Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

The presence of Leptospira spp. in the follicular fluid of naturally infected cows affects the overall efficiency of the in vitro embryo production technique.

The relationship between Leptospira infection and reproductive failures, as well as the mechanisms that lead to it, has not yet been fully established. It has been hypothesized that the presence of Leptospira spp. in the follicular fluid (FF) could impair the oocyte developmental competence. Thus, the impact of the presence of Leptospira spp. in the FF on in vitro embryo production (IVEP) outcomes was assessed. Dairy cows (n=244) from different farms were subjected to ovum pick-up for cumulus-oocyte complexes (COCs) collection. After PCR analysis of the FF, cows were retrospectively allocated into either: positive (POS-FF) or negative (NEG-FF) group. Statistical modeling was conducted using the farm, PCR result, and laboratory in which the IVEP was performed as effects. Noteworthy, 26.6% of the animals were positive for Leptospira spp., and 70% of farms had at least one POS-FF cow in the herd. POS-FF cows had a lower number of COCs recovered (22.6 ± 1.2 vs 15.0 ± 2.8, P=0.036), rate of viable COCs (85.6 ± 0.9% vs 78.1 ± 2.8%, P=0.015), number of good-quality COCs (16.0 ± 0.9 vs 9.8 ± 2.1, P=0.026), cleaved embryos (11.9 ± 0.7 vs 7.5 ± 1.5, P=0.032), and blastocysts (7.3 ± 0.4 vs 2.3 ± 0.7, P=0.044) yielded per cow. In conclusion, the presence of Leptospira spp. in the FF of naturally infected cows impaired the amount of COCs recovered, decreasing the overall IVEP efficiency.

Oocyte competence and gene expression in parthenogenetic produced embryos from repeat breeder and normally fertile buffaloes (Bubalus bubalis) raised in sub-humid tropical climate.

The reproductive management of the buffalo species still faces several unresolved problems, which directly affect the productivity of the herd, one of them being the presence of repeat breeder females. Given this scenario, this study aimed to verify the developmental competence of oocytes obtained from repeat breeder females and submitted to parthenogenetic activation. In addition, embryo gene expression was compared to normally fertile females. Murrah buffaloes were divided into two groups: repeat breeder (RB, n = 8) and normally fertile or control (CR, n = 7). Cumulus-oocyte complexes (COCs) were aspirated by transvaginal ovum pick-up from estrus synchronized females. The COCs were submitted to IVM for 24 h, and subsequently, the oocytes were activated using ionomycin, followed by 6-DMAP. Afterwards, the presumptive parthenotes were cultured for six or seven days in a microenvironment of 5 % CO2, 5 % O2, and 90 % N2 at 38.5 °C. The expression of OCT4, GLUT1, BCL2 and TFAM genes from blastocysts was evaluated. The overall COCs recovery rate was 70.9 % (190/268). The maturation (57.8 vs 71.1), cleavage (45.2 vs 62.2) and blastocyst (30.1 vs 45.9) rates did not differ (P > 0.05) between RB and CR females, respectively. Similarly, no significant difference (P > 0.05) was observed for the expression of studied genes in both RB and CR females. In conclusion, oocytes obtained from RB were as developmentally competent as those collected from CR females, with similar energy metabolism and in vitro development capacity. Thus, the low fertility rate of repeat breeder buffaloes, when compared to normal cyclic females, must be due to subsequent events to the blastocyst stage.

Supraphysiological doses of nandrolone decanoate disrupts spermatogenesis but did not interfere on embryo rate.

The aim of this study was to investigate the effects of 10 mg/kg/week of nandrolone decanoate (DECA - Deca Durabolin®) on body composition, hormonal levels, spermatic parameters, redox status, and morphometric parameters of testicle and epididymis; furthermore, the fertility capacity of Wistar rats was measured thought in vitro fertilization (IVF). The animals (n = 16) were divided into two groups: control group (CTRL, n = 8), which received only vehicle composed by peanut oil and 10% of the benzoic alcohol and nandrolone decanoate group (DECA, n = 8), which received intramuscular injections of DECA for 8 weeks, both groups were treated for 8 weeks. The results demonstrate significative decrease in visceral fat, testosterone levels, and thiol content on epididymis, reduction on normal sperm parameters, and deleterious effect on testicles and epididymis tissue morphology showing reduction of germ height and luminal diameter on the DECA group. Thus, it can be concluded that high doses of nandrolone decanoate impairs male reproductive parameters.

Micro-vibration results in vitro-derived bovine blastocysts with greater cryotolerance, epigenetic abnormalities, and a massive transcriptional change.

Much effort has been employed to improve the quality of embryos obtained by in vitro production (IVP) given the relevance of this technology to current livestock systems. In this context, dynamic IVP systems have proved beneficial to the embryo once they mimic fluid flows and mechanical forces resulting from the movement of ciliated cells and muscle contraction in the reproductive tract. In the present study, we sought to confirm these initial findings as well as assess potential molecular consequences to the embryo by applying micro-vibration (45 Hz for 5 s once per 60 min) during both oocyte maturation and embryo culture in cattle. As a result, micro-vibration led to lower incidence of apoptosis in blastocysts following vitrification-thawing. Further analyses revealed epigenetic and transcriptional changes in blastocysts derived from the micro-vibration treatment, with a total of 502 differentially expressed genes. Enrichment analyses linked differentially expressed genes to 'Oxidative phosphorylation', 'Cytokine-cytokine receptor interaction', and 'Signaling pathways regulating pluripotency of stem cells'. Yet, a meta-analysis indicated that the transcriptional changes induced by micro-vibration were not toward that of in vivo-derived embryos. In conclusion, micro-vibration increases the cryoresistance of bovine embryos, but caution should be taken given the unclear consequences of epigenetic and transcriptional abnormalities induced by the treatment.

Effect of Nellore (Bos indicus) donor age on in-vitro embryo production and pregnancy rate.

The aim of this study was to evaluate the effect of age of Nellore (Bos indicus) donors on the efficiency of in vitro embryo production (IVEP) and pregnancy rate. Thirty-six donors, including 11 female calves (13 ± 0.61 months), 17 prepubertal heifers (25 ± 0.78 months) and 8 cows (83 ± 28 months), were submitted to 3 procedures of ovum pickup (OPU) on random days of the estrous cycle at intervals of 21 days. Caspase-3 and IGFBP2 were quantified in oocytes and blastocysts for the evaluation of oocyte and embryo quality. The produced embryos were vitrified (n = 445) and transferred to synchronized recipients. Cows produced a larger number of follicles (cows: 54.5 ± 6.2; calves: 20.0 ± 0.57; prepubertal heifers: 20.8 ± 0.46), total oocytes (cows: 45.97 ± 7.22; calves: 28.93 ± 6.14; prepubertal heifers: 27.21 ± 4.94) and cleaved oocytes (cows: 21.14 ± 4.22; calves: 13.09 ± 3.72; prepubertal heifers: 12.4 ± 3.19). The cleavage rate was similar between age categories; however, cows tended (p < 0.07) to produce a larger number of blastocysts (9.74 ± 2.26) per OPU than calves (5.57 ± 1.99) and prepubertal heifers tended to have a higher blastocyst yield (35.4%) than calves (27.1%) (p < .07). The expression levels of IGFBP2 and caspase-3 were higher in oocytes derived from calves compared to the other two categories. The pregnancy rate was higher in calves (43.1%) and cows (40.4%) than in prepubertal heifers (33.8%) (p = .03). Despite the larger numbers of follicles and viable oocytes in cows, the blastocyst production results and pregnancy rates obtained indicate that the use of young females as oocyte donors in IVEP is feasible and may contribute to reduce the generation interval.

Antral follicle count, oocyte production and embryonic developmental competence of senescent Nellore (Bos indicus) cows.

Information on the follicular population and oocyte quality of cows in the final period of reproductive life is scarce. The present study aimed to compare the antral follicle count (AFC), oocyte production and embryonic developmental competence of young versus long-lived and senescent Bos indicus beef cows. Nellore cows (Bos indicus) were classified into three groups according to age: young (4-9 years, n = 10), long-lived (14-17 years, n = 10) and senescent (17-23 years, n = 10). At a random time in the estrus cycle, the cows received cloprostenol sodium salt (0.5 mg, IM), estradiol benzoate (1 mg, IM) and an intravaginal P4 device (1.4 g). Five days later, the P4 devise was removed and oocyte collection (OPU1) was performed. A second OPU (OPU2) was performed 5 days after the first in order to aspirate only growing follicles. During each OPU, AFC and the number and quality of cumulus-oocyte complexes (COCs) were evaluated. Then, the COCs were placed in standard maturation medium (IVM), fertilized and incubated for 9 days. The data were subjected to ANOVA and Multinomial Logistic Regression. The AFC was smaller in long-lived and senescent cows in both OPU1 and OPU2 when compared to younger cows. There was no difference in AFC between OPU1 (19.9 ± 1.8) and OPU2 (17.6 ± 1.9) in young cows, however, more follicles were punctured in long-lived and senescent cows in OPU1 (12.0 ± 2.6 and 19.3 ± 4.6) than in OPU2 (9.2 ± 1.9 and 10.3 ± 2.3), respectively (P < 0.01). The numbers of COCs recovered from young cows (OPU1 = 14.2 ± 1.8; OPU2 = 8.4 ± 0.9) were higher than those obtained from long-lived cows (OPU1 = 5.9 ± 2.3; OPU2 = 4.3 ± 1.0) and senescent cows (OPU1 = 7.2 ± 3.0; OPU2 = 4.1 ± 1.7), respectively (P < 0.05). The cleavage rate did not differ between groups. However, the rate of blastocyst formation was higher for young (64.8%) and long-lived (65.0%) compared to senescent (16.5%) cows (P < 0.01). In conclusion our results indicate that the AFC is lower in long-lived and senescent cows compared with young cows. However, unlike in senescent cows, the embryonic development of long-lived cows is similar to that of young cows. This suggests that Nellore cows aged >17 years begin to have reduced embryonic development capacity due to ovarian aging.

Silvopastoral system is an alternative to improve animal welfare and productive performance in meat production systems.

Climate change is a reality and global surface temperature is projected to rise substantially in the next 80 years. Agriculture practices will have to adapt to climate change, and also help to mitigate this effect using, among other strategies, forest conservation and management. Silvopastoral systems have been adopted in tropical climate livestock areas but their benefits on thermal comfort and reproductive performance of beef cows are not completely known. Therefore, our aims were to compare the microclimate of silvopastoral and intensive rotational unshaded grazing systems in different months and to evaluate physiological variables (Exp. 1 and 2), metabolism, and in vitro embryo production (Exp. 2) in crossbred beef females. Our hypothesis is that the silvopastoral system can improve the thermal comfort of beef heifers and cows and, consequently, also improve dry matter intake, body weight gain, and in vitro embryo production when compared to the unshaded rotational grazing system. In Exp 1, the silvopastoral system decreased body temperature and increased welfare and performance of heifers. In Exp. 2, the silvopastoral system enhanced the body weight but did not affect metabolism and the general reproductive performance, but increased the recovery rate of oocytes in primiparous cows.

Superstimulation prior to the ovum pick-up improves the in vitro embryo production in nulliparous, primiparous and multiparous buffalo (Bubalus bubalis) donors.

The aim of this study was to evaluate the ovarian follicular population, the oocyte yield and the in vitro embryo production (IVEP) of nulliparous (NU), primiparous (PR) and multiparous (MU) buffalo donors submitted to the superstimulation with FSH prior to the ovum pick-up (OPU). A total of 54 buffalo donors (18 NU, 15 PR and 21MU) received an intravaginal progesterone device (1.0 g) plus estradiol benzoate [2.0 mg, intramuscular (im)] at random stage of the estrous cycle (Day 0) during the breeding season (autumn and winter). Buffaloes from different categories were then randomly allocated to one of two groups (Control or FSH), in a cross-over experimental design. Buffalo donors in the Control group received no further treatment, whereas buffalo donors in the FSH group received a total dosage of 200 mg im of FSH on Days 4 and 5, in four decreasing doses 12 h apart (57, 57, 43 and 43 mg). On Day 7, the progesterone device was removed and the OPU procedure was performed in both groups. The same semen was used across all replicates and donor category. Data were analyzed by the GLIMMIX procedure of SAS 9.4®. There was no interaction between FSH treatment and animal category for all analyzed variables. Furthermore, no differences between animal category (P = 0.73) and FSH treatment (P = 0.53) were observed regarding the total follicles aspirated. However, the FSH treatment increased (P < 0.001) the proportion of large (>10 mm; FSH = 16.2% and Control = 2.0%) and medium-sized follicles (6-10 mm; FSH = 36.3% and Control = 6.1%) available for the OPU procedure. The total of recovered oocytes was greater in NU than in MU, and PR were similar to NU and MU (P = 0.05). No effect of FSH treatment was observed (P = 0.85) for this variable. Buffalo donors treated with FSH had a greater viable oocytes rate (P = 0.03), blastocyst rate (P = 0.03) and embryo yield per OPU-IVEP session (P = 0.07), however, no category effects were observed for these variables. These results provided evidence that superstimulation with FSH increased the proportion of large and medium-sized follicles available for the OPU procedure. Consequently, the FSH treatment enhanced the proportion of viable oocytes for culture and resulted in greater blastocyst rates and embryo yield per OPU-IVEP session in all buffalo donors categories.

Influence of post-thawing thermal environment on bovine sperm characteristics and in vitro fertility.

Our aim was to evaluate the effects of three thermal environments over time on kinetics, functionality and in vitro fertility of cryopreserved bovine spermatozoa. Four ejaculates from five bulls (n = 20) were cryopreserved. After thawing, semen was evaluated (0 hr), incubated for 4 hr in T36.0 (36.0°C), T38.0 (38.0°C) and T39.5 (39.5°C), and analysed every hour (1 hr, 2 hr, 3 hr, 4 hr). In vitro production of embryos was performed at 0 hr and 4 hr. Sperm motility and cell kinetics (Computer-Assisted Sperm Analysis) were impaired after 2 hr at T38.0 and T39.5 (p < 0.05). Flow cytometry revealed an increase in the cells with injured plasma membrane to 39.5°C and a general reduction in the mitochondrial potential over time (p < 0.05). In vitro fertility was impaired in all temperatures after 4 hr, but there was no difference between 36.0°C and 38.0°C. Our results suggest that the ex situ resilience of semen at 36.0°C after thawing with no major damage to the quality is limited to 3 hr. In normothermia or in thermal stress, sperm cells present a gradual reduction of movement and functionality, which were more significant after 1 hr of incubation. The in vitro production of embryos is impaired when the semen is kept in a thermal environment ≥36.0°C for 4 hr.

Effects of melatonin during IVM in defined medium on oocyte meiosis, oxidative stress, and subsequent embryo development.

Melatonin may have beneficial effects when used in oocyte maturation and embryo development culture. The effect of melatonin during IVM on meiosis resumption and progression in bovine oocytes and on expression of antioxidant enzymes, nuclear fragmentation and free radicals, as well as on embryo development were assessed. Cumulus-oocyte complexes were matured in vitro with melatonin (10(-9) and 10(-6) M), FSH (positive control), or without hormones (negative control) in defined medium. Maturation rates were evaluated at 6, 12, 18, and 24 hours. Transcripts for antioxidant enzymes (CuZnSOD, MnSOD, and glutathione peroxidase 4 (GPX4)) in oocytes and cumulus cells, nuclear fragmentation in cumulus cells (TUNEL) and reactive oxygen species levels in oocytes (carboxy-H2 difluorofluorescein diacetate) were determined at 24 hours IVM. Effect of treatments on embryo development was determined after in vitro fertilization and culture. At 12 hours, meiosis resumption rates in FSH and melatonin-treated groups were similar (69.6%-81.8%, P > 0.05). At 24 hours, most oocytes were in metaphase II, with FSH showing highest rates (90.0%, P < 0.05) compared with the other groups (51.6%-69.1%, P > 0.05). In cumulus cells, MnSOD expression was higher in FSH group (P < 0.05) whereas Cu,ZnSOD transcripts were more abundant in melatonin group (10(-6)M; P < 0.05). Nuclear fragmentation in cumulus cells was highest in controls (37.4%/10,000 cells; P < 0.05) and lower in FSH and 10(-6)M melatonin (29.4% and 25.6%/10,000 cells, respectively). Reactive oxygen species levels were lower in oocytes matured with 10(-6)M melatonin than in control and FSH groups (P < 0.05). Embryo development from oocytes matured only with melatonin was similar to those matured in complete medium (P > 0.05). In conclusion, although melatonin during IVM in a defined medium does not stimulate nuclear maturation progression it does stimulate meiosis resumption and such treated oocytes support subsequent embryo development. Melatonin also shows cytoprotective effects on cumulus-oocyte complexes.

The Infertility of Repeat-Breeder Cows During Summer Is Associated with Decreased Mitochondrial DNA and Increased Expression of Mitochondrial and Apoptotic Genes in Oocytes.

Oocyte quality is known to be a major cause of infertility in repeat-breeder (RB) and heat-stressed dairy cows. However, the mechanisms by which RB oocytes become less capable of supporting embryo development remain largely unknown. Thus, the aim of this study was to investigate whether the decreased oocyte competence of RB cows (RBs) during summer is associated with an altered gene expression profile and a decrease in mitochondrial DNA (mtDNA) copy number. Therefore, oocytes collected from heifers, non-RBs in peak lactation (PLs), and RBs were used to evaluate mtDNA amounts as well as the expression levels of genes associated with the mitochondria (MT-CO1, NRF1, POLG, POLG2, PPARGC1A, and TFAM), apoptosis (BAX, BCL2, and ITM2B), and oocyte maturation (BMP15, FGF8, FGF10, FGF16, FGF17, and GDF9). The oocytes retrieved from RBs during winter contained over eight times more mtDNA than those retrieved from RBs during summer. They also contained significantly less mtDNA than oocytes retrieved from heifers and PLs during summer. Moreover, the expression of mitochondria- (NRF1, POLG, POLG2, PPARGC1A, and TFAM) and apoptosis-related (BAX and ITM2B) genes, as well as of GDF9, in RB oocytes collected during summer was significantly greater than that in oocytes collected from heifers and PLs during the same season. In oocytes from heifers and PLs, the expression levels of these genes were lower in those collected during summer compared with winter, but this difference was not observed in oocytes collected from RBs. Altogether, these data provide evidence of altered gene expression and reduced mtDNA copy number in the oocytes collected from RBs during summer. This indicates a loss of fertility in RBs during summer, which might be caused by a possible mitochondrial dysfunction associated with a greater chance of oocytes to undergo apoptosis.

Parthenogenesis and Human Assisted Reproduction.

Parthenogenetic activation of human oocytes obtained from infertility treatments has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself, somatic cell, and nuclear transfer experiments and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. Different activating methods have been tested on human and nonhuman oocytes, with varying degrees of success in terms of parthenote generation rates, embryo development stem cell derivation rates. Success in achieving a standardized artificial activation methodology for human oocytes and the subsequent potential therapeutic gain obtained from these embryos depends mainly on the availability of gametes donated from infertility treatments. This review will focus on the creation of parthenotes from clinically unusable oocytes for derivation and establishment of human parthenogenetic stem cell lines and their potential applications in regenerative medicine.

The interval between the emergence of pharmacologically synchronized ovarian follicular waves and ovum pickup does not significantly affect in vitro embryo production in Bos indicus, Bos taurus, and Bubalus bubalis.

The aim of the present study was to determine the optimal phase of the follicular wave to perform ovum pickup (OPU) for in vitro embryo production (IVEP) in various genetic groups. For this purpose, 27 heifers-nine Bos taurus (Holstein), nine Bos indicus (Nelore), and nine Bubalus bubalis (Mediterranean)-were maintained under the same nutritional, management, and environmental conditions. Heifers within each genetic group were submitted to six consecutive OPU trials with 14-day intersession intervals, at three different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5 after follicular wave emergence), in a 3 × 3 crossover design. When OPU was performed at different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5), no differences were found in the percent of oocytes recovered (70.5 ± 3.1%, 75.0 ± 3.1%, 76.0 ± 3.2%, respectively; P = 0.41) or blastocyst production rates (19.4 ± 2.9%, 16.6 ± 2.9%, 15.9 ± 2.6%, respectively; P = 0.36). Comparing genetic groups, B indicus showed a higher blastocyst rate (28.3(a) ± 2.8%; P < 0.01) than B taurus and B bubalis (14.1(b) ± 2.9% and 10.2(b) ± 2.0%, respectively). However, only B indicus heifers showed a variation in the number of visualized follicles and the total and viable oocytes along consecutive OPU sessions. In conclusion, different phases of the pharmacologically synchronized ovarian follicular wave did not affect OPU-IVEP in B indicus, B taurus, and B bubalis heifers. Additionally, B indicus heifers showed greater OPU-IVEP efficiency than did the other genetic groups, under the same management conditions.

Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.

Differential gene expression and developmental competence in in vitro produced bovine embryos.

The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (S4) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.

Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro.

Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.

Sexagem de embriöes bovinos fecundados in vitro pela técnica de PCR multiplex / Sexing of in vitro fertilized bovine embryos by multiplex PCR

Neste trabalho, a técnica de PCR ("polymerase chain reaction") foi utilizada para a sexagem de 92 embriöes bovinos fertilizados in vitro. Os embriöes originaram-se de fertilizaçäo in vitro de oócitos aspirados de ovários de fêmeas bovinas, provenientes de abatedouros comerciais. Os oócitos foram maturados, fertilizados e cultivados até o estádio de blastocisto. Os embriöes foram lavados em soluçäo de PBS, transferidos para tubos de polipropileno contendo água ultrapura, e imediatamente congelados a -196§C. Os embriöes foram descongelados sobre isopor contendo gelo picado e tratados com proteinase K. Para a reaçäo de PCR, utilizaram-se alíquotas de 34 µl de cada tudo, onde foram acrescidos dois pares de primers, seqüência BC1.2 e seqüência satélite 1.715, desoxinucleotídeos, MgCl2, tampao PCR 10X, TaqDNA polimerase e água, em um volume final de 50 µl. As amostras foram amplificadas e a eletroforese realizada em gel de poliacrilamida a 8 por cento. Os géis foram corados com soluçäo de brometo de etídio e analisados em transiluminador de luz ultravioleta. Um índice de 93,47 por cento de amplificaçäo foi atingido, com 41 embriöes (47,67 por cento) machos e 45 (52,32 por cento) embriöes fêmeas. O uso de gel de poliacrilamida a 8 por cento foi eficaz na separaçäo de fragmentos de DNA muito próximos