Blastocyst Production Rate Research Articles

Hand-Made Cloned Goat (Capra hircus) Embryos—A Comparison of Different Donor Cells and Culture Systems

Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p < 0.05) increase in blastocyst production in goats when cultured on a flat surface (FS) (23.78 ± 3.33 %) than well of wells (WOW) (15.84 ± 2.12 %) and microdrops (MD) (0.7 ± 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 ± 2.12%) than the MD (0.7 ± 0.7%) system. The quality of HMC blastocysts was studied by differential staining. Genetic similarity was confirmed by polymerase chain reaction (PCR)-based amplification of the second exon of the MHC class II DRB gene, which gave similar bands in electrophoresis (286 bp) both in cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.

Comparative Potential of Cultured Skin Fibroblast, Cumulus, and Granulosa Cell to Produce Somatic Cell Nuclear Transfer (SCNT) Preimplantation Embryos in Buffaloes (Bubalus bubalis) in Relation to Gene Expressions

To improve the efficiency of somatic cell nuclear transfer (SCNT)-derived embryos in buffaloes (Bubalus bubalis), skin fibroblast, cumulus, and granulosa cells were cultured up to the 15th passage and cloned embryos were produced from each cell type. At the 15th passage the cumulative population doublings (CPDs) in cumulus cells was higher (60.78) than skin fibroblast (57.12) and granulosa (56.05) cell lines. Gene expression of chromatin remodelling proteins, that is, HDAC1, DNMT1, DNMT3a, and DNMT3b, were comparable at all five passages (P-3, P-6, P-9, P-12, and P-15) groups in cumulus cells but different in skin fibroblast and granulosa cells. Cleavage and blastocyst production rate in cumulus (65.9 and 27.4%)-derived embryos was higher than skin fibroblast (63.8 and 24.3%) and granulosa (62.5 and 22.3%)-derived embryos. Expressions of HDAC1, DNMT1, and DNMT3a mRNA in cumulus-derived blastocysts were similar to IVF blastocysts (control), whereas skin fibroblast and granulosa-derived blastocysts expression was significantly different (p < or = 0.05). DNMT3b mRNA expression in all the three donor cell types and IVF control were similar. The expression pattern of these genes showed the effect of donor cell type with different epigenetic reprogramming capabilities for SCNT embryo production rate. Overall, results indicated that cumulus cells are the best nuclear donor for SCNT.

Effects of Recombinant Relaxin on In Vitro Maturation of Porcine Oocytes

Relaxin, a member of the insulin superfamily, has diverse functions in both reproductive and nonreproductive tissues. The aim of the present study was to evaluate the effects of recombinant relaxin on the in vitro maturation of porcine oocytes and their subsequent embryonic development following in vitro fertilization. Three concentrations of relaxin (1, 10, and 100 ng/ml) were used in the in vitro maturation (IVM) medium [TCM supplemented with 10% (v/v) porcine follicular fluid, 10 ng/ml of epidermal growth factor, 4 IU/ml of pregnant mare serum gonadotropin, and (only for the first 22 hr) 4 IU/ml of human chorionic gonadotropin]. Relaxin was used during the entire IVM period. Nuclear maturation of oocytes was examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) for 5 min and mounted on a glass slide. The glutathione (GSH) content in oocytes, an important indicator of cytoplasmic maturity, was measured using a micro-glutathione assay. Cryopreserved boar semen was used for in vitro fertilization. Embryos were cultured in modified NCSU-23 medium supplemented with 0.5 mM pyruvate and 5 mM lactate. Although nuclear maturation of oocytes did not vary, the GSH content in oocytes was significantly higher when cultured with 1 ng/ml (7.9 pmol/oocyte) and 10 ng/ml (8.47 pmol/oocyte) compared to a control group. However, no additional beneficial effect was observed when 100 ng/ml of relaxin was added to the IVM medium. A significantly higher rate of blastocyst formation was observed with 10 ng of relaxin (32.4%) compared to the control (14.4%) or 100 ng of relaxin (21.4%). No difference between 1 ng and 10 ng was observed in terms of the blastocyst production rate. The inner cell mass cell numbers in relaxin-treated groups were significantly higher than control, and trophectoderm cell number was the highest in the 10 ng relaxin group. Relaxin (10 ng/ml) can be supplemented in IVM medium to support the maturation of porcine oocytes.

363 ENHANCEMENT OF COMMERCIAL BOVINE IN VITRO EMBRYO PRODUCTION BY SEXING FROZEN SEMEN PRIOR TO INSEMINATION

The high commercial demand on production of genetically valuable female cattle was emphasized after use of sex-sorted semen in commercial in vitro embryo production (IVEP) programs and a reduction of interest in the use of embryo biopsy followed by PCR. Brazil is a leader in IVEP of bovine embryos, and frozen-thawed, sex-sorted samples become a new option for embryo sexing. There are only a few reports in the literature using this new technique for IVEP. Most reports have involved the use of frozen-thawed then sorted for use with AI in Europe. In the present study, frozen semen from three Nelore bulls was used and the blastocyst production rate was evaluated at Day 7 of IVP and compared to previous data from the same bulls and a batch of non-sorted semen. Cumulus oocyte complexes obtained from high genetic merit donors by ovum pickup were matured (TCM-199, supplemented with FCS, LH, FSH, estradiol, pyruvate, and antibiotics) for 24 h and fertilized (Fert-TALP supplemented with BSA, phenylalanine, and heparin) for 18-22 h (Day 0) in vitro. Frozen semen straws were thawed at 35°C for 30 s in a water bath and then selected by centrifugation at 800 g on discontinuous Percoll™ gradients (45 :90%). Samples of frozen-thawed sex-sorted semen were processed from straws from bulls that in some cases may already be dead. Frequently, 3 to 5 straws were thawed, flow-sorted, and then centrifuged to provide sufficient sperm to fertilize 100 oocytes. On Day 1, presumptive zygotes were transferred to culture media (SOFaa supplemented with BSA and FCS) and on Day 7 blastocyst production rate was evaluated. The present results were commercially satisfactory because the frozen-thawed sex-sorted semen from all three bulls performed comparably and, in one case, better than non-sorted semen from the same bulls. Therefore, its use is extremely attractive in commercial bovine IVEP systems and is a more efficient system than embryo sexing. Table 1.Embryo production rates at 7 days post insemination

50 PRE-MATURATION OF BOVINE OOCYTES SUBMITTED TO NUCLEAR TRANSFER: EFFECTS ON IN VIVO DEVELOPMENT

In vitro embryo production by somatic cell nuclear transfer (SCNT) still presents low efficiency and blastocyst production rates are around 20%. Pre-maturation with cell cycle inhibitors is one alternative that has been studied to improve oocyte competence for use in in vitro production systems. The neurotrophin brain-derived neurotrophic factor (BDNF) has been reported to improve oocyte maturation. The aim of this work was to optimize the in vitro pre-maturation culture of bovine oocytes and its use in SCNT. Oocytes that were submitted to meiosis block before IVM (BL group) were cultured in TCM-199 medium supplemented with 10 ng mL-1 BDNF and 10 μM butyrolactone I for 24 h and then matured in IVM medium (TCM-199, 10% FCS, 0.5 μg mL-1 FSH, 5.0 μg mL-1 LH, 2.0 mM pyruvate, and 50 μg mL-1 gentamicin). Control oocytes (control group) were matured in IVM medium. After 19 h of IVM, oocytes from both groups were denuded with 0.2% hyarulonidase, enucleated, and reconstructed. Reconstructed embryos were chemically activated with ionomycin (5 min) and 6-DMAP (3h) and cultured in vitro in SOF medium for 7 or 8 days. Statistical analyses were performed by using BIOSTATS v.4.0 software. In vitro development variables [1st polar body (PB), fusion, cleavage, and blastocyst rates on Days 7 and 8] and in vivo development rates on Days 35, 60, 90, and 120 of pregnancy were analyzed by chi-square test. Total cell numbers and cells with fragmented DNA were analyzed by ANOVA. A level of 5% significance was considered. Extrusion of 1st PB (BL: n = 693; 69.3% and control: n = 639; 63.5%) and fusion rates (BL: n = 397; 79.2% and control: n = 345; 72.9%) were higher (P &lt; 0.05) in the BL group. There were no differences between treatments for cleavage rates (BL: n = 268; 67.5% and control: n = 228; 66.1%) or blastocyst rates on Day 7 (BL: n = 77; 19.4% and control: n = 69; 20.0%) and Day 8 (BL: n = 81; 20.4% and control: n = 73; 21.2%). Cloned blastocysts from both groups were submitted to TUNEL reaction (Day 8 blastocysts, n = 15 for BL and control groups) for DNA fragmentation analysis or were transferred to synchronized recipients (Day 7 blastocysts, n = 26 and n = 28 for BL and control groups, respectively) for in vivo development analysis. No differences were observed (P &gt; 0.05) between BL and control groups for total cell numbers (n = 127 and n = 138, respectively) and cells with fragmented DNA (0.0209 and 0.0188, respectively). Pregnancy rates at 35 (BL: n = 5; 19.2% and control: n = 9; 32.1%), 60 (BL: n = 3; 11.5 and control: n = 3; 10.7), 90 (BL: n = 3; 11.5 and control; n = 3; 10.7), and 120 days (BL: n = 3; 11.5 and control: n = 3; 10.7) also did not differ (P &gt; 0.05) between treatments. In conclusion, pre-maturation enhanced 1st PB extrusion and fusion rates of oocytes submitted to SCNT, and moreover, it was able to establish pregnancies until 120 days, similarly to the control group. Financial support: FAPESP and CNPQ, Brazil.

300 CASE REPORT: MORE THAN ONE CALF PER WEEK PRODUCED BY NELORE DONOR

According to the animal reproduction biotechnologies, in vitro fertilization is considered one of the most important tools to improve genetic gain as well as reduce bovine herd generations. Brazil is recognized as having the greatest commercial bovine herd in the world as well as by its use of IVP; however, this biotechnology needs more studies to improve not only the oocyte-embryo conversion rate but also the competent oocyte recovery from donor cows. Recently, one Nelore donor cow in 3 sessions was able to convert 233 (53.81%) embryos from 433 oocytes recovered, which resulted in 156 pregnancies (55.95%), which means one pregnancy per 2.7 oocytes or 3.05 pregnancies per week (when 51 weeks are admitted in one year). The COC obtained from this donor were matured (TCM-199, supplemented with FCS, LH, FSH, E2, pyruvate, and antibiotic) for 24 h and fertilized (Fert-TALP supplemented with BSA, PHE, and heparin) for 18 to 22 h (Day 0) in vitro. On Day 1, presumptive zygotes were transferred to development media (SOFaa supplemented with BSA and FCS) and on Day 7 the blastocyst production rate was evaluated. Following embryo transfer, the pregnancy diagnosis was performed at 30 days and confirmed at 60 days after ET. The possibility of identification of factors related to high performance and its selection could improve the formation of a homogeneous bovine herd, highly correlated with embryo production, which numerically could promote more than fifty calves per year (more than one calf per week). This case report refers to an animal highly superior to the average and therefore needs more investigation, mainly with molecular markers able to identify and select this class of high genetic animal. Table 1.Embryo production performance

46 RECLONING USING TRANSGENIC FETAL FIBROBLASTS AS NUCLEI DONORS INCREASES DEVELOPMENTAL POTENTIAL OF RECONSTRUCTED EMBRYOS IN CATTLE

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. Gene expression studies and adequate selection of the nuclei donor cell for NT guarantees the presence of the gene construction in the offspring and the absence of deleterious mutations caused by the random insertion of transgenes in functional areas of the genome. Embryonic development after NT requires a change in the transcriptome of the donor cell from a somatic to an embryonic pattern, causing cloning efficiencies to be low because of incomplete or defective nuclear reprogramming. Therefore, the establishment of methodologies able to increase cloning success is highly desirable. The experiment was designed to test if recloning of transgenic fetal fibroblasts increases cloned blastocyst production and the pregnancy rates of transgenic cloned embryos produced by NT. This study compared the developmental potential of cloned embryos reconstructed with fetal fibroblasts genetically modified by lentivirus random integration (control group) expressing the green fluorescent protein gene (eGFP), with a transgenic fetal fibroblast cell line established from a 30-day transgenic pregnancy (recloning group). Fusion, cleavage (72 h post-activation, hpa), blastocyst production (168 hpa), and 30-day pregnancy rates were analyzed. A total of 1213 embryos were reconstructed; 884 (10 replicates) with random transgene insertion fibroblasts and 329 (4 replicates) with cells derived from the cloned fetus. Results were analyzed by chi-square test at 5% significance. No difference was observed (P &gt; 0.05) between control and recloned groups regarding fusion rate (n = 550, 62.22% and n = 189, 57.25%; respectively) or cleavage rate (n = 383, 69.45% and n = 132, 69.84%, respectively). The recloned group, however, showed a higher blastocyst development rate (P &lt; 0.01) compared with the control group (n = 51, 26.98%, and n = 79, 14.36%, respectively) and a higher 30-day pregnancy rate (n = 6, 15.38% and n = 3, 5.56%, respectively). In conclusion, recloning of transgenic fibroblasts from a successfully established pregnancy augments the efficiency in the production of embryos and pregnancy establishment compared with the control group. We speculate that a second round of NT enhances the nuclear reprogramming of donor cells, and moreover, the use of a transgenic cell lineage already proven to be successfully reprogrammed may indicate that transgene integration is not deleterious in that specific cell lineage, resulting in a good source of donor cells to be used in order to produce a homogeneous bioreactor herd. Financial support: FAPESP, Brazil.

150 DEVELOPMENT OF ZONA-FREE AND WITH ZONA PARTHENOGENETIC GOAT EMBRYOS IN DIFFERENT MEDIA

The present study was carried out to see the developmental efficiency of zona-free and with zona parthenogenetic goat embryos cultured in Research Vitro Cleave from Cook Australia (RVCL), Embryo Development Media (EDM), modified synthetic oviductal fluid (mSOF), and modified Charles Rosenkrans media (mCR2a). Zona-free embryos were cultured in 4 media, whereas with zona embryos were cultured in 3 media except mCR2a. Ovaries were collected from slaughterhouse and oocytes were isolated by puncturing the follicles in medium containing Dulbecco’s phosphate-buffered saline, 3% BSA, and 50 μg mL-1 gentamicin. Oocytes were matured in maturation medium containing TCM-199 (HEPES modified), 0.05 mg mL-1 Na pyruvate, 0.003 mg mL-1 L-glutamine, 5.5 mg mL-1 glucose, 3 mg mL-1 BSA, 5 μg mL-1 FSH, 10 μg mL-1 LH, 1 μg mL-1 estradiol-17β, 50 μg mL-1 gentamicin, and 10% FBS in 5% CO2 in air at 38.5°C. The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 27 h. Matured oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona-free by pronase (2 mg mL-1) in zona-free parthenogenesis. Then the oocytes were activated by 5 μM Ca ionophore for 5 min in a CO2 incubator and then treated with 2 mM 6-DMAP for 4 h. Activation was also done by electrical activation with DC 1.78 kV cm-1, 20 μs, and 2 pulses. Then the zona-free oocytes were kept for in vitro culture in 4 types of media such as RVCL, EDM, mSOF, andm CR2a for 7 days in 5% CO2 in air at 38.5°C. The cleavage rate andmorulae formation were observed in RVCL 40.95%, 13.95%, in EDM 46.92%, 14.75%, in mCR2a 56.66%, 5.88%, and in mSOF 48.23%, 14.63%, respectively. The cleavage rate and morulae formation were also found 55.9%, 14.63% during chemical activation and 32%, 12.5% in electrical activation. Hence, better result was found in chemical activation than electrical activation. For with zona parthenogenesis, the matured oocytes were chemically activated by 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. Then the oocytes were cultured in RVCL, EDM, and mSOF in 100-μL micro-drops media for 7 days. The cleavage, morulae, and early blastocyst production rate were as follows: cleavage rate 75.68%, 72.03%, and 57.11%; morulae 44.61%, 30.29%, and 40.22%; and early blastocyst 17.49%, 11.88%, and 25.01% in RVCL, EDM, and mSOF, respectively. Hatched blastocyst formation rate was 6.75%, 5.48%, and 1.15% in RVCL, EDM, and mSOF, respectively. It could be concluded that zona-free parthenogenetic embryos were produced better in EDM medium and with chemical activation. With zona parthenogenetic embryo development was significantly (P &lt; 0.05) higher in RVCL and EDM media.

286 ASSESSMENT OF NUCLEAR AND CYTOPLASMATIC MATURATION IN VITRO OF OOCYTES COLLECTED AT DIFFERENT STAGES OF FOLLICULAR WAVE FROM NELORE COWS (BOS TAURUS INDICUS)

The objective of experiment 1 was to evaluate the influence of follicular wave moment on the quality and number of recovered oocytes through ovum pick-up (OPU), as well as blastocyst rate, in Nelore cows. Two hypotheses were tested: (1) the number of recovered viable COC would be higher at Days 3 and 5 than at Days 7 and 9; (2) the COC aspirated at the beginning of the follicular wave (Day 3 or Day 5) would be more competent and would produce more blastocysts than COC obtained at the end of the wave (Day 7 and Day 9), because these would be in a certain degree of advanced atresia. Cows (n = 10) received 2 mL of estradiol benzoate (Estrogin®), an auricular progestagen implant (Crestar®) for 7 days, and 2 mL of PGF2 analog (Preloban®) at the moment of implant retrieval. After 24 h animals were submitted to aspiration of the dominant follicle (ADF), this moment being considered Day 0. After ADF the donors were submitted to OPU on Days 3, 5, 7, and 9 (groups D3, D5, D7, and D9, respectively) of the new follicular wave. Each animal was submitted to all treatments. In each phase the protocol began with a 2-day interval from the group D9, D7, D5, and D3. Recovered COC went to IVM, IVF, and IVC in the same session of IVP. Statistical analysis were performed with SAS software (SAS Institute, Cary, NC, USA), using ANOVA at a 5% level of significance. There was no difference in the total number of follicles visualized at the moment of OPU among groups (D3:195, D5:189, D7:161, D9:151) or in the recovery rate (77.94; 78.30; 72.04; 73.50) and number of recovered total structures (152, 148, 116, 111). There was no difference in the total number of viable COC (133, 137, 103, 97) or in cleavage rate of COC in the 4 groups (79.69; 83.45; 71.84; 76.28). The total number and the blastocyst rates were higher in groups D3 and D5 (66, 49%, and 62, 45.25%) than in groups D7 and D9 (34, 33%, and 35, 36.08%). Statistical analysis was performed with the chi-square test, with a significance level of 5%. In experiment 2 nuclear and cytoplasmatic maturation were evaluated in COC recovered at Days 4 and 8 regarding nuclear maturation (progression to metaphase II stage) and cortical granule migration, respectively. The hypothesis tested was that COCs recovered at the beginning of the follicular wave would have a higher competence and therefore higher maturation rate compared with those the aspirated at the end. There was no difference in the number of viable COC (78 v. 97), percentages of nuclear maturation (74.35 v. 53.6), and cytoplasmatic maturation (48.71 v. 30.92). The results of the 2 experiments demonstrated that the beginning wave (Days 3 to 5) is the most appropriate moment for obtaining oocytes destined for IVP because of the greater oocyte competence and consequently, larger number and increased blastocyst production rates. Moreover, the results obtained in experiment 2 indicate that the low rate of cytoplasmic maturation can be a major limiting factor of IVP in Nelore cows. FAPESP.

Recovery and Cryopreservation of Epididymal Sperm of Plains Bison (Bison bison bison) as a Model for Salvaging the Genetics of Wood Bison (Bison bison athabascae)

The objective of this study was to optimize recovery and cryopreservation of epididymal sperm from plains bison, as a model for wood bison. In Phase 1, cauda epididymides were recovered from bison (n = 14) immediately after slaughter, minced and incubated in Sp-TALPH buffer for 3 h at 36 degrees C. The resulting sperm suspensions were cryopreserved in Triladyl, using a protocol for bovine semen. In Phase 2, epididymal sperm were cryopreserved in either Triladyl or Andromed. The mean (+/-SD) estimated number of sperm recovered was 468 +/- 207 x 10(6). There was an increase (p < 0.05) in the proportion of sperm with normal morphology between initial recovery and after extension (52.4 +/- 4.6 vs 69.7 +/- 2.4%), with a concurrent decrease (p < 0.05) in the proportion of sperm with distal droplets. Median values for progressively motile sperm in post-thaw samples (60%) were lower (p < 0.05) than that after extension or after chilling (70% for both). The mean percentages of viable sperm and of sperm with an intact acrosome were lower (p < 0.05) for frozen-thawed samples (38.7 +/- 2.8 and 85.2 +/- 1.1) compared with extended (66.2 +/- 2.2 and 92.4 +/- 0.9) or chilled (63.7 +/- 2.5 and 90.0 +/- 1.0) samples. Rates of cleavage, morulae and blastocyst production were not significantly different for chilled (70.9, 38.7 and 8.0%) vs post-thaw sperm (73.0, 46.0 and 6.3%). There was no significant difference between extenders for most sperm characteristics. In conclusion, we developed a functional protocol for the recovery and cryopreservation of epididymal sperm from plains bison, which may have implications for the genetic preservation of wood bison.

Fibroblasts from the new-born male testicle of Guangxi Bama mini-pig (Sus scrofa) can support nuclear transferred embryo development in vitro

Miniature pigs are valuable for research in xenotransplantation and as models for investigating human diseases. Although many mammalian species have been cloned, the success rates have been very low, especially in the pig. In the present study, an attempt was made to optimize somatic cell nuclear transfer (SCNT) protocols for use in the production of the Guangxi Bama mini-pig. Firstly, mini-pig fibroblast cells from a new-born Guangxi Bama piglet were isolated and cultured. Cell type was identified by fluorescence immunocytochemistry (ICC); the cells expressed cimentin, but not cytoceratin and follicular stimulation hormone receptor (FSHR). Secondly, the optimal cell cycle synchronization protocol for treating fibroblast cells from the newborn piglet's testicle was investigated by contact inhibition and serum starvation. When fibroblast cells were treated by contact inhibition, a higher fusion (66.0% vs. 58.3%, p > 0.05) and blastocyst production (20.8% vs. 15.1, p > 0.05) rates were obtained than with serum starvation. Thirdly, to examine the ability of old cells to be morphologically remodelled after activation, testicular fibroblasts (passage 10-14) were introduced into enucleated oocytes; enlarged nuclei were formed in most of the reconstructed embryos at 6 h and enlarged nuclei or distinct pseudopronuclei were formed in nearly all the reconstructed embryos at 12 h. The old donor cell could be morphologically remodelled correctly and was competent to support embryo development to the blastocyst in vitro. Fourthly, the in vitro development potential of the cloned embryos was investigated using two types of donor cell: ear fibroblasts and low or high passage testicular fibroblasts. The rate of fusion was highest using low passage testicle fibroblasts (84.5% vs. 69.8% and 80.0%, p < 0.05), as was development to the blastocyst stage (14.6% vs. 7.7% and 6.3%, p < 0.05). Finally, the effect of phytohaemagglutinin (PHA) on parthenogenetic and cloned embryo development was examined. The PHA had no significant effect on the parthenogenetic embryos, but cloned embryo development to the blastocyst stage was significantly increased by PHA (10 microg/ml), (13.4% vs. 5.6% and 5.6%, p < 0.05).

Effect of repeated gonadotropin stimulation on ovarian reserves and proliferation of ovarian surface epithelium in mice

This study aimed to evaluate the effect of repeated ovarian stimulation (OS) on the ovarian follicular population and morphology in female mice and its influence on the embryo’s developmental ability, and the profile of the ovarian surface epithelium (OSE). A total of 75 mice were enrolled in this experiment and randomly assigned into three groups: repeated ovarian stimulated group [n = 25; receiving 5 IU pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotropin (hCG) at 6 day intervals for 5 cycles]; single ovarian stimulated group (n = 25; receiving 5 IU PMSG and hCG for 1 cycle), and control group (n = 25; without additional treatment). The follicle number at various stages and the morphologies were recorded respectively in the three groups. The harvested oocytes or embryos, cleavage rate, good quality embryo rate, and blastocyst production rate were counted and calculated, and the proliferations of ovarian surface epithelium were evaluated respectively. In the three groups, the single ovarian stimulation treatment significantly increased the mean number of ovarian oocytes or embryos (39.25±10.77 one-cell embryos/female); on the other hand, repeated gonadotropin stimulation obtained the lowest mean number (5.15± 2.81 eggs/female, P<0.01). Repeated ovarian stimulation also tended to decrease normal follicles of primary follicles (66.67%) and secondary follicles (72.86%), and got the lowest cleavage rate (67.47%), lowest good quality embryo rate (2.41%), and lowest blastocyst production rate (0). The OSE cells adjacent to the antral follicles and corpus luteum (CL) in the repeated ovarian stimulated group (81.8%) had a significantly higher proliferation rate than the other groups. The proliferation rate of the OSE in the single ovarian stimulated group (56.4%) was significantly higher than that in the control group (37.5%) (P<0.01). In conclusion, single ovarian stimulation may produce more oocytes/embryos. However, repeated gonadotropin stimulation may have a negative effect on the ovarian follicular quality, the number of mature retrieved oocytes, and the embryo quality, even increasing the chance of ovarian cancer.

Efficiency of SCNT Buffalo (Bubalus bubalis) Embryos in Different Culture Medium and Analysis of mRNA Expression of Insulin-like Growth Factors During Embryogenesis

Growth factors in culture media are known to affect the embryo production rates in in vitro production cultures. To improve the efficiency of somatic cell nuclear transfer (SCNT) derived embryos in Indian buffaloes (Bubalus bubalis), embryos were cultured in three different culture mediums viz. Group-A; TCM-199 + FBS, Group-B; TCM-199 + Poly vinyl alcohol (PVA) and Group-C; CR1aa + BSA. Embryo production rate and expression level of insulin-like growth factor genes (IGF-1, IGF-1R, IGF-2 and IGF-2R) were analysed in embryo culture. Cleavage and blastocyst production rates were 62.5% and 22.3% in Group-A, 53.8% and 13.0% in Group-B and 62.0% and 19.2% in Group-C respectively, whereas in in vitro fertilization (IVF) control cultured in TCM-199 plus 10% FBS, rates were 79.1% and 29.4%. Relative gene expression of SCNT embryos was compared with that in IVF control. IGF-1 and IGF-2 mRNA expression at blastocyst stage was up-regulated (p ≤ 0.05) in all culture groups, while IGF-1R and IGF-2R expression was down regulated (p ≤ 0.05) in Group-B and Group-C. In conclusion, the higher mRNA levels at certain stages in different culture conditions affected in vitro development of SCNT embryos. These results show that the transcript level of the insulin-like growth factor genes was significantly altered by in vitro culture condition. Culture medium TCM-199 with 10% FBS produced higher number of embryos and was able to co-op with gene expression of IVF control. Differences continue to be observed between SCNT cultured and IVF embryos, and until these differences are minimized, aberrations in SCNT embryonic development will continue to arise.

Pregnancies established from handmade cloned blastocysts reconstructed using skin fibroblasts in buffalo ( Bubalus bubalis)

Handmade cloning (HMC), a simple, micromanipulation-free cloning technique, has been applied for the production of cloned embryos and offspring in many livestock species. The objective of the present study was to compare the effect of donor cell type on developmental competence of HMC embryos and to explore the possibility of establishing pregnancies using these embryos in buffalo. After technical optimization of the HMC procedure for in vitro development of cloned blastocysts, various donor cells were compared for their developmental efficiency. Using buffalo fetal-, newborn-, adult fibroblasts and cumulus cells, blastocyst production rates obtained from reconstructed embryos were 24.0 ± 1.8% (35/145), 33.0 ± 8.0% (56/163), 21.0 ± 9.3% (29/133) and 49.6 ± 1.9% (77/154), respectively. Blastocyst rates were higher ( P < 0.05) in cumulus cell reconstructed embryos in comparison to those derived from fetal or adult fibroblasts. Pregnancy diagnosis (transrectal ultrasonography) was carried out at Day 40 of gestation. Following transfer of HMC embryos reconstructed using newborn fibroblasts 25% (2/8) buffaloes were pregnant and are at Days 201 and 94 of gestation, whereas after transfer of HMC embryos reconstructed using fetal fibroblasts, 20% (1/5) buffaloes were pregnant and are at Day 73 of gestation. In conclusion, HMC could be a simple and efficient technique for the production of cloned embryos for establishing pregnancies in buffalo.

209 A COMPARISON OF THE EFFECTS OF DEFINED, SEMI-DEFINED, AND UNDEFINED MATURATION MEDIA ON CLEAVAGE AND SUBSEQUENT EMBRYO DEVELOPMENT OF SHEEP OOCYTES

The objectives were to determine the effects of addition of growth factor(s) and antioxidant to defined (DMM), semi-defined (SDMM), or undefined (UDMM) sheep oocyte maturation media on cleavage and subsequent embryo development. Exogenous epidermal growth factor (EGF, 10 ng mL–1), insulin-like growth factor-I (IGF-I, 50 ng mL–1), or cysteamine (Cys, 100 m) was added to DMM, SDMM, or UDMM. In the experiments, 967 oocytes were collected from 1160 ovaries (Experiment 1), 770 oocytes were collected from 831 ovaries (Experiment 2), and 778 oocytes were collected from 845 ovaries (Experiment 3). After in vitro fertilization with fresh ram semen in SOF medium, groups of 10 oocytes were stripped free of cumulus cells and transferred into 50 μL of a 2-step SOF medium. The incubations were conducted under paraffin oil at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. In all experiments, data were analyzed by ANOVA. Duncan’s multiple range test was used to determine the difference between 2 means after ANOVA. Results were expressed as mean ± SEM, and statistical significance was accepted from P &lt; 0.05. In Experiment 1, the effects of IGF-I and EGF in DMM (TCM-199 supplemented with FSH, LH, 17β-estradiol, Na pyruvate, gentamycin sulfate, and polyvinyl alcohol) were evaluated in 4 treatment groups (DMM as a control, DMM + EGF, DMM + IGF-I, and DMM + EGF + IGF-I). Cleavage, morula, and blastocyst production rates were higher in the DMM + EGF + IGF-I (83.1 ± 0.5, 47.7 ± 0.5, and 30.8 ± 1.2) than in the DMM (72.9 ± 1.3, 37.0 ± 1.1, and 20.2 ± 1.2), DMM + EGF (77.2 ± 1.0, 43.2 ± 2.1, and 26.1 ± 1.8), or DMM + IGF-I (79.9 ± 0.6, 43.3 ± 1.2, and 25.7 ± 1.3) groups (P &lt; 0.05). In Experiment 2, the effects of Cys in DMM were evaluated. The addition of Cys to DMM + EGF + IGF-I resulted in a mean blastocyst rate of 35.0 ± 0.9% compared with 31.5 ± 1.3% in DMM + EGF + IGF-I alone (P &lt; 0.05); however, mean cleavage rates (84.5 ± 1.3 v. 82.8 ± 1.0) did not differ (P = 0.32). In Experiment 3, the effects of DMM + EGF + IGF-I + Cys, SDMM (TCM-199 supplemented with FSH, LH, 17β-estradiol, Na pyruvate, gentamycin sulfate, and BSA) + EGF + IGF-I + Cys, and UDMM (TCM-199 supplemented with FSH, LH, 17β-estradiol Na pyruvate, gentamycin sulfate, and FBS) + EGF + IGF-I + Cys on cleavage and embryo developmental were compared. The UDMM supplemented with EGF, IGF-I, and Cys resulted in higher proportions of cleavage, morula yields, and blastocyst yields (P &lt; 0.05) than the DMM or SDMM supplemented with EGF + IGF-I + Cys. There was no significant difference between DMM and SDMM in the proportions of oocytes reaching the morula and blastocyst stages. In conclusion, an efficient system for in vitro production of sheep blastocysts was developed by using a defined oocyte maturation system combined with growth factors, hormones, and an antioxidant, but the UDMM resulted in higher cleavage, morula yields, and blastocyst yields than the DMM or SDMM.

30 IN VITRO EXPRESSION OF BAX AND BCL2 GENES IN NUCLEAR DONOR SKIN FIBROBLAST, CUMULUS, AND GRANULOSA CULTURED CELLS DURING CULTURE AND THEIR SOMATIC CELL NUCLEAR TRANSFER-CLONED EMBRYOS IN INDIAN BUFFALOES (BUBALUS BUBALIS)

Somatic cell nuclear transfer (SCNT) involves functional changes in the genome which result in low efficiency for the production of viable and cloned embryos. It is primarily due to incomplete reprogramming of genome of donor cell nuclei in the reconstructed embryos (Vassena et al. 2007 Dev. Biol. 304, 75–89). Expression of BCL2 and Bax can be correlated with apoptosis. BCL2 inhibits apoptosis by regulating the release of cytochrome-c and other proteins from mitochondria (Keep et al. 2007 EMBO J. 26, 825–834). Antiapoptotic BCL2 is antiproliferative by facilitating G0. Bax is proapoptotic and accelerates S-phase progression. The dual functions in apoptosis and cell cycle are coordinately regulated by the BCL2 family and suggest that survival is maintained at the expense of proliferation (Zinkel et al. 2006 Cell Death Differ. 13, 1351–1359). The aim of this study was to estimate the relative expression of BCL2 oncogene and Bax gene in regulating apoptosis, in skin fibroblast, cumulus, and granulosa cells in culture, so that ideal-type donor cell lines are developed for higher success rates in SCNT-derived buffalo cloning. The cell lines up to 25th passage were from all the 3 tissue types by previous method (Gupta et al. 2007 Cell Biol. Int. 31, 1257–1264). The cells between passages 5th to 15th were selected as competent donor cells and transferred into enucleated in vitro-matured oocytes from slaughter ovaries. The couplets were activated electrically (1.5 kV cm–2, 15 μs) and chemically (ionomycin, 6-DMAP, CHX, and Cyto-B) and were cultured up to blastocyst. The cDNA were prepared from the growing cells in culture at 5, 10, and 15 passages from all cell lines and SCNT-cloned blastocysts from these cell lines at respective passages for Bax and BCL2 gene expression analysis. Relative expression of these candidate genes was quantified using real-time PCR. The data was analyzed for 1-way ANOVA and post-hoc Duncan multiple range test at P ≤ 0.05 level of significance. The cell proliferation rate in cultured cells at fifth passage was higher in all the 3 cell lines and declined in subsequent passages (range from 1.06 to 0.67). The relative abundance of Bax mRNA in granulosa cell was comparable with skin fibroblasts but significanly higher than cumulus cells at respective passages. BCL2 mRNA expression was significantly upregulated in cumulus cells as compared to granulosa cells but not with skin fibroblasts. The SCNT blastocyst production rates from granulosa were highest (24.28%) as compared to fibroblast (22.6%) and cumulus (21.4%) at passage 10. Level of Bax and BCL2 mRNA in granulosa and fibroblast SCNT blastocysts was not significantly different from IVF (control), whereas cumulus-derived blastocyst showed abnormal patterns with downregulated expression of Bax mRNA and upregulated expression of BCl2 mRNA. Identification of expressed genes in cells and cloned embryos will help to investigate the causes of developmental abnormality due to deregulation of expression of important gene associated with ART.

Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST ( n = 5; receiving 500 mg of bST in regular intervals) and control group ( n = 5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (>3 mm in diameter; 12.2 compared with 8.7; p < 0.05) and of small antral follicles (<5 mm; 9.1 compared with 6.5; p < 0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p = 0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p = 0.07). bST showed no effect on cleavage and blastocyst production rates ( p > 0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population ( p < 0.001) as well as the number of follicles aspirated ( p < 0.001), and oocytes recovered ( p < 0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs.

Effect of breeding season on in vivo oocyte recovery and embryo production in non-descriptive Indian river buffaloes ( Bubalus bubalis)

The present study was carried out to examine the effect of season on in vivo oocyte recovery and embryo production in non-descriptive, Indian river buffaloes ( Bubalus bubalis). Ovum pick up (OPU) was conducted twice a week for 8 weeks during peak (October–March) and low (April–September) breeding season in live buffaloes ( n = 6). OPU was performed using ultrasound equipment with a 5 MHz transvaginal transducer, a single lumen 18-gauge, 55-cm long needle and a constant vacuum pressure of 110 mmHg. The number and size of follicles was determined before puncture. The recovered oocytes were graded and only grade A and grade B oocytes were used for in vitro production (IVP) of embryos. The mean number of follicles observed per animal per session did not differed ( P < 0.05) between animals or between puncture sessions in both low and peak breeding seasons. Higher ( P < 0.05) number of follicles were observed (4.8 ± 0.2 versus 3.1 ± 0.3) and punctured (4.0 ± 0.2 versus 2.4 ± 0.2) during peak breeding season when compared to low breeding season. Oocytes recovered (1.6 ± 0.1 versus 1.0 ± 0.3) per animal per session were higher ( P < 0.05) in peak breeding season than low breeding season. During the peak breeding season, the blastocyst yield per animal per session (0.3 ± 0.4 versus 0.18 ± 0.4) was higher ( P < 0.05) than the low breeding season. However, season did not significantly affect the percentage of oocytes suitable for IVP (grade A + B) and blastocyst production rate. In conclusion, the efficiency of OPU combined with IVP was higher during the peak breeding season than the low breeding season in buffaloes.

Effects of insulin-like growth factor-I, epidermal growth factor and cysteamine on the in vitro maturation and development of oocytes collected from 6- to 8-week-old Merino lambs

To improve the viability of embryos produced in vitro from lamb oocytes, maturation medium was supplemented with insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), cysteamine, and combinations thereof. Experiment 1 examined the effects of IGF-I supplementation and duration of oocyte maturation on nuclear maturation and embryo development while Experiments 2 and 3 examined the effects of cysteamine and EGF supplementation respectively on embryo development. In Experiment 4, embryo development was examined after maturation with various combinations of supplements. IGF-I supplementation increased cleavage rate (P < 0.05) but its effect on the rate of blastocyst production from original oocytes was variable. Supplementation with IGF-I increased (P < 0.01) the proportion of oocytes at Metaphase II (MII) after 18 h of maturation but not at later times. EGF either alone or combined with IGF-I significantly (P < 0.05) increased cleavage rates compared with other treatment groups but EGF consistently failed to improve blastocyst production rates. Cysteamine improved hatching rates but only when supplemented alone. Maturation of lamb oocytes for 22 h in medium supplemented with 100 ng mL(-1) IGF-I and 100 microm cysteamine resulted in the production of 16.0 lambs per donor lamb after embryos were transferred to recipient ewes. It is concluded that EGF and, to a lesser extent, IGF-I, whilst beneficial to initial cleavage, can adversely influence subsequent embryo development. Improvements in embryo viability may more likely be obtained by addressing issues that influence fetal oocyte quality than by modifying in vitro methodology.

The Blastocyst Production Rate and Incidence of Chromosomal Abnormalities by Developmental Stage in In Vitro Produced Porcine Embryos

The present study was conducted to determine the relationship between embryonic development speed at different stages (the cleaved stage at 52 h and the blastocyst stage at 6 days post insemination) and incidences of chromosome abnormalities in in vitro produced porcine embryos. Porcine oocytes were collected from 3-6-mm ovarian follicles obtained at a slaughterhouse and matured in modified NCSU-37 medium for 44-46 h. Following in vitro fertilization with a final concentration of 1 x 10(5) sperm/ml for 3 h, all oocytes were cultured in vitro for 52 h. Day-2 (52 h after insemination) embryos were classified according to their cleaved stages into 2-cell, 3- to 4-cell, 5- to 8-cell, and >8-cell stages; these were cultured separately for additional 4 days (Day 6). The resultant Day-6 blastocysts were classified according to the morphological diameter into 3 grades: Grade A, expanded blastocysts; Grade B, expanding blastocysts; and Grade C, early blastocysts. They were then analyzed chromosomally. The 3- to 4-cell and 5- to 8-cell embryos had significantly high blastocyst development rates (46.1 and 36.9%, respectively), and these blastocysts contained significantly more cells (40.2 and 42.4 cells, respectively) than those derived from 2-cell embryos and >8-cell embryos (28.6 and 26.5 cells, respectively). The incidence of chromosomal abnormalities was significantly higher in the blastocysts derived from 2-cell and >8-cell stage embryos than in the blastocysts derived from the other stage embryos. Furthermore, the grade A blastocysts had the lowest incidence of chromosomal abnormalities (35.3%) and contained the most cells (48.7 cells). Porcine in vitro production (IVP) yielded a high blastocyst rate and an excellent embryo quality when 3- to 4-cell and 5- to 8-cell stage embryos were selected on Day 2 after insemination. The same criteria yielded a higher quality of expanded blastocysts based on the stage of embryo development and morphology.