In-Vitro Growth Kinetics of Mesenchymal Stem Cells in Cytotoxicity Tests Using Low-Diluted Viscum Album.

INTRODUCTION: The use of mesenchymal stem cells (MSC) in cytotoxicity tests is an in-vitro alternative model for predicting initial doses. Homeopathic medicines may stimulate the immune system to combat a pathology effectively and have been used for over two centuries. Viscum album (VA) extracts are widely used in the treatment of cancer, due to their immunomodulatory, cytotoxic and pro-apoptotic properties. OBJECTIVE: This study aimed to evaluate the in-vitro growth kinetics of canine MSC in relation to cytotoxicity, cell differentiation and expression of pluripotentiality markers, using a VA preparation at the D1D2 (1×10-1, 1×10-2 potency (VAD1D2). METHODS: MSC were obtained from adipose tissue sampled from a healthy dog that was undergoing an elective veterinary procedure and with its owner's permission. The experiments were performed in three groups: MSC treated with VAD1D2 or diluent or untreated (control). The cytotoxicity was evaluated by MTT assay. The differentiation was induced in three lineages, and apoptotic cell labeling was performed by an Annexin-V test. RESULTS: At the concentration of 10 µL/mL of VA, the number of cells after in-vitro culture was maintained when compared with the control (untreated) group. A significant and gradual decrease in cell viability was recorded as VA concentrations increased. The apoptosis analysis showed that VA at 20 µL/mL presented absolute percentages of initial apoptosis twice as high as at 10 µL/mL, which was similar to the control (untreated group). CONCLUSION: The results suggest that the use of efficient methods to assess the in-vitro cytotoxicity of VA-based homeopathic medicines using MSC lineages may predict the potential action at different concentrations. These findings demonstrated that VAD1D2 interferes with canine MSC growth kinetics.

Impact of donor nutritional balance on the growth and development of mesenchymal stem cells from caprine umbilical cord Wharton´s jelly.

Mesenchymal stem cells (MSCs) from the umbilical cord (UC) have aroused considerable interest. However, little is known about the maternal effect on these cells. The aim of this study was to verify the impact of the nutritional status of donor goats on the growth and differentiation of MSCs from the UC. At parturition, 19 goats were grouped based on their low or high body mass index (low BMI, LBMI, n = 9; and high BMI, HBMI, n = 10). UCs were collected during delivery and Wharton's jelly (WJ) fragments cultured. WJ-MSCs were differentiated into osteocytes, adipocytes, chondrocytes, and the population doubling time (PDT) was determined. Samples of WJ-MSCs were also used to verify the expression of the CD90, CD73, CD34, CD45, and CD105 genes. Media used for WJ-MSC primary cultures were analyzed using near-infrared spectroscopy. The lag phase was 7.5 ± 0.6 days and the entire culture took 26.7 ± 1.3 days, with a cell proliferation rate of 8.500 cells/day. The mean PDT from subculture was 30.0 ± 0.7 h. The CD105 gene was sub-expressed in LBMI, and the spectra of the spent media from the second to fourth day of WJ-MSC primary culture were segregated into negative scores by multivariate analysis. We conclude that, in goats, the nutritional balance of the donor did not affect the in vitro growth of MSCs derived from the UC. However, the molecular profile observed in the low BMI group suggests that the use of MSCs for therapeutic purposes should be considered more carefully.

Effectiveness of near-infrared spectroscopy as a non-invasive tool to discriminate spectral profiles of in vitro cultured oocytes from goats.

Here, we aimed to discriminate between the spectral profiles of spent culture media after oocyte in vitro maturation (IVM) and culture (IVC) from goats of different ages subjected to repeated hormonal treatments. The profiles were discriminated using near infrared (NIR) spectroscopy combined with multivariate methods. A total of 19 goats (young = 10; old = 9) were subjected to serial hormonal stimulation (HS) with gonadotropins. Cumulus oophorus complexes (COCs) were collected using laparoscopic ovum pick-up (LOPU) and subjected to IVM and parthenogenetic activation. The initial embryos were subjected to IVC. Spent culture media were collected after oocyte IVM and on day 2 of IVC and analyzed using NIR spectroscopy. NIR spectral data were interpreted through chemometric methods, such as principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The results of PCA analysis clearly showed a separation in the spectral profiles between the experimental groups (HS sessions; young and old animals) both after IVM and IVC. Overall, the main absorption bands were attributed to the C-H group second overtone, first overtone of O-H and N-H, and C-H combinations and may serve as molecular markers. On the other hand, the spectral data obtained using PLS-DA models provided a better classification of the groups. The results showed the possibility of discriminating young and old groups as well as the three HS sessions with high specificity, sensitivity, and accuracy using NIR spectra. Thus, the culture medium analysis using NIR spectroscopy combined with multivariate methods indicated the dissimilarities between the groups and provided an insight into the in vitro development of goat oocytes. This technique serves as an efficient, objective, rapid, and non-invasive method to discriminate spectral profiles.

Virtual Reality Applied in a Thermoelectric Power Plant Planning

Abstract The planning of a new thermal power plant is linked to the various decision elements and evaluation criteria. Factors such as the plant's geographic positioning, primary energy supply points, paths, and means of delivery of this primary energy should be analyzed. Similar studies are imposed when studying the change of a thermoelectric plant's primary energy source occurs. In Brazil, several plants are currently investigating the exchange of their primary fuel from oil to gas due to the decrees issued by ANEEL. This paper presents software, which uses virtual reality to assist in the various stages of the planning process and in the analyses that must be performed. This software was developed for the Hidrotermica Group and had as the primary target the Borborema Thermoelectric Power Plant.

Effect of Continuous Administration of Enalapril Maleate on the Oocyte Quality and In Vitro Production of Parthenote Embryos in Nulliparous and Multiparous Goats Undergoing Serial Laparoscopic Ovum Pick-Up.

The aim of this work was to determine the effect of enalapril maleate administration, during oocyte recovery by serial laparoscopic ovum pick-up (LOPU), on the ovarian response and in vitro embryo production (IVP). Twenty cross-bred goats were allocated equally into two groups: Nulliparous and Multiparous. In each group, five animals were selected to receive daily doses of enalapril maleate during the hormonal protocol. Estrus was synchronized by a PGF2α analog, followed 48 h later by insertion of an intravaginal device with progesterone. Forty-eight hours after, a single dose of FSH/eCG was administered. The FSH/eCG doses were repeated three times, on every four day. Oocytes were recovered by LOPU 24 h after each FSH/eCG dose. Viable oocytes were matured in vitro, to be parthenogenetically activated and cultured for 72 h to the cleavage stage. The drug treatment increased the proportion of total follicles observed at LOPU (p < 0.01) in multiparous goats. In both parity groups, enalapril administration had no effect on the proportion or quality of oocytes recovered. Furthermore, the number of embryos cleaved was similar between the groups. Thus, enalapril maleate affected the ovarian response in multiparous animals only and had no effect on the oocyte quality or IVP.

Impact of short nutrient stimuli with different energy source on follicle dynamics and quality of oocyte from hormonally stimulated goats.

The aim of this study was to verify the effect of the energy source for a short-term diet supplementation on follicular dynamics, ovarian response and oocyte recovery in goats. Thirty Anglo Nubian crossbred does received a diet for 4 weeks to satisfy the nutritional requirements of breeding for adult non-dairy goats. Seven days prior to oocyte recovery (OR), a group of does (n = 10) was supplemented with ground full-fat linseed in the diet (Diet A), whereas a second group of does (n = 10) received crude glycerine in the diet (Diet B). The total mixed ration (TMR) diet was maintained as the Control Diet (n = 10). All animals were oestrous-synchronized by the use of a progesterone insert for 12 days prior to OR. Follicles were stimulated by using pFSH (five 40-mg/ml doses) during the supplementation time. At OR, follicles were counted and recovered oocytes were classified as viable or degenerated. Follicular dynamics was monitored by ultrasonography, and plasma glucose, cholesterol and triglyceride levels were measured during supplementation. Glucose was higher in Diet B and cholesterol in Diet A. Diet B had a lower proportion of small (<3 mm) and large follicles (≥3 mm; p = 0.01). The follicular growth rate was higher in Diet A (p < 0.01), with follicles emerging in the 5th day of supplementation. No differences were observed for follicles counted and oocytes recovered. Thus, the type of energy source supplemented for a short term was capable to alter the follicular dynamics, without affecting the proportion of morphologically viable oocytes upon recovery.

Proteome of the periovulatory oviduct and uterus of goats as related to nutritional balance.

This study was conducted to evaluate the effects of different feeding levels on the proteome of oviduct and uterus tissues of hormonally stimulated goats during the periovulatory period. Forty goats were separated into four different diet groups: Diet 1.0 M (n = 11), Diet 1.3 M (n = 10), Diet 1.6 M (n = 9), Diet 1.9 M (n = 10), fed with 1.0, 1.3, 1.6 and 1.9 times live weight maintenance, respectively. After four weeks of treatment, six hormonally stimulated females per treatment group were randomly selected for collection of uterine and the oviduct tissue samples. Samples were collected after animals were slaughtered in a commercial unit. Feeding goats with 1.3 to 1.9 times more nutrients than a control group directly influenced the proteome of the oviduct and uterus, altering the expression of proteins that participate in biological processes such as apoptosis, antioxidant, and immunological activities. These events are crucial for fertilization and early embryonic survival. Expression of oviduct proteins such as Tubulin Beta 2B, Transferrin and Disulphide-isomerase A3 increased in the 1.9 M group in relation to the other feeding levels. Disulphide-isomerase A4 showed higher expression in the 1.0 M group compared to diets with higher energetic levels. As energy intake increased in the diets, there was higher expression of Alpha-1-antitrypsin and downregulation of Profilin-1 in the uterus of the goats. In conclusion, this study showed that specific proteins of the goat oviduct and uterus expressed during the periovulatory period are modified as the result of nutritional balance.

Impact of cumulative gain in expertise on the efficiency of handmade cloning in cattle.

The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency.

Developmental Outcome and Related Abnormalities in Goats: Comparison Between Somatic Cell Nuclear Transfer- and In Vivo-Derived Concepti During Pregnancy Through Term.

Cloning by somatic cell nuclear transfer (SCNT) is characterized by low efficiency and the occurrence of developmental abnormalities, which are rather poorly studied phenomena in goats. This study aimed at comparing overall SCNT efficiency in goats by using in vitro-matured (IVM) or in vivo-matured oocytes and fibroblast donor cells (mock transfected, transgenic, or wild type), also characterizing symptoms of the Abnormal Offspring Syndrome (AOS) in development, comparing results with pregnancies produced by artificial insemination (AI) and in vivo-derived (IVD) embryos. The SCNT group had lower pregnancy rate (18.3%, 11/60), total number of concepti (20.0%, 12/60), term births (3.3%, 2/60), and live births (1.7%, 1/60) than both the IVD (77.8%, 7/9; 155.5%, 14/9; 122.2%, 11/9; 88.8%, 8/9) and the AI (71.4%, 10/14; 121.4%, 17/14; 100%, 14/14; 78.5%, 11/14) groups, respectively (p < 0.05). No SCNT pregnancies reached term using IVM oocytes, but in vivo-matured oocytes resulted in two term transgenic cloned kids. The proportion fetal membrane (FM) weight/birth weight reflected an increase in FM size and cotyledonary enlargement in clones, for disproportionally bigger newborns in relation to cotyledonary numbers. Overall, goat cloning showed losses and abnormality patterns similar to the AOS in cloned cattle and sheep, which have not been previously well recognized in goats.

Assessment of DNA damage in goat preantral follicles after vitrification of the ovarian cortex.

Effective methods for gamete preservation should have low impact on DNA integrity. The present study investigated the effects of vitrification of goat ovarian tissues on the occurrence of DNA fragmentation and DNA double-stand breaks using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and detection of phosphorylated histone H2AX (γH2AX), respectively. Goat ovaries were collected at a local abattoir and 12 tissue fragments were prepared from each ovarian pair. Tissue fragments were used as fresh control samples or were cultured in vitro, vitrified or vitrified and cultured. Vitrification was performed using the Ovarian Tissue Cryosystem. Fragments from all groups (control and treatments) were processed for histology, transmission electron microscopy, TUNEL assay and immunofluorescence. Compared with fresh control samples, a lower percentage of morphologically normal follicles was detected in the vitrification followed by culture treatment group (P<0.05). Normal follicular ultrastructure was observed in all groups. Immunofluorescence revealed the presence of γH2AX foci in few oocytes and ovarian stromal cells. TUNEL-positive follicles were found in samples without significant differences among groups (P>0.05). In conclusion, the vitrification protocol used in the present study did not increase DNA damage in preantral follicles enclosed in goat ovarian tissues.

Activin-A promotes the development of goat isolated secondary follicles in vitro.

The role of activin-A in follicular development and on the mRNA expression levels of different genes in goat secondary follicles was evaluated. Goat secondary follicles (≥ 150 µm) were cultured for 18 days under control conditions or with the addition of either 50 or 100 ng/ml activin-A (Experiment 1). The mRNA levels for the genes that code for activin-A, ActR-IA, ActR-IB, ActR-IIA, ActR-IIB, follicle stimulating hormone receptor (FSH-R) and P450 aromatase were measured in each condition (Experiment 2). We observed that after 6 days of culture, the antrum formation rate was higher in cultures with added activin-A than in the cultured control (P < 0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate in the final third of the culture (days 12-18), resulting in a higher percentage of meiosis resumption (P < 0.05). On day 6, the addition of activin-A (50 ng/ml) increased the levels of ActR-IA mRNA compared with the cultured control (P < 0.05). After 18 days, the addition of 50 ng/ml activin-A significantly increased the levels of its own mRNA compared with the non-cultured control. Moreover, this treatment reduced the mRNA levels of P450 aromatase in comparison with the cultured control (P < 0.05). Higher levels of P450 aromatase mRNA were found for both activin-A treatments compared with the non-cultured control (P < 0.05). No difference in estradiol levels was detected among any of the tested treatments. In conclusion, the addition of activin-A to culture medium stimulated early antrum formation as well as an increase in the daily follicular growth rate and the percentage of meiosis resumption.

Effect of medium composition on the in vitro culture of bovine pre-antral follicles: morphology and viability do not guarantee functionality.

Summary This study investigated the effect of three different culture media (α minimum essential medium (α-MEM), McCoy or TCM199 during the in vitro culture (IVC) of bovine isolated pre-antral follicles. Pre-antral follicles greater than 150 µm in size were isolated and cultured for 0 (control), 8 or 16 days in one of the abovementioned culture media. Follicles were evaluated for survival, growth and antrum formation at days 8 and 16. The results showed that TCM199 was the most suitable medium to preserve follicular viability and ultrastructure, resulting in the highest rates of antrum formation. In conclusion, TCM199 promotes the in vitro development of isolated pre-antral follicles without hampering follicular functionality by sustaining in vitro growth and antrum formation.

Progesterone and Follicle Stimulating Hormone interact and promote goat preantral follicles survival and development in vitro / Progesterona e Hormônio Folículo-Estimulante interagem e promovem a sobrevivência e o desenvolvimento in vitro de folículos pré-antrais caprinos

We investigated the effects of progesterone and follicle stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) alone or containing progesterone (1, 2.5, 5, 10 or 20ng/mL), FSH (50ng/mL) or the interaction between progesterone and FSH. Fresh (non-cultured control) and cultured ovarian tissues were processed for histological and ultrastructural studies. After 7 days the addition of FSH to all progesterone concentrations maintained the percentage of normal follicles similar to fresh control. At day 7 of culture, a higher percentage of developing follicles was observed only in 2.5ng/ml of progesterone associated with FSH or 10ng/ml of progesterone alone when compared with control. From day 1 to day 7 of culture, a significant increase in the percentage of developing follicles was observed in MEM and 2.5ng/ml of progesterone + FSH. In addition, after 7 days, in all treatments, there was a significant increase in follicular diameter when compared with control, except for MEM alone and in 5ng/ml of progesterone + FSH or 10ng/ml of progesterone alone. Ultrastructural studies confirmed follicular integrity after 7 days of culture in 2.5ng/ml of progesterone with FSH. In conclusion, this study demonstrated that the interaction between progesterone and FSH maintains ultrastructural integrity, stimulates primordial follicles activation and further growth of cultured caprine preantral follicles.

Este trabalho verificou os efeitos da progesterona e do hormônio folículo-estimulante (FSH) na sobrevivência e no crescimento de folículos pré-antrais caprinos. Fragmentos de tecido ovariano foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (MEM) sozinho ou contendo progesterona (1, 2.5, 5, 10 ou 20ng/mL), FSH (50ng/mL) ou a combinação entre esses dois hormônios. O tecido fresco (controle não-cultivado) e o cultivado foram processados para análise histológica e ultra-estrutural. Após 7 dias a adição de FSH a todas as concentrações de progesterone manteve o percentual de folículos normais similar ao controle fresco. No dia 7 de cultivo, um alto percentual de folículos em desenvolvimento foi observado somente no tratamento com 2,5ng/ml de progesterona associada ao FSH ou com 10ng/ml de progesterona sozinha, em relação ao controle fresco. Do dia 1 para o dia 7 de cultivo, um aumento significativo no percentual de folículos em desenvolvimento foi observado no MEM sozinho e adicionado de 2,5ng/ml de progesterona + FSH. Além disso, após 7 dias, em todos os tratamentos, houve um aumento significativo no diâmetro folicular em relação ao controle, exceto nos tratamentos com MEM sozinho, 5ng/ml de progesterona + FSH ou 10ng/ml de progesterona sozinha. A análise ultra-estrutural confirmou a integridade follicular após 7 dias de cultivo no tratamento com 2,5ng/ml de progesterona + FSH. Em conclusão, este estudo demonstrou que a interação entre progesterona e FSH mantém a integridade ultra-estrutural, estimula a ativação de folículos primordiais e o posterior crescimento de folículos pré-antrais caprinos cultivados in vitro.

BMPRIB and BMPRII mRNA expression levels in goat ovarian follicles and the in vitro effects of BMP-15 on preantral follicle development.

This study evaluated the levels of bone morphogenetic protein receptors BMPRIB and BMPRII mRNA in goat follicles and the effects of bone morphogenetic protein-15 (BMP-15) on the in vitro development of cultured preantral follicles. Real-time polymerase chain reaction (PCR) was used to analyze the levels of BMPRIB and BMPRII mRNA in caprine preantral follicles and in small and large antral follicles. Preantral follicles (≥150 µm) were also isolated from goat ovaries and cultured for 18 days in α-MEM(+) supplemented with or without BMP-15 (10, 50, or 100 ng/ml). At the end of culture, some follicles were fixed for ultrastructural evaluation. Real-time PCR showed a reduction in BMPRII mRNA levels from the primary to secondary follicles. Higher levels of BMPRIB mRNA were observed in granulosa/theca cells from large antral follicles compared with small antral follicles. Moreover, BMPRII mRNA was expressed to a greater extent in cumulus-oocyte complexes from large antral follicles than in their respective granulosa/theca cells. In culture, 50 ng/ml BMP-15 positively influenced antral cavity formation and follicle growth after 18 days and also maintained follicular integrity. Thus, BMPRIB and BMPRII mRNAs are present in all follicular categories. BMP-15 (50 ng/ml) stimulates growth, antrum formation and the ultrastructural integrity of isolated caprine preantral follicles after 18 days of culture.

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro.

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.