C. S. Oliveira

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.

Molecular demonstration of intermittent shedding of Leptospira in cattle and sheep and its implications on control

For a long time, it has been stated that urine leptospiral shedding is intermittent, which was observed primarily by culturing. However, culturing presents serious limitations, mainly low sensitivity, and failure on detection of leptospires cannot be neglected. PCR presents several advantages, mainly higher sensitivity. The present study aimed to analyze the occurrence of intermittency on leptospiral shedding by PCR in naturally and experimentally infected animals. In this study two experiments were conducted, the first with 60 cows naturally infected from an endemic herd. The second one was conducted in three sheep experimentally infected, each one with a different strain of Leptospira (strains Copenhageni L1-130, Canicola LO-4 and Pomona Fromm). Considering cattle, 43.3% presented negative in all tests, the remaining (56.7%) were positive at least once. From these, only one (1.6%) was positive in all samples, and seven (11.8%) were positive only in the last sampling, making it impossible to evaluate the intermittency. Noteworthy, 26 cows (43.3%) presented the typical intermittent pattern of leptospiral shedding in urine. In sheep, all experimentally infected animals presented the typical intermittent shedding patterns, independently of the inoculated leptospiral strain. We considered that a careful serial analysis of urine samples for a more definitive and reliable individual diagnosis would be required for a successful antimicrobial therapy and control of leptospirosis on a herd.

Cell death is involved in sexual dimorphism during preimplantation development

In bovine preimplantation development, female embryos progress at lower rates and originate smaller blastocysts than male counterparts. Although sex-specific gene expression patterns are reported, when and how sex dimorphism is established is not clear. Differences among female and male early development can be useful for human assisted reproductive medicine, when X-linked disorders risk is detected, and for genetic breeding programs, especially in dairy cattle, which requires female animals for milk production. The aim of this study was to characterize the development of female and male embryos, attempting to identify sex effects during preimplantation development and the role of cell death in this process. Using sex-sorted semen from three different bulls for fertilization, we compared kinetics of bovine sex-specific embryos in six time points, and cell death was assessed in viable embryos. For kinetics analysis, we detected an increased population of female embryos arrested at 48 and 120h.p.i., suggesting this time points as delicate stages of development for female embryos that should be considered for testing improvement strategies for assisted reproductive technologies. Assessing viable embryos quality, we found 144h.p.i. is the first time point when viable embryos are phenotypically distinct: cell number is decreased, and apoptosis and cell fragmentation are increased in female embryos at this stage. These new results lead us to propose that sex dimorphism in viable embryos is established during morula-blastocyst transition, and cell death is involved in this process.

Efeitos da redução ou substituição do soro fetal bovino por outros compostos na maturação in vitro de oócitos bovinos

A utilizacao do soro fetal bovino (SFB), embora bastante disseminada na producao in vitro (PIV) de embrioes bovinos, apresenta limitacoes por ser um meio indefinido e por causar efeitos que prejudicam a qualidade desses embrioes. Por esse motivo, nos ultimos anos, grande parte das pesquisas relacionadas a PIV esta voltada para a substituicao do SFB por outros compostos nos meios de cultura. No presente estudo, foram utilizados como compostos proteicos a albumina serica bovina livre de acidos graxos (BSA-FAF) e um produto comercial denominado fluido embrionico (FE) de maneira isolada ou em diferentes combinacoes e concentracoes, com objetivo de substituir ou diminuir a concentracao do SFB durante a maturacao in vitro (MIV). Para isso, oocitos bovinos foram maturados in vitro nos seguintes grupos (G) que foram delineados de acordo com a suplementacao proteica recebida: G1 (controle) = 10% de SFB, G2 = 8mg/mL de BSA-FAF, G3 = 10% de FE, G4 = 6mg/mL de BSA-FAF + 5% de SFB, G5 = 6mg/mL de BSA-FAF + 3,5% de SFB + 1,5% de FE, G6 = 6mg/mL de BSA-FAF + 1,5% de SFB + 3,5% de FE, G7 = 6mg/mL de BSA-FAF + 5% de FE e G8 = 5% de SFB + 5% de FE. Apos 24 horas de MIV, os oocitos foram classificados de acordo com a progressao meiotica e migracao dos grânulos corticais (GC). As taxas de maturacao foram avaliadas pelo teste do Qui-Quadrado (χ2) ou, quando apropriado, pelo teste exato de Fisher, e para o estudo dos efeitos dos suplementos foram realizados contrastes ortogonais. O grupo suplementado com BSA-FAF (G2) mostrou diminuicao na taxa de oocitos que atingiram MII (75%) em comparacao aos grupos G1, G4, G5 e G8 (88,9%, 89,6%, 87% e 86,8%, respectivamente), sem diferir do do G3 (79,8%), G6 (82,9%) e G7 (82,9%). Ademais, o G3 tambem apresentou diminuicao na taxa de maturacao nuclear quando comparado ao G4. Quanto a maturacao citoplasmatica, nos grupos G2, G7, G6 e G3, houve reducao (p<0,05) das taxas para 43,9%, 43,2%, 43,1% e 36,5%, respectivamente, quando comparadas ao meio controle (G1), que permitiu a obtencao de valores medios de 62,4%. Por outro lado, nos grupos G8, G4 e G5, a taxa de maturacao citoplasmatica nao foi afetada com a reducao do SFB, onde 59,3%, 51,3% e 50,8% dos oocitos apresentaram os GC dispostos na periferia, respectivamente. Os resultados obtidos pelo teste de contrastes ortogonais complementam os obtidos na avaliacao da maturacao nuclear e migracao de grânulos corticais, mostrando a necessidade do SFB durante a MIV, mesmo que em baixas concentracoes, e a possibilidade de diminuir a sua concentracao associando-o a BSA-FAF e/ou FE. Dessa forma, conclui-se que e possivel reduzir a concentracao de SFB no meio de MIV para ate 3,5% sem prejuizo significativo aos indices de maturacao nuclear e citoplasmatica.

Variants in the CYP19A1 gene can affect in vitro embryo production traits in cattle

PurposeThis study aimed to associate DNA variants in promoter and exon flanking regions of the CYP19A1 gene with in vitro embryo production traits in cattle. The role of transcription factor binding sites created or lost due to DNA sequence variation and their possible effect on gene expression was also evaluated.MethodsWe collected date from Gyr dairy oocyte donor cows (Bos taurus indicus) at a commercial in vitro embryo production farm and analyzed the genotype–phenotype association with in vitro production traits. Using Sanger sequencing and web-based software, we assessed important CYP19A1 gene regions in oocyte donor cows and analyzed the effects of variants on the transcription factor binding sites.ResultsTwo SNP mutations significantly associated with oocyte production, oocyte viability, embryo development, and pregnancies were found (T > C in the untranslated exon 1 flanking region ([GenBank: AJ250379.1]: rs718446508 T > C), and a T > C in the 5′-upstream region (1.1 promoter) ([GenBank: AC_000167.1]: rs41651668 T > C). Six new transcription factor binding sites were created. A binding site for transcription factors associated with the development of the placenta and embryo implantation was eliminated due to variations in the DNA sequence identified.ConclusionsThe CYP19A1 gene contributes to genetic variation of in vitro embryo production traits in cattle. The complexity of the physiological phenomena related to estrogen pathways and their influence on reproduction in cattle allow indication of the mutations evaluated here as possible genetic markers for embryo production traits, which should be validated in the next steps of marker-assisted selection.

Evaluation of the simulated physiological oocyte maturation (SPOM) system on F1 Gyr × Holstein oocytes and embryos

The present study aimed to evaluate the effect of the simulated physiological oocyte maturation (SPOM) system on F1 Gyr × Holstein oocytes and embryos by evaluating the meiotic arrest, embryo production rates, total number of cells and lipid score. Three experiments were conducted and the following three experimental groups were formed according to in vitro maturation (IVM) treatments: CONTROL 1 (TCM 199 medium without FBS), CONTROL 2 (commercial medium) and SPOM (TCM 199 medium with forskolin and 3-isobutyl-1-methylxanthine (IBMX) in pre-IVM and extended IVM with cilostamide). In the first experiment (ovum pick-up), a significant (P 0.05) difference in total number of cells among the groups. No difference (P > 0.05) was found on lipid score among the groups at Day 7 of development, in both Experiments 2 and 3. At Day 9 (Experiment 2), only the CONTROL 2 showed a significant increase (P > 0.05) compared with the other treatments. It was concluded that under our conditions, the SPOM system was efficient in prolonging meiotic arrest on Gyr × Holstein oocytes, offering the oocytes in vitro conditions more similar to those found in vivo; however, it adversely affected embryo production rates and promoted no beneficial effect on the total number of cells and the lipid score.

Dynamics of humoral response in naturally-infected cattle after vaccination against leptospirosis

Vaccination is one of the most important measures for the control of bovine leptospirosis. Despite the broad usage of vaccination against leptospirosis in cattle worldwide, the dynamics of the post-vaccine immune response remain controversial and many aspects are still unclear, particularly in naturally-infected animals. Thus, the objective of this study is to describe the dynamics of humoral response in naturally-infected cattle after vaccination against leptospirosis. A total of 162 cows were studied, consisting of 129 included in the experimental group (G1), and subdivided into two groups, vaccinated with two different brands of bacterins, as well as 33 in the control group (G2). Serology (MAT) was performed in all cows on D0 (vaccination), then 60 and 120 days post-vaccination. Vaccination significantly elicited the production of anti-leptospiral antibodies. Seroreactivity increased rapidly but was of short duration (up to D60). Significantly, that increase was notably higher in the vaccinated group than in the controlled. Both vaccines elicited a similar response with a higher rate of seroreactive animals, but predominately against different serogroups. In this context, our results reinforce that, although of limited duration, vaccination against leptospirosis significantly elicits a specific humoral response in naturally-infected animals. The two studied vaccines presented similar seroconversion levels, but predominantly to different serogroups, being one against Icterohaemorrhagiae and the other against Sejroe.

Post implantation development reveals that biopsy procedure can segregate “healthy” from “unhealthy” bovine embryos and prevent miscarriages

Embryo biopsy has been performed in bovine in vivo produced embryos for the last twenty years, but little could be done with few embryonic cells in the past. Recently, advances in single cell analysis enabled a wide range of applications using embryo biopsy, from morphology to genetics analysis and different omics-techniques, which are promising for in vitro-fertilized (IVF) embryos. The aim of this study was to address if biopsy procedure would affect post implantation development of IVF blastocyts. Here we show that blastocyst stage do not affect re-expansion of biopsied embryos (regular blastocyst: 73.7%; expanded blastocyst: 73.1%), but affects (p<0.05) implantation (regular blastocyst: 37.8%, expanded blastocyst: 61.0%), so ideally biopsy should be performed in expanded blastocysts. No detrimental effect of biopsy procedure was detected for post-implantation development (calving rates, Biopsy: 47.1%, Control: 41.9%), and normal calves were born (Birth weight, Biopsy: 32.10±7.20kg; Control: 30.95±5.43kg). Surprisingly, we found interesting results suggesting embryo survival can be increased with aggressive procedures (such as embryo biopsy), and this is highly associated with early pregnancy loss (Biopsy: 0%, Control: 17.4%). This finding also suggests morphological classification of day 7 blastocysts is far from ideal, and supposedly, unhealthy embryos can implant but are bound to miscarriage during the first trimester (non-biopsied embryos). Our results show biopsy procedure is safe for bovine IVF embryos, and shed new light into the importance of conceptus in early pregnancy loss in cattle.

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.

53 EFFECTS OF DEMECOLCINE ON MICROTUBULE COMPOSITION AND CHEMICALLY ASSISTED ENUCLEATION OF BOVINE OOCYTES

The developmental competence of enucleated oocytes is a key factor that determines the overall success of animal cloning. Enucleation is an invasive procedure in traditional nuclear transfer (NT). The objective of this work was to evaluate the effects of demecolcine, a microtubule-depolymerizing agent, on metaphase II (MII) bovine oocytes and to verify the capacity of embryonic development after NT using chemically assisted enucleation. In the first experiment, oocytes after 21 h of IVM were exposed for 2 h to several concentrations of demecolcine: 0 (control), 0.025, 0.05, 0.2, and 0.4 µg mL–1, and evaluated in relation to membrane protrusion formation. After the best concentration of demecolcine was determined, the nuclear and microtubular dynamics of the treated oocytes were evaluated by immunofluorescence microscopy of tubulin and chromatin (Liu et al. 1998 Biol. Reprod. 5, 537–545) in a second experiment. The results were analyzed by Duncan and Tukey tests in Experiments I and II, respectively, and a level of 5% significance was used. In Experiment III, embryonic development following NT was determined using adult donor cells, derived from a skin biopsy. After 19 h of IVM, oocytes were exposed to demecolcine (0.05 µg mL–1) for 2 h, and enucleation was performed in oocytes that presented membrane protrusions. Samples of cytoplasts were stained with Hoechst 33342 for 10 min (10 µg mL–1) for evaluation of enucleation effectiveness. The remaining oocytes were reconstituted by NT, fused (two pulses of 2.0 kV cm–1 for 20 µs each, in 0.28 m mannitol solution), chemically activated (5 mm ionomycin for 5 min and 2 mm 6-DMAP for 4 h), and cultured in SOF medium with 2.5% FCS and 0.5% BSA. The 0.05 µg mL–1 concentration resulted in greater protrusion rates (55.1%; 211/388) in comparison to 0, 0.025, 0.2, and 0.4 µg mL–1 concentrations (0, 42.6, 45.1, and 39.3%, respectively). In Experiment II, at the beginning of treatment, the majority of the oocytes were in MII (40.7%) or anaphase I/telophase I (AI/TI; 22.4%). Effects of demecolcine occurred within only 0.5 h of treatment, with a significant increase in oocytes with complete depletion of microtubules (21.8%; initial average: 1.9%) and a reduction in the proportion at MII (13.5%) and AI/TI (8.2%). New polymerization of microtubules was observed when treated oocytes were then cultured in drug-free medium for 6 h (42.4% oocytes with two evident sets of microtubules). In Experiment III, we verified the effectiveness of the chemically assisted enucleation technique (90.6%; 77/85). We evaluated 515 oocytes, of which 219 (42.5%) had protrusions and were enucleated. After losses in the NT procedure and fusion, 58 reconstructed 1-cells were cultured resulting in cleavage and blastocyst rates of 84.5% (49/58) and 27.6% (16/58), respectively, with great variation in blastocyst production between the three replicates (12.5% to 47%). In conclusion, demecolcine can be used at lower concentrations than those used routinely, and the chemically assisted enucleation method has proven highly efficient and does not appear to inhibit embryonic development in the bovine. This work was supported financially by FAPESP.