Genomic and phenotypic features of Acinetobacter baumannii isolated from oil reservoirs reveal a novel subspecies specialized in degrading hazardous hydrocarbons.

The genus Acinetobacter encompasses biotechnologically relevant species and nosocomial pathogens. In this study, nine isolates recovered from different oil reservoir samples showed the ability to grow with petroleum as the only carbon source and possessed the ability to emulsify kerosene. The whole genomes of the nine strains were sequenced and analyzed. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of all strains were compared to the reference strains, and the results were below the reference values (<97.88 and 82, respectively), suggesting that the isolates belong to a new subspecies of Acinetobacter baumannii. The name Acinetobacter baumannii oleum ficedula is proposed. A comparison of the whole genome repertoire of 290 Acinetobacter species indicated that the strains in this study resemble non-pathogenic Acinetobacter strains. However, the new isolates resemble A. baumannii when comparing virulence factors. The isolates in this study carry many genes involved in hydrocarbon degradation, indicating the potential to degrade most toxic compounds listed by environmental regulatory agencies such as ATSDR, EPA, and CONAMA. In addition, despite the absence of known biosurfactant or bioemulsifier genes, the strains showed emulsifying activity, suggesting the presence of new pathways or genes related to this process. This study investigated the genomic, phenotypic, and biochemical features of the novel environmental subspecies A. baumannii oleum ficedula, revealing their potential to degrade hydrocarbons and to produce biosurfactants or bioemulsifiers. Applying these environmental subspecies in bioaugmentation strategies sheds light on future approaches to bioremediation. The study shows the importance of genomic analysis of environmental strains and their inclusion in metabolic pathways databases, highlighting unique enzymes/alternative pathways for consuming hazardous hydrocarbons.

The Influence of the Synthesis Method on the Characteristics of BaTiO3.

In this work, barium titanate powders were produced by sol-gel and sol-precipitation methods from metal alkoxides. In the sol-gel method, tetraisopropyl orthotitanate was mixed with 2-propanol, acetic acid and barium acetate, and the gel samples obtained were calcined at 600 °C, 800 °C and 1000 °C. Through the sol-precipitation method, tetraisopropyl orthotitanate was mixed with acetic acid and deionized water and precipitated by the addition of a concentrated solution of KOH. The products were calcined at various temperatures, and the microstructural and dielectric properties of the BaTiO3 prepared for the two processes were analyzed and compared. The results of these analyses allowed us to observe an increase in the tetragonal phase and the dielectric constant (15-50 at 20 kHz) with increasing temperatures in the samples produced by the sol-gel method, while the sample obtained by sol precipitation was cubic. The presence of BaCO3 is more evident in the sample produced by sol-precipitation, and the band gap of the products obtained did not show significant variation, changing the synthesis method (3.363-3.594 eV).

Effect of carvacrol antioxidant capacity on oocyte maturation and embryo production in cattle.

Carvacrol (C10H14O), an efficient phenolic antioxidant substance for several cell types, may become a useful antioxidant for female germ cells and embryo culture. This study investigates the effects of carvacrol supplementation on bovine oocytes in in vitro maturation (IVM) and embryo production. In total, 1222 cumulus-oocyte complexes were cultured in TCM-199+ alone (control treatment) or supplemented with carvacrol at the concentrations of 3 µM (Carv-3), 12.5 µM (Carv-12.5), or 25 µM (Carv-25). After IVM, the oocytes were subjected to in vitro fertilization and embryo production, and the spent medium post-IVM was used for evaluating the levels of reactive oxygen species and the antioxidant capacity (2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2'-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid quantification). A greater (P < 0.05) antioxidant potential was observed in the spent medium of all carvacrol-treated groups compared with the control medium. Moreover, the addition of carvacrol to the maturation medium did not affect (P > 0.05) blastocyst production on days 7 and 10 of culture; however, the total number of cells per blastocyst was reduced (P < 0.05) in two carvacrol-treated groups (Carv-3 and Carv-25). In conclusion, carvacrol demonstrated a high antioxidant capacity in the spent medium after oocyte maturation; however, although embryo production was not affected, in general, carvacrol addition to IVM medium reduced the total number of cells per blastocyst. Therefore, due to the high antioxidant capacity of carvacrol, new experiments are warranted to investigate the beneficial effects of lower concentrations of carvacrol on embryo production in cattle and other species.

Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst.

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.

In vitro- and in vivo-derived early antral follicles have comparable in vitro follicular growth and oocyte maturation rates in goats.

Recent in vitro follicle culture (IVFC) studies in caprine have yielded lower maturation rates using late preantral follicles compared to early antral follicles. Thus, research focusing on developing stage-specific customized culture systems able to improve the efficiency of IVFC for late preantral follicles are warranted. This study aimed to compare the morphometric features, estradiol production, and gene expression between early antral caprine follicles produced in vitro and in vivo. In vitro-derived early antral follicles were produced after a 6-day in vitro culture of late preantral follicles, while in vivo-derived early antral follicles were yielded immediately after isolation from the ovaries; antral follicles were, thereafter, cultured for 18 days. In vitro-derived antral follicles were cultured either in a medium developed for preantral follicles (PF medium) or in a medium developed for antral follicles (AF medium). In vivo-derived early antral follicles, on the other hand, were cultured in AF medium (Control treatment). Results demonstrated that in vitro-derived antral follicles cultured in PF medium produced higher estradiol concentration, and m-RNA expression for matrix metalloproteinase-9 (MMP-9), and insulin receptor when compared to both in vitro- and in vivo-derived antral follicles cultured in AF medium. Remarkably, in vitro-derived antral follicles cultured in PF medium had similar MII and oocytes ≥110 µm rates compared with in vivo-derived antral follicles (Control treatment). In conclusion, when cultured in a single and appropriate medium (i.e., PF medium), in vitro-derived early antral follicles had comparable oocyte maturation rates to the in vivo-derived early antral follicles.

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue.

This study evaluated the effects of different concentrations (10, 20, or 40 µM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 µg/mL; AA) or anethole (300 µg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.

Impact of ethanol and heat stress-dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS.

Qualidade da água natural produzida em uma indústria no município de São José de Ribamar MA / Quality of natural water produced in an industry the municipality of São José de Ribamar MA

O presente trabalho teve por objetivo avaliar a qualidade físico-química e microbiológica da água natural de uma indústria produtora localizada no município de São José de Ribamar - MA e a implantação das Boas Práticas de Fabricação (BPF). Foram coletadas amostras de água em pontos específicos da indústria sendo eles: poço, filtro I tipo bag, filtro II tipo bag, filtro III tipo polidor, reservatório I, reservatório II, filtro IV tipo polidor, área de envase e laboratório, e realizadas análises microbiológicas por meio do teste rápido Colilert® (pesquisa de coliformes a 35ºC e Escherichia coli) e análises físicoquímicas (condutividade elétrica, potencial hidrogeniônico, sólidos totais e turbidez). A aplicação do checklist foi baseado na Resolução RDC n° 173 de setembro de 2006 da Agência Nacional de Vigilância Sanitária (ANVISA). O resultado das análises físico-químicas e microbiológicas apresentaram-se dentro dos padrões estabelecidos pela RDC nº 274 de 22 de setembro de 2005 da ANVISA.(AU)

The aim of the present work was to evaluate a physical-chemical and microbiological quality of natural water industries from one located in the city of São José de Ribamar - MA and the evaluation of Good Manufacturing Practices (GMPs). Water collections were done at several specific points in the industry, namely: well, filter I type bag, filter II type bag, filter III type polisher, reservoir I, reservoir II, filter IV type polisher, filling area and laboratory, and microbiological analysis were performed using the Colilert® rapid test (research of 35ºC coliforms as well as Escherichia coli) and physical-chemical analysis (electrical conductivity, hydrogen potential, total solids and turbidity). The application of checklist was based in the Resolution RDC No . 173 of September 2006 from ANVISA. The result of analyzes physical-chemical and microbiological are within normal limits and in accordance with the parameters changed by the RDC legislation No . 274/2005 of National Health Surveillance Agency (ANVISA).(AU)

Qualidade da água natural produzida em uma indústria no município de São José de Ribamar – MA / Quality of natural water produced in an industry the municipality of São José de Ribamar – MA

O presente trabalho teve por objetivo avaliar a qualidade físico-química e microbiológica da água natural de uma indústria produtora localizada no município de São José de Ribamar - MA e a implantação das Boas Práticas de Fabricação (BPF). Foram coletadas amostras de água em pontos específicos da indústria sendo eles: poço, filtro I tipo bag, filtro II tipo bag, filtro III tipo polidor, reservatório I, reservatório II, filtro IV tipo polidor, área de envase e laboratório, e realizadas análises microbiológicas por meio do teste rápido Colilert® (pesquisa de coliformes a 35ºC e Escherichia coli) e análises físicoquímicas (condutividade elétrica, potencial hidrogeniônico, sólidos totais e turbidez). A aplicação do checklist foi baseado na Resolução RDC n° 173 de setembro de 2006 da Agência Nacional de Vigilância Sanitária (ANVISA). O resultado das análises físico-químicas e microbiológicas apresentaram-se dentro dos padrões estabelecidos pela RDC nº 274 de 22 de setembro de 2005 da ANVISA.

The aim of the present work was to evaluate a physical-chemical and microbiological quality of natural water industries from one located in the city of São José de Ribamar - MA and the evaluation of Good Manufacturing Practices (GMP’s). Water collections were done at several specific points in the industry, namely: well, filter I type bag, filter II type bag, filter III type polisher, reservoir I, reservoir II, filter IV type polisher, filling area and laboratory, and microbiological analysis were performed using the Colilert® rapid test (research of 35ºC coliforms as well as Escherichia coli) and physical-chemical analysis (electrical conductivity, hydrogen potential, total solids and turbidity). The application of checklist was based in the Resolution RDC No . 173 of September 2006 from ANVISA. The result of analyzes physical-chemical and microbiological are within normal limits and in accordance with the parameters changed by the RDC legislation No . 274/2005 of National Health Surveillance Agency (ANVISA).

The impact of two root canal treatment protocols on the oral health-related quality of life: a randomized controlled pragmatic clinical trial.

AIM: To assess the impact of two root canal treatment protocols on the oral health-related quality of life (OHRQoL) of patients in need of root canal treatment on their anterior teeth. METHODOLOGY: The sample consisted of 120 participants (mean age: 34 years) enrolled in a pragmatic randomized clinical trial evaluating two root canal treatment protocols. Anterior teeth with nonvital pulps were allocated for root canal preparation with either hand files and filled with lateral compaction of gutta-percha (manual protocol) or canal preparation with a single file in a reciprocating movement and filled with a single cone technique (Reciproc protocol). OHRQoL data were assessed using the Oral Health Impact Profile instrument (OHIP-14), which was administered before the root canal intervention (baseline), and 6 and 12 months after treatment. Demographic and clinical characteristics of participants were collected at baseline. Data were analysed using bivariate analyses, Poisson univariate and multiple regression (α = 0.05). RESULTS: The drop-out rate from baseline was 27% and 28% at 6 and 12 months after treatment, respectively. Both root canal protocols significantly enhanced patients' OHRQoL, regardless of the follow-up time (P < 0.001). After 6 months, patients treated with the Reciproc protocol had significantly lower OHIP-14 overall scores (P = 0.030), as well as significantly lower scores for psychological discomfort (P = 0.031) and social disability (P = 0.013). After 12 months, no significant difference was observed between the two root canal protocols for OHIP-14 overall scores (P = 0.174). Either large or moderate effect sizes were observed for all domains and overall scores at both evaluation times, irrespective of the protocol. Low-income persons (RR = 2.03) and the Reciproc protocol (RR = 1.52) had a higher likelihood of a positive impact on OHRQoL 12 months after root canal treatment. CONCLUSIONS: The two root canal protocols improved the OHRQoL and differences in scores were observed only after 6 months with poorer OHRQoL for the manual protocol. After 12 months, patients with low-income status and treated with Reciproc were associated with a greater improvement in OHRQoL scores.

Detection of IgG3 antibodies specific to the human immunodeficiency virus type 1 (HIV-1) p24 protein as marker for recently acquired infection.

Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

Interactions between different media and follicle-stimulating hormone supplementation on in vitro culture of preantral follicles enclosed in ovarian tissue derived from collared peccaries (Pecari tajacu Linneaus, 1758).

The aim was to verify the effect of follicle-stimulating hormone (FSH) supplementation to α-MEM+ or TCM199+ media on the in vitro development of ovarian preantral follicles (PFs) derived from collared peccaries. Ovaries (n = 5 pairs) were collected and divided into fragments destined to control group (non-cultured) or treatments that were cultured for 7 days. The PFs morphology, growth and activation were evaluated by classical histology. The immunohistochemistry markers Ag-NOR and PCNA were used for nuclear proliferation analysis, and the picrosirius red labelling was used for ovarian extracellular matrix (ECM) evaluation. After 7-day culture, only the TCM199+ treatment maintained the proportion of intact PFs similar to day 1 (63.2%), but no differences were found among treatments (p > .05). In addition, a significant increase in the growing follicles proportion was verified for all the treatments, indicating follicular activation (p > .05). By the Ag-NOR analysis, only the TCM199+/FSH maintained the nuclear proliferation similar to the first day (p > .05). The picrosirius red staining revealed that the ECM remained intact in all the treatments (p > .05). We suggest the use of TCM199+ medium supplemented of FSH for the in vitro development of peccaries PFs under 7-day culturing conditions.

Role of EGF on in situ culture of equine preantral follicles and metabolomics profile.

The effects of epidermal growth factor (EGF) concentrations (0, 10, 50, and 100ng/ml) on in vitro culture (IVC) of equine preantral follicles were evaluated using histology, estradiol and reactive oxygen species (ROS) production and metabolomics. After IVC, the percentage of normal follicles was lower (P<0.05) for all treatments when compared to non-cultured control. EGF 50ng/ml treatment had more (P<0.05) normal follicles at Day 7 of culture when compared with EGF 0 and 100ng/ml. EGF 50ng/ml had more (P<0.05) developing follicles than the 0ng/ml and 10ng/ml EGF treatments. Follicular and oocyte diameters were greater (P<0.05) with EGF 50ng/ml than the other cultured treatments, but similar (P>0.05) to the non-cultured control. From Day 1 to Day 7 estradiol production increased (P<0.05) in all EGF treatments. EGF 50ng/ml was the only treatment that maintained ROS production through IVC. Metabolomics profiles of the spent media indicated that eleven ions from variable influence in the projection (VIP) scores were higher represented in the EGF 50ng/ml treatment. In conclusion, EGF 50ng/ml treatment maintained follicle survival and ROS production, and promoted activation of cultured equine preantral follicles enclosed in ovarian tissue.

In vitro growth and maturation of isolated caprine preantral follicles: Influence of insulin and FSH concentration, culture dish, coculture, and oocyte size on meiotic resumption.

The aims of this study were: (1) to evaluate the effect of different insulin concentrations, alone or in combination with either a fixed FSH concentration or increasing FSH concentrations on the in vitro culture of isolated caprine preantral follicles and (2) to analyze the efficiency of two IVM media and maturation culture systems (with or without coculture with in vivo grown oocytes) on the meiosis resumption. Secondary follicles were cultured for 18 days in a basic medium supplemented with low- or high-insulin concentration alone or with a fixed FSH concentration or with increasing FSH concentrations. Oocytes grown in vivo or in vitro were matured alone or cocultured. The high-insulin concentration associated with fixed FSH treatment had higher meiotic resumption rate (P < 0.05) and was the only treatment capable of producing oocytes in metaphase II. The rates of germinal vesicle, germinal vesicle breakdown, metaphase I, metaphase II (MII), meiotic resumption, and oocyte diameter were similar between the maturation media. In conclusion, a basic medium supplemented with 10-µg/mL insulin and 100-µg/mL FSH throughout the culture period improved meiotic resumption rate and produced MII oocytes from caprine preantral follicles cultured in vitro. The MII rate was similar between in vivo and in vitro grown oocytes ≥110 µm.

Anti-Müllerian hormone reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro.

The aim of the present study was to evaluate the effect of anti-Müllerian hormone (AMH), with and without FSH, on the in vitro development of isolated caprine preantral follicles, as well as follicular steroid production and mRNA levels of AMH, hormone receptors (AMH and FSH), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP17 (cytochrome P450, family 17, subfamily A, polypeptide 1), HSD3B (3-beta-hydroxysteroid dehydrogenase) and Myc (myelocytomatosis oncogene). Isolated secondary follicles were cultured in minimum essential medium alpha (α-MEM+) alone or supplemented with 50ng mL-1 AMH and/or 100ng mL-1 FSH added sequentially on different days of culture. Follicles were cultured for a total of 18 days, with different media during the first (Days 0-9) and second (Days 10-18) halves of the culture period, resulting in six treatment groups, as follows: α-MEM+/α-MEM+, FSH/FSH, AMH/AMH, AMH+FSH/AMH+FSH, AMH/FSH, and FSH/AMH. Follicle development was evaluated on the basis of follicular growth, oocyte maturation and steroid secretion. There was a decrease in follicular growth rate in the AMH, AMH+FSH and AMH/FSH treatment groups compared with α-MEM+ and FSH treatment groups (P<0.05). However, the different culture conditions had no effect on rates of meiotic resumption and steroid secretion (P>0.05). Moreover, follicles cultured in the presence of FSH had lower levels of AMH receptor type II (AMHRII) mRNA compared with non-cultured control (freshly isolated follicles), and the AMH and AMH/FSH treatment groups. In conclusion, AMH reduces the follicular growth rate of isolated goat preantral follicles in vitro without affecting follicular survival.

Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture.

Our aim has been to evaluate the effect of cryoprotective agents (CPAs) on the exposure, vitrification (VIT), and in vitro culture (IVC) of ovarian tissue with regard to the expression and immunolocalization of aquaporins (AQPs) 3 and 9 in ovine preantral follicles. Tissues were treated as follows: Experiment I: (1) control (without exposure to CPAs), (2) e-EG (exposure to ethylene glycol), (3) er-EG (exposure to and removal of EG), (4) e-DMSO (exposure to dimethyl sulfoxide), (5) er-DMSO (exposure to and removal of DMSO), (6) e-EG+DMSO (exposure to EG+DMSO), (7) er-EG+DMSO (exposure to and removal of EG+DMSO); Experiment II: (1) control, (2) VIT, (3) IVC, (4) VIT-IVC. In Experiment I, following er-EG or er-DMSO, tissue showed the down-regulation (P < 0.05) of AQP3 mRNA. The mRNA transcript levels were reduced (P < 0.05) for AQP9 in tissue following er-EG+DMSO. Immunolocalization was positive for both proteins (AQP3 and AQP9) on ovine preantral follicles following all treatments, except in the e-EG+DMSO group. In Experiment II, the mRNA levels of AQP3 and AQP9 following VIT treatment were similar (P > 0.05) to that of the control group. Nevertheless, VIT-IVC treatment led to the down-regulation of mRNA of AQP3 and AQP9. Thus, AQP3 and AQP9 act in a mutually dependent way, maintaining the cell homeostasis that is essential for the ovary cryopreservation process. Furthermore, the changes in the expression profiles of mRNA and protein after culture are a strong indicator that in vitro conditions have to be strictly controlled to ensure follicle viability and functionality.

Immunolocalization of the Anti-Müllerian Hormone (AMH) in Caprine Follicles and the Effects of AMH on In Vitro Culture of Caprine Pre-antral Follicles Enclosed in Ovarian Tissue.

The aims of this study were to evaluate the localization, by immunohistochemistry, of the anti-Müllerian hormone (AMH) in goat ovaries and to investigate its effects on the in vitro survival and development of caprine pre-antral follicles enclosed in fragments of ovarian tissue. Pre-antral follicles were cultured in vitro for 1 or 7 days in α-MEM(+) in the absence or presence of kit ligand (KL; 50 ng/ml, positive control) or AMH (50 or 150 ng/ml). The results showed that AMH was localized in oocytes and granulosa cells from the primordial follicle to antral follicle stages. Addition of AMH maintained the percentage of developing follicles, similar to that in the uncultured control; however, the percentage of developing follicles was significantly lower than that in the cultured control and KL. Nonetheless, addition of AMH to the culture medium did not affect survival rates and follicular growth. In conclusion, this study demonstrated that the expression of AMH varies according to the compartment and stage of follicular development. Furthermore, AMH inhibits the activation of caprine primordial follicles.

FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue.

This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture.

Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture.

This study investigated the effect of insulin concentration on the in vitro culture of equine preantral follicles enclosed in ovarian tissue. Ovarian tissue samples were immediately fixed (noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0 ng/mL, 10 ng/mL, or 10 µg/mL insulin. Ovarian tissues were processed and analyzed by classical histology. Culture medium samples were collected after 1 and 7 days of culture for steroid and reactive oxygen species (ROS) analyses. The percentage of morphologically normal follicles was greater (P < 0.001) in insulin-treated groups after 1 day of culture; likewise, more (P < 0.02) normal follicles were observed after 7 days of culture in medium supplemented with 10-ng/mL insulin. Furthermore, an increase (P < 0.01) in developing (transition, primary, and secondary) follicles between Days 1 and 7 of culture was observed only with the 10-ng/mL insulin treatment. ROS production after 1 or 7 days of culture was lower (P < 0.0001) in medium with 10-ng/mL insulin than the other treatments. Ovarian tissues containing preantral follicles were able to produce estradiol and progesterone after 1 and 7 days of culture; however, treatments did not differ in steroid production. In conclusion, the use of a physiological concentration (10 ng/mL) of insulin rather than the previously reported concentration (10 µg/mL) for in vitro culture of equine preantral follicles improved follicular survival and growth and lowered oxidative stress. Results from this study shed light on new perspectives for producing an appropriate medium to improve equine preantral follicle in vitro survival and growth.