The polyspermy occurrence is considerably lower under in vivo compared to in vitro embryo culture conditions, suggesting that the presence of some factors in the maternal environment is responsible for this. The α-L-fucosidase (FUCA) is a natural glycosidase present in the oviductal fluid, therefore, this study aimed at investigating the effect of adding FUCA to the hardening of the zona pellucida (ZP), polyspermy control, and embryonic yield and quality of bovine blastocysts produced in vitro. In the first experiment, the effect of FUCA (0.125 U/mL) was evaluated during the entire in vitro fertilization (IVF). However, it was demonstrated to be embryotoxic by completely inhibiting the blastocyst formation. In the second experiment, the FUCA (0.125 U/mL) was tested as short-term incubation before IVF (pre-fertilization step) for 30 min or 2 h, which demonstrated that FUCA treatment for 30 min resulted in ZP hardening. In the third experiment, a pre-fertilization FUCA treatment (1 h) at different concentrations (0, 0.0625, and 0.125 U/mL) showed that FUCA (0.0625 U/mL) improved pre-fertilization ZP hardening and tended to increase monospermic fertilization rates but did not improve embryo yield and quality. Together, it has been demonstrated that FUCA can induce oocyte pre-fertilization ZP hardening and might improve monospermic fertilization performance, and this effect is dependent on both variables (protein concentration and incubation time).

Antral follicle count (AFC) was phenotypically and genetically evaluated in a Bos taurus cow population. A total of 257 Aberdeen Angus cows underwent an ovarian ultrasound evaluation and hormonal synchronization for timed artificial insemination (TAI). These animals were between 40 and 70 d postpartum with a body score condition (BSC) between 2 and 4 (average = 2.7) on a scale of 1-5. The number of antral follicles was assessed 10 d before (D-10) the beginning of TAI. Visible follicles ( 24 follicles and 16 follicles (n = 97); and high AFC as > 25 follicles (n = 67). The comparison between the average AFC and pregnancy rate was performed using the chi-square test in the Minitab (R) 19.2 statistical software, adopting a Pvalue < 0.05. Hair samples of Aberdeen Angus cows were collected for genomic DNA extraction and subsequent genotyping analysis using the GGP HD array. The associations between SNP markers and phenotypic information (AFC) were determined using the GBLUP methodology. Windows explaining more than 1% of the total additive genetic variance were selected as candidates for functional prospection using QTL colocalization and enrichment analysis using a hypergeometric test. The GWAS identified seven candidate genomic windows and 54 annotated genes within them. Among these genes, DUSP1, SOX17, XKR4, PLAG1, HFM1, PPM1K, IBSP, NCAPG-LCORL, and HMGHZ were associated with biological processes that might be related to follicle count in Angus cows. Enrichment analysis of the reproduction-related traits found enriched QTLs in BTA5 ("Inhibin level", "Insulinlike growth factor 1 level"), BTA6 ("Calving ease", "Calf size", "Calving index"), and BTA14 ("Insulin-like growth factor 1 level"; "Age at puberty", "Interval to first estrus after calving"). This study brings new knowledge of the genomic background of AFC and may lead to further investigations regarding physiological explanations for different AFCs and their relationship to fertility.

Sample types such as those from reproductive systems often yield scarce material, which limits RT-qPCR analysis to only a few targets. Recently developed high-throughput systems can potentially change this scenario, however, the nanoliter scale of such platforms requires extra processing, e.g., preamplification, which needs to be defined through observation and experience. In order to establish best practices in high-throughput PCR approaches using samples from reproductive systems, we evaluated the Biomark™ HD performance using 11 different sample types from the bovine reproductive system: blastocyst (single/pool), oocyte (pool), cumulus, granulosa, and theca cells, oviduct tissue, fetal ovary, testicle (adult/fetal), and uterine horn. We observed that the preamplification step is not just reliable, but mandatory. Our results indicated that 14-preamplification cycles associated to 5- and 7-fold-dilution is the best approach for those samples. Additionally, the Biomark™ HD system has a high intra and inter reproducibility, therefore its performance in duplicate is unnecessary for the ΔCq analysis, taking in consideration the cutoff value 4 < Cq < 22. In summary, this high-throughput approach is a reliable and excellent tool for studying the bovine reproductive system, especially using quantitatively-limited samples, as a larger number of target genes can be assessed from a very low amount of starting material.

Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently “primed” for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence.

Studies have shown that the use of equine chorionic gonadotropin (eCG), which binds both follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors, could modify the female reproductive tract. We, thus, aimed to quantify the messenger RNA (mRNA) abundance of genes related to cumulus-oocyte complexes (COCs) and embryo quality in Nelore cows (Bos taurus indicus) submitted to ovarian superstimulation using only FSH (FSH group; n = 10) or replacement of the last two doses of FSH by eCG (FSH/eCG group; n = 10). All animals were slaughtered and the ovarian antral follicles from both groups (10-14 mm in diameter) were aspirated for cumulus, oocyte and in vitro embryo production gene expression analysis. The relative mRNA abundance of 96 genes related to COCs development and embryo quality was measured by RT-qPCR. We found that oocytes are more affected by eCG use and that 35 genes involved in lipid metabolism, oxidative stress, transcriptional control, and cellular development were upregulated in the FSH/eCG group. In blastocysts, lipid metabolism seems to be the main pathway regulated by eCG use. We suggest that these multiple effects could be due to the ability of eCG to bind LHR and FSHR, which could activate multiple signal transduction pathways in the superstimulated ovary, further impacting the transcriptional profile of COCs and blastocysts.

In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule involved in ovarian follicle development, which acts through two transmembrane receptors. Within the ovarian follicle, there appear to be species differences in the precise pattern of localization of AGTR2 protein and it has an important role in in vitro maturation of oocytes in mammals. The infusion of ANG II induced ovulation in rabbits and the use of ANG II antagonists inhibited ovulation in rabbits, rats, and cattle. In fetal ovaries, AGTR2 protein was detected in ovigerous cords and preantral follicles throughout porcine and bovine gestation. In the oviduct, ANG II is responsible for the orchestration of the transport of gametes. In the male reproductive system, there is considerable evidence for the local synthesis of components of renin-angiotensin system (RAS) in male reproductive tissues. The roles of RAS in local processes at these sites are still uncertain, although there is evidence for involvement in tubular contractility, spermatogenesis, sperm maturation, capacitation, acrosomal exocytosis, and fertilization.

In the bovine oviduct, estradiol (E2) stimulates secretion and cell proliferation, whereas progesterone (P4) suppresses them. In this study, we have evaluated the effect of two superstimulatory protocols (follicle-stimulating hormone [FSH] or FSH combined with equine chorionic gonadotropin [eCG]) on the oviductal levels of E2 and P4 and its outcome on oviductal cells. Compared with the control group (a single pre-ovulatory follicle), we have observed that the cows submitted to FSH/eCG treatment showed a higher concentration of E2 in the oviduct tissue, together with a higher abundance of messenger RNA encoding steroid receptors (ESR1 and progesterone receptor), and genes linked to gamete interactions and regulation of polyspermy (oviduct-specific glycoprotein 1, heat-shock protein family A member 5, α-l-fucosidase 1 [FUCA1], and FUCA2) in the infundibulum and ampulla segments of the oviduct. However, we did not observe any modulation of gene expression in the isthmus segment. Even though the FSH protocol upregulated some of the genes analyzed, we may infer that the steady effect of FSH combined with eCG on oviduct regulation might benefit fertilization and may potentially increase pregnancy rates.

Insulin-like growth factor 1 (IGF-1) activity is established by the regulation of IGF binding protein activity, which blocks IGF-1 functions, whereas pregnancy-associated plasma protein-A (PAPP-A) improves IGF-1 bioavailability and facilitates binding to IGF receptors. To further extend our understanding of the effect of exogenous PAPP-A on bovine embryo production, we added this protein during in vitro maturation of cumulus-oocyte complexes (COCs); moreover, we assessed its effects on IGF-1 quantity in the maturation medium, embryonic yield and postwarming survival, blastocyst quality, and transcript abundance. Bovine COCs were matured in a serum-free medium, either with PAPP-A supplementation (100 ng/ml) or without (control). The treatment group produced higher IGF-1 concentrations in the maturation medium; however, showed no difference on cleavage, blastocysts rates, and embryonic survival 3 and 24 hr postcryopreservation. Regarding gene expression, VNN1 was upregulated, whereas AGPAT9, FASN, EGFR, HAS2, and IMPDH1 were downregulated in PAPP-A treated. PAPP-A treated, CPT2, DNMT3A, and TFAM were upregulated, whereas ATF4 and IFITM3 were downregulated. We concluded that although the addition of PAPP-A did not affect embryo yield and blastocyst survival, higher IGF-1 levels may affect embryo competence through differential expression of genes involved in lipid metabolism, oocyte competence, and mitochondrial function.

Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus-oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2h and 16 transcripts after 24h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos.

In our study, we added natriuretic peptide type C (NPPC) and/or sildenafil during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) followed by in vitro culture (IVC) of embryos with or without sildenafil. We evaluated the effects on the lipid content (LC) of oocytes and embryos and also verified the expression of 96 transcripts related to competence in matured COCs and 96 transcripts related to embryo quality in blastocysts. After IVM, LC was decreased in oocytes by NPPC while sildenafil did not affect LC in oocytes. The genes involved in lipid metabolism and lipid accumulation (DGAT1, PLIN2and PLIN3) were not affected in COCs after treatment during IVM, although the expression of PTX3 (a cumulus cells expansion biomarker) was increased and the hatched blastocyst rate was increased by NPPC during IVM. During IVM, sildenafil increased the mRNA relative abundance of HSF1 and PAF1 and decreased REST in blastocysts. The use of sildenafil in IVC increased the LC of blastocysts. The mRNA abundance in blastocysts produced during IVC with sildenafil was changed for ATF4, XBP1, DNMT3A, DNMT3B, COX2, and SOX2. Although NPPC reduced the LC of oocytes after IVM and upregulated markers for cumulus expansion, embryo production was not affected and the produced blastocysts were able to regain their LC after IVC. Finally, the use of sildenafil during IVC increased the cytoplasmic LC of embryos but did not affect embryo quality, as measured by analysis of 96 transcripts related to embryo quality.

New pasture legumes need to be tested for deleterious effects on reproduction in grazing livestock. Measurement of these effects in grazing animals is difficult, so in vitro techniques are preferred for initial screening. Using ovine oocytes and embryos, we have previously observed the effects of the isoflavones responsible for clover disease and the effects of a crude extract of B. pelecinus . Here, we go to the next stage by attempting to identify the plant secondary metabolites produced by B. pelecinus that could be responsible for the effect. Experiment 1: B. pelecinus was extracted with 1:1 (v/v) MeOH/CHCl 3 and the extract was fractionated using rapid silica filtration (RSF) with solvents of varying polarities; dried fractions were added, at final concentrations of 0, 100 or 200 µg mL -1 , to the medium used for in vitro maturation of cumulus-oocyte complexes (COCs) derived from abattoir-sourced

Testosterone is often recommended in the treatment of several aging-related conditions. However, there are still questions about the consequences of this therapy in terms of hormonal and inflammatory parameters that are crucial for prostate homeostasis. Thus, we investigate if the testosterone therapy (TT) modulates the hormone receptors and inflammatory cytokines in the ventral prostate of adult rats. Wistar rats aging 150 days were divided into two experimental groups (n = 10/group): T: received subcutaneous injections of testosterone cypionate (5 mg/kg body weight) diluted in corn oil every other day for 4 weeks; and C: received corn oil as vehicle. Animals were euthanized at 180 days old by decapitation. Blood was collected to obtain hormone and cytokines concentrations. The ventral prostate was dissected and processed for light microscope and molecular analyses. Relative ventral prostate weight and epithelial compartment were increased after TT. The number of intact and degranulated mast cells was reduced in the T group. Plasma testosterone, DHT and intraprostatic testosterone concentrations were higher in the T group. TT leads to an increase in cell proliferation and up-regulation of AR, ERβ, PAR-4, and NRF2. Importantly, plasma concentration and tissue expression of IL-10 and TNF-α were higher after TT. In summary, these results indicate that TT can regulate inflammatory response, with impacts in cytokines and mast cell population, and modulates steroids receptors, important parameters for prostatic homeostasis.

To better understand the impact of ovarian superstimulation on bovine follicular microenvironment, Nelore cows (Bos taurus indicus) were subjected to ovarian superstimulation with follicle stimulating hormone (FSH, n = 10; P-36 protocol) or FSH combined with eCG (n = 10; P-36/eCG protocol). Follicular fluid was analyzed for cholesterol concentration. Granulosa cells were analyzed by RT-qPCR to assess the expression of genes involved in steroidogenic and ovulatory and expression of microRNAs involved in final follicular development and luteinizing hormone/choriogonadotropin receptor (LHCGR) expression. Plasma concentration of estradiol was also measured. Follicular fluid from the P-36 group showed higher concentration of cholesterol than that of control (non-superstimulated) cows. Plasma concentration of estradiol was higher in the P-36/eCG group. Abundance of STAR and FSHR mRNAs were lower in granulosa cells from the P-36/eCG group. In contrast, LHCGR mRNA abundance was higher in superstimulated granulosa cells from the P-36 group and showed a pattern opposite to that of miR-222 expression. Ovarian superstimulation did not affect the expression of other markers (mmu-miR-202-5p, has-miR-873, has-miR-144, and their target genes, CREB, TGFBR2, and ATG7) of antral follicle development. However, the mRNA expression of VEGF pathway components was modulated by P-36 treatment. Taken together, these results demonstrate that superstimulatory protocols modify steroidogenic capacity, increase plasma estradiol, and regulate the abundance of VEGF system, LHCGR mRNA and suppress the expression of miR-222 in bovine granulosa cells.

In vitro production (IVP) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level, and relate these finding to previous data on oocyte developmental competence and ultrastructure. The cumulus/granulosa cell gene expression patterns indicate that early follicular atresia is associated with increased blastocyst yield and this hypothesis is supported by previous data on oocyte competence and ultrastructure.

In the female reproductive system, angiotensin II (ANG II) is a potential signaling molecule involved in ovarian follicle development, which acts through two transmembrane receptors. Within the ovarian follicle, there appear to be species differences in the precise pattern of localization of AGTR2 protein and it has an important role in in vitro maturation of oocytes in mammals. The infusion of ANG II induced ovulation in rabbits and the use of ANG II antagonists inhibited ovulation in rabbits, rats, and cattle. In fetal ovaries, AGTR2 protein was detected in ovigerous cords and preantral follicles throughout porcine and bovine gestation. In the oviduct, ANG II is responsible for the orchestration of the transport of gametes. In the male reproductive system, there is considerable evidence for the local synthesis of components of renin-angiotensin system (RAS) in male reproductive tissues. The roles of RAS in local processes at these sites are still uncertain, although there is evidence for involvement in tubular contractility, spermatogenesis, sperm maturation, capacitation, acrosomal exocytosis, and fertilization.

Several discoveries have been described recently (5-10 years) about the biology of ovarian follicles (oocyte, cumulus cells and granulosa cells), including new aspects of cellular communication, the control of oocyte maturation and the acquisition of oocyte competence for fertilization and further embryo development. These advances are nourishing assisted reproduction techniques (ART) with new possibilities, in which novel culture systems are being developed and tested to improve embryo yield and quality. This mini-review aims to describe how the recent knowledge on the physiological aspects of mammalian oocyte is reflecting as original or revisited approaches into the context of embryo production. These new insights include recent findings on the mechanisms that control oocyte maturation, especially modulating intraoocyte levels of cyclic nucleotides during in vitro maturation using endogenous or exogenous agents. In this mini-review we also discuss the positive and negative effects of these manipulations on the outcoming embryo.

Assisted reproductive technologies (ARTs) have a strong impact on breeding especially when coupled with genomic selection (GS). The routine implementation of in vitro production (IVP) and GS of embryos before embryo transfer (ET) in breeding companies is not yet possible. Improvement of oocyte donor and embryo recipient quality is needed to make realistic a commercialization of these procedures in the near future. A better understanding of both biological mechanisms and molecular markers associated to IVPET related traits is necessary to improve the prediction of donor and recipient cow quality for IVP procedures. The huge amount of data generated from high throughput technologies has a tremendous impact in the search for biomarkers of complex traits. This paper reviews integrative genomics and systems biology approaches as applied to both Bos indicus and Bos taurus cattle reproduction by both conventional and ARTs such as OPU-IVP. The integration of systems biology information across different biological layers generates a complete view of the different molecular networks that control complex traits and can provide a strong contribution to the understanding of traits related to ARTs.

Oocytes resume meiosis spontaneously when subjected to in vitro maturation (IVM). Cyclic adenosine monophosphate (cAMP) inductors have been used for artificial blocking of meiotic resumption to improve developmental competence of IVM oocytes. We tested a prematuration system that uses forskolin (FSK) and 3- isobutyl-1-methylxanthine (IBMX) to maintain higher concentrations of cAMP in the oocyte and sustain transzonal projections with gap junction communication within the cumulus-oocyte complex (COC). This system has been proved to prevent spontaneous meiosis resumption that usually occurs in the conventional IVM systems. Lipid accumulation in oocytes and IVP-embryos has been related to lower developmental potential and cryopreservation inability, especially when compared to their in vivo counterparts. We aimed to evaluate the effect of the in vitro prematuration system in the rates of blastocyst and on embryo quality by the semi-quantitative lipid content in oocytes and blastocysts from three systems: prematuration, conventional IVM and the laboratory control. Bovine immature oocytes were subjected to the prematuration system, in which COCs were cultured in the first 2h in TCM199 media with FSK (100 μM) and IBMX (500 μM), followed by 24 h of conventional IVM [TCM199, rhFSH (0.1 IU/mL), bovine serum albumine (4 mg/mL), pyruvate (0.011 g/mL) and amikacin (16.67 mg/μL) – Pre-IVM group]. Simultaneously, COCs were cultured for 24 h with the same IVM media described above for the conventional IVM (Con-IVM group). There was a third group as laboratory control cultured with the same media of the conventional IVM with the addition of 10% of fetal calf serum (FCS - Lab-Ctrl group). Matured oocytes were equally fertilized and cultured to the blastocyst stage in serum-free media, except for the Lab-Ctrl group where culture media was enriched with 2.5% of FCS. To evaluate the lipid content, we collected oocytes at different stages: immature, after 2h of prematuration and matured oocytes of the three different groups (Pre-IVM, Con-IVM and Lab-Ctrl). After assessing the blastocyst rates (day 7-8 after fertilization), expanded blastocysts from all groups were collected as well. Samples (oocytes and blastocysts) were fixed in formaldehyde (10% v/v) and stained in 1% Sudan black B/70% ethanol (w/v). The relative amount of lipid content was estimated by a digital image of each sample and examined in Image J software (v.1.41). Images were converted to a gray-scale pattern and for each structure we calculated the gray intensity per volume (arbitrary units/μm3 ). There was no difference related to rates of cleavage (data not shown) or blastocyst (percentage in relation to oocyte number ± standard error of the mean) among groups (26.52±3.86, 22.22±4.45 and 32.89±4.99, respectively to Pre-IVM, Con-IVM and Lab-Ctrl). Lipid semi-quantification of oocytes indicated that immature oocytes had the lowest lipid content (P 0.05). We can infer that the prematuration system did not affect the in vitro performance (blastocyst rates), but was able to affect the lipid accumulation differently in oocytes and blastocyst, in what seems to be a compensation mechanism during the preimplantation embryo development in vitro.

The Insulin-like Growth Factor (IGF) system has been demonstrated as an important factor to cumulus oocyte complex (COC) maturation and early embryo development. Studies demonstrate benefits of IGF-I addition during the in vitro maturation of oocytes in different species, including cattle. The IGF availability is controlled by two mechanisms: IGF Binding Proteins (IGFBPs), which inhibit IGF actions; and Pregnancy-Associated Plasma ProteinA (PAPP-A), responsible to cleavage the IGFBP and to release free IGF. The aim of this study was to investigate the effects of PAPP-A on cellular aspects of in vitro-matured COCs and further, to explore their impacts in the blastocysts’ yield and pattern of transcription. Bovine COCs (5 replicates/20 COC per replicate) from ovaries obtained in a local abattoir, predominantly from Nelore cows, were aspirated and matured in serum free medium in the absence (control group) or presence of 1 ng/mL (P1 group), 10 ng/mL (P10 group) or 100 ng/mL (P100 group) of PAPP-A. After 24 h of in vitro maturation (IVM), we assessed the meiosis progression by Hoescht staining and DNA fragmentation (apoptosis) by TUNEL assay, Matured COCs (5 replicates/20 COC per replicate) from each group were fertilized and cultured until blastocyst stage. On day seven, the blastocyst rate was analyzed and pools of three blastocysts were submitted to assess embryonic gene expression. The transcription pattern of 91 genes were analyzed by RT-qPCR using Taqman® assays in the HD-Biomark System® (96.96 dynamic array IFC) in four pools for control, P1 and P10 groups and five pools for P100 group. The statistical analysis was tested by ANOVA using PROC GLM procedure of SAS (SAS, 9.2, SAS Inst., Cary, NC, USA). Individual differences were analyzed through pair-wise comparisons (SAS). The addition of PAPP-A did not affect apoptotic rates: 42% (control), 50% (P1), 55 % (P10) and 45 % (P100). Moreover, the presence of PAPP-A during IVM did not affect the percentage of oocytes in metaphase II as compared to control group; however, it increased the number of oocytes with complete extrusion of the first polar body in the P100 group (90%) when compared to P1 group (71%). Also, no differences were demonstrated on embryo yield: cleavage rate (83%, 84%, 84% and 79%, in control, P1, P10 and P100 groups, respectively) and blastocyst rate (12%, 15%, 16% and 17%, in control, P1, P10 and P100 groups, respectively). However, the embryonic pattern of transcription was affected, demonstrating higher mRNA abundance of DNMT3A in the P1 and P100 when compared to control and P10 groups; higher mRNA abundance of ATF4 and POU5F1 in the P100 as compared to control group; higher mRNA abundance of STAT3 and PAF1 in P100 than the P10 group and higher mRNA abundance of TFAM in P100 then the control and P1 groups. Taken together, these data demonstrate an important role of PAPP-A in the control of oocyte maturation and further effects on embryonic gene expression. Specifically, the addition of 100 ng/mL of PAPP-A during the IVM of bovine COCs was able to positively influence the meiosis progression and affected the abundance of important genes related to embryo quality, possibly, by increasing the IGF bioavailability, which is known to support embryo development and improve embryo quality