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Macabelli C.H.,Federal University of Sao Carlos | Macabelli C.H.,University of Sao Paulo | Ferreira R.M.,University of Sao Paulo | Gimenes L.U.,Sao Paulo State University | And 12 more authors.
PLoS ONE | Year: 2014

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis. © 2014 Macabelli et al. Source

Baruselli P.S.,University of Sao Paulo | Gimenes L.U.,University of Sao Paulo | Carvalho N.A.T.,APTA UPD Registro | Sa Filho M.F.,University of Sao Paulo | Ferraz M.L.,Vida Reprodutiva Consultoria
Revista Veterinaria | Year: 2010

Our expanding knowledge of ovarian function during the buffalo estrous cycle has given us new approaches for the precise synchronization and control of ovulation. Recent protocols, designed to control both luteal and follicular function, permit fixed-time AI with high pregnancy rates. Furthermore, allow the initiation of superstimulatory treatments at a self-appointed time, and finally, provide opportunities to do fixed-time AI in donors and fixed-time embryo transfer in recipients. The intention of the following review is to discuss how these events impact the application of assisted reproductive technologies in buffalo. Source

Gimenes L.U.,Sao Paulo State University | Ferraz M.L.,Vida Reprodutiva Consultoria | Fantinato-Neto P.,University of Sao Paulo | Chiaratti M.R.,Federal University of Santa Catarina | And 7 more authors.
Theriogenology | Year: 2015

The aim of the present study was to determine the optimal phase of the follicular wave to perform ovum pickup (OPU) for invitro embryo production (IVEP) in various genetic groups. For this purpose, 27 heifers-nine Bos taurus (Holstein), nine Bos indicus (Nelore), and nine Bubalus bubalis (Mediterranean)-were maintained under the same nutritional, management, and environmental conditions. Heifers within each genetic group were submitted to six consecutive OPU trials with 14-day intersession intervals, at three different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5 after follicular wave emergence), in a 3×3 crossover design. When OPU was performed at different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5), no differences were found in the percent of oocytes recovered (70.5±3.1%, 75.0±3.1%, 76.0±3.2%, respectively; P=0.41) or blastocyst production rates (19.4±2.9%, 16.6±2.9%, 15.9±2.6%, respectively; P=0.36). Comparing genetic groups, B indicus showed a higher blastocyst rate (28.3a±2.8%; P<0.01) than B taurus and B bubalis (14.1b±2.9% and 10.2b±2.0%, respectively). However, only B indicus heifers showed a variation in the number of visualized follicles and the total and viable oocytes along consecutive OPU sessions. In conclusion, different phases of the pharmacologically synchronized ovarian follicular wave did not affect OPU-IVEP in B indicus, B taurus, and B bubalis heifers. Additionally, B indicus heifers showed greater OPU-IVEP efficiency than did the other genetic groups, under the same management conditions. © 2015 Elsevier Inc. Source

Chiaratti M.R.,University of Sao Paulo | Ferreira C.R.,University of Sao Paulo | Ferreira C.R.,Sao Paulo State University | Meirelles F.V.,University of Sao Paulo | And 10 more authors.
Cellular Reprogramming | Year: 2010

Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p=0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication. Copyright 2010, Mary Ann Liebert, Inc. 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 | Year: 2011

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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