Quantitative evaluation of very early cartilage damage in experimental osteoarthritis using scanning electron microscopy.

OBJECTIVE: To describe parametric changes observed using scanning electron microscopy (SEM) in very early stages in posttraumatic osteoarthritis (OA) models in mice. METHODS: Mice (5/group) had their knees subjected to anterior cruciate ligament transection (ACLT), ACLT plus meniscectomy (MNCT) or sham surgery, sacrificed after 3, 7 or 14 days, had the articular cartilage evaluated under optical microscopy using Osteoarthritis Research Society International (OARSI) parameters as well as cartilage thickness, roughness, and a damage index using SEM. RESULTS: Alterations of the cartilage under optical microscopy were not significantly relevant among groups. SEM analysis revealed reduction of femoral and tibial cartilage thickness in ACLT and MNCT groups at 7 and 14 days, with increased cartilage roughness in MNCT group as early as 3 days postsurgery, being sustained up to 14 days. Articular damage index was significantly higher at 14 days post surgery in ACLT and MNCT vs control groups. CONCLUSION: This is the first demonstration of very early quantitative changes in the cartilage of mice subjected to posttraumatic experimental OA using SEM, revealing increased roughness and thickness as early as 3 days post surgery. These changes may be used as early surrogates for later joint damage in experimental OA.

Sheep Isolated Secondary Follicles Are Able to Produce Metaphase II Oocytes After Vitrification and Long-Term In Vitro Growth.

The vitrification of preantral follicles followed by in vitro growth (IVG) could be valuable to produce fertilizable oocytes. However, the meiotic resumption rates of oocytes cultured from vitrified secondary follicles (SF) have been reported as suboptimal. This study aimed to verify two base media (alpha modification of minimum essential medium, α-MEM, and tissue culture medium 199, TCM199) on vitrified SF regarding different requirements during IVG. Sheep ovarian fragments were divided in six groups: (1) Fresh groups (Control α-MEM and TCM199): SF without vitrification; (2) Follicle-Vitrified (Follicle-Vit α-MEM and TCM199): SF vitrified after isolation; and (3) Tissue-Vitrified (Tissue-Vit α-MEM and TCM199): SF vitrified enclosed in ovarian fragments and, subsequently, isolated. The isolated SF were submitted to IVG for 18 days. Thereafter, the recovered cumulus-oocyte complexes (COCs) underwent in vitro maturation (IVM) and evaluation of chromatin configuration. Follicular granulosa cells were analyzed for their gene expression of Bax, Bcl2, and Connexins (CX) 37 and 43. COCs from in vivo antral follicles were used as in vivo control. Data were analyzed by analysis of variance, Tukey, and chi-square tests. Differences were considered significant if p-value is <0.05. Follicle-Vit groups had higher (p < 0.05) percentage of antrum formation compared with Tissue-Vit groups. Vitrification did not affect (p > 0.05) oocyte diameter postmaturation. Oocytes from Follicle-Vit in α-MEM reached metaphase II stage after IVM. Gene expression for CX37, CX43, and Bax was lower in Tissue-Vit groups. For Bcl2, the gene expression was the opposite. In conclusion, during IVG for 18 days, maximal oocyte meiotic resumption was not negatively impacted by vitrification and was greatest for isolated SF using α-MEM as a medium.

Fraction of Auxemma oncocalyx and Oncocalyxone A Affects the In Vitro Survival and Development of Caprine Preantral Follicles Enclosed in Ovarian Cortical Tissue.

BACKGROUND: Auxemma oncocalyx and its main component oncocalyxone A (onco A) have a high level of antioxidant and antitumor activity, but there are no studies on the action of both of these drugs regarding folliculogenesis. MATERIAL AND METHODS: Caprine ovarian tissue fragments were fixed (non-cultured control) or cultured for 1 or 7 days in α-MEM+ alone (cultured control) or supplemented with dimethyl sulfoxide (DMSO; 20% v/v), bone morphogenetic protein 15 (BMP-15; 100 ng/ml), doxorubicin (DXR; 0.3 g/ml), or different concentrations of A. oncocalyx (1.2, 12, or 34 g/ml) or onco A (1, 10, or 30 g/ml). We analyzed for follicular morphology and growth, apoptosis (terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay), and cell proliferation (silver staining of argyrophilic nucleolus organizer regions (AgNOR) and test for proliferating cell nuclear antigen (PCNA)). RESULTS: A. oncocalyx and onco A (in a concentration-dependent manner) and DXR decreased (P < 0.05) the number of morphologically normal follicles, with no effect (P > 0.05) on follicular growth. A. oncocalyx reduced (P < 0.05) the percentage of normal follicles compared to onco A, whereas DXR, A. oncocalyx 1.2 g/ml, and onco A 1 g/ml increased (P < 0.05) the percentage of TUNEL-positive follicles. DXR decreased (P < 0.05) the number of nucleolus organizer regions. CONCLUSION: A. oncocalyx and onco A affected the in vitro caprine folliculogenesis in a concentration-dependent manner. Onco A (1 g/ml) has a less harmful effect than DXR on goat preantral follicle survival.

In situ cultured preantral follicles is a useful model to evaluate the effect of anticancer drugs on caprine folliculogenesis.

Despite the increase in the incidence of cancer, the number of women who survive cancer treatment is growing. However, one of the principal results of chemotherapy is premature ovarian failure (POF). The aim of this study was to use the in situ culture preantral follicles as an in vitro model to evaluate the toxicity of two anticancer drugs, doxorubicin (DXR) and paclitaxel (PTX), on the integrity and development of ovarian follicles. Fragments of the ovarian cortex of goats were cultured in vitro for 1 or 7 days in α-MEM(+) supplemented with different concentrations of DXR (0.003, 0.03, or 0.3 µg/mL) and PTX (0.001, 0.01, or 0.1 µg/mL). Analyses were performed before and after culture to evaluate tissue integrity by classical histology, apoptosis by TUNEL assay, DNA laddering kit and the detection of activated caspase 3, and DNA damage by the immune detection of phosphorylated histone H2A.x (H2AXph139). Both DXR and PTX reduced the number of morphologically normal primordial and developing follicles. Positive staining for TUNEL and active caspase 3 was detected in all the samples (P < 0.05). Therefore, we propose the in situ culture of caprine preantral follicles as a useful experimental model for assessing the toxic effects of the chemotherapeutic agents on ovarian folliculogenesis. Microsc. Res. Tech. 79:773-781, 2016. © 2016 Wiley Periodicals, Inc.

Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments.

Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissue-vitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chi-square. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in follicle-vit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissue-vit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between treatments except for in vivo grown. After vitrification, only follicle-vit showed metaphase I oocyte. We conclude that secondary follicles vitrified after isolation displayed a better follicular growth rate, oocyte viability, percentage of oocytes reaching the metaphase I stage, and fewer follicles with decreased diameter after IVC.

Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue.

Cryoinjuries caused by vitrification of tissues and organs lead to the loss of membrane proteins that mediate intercellular communications, such as connexins 37 (Cx37) and 43 (Cx43). Thus, the present study aimed to evaluate ovine Cx37 and Cx43 gene and protein expressions and developmental competence by in vitro-cultured secondary follicles retrieved from vitrified ovarian tissue. Ovarian fragments for the same ovary pair were distributed into six treatments: (1) fresh ovarian tissue (FOT); (2) vitrified ovarian tissue (VOT); (3) isolated follicles from fresh ovarian tissue (FIF); (4) isolated follicles from vitrified ovarian tissue; (5) isolated follicles from fresh ovarian tissue followed by in vitro culture (CFIF); (6) isolated follicles from vitrified ovarian tissue followed by in vitro culture (CVIF). In all treatments, Cx37 and Cx43 gene and protein expression patterns were evaluated by reverse transcription polymerase chain reaction and immunocytochemistry. In addition, secondary follicles were analyzed according to follicular integrity and growth, apoptosis, and cell proliferation. In vitro-cultured secondary follicles (CFIF and CVIF) were evaluated based on morphology (extruded follicles), antrum formation, and viability. The percentage of intact follicles was higher, whereas antrum formation, oocyte extrusion rate, and follicle viability were lower in CVIF than in CFIF treatment (P < 0.05). Terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling assay demonstrated that apoptosis was absent in FIF, whereas follicles from all other treatments showed positive labeling. Cell proliferation index was higher in isolated follicles from vitrified ovarian tissue and CVIF treatments than in follicles from FIF. Expression of Cx43 messenger RNA was lower in CVIF treatment when compared with follicles from all other treatments (P < 0.05). Follicle Cx37 messenger RNA levels did not show alterations in any treatment (P > 0.05). Cx37 and Cx43 immunolabeling was localized mainly on granulosa cells and oocytes, respectively. In conclusion, isolation of ovine secondary follicles could be done successfully after vitrification of ovarian tissue, and the basement membrane integrity remained intact after in vitro culture. Although the gene and protein expression of Cx37 did not change after vitrification of ovarian tissue, Cx43 turned out to be altered in secondary follicles after vitrification and in vitro culture.

Intervalos de referência sanguíneos e a influência da idade e sexo sobre parâmetros hematológicos e bioquímicos de ovinos da raça Santa Inês criados na Amazônia Oriental / Blood reference intervals and the influence of age and gender on hematologic and biochemical parameters of Santa Ines sheep bred in the eastern Amazon

Blood count and biochemical tests are used to diagnose diseases in domestic animals. These tests can be influenced by age, gender, nutrition, breed, species and environmental conditions. Thus, data from one region should not be extrapolated to animals raised in other regions. The objective of this study was to determine the hematological and biochemical values of different ages and genders of Santa Inês sheep, raised in the eastern Amazon. There were examined 91 sheep that were assigned to three groups: G1 (three to six months old, n = 31); G2 (seven to 24 months old, n = 30) and G3 (above 24 months old, n = 30). The blood cell count, white blood count and biochemical determinations were made with an automatic counter and a semi-automatic analyzer, respectively. Mean values were compared using the Tukey test. The number of erythrocytes, the red blood indices, the thrombogram, eosinophils, total protein, urea and creatinine concentrations were influenced by the age of the animals. The erythrocyte coefficient of variation and the creatinine concentration were influenced by gender, and were greater in males. The neutrophil:lymphocyte (N:L) ratio was greater than one for all age groups. This study led to the determination of reference values for sheep raised in the Eastern Amazon and demonstrated that when interpreting hematological and biochemical tests of sheep, age and gender must be considered.

O hemograma e o perfil bioquímico são usados para diagnosticar doenças em animais domésticos. Esses exames podem ser influenciados pela idade, sexo, nutrição, raça, espécie e condições ambientais. Portanto, dados de uma região não podem ser totalmente extrapolados para animais criados em regiões geograficamente distintas. Isso se aplica a ovinos criados no Bioma Amazônico. O objetivo deste estudo foi determinar os valores hematológicos e bioquímicos de ovinos Santa Inês, de diferentes idades e gêneros criados na Amazônia oriental. Foram examinados 91 ovinos divididos em três grupos: G1 (3-6 meses de idade, n = 31); G2 (7 a 24 meses de idade, n = 30) e G3 (mais de 24 meses de idade, n = 30). O hemograma e as determinações bioquímicas foram realizados com um contador automático e um analisador semi-automático, respectivamente. Os resultados foram comparados pelo teste de Tukey. O número de eritrócitos, índices eritrocitários, plaquetograma, número de eosinófilos, teor de proteína total, de ureia e de creatinina foram influenciados pela idade dos animais. O coeficiente de variação dos eritrócitos e a concentração de creatinina foram influenciados pelo sexo, sendo maiores nos machos. A relação neutrófilos:linfócitos (N:L) foi maior que um para todos os grupos etários. Neste estudo foram determinados valores de referência para ovinos criados na Amazônia Oriental. Além disso, demonstrou-se que ao interpretar exames hematológicos e bioquímicos de ovinos, a idade e o sexo devem ser considerados.

Vitrified sheep isolated secondary follicles are able to grow and form antrum after a short period of in vitro culture.

The risk of reintroducing malignant cells after ovarian graft into patients following post-cancer treatment is an obstacle for clinical applications (autotransplantation). In this context, in vitro follicle culture would be an alternative to transplantation in order to minimize such risks. Therefore, the aim of this study was to compare the development of secondary follicles after vitrification in isolated form (without stroma) with vitrification in in situ form (within fragments of ovarian tissue). Follicles were first isolated from ovarian fragments from mixed-breed ewes and then vitrified; these comprised the Follicle-Vitrification group (Follicle-Vit), or fragments of ovarian tissue were first vitrified, followed by isolation of the follicles, resulting in the Tissue-Vitrification group (Tissue-Vit). Control and vitrified groups were submitted to in vitro culture (6 days) and follicular morphology, viability, antrum formation, follicle and oocyte diameter, growth rate, ultrastructural characteristics and cell proliferation were evaluated. The percentages of morphologically normal follicles and antrum formation were similar among groups. Follicular viability and oocyte diameter were similar between Follicle-Vit and Tissue-Vit. The follicular diameter and growth rate of Follicle-Vit were similar to the Control, while those of Tissue-Vit were significantly lower compared to the Control. Both vitrified groups had an augmented rate of granulosa cellular proliferation compared to Control. Secondary follicles can be successfully vitrified before or after isolation from the ovarian tissue without impairing their ability to survive and grow during in vitro culture.

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.

Thymol and eugenol derivatives as potential antileishmanial agents.

In Northeastern Brazil visceral leishmaniasis is endemic with lethal cases among humans and dogs. Treatment is toxic and 5-10% of humans die despite treatment. The aim of this work was to survey natural active compounds to find new molecules with high activity and low toxicity against Leishmania infantum chagasi. The compounds thymol and eugenol were chosen to be starting compounds to synthesize acetyl and benzoyl derivatives and to test their antileishmanial activity in vitro and in vivo against L. i. chagasi. A screening assay using luciferase-expressing promastigotes was used to measure the growth inhibition of promastigotes, and an ELISA in situ was performed to evaluate the growth inhibition of amastigote. For the in vivo assay, thymol and eugenol derivatives were given IP to BALB/c mice at 100mg/kg/day for 30 days. The thymol derivatives demonstrated the greater activity than the eugenol derivatives, and benzoyl-thymol was the best inhibitor (8.67 ± 0.28 µg/mL). All compounds demonstrated similar activity against amastigotes, and acetyl-thymol was more active than thymol and the positive control drug amphotericin B. Immunohistochemistry demonstrated the presence of Leishmania amastigote only in the spleen but not the liver of mice treated with acetyl-thymol. Thus, these synthesized derivatives demonstrated anti-leishmanial activity both in vitro and in vivo. These may constitute useful compounds to generate new agents for treatment of leishmaniasis.

Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

Insulin-like growth factor II (IGF-II) and follicle stimulating hormone (FSH) combinations can improve the in vitro development of grown oocytes enclosed in caprine preantral follicles.

OBJECTIVE: Evaluate the possible role of IGF-II alone or in association with FSH on in vitro development of isolated caprine preantral follicles. METHODS: Preantral follicles (≥150 µm) were isolated from goat ovaries and cultured for 18 days in basic αMEM medium (control) or supplemented with IGF-II alone at 20 or 50 ng/ml, named IGF20 and IGF50, respectively, or in combination with recombinant FSH (FSH, IGF20F or IGF50F). During in vitro culture, the follicles were analyzed by using morphology criteria, antrum formation and growth rate as parameters. After 18 days of follicular culture, oocytes equal to or larger than 110 µm were used for in vitro maturation (IVM). Oocyte viability and meiosis resumption were assessed by fluorescence microscopy after labeling with calcein-AM, ethidium homodimer and Hoechst 33342. RESULTS: The IGF20 treatment was the only treatment capable of maintaining the percentage of morphologically normal follicles from D0 until D6 and from D12 to D18 (p>0.05), while in all other treatments the percentage of morphologically normal follicles decreased progressively during 18 days of in vitro culture (p<0.05). At D18, all treatments with IGF-II or FSH resulted in a significantly higher percentage of normal follicles when compared to αMEM alone. The IGF50F treatment provided a significantly higher early antrum formation rate when compared to αMEM and FSH alone. The addition of IGF-II alone (20 or 50 ng/ml) or in combination with FSH prevented oocyte degeneration after IVM. Moreover, the FSH treatment demonstrated a lower percentage of oocyte degeneration when compared to control (4.35% vs. 26.3%, respectively; p<0.05). Regarding meiosis resumption, the IGF20F treatment was the only treatment that significantly differed from αMEM alone. All treatments except the control (αMEM alone) presented oocytes at metaphase II. CONCLUSION: IGF-II associated with FSH stimulated in vitro follicular development, oocyte viability and meiotic resumption of caprine oocytes after IVM.

Dynamized follicle-stimulating hormone affects the development of ovine preantral follicles cultured in vitro.

OBJECTIVE: To evaluate the effect of dynamized follicle-stimulating hormone (FSH) on the survival, activation and growth of ovine preantral follicles (PFs) in vitro. METHODS: Ovarian fragments were cultured for 1 or 7 days in alpha minimum essential medium (α-MEM(+)) control in the absence or presence of alcohol (Al control) or FSH (6cH, 12cH and 30cH) added at intervals of 24 or 48 h. The ovarian fragments were processed, coded and analyzed by a blinded observer by classical histology (CH), fluorescence microscopy (FM) and transmission electron microscopy (TEM). RESULTS: After 7 days of culture, the group which to which FSH 6cH was added at 24 h intervals showed better rates of follicle survival and activation compared to α-MEM(+) control or Al control (p < 0.05). This group also showed higher follicle and oocyte growth than α-MEM(+) control (p < 0.05). FM and TEM techniques confirmed that FSH 6cH promoted viability and ultrastructural integrity of follicles after 7 days of culture. CONCLUSIONS: FSH 6cH (24 h) treatment maintained the viability, and promoted the activation and in vitro growth of ovine PFs.

Different susceptibilities of Leishmania spp. promastigotes to the Annona muricata acetogenins annonacinone and corossolone, and the Platymiscium floribundum coumarin scoparone.

Leishmaniasis is a zoonotic disease that can manifest itself in visceral and cutaneous form. The aim of this study was to search for new leishmanicidal compounds. Preliminarily, Artemia salina assay was applied to compounds from two plants found in Northeastern Brazil, Platymiscium floribundum and Annona muricata. Then these compounds were tested against three Leishmania species (Leishmania donovani, Leishmania mexicana and Leishmania major). A screening assay using luciferase-expressing promastigote form were used to measure the viability of promastigote One coumarin, scoparone, isolated from P. floribundum and two acetogenins, annonacinone and corossolone isolated from A. muricata showed leishmanicidal activity in all species tested. Nevertheless, Leishmania species indicated different susceptibilities in relation to the tested compounds: L. mexicana was more sensitive to scoparone followed by L. major and L. donovani. The three species presented similar inhibition to corossolone and annonacinone. Acetogenin annonacinone (EC(50)=6.72-8.00 µg/mL) indicated high leishmanicidal activity; corossolone (EC(50)=16.14-18.73 µg/mL) and scoparone (EC(50)=9.11-27.51 µg/mL) moderate activity. A. saline larvae were less sensitive to the coumarin scoparone and acetogenin corossolone was the most toxic. In conclusion, the leishmanicidal activity demonstrated by the coumarin and acetogenins indicate these compounds for further studies aiming the development of new leishmanicidal agents.

Study of the development of uteroplacental and fetal feline circulation by triplex Doppler.

The objective was to evaluate blood flow in fetal and maternal vessels by Triplex Doppler and its association with development of blood vessels during gestation in the domestic cat. Ten queens were examined weekly from 14 to 63 d after mating. Peak systolic velocity (PSV), end diastolic velocity (EDV), resistance index (RI) and pulsatility index (PI) of uteroplacental, aorta and umbilical fetal arteries and caudal vena cava of the fetus were evaluated. Throughout pregnancy, there was an increase in PSV and EDV in the aorta and umbilical arteries. In the caudal vena cava, there was an increase in PSV, whereas the EDV was constant, with a significant increase on Day 63. Peak systolic velocity and EDV of the uteroplacental artery reduced significantly on Day 63. Resistance index of the umbilical artery progressively decreased. In the aorta, this reduction was detected only on Day 42, with no defined pattern in the caudal vena cava and uteroplacental artery. Pulsatility index of the aorta varied. Although pulsatility increased in the caudal vena cava on Day 35 and remained elevated, pulsatility was significantly reduced in the umbilical artery by Day 63. The pulsatility index of the uteroplacental artery was constant (increased only on Day 63). Triplex Doppler evaluation could be a useful adjunct for prenatal care of pregnant queens, including assessment of vascular gestational development and prediction of gestational age.

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol.

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.

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.

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue.

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.

Effect of six tropical tanniferous plant extracts on larval exsheathment of Haemonchus contortus.

Tanniferous plants represent a promising alternative for controlling gastrointestinal nematodes of small ruminants. This experiment evaluated the effects of extracts from the leaf and stem of Anadenanthera colubrina, Leucaena leucocephala and Mimosa tenuiflora on larval exsheathment of Haemonchus contortus in vitro and verified the role of tannins in this process. Third-stage larvae of H. contortus were incubated with extracts for 3 hours and were exposed to sodium hypochlorite solution. The extracts were tested at 300 µg.mL(-1) and accompanied by controls: phosphate buffer solution (PBS) and polyvinyl polypyrrolidone (PVPP). The larval exsheathment was evaluated for 60 minutes, and the results were subjected to the Kruskal-Wallis test (p < 0.05). The six extracts blocked larval exsheathment. After PVPP addition, a tannin inhibitor, the exsheathment percentage was similar to the PBS (p > 0.05), except for L. leucocephala and M. tenuiflora leaf extracts. However, pre-incubation with PVPP of these two extracts significantly changed larval exsheathment when compared to the non-treated extracts (p < 0.05). These results suggest that A. colubrina, L. leucocephala and M. tenuiflora could be useful in gastrointestinal nematode control and that tannins are probably the main compounds involved in the observed effects. However, in vivo and toxicological studies should be conducted.

Effect of six tropical tanniferous plant extracts on larval exsheathment of Haemonchus contortus / Efeito de seis extratos de plantas taniníferas tropicais sobre o desembainhamento larvar de Haemochus contortus

Tanniferous plants represent a promising alternative for controlling gastrointestinal nematodes of small ruminants. This experiment evaluated the effects of extracts from the leaf and stem of Anadenanthera colubrina, Leucaena leucocephala and Mimosa tenuiflora on larval exsheathment of Haemonchus contortus in vitro and verified the role of tannins in this process. Third-stage larvae of H. contortus were incubated with extracts for 3 hours and were exposed to sodium hypochlorite solution. The extracts were tested at 300 µg.mL-1 and accompanied by controls: phosphate buffer solution (PBS) and polyvinyl polypyrrolidone (PVPP). The larval exsheathment was evaluated for 60 minutes, and the results were subjected to the Kruskal-Wallis test (p < 0.05). The six extracts blocked larval exsheathment. After PVPP addition, a tannin inhibitor, the exsheathment percentage was similar to the PBS (p > 0.05), except for L. leucocephala and M. tenuiflora leaf extracts. However, pre-incubation with PVPP of these two extracts significantly changed larval exsheathment when compared to the non-treated extracts (p < 0.05). These results suggest that A. colubrina, L. leucocephala and M. tenuiflora could be useful in gastrointestinal nematode control and that tannins are probably the main compounds involved in the observed effects. However, in vivo and toxicological studies should be conducted.

Plantas taniníferas representam uma promissora alternativa de controle dos nematóides gastrintestinais de pequenos ruminantes. Esse experimento avaliou in vitro os efeitos dos extratos das folhas e caules de Anadenanthera colubrina, Leucaena leucocephala e Mimosa tenuiflora sobre o desembainhamento larvar de Haemonchus contortus e verificou o papel dos taninos nesse processo. Larvas de terceiro estádio de H. contortus foram incubadas com 300 µg.mL-1 de extrato por 3 horas e expostas a uma solução de hipoclorito de sódio. O ensaio foi acompanhado por controles: solução salina tamponada com fosfato (PBS) e polivinilpolipirrolidona (PVPP). O desembainhamento larvar foi avaliado durante 60 minutos e os resultados submetidos ao teste Kruskal-Wallis (p < 0,05). Os seis extratos bloquearam o desembainhamento larvar. Após adição de PVPP, um inibidor de taninos, o percentual de desembainhamento foi similar ao PBS (p > 0,05), exceto nos extratos das folhas de L. leucocephala e M. tenuiflora. Entretanto, a pré-incubação desses dois extratos com PVPP alterou significativamente o desembainhamento quando comparado com extratos não-tratados (p < 0,05). Esses resultados sugerem que A. colubrina, L. leucocephala e M. tenuiflora podem ser úteis no controle de nematóides gastrintestinais e que os taninos são provavelmente os principais compostos envolvidos nos efeitos. Contudo, estudos toxicológicos e in vivo são necessários.