Comparative electron microscopy study of spermatozoa in snakes (Lepidosauria, Squamata).

The ultrastructure of snake sperm has received substantial attention primarily because snakes exhibit considerable variability in reproductive characteristics between species, with a wide range of mating systems and reproductive behaviors. Variability of sperm morphology among snake species may be associated with the reproductive strategies of each taxon, such as competition or sperm storage. We provide a detailed description of the sperm ultrastructure of nine snake species (Anilius scytale, Tropidophis paucisquamis, Bothrops jararaca, Oxyrhopus guibei, Dipsas mikanii, Micrurus corallinus, Xenopholis scalaris, Acrochordus javanicus, and Cylindrophis ruffus) and compared this with sperm data from the literature for the following taxa: Liotyphlops beui, Amerotyphlops reticulatus, Trilepida koppesi, Anilios waitii, Anilios endoterus, Aspidites melanochephalus, Boa constrictor amarali, Corallus hortulana, Epicrates cenchria, Boa constrictor occidentalis, Eryx jayakari, Micrurus corallinus, Micrurus surinamensis, Micrurus frontalis, Micrurus altirostris, Oxyuranus microlepidotus, Bothrops alternatus, Bothrops diporus, Crotalus durissus, Agkistrodon contortrix, Vipera aspis, Boiga irregularis, Zamenis schrenckii, Zamenis scalaris, Stegonotus cuculatus, Nerodia sipedon, Liodytes pygaea, and Myrrophis chinensis. We found twelve polymorphic characters in the ultrastructure of sperm among the described snakes. Our work supports the importance of ultrastructural analysis of sperm morphology to understand snake reproduction, and provides sperm-derived morphological characters for phylogenetic analysis.

Cobalamin F deficiency in a girl with severe skin hyperpigmentation and a homozygous LMBRD1 variant.

Cobalamin (vitamin B12) is important in gastrulation, nervous system development and haemoglobin formation. Mutations of the ABCD4 or LMBRD1 genes can lead to cobalamin-related disorders. We report a patient with disseminated skin hyperpigmentation caused by a homozygous LMBRD1 variant. Genetic disorders of cobalamin metabolism caused by variants in the ABCD4 or LMBRD1 genes should be considered in patients presenting with cutaneous hyperpigmentation. Click https://www.wileyhealthlearning.com/#/online-courses/a6ef1275-8325-4834-89d2-aa18fa31e63f for the corresponding questions to this CME article.

A Study of the Pupal Development of Five Forensically Important Flies (Diptera: Brachycera).

Holometabolous insects undergo complete metamorphosis, and hence, they have different phases of development (egg, larva, pupa, and adult), which occupy distinct ecological niches. The pupae of several fly species are surrounded by the puparium, which is a rigid structure, usually formed by the integument of the last larval instar. The puparium presents unique characteristics distinct from those of the larval and adult phases. During intrapuparial development, it is possible to distinguish at least four fundamental and continuous steps, namely: 1) larval-pupal apolysis, 2) cryptocephalic pupa, 3) phanerocephalic pupa, and 4) pharate adult. The objective of this work was to describe the external morphology of the distinct phase of development for five species that were collected, identified, and raised in the laboratory; intrapuparial development was studied by fixing immature specimens at regular intervals; the morphological analyses were performed with the aid of both light and scanning electron microscopy. Under the conditions established (27 ± 1.0 or 23 ± 1.0°C, 60 ± 10% relative humidity, 12 h of photoperiod), the minimum time for intrapuparial development was: 252 h for Megaselia scalaris (Loew 1966) (Phoridae), 192 h for Piophila casei (Linnaeus 1758) (Piophilidae), Fannia pusio (Wiedemann 1830) (Fanniidae), and Musca domestica (Linnaeus 1758) (Muscidae), and 96 h for Chrysomya megacephala (Fabricius 1794) (Calliphoridae). Intrapuparial development has defined steps, and distinct species responded differently to the same environmental conditions. In addition, it is possible to establish a sequential rule without ignoring the specific characteristics of each taxon.

Analysis of Paracoccidioides lutzii mitochondria: a proteomic approach.

The genus Paracoccidioides is composed of thermal dimorphic fungi, causative agents of paracoccidioidomycosis, one of the most frequent systemic mycoses in Latin America. Mitochondria have sophisticated machinery for ATP production, which involves metabolic pathways such as citric acid and glyoxylate cycles, electron transport chain and oxidative phosphorylation. In addition, this organelle performs a variety of functions in the cell, working as an exceptional metabolic signalling centre that contributes to cellular stress responses, as autophagy and apoptosis in eukaryotic organisms. The aim of this work was to perform a descriptive proteomic analysis of mitochondria in Paracoccidioides lutzii yeast cells. After mitochondria fractionation, samples enriched in mitochondrial proteins were digested with trypsin and analysed using a NanoUPLC-MSE system (Waters Corporation, Manchester, UK). Ours results revealed that the established protocol for purification of mitochondria was very effective for P. lutzii, and 298 proteins were identified as primarily mitochondrial, in our analysis. To our knowledge, this is the first compilation of mitochondrial proteins from P. lutzii, to date. Copyright © 2016 John Wiley & Sons, Ltd.

Structure and ultrastructure of spermatozoon in six species of Drosophilidae (Diptera).

The Drosophilidae family is formed by Brachycera Diptera distributed widely across different regions of the planet. It is composed of about 4000 species, 304 of which are found in Brazil. The objective of this work was to characterize morphologically the structure of the male internal reproductive apparatus and the ultrastructure of the spermatozoon in four Neotropical (Drosophila cardini, D. mercatorum, D. nebulosa and D. sturtevanti) and two invasive (D. simulans and Zaprionus indianus) species of drosophilids. The structural aspect of the internal reproductive apparatus corresponds with that described for other drosophilids; however, there are differences in the size and coloration of the structures, such as the testes, in each species analyzed. The spermatozoon of these species was seen to be long and fine, presenting morphological variation. The ultrastructure of the spermatozoon revealed that the morphological pattern is similar to that found in the majority of insects. The head region presents a nucleus with condensed chromatin and the acrosome positioned laterally to the nucleus. In the tail region, the axoneme presents the 9+9+2 pattern commonly described for other species of Diptera. The species presented differences regarding the shape and size of the mitochondrial derivatives. Cytochemical analysis using EPTA also revealed differences in terms of the location of the basic proteins in the mitochondrial derivates. The results obtained contribute to expanding the database for the Drosophilidae family, providing information that may contribute to intra- and inter-specific identification and supplying phylogenetic analyses.

The complete genome of a baculovirus isolated from an insect of medical interest: Lonomia obliqua (Lepidoptera: Saturniidae).

Lonomia obliqua (Lepidoptera: Saturniidae) is a species of medical importance due to the severity of reactions caused by accidental contact with the caterpillar bristles. Several natural pathogens have been identified in L. obliqua, and among them the baculovirus Lonomia obliqua multiple nucleopolyhedrovirus (LoobMNPV). The complete genome of LoobMNPV was sequenced and shown to have 120,022 bp long with 134 putative open reading frames (ORFs). Phylogenetic analysis of the LoobMNPV genome showed that it belongs to Alphabaculovirus group I (lepidopteran-infective NPV). A total of 12 unique ORFs were identified with no homologs in other sequenced baculovirus genomes. One of these, the predicted protein encoded by loob035, showed significant identity to an eukaryotic transcription terminator factor (TTF2) from the Lepidoptera Danaus plexippus, suggesting an independent acquisition through horizontal gene transfer. Homologs of cathepsin and chitinase genes, which are involved in host integument liquefaction and viral spread, were not found in this genome. As L. obliqua presents a gregarious behavior during the larvae stage the impact of this deletion might be neglectable.

Impact of insulin concentration and mode of FSH addition on the in vitro survival and development of isolated bovine preantral follicles.

UNLABELLED: The insulin and FSH are two important substances in the folliculogenesis process. Thus, the hypothesis of this experiment is that insulin concentration and the form of FSH addition affect the in vitro survival, growth, and estradiol production after culture of isolated bovine preantral follicles. The effects of insulin concentration (experiment 1) and the influence of both fixed and sequential concentrations of FSH (experiment 2) on the in vitro survival and development of bovine preantral follicles were investigated in this study by IVC for 18 days. In experiment 1, on Day 18 of culture, the addition of insulin at all concentrations promoted follicular survival rates significantly higher than that of the control, with the 10-ng/mL insulin treatment showing values significantly higher than the other treatments. The addition of 5- and 10-ng/mL insulin promoted higher follicular growth than the control and other treatments. In experiment 2, FSH 100 had a higher percentage of follicular viability compared with the control. FSH 100 produced follicle diameters significantly higher than those of the control and FSH seq. TREATMENT: Estradiol levels in the presence of FSH (fixed concentration) were significantly higher than the other treatments. In conclusion, the association of insulin (10 ng/mL) and fixed concentration FSH (100 ng/mL) provides high rates of survival, growth, and estradiol production in bovine preantral follicles.

Comparison between the additive effects of diluted (rFSH) and diluted/dynamized (FSH 6 cH) recombinant follicle-stimulating hormone on the in vitro culture of ovine preantral follicles enclosed in ovarian tissue.

OBJECTIVE: This study compared 2 types of recombinant follicle stimulating hormone (rFSH): diluted and diluted/dynamized, on in vitro development of ovine follicles. METHODS: In experiment 1, ovarian fragments were cultured for 1 or 7 days in α-MEM(+) in the absence or presence of different concentrations of diluted rFSH to determine the best concentration. In experiment 2, the effect of diluted and diluted/dynamized rFSH (rFSH 6 cH--ultradiluted and succussioned), alone or in combination, was studied. RESULTS: In experiment 1, compared to control, 50ng/mL of diluted rFSH induced higher rates of follicular survival after 7 days of culture and higher percentages of growing follicles at day 1 of culture (P<0.05). In experiment 2, compared to control, diluted/dynamized rFSH induced higher follicular diameter and survival rate after 7 days and early follicle activation at day 1 of culture (P<0.05). Compared to diluted rFSH, diluted/dynamized rFSH induced higher rates of follicle activation at day 1 of culture (P<0.05). CONCLUSION: In conclusion, compared to the control medium, diluted/dynamized rFSH promoted survival and early activation of follicles, while diluted rFSH promoted higher activation later in the culture. Thus, diluted/dynamized rFSH may be used as an alternative to diluted rFSH for the in vitro culture of ovine preantral follicles.

Suitability of Corneal Tissue for Transplantation Derived From Violent Death: A 10-Year Analysis.

Trauma is a leading cause of death and disability worldwide. Corneal tissue donors generally are those who suffered an injury to the brain or fatal trauma caused by stroke, vehicle/motorbike accidents, gunshot wounds, and drowning or cardiovascular death. In Brazil, the Distrito Federal (DF) Eye Bank, located within a trauma center hospital, and the Secretariat of Public Security have collaborated with the aim of increasing the overall number of cornea donations from fatal trauma victims. The purpose of this study was to determine the suitability of cornea tissue for transplantation derived from trauma-related death. The records of eyes donated in the DF Eye Bank were analyzed retrospectively for the period from 2004-2013. We had 3388 cornea donors, the majority of which were between 21 and 30 years old (17.4%), which were derived from violent death (84.1%; P = .00) and were predominately male (73.5%). Among the donated corneas, 54.0% were used for optic purposes. Mechanical trauma caused by gunshot, stabbing or blunt force (23.7%), and road traffic injuries (11%) were the main causes of violent death. Another common cause of death was cardiovascular disease (26.3%). Donor tissue derived from violent death had no statistical interference on tissue suitability for transplantation (P = .06). Because of the large waiting lists, and waiting times for transplants, it is advisable to increase the available tissue from corneas donors derived from violent death through the implementation of this interagency model of collaboration and by the practicing of active tissue donor screening in trauma center hospitals.

Activity and in vivo tracking of Amphotericin B loaded PLGA nanoparticles.

The development of biocompatible polymeric nanoparticles has become an important strategy for optimizing the therapeutic efficacy of many classical drugs, as it may expand their activities, reduce their toxicity, increase their bioactivity and improve biodistribution. In this study, nanoparticles of Amphotericin B entrapped within poly (lactic-co-glycolic) acid and incorporated with dimercaptosuccinic acid (NANO-D-AMB) as a target molecule were evaluated for their physic-chemical characteristics, pharmacokinetics, biocompatibility and antifungal activity. We found high plasma concentrations of Amphotericin B upon treatment with NANO-D-AMB and a high uptake of nanoparticles in the lungs, liver and spleen. NANO-D-AMB exhibited antifungal efficacy against Paracoccidioides brasiliensis and induced much lower cytotoxicity levels compared to D-AMB formulation in vivo and in vitro. Together, these results confirm that NANO-D-AMB improves Amphotericin B delivery and suggest this delivery system as a potential alternative to the use of Amphotericin B sodium deoxycholate.

Cryopreservation of boar sperm comparing different cryoprotectants associated in media based on powdered coconut water, lactose and trehalose.

In swine spermatozoa, the damage caused by cryopreservation is more severe than other species, provoking reduced potential for fertilization. Adjustments in the freezing extender composition may be an important alternative to increase its efficiency. The objective of this study was to test the efficiency of different cryoprotectant solutions during cryopreservation of swine semen with a controlled cooling curve. Three cryoprotectant solutions (5% dimethylformamide, 3% glycerol and the combination of these two cryoprotectants) were used in association with three base media (powdered coconut water, lactose and trehalose), constituting nine different treatments. The semen was frozen using a controlled-rate freezer (TK-3000). After thawing, semen was evaluated for total sperm motility, vigor, morphology, plasma membrane integrity and acrosome integrity. Cryopreservation with the controlled curve using an automated system showed satisfactory results, guaranteeing practicality and repeatability for the process of freezing swine sperm. With this curve, the solutions of lactose, trehalose and powdered coconut water associated with glycerol, as well as the solution of coconut water containing dimethylformamide, presented higher quality of sperm compared to the other solutions. Powdered coconut water associated with dimethylformamide appears as a new solution for swine sperm cryopreservation. The freezing controlled curve used in this study allowed standardization of the cryopreservation technique.

Effects of prolonged in vitro culture and cryopreservation on viability, DNA fragmentation, chromosome stability and ultrastructure of bovine cells from amniotic fluid and umbilical cord.

The objective of this work was to study cellular types that did not participated in the gastrulation process, amniotic fluid cells (AFCs) and umbilical cord cells (UCCs), in conditions of long-term culture and cryopreserved with different solutions. The AFCs and UCCs were used in a comparative study with ear fibroblast cells (EFCs) that were cultured in vitro until 20 cellular passages and cryopreserved in 10% dimethylsulphoxide (DMSO), 5% dimethyl formamide (DMF) and 7% glycerol (Gly) solutions. The cellular viability, ultrastructure, DNA fragmentation and chromosome stability were evaluated to determine the cellular type most resistant. In all cell types, it was possible to evaluate the AFCs until 15 passages and UCCs until 20 passages with different periods of cellular growth to reach the confluence phase. Solutions containing 10% DMSO ensured viability of 90.33 ± 5.58%, 90.56 ± 4.40% and 81.90 ± 3.31%, respectively for EFCs, AFCs and UCCs, being significantly more efficient and with less variation than other cryoprotectant solutions. The AFCs were more sensitive to cryopreservation and presented low viability rate at the passage 20 (17.2 ± 8.87%). There was no change in karyotype and nuclear fragmentation was low in all cellular passages studied. With the scanning electron analysis was possible the characterization of AFCs and UCCs in suspension. The three cellular types of cells presented different shapes and characteristics on the surface. The results demonstrate that bovine AFCs and UCCs can be isolated, cultured in vitro and cryopreserved in 10% DMSO, not causing damage to DNA and chromosomes. The UCCs were more resistant than AFCs in all aspects.

Sphingosine 1-phosphate promotes activation of aprine preantral follicle in vitro / Esfingosina 1-fosfato promove ativação de folículos pré-antrais de caprinos in vitro

This study describes the effect of sphingosine 1-phosphate (S1P) for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL). All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05) in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC) and Control Culture (CC), which was followed by an increase of activated follicles (intermediary, primary and secondary). In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability...

Este estudo descreve o efeito da esfingosina 1-fosfato (S1P) no desenvolvimento de folículos pré-antrais, portanto da ativação e viabilidade de folículos caprinos cultivados in vitro. Fragmentos de ovários foram cultivados por um ou sete dias em meio essencial mínimo com diferentes concentrações de S1P (0, 1, 10, 50, 100 ou 200ng/mL). Os fragmentos de ovário foram processados para análise histológica em microscopia óptica, microscopia eletrônica e microscopia de fluorescência. O tratamento usando 1ng/mL de S1P foi capaz de manter a porcentagem de folículos normais durante o período de cultivo de sete dias. Ao final do período de cultivo, houve uma redução significativa (p<0,05) na porcentagem de folículos primordiais em todos os grupos tratados com S1P, comparados com os grupos controle (FC e CC), seguida por um aumento do número de folículos ativados (intermediários, primários e secundários). Adicionalmente, na cultura por sete dias com meio suplementado com S1P (1ng/mL), houve preservação da ultraestrutura das organelas e manteve-se a viabilidade dos folículos pré-antrais avaliados por microscopia de fluorescência. Em conclusão, após sete dias de cultura, o meio suplementado com 1ng/mL de S1P ativa o desenvolvimento de folículos pré-antrais de caprino, cultivados in situ e mantém as viabilidades oocitária e folicular...

Transforming growth factor-β (TGF-β) maintains follicular ultrastructure and stimulates preantral follicle growth in caprine ovarian tissue cultured in vitro / Fator de crescimento transformante-β (TGF-β) mantém a ultraestrutura folicular e estimula o crescimento de folículos pré-antrais caprinos inclusos em tecido ovariano cultivado in vitro

The objectives of this study were to investigate whether TGF-β affect the survival, activation and further growth of goat primordial follicles enclosed in ovarian cortex after in vitro culture. Goat ovaries were collected from an abattoir and pieces of ovarian tissues were cultured for one or seven days in a supplemented alpha Minimum Essential Medium, alone or containing TGF-β (1, 5, 10 or 50ng/mL). Ovarian tissues from the fresh control as well as those cultured were processed for histological and ultrastructural studies. The results showed that when compared with fresh control, there was decrease in the percentages of histologically normal follicles in all treatments only after seven days culture. TGF-β did not affect the activation of preantral follicles regardless of its concentration, however, larger follicles diameter (P<0.05) was observed using 10ng/mL TGF-β than in the fresh control and other treatments. Moreover, this concentration maintained the normal ultrastructure after seven days of culture. In conclusion, TGF-β showed additional effect on the follicle growth and the maintenance of ultrastructural integrity of goat preantral follicles enclosed in ovarian tissue when used at 10ng/mL during seven days of culture...

O objetivo desse estudo foi investigar se o TGF-β afeta a sobrevivência, ativação e crescimento de folículos primordiais caprinos inclusos no córtex ovariano após o cultivo in vitro. Ovários de cabras foram coletados em abatedouro e fragmentos de tecido ovariano foram cultivados por um e sete dias em meio essencial mínimo alfa (α-MEM+) sozinho ou suplementado com TGF-β (1, 5, 10 ou 50ng/mL). Fragmentos ovarianos não cultivados e cultivados foram processados para análise histológica e ultraestrutural. Os resultados mostraram que, comparado ao controle fresco, houve diminuição no percentual de folículos morfologicamente normais em todos os tratamentos somente após sete dias de cultivo. O TGF-β não afetou a ativação folicular independente da concentração testada, contudo, o diâmetro folicular foi superior (P<0.05) no tratamento com 10ng/mL de TGF-β quando comparado ao controle fresco e aos demais tratamentos. Além disso, essa mesma concentração manteve a ultraestrutura normal dos folículos após sete dias de cultivo. Em conclusão, o TGF-β apresentou efeito adicional no crescimento folicular e na manutenção da integridade ultraestrutural de folículos pré-antrais caprinos inclusos no tecido ovariano quando utilizado na concentração de 10ng/mL durante sete dias de cultivo...

Identification and Expression Profile of Two Putative Odorant-Binding Proteins from the Neotropical Brown Stink Bug, Euschistus heros (Fabricius) (Hemiptera: Pentatomidae).

This work presents the first identification of putative odorant-binding proteins (OBPs) from a member of the Pentatomidae, i.e. the brown stink bug Euschistus heros (Fabricius), an important pest of soybean in Brazil. Antennae from both sexes of E. heros adults (12 days old and unmated) were used to construct a cDNA library, from which two transcripts encoding putative E. heros OBPs (EherOBPs) were identified. The expression levels of EherOBP1 and EherOBP2 were found to be higher in male antennae than in female and there was difference in expression in legs, wings, and abdomens of the two sexes. The histolocalization of EherOBP1 and EherOBP2 transcripts in antennae also showed a sexual dimorphism in the chemoreception system, with different expression sites in the antennal segments between males and females, occurring predominantly at the base of the sensillum. The implications of these findings for stink bug chemoreception are discussed.

Limb immobilization alters functional electrophysiological parameters of sciatic nerve.

Immobilization, used in clinical practice to treat traumatologic problems, causes changes in muscle, but it is not known whether changes also occur in nerves. We investigated the effects of immobilization on excitability and compound action potential (CAP) and the ultrastructure of the rat sciatic nerve. Fourteen days after immobilization of the right leg of adult male Wistar rats (n=34), animals were killed and the right sciatic nerve was dissected and mounted in a moist chamber. Nerves were stimulated at a baseline frequency of 0.2 Hz and tested for 2 min at 20, 50, and 100 Hz. Immobilization altered nerve excitability. Rheobase and chronaxy changed from 3.13 ± 0.05 V and 52.31 ± 1.95 µs (control group, n=13) to 2.84 ± 0.06 V and 59.71 ± 2.79 µs (immobilized group, n=15), respectively. Immobilization altered the amplitude of CAP waves and decreased the conduction velocity of the first CAP wave (from 93.63 ± 7.49 to 79.14 ± 5.59 m/s) but not of the second wave. Transmission electron microscopy showed fragmentation of the myelin sheath of the sciatic nerve of immobilized limbs and degeneration of the axon. In conclusion, we demonstrated that long-lasting leg immobilization can induce alterations in nerve function.

Limb immobilization alters functional electrophysiological parameters of sciatic nerve

Immobilization, used in clinical practice to treat traumatologic problems, causes changes in muscle, but it is not known whether changes also occur in nerves. We investigated the effects of immobilization on excitability and compound action potential (CAP) and the ultrastructure of the rat sciatic nerve. Fourteen days after immobilization of the right leg of adult male Wistar rats (n=34), animals were killed and the right sciatic nerve was dissected and mounted in a moist chamber. Nerves were stimulated at a baseline frequency of 0.2 Hz and tested for 2 min at 20, 50, and 100 Hz. Immobilization altered nerve excitability. Rheobase and chronaxy changed from 3.13±0.05 V and 52.31±1.95 µs (control group, n=13) to 2.84±0.06 V and 59.71±2.79 µs (immobilized group, n=15), respectively. Immobilization altered the amplitude of CAP waves and decreased the conduction velocity of the first CAP wave (from 93.63±7.49 to 79.14±5.59 m/s) but not of the second wave. Transmission electron microscopy showed fragmentation of the myelin sheath of the sciatic nerve of immobilized limbs and degeneration of the axon. In conclusion, we demonstrated that long-lasting leg immobilization can induce alterations in nerve function.

Novel wide-capacity method for vitrification of caprine ovaries: Ovarian Tissue Cryosystem (OTC).

In this study we aimed testing the efficiency of a newly developed device for vitrification of ovaries without contact with liquid nitrogen, Ovarian Tissue Cryosystem (OTC). From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification (fragments, hemi-ovary or whole ovary), either or not followed by in vitro culture for two days. Vitrification was performed using the OTC system. The OTC is a cylindrical structure made by stainless steel and composed by three pieces (basis, insert and cover), which can be hermetically closed avoiding contact of the tissue with liquid nitrogen during vitrification. Before and after culture, the ovarian tissue was histologically evaluated. Independently from the size of the ovarian tissue, it was observed a decrease (P<0.05) in the rates of normal preantral follicles when fragments (58.1%), hemi-ovary (54.4%) and whole ovary (54.3%) were vitrified, in comparison with fresh control (68.1%). These data were confirmed by ultrastructural analysis, which showed a great extension of degeneration in follicles vitrified in the whole ovary. Follicular survival after vitrification followed by culture was higher (P<0.05) when ovarian fragments were vitrified (36.1%) than in those enclosed in vitrified hemi-ovary (22.3%) or whole ovary (18.4%). In conclusion, the Ovarian Tissue Cryosystem (OTC) opens a new possibility for successful vitrification of caprine ovarian fragments.

Dynamic medium containing growth differentiation factor-9 and FSH maintains survival and promotes in vitro growth of caprine preantral follicles after long-term in vitro culture.

The aim of the present study was to evaluate the effects of growth differentiation factor 9 (GDF-9) and FSH on the in vitro development of caprine preantral follicles cultured for 16 days. Ovarian fragments were cultured in αMEM⁺ (α-minimum essential medium, pH 7.2-7.4, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5.0 ng mL⁻¹ selenium, 2 mM glutamine, 2 mM hypoxanthine and 1.25 mg mL⁻¹ bovine serum albumin) in the absence or presence of 200 ng mL⁻¹ GDF-9 and/or 50 ng mL⁻¹ FSH added during the first (Days 0-8) and/or second (Days 8-16) half of the culture period. Non-cultured and cultured fragments were processed for histological and ultrastructural analyses. After 16 days, all treatments using GDF-9 or FSH showed higher rates of follicular survival compared with αMEM⁺ alone. Compared with non-cultured control, sequential culture media containing GDF-9 and/or FSH significantly increased the percentage of developing follicles and follicle diameter. Moreover, a progressive increase in oocyte diameter was observed only with sequential culture medium containing GDF-9 until Day 8 followed by FSH (GDF-9/FSH) in the second half of the culture period. After 16 days of culture, ultrastructural analysis confirmed the integrity of follicles cultured in the presence of GDF-9/FSH. In conclusion, a dynamic medium containing GDF-9 and FSH (GDF-9/FSH) maintained follicular integrity and promoted activation of primordial follicles and growth during long-term in vitro culture of goat preantral follicles.

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles.

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM(+)) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM(+) alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM(+) alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.