Reliability and validity of the T-shirt test for the assessment of unsupported sitting in manual wheelchair users with spinal cord injury.

This study aimed to evaluate the validity and reliability of the T-shirt test (TST) in assessing sitting stability under three thigh support conditions and with timed outcomes derived in six ways among individuals with a spinal cord injury (SCI). The TST was performed five times under three thigh support conditions (85%, 55% and 25% of total thigh length) in two evaluations spaced between 7-14 days. For each thigh condition, six different outcomes were derived (average or best time from 2, 3, and 5 trial). All outcomes derivation showed excellent reliability on test day (intraclass correlation coefficient; ICC  ≥ 0.997) and excellent test-retest reliability (ICC ≥ 0.874) for each thigh support condition. The TST showed high inverse correlations with the Spinal Cord Independence Measure III (SCIM)-mobility score for all outcomes and support conditions (ρ≥-0.706), except for Best-5; moderate inverse correlations with total SCIM-total scores for most outcome derivations and support conditions (ρ≥-0.636); and a moderate inverse correlation with confidence and capacity domains of Wheelchair Skills Test-Questionnaire for most outcome derivation and support conditions (ρ≥-0.504). The TST could discriminate cervical from high and low thoracic levels of injuries under minimal thigh support condition. Overall, all the TST-derived outcomes and support conditions showed adequate validity and test-retest reliability, but Best-5 had inconsistency. Under the minimal thigh support condition, all outcome derivations except Best-3 could discriminate cervical from other injury-level groups. Although all outcome derivations and thigh support conditions provided reliable results, we recommend using the average of 3 trials under the maximal thigh support condition.

Nuclear transfer alters placental gene expression and associated histone modifications of the placental-specific imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2) in cattle.

Abnormal placental development is frequent in nuclear transfer (NT) pregnancies and is likely to be associated with altered epigenetic reprogramming. In the present study, fetal and placental measurements were taken on Day 60 of gestation in cows with pregnancies produced by AI, IVF and NT. Placentas were collected and subjected to histological evaluation, the expression of genes important in trophoblast differentiation and expression of the placental imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2), as well as chromatin immunoprecipitation (ChIP) for histone marks within the promoter of PHLDA2. Fewer binucleated cells were observed in NT cotyledons, followed by IVF and AI cotyledons (P<0.05). Expression of heart and neural crest derivatives expressed 1 (HAND1), placental lactogen (PL), pregnancy-associated glycoprotein 9 (PAG-9) and PHLDA2 was elevated in NT cotyledons compared with AI cotyledons. Expression of PHLDA2 was higher in IVF than AI samples (P<0.05). ChIP revealed an increase in the permissive mark dimethylation of lysine 4 on histone H3 (H3K4me2), surprisingly associated with the silent allele of PHLDA2, and a decrease in the inhibitory mark H3K9me2 in NT samples. Thus, genes critical for placental development were altered in NT placentas, including an imprinted gene. Allele-specific changes in the permissive histone mark in the PHLDA2 promoter indicate misregulation of imprinting in clones. Abnormal trophoblast differentiation could have resulted in lower numbers of binucleated cells following NT. These results suggest that the altered expression of imprinted genes associated with NT are also caused by changes in histone modifications.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos / Suplementação com soro fetal bovino altera a modificação de histona H3R26me2 durante o período pré-implantacional em embriões bovinos produzidos in vitro

In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation...

A produção in vitro (PIV) de embriões de bovinos não é apenas de grande importância econômica para a pecuária, mas é também um importante modelo para estudar o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a modificação de histona, H3R26me2 durante o desenvolvimento pré-implantacional em embriões bovinos produzidos in vitro, cultivados com ou sem suplementação de soro fetal bovino (SFB), bem como comparar essa modificação específica entre mórulas produzidas in vitro e in vivo. Após a maturação in vitro e fertilização, embriões foram cultivados com suplementação de 0 ou 2,5% SFB. O desenvolvimento embrionário foi avaliado e embriões foram coletados e fixados em diferentes fases durante o desenvolvimento (2, 4, 8 e 16 células, mórula e blastocisto). Os embriões fixados foram avaliados por imunofluorescência utilizando um anticorpo para H3R26me2. Imagens de embriões corados foram analisadas baseadas na porcentagem do DNA total. Embriões cultivados com 2,5% SFB tiveram uma taxa de desenvolvimento ao estágio de blastocisto maior que o grupo que não recebeu suplementação com SFB (34.85±5,43% vs 23.38±,93%; P<0,05). Níveis de H3R26me2 variaram para ambos os grupos ao longo do desenvolvimento. No grupo 0% SFB, a marcação para H3R26me2 foi mais intensa nos estágios de 4 células (P<0,05), 16 células (P<0,05) e mórula (P<0.05). No grupo 2.5% SFB, apenas os embriões de 4 células tiveram marcação significativamente maior que todas as outras fases (P<0,01). Mórulas produzidas in vivo apresentaram níveis de H3R26me2 semelhantes ao grupo 0% SFB, e ambos foram significativamente maiores que o grupo 2.5% SFB. Estes resultados sugerem que a modificação de histona H3R26me2 é regulada durante o desenvolvimento pré-implantacional de embriões bovinos, e que as condições de cultura alteram de maneira importante esta regulação...

Influence of bovine serum albumin and fetal bovine serum supplementation during in vitro maturation on lipid and mitochondrial behaviour in oocytes and lipid accumulation in bovine embryos.

Proper oocyte maturation is crucial for subsequent embryo development; however, oocyte mitochondrial and lipid-droplet behaviour are still poorly understood. Although excessive lipid accumulation during in vitro production (IVP) of bovine embryos has been linked with impaired cryotolerance, lipid oxidation is essential for adequate energy supply. Fetal bovine serum (FBS) and bovine serum albumin (BSA) are supplements used during IVP, containing high and low lipid content, respectively. This study aimed to understand how these supplements influence oocyte mitochondrial and lipid behaviour during in vitro maturation (IVM) in comparison to in vivo maturation, as well as their influence on development rates and embryo lipid accumulation during IVP. We demonstrate that only in vivo-matured oocytes maintained correlation between lipid content and active mitochondria. IVM media containing FBS increased total lipid content 18-fold and resulted in higher lipid accumulation in oocytes when compared with media with BSA. IVM using a lower FBS concentration combined with BSA resulted in satisfactory maturation and embryo development and also reduced lipid accumulation in blastocysts. In conclusion, IVM causes changes in mitochondrial and lipid dynamics, which may have negative effects on oocyte development rates and embryo lipid accumulation. Moreover, decreasing FBS concentrations during IVM may reduce embryo lipid accumulation without affecting production rates.

Oxygen tension affects histone remodeling of in vitro-produced embryos in a bovine model.

In vitro production of bovine embryos is a biotechnology of great economic impact. Epigenetic processes, such as histone remodeling, control gene expression and are essential for proper embryo development. Given the importance of IVP as a reproductive biotechnology, the role of epigenetic processes during embryo development, and the important correlation between culture conditions and epigenetic patterns, the present study was designed as a 2 × 2 factorial to investigate the influence of varying oxygen tensions (O2; 5% and 20%) and concentrations of fetal bovine serum (0% and 2.5%), during IVC, in the epigenetic remodeling of H3K9me2 (repressive) and H3K4me2 (permissive) in bovine embryos. Bovine oocytes were used for IVP of embryos, cleavage and blastocyst rates were evaluated, and expanded blastocysts were used for evaluation of the histone marks H3K9me2 and H3K4me2. Morulae and expanded blastocysts were also used to evaluate the expression of remodeling enzymes, specific to the aforementioned marks, by real-time polymerase chain reaction. Embryos produced in the presence of fetal bovine serum (2.5%) had a 10% higher rate of blastocyst formation. Global staining for the residues H3K9me2 and H3K4me2 was not affected significantly by the presence of serum. Notwithstanding, the main effect of oxygen tension was significant for both histone marks, with both repressive and permissive marks being higher in embryos cultured at the higher oxygen tension; however, expression of the remodeling enzymes did not differ in morulae or blastocysts in response to the varying oxygen tension. These results suggest that the use of serum during IVC of embryos increases blastocyst rate without affecting the evaluated histone marks and that oxygen tension has an important effect on the histone marks H3K9me2 and H3K4me2 in bovine blastocysts.

Efeitos da redução ou substituição do soro fetal bovino por outros compostos na maturação in vitro de oócitos bovinos / Effects of reduction or replacement of fetal calf serum by other compounds during in vitro maturation of bovine oocytes

A utilização do soro fetal bovino (SFB), embora bastante disseminada na produção in vitro (PIV) de embriões bovinos, apresenta limitações por ser um meio indefinido e por causar efeitos que prejudicam a qualidade desses embriões. Por esse motivo, nos últimos anos, grande parte das pesquisas relacionadas à PIV está voltada para a substituição do SFB por outros compostos nos meios de cultura. No presente estudo, foram utilizados como compostos protéicos a albumina sérica bovina livre de ácidos graxos (BSA-FAF) e um produto comercial denominado fluido embriônico (FE) de maneira isolada ou em diferentes combinações e concentrações, com objetivo de substituir ou diminuir a concentração do SFB durante a maturação in vitro (MIV). [...] Ademais, o G3 também apresentou diminuição na taxa de maturação nuclear quando comparado ao G4. Quanto à maturação citoplasmática, nos grupos G2, G7, G6 e G3, houve redução (p<0,05) das taxas para 43,9por cento, 43,2 por cento, 43,1 por cento e 36,5 por cento, respectivamente, quando comparadas ao meio controle (G1), que permitiu a obtenção de valores médios de 62,4 por cento. Por outro lado, nos grupos G8, G4 e G5, a taxa de maturação citoplasmática não foi afetada com a redução do SFB, onde 59,3 por cento, 51,3 por cento e 50,8 por cento dos oócitos apresentaram os GC dispostos na periferia, respectivamente. Os resultados obtidos pelo teste de contrastes ortogonais complementam os obtidos na avaliação da maturação nuclear e migração de grânulos corticais, mostrando a necessidade do SFB durante a MIV, mesmo que em baixas concentrações, e a possibilidade de diminuir a sua concentração associando-o a BSA-FAF e/ou FE. Dessa forma, conclui-se que é possível reduzir a concentração de SFB no meio de MIV para até 3,5% sem prejuízo significativo aos índices de maturação nuclear e citoplasmática.

The use of fetal calf serum (FCS), although widely employed during in vitro production (IVP) of bovine embryos, has limitations. FCS is an undefined media and may have harmful effects on the quality of embryos. For this reason, in recent years, research efforts aimed at improving IVP of bovine embryos, have focused at the replacement of FCS by alternative compounds in culture media. In this study, fatty acid free bovine serum albumin (BSA-FAF) and embryonic fluid (EF) were used separately or in combination, in different concentrations, to replace or reduce the concentration of FCS during in vitro maturation (IVM). [...] Moreover, G3 also showed inferior nuclear maturation rate when compared to G4. Regarding cytoplasmic maturation, the rates were reduced to 43.9 percent, 43.2 percent, 43.1 percent and 36.5 percent in G2, G7, G6 and G3 groups, respectively, compared to the control group (G1; 62.4 percent). On the other hand, in the groups G8, G4 and G5, maturation rates were not affected by reduction of FCS, where 59.3 percent, 51.3 percent and 50.8 percent of the oocytes displayed CG arranged peripherally, respectively. The results obtained by the orthogonal contrast test are in accordance with the ones from the evaluation of the nuclear maturation and cortical granules migration. These data show the need of FCS on the MIV, even in low concentrations, and the possibility of decrease its concentration by associating it with BSA-FAF and/or EF. Therefore, we concluded that it is possible to reduce the concentration of FCS in IVM medium to a concentration of 3.5 percent without affecting nuclear and cytoplasmic maturation rates.