Cytoplasmic granules in bovine oocytes do not affect embryonic or fetal development.

Oocyte cytoplasmic evaluation is based on homogeneity and granular appearance. Our study investigated if a granular cytoplasm, highly heterogeneous, would affect oocyte competence in bovine. In two experiments, bovine cumulus-oocyte complexes (COCs) with homogeneous cytoplasm (control, CC) and granulated cytoplasm (granular, GC) were selected from a regular pool of COCs. Experiment 1 was performed with slaughterhouse ovaries, and Experiment 2 was carried out in Girolando COCs obtained from ovum pick-up. Granular oocytes had higher caspase 3 levels (66.17 ± 11.61 vs 172.08 ± 16.95, P < 0.01) and similar GAP junction activity (5.64 ± 0.45 vs 6.29 ± 0.29). ZAR1 relative mRNA amount was lower in granular oocytes (178.27 ± 151.63 vs 0.89 ± 0.89, P = 0.01) and no effect was detected for MATER, PPP2R1A, ENY2, IGF2R, and BMP15 genes. Despite molecular differences, no detrimental effect was detected on oocyte competence in GC oocytes. Cleavage (Experiment 1: 59.52 ± 7.21% vs 59.79 ± 6.10% and Experiment 2: 68.88 ± 4.82 vs 74.41 ± 5.89%) and blastocyst (Experiment 1: 29.28 ± 4.14% vs 23.15 ± 2.96% and Experiment 2: 21.11 ± 3.28% vs 21.02 ± 6.08%) rates were similar between CC and GC (Experiments 1 and 2, respectively). Post-transfer embryo development revealed that pregnancy (CC: 24.27 ± 9.70% vs GC: 26.31 ± 7.23%) and calving (23.68% vs 33.33%) rates and fetal growth were not affected by the presence of cytoplasmic granules. Our results demonstrated that oocytes with granular cytoplasm present equivalent efficiency for IVF and calf production compared with homogenous cytoplasm oocytes. This could be observed through similar cleavage, blastocyst rates, and fetal growth development. In addition to differences in oocyte gene expression related to oocyte quality, it seems not to affect oocyte developmental competence.

Chemically induced enucleation of activated bovine oocytes: chromatin and microtubule organization and production of viable cytoplasts.

As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microtubules was observed in most oocytes, these structures did not disappear completely during assessment. Approximately 50% of treated oocytes presented microtubule reduction at the end of the evaluation period, while 23% of oocytes were observed to exhibit the complete disappearance of these structures and 28% exhibited visible microtubules. These findings indicated the lack of immediate microtubule repolymerization after culture in demecolcine-free medium, a fact that may negatively influence embryonic development. However, cleavage rates of 63.6-70.0% and blastocyst yield of 15.5-24.2% were obtained in the final experiment, without significant differences between techniques, indicating that chemically induced enucleation produces normal embryos.

HDAC inhibition decreases XIST expression on female IVP bovine blastocysts.

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, although in vitro culture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development. We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and the same was detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessed XIST and G6PD expression in individual female bovine blastocysts by quantitative real-time PCR. Even though G6PD expression remained unaltered after TSA exposure, XIST expression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectable XIST levels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrant XIST expression described for IVF embryos.

Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos.

Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA.

Placental scaffolds as a potential biological platform for embryonic stem cells differentiation into hepatic-like cells lineage: A pilot study.

Hepatic microenvironment plays an essential role in liver regeneration, providing the necessary conditions for cell proliferation, differentiation and tissue rearrangement. One of the key factors for hepatic tissue reconstruction is the extracellular matrix (ECM), which through collagenous and non-collagenous proteins provide a three-dimensional structure that confers support for cell adhesion and assists on their survival and maintenance. In this scenario, placental ECM may be eligible for hepatic tissue reconstruction, once these scaffolds hold the major components required for cell support. Therefore, this preliminary study aimed to access the possibility of mouse embryonic stem cells differentiation into hepatocyte-like cells on placental scaffolds in a three-dimensional dynamic system using a Rotary Cell Culture System. Following a four-phase differentiation protocol that simulates liver embryonic development events, the preliminary results showed that a significant quantity of cells adhered and interacted with the scaffold through outer and inner surfaces. Positive immunolabelling for alpha fetus protein and CK7 suggest presence of hepatoblast phenotype cells, and CK18 and Albumin positive immunolabelling suggest the presence of hepatocyte-like phenotype cells, demonstrating the presence of a heterogeneous population into the recellularized scaffolds. Periodic Acid Schiff-Diastase staining confirmed the presence of glycogen storage, indicating that differentiate cells acquired a hepatic-like phenotype. In conclusion, these preliminary results suggested that mouse placental scaffolds might be used as a biological platform for stem cells differentiation into hepatic-like cells and their establishment, which may be a promissing biomaterial for hepatic tissue reconstruction.

Recellularized rat testis scaffolds with embryoid bodies cells: a promising approach for tissue engineering.

Tissue engineering is gaining use to investigate the application of its techniques for infertility treatment. The use of pluripotent embryonic cells for in vitro production of viable spermatozoa in testicular scaffolds is a promising strategy that could solve male infertility. Due to cell-extracellular matrix (ECM) interactions, here we aim to investigate the differentiation of embryoid bodies (EBs) in cultured into decellularized rat testis scaffolds. Decellularized testis (P = 0.019) with a low concentration of gDNA (30.58 mg/ng tissue) was obtained by sodium dodecyl sulfate perfusion. The structural proteins (collagens type I and III) and the adhesive glycoproteins of ECM (laminin and fibronectin) were preserved according to histological and scanning electron microscopy (SEM) analyses. Then, decellularized rat testis were cultured for 7 days with EB, and EB mixed with retinoic acid (RA) in non-adherent plates. By SEM, we observe that embryonic stem cells adhered in the decellularized testis ECM. By immunofluorescence, we verified the positive expression of HSD17B3, GDNF, ACRV-1, and TRIM-36, indicating their differentiation using RA in vitro, reinforcing the possibility of EB in male germ cell differentiation. Finally, recellularized testis ECM may be a promising tool for future new approaches for testicular cell differentiation applied to assisted reproduction techniques and infertility treatment.Abbreviations: ACRV-1: Acrosomal vesicle protein 1; ATB: Penicillin-streptomycin; DAPI: 4,6-Diamidino-2-phenylindole; EB: Embryoid bodies; ECM: Extracellular matrix; ESCs: Pluripotent embryonic stem cells; GAGs: Glycosaminoglycans; gDNA: Genomic DNA; GDNF: Glial cell line-derived neurotrophic factor; H&E: Hematoxylin and eosin; HSD17B3: 17-beta-Hydroxysteroid dehydrogenase type 3; PBS: Phosphate-buffered saline; PGCLCs: Primordial germ-cell-like cells; RA: Retinoic acid; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SSCs: Spermatogonial stem cells; TRIM-36: Tripartite Motif Containing 36.

Growth performance, reproductive parameters and fertility measures in young Nellore bulls with divergent feed efficiency.

The growth, sexual maturity and fertility-related parameters related of young Nellore bulls with divergent residual feed intake (RFI) raised on pasture were evaluated. After classification of 48 young males as low and high RFI (more and less efficient, respectively), the animals were evaluated for growth and reproductive parameters at 28-day intervals from 14.3 to 24.6 months of age. The semen was cryopreserved in the last sampling and fresh and post-thaw semen samples were evaluated. Low RFI bulls exhibited higher initial and final body weight (P < 0.05), but feed intake, body condition score and growth measures evaluated by carcass ultrasound were unaffected by RFI (P > 0.05). The scrotal circumference, sperm concentration, defects, and quality of fresh semen, and ultrasonographic testicular characteristics were unaffected by RFI (P > 0.05). However, velocity parameters such as average path and curvilinear velocities determined by computer-assisted sperm analysis of thawed semen submitted to the rapid thermoresistance test were improved (P < 0.05) in low RFI bulls, but this improvement in quality did not enhance in vitro sperm fertilizing ability. Our results demonstrated significant differences in metabolism and growth performance between bulls of divergent RFI. In addition, there was slight improvement in the semen quality of bulls with low RFI bulls, but this did not enhance in vitro fertilizing ability. Selection of beef bulls for RFI can be performed, which will result in economic benefits by improving the growth performance of the animals without affecting reproductive parameters.

Ooplast-mediated developmental rescue of bovine oocytes exposed to ethidium bromide.

Ooplasm transfer has been used successfully to treat infertility in women with ooplasmic insufficiency and has culminated in the birth of healthy babies. To investigate whether mitochondrial dysfunction is a factor in ooplasmic insufficiency, bovine oocytes were exposed to ethidium bromide, an inhibitor of mitochondrial DNA replication and transcription, during in-vitro maturation (IVM). Exposure of immature oocytes to ethidium bromide for 24h during IVM hampered meiotic resumption and the migration of cortical granules. However, a briefer treatment with ethidium bromide during the last 4h of IVM led to partial arrest of preimplantation development without affecting oocyte maturation. Ooplasm transfer was then performed to rescue the oocytes with impaired development. In spite of this developmental hindrance, transfer of normal ooplasm into ethidium bromide-treated oocytes resulted in a complete rescue of embryonic development and the birth of heteroplasmic calves. Although this study unable to determine whether developmental rescue occurred exclusively through introduction of unaffected mitochondria into ethidium bromide-damaged oocytes, e.g. ethidium bromide may also affect other ooplasm components, these results clearly demonstrate that ooplasm transfer can completely rescue developmentally compromised oocytes, supporting the potential use of ooplasm transfer in therapeutic applications.

Meiotic inhibition of bovine oocytes in medium supplemented with a serum replacer and hormones: effects on meiosis progression and developmental capacity.

Aiming to improve the developmental competence of bovine oocytes during meiotic block, this study evaluated the effects of a serum replacer (Knockout SR®) and hormones (gonadotropins and estradiol) supplementation of prematuration medium (TCM119 with 0.5 mM IBMX [IBMX group] or 25 µM roscovitine [ROSC group]) on the kinetics of oocyte nuclear maturation and embryo development. Most IBMX and ROSC oocytes prematured for 8 h culture remained in the GV stage (70.3% and 73.1%, respectively; p > 0.05) similar to Control 8 h (63.5%) and to control immature oocytes (Control 0 h, 92.5%). After prematuration for 16 h, no oocytes remained in the GV stage at similar rates to those recently aspirated (p < 0.05); GV rates in ROSC (32.4%) were higher (p < 0.05) than in the Control 16 h group (8.6%), but similar (p > 0.05) to IBMX (9.7%). After in vitro maturation (IMV) for 24 h, metaphase II (MII) rates for oocytes prematured during 8 h were similar (p > 0.05) between control and treatments (65.0-71.7%). Similarly, MII rates oocytes prematured during 16 h were similar (p > 0.05) between all groups (45.9-60.4%). Cleavage rates (67.8-78.2%), embryonic development in day-7 (25.0-35.6%) and hatching rates in day-8 (2.5-11.3%) oocytes blocked during 8 h were similar for all groups (p > 0.05). Results indicate that addition of Knockout SR® and hormones to meiotic block culture with IBMX and roscovitine negatively affected meiotic arrest, but did not impair oocyte nuclear maturation and acquisition of developmental competence.

Pronounced segregation of donor mitochondria introduced by bovine ooplasmic transfer to the female germ-line.

Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offspring's oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygote's ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% +/- 9.6% versus 12.1% +/- 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents-the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r(2) = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.

Applications of mesenchymal stem cell technology in bovine species.

Mesenchymal stem cells (MSCs) have received a great deal of attention over the past 20 years mainly because of the results that showed regeneration potential and plasticity that were much stronger than expected in prior decades. Recent findings in this field have contributed to progress in the establishment of cell differentiation methods, which have made stem cell therapy more clinically attractive. In addition, MSCs are easy to isolate and have anti-inflammatory and angiogenic capabilities. The use of stem cell therapy is currently supported by scientific literature in the treatment of several animal health conditions. MSC may be administered for autologous or allogenic therapy following either a fresh isolation or a thawing of a previously frozen culture. Despite the fact that MSCs have been widely used for the treatment of companion and sport animals, little is known about their clinical and biotechnological potential in the economically relevant livestock industry. This review focuses on describing the key characteristics of potential applications of MSC therapy in livestock production and explores the themes such as the concept, culture, and characterization of mesenchymal stem cells; bovine mesenchymal stem cell isolation; applications and perspectives on commercial interests and farm relevance of MSC in bovine species; and applications in translational research.

Sexual maturity and fertility-related measures in young Nellore bulls receiving long-term dietary supplementation with rumen-protected polyunsaturated fatty acids.

The objective of this study was to evaluate the effects of long-term supplementation with rumen-protected fatty acids (FA) on growth and reproductive parameters of young Nellore bulls in a grazing regime. Forty-eight young bulls were distributed into two groups: FA (supplemented with rumen-protected polyunsaturated FA); and control (control fat-free supplement). The animals were supplemented from 14.3 to 24.6 months of age and growth and reproductive parameters were evaluated at 28-day intervals. The semen was cryopreserved in the last collection and fresh and post-thaw semen samples were evaluated. Feeding FA did not affect (P > 0.05) growth, reproductive parameters (scrotal circumference, sperm concentration per mL of ejaculate, percentage of sperm defects, sperm quality and fertility in vitro), or testicular ultrasonographic characteristics. However, thawed semen from bulls fed FA exhibited better quality (P < 0.05) than control semen for the following parameters evaluated by computer-assisted sperm analysis: average path velocity [µm/s: 90.48 vs. 79.66 post-thaw and 74.81 vs. 72.80 post-rapid thermoresistance test (TRT)], straight-line velocity (µm/s: 72.37 vs. 65.20 post-thaw and 64.96 vs. 63.25 post-TRT), and curvilinear velocity (µm/s: 148.44 vs. 131.31 post-thaw and 115.68 vs. 113.35 post-TRT). In addition, feeding FA increased peripheral concentrations of testosterone, leptin, total cholesterol and high-density lipoprotein. In conclusion, the increase in testosterone concentrations in bulls fed FA was not related to variations in growth parameters and sexual maturity. In addition, post-thawing sperm velocities were enhanced by diet, however, such increases were not related to better in vitro embryo production rates.

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos.

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility.

Cryopreservation of bull semen is a common biotechnology procedure in cattle breeding. However, when the ejaculate is obtained by electroejaculation, wide variation is observed in the sperm/seminal plasma (SP) ratio that can affect the freezability of semen in this species. The removal of SP may improve the quality of frozen bull semen. The objective of this study was to evaluate the effect of SP removal from the ejaculate on the cryopreservation of semen from 38 Nellore bulls collected by electroejaculation. After collection, the ejaculate was divided into three aliquots: (1) control (N) diluted to a concentration of 60 × 106 spermatozoa/mL and frozen with SP; (2) centrifugation (C) at ×600g for 10 minutes and the pellet resuspended and frozen at the same concentration as N; and (3) filtration (F) through SpermFilter and sperm recovered and frozen at the same concentration as N. After thawing, sperm kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, oxidative stress, and in vitro fertility were evaluated. Statistical analysis was performed using the SAS 9.2 package, and differences were considered significant when P < 0.05. Higher average path velocity and straight-line velocity were observed in the groups submitted to SP removal compared to the control group (P < 0.01). In contrast, filtered samples exhibited higher beat cross frequency, straightness, and linearity compared to the other groups. Plasma membrane integrity was reduced when SP was removed, but lower oxidative stress was observed in groups C and F (34.91 ± 2.95% and 31.63 ± 2.95%, respectively) compared to group N (57.39 ± 2.95%). However, the percentage of hatched blastocysts was similar in the N and F groups (21.22 ± 1.05% and 24.00 ± 1.05%, respectively) and higher compared to group C (18.83 ± 1.05%). In conclusion, removal of SP by centrifugation for bull semen freezing reduced the rate of in vitro-produced embryos, whereas filtration of prefrozen semen was found to be an efficient alternative in terms of semen freezability and in vitro production of bovine embryos.

Use of strontium for bovine oocyte activation.

Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50 mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca2+- and Mg2+-free TALP for 6 h), ionomycin+strontium (IS), and strontium+ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 microM for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to I, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were similar for S, I, IS, and SI treatments (61.7-76.7, and 8.3-13.3%, respectively) and the same was observed for ICM, TE, and total cell number, and ICM/total cell ratio (22-25, 64-69, and 86-95, and 0.26-0.27). In conclusion, strontium may be efficiently applied for bovine oocyte activation at 20 mM in Ca2+- and Mg2+-free TALP medium for 6 h.

Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium.

Efficient artificial activation is indispensable for the success of cloning programs. Strontium has been shown to effectively activate mouse oocytes for nuclear transfer procedures, however, there is limited information on its use for bovine oocytes. The present study had as objectives: (1). to assess the ability of strontium to induce activation and parthenogenetic development in bovine oocytes of different maturational ages in comparison with ethanol; and (2). to verify whether the combination of both treatments improves activation and parthenogenetic development rates. Bovine oocytes were in vitro matured for 24, 26, 28, and 30 h, and treated with ethanol (E, 7% for 5 min) or strontium chloride (S, 10mM SrCl(2) for 5h) alone or in combination: ethanol+strontium (ES) and strontium+ethanol (SE). Activated oocytes were cultured in vitro in synthetic oviductal fluid (SOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage (D7). Treatment with ethanol and strontium promoted similar results regarding pronuclear formation (E, 20-66.7%; S, 26.7-53.3%; P>0.05) and cleavage (E, 12.8-40.6%; S, 16.1-41.9%; P>0.05), regardless of oocyte age. The actions of both strontium and ethanol were influenced by oocyte age: ethanol induced greater activation rates after 28 and 30 h of maturation (48.4 and 66.7% versus 20.0 and 23.3% for 24 and 26 h, respectively; P<0.05) and strontium after 30 h (53.3%) was superior to 24 and 26 h (26.7% for both). Blastocyst development rates were minimal in all treatments (0.0-6.3%; P>0.05), however, when the mean (+/-S.D.) cell number in blastocysts at the same maturational period was compared, strontium treatment was superior to ethanol for activation rates (82+/-5.7 and 89.5+/-7.8 versus 54 and 61, at 28 and 30 h, respectively). Improved results were obtained by combined treatments. The combination of ethanol and strontium resulted in similar pronuclear formation (ES, 36.7-83.9%; SE, 53.1-90.3%) and cleavage rates (ES, 31.3-81.3%; SE, 65.6-80.7%). Regarding embryo development, there was no difference (P>0.05) between treatments, and blastocysts were only obtained in treatment SE at 24 and 26 h (6.5% for both). It is concluded that, SrCl(2) induces activation and parthenogenetic development in bovine oocytes.

Chemically assisted enucleation results in higher G6PD expression in early bovine female embryos obtained by somatic cell nuclear transfer.

Despite extensive efforts, low efficiency is still an issue in bovine somatic cell nuclear transfer (SCNT). The hypothesis of our study was that the use of cytoplasts produced by chemically assisted enucleation (EN) would improve nuclear reprogramming in nuclear transfer (NT)-derived embryos because it results in lower damage and higher cytoplasm content than conventional EN. For that purpose, we investigated the expression of two X-linked genes: X inactive-specific transcript (XIST) and glucose 6-phosphate dehydrogenase (G6PD). In the first experiment, gene expression was assessed in day-7 female blastocysts from embryonic cell NT (ECNT) groups [conventional, ECNT conv; chemically assisted, ECNT deme (demecolcine)]. Whereas in the ECNT conv group, only one embryo (25%; n=4) expressed XIST transcripts, most embryos showed XIST expression (75%; n=4) in the ECNT deme group. However, no significant differences in transcript abundance of XIST and G6PD were found when comparing the embryos from all groups. In a second experiment using somatic cells as nuclear donors, we evaluated gene expression profiles in female SCNT-derived embryos. No significant differences in relative abundance (RA) of XIST transcripts were observed among the groups. Nonetheless, higher (p<0.05) levels of G6PD were observed in SCNT deme and in vitro-derived groups in comparison to SCNT conv. To know whether higher G6PD expression in embryos derived from SCNT chemically assisted EN indicates higher metabolism in embryos considered of superior quality or if the presence of higher reactive oxygen species (ROS) levels generated by the increased oxygen consumption triggers G6PD activation, the expression of genes related to stress response should be investigated in embryos produced by that technique.

The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development.

Aiming to standardize in vitro production of bovine embryos and to obtain supplements to replace serum in culture media, this study evaluated the nuclear maturation kinetics and embryonic development in bovine after in vitro maturation (IVM) and culture (IVC) with several macromolecules (animal origin: bovine serum albumin (BSA), fetal calf serum (FCS); synthetic: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Ficoll, and Knockout) at two oxygen tensions (20% and 5% O(2)). Regarding nuclear kinetics, neither the presence of the expected stage (metaphase I, transition anaphase to telophase, and metaphase II) at each evaluation moment (6, 18, and 24 h after IVM, respectively) nor the accelerated polar body emission (at 18 h after IVM) related developmental competence to blastocyst stage when different supplements were compared. Independently of supplement, cleavage rates at 20% O(2) (61.6-79.2%) were higher than at 5% O(2) (38.9-58.7%). At 20% O(2), higher blastocyst and hatching rates, respectively, were obtained in treatments BSA, FCS, Knockout, and control group (IVM with FCS and IVC with BSA + FCS, 14.0-23.5% and 6.8-15.4%) in comparison to PVA, PVP, and Ficoll (0%). The same was observed at 5% O(2) for blastocyst rates with BSA, FCS, Knockout, and control (5.4-16.8%) and for hatching rates with BSA, FCS, and control (2.0-11.1%). We can conclude that producing bovine embryos at 20% O(2) during the entire IVP process resulted in higher developmental rates than at 5% O(2). In addition, while defined macromolecules PVA, PVP, and Ficoll were not suitable for embryonic development, the synthetic serum Knockout was able to replace serum and albumin for IVP in bovine at 20% O(2).

Conception rate of lactating cows and heifers (Bos taurus X Bos indicus) for sexed and conventional semen in artificial insemination / Taxa de concepção de vacas lactantes e novilhas (Bos taurus X Bos indicus) com o uso de sêmen sexado e convencional na inseminação artificial

The objective of this study was to evaluate the conception rate of crossbred heifers (n=50) and cows (n=50) inseminated with sexed and conventional semen between 18 and 24 hours after estrous detection. The synchronization protocol of the estrous cycle started on day zero (D0) by inserting the intravaginal device with 1g progesterone (Sincrogest® Ourofino, Brazil) and injecting 2 mg of estradiol benzoate, intramuscularly (Sincrodiol® Ourofino, Brazil). On the fifth day (D5), 200 IU of equine chorionic gonadotrophin was injected intramuscularly (Folligon®, Intervet, Brazil). On the eighth day (D8), after removing the progesterone device, 500 g of sodium cloprostenol was injected intramuscularly (Sincrocio®, Ourofino, Brazil). After that, the animals were checked for estrus 3 times daily, and inseminated 18 to 24 hours after estrus detection. Pregnancy diagnosis was performed 30 to 40 days after insemination. Conception rate did not differ (P> 0.05) according to animal category, but was higher for conventional semen compared to sexed semen when evaluating the total of animals and lactating cows (P <0.05). Artificial insemination of heifers with sexed semen 18 to 24 hours after estrus detection was effective, however, conventional semen was more efficient in lactating cows.

Considerando os benefícios do uso de sêmen sexado e também os danos causados pelo processo de separação dos espermatozoides, o objetivo do presente estudo foi avaliar a taxa de concepção de novilhas (n=50) e vacas (n=50) mestiças inseminadas com sêmen sexado e convencional após 18 a 24 horas a observação do cio. O protocolo de sincronização do ciclo estral consistiu em inserção de dispositivo intravaginal com 1g de progesterona (Sincrogest® Ourofino, Brasil) e aplicação intramuscular de 2mg de benzoato de estradiol (Sincrodiol® Ourofino, Brasil) no dia zero (D0). No quinto dia (D5), foi realizada uma aplicação intramuscular de 200UI de gonadotrofina coriônica equina (Folligon®, Intervet, Brasil). No oitavo dia (D8), o dispositivo de progesterona foi retirado, e aplicado por via intramuscular 500µg de cloprostenol sódico (Sincrocio®, Ourofino, Brasil). A partir deste momento, o estro foi observado 3 vezes ao dia e os animais foram inseminados 18 a 24 após a detecção do cio. O diagnóstico de gestação foi realizado 30 a 40 dias após a inseminação. Não foi observada diferença na taxa de concepção de acordo com a categoria animal (P > 0,05), entretanto, animais inseminados com sêmen convencional apresentaram melhor taxa de concepção do que com sêmen sexado quando se avaliou o total de animais e vacas lactantes (P < 0,05). A inseminação artificial de novilhas com sêmen sexado 18 a 24 horas após detecção de estro mostrou-se eficaz, entretanto, para vacas lactantes não foi observada a mesma eficiência ao se comparar com o sêmen convencional.

Chromatin modifying agents in the in vitro production of bovine embryos.

The low efficiency observed in cloning by nuclear transfer is related to an aberrant gene expression following errors in epigenetic reprogramming. Recent studies have focused on further understanding of the modifications that take place in the chromatin of embryos during the preimplantation period, through the use of chromatin modifying agents. The goal of these studies is to identify the factors involved in nuclear reprogramming and to adjust in vitro manipulations in order to better mimic in vivo conditions. Therefore, proper knowledge of epigenetic reprogramming is necessary to prevent possible epigenetic errors and to improve efficiency and the use of in vitro fertilization and cloning technologies in cattle and other species.