which act in a complementary manner to those secreted by cumulus cells and those supplemented with serum and result in reliably high blastocyst rates in the range of 40%. Since contamination of trophoblastic vesicle-conditioned medium with serum proteins can be avoided, this medium may be a reasonable source for the purification of specific embryotrophic factors.

Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning

To establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25-34), intermediate (40-55), and high (60-81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50-83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40-55) or high (60-81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40-55 cells) and 41% versus 25% (60-81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning.