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Merighe G.K.F.,University of Sao Paulo | dos Santos Miranda M.,University of Sao Paulo | Camara de Bem T.H.,University of Sao Paulo | Watanabe Y.F.,Vitrogen Pesquisa e Desenvolvimento em Biotecnologia da Reproducao S C Ltda. | Meirelles F.V.,University of Sao Paulo
Revista Brasileira de Zootecnia

The objective of this study was to evaluate the effects of culture time and sex of nuclei donor cells on embryo and fetal development after nuclear transfer. Thus, bovine oocytes were matured, enucleated and reconstructed with somatic cells from an adult animal. After fusion and chemical activation, the reconstituted zygotes were cultured in Charles Rosenkranz 2 (CR2) on a granular monolayer cell at 38.8°C in a humidified atmosphere 5% CO2 in air for seven days, and transferred to synchronized receptors. Cleavage rates and development to blastocyst of embryo reconstructed with cells cultured for a longer time were lower than rates obtained with other culture times. Moreover, these produced blastocysts did not result in the development of full term pregnancy. Although cleavage rates were higher in female embryos, the number of embryos that reached blastocyst stage was higher in male embryos. During gestation period, females showed higher abortion rates from 90 to 120 days of gestation. These results indicate that cells donnors of nuclei cultured for long periods make the production of blastocysts difficult and increase the chances of losses during pregnancy. Cloned male embryos are more succesful in becoming blastocysts and result in lower gestational loss rate. © 2010 Sociedade Brasileira de Zootecnia. Source

Santos-Biase W.K.F.,University of Sao Paulo | Biase F.H.,University of Sao Paulo | Biase F.H.,University of Illinois at Urbana - Champaign | Buratini J.,Sao Paulo State University | And 7 more authors.
Animal Reproduction Science

The number of follicles recruited in each estrous cycle has gained practical importance in artificial reproductive technology, as it determines the oocyte yield from ultrasound-guided ovum pickup for in vitro embryo production. We aimed to identify single nucleotide polymorphisms (SNPs) in bovine genes related to reproductive physiology and evaluate the association between the candidate SNPs and the number of oocytes collected from ultrasound-guided ovum pickup. We sequenced genomic segments of GDF9, FGF8, FGF10 and BMPR2 and identified seventeen SNPs in the Bos taurus and Bos indicus breeds. Two SNPs cause amino acid changes in the proteins GDF9 and FGF8. Three SNPs in GDF9, FGF8 and BMPR2 were genotyped in 217 Nelore cows (B. indicus), while two previously identified mutations in LHCGR and mitochondrial DNA (mtDNA) were genotyped in the same group. The polymorphisms in GDF9, FGF8, BMRP2 and LHCGR were significantly associated (P<0.01) with the number of oocytes collected by ovum pickup, whereas the SNP in the mtDNA was not. In addition, we estimated an allelic substitution effect of 1.13 ± 0.01 (P<0.01) oocytes for the SNP in the FGF8 gene. The results we report herein provide further evidence to support the hypothesis that genetic variability is an important component of the number of antral follicles in the bovine ovary. © 2012 Elsevier B.V. Source

Ruggeri R.R.,Federal University of Rio Grande do Sul | Watanabe Y.,Vitrogen Pesquisa e Desenvolvimento em Biotecnologia da Reproducao S C Ltda. | Meirelles F.,University of Sao Paulo | Bressan F.F.,University of Sao Paulo | And 2 more authors.
Journal of Assisted Reproduction and Genetics

Purposes: Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. Methods: Cumulus-oocyte- complexes were in vitro matured and activated using Ca2+ Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in Stem-Pro® medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. Results: Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39%and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. Conclusion: Our data show that Ca 2+ Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions. © Springer Science+Business Media New York 2012. Source

Ferraz M.L.,Vida Reprodutiva Consultoria | Araujo A.B.,Vida Reprodutiva Consultoria | Rodrigues C.A.,Clinica Veterinaria SAMVET de Sao Carlos Ltda | Watanabe Y.F.,Vitrogen Pesquisa e Desenvolvimento em Biotecnologia da Reproducao S C Ltda. | And 3 more authors.
Journal of Dairy Science

It was hypothesized the lower fertility of repeat-breeder (RB) Holstein cows is associated with oocyte quality and this negative effect is enhanced during summer heat stress (HS). During the summer and the winter, heifers (H; n. =36 and 34, respectively), peak-lactation (PL; n. =37 and 32, respectively), and RB (n. =36 and 31, respectively) Holstein cows were subjected to ovum retrieval to assess oocyte recovery, in vitro embryonic developmental rates, and blastocyst quality [terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and total cell number]. The environmental temperature and humidity, respiration rate, and cutaneous and rectal temperatures were recorded in both seasons. The summer HS increased the respiration rate and the rectal temperature of PL and RB cows, and increased the cutaneous temperature and lowered the in vitro embryo production of Holstein cows and heifers. Although cleavage rate was similar among groups [H. =51.7% ± 4.5 (n. =375), PL. =37.9% ± 5.1 (n. =390), RB. =41.9% ± 4.5 (n. =666)], blastocyst rate was compromised by HS, especially in RB cows [H. =30.3% ± 4.8 (n. =244) vs. 23.3% ± 6.4 (n. =150), PL. =22.0% ± 4.7 (n. =191) vs. 14.6% ± 7.6 (n. =103), RB. =22.5% ± 5.4 (n. =413) vs. 7.9% ± 4.3 (n. =177)]. Moreover, the fragmentation rate of RB blastocysts was enhanced during the summer, compared with winter [4.9% ± 0.7 (n. =14) vs. 2.2% ± 0.2 (n. =78)] and other groups [H. =2.5% ± 0.7 (n. =13), and PL. =2.7% ± 0.6 (n. =14)] suggesting that the association of RB fertility problems and summer HS may potentially impair oocyte quality. Our findings provide evidence of a greater sensitivity of RB oocytes to summer HS. © 2011 American Dairy Science Association. Source

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