Nuclear maturation kinetics and in vitro fertilization of immature bovine oocytes injected into pre-ovulatory follicles.

The maturation kinetics and in vitro fertilization of immature bovine oocytes injected by the intra-follicular oocyte injection (IFOT) technique into pre-ovulatory follicles of previously synchronized cows were evaluated. In Experiment 1, grade I, II and III cumulus-oocyte complexes (COCs) were randomly distributed to one of three Groups: Matvitro22 (COCs matured in vitro for 22 h), MatFol20 and MatFol28 (COCs matured in vivo after being injected into a pre-ovulatory follicle of previously synchronized cows for 19.8 ± 0.1 h and 28.3 ± 0.1 h, respectively). Cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at the time of IFOT in the MatFol20 Group or 10 h after IFOT in the MatFol28 Group. MatFol20 and MatFol28 COCs were aspirated approximately 20 h after the LH injection for nuclear maturation kinetics and recovery rate assessment. In Experiment 2, grade I, II, and III COCs were randomly distributed into two Groups: Matvitro22 Group, COCs were matured and fertilized in vitro, and MatFol20 Group, COCs were matured as in the MatFol20 Group in Experiment 1, but COCs were fertilized in vitro. Putative zygotes were classified as fertilized, unfertilized or polyspermic. In Experiment 1, the recovery rate was lower (P < 0.001) in the MatFol20 Group (52.9%, 91/172) compared with MatFol28 (72.9%, 113/155). Rate of oocytes in germinal vesicle stage, metaphase I, anaphase I and telophase I were similar among Groups. However, oocytes matured in vivo for 28.3 h had lower rate of metaphase II (P = 0.001) and greater rates of degenerated (P = 0.001) and parthenogenetically activated (P = 0.001) oocytes. In experiment 2, the rates of polyspermy and degenerated were similar between Groups. However, the rate of fertilized oocytes was greater (P = 0.05) in oocytes in the MatFol20 Group. It is concluded that oocyte in vivo maturation for 19.8 h after IFOT does not compromise the nuclear maturation kinetics and increases in vitro fertilization rates. However, the extra 10 h of intra-follicular incubation time decreased oocyte viability.

Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization / [Lentivírus falham na entrega de transgenes em zigotos bovinos após coincubação com espermatozoide, durante fertilização in vitro]

As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)

Quantificação do DNA e genotipagem de biópsias de embriões bovinos Gir e Girolando produzidos in vitro / DNA quantification and genotyping of biopsies obtained from Gyr and Girolando in vitro produced bovine embryos

A biópsia embrionária associada à genotipagem permite a obtenção de informações genômicas antes mesmo da transferência dos embriões. Neste estudo, foram avaliadas amostras biopsiadas de blastocistos bovinos transferidos para receptoras (n=47), sob a hipótese de que a raça (Gir ou Girolando), o estádio embrionário (blastocisto ou blastocisto expandido) e a competência para estabelecimento de prenhez (positiva ou negativa) afetariam a quantidade e a qualidade do DNA da amostra obtida. O DNA foi extraído, amplificado, quantificado por eletroferograma e genotipado. O parâmetro call rate (CR) foi adotado para mensurar a qualidade da genotipagem. Obteve-se concentração de DNA de 86,07±171,66ng/µL e CR 0,73±0,17. O CR não variou em função da quantidade de DNA nas amostras. As variáveis raça e estádio embrionário não influenciaram a concentração de DNA, nem o CR. Houve efeito da prenhez sobre o CR (P=0,0187), mas, como houve maior CR nas amostras provenientes do grupo prenhez negativa, não foi possível associar esse parâmetro à qualidade embrionária. Concluiu-se que a raça e a qualidade embrionária não afetam os parâmetros aqui estudados em amostras embrionárias, ou seja, embriões com maiores chances de implantação não refletem alta qualidade nas amostras de biópsia genotipadas.(AU)

Embryo biopsy associated with genotyping allows genomic information before embryo transfer. In this study, blastocyst biopsy samples from embryos transferred to recipients (n= 47) were evaluated, under the hypothesis that breed (Gyr or Girolando), embryonic stage (blastocyst or expanded blastocyst) and competence to establish pregnancy (positive or negative) would affect the quantity and DNA quality of samples. DNA was extracted, amplified, quantified by electropherogram and genotyped. The parameter call rate (CR) was used to measure the quality of genotyping. DNA concentration of 86.07±171.66ng/µl, and CR 0.73±0.17 was obtained. CR did not vary according to the amount of DNA in the samples. The variables breed and embryonic stage had no influence on DNA concentration or CR. There was pregnancy effect on the CR (P= 0.0187), but since there was a higher CR in the samples from the negative pregnancy group, it was not possible to associate this parameter with the embryonic quality. We conclude that the breed and embryo quality do not affect the evaluated parameters in embryonic samples. Embryos with higher chances of implantation do not reflect high quality in embryo biopsy genotyped samples.(AU)

Addition of insulin-like growth factor I to the maturation medium of bovine oocytes subjected to heat shock: effects on the production of reactive oxygen species, mitochondrial activity and oocyte competence.

This study was performed to investigate the effects of insulin-like growth factor-I (IGF-I) addition to in vitro maturation (IVM) medium on apoptosis, mitochondrial membrane potential, ROS production, and developmental competence of bovine oocytes subjected to heat shock. Two temperatures (conventional: 24 h at 38.5°C, or heat shock: 12 h at 41°C followed by 12 h at 38.5°C) and 3 IGF-I concentrations (0, 25, and 100 ng/mL) were tested during IVM. The oocytes were then fertilized in vitro, and the presumptive zygotes were cultured until reaching the blastocyst stage. There was no interaction between temperature and IGF-I concentration for any variable evaluated (P > 0.05). The addition of IGF-I did not alter the proportion of nuclear maturation, TUNEL-positive oocytes and caspase-3 activity, or blastocyst proportion on Days 7 and 8 post-fertilization. Furthermore, the total number of cells and the number of cells in the inner cell mass (ICM) in the blastocyst were not altered (P > 0.05). However, IGF-I increased (P < 0.05) the mitochondrial membrane potential and the production of ROS in oocytes and decreased (P < 0.05) the proportion of apoptotic cells in the ICM in blastocysts. Heat shock increased (P < 0.05) the proportion of TUNEL-positive oocytes and ROS production and reduced (P < 0.05) the mitochondrial membrane potential. Moreover, heat shock increased (P < 0.05) the apoptosis proportion in the ICM cells. In conclusion, supplementing IVM medium with IGF-I may increase the mitochondrial membrane potential and ROS production in oocytes and decrease apoptosis in the ICM in blastocysts. Heat shock for 12 h compromised oocyte developmental competence and increased apoptosis within the ICM cells of the blastocysts.

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre-Implantation Stage.

Oocyte has been considered the major contributor for embryo thermo-tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo-tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo-sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post-insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat-shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat-shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.

Effects of a high-energy diet on oocyte quality and in vitro embryo production in Bos indicus and Bos taurus cows.

The effects of different dietary energy levels [100 and 170% for maintenance (M) and high energy (1.7M), respectively] on metabolic, endocrine, and reproductive parameters were evaluated in nonlactating Bos indicus (Gir; n=14) and Bos taurus (Holstein; n=14) cows submitted to ultrasound-guided ovum pick-up followed by in vitro embryo production. The oocyte donor cows were housed in a tiestall system and fed twice daily (0800 and 1600 h). Twenty-one days before the beginning of the experiment, the animals were fed with a maintenance diet for adaptation followed by the experimental diets (M and 1.7M), and each cow underwent 9 ovum pick-up procedures 14 d apart. The recovered oocytes were cultured in vitro for 7 d. We measured glucose and insulin concentrations and performed glucose tolerance tests and the relative quantification of transcripts (PRDX1, HSP70.1, GLUT1, GLUT5, IGF1R, and IGF2R) from the oocytes recovered at the end of the experimental period. No interactions were observed between the effects of genetic groups and dietary energy level on the qualitative (viable oocytes, quality grade, and oocyte quality index) and quantitative (oocytes recovered) oocyte variables. There were no effects of dietary energy level on the qualitative and quantitative oocyte variables. However, Bos indicus cows had greater numbers of recovered structures, viable oocytes, and A and B oocyte grades as well as better oocyte quality index scores and lower DNA fragmentation rates compared with Bos taurus donors. In vitro embryo production (cleavage and blastocyst rates and number of embryos) was similar between diets, but the 1.7M diet reduced in vitro embryo production in Bos indicus cows after 60 d of treatment. Moreover, Bos indicus cows on the 1.7M diet showed lower transcript abundance for the HSP70.1, GLUT1, IGF1R, and IGF2R genes. All cows fed 1.7M diets had greater glucose and insulin concentrations and greater insulin resistance according to the glucose tolerance test. In conclusion, increasing dietary energy did not interfere with oocyte numbers and quality, but the 1.7M diet reduced in vitro embryo production in Bos indicus cows after 60 d of treatment. Finally, Bos indicus cows had greater oocyte quality, greater numbers of viable oocytes and greater in vitro embryo yield than Bos taurus.

Cytotoxicity and expression of genes involved in the cellular stress response and apoptosis in mammalian fibroblast exposed to cotton cellulose nanofibers.

Cellulose nanofibers (CNF) have mechanical properties that make them very attractive for applications in the construction of polymeric matrices, drug delivery and tissue engineering. However, little is known about their impact on mammalian cells. The objective of this study was to evaluate the cytotoxicity of CNF and their effect on gene expression of fibroblasts cultured in vitro. The morphology of CNF was analyzed by transmission electron microscopy and the surface charge by Zeta potential. Cell viability was analyzed by flow cytometry assay and gene expression of biomarkers focused on cell stress response such as Heat shock protein 70.1 (HSP70.1) and Peroxiredoxin 1 (PRDX1) and apoptosis as B-cell leukemia (BCL-2) and BCL-2 associated X protein (BAX) by RT-PCR assay. Low concentrations of CNF (0.02-100 µg ml(-1)) did not cause cell death; however, at concentrations above 200 µg ml(-1), the nanofibers significantly decreased cell viability (86.41 ± 5.37%). The exposure to high concentrations of CNF (2000 and 5000 µg ml(-1)) resulted in increased HSP70.1, PRDX1 and BAX gene expression. The current study concludes that, under the conditions tested, high concentrations (2000 and 5000 µg ml(-1)) of CNF cause decreased cell viability and affect the expression of stress- and apoptosis-associated molecular markers.