Low levels of sulfur and cobalt during the pre- and periconceptional periods affect the oocyte yield of donors and the DNA methylome of preimplantation bovine embryos.

Maternal nutrition is critical in mammalian development, influencing the epigenetic reprogramming of gametes, embryos, and fetal programming. We evaluated the effects of different levels of sulfur (S) and cobalt (Co) in the maternal diet throughout the pre- and periconceptional periods on the biochemical and reproductive parameters of the donors and the DNA methylome of the progeny in Bos indicus cattle. The low-S/Co group differed from the control with respect to homocysteine, folic acid, B12, insulin growth factor 1, and glucose. The oocyte yield was lower in heifers from the low S/Co group than that in the control heifers. Embryos from the low-S/Co group exhibited 2320 differentially methylated regions (DMRs) across the genome compared with the control embryos. We also characterized candidate DMRs linked to the DNMT1 and DNMT3B genes in the blood and sperm cells of the adult progeny. A DMR located in DNMT1 that was identified in embryos remained differentially methylated in the sperm of the progeny from the low-S/Co group. Therefore, we associated changes in specific compounds in the maternal diet with DNA methylation modifications in the progeny. Our results help to elucidate the impact of maternal nutrition on epigenetic reprogramming in livestock, opening new avenues of research to study the effect of disturbed epigenetic patterns in early life on health and fertility in adulthood. Considering that cattle are physiologically similar to humans with respect to gestational length, our study may serve as a model for studies related to the developmental origin of health and disease in humans.

Effects of Prostaglandins E2 and F2α on the in vitro maturation of bovine oocytes.

We aimed to elucidate the effects of PGE2 and PGF2α on the in vitro maturation (IVM) of bovine oocytes. First, cumulus-oocyte complexes were matured in the media supplemented with or without PGE2, PGF2α, or PGE2 plus PGF2α for the final 24, 12, or 6 h of culture. Then, the cumulus-oocyte complexes were matured in the absence or presence of a PG endoperoxide synthase 2 (PTGS2) enzyme inhibitor (NS398) supplemented with PGE2, PGF2α, or PGE2 plus PGF2α. Finally, the expression of genes associated with PGs activity in cumulus cells (PTGS2, PG E-synthase-1 [PTGES1], and aldo-keto reductase 1 [AKR1B1]) or oocytes (receptors for PGE2 [PTGER2] and PGF2α [PTGFR]) of different competencies was quantified. Supplementation of the IVM medium with PGs did not improve in vitro embryo production or embryo quality (P > 0.05). During maturation, the relative abundance of PTGS2 transcripts increased (P < 0.05) only in the less-competent group, whereas those of PTGES1 increased in the less-competent and in the more-competent groups. Conversely, AKR1B1 expression decreased only in the less-competent group (P < 0.05). Receptors for the PGE2 and PGF2α genes were very low or undetectable in oocytes. In conclusion, PGE2 and PGF2α are not recommended for media supplementation during maturation because they have no effect on embryo development. Although genes related to PGs activity are differentially expressed in cumulus cells of cumulus-oocyte complexes of different competence during maturation, the expression of PGE2 and PGF2α receptor genes was either not detectable or was detected at low levels in oocytes.

Histone acetylation during the in vitro maturation of bovine oocytes with different levels of competence.

We aimed to analyse the histone acetylation status and expression profile of genes involved in histone acetylation (histone acetyltransferase 1 (HAT1), lysine acetyltransferase 2A (KAT2A), histone deacetylase 1(HDAC1), HDAC2 and HDAC3) in bovine oocytes of different competences during invitro maturation (IVM). Cumulus-oocyte complexes were recovered from two groups of follicles: minor follicles (1.0-3.0mm in diameter), classified as low competence (LC) and large follicles (6.0-8.0mm in diameter) classified as high competence (HC). Oocytes were submitted to IVM for 0, 8 and 24h and stored for analysis. Acetylation status of histone H4 on lysine K5, K6, K12 and K16 was assessed by immunohistochemistry. For gene expression, mRNA levels were determined by real-time quantitative polymerase chain reaction. All oocytes, regardless of their competence, showed a gradual decrease (P<0.05) in acetylation signals during IVM. From 0 to 8h of maturation, an increase (P<0.05) in the relative abundance of HAT1 mRNA was observed only in the HC oocytes. In this group, higher (P<0.05) mRNA levels of HDAC1 at 8h of maturation were also observed. In conclusion, in the present study, LC oocytes were shown to have adequate acetylation levels for the resumption and progression of meiosis; however, these oocytes do not have the capacity to synthesise RNA during IVM as the HC oocytes do.

The Replacement of Fetal Bovine Serum with Bovine Serum Albumin During Oocyte Maturation and Embryo Culture Does Not Improve Blastocyst Quality After Slow Freezing Cryopreservation.

In the present study, four experimental groups were used: fresh embryos, cultured during in vitro maturation and in vitro culture in media supplemented with bovine serum albumin (BSA) (fresh BSA) or fetal bovine serum (FBS) (fresh FBS); and two groups of cryopreserved and thawed embryos, produced under the same conditions (frozen BSA and frozen FBS). Experiment 1 evaluated the protein source effect on embryo development and response to cryopreservation. At day 7, half of the expanded blastocysts (Bx) from each group were cryopreserved and warmed and the other half were used as controls. After warming, embryos were incubated under the same conditions for 48 hours, and the hatching rate was measured at 24 and 48 hours. The total and the apoptotic cell numbers were measured in a subset of Bx after 24 hours. Experiment 2 used the Bx of experiment 1 to compare the expression of KRT8, PLAC8, FOSL1, HSP1A1, and HSPA5 genes in hatched blastocysts at 24 and 48 hours for all groups. The FBS group showed a higher percentage (p < 0.05) of embryos (42.8% vs. 27.9%) and higher rates of Bx (75.0% vs. 63.8%) on day 7, compared with the BSA group. At 24 hours postwarming, the fresh FBS group showed the highest hatching rate (p < 0.05) in comparison with other treatments. However, at 48 hours, the hatching rate was similar (p > 0.05) among groups: fresh FBS (68.1% ± 23.3%), fresh BSA (70.0% ± 31.0%), frozen FBS (39.2 ± 27.1), and frozen BSA (38.2 ± 23.9). After 24 hours, frozen BSA showed a higher number of cells compared with frozen FBS (p < 0.05). The expression of the PLAC8 gene was higher (p < 0.05) in fresh BSA embryos compared with frozen FBS embryos at 24 hours. In the present study, BSA replacement reduced embryo development, but did not affect the response to cryopreservation. However, upregulation of the PLAC8 gene suggests that embryos cultured in BSA might have better quality to support further development.

Procaine and S-Adenosyl-l-Homocysteine Affect the Expression of Genes Related to the Epigenetic Machinery and Change the DNA Methylation Status of In Vitro Cultured Bovine Skin Fibroblasts.

Cloning using somatic cell nuclear transfer (SCNT) has many potential applications such as in transgenic and genomic-edited animal production. Abnormal epigenetic reprogramming of somatic cell nuclei is probably the major cause of the low efficiency associated with SCNT. Strategies to alter DNA reprogramming in donor cell nuclei may help improve the cloning efficiency. In the present study, we aimed to characterize the effects of procaine and S-adenosyl-l-homocysteine (SAH) as demethylating agents during the cell culture of bovine skin fibroblasts. We characterized the effects of procaine and SAH on the expression of genes related to the epigenetic machinery, including the DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3 alpha (DNMT3A), DNA methyltransferase 3 beta (DNMT3B), TET1, TET2, TET3, and OCT4 genes, and on DNA methylation levels of bovine skin fibroblasts. We found that DNA methylation levels of satellite I were reduced by SAH (p = 0.0495) and by the combination of SAH and procaine (p = 0.0479) compared with that in the control group. Global DNA methylation levels were lower in cells that were cultivated with both compounds than in control cells (procaine [p = 0.0116], SAH [p = 0.0408], and both [p = 0.0163]). Regarding gene expression, there was a decrease in the DNMT1 transcript levels in cells cultivated with SAH (p = 0.0151) and SAH/procaine (0.0001); a decrease in the DNMT3A transcript levels in cells cultivated with SAH/procaine (p = 0.016); and finally, a decrease in the DNMT3B transcript levels in cells cultivated with procaine (p = 0.0007), SAH (p = 0.0060), and SAH/procaine (p = 0.0021) was found. Higher levels of TET3 transcripts in cells cultivated with procaine (p = 0.0291), SAH (p = 0.0373), and procaine/SAH (p = 0.0013) compared with the control were also found. Regarding the OCT4 gene, no differences were found. Our results showed that the use of procaine and SAH during bovine cell culture was able to alter the epigenetic profile of the cells. This approach may be a useful alternative strategy to improve the efficiency of reprogramming the somatic nuclei after fusion, which in turn will improve the SCNT efficiency.

Production of transgenic cattle by somatic cell nuclear transfer (SCNT) with the human granulocyte colony-stimulation factor (hG-CSF).

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.

Bovine epididymal spermatozoa treatment for in vitro fertilization: Heparin accelerates fertilization and enables a reduction in coincubation time.

This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P<0.05) than that in the EJ (19%) and EP- (42%) groups. At 12 and 18 h, FR remained higher in the EP+ group, and a gradual increase in polyspermy was observed. The use of a P or PS gradient yielded a similar BR on D7 (54% and 52%), which was higher than the rate obtained using the washing method (37%). The embryos produced by EP and selected in a P or PS gradient resulted in a sex deviation in favor of male embryos (P>0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient.

Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming.

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.