Fernando Henrique Biase

Global poly(A) mRNA expression profile measured in individual bovine oocytes and cleavage embryos.

The objective of this article was to estimate quantitative differences for GAPDH transcripts and poly(A) mRNA: (i) between oocytes collected from cumulus-oocyte complexes (COCs) qualified morphologically as grades A and B; (ii) between grade A oocytes before and after in vitro maturation (IVM); and (iii) among in vitro-produced embryos at different developmental stages. To achieve this objective a new approach was developed to estimate differences between poly(A) mRNA when using small samples. The approach consisted of full-length cDNA amplification (acDNA) monitored by real-time PCR, in which the cDNA from half of an oocyte or embryo was used as a template. The GAPDH gene was amplified as a reverse transcription control and samples that were not positive for GAPDH transcripts were discarded. The fold differences between two samples were estimated using delta Ct and statistical analysis and were obtained using the pairwise fixed reallocation randomization test. It was found that the oocytes recovered from grade B COCs had quantitatively less poly(A) mRNA (p<0.01) transcripts compared with grade A COCs (1 arbitrary unit expression rate). In the comparison with immature oocytes (1 arbitrary unit expression rate), the quantity of poly(A) mRNA did not change during IVM, but declined following IVF and varied with embryo culture (p<0.05). Amplification of cDNA by real-time PCR was an efficient method to estimate differences in the amount of poly(A) mRNA between oocytes and embryos. The results obtained from individual oocytes suggested an association between poly(A) mRNA abundance and different morphological qualities of oocytes from COCs. In addition, a poly(A) mRNA profile was characterized from oocytes undergoing IVM, fertilization and blastocyst heating.

Association of the estrogen receptor gene Pvu II restriction polymorphism with expected progeny differences for reproductive and performance traits in swine herds in Brazil

Estrogen has an important function in swine reproduction and growth. A Pvu II restriction enzyme polymorphism has been proven to be an important genetic variation in the estrogen receptor gene (ESR) and may be considered as a candidate gene for use in pig production but there is no data regarding the prevalence of this polymorphism in the Brazilian pig population. We used DNA samples from the following three purebred pig breeds: Large White (336 females and 26 males), Landrace (304 females and 27 males) and Pietrain (125 females and 11 males). The ESR genotyping was performed using PCR-RFLP. For each breed, genotypes for the ESR gene were compared independently for expected progeny differences (EPD) in litter size (LS), average daily weight gain (DWG) (g/day) and back fat thickness (BT) as measured in mm by ultrasound. In the Large White breed, but not the other breeds, the ESR genotype was significantly (p < 0.05) associated to LS, DWG and BT. Large Whites genotyped as AA or AB had higher EPD values for the LS and BT traits compared to BB Large Whites, while AA Large Whites had higher DWG EPD values than BB Large Whites. Our results for the Large White population showed that the A allele has a beneficial effect on LS, DWG and BT expected progeny differences.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

OBJECTIVE To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. DESIGN Comparison between oocyte groups and between early embryo stages. SETTING Laboratories of embryo manipulation and molecular biology from Departamento de Genética (FMRP) and Departamento de Ciências Básicas (FZEA)--University of São Paulo. SAMPLE(S) Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. MAIN OUTCOME MEASURE(S) Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). RESULT(S) Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good- and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. CONCLUSION(S) Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome.

SummaryOocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

Mitochondrial DNA single nucleotide polymorphism associated with weight estimated breeding values in Nelore cattle (Bos indicus)

We sampled 119 Nelore cattle (Bos indicus), 69 harboring B. indicus mtDNA plus 50 carrying Bos taurus mtDNA, to estimate the frequencies of putative mtDNA single nucleotide polymorphisms (SNPs) and investigate their association with Nelore weight and scrotal circumference estimated breeding values (EBVs). The PCR restriction fragment length polymorphism (PCR-RFLP) method was used to detect polymorphisms in the mitochondrial asparagine, cysteine, glycine, leucine and proline transporter RNA (tRNA) genes (tRNAasn, tRNAcys, tRNAgly, tRNAleu and tRNApro). The 50 cattle carrying B. taurus mtDNA were monomorphic for all the tRNA gene SNPs analyzed, suggesting that they are specific to mtDNA from B. indicus cattle. No tRNAcys or tRNAgly polymorphisms were detected in any of the cattle but we did detect polymorphic SNPs in the tRNAasn, tRNAleu and tRNApro genes in the cattle harboring B. indicus mtDNA, with the same allele observed in the B. taurus sequence being present in the following percentage of cattle harboring B. indicus mtDNA: 72.46% for tRNAasn, 95.23% for tRNAleu and 90.62% for tRNApro. Analyses of variance using the tRNAasn SNP as the independent variable and EBVs as the dependent variable showed that the G ® T SNP was significantly associated (p < 0.05) with maternal EBVs for weight at 120 and 210 days (p < 0.05) and animals EBVs for weight at 210, 365 and 455 days. There was no association of the tRNAasn SNP with the scrotal circumference EBVs. These results confirm that mtDNA can affect weight and that mtDNA polymorphisms can be a source of genetic variation for quantitative traits.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.