ß-Mercaptoethanol in culture medium improves cryotolerance of in vitro-produced bovine embryos.

The objective of this study was to investigate the effects of adding ß-mercaptoethanol (ßME) to culture medium of bovine in vitro-produced (IVP) embryos prior to or after vitrification on embryo development and cryotolerance. In Experiment I, Day-7 IVP blastocysts were vitrified and, after warming, cultured in medium containing 0, 50 or 100 µM ßME for 72 h. Embryos cultured in 100 µM ßME attained higher hatching rates (66.7%) than those culture in 0 (47.7%) and 50 (52.4%) µM ßME. In Experiment II, IVP embryos were in vitro-cultured (IVC) to the blastocyst stage in 0 (control) or 100 µM ßME, followed by vitrification. After warming, embryos were cultured for 72 h (post-warming culture, PWC) in 0 (control) or 100 µM ßME, in a 2 × 2 factorial design: (i) CTRL-CTRL, control IVC and control PWC; (ii) CTRL-ßME, control IVC and ßME-supplemented PWC; (iii) ßME-CTRL, ßME-supplemented IVC and control PWC; or (iv) ßME-ßME, ßME-supplemented IVC and ßME-supplemented PWC. ßME during IVC reduced embryo development (28.0% vs. 43.8%) but, following vitrification, higher re-expansion rates were seen in ßME-CTRL (84.0%) and ßME-ßME (87.5%) than in CTRL-CTRL (71.0%) and CTRL-ßME (73.1%). Hatching rates were higher in CTRL-ßME (58.1%) and ßME-ßME (63.8%) than in CTRL-CTRL (36.6%) and ßME-CTRL (42.0%). Total cell number in hatched blastocysts was higher in ßME-ßME (181.2 ± 7.4 cells) than CTRL-CTRL (139.0 ± 9.9 cells). Adding ßME to the IVC medium reduced development but increased cryotolerance, whereas adding ßME to the PWC medium improved embryo survival, hatching rates, and total cell numbers.

Promoter-specific expression of the imprinted IGF2 gene in bovine oocytes and pre-implantation embryos.

The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter-specific expression patterns in oocytes and pre-implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre-implantation embryos at the 1-, 2-, 4-, 8- and 16-cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT-qPCR, using four sets of IGF2 promoter-specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2- and P4-derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2-cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1-derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology.

In vitro development of IVF-derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene.

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus-oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/µl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/µl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/µl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.

Effects of follicle-stimulating hormone, insulin-like growth factor 1, fibroblast growth factor 2, and fibroblast growth factor 9 on sirtuins expression and histone deacetylase activity in bovine granulosa cells.

Granulosa cells (GC) are critical regulators of fertility. During the process of ovarian folliculogenesis, these cells undergo profound changes while producing steroid hormones that are important to control follicular growth, oocyte maturation, and ovulation. Sirtuins are enzymes that regulate several biological processes and have been associated with control of GC function. However, how sirtuins are regulated in GC during ovarian folliculogenesis remains to be unveiled. The present study was designed to investigate effects of hormones that control GC proliferation, differentiation, and steroidogenesis on expression of the seven members of the mammalian sirtuins family (SIRT1-7) and on histone deacetylase activity of nuclear sirtuins (SIRT1, 6, and 7) in GC. Bovine granulosa cells were isolated from small antral follicles (1-5 mm) and were treated with or without follicle-stimulating hormone (FSH), insulin-like growth factor 1 (IGF-1), and fibroblast growth factors 2 (FGF2) and 9 (FGF9). Following treatments, cell proliferation was determined via a cell analyzer, estradiol synthesis and histone deacetylase activity were determined via ELISA, and sirtuins mRNA expression was determined via qPCR. Treatments with FSH and IGF-1 stimulated cell proliferation while addition of FGF2 or FGF9 suppressed estradiol production stimulated by FSH plus IGF-1. In terms of treatments that regulated sirtuins expression in GC, fibroblast growth factors were the most impactful: FGF2 alone increased SIRT1 mRNA expression in comparison to several treatments and increased mRNA abundance of SIRT2 and SIRT7 when added to the combination of FSH and IGF-1; the addition of FGF9 to the combination of FSH and IGF-1 increased mRNA expression of SIRT2, SIRT3, SIRT4, SIRT6, and SIRT7 and increased mRNA expression of SIRT5 in comparison to the negative control group that received no treatment. Also, FGF2 alone increased histone deacetylase activity of sirtuins in comparison to all treatments that contained FSH and/or IGF-1. Furthermore, several correlations were observed between treatments and sirtuins expression and activity, between estradiol or GC numbers and sirtuins expression, and between expression of sirtuins. As FGF2 and FGF9 are considered anti-differentiation factors of GC that stimulate GC proliferation while suppressing estradiol production in combination with FSH and IGF-1, data of this study suggest that sirtuins are associated with control of differentiation of bovine GC.

Effects of asprosin on estradiol and progesterone secretion and proliferation of bovine granulosa cells.

Asprosin is an adipokine synthesized by the white adipose tissue that regulates glucose homeostasis and that has been reported to affect bovine theca cell function and follicular growth, but its role on granulosa cell functions remains to be unveiled. Hence, the objective of this study was to investigate asprosin impacts on granulosa cell steroidogenesis. Bovine granulosa cells from small ovarian follicles were cultured in vitro to investigate the effects of asprosin on cell proliferation, production of steroids, mRNA abundance of genes that encode steroidogenic enzymes and cell cycle regulators, and protein relative abundance of steroidogenic signaling pathways. Asprosin was shown to affect granulosa cell functions in a dose-dependent manner. In the presence of FSH, asprosin enhanced estradiol production and stimulated an increase in mRNA expression of FSHR and CYP19A1 in a dose-dependent manner. In the presence of IGF1, asprosin decreased estradiol production, increased progesterone production, altered PKA relative protein expression, and tended to alter the ratio of p-ERK1/2/total ERK1/2 protein expression in a dose-dependent manner. Furthermore, asprosin increased p-53 gene expression in basal culture conditions and with or without FSH and IGF1. Taken together, findings of this study show that asprosin is a regulator of granulosa cell functions and the effects of asprosin depend on dose and cell culture conditions.

Sperm DNA 5-methyl cytosine and RNA N6-methyladenosine methylation are differently affected during periods of body weight losses and body weight gain of young and mature breeding bulls.

Aiming to characterize the effects of nutritional status on epigenetic markers, such as DNA 5-methyl cytosine (mC) methylation and RNA N6-methyladenosine (m6A) methylation, of bovine sperm, 12 Angus × Hereford crossbred breeding bulls were submitted to nutritional changes for a period of 180 d: no change in body weight (BW) (phase 1 = 12 d), BW loss (phase 2 = 78 d), and BW gain (phase 3 = 90 d) in a repeated measures design. Animals were fed Beardless wheat (Triticum aestivum) hay and mineral mix. Statistical analyses were performed using SAS 9.4 (SAS Inst., Cary, NC). Higher levels of RNA m6A (P = 0.004) and DNA methylation (P = 0.007) of spermatic cells were observed at phase 2 compared with phase 1. In phase 3, sperm RNA m6A methylation levels continued to be higher (P = 0.004), whereas the DNA of sperm cells was similar (P = 0.426) compared with phase 1. Growing bulls had a tendency (P = 0.109) of higher RNA m6A methylation levels than mature bulls. Phase 2 altered scrotal circumference (P < 0.001), sperm volume (P = 0.007), sperm total motility (P = 0.004), sperm progressive motility (P = 0.004), total sperm count (P = 0.049), normal sperm (P < 0.001), abnormal sperm (P < 0.001), primary sperm defects (P = 0.039), and secondary sperm defects (P < 0.001). In phase 3, bulls had scrotal circumference, sperm volume, sperm motility, sperm progressive motility, total sperm count, normal and abnormal spermatozoa, and primary and secondary spermatozoa defects similar to phase 1 (P > 0.05). Serum concentrations of insulin-like growth factor-1 and leptin decreased during phase 2 (P = 0.010), while no differences (P > 0.05) were detected between phases 3 and 1; growing bulls tended (P = 0.102) to present higher leptin levels than mature bulls. Specific for mature bulls, DNA methylation was positively correlated with leptin concentration (0.569, P = 0.021), whereas for young bulls, DNA methylation was positively correlated with abnormal spermatozoa (0.824, P = 0.006), primary spermatozoa defect (0.711, P = 0.032), and secondary spermatozoa defect (0.661, P = 0.052) and negatively correlated with normal spermatozoa (-0.824, P = 0.006), total sperm count (-0.702, P = 0.035), and sperm concentration (-0.846, P = 0.004). There was no significant correlation (P > 0.05) between RNA m6A and hormones and semen traits. In conclusion, the nutritional status of breeding bulls alters epigenetic markers, such as DNA methylation and RNA m6A methylation, in sperm, and the impact of change seems to be age dependent. These markers may serve as biomarkers of sperm quality and fertility of bulls in the future. Detrimental effects on sperm production and seminal quality are observed at periods and places when and where environmental and nutritional limitations are a year-round reality and may carry hidden players that may influence a lifetime of underperformance.

Characterization of body composition and liver epigenetic markers during periods of negative energy balance and subsequent compensatory growth in postpubertal beef bulls.

This study aimed to characterize the effects of dietary restriction and subsequent re-alimentation on body composition and hepatic gene expression of epigenetic markers of DNA methylation, RNA m6A methylation, and histone acetylation in the liver of postpubertal beef bulls. Twelve Angus × Hereford crossbred bulls (n = 6, 23 ± 0.55 mo [young bulls], 558 ± 6.1 kg; and n = 6, 47 ± 1.2 mo [mature bulls], 740 ± 30.5 kg) were submitted to two dietary regimes per offering of the same hay: low plane of nutrition (90 d) and compensatory growth (90 d). Each animal acted as its own control and were fed Beardless wheat (Triticum aestivum) hay and mineral mix during the trial. Statistical analyses were performed using SAS 9.4 following a pre-post repeated measures design. Bulls in negative energy balance (NEB) decreased (P < 0.001) empty body weight (EBW; 23.1% [-139.1 kg]), empty body fat (EBF; 39.8% [-85.4 kg]), and empty body protein (EBP; 14.9% [-13.5 kg]) and fully recovered at the end of the trial. Body fat accounted for 77.1% of daily changes in body energy status, whereas body protein accounted for only 22.9% (P < 0.001). Relative abundance of epigenetic markers transcripts was analyzed via qPCR. Bulls at NEB tended (P ≤ 0.097) to increase gene expression of epigenetic markers of RNA m6A methylation (METTL14, VIRMA, and WTAP) and increased (P ≤ 0.050) the gene expression of epigenetic markers of DNA methylation (DNMT3A) and histone-acetylation (SIRT3 and SIRT7). Young bulls had a tendency (P ≤ 0.072) of higher RNA m6A methylation, VIRMA, and WTAP than mature bulls. Effect of diet × age interaction was not detected (P ≥ 0.137) for METTL14, VIRMA, WTAP, DNMT3A, SIRT3, or SIRT7. Younger bulls tended to have greater RNA m6A methylation levels than mature bulls, indicating that, while contemporaneously fed the same diet during periods of undernourishment followed by compensatory growth, age has an impact on this epigenetic mechanism. In conclusion, metabolic status seems to carry a greater impact on regulating bovine hepatic epigenetic mechanisms that modulate gene transcription, such as DNA methylation and histone acetylation, than on epigenetic mechanisms that regulate gene translation, such as RNA m6A methylation. During periods of undernourishment followed by compensatory growth, body fat pools appear to change more dynamically and are easily detected having a greater impact on epigenetic markers that modulate hepatic gene transcription rather than translation.

Epigenetics refers to heritable modifications that regulate gene expression without altering DNA sequence, hence, acting on top of the genes. Epigenetic markers change in response to stressors such as environmental factors, nutritional challenges, among other overlooked players that altogether could drastically impair animal performance. During periods of undernourishment followed by fast weight gain, dynamic changes in body composition, especially fat, appear to trigger an increased action of such physiological markers that modulate hepatic gene expression. Findings of this study unveil epigenetic metabolic pathways that deserve further investigation for proper quantification of potential consequences of metabolic stress on the liver of bovines that suffer significant loss of body weight followed by recovery. The alterations at the molecular level shown in this study provide a picture of silent metabolic changes that have not been detected previously in liver metabolism studies of cattle. Therefore, the impact of nutritional management and metabolic stress may be greater than previously expected and differently controlled than previously assumed.

A blueprint to establish a four-bed obstetric critical care unit in the labor ward of a central hospital.

Patients at risk of organ dysfunction or with established organ dysfunction should be referred to central or tertiary-level hospitals. However, even in central hospitals, intensive care unit (ICU) beds are often unavailable, which may contribute to maternal deaths. One pragmatic solution is to establish obstetric critical care units (OCCUs) in the labor wards of central hospitals; however, specific guidance on how to do this is limited. In addition, globally applicable standards of care are lacking, with uncertainty regarding who should lead obstetric critical care. In this article the specific OCCU infrastructure, equipment and human resources required to establish such units in central hospitals in low- and middle-income countries are described in sufficient detail for easy replication. Admission and discharge guidelines and operational recommendations that include quality indicators are also provided.

The effect of guideline variations on the implementation of active management of the third stage of labor.

OBJECTIVE: To determine whether healthcare providers performed active management of the third stage of labor (AMTSL) as defined by FIGO/ICM and WHO, and as described by the Cochrane Collaboration. METHODS: In a prospective observational study, a questionnaire regarding knowledge of AMTSL was administered to healthcare providers in the largest maternity teaching center in Colombia. It was subsequently observed whether and how the healthcare providers performed AMTSL in practice. The percentage of correct use of AMTSL was calculated. RESULTS: Healthcare providers indicated they knew what AMTSL was but disagreed on the timing of prophylactic oxytocin use. In total, 241 deliveries were observed. Oxytocin at varying doses and routes was used in 239 (99.2%) deliveries. In all deliveries, the umbilical cord was clamped early. In 49 (20.3%) deliveries, controlled cord traction was performed. Uterine massage was carried out in 213 (88.4%) deliveries. According to the FIGO/ICM and WHO definitions, and the Cochrane Collaboration description, correct use of AMTSL occurred in 0.8%, 0.0%, and 8.3%, of cases, respectively. CONCLUSION: Correct use of AMTSL is low at the largest maternity teaching center in Colombia. There is an urgent need for a single definition of AMTSL, which could be used globally for research, training, and scaling-up the performance of AMTSL.