This study aimed to evaluate the epigenetic reprogramming of ICR1 (KvDMR1) and ICR2 (H19DMR) and expression of genes controlled by them as well as those involved in methylation, demethylation, and pluripotency. We collected germinal vesicle (GV) and metaphase II (MII) oocytes, and preimplantation embryos at five stages [zygote, 4–8 cells, 8–16 cells, morula, and expanded blastocysts (ExB)]. DNA methylation was assessed by BiSeq, and the gene expression was evaluated using qPCR. H19DMR showed an increased DNA methylation from GV to MII oocytes (68.04

Embryos that are produced in vitro frequently present epigenetic modifications. However, maternal supplementation with folic acid (FA) may improve oocyte maturation and embryo development, preventing epigenetic errors in the offspring. We sought to evaluate the influence of FA supplementation during in vitro maturation of grade I (GI) and grade III (GIII) bovine oocytes on embryo production rate and the expression of IGF2 and KCNQ1OT1 genes. The oocytes were matured in vitro with different concentrations of FA (0, 10, 30 and 100 μM), followed by in vitro fertilization and embryo culture. On the seventh day (D7) of culture, embryo production was evaluated and gene expression was measured using real-time qPCR. Supplementation with 10 μM of FA did not affect embryo production for GI and GIII oocytes. Moderate supplementation (30 μM) seemed to be a positive influence, increasing embryo production for GIII (P = 0.012), while the highest dose (100 μM) reduced embryo production (P = 0.010) for GI, and IGF2 expression was not detected. In GIII, only embryos whose oocyte maturation was not supplemented with FA demonstrated detected IGF2 expression. The lowest concentration of FA (10 μM) reduced KCNQ1OT1 expression (P = 0.05) on embryos from GIII oocytes. Different FA concentrations induced different effects on bovine embryo production and gene expression that was related to oocyte quality. Despite the epigenetic effects of FA, supplementation seems to be a promising factor to improve bovine embryo production if used carefully, as concentration is an important factor, especially in oocytes with impaired quality.

Soro fetal bovino (SFB) e albumina sérica bovina (BSA) são componentes importantes do cultivo in vitro (CIV) de embriões bovinos, porém são frequentemente associados ao acúmulo excessivo de lipídios, podendo prejudicar o desenvolvimento embrionário.Este estudo teve como objetivo substituir parcialmente o SFB por BSA V FAF durante o CIV de embriões bovinos, avaliar a produção embrionária e quantificar os lipídios dos embriões, SFB e dos meios de cultivo.Para isto, os embriões desenvolveram

Fetal bovine serum (FBS) and bovine serum albumin (BSA) are important components during bovine embryo in vitro culture (IVC), but they are associated with excess of embryonic lipid, which might impair embryo development. This study aimed to partially replace FBS by BSA V FAF during bovine IVC, evaluate embryo production and quantify the phospholipid content in produced embryos, SFB and IVC medium. The embryos were in vitro cultured in medium supplied with 10% of FBS (FBS10%) or with 5% of FBS plus 0.03 g BSA V FAF (FBS5%/BSA). The lipid content was evaluated using UHPLC-MS/MS and statistical analysis was performed using t-test and ANOVA. The partial replacement of FBS by BSA V FAF did not alter embryo production. Ten phospholipids were identified in both groups and three of them, DOPC (p=0.037), POPG (p=0.046) and C24: 1-SM (p=0.009) presented lower concentration in FBS5%/BSA culture medium. The phospholipids identified on embryos matches with those found on SFB and culture medium and four of them DOPC (p=0.013), DPPC (p=0.004), POPG (p=0.05) and C24:1SM (p=0.003) reduced its concentration when FBS was reduced. Theses founds shown that the FBS partial replacement reduces phospholipids content in embryos but do not decrease embryo production, suggesting a technical improvement.

We assessed whether single nucleotide polymorphisms (SNPs) in the genes beta 1,4galactosyltransferase (B4GALT1), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR) and insulin-like growth factor 2 (IGF2) could be molecular markers for scrotal circumference (SC) in Nellore bulls. Animals with positive (+, n = 104) and negative (-, n = 74) expected progeny difference for scrotal circumference at 365 days (EPD SC 365) were selected and their SNPs were analyzed by restriction fragment length polymorphism (RFLP). The correlation between EPD SC 365 and expected progeny difference for age at first birth (EPD AFB) was also investigated. The SNPs in B4GALT1 and FSHR was not different between two groups analyzed. The CC genotype for LHR gene was most frequent in animals with EPD SC 365(+), whereas the TT was most frequent in the EPD SC 365(-). For IGF2 the CT and CC were the most frequent genotypes observed in animals with positive and negative EPD SC 365, respectively. The EPD SC 365 was negatively correlated with the EPD AFB (r = 0.23). We suggest that CC and TT genotypes for LHR and IGF2, respectively, could be possible molecular markers for SC selection in Nellore bulls, that can also predict for AFB.

Um dos mecanismos epigeneticos relacionados a especializacao celular e a metilacao do DNA, preferencialmente nas regioes promotoras dos genes. Uma assinatura epigenetica e caracteristica de cada linhagem celular e pode mudar ao longo da vida de um organismo. Desta maneira, este trabalho teve como objetivo avaliar o nivel de metilacao do gene de crescimento PTPRS em diferentes tecidos ao longo da vida de galinhas ( Gallus gallus domesticus ). Para isto, amostras de DNA do coracao, musculo esqueletico, encefalo e sangue de cinco filhotes e cinco adultos foram utilizadas para quantificar a metilacao pelo Metodo de Digestao Enzimatica Sensivel a Metilacao associado a qPCR utilizando a enzima HpaII . Foi possivel identificar que a porcentagem de metilacao do gene PTPRS foi maior em adultos (sangue: 0,62±0,05; coracao: 0,42±0,09; musculo: 0,19±0,04; encefalo: 0,25±0,01), quando comparado com os filhotes (sangue: 0,52±0,02; coracao: 0,23±0,06; musculo: 0,18±0,03; encefalo: 0,12±0,01), em todos os tecidos analisados. Observou-se tambem que os tecidos encefalo e musculo, onde o gene e determinante no desenvolvimento, apresentaram os menores niveis de metilacao do gene PTPRS . Desta maneira, o presente estudo reforca a existencia de niveis de metilacao especificos de cada linhagem celular para o gene PTPRS , e que ao longo da vida da ave estes niveis podem ser alterados, reforcando a necessidade de estudos exploratorios previos a analises epigeneticas.

The bovine IVP faces many obstacles that interfere negatively in the production and survival of embryo, like oocytes with morphological differences (grade I, II and III) and composition of IVM, IVF and IVC medium. The components present in the culture medium, as well as those stored by the oocyte during maturation process, are associated to genetics and epigenetics alterations, influencing the embryo quality and the success of bovine embryo IVP. The FCS seems to influence the embryo production and has been related with the abnormal accumulation of lipids that affect the process of cryopreservation. (Rumpf. Rev. Bra. de Zoo. 36, 229-233. 2007). The folic acid (FA), in turn, seems to help the IVP of embryos, correcting possible epigenetics alterations related to assisted reproduction biotechnologies (Kim et al. Journal of Nutritional Biochemistry. 20, 917-926. 2009). The aim of this work was to evaluate the effect of partial replacement of FCS for BSA in the embryonic lipid composition and, posteriorly, evaluate the influence of FA in the IVM of grades I and III oocytes, aiming to increase the production of embryos derived from grade III oocytes. The study was performed in two experiments, both in triplicates. In the first experiment COCs (n = 297) were matured in IVM medium (TCM199 with Earles’ salt, glutamin, FCS, FSH, LH, amikacin and estradiol). Embryos were cultured in two different mediums: CR2 control (10% FCS, 0.03 g BSA V, alanine, glycine and amikacin in 10 mL of medium) and CR2 modified (5% of FCS, 0.03 g of BSA V FAF, 0.03 g BSA V, alanine, glycine and amikacin in 10mL of medium). Embryo production was evaluated using Student’s t test (P < 0.05) and the lipid composition of embryos, classified as expanded blastocysts on day 7 (D7), was determined by Ultra High Performance Liquid Chromatography Mass Spectrometry (UHPLC-MS). The reduction of FCS did not influence (P = 0.35) embryonic production on D7 on control group (51.5% ± 6.1) and modified (44.9% ± 6.3). The lipid analysis revealed that the embryonic culture in modified CR2 medium reduced the concentration of three phospholipids classes, the dioleoil phosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] (POPG) and sphingomyelin (SM2) (P = 0.04, P = 0.05 and P = 0.01, respectively). This result corroborates with the hypothesis that the embryo can absorb lipids from FCS. On the second experiment, 360 oocytes (180 grade I and 180 grade III) were matured on IVM medium and 350 oocytes (180 grade I and 170 grade III) were matured on IVM medium (supplemented with 10 mM FA in final volume of 10 mL). The embryonic culture was performed in modified CR2 medium. The embryonic production was evaluated using Student’s t test (P < 0.05). The addition of FA on IVM medium increased (P = 0.02) the embryo production derived from grade III oocytes, with a total of 56 embryos (32.6% ± 1.6) without FA and 64 embryos (42.3% ± 3.0) with FA. There was no influence (P = 0.29) of FA supplementation for grade I oocytes, presenting, on D7, 88 embryos (52.7% ± 6.9) without FA and 84 embryos (45.4% ± 4.6) with FA. However, more studies should be performed to determine the effects of FA supplementation during IVM on gene expression in oocytes with different morphological qualities.

The chicken (Gallus gallus) embryo has been used as a classic model system for developmental studies because of its easy accessibility for surgical manipulation during embryonic development. Sex determination in birds is chromosomally based (ZZ for males and ZW for females); however, the basic mechanism of sex determination is still unknown. Here, the dynamics of expression of candidate genes implicated in vertebrate sex determination and differentiation were studied during embryonic chicken gonadal development. Gene expression profiles were obtained before, during, and after gonadal sex differentiation in females and males for DMRT1, SOX3, SOX9, DAX1, SCII, HINTZ, HINTW, and the male hypermethylated (MHM) region. Transcripts for the HINTZ, DMRT1, DAX1, SCII, and SOX9 genes were observed in both sexes, but expression was higher in male gonads and may be correlated with testicular differentiation. The expression patterns of HINTW, SOX3, and MHM suggest that they may act in ovary development and may be involved in meiosis entry. MHM was upregulated and DMRT1 was downregulated in females at the same developmental stage. This may indicate a regulation of DMRT1 by MHM ncRNA. Similar dynamics were observed between HINTW and HINTZ. This study reports on the MHM expression profile during gonadal development and its correlation with the expression of genes involved in vertebrate sex determination.