Seminal cell-free DNA as a potential marker for in vitro fertility of Nellore bulls.

PURPOSE: This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing. METHODS: Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12 h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated. RESULTS: Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12 h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups. CONCLUSION: The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

Cumulus-oocyte complexes from sows show differences in lipid metabolism compared to cumulus-oocyte complexes from prepubertal gilts during in vitro maturation.

This study aimed to evaluate the effects of donor age on lipid metabolism during in vitro maturation (IVM) of pigs cumulus-oocyte complexes (COCs). We evaluated transcript levels of genes, the percentage of ooplasm occupied by lipid droplets (LD) and evaluated DNA methylation in COCs from sows and prepubertal gilts. Transcript levels of six genes (ACACA, ACSS2, FASN, FABP3, SLC27A4, PLIN2), which were analyzed in cumulus cells (CCs), increased after 44 h of IVM in the sow group. In the gilt group, only FASN expression increased, while NR3C1 expression decreased after IVM. The measurement of LD in oocytes showed an accumulation of lipids in sow oocytes during IVM, while gilt oocytes showed a decrease in LD. FABP3 and NR3C1 methylation patterns exhibited a demethylation pattern in CCs and oocytes from gilts and sows and showed statistical differences between groups. CCs from sows had a better capacity to change transcription levels of the major genes involved in lipid metabolism during IVM than CCs from gilts. This difference may be involved in accumulation of lipids, acquisition of competence, and maturation of enclosed oocytes. Our results contribute to a better understanding of mechanisms involved in lipid metabolism and acquisition of competence in porcine COCs.

Characterization of the regulatory 5'-flanking region of bovine mucin 2 (MUC2) gene.

Throughout the intestinal epithelium surface there is an intricate polymer network composed by gel-forming mucins, which plays a protective role due to the formation of a physical, chemical and immunological barrier between the organism and the environment. Mucin 2 (MUC2) is the main mucin in the small and large intestine, and it is expressed specifically in the gastrointestinal tract (GIT), which makes its promoter region an important candidate for expression of heterologous genes of biotechnological interest in the GIT of bovine and other ruminants. In order to characterize the bovine MUC2 promoter we designed primers to amplify and isolate a candidate region for this promoter. The amplified sequence was confirmed by sequencing and cloned into a plasmid vector containing the luciferase (LUC) reporter gene. The regulatory sites of the MUC2 promoter already described in the literature were used to find the putative regulatory sites in the bovine MUC2 promoter region. With these data, some deletions were performed in order to find the promoter sequence with greatest expression capacity and specificity. The constructions were tested by transient transfection assays in LoVo cells (human colorectal adenocarcinoma) and bovine fibroblasts. The quantification of the relative expression of the promoter was measured using dual-luciferase assays. Real-time PCR was performed to analyze the expression of endogenous MUC2. The results presented herein prove that the isolated sequence corresponds to the promoter of bovine MUC2 gene, since it was able to induce expression of a reporter gene in an in vitro cell culture experimental platform.

Differentially methylated regions identified in bovine embryos are not observed in adulthood.

The establishment of epigenetic marks during the reprogramming window is susceptible to environmental influences, and stimuli during this critical stage can cause altered DNA methylation in offspring. In a previous study, we found that low levels of sulphur and cobalt (low S/Co) in the diet offered to oocyte donors altered the DNA methylome of bovine embryos. However, due to the extensive epigenetic reprogramming that occurs during embryogenesis, we hypothesized that the different methylation regions (DMRs) identified in the blastocysts may not maintain in adulthood. Here, we aimed to characterize DMRs previously identified in embryos, in the blood and sperm of adult progenies of two groups of heifers (low S/Co and control). We used six bulls and characterized the DNA methylation levels of KDM2A, KDM5A, KMT2D, and DOT1L genes. Our results showed that all DMRs analysed in both groups and tissues were hypermethylated unlike that noticed in the embryonic methylome profiles. These results suggest that embryo DMRs were reprogrammed during the final stages of de novo methylation during embryogenesis or later in development. Therefore, due to the highly dynamic epigenetic state during early embryonic development, we suggest that is essential to validate the DMRs found in embryos in adult individuals.

Dynamics of the Reproductive Changes and Acquisition of Oocyte Competence in Nelore (Bos taurus indicus) Calves during the Early and Intermediate Prepubertal Periods

The purpose of this study was to characterize the reproductive physiology, oocyte competence, and chromatin compaction in Nelore calves in the early-prepubertal period (EPP) and the intermediate-prepubertal period (IPP). Calves aged 2-5 (EPP) and 8-11 months old (IPP) were assigned to Trial 1 (morpho-physiological-endocrine evaluations, n = 8) or Trial 2 (oocyte donors, n = 8) vs. the respective control groups of cows (n = 8, each). All morphological endpoints, except the antral follicle count, increased from the EPP to the IPP. The EPP LH-FSH plasma concentrations were similar to cows, whereas LH was lower and FSH was higher in the IPP than in cows. . Cows produced more Grade I (12.9% vs. 4.1% and 1.7%) and fewer Grade III COC (30.1% vs. 44.5% and 49.0%) than the EPP and IPP calves, respectively. The IPP calves' oocyte diameter was similar to those from cows but greater than those from EPP females (124.8 ± 8.5 and 126.0 ± 7.5 µm vs. 121.3 ± 7.5 µm, respectively). The expression of the chromatin compaction-related gene HDAC3 was downregulated in calves. The proportion of the blastocyst rate to the controls was lower in EPP than in IPP calves (43.7% vs. 78.7%, respectively). Progressive oocyte competence was found during the prepubertal period, which can help to decide whether to recover oocytes from calves.

DNA methylation of the endogenous retrovirus Fematrin-1 in fetal placenta is associated with survival rate of cloned calves.

INTRODUCTION: The expression of retroviral envelope proteins in the placenta facilitates generation of the multinuclear syncytiotrophoblast as an outer cellular layer of the placenta by fusion of the trophoblastic cells. This process is essential for placenta development in eutherians and for successful pregnancy. METHODS: We tested the hypothesis that alterations in DNA methylation and gene expression profiles of the endogenous retroviruses (ERVs) and genes related to epigenetic reprogramming in placenta of cloned calves result in abnormal offspring phenotypes. The fetal cotyledons in 13 somatic cell nuclear transfer (SCNT) pregnancies were collected. DNA methylation level of Fematrin-1 was analyzed using bisulfite PCR and mRNA levels of Fematrin-1, Syncytin-Rum1, DNMT1, DNMT3A, DNMT3B, TET1, TET2 and TET3 measured by RT-qPCR. RESULTS: Methylation of Fematrin-1 in placenta of control animals produced by artificial insemination (AI) was similar to live SCNT-produced calves, but hypermethylated than dead SCNT-produced calves. The levels of mRNA differed between SCNT-produced calves and AI animals for all genes, except TET3. However, no differences were observed between the live and dead cloned calves for all genes. Moreover, no differences were found between mRNA levels of Fematrin-1 and Syncytin-Rum1. DISCUSSION: Our results suggest that this altered DNA methylation, deregulation in the expression of ERVs and in the genes of epigenetic machinery in fetal cotyledons of cloned calves may be associated with abnormal placentogenesis found in SCNT-produced animals. Further studies characterizing other mechanisms involved in the regulation of ERVs are important to support the development of new strategies to improve the efficiency of cloning.

DNA methylation profile at a satellite region is associated with aberrant placentation in cloned calves.

INTRODUCTION: Cloning via somatic cell nuclear transfer (SCNT) has been associated with a variety of pathologies, primarily in the placenta, and these alterations may be associated with aberrant epigenetic reprogramming of the donor cell genome. We tested the hypothesis that DNA methylation patterns are not appropriately established after nuclear transfer and that those altered patterns are associated with specific aberrant phenotypes. METHODS: We compared global and specific placental DNA methylation patterns between aberrant and healthy SCNT-produced calves. Foetal cotyledon samples of ten SCNT pregnancies were collected. Global DNA methylation and hydroxymethylation levels were measured using an ELISA-based assay and specific DNA methylation of satellite I, and α-satellite repeat elements were measured using bisulfite PCR. RESULTS: Our analysis revealed that the SCNT-produced calves, which showed aberrant phenotypes, exhibited a reduced methylation pattern of the satellite I region compared to that of healthy calves. In contrast, global methylation and hydroxymethylation analyses showed higher levels for both cytosine modifications in SCNT-produced female calves with aberrant phenotypes. The satellite I region showed most of the sequences to be hypermethylated in live cloned calves compared with those in deceased calves. DISCUSSION: Our results suggest that this satellite I region could be used as an epigenetic biomarker for predicting offspring viability. Studies evaluating DNA methylation patterns of this satellite region in the donor cell genome or embryo biopsies could shed light on how to improve the efficiency of SCNT cloning.