Contribution of lipids to the organelle differential profile of in vitro-produced bovine embryos.

Each living organism is unique because of the lipid identity of its organelles. The diverse distribution of these molecules also contributes to the role of each organelle in cellular activity. The lipid profiles of whole embryos are well documented in the literature. However, this approach can often lead to the loss of relevant information at the subcellular and consequently, metabolic levels, hindering a deeper understanding of key physiological processes during preimplantation development. Therefore, we aimed to characterize four organelles in vitro-produced bovine embryos: lipid droplets (LD), endoplasmic reticulum (ER), mitochondria (MIT), and nuclear membrane (NUC), and evaluate the contribution of the lipid species to each organelle evaluated. Expanded blastocysts were subjected to cell organelle isolation. Thereafter, lipid extraction from cell organelles and lipid analysis using the Multiple Reaction Monitoring (MRM) profiling method were performed. The LD and ER displayed a greater number of lipids (Phosphatidylcholine - PC, Ceramide - Cer, and Sphingomielin - SM) with high signal-to-noise intensities. This result is due to the high rate of biosynthesis, lipid distribution, and ability to store and recycle lipid species of these organelles. The NUC had a more distinct lipid profile than the other three organelles, with high relative intensities of PC, SM, and triacylglycerols (TG), which is consistent with its high nuclear activity. MIT had an intermediate profile that was close to that of LD and ER, which aligns with its autonomous metabolism for some classes of phospholipids (PL). Our study revealed the lipid composition of each organelle studied, and the roles of these lipids could be associated with the characteristic organellar activity. Our findings highlight the lipid species and classes that are relevant for the homeostasis and function of each associated organelle and provide tentative biomarkers for the determination of in vitro embryonic development and quality.

Effects of the addition of oocyte meiosis-inhibiting drugs on the expression of maturation-promoting factor components and organization of cytoplasmic organelles.

The present study evaluated the effects of the blockade of meiosis in bovine oocytes by the cyclin-dependent kinase inhibitors roscovitine (ROS) and butyrolactone-I (BL-I) on nuclear maturation and extracellular signal-regulated kinase 1/2 (ERK1/2), cyclin B1 and p34cdc2 protein expression and localization. We also evaluated ultrastructural changes in oocytes. Immature oocytes were obtained from slaughtered bovines and divided into: (1) control (oocytes for in vitro maturation only in tissue culture medium-199 for 24 h), (2) oocytes that were treated with 12.5µMROS for 6 h, (3) oocytes that were treated with 50µMBL-I for 6 h and (4) oocytes that were treated with 6.25 µMROS+25 µMBL-I for 6 h. Incubation with inhibitors was followed by the reversal of blockade for 18 h. Oocytes then underwent immunohistochemical analysis to visualize chromatin and assess ERK1/2, cyclin B1 and p34cdc2 localization/expression, followed by preparation of the cells for ultrastructure analysis by electron microscopy. The groups at 6 h of maturation and before IVM exhibited the lowest number of oocytes in metaphase I. ROS group had the highest number of degenerating oocytes (p < 0.05). After maturation, majority of oocytes were in metaphaseII with no differences among groups (p> 0.05). ERK1/2, cyclin B1 and p34cdc2 expression differed throughout inhibition and oocyte maturation (p < 0.05). No difference was observed in the localization of these proteins in the ooplasm. No ultrastructural changes in oocytes were observed between treatments, with the exception of treatment with drugs that augmented lipid metabolism (p < 0.05). Results indicate that the effects of CDK1 inhibitors are reversible in bovine oocytes, indicated by nuclear, cytoplasmic, and molecular maturation parameters.

Clinical safety of intratesticular transplantation of allogeneic bone marrow multipotent stromal cells in stallions.

Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.

Treatment with roscovitine and butyrolactone I prior to in vitro maturation alters blastocyst production.

This study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.

Modulation of long-chain Acyl-CoA synthetase on the development, lipid deposit and cryosurvival of in vitro produced bovine embryos.

In this study, we evaluated the modulation effect of long-chain Acyl-CoA synthetase during early embryo development. Bovine embryos were cultured in four groups: positive modulation (ACS+) with GW3965 hydrochloride, negative modulation (ACS-) with Triacsin C, association of both modulators (ACS±), and control. Embryo development rates were not altered (P>0.05) by treatments. Embryonic cytoplasmic lipid content increased in ACS+ but reduced in ACS- compared to the control (P < 0.05), whereas the membrane phospholipids profile was not altered by treatments. The total number of blastomeres did not differ (P > 0.05) between groups; however, an increased apoptotic cells percentage was found in ACS- compared to control. Twenty-four hours after warming, ACS+ and control grade I embryos presented the best hatching rates, whereas the ACS+ group equaled the hatching rates between their embryos of grades I, II and III 48 hours after warming. The relative abundance of transcripts for genes associated with lipid metabolism (ACSL3, ACSL6, ACAT1, SCD, and AUH), heatshock (HSP90AA1 and HSF1), oxidative stress (GPX4), and angiogenesis (VEGF), among other important genes for embryo development were affected by at least one of the treatments. The treatments were effective in modulating the level of transcripts for ACSL3 and the cytoplasmic lipid content. The ACS- was not effective in increasing embryonic cryosurvival, whereas ACS+ restored survival rates after vitrification of embryos with low quality, making them equivalent to embryos of excellent quality.

New Protocol for Cell Culture to Obtain Mitotic Chromosomes in Fishes.

Cell culture is an excellent alternative for the maintenance of cell lines and to obtain quality chromosome preparations of fishes. However, this methodology is still little employed, mainly because of the difficulty of standardization of cell cultures. In this study, we describe a methodology for the rapid acquisition of cell lineages and mitotic chromosomes for cytogenetic studies of fish species from muscle tissue cells. Our methodology is based on the use of a gelatin film, which provides better adhesion of a large number of cells and appropriate conditions for multiplication. The cells of Astyanax altiparanae, used as an experimental model, with fibroblast-like morphology, showed rapid cellular proliferation, resulting in a great number of cells. Chromosomal preparations of cultured cells showed the diploid number of the species, 2n = 50 chromosomes, in 80% of the cells examined, with chromosomes intact and distended. Cell populations were cryopreserved and after being recovered, these cells maintained their proliferative capacity. The development of this methodology represents an innovation for the fish cytogenetics area and it may bring a significant contribution to the conservation and study of several groups due to the difficulty of obtaining good-quality chromosome preparations.

Cell apoptosis and lipid content of in vitro-produced, vitrified bovine embryos treated with forskolin.

The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 µM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 µM: 128.5; P < 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 µM: 173.5; P < 0.05) than controls for 2.5 µM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 µM: 101.3, F 5 µM: 115.4, F 10 µM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 µM: 16.7%, F 5 µM: 11.1%, F 10 µM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 µM: 37.3%, F 5 µM: 33.2%, F 10 µM: 30.3%; P < 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells.

Bovine endometrial cells: a source of mesenchymal stem/progenitor cells.

Endometrial mesenchymal stem/progenitor cells (eMSCs) are multipotent cells known to modulate the immune system and have clinical application for human and animal health. This makes these bovines cells attractive for dual use as cellular therapy and experimental model. The aim of this study was to isolate, evaluate the differentiation potential, immunophenotypic and immunocytochemistry characteristics, chromosomal stability, cloning efficiency, and cryopreservation response of bovine eMSCs collected in two phases of the estrous cycle. For this, cells were isolated and submitted to differentiation for adipogenic and osteogenic lineage. The cells were then characterized by flow cytometer (FC) (vimentin, CD29, CD44, MHC-II, CD34) and immunocytochemistry (vimentin, pan-cytokeratin, CD44) and submitted to cytogenetic and cloning efficiency assay. The cells were also cryopreserved using two different medium of cryopreservation and analyzed by FC for viability, necrosis, late-apoptosis + necrosis, and initial apoptosis rates before and after cryopreservation. We obtained homogeneous cell populations which have fibroblastic morphology and adherence to plastic. These cells expressed high levels of markers CD29, CD44, and vimentin, low expression levels for CD34 and no MHC-II. The cells were chromosomally stable (2n = 60) with high cloning efficiency and no difference (P > 0.05) between medium of cryopreservation or phase was observed after thawing. We showed the presence and differentiation potential of bovine eMSCs, with chromosomal stability and great response to cryopreservation with both medium, which has implications for build biobanks or development of new therapeutic approaches to combat uterine diseases or to study.

Intramuscular Transplantation of Allogeneic Mesenchymal Stromal Cells Derived from Equine Umbilical Cord.

BACKGROUND AND OBJECTIVES: Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. METHODS AND RESULTS: For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs diluted in Hank's Balanced Solution (HBSS) was performed on right and left sides of the rump muscle. As a control, HBSS injections were performed caudally in the same muscle. Muscle biopsies were obtained as a control 30 days before transplantation (D-30). The biopsies were collected again on day 2 (left side) and day 7 (right side) post transplantation and examined histologically. All procedures were preceded by ultrasound examination and blood sampling. Hematologic evaluation remained within normal limits and no differences were observed between time points (p>0.05). Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups (control and treated). At histological evaluation it was found only discrete inflammation signals between D-30×D2 (p<0.05) in the treated group, without differences (p> 0.05) between the groups at different time points. CONCLUSIONS: Equine UC-MSCs under the experimental conditions did not promote severe inflammation that causes tissue damage or lead to its rejection by the host organism and therefore has a good potential for clinical use.

Time course of the meiotic arrest in sheep cumulus-oocyte complexes treated with roscovitine.

Temporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus-oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 µM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

In vitro embryos production after oocytes treatment with forskolin.

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.

Influence of culture medium and age of bovine blastocysts in established colonies of embryonic stem cells.

The objective of this study was to evaluate the potential of bovine IVF blastocysts at different stages of embryonic development in establishing ESC-like. Furthermore, blastocysts cultured in medium containing (10%) and (2.5%) fetal calf serum (FCS) were compared to determine if the serum concentration during in vitro culture alters the blastocyst's potential to establish ESC-like culture. It was observed that only ICM's from expanded blastocysts adhered to the monolayer (n=160) independent of the concentration of serum used during IVF culture. There were no observable differences in potential to establish ESC-like in embryos cultured with 2,5% or 10% FCS . The bFGF didn´t seems to be required for maintenance of bovine ESC-like regardless of culture conditions. Blastocysts and colonies in primary culture and after the first passage were positive for Oct4, Nanog, SSEA-3 and TRA-1-81. Expanded blastocysts gave rise to ESC-like colonies, and the addition of LIF was sufficient to maintain cells undifferentiated in culture.

In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells.

PURPOSE: To evaluate in vitro ability the of three different biomaterials - purified hydroxyapatite, demineralized bone matrix and castor oil-based polyurethane - as biocompatible 3D scaffolds for canine bone marrow mesenchymal stem cell (MSC) intending bone tissue engineering. METHODS: MSCs were isolated from canine bone marrow, characterized and cultivated for seven days with the biomaterials. Cell proliferation and adhesion to the biomaterial surface were evaluated by scanning electron microscopy while differentiation into osteogenic lineage was evaluated by Alizarin Red staining and Sp7/Osterix surface antibody marker. RESULTS: The biomaterials allowed cellular growth, attachment and proliferation. Osteogenic differentiation occurred in the presence of hydroxyapatite, and matrix deposition commenced in the presence of the castor oil-based polyurethane. CONCLUSION: All the tested biomaterials may be used as mesenchymal stem cell scaffolds in cell-based orthopedic reconstructive therapy.

In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells

PURPOSE: To evaluate in vitro ability the of three different biomaterials - purified hydroxyapatite, demineralized bone matrix and castor oil-based polyurethane - as biocompatible 3D scaffolds for canine bone marrow mesenchymal stem cell (MSC) intending bone tissue engineering. METHODS: MSCs were isolated from canine bone marrow, characterized and cultivated for seven days with the biomaterials. Cell proliferation and adhesion to the biomaterial surface were evaluated by scanning electron microscopy while differentiation into osteogenic lineage was evaluated by Alizarin Red staining and Sp7/Osterix surface antibody marker. RESULTS: The biomaterials allowed cellular growth, attachment and proliferation. Osteogenic differentiation occurred in the presence of hydroxyapatite, and matrix deposition commenced in the presence of the castor oil-based polyurethane. CONCLUSION: All the tested biomaterials may be used as mesenchymal stem cell scaffolds in cell-based orthopedic reconstructive therapy.

Use of bayesian inference to correlate in vitro embryo production and in vivo fertility in zebu bulls.

The objective of this experiment was to test in vitro embryo production (IVP) as a tool to estimate fertility performance in zebu bulls using Bayesian inference statistics. Oocytes were matured and fertilized in vitro using sperm cells from three different Zebu bulls (V, T, and G). The three bulls presented similar results with regard to pronuclear formation and blastocyst formation rates. However, the cleavage rates were different between bulls. The estimated conception rates based on combined data of cleavage and blastocyst formation were very similar to the true conception rates observed for the same bulls after a fixed-time artificial insemination program. Moreover, even when we used cleavage rate data only or blastocyst formation data only, the estimated conception rates were still close to the true conception rates. We conclude that Bayesian inference is an effective statistical procedure to estimate in vivo bull fertility using data from IVP.

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification.

UNLABELLED: The objective was to evaluate supplementation of fetal calf serum (FCS) and phenazine ethosulfate (PES), a metabolic regulator that inhibits fatty acid synthesis, in culture media during in vitro production (IVP) of bovine embryos. Taking oocyte fertilization (n = 4,320) as Day 0, four concentrations of FCS (0, 2.5, 5, and 10%) and three periods of exposure to PES (without addition-CONTROL; after 60 h-PES Day 2.5 of embryo culture; and after 96 h-PES Day 4) were evaluated. Increasing FCS concentration in the culture media enhanced lipid accumulation (P < 0.05), increased apoptosis in fresh (2.5%: 19.1 ± 1.8 vs 10%: 28.4 ± 2.3, P < 0.05; mean ± SEM) and vitrified (2.5%: 42.8 ± 2.7 vs 10%: 69.2 ± 3.4, P < 0.05) blastocysts, and reduced blastocoele re-expansion after vitrification (2.5%: 81.6 ± 2.5 vs 10%: 67.3 ± 3.5, P < 0.05). The addition of PES in culture media, either from Days 2.5 or 4, reduced lipid accumulation (P < 0.05) and increased blastocoele re-expansion after vitrification ( CONTROL: 72.0 ± 3.0 vs PES Day 2.5: 79.9 ± 2.8 or PES Day 4: 86.2 ± 2.4, P < 0.05). However, just the use of PES from D4 reduced apoptosis in vitrified blastocysts ( CONTROL: 52.0 ± 3.0 vs PES Day 4: 39.2 ± 2.4, P < 0.05). Independent of FCS withdrawal or PES addition to culture media, the in vivo control group had lesser lipid accumulation, a lower apoptosis rate, and greater cryotolerance (P < 0.05). The increased lipid content was moderately correlated with apoptosis in vitrified blastocysts (r = 0.64, P = 0.01). In contrast, the increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts (r = 0.94, P < 0.0001). Therefore, using only 2.5% FCS and the addition of PES from Day 4, increased the survival of IVP embryos after vitrification. Moreover, embryo quality, represented by the fresh apoptosis rate, was better than lipid content for predicting embryo survival after vitrification.

Ultrastructure of Sheep Primordial Follicles Cultured in the Presence of Indol Acetic Acid, EGF, and FSH.

The aim of this study was to investigate the ultrastructural characteristics of primordial follicles after culturing of sheep ovarian cortical slices in the presence of indol acetic acid (IAA), Epidermal Growth Factor (EGF), and FSH. To evaluate ultrastructure of primordial follicles cultured in MEM (control) or in MEM containing IAA, EGF, and FSH, fragments of cultured tissue were processes for transmission electron microscopy. Except in the control, primordial follicles cultured in supplemented media for 6 d were ultrastructurally normal. They had oocyte with intact nucleus and the cytoplasm contained heterogeneous-sized lipid droplets and numerous round or elongated mitochondria with intact parallel cristae were observed. Rough endoplasmic reticulum (RER) was rarely found. The granulosa cells cytoplasm contained a great number of mitochondria and abundant RER. In conclusion, the presence of IAA, EGF, and FSH helped to maintain ultrastructural integrity of sheep primordial follicles cultured in vitro.

Viability of primordial follicles derived from cryopreserved ovine ovarian cortex tissue.

OBJECTIVE: To develop an efficient technique to preserve primordial follicles from cryopreserved ovarian tissue. DESIGN: Frozen-thawed and fresh preantral follicles were mechanically isolated for viability testing, and their morphology was histologically analyzed. SETTING: Laboratory of Animal Reproduction, Faculty of Veterinary Medicine and Zootechny, University State of São Paulo, Brazil. ANIMAL(S): Lambs 12-24 months of age. INTERVENTION(S): Ovarian cortical fragments were prepared for cryoprotectant toxicity testing, freezing and thawing procedures, and in vitro culture. MAIN OUTCOME MEASURE(S): Histologic structure and follicular viability. RESULT(S): On day zero, no morphologic differences were observed between follicles isolated from fresh tissue and those treated with the cryopreservatives ethylene glycol (EG) and dimethyl sulfoxide (DMSO) and subjected to freezing. Even so, frozen follicles treated with DMSO + EG showed dark staining, indicating degeneration. On day zero, the follicular viability was similar between the control group (78.9%) and those treated with EG (77%) and frozen with EG (75%). After 10 days in culture, a reduced percentage of follicles was considered viable in all groups. This decrease was accentuated in those treated with DMSO (37.5% and 35.2% in those exposed to and frozen with DMSO, respectively) and DMSO + EG (33.9% and 30% in those exposed to and frozen with DMSO + EG, respectively) as compared with the control group (45%) and EG-treated groups (40.1% and 40% for those exposed to and frozen with EG, respectively). CONCLUSION(S): Ethylene glycol seems to be the best cryoprotectant for the cryopreservation of ovine ovarian tissue.

Infertilidade associada a defeito microtubular dos espermatozóides de jumento (Equus asinus) avaliados por microscopia eletrônica de transmissão / Infertility associated with microtubular defect in a donkey (Equus asinus) spermatozoa evaluated by transmission electron microscopy

Descreve-se um caso de infertilidade de um jumento SRD confirmada por meio de microscopia eletrônica de transmissão (MET). O espermiograma, avaliado sob microscopia ótica, revelou baixa motilidade e alta concentração de anormalidades espermáticas do tipo gota citoplasmática proximal. O material foi avaliado por MET, observando-se um acúmulo desordenado de microtúbulos causando protusões irregulares na região do colo espermático. O último teste realizado correspondeu ao de fertilidade in vivo, utilizando-se quatro éguas portadoras de bom histórico reprodutivo, nas quais não foi possível confirmar nenhuma prenhez. Frente aos resultados obtidos, associados aos achados da MET, estabeleceu-se o diagnóstico de infertilidade associada a defeito microtubular dos espermatozóides.

A donkey infertility was described by transmission electron microscopy (MET). The spermiogram evaluated by light microscopy showed low sperm motility and high concentration of abnormal sperm with proximal cytoplasmic droplets. The material was evaluated by MET, where it was observed disarrangement of microtubules, causing irregular protrusions in the spermatic neck. The last test done was the in vivo fertility, using four mares, with a reproductive healthy historic, where no pregnancy occurred. Facing the results that we had in vivo, associated with the MET findings, we diagnosed infertility associated with microtubular defect of the spermatozoa.

Utilização do primeiro ciclo ovulatório da estação reprodutiva para produção de embriões em éguas sob condições tropicais / Utilization of the first ovulatory cycle of seasonal breeding for embryo production in mares in tropical conditions

A eficiência da primeira ovulação da estação reprodutiva para a produção de embriões foi determinada através da taxa de recuperação embrionária, da viabilidade dos embriões recuperados, da concentração sérica de progesterona, da resposta do primeiro folículo pré-ovulatório da estação reprodutiva ao hCG e da resposta do primeiro CL a PGFa alfa. Treze éguas que estavam no periodo de anestro ou inicial da transição de primavera foram acompanhadas por ultra-sonografia até a detecção do primeiro folículo pré-ovulatório. Neste momento a ovulação foi induzida com 2500 UI de hCG (IV) e elas foram inseminadas em dias alternados até a ovulação. Sete dias após a detecção da ovulação foi realizada coleta de embrião e a concentração de P 4 foi determinada. A partir da detecção do primeiro folículo > e =25mm da estação, as éguas demoraram em média 14,92± 10,80 dias para alcançarem o primeiro folículo pré-ovulatório e 18,00 ±11,08 dias para ovularem, sendo que 11/13 éguas ovularam em até 48 horas após a administração do hCG. Estes folículos cresceram em média 2,19± 0,86 mm/dia. Nove das 13 éguas (69,2%) produziram embriões e todos foram considerados viáveis após avaliação visual e pelo exame de fluorescência. Os corpos lúteos formados mostraram-se funcionalmente competentes produzindo em média 7,39 ± 2,11 ng/ ml de P 4, além de serem responsivos à PGF2alfa. Deste modo, o primeiro ciclo ovulatório do ano pode ser utilizado com sucesso para a produção de embriões viáveis.

The efficacy of the first ovulation of the breeding season was determined through the response of first pre-ovulatory follicle of the breeding season to hCG, embryo recovery rate, viability of recovered embryos, serum concentrations of progesterone, and response of first CL to PGF2 alpha. Thirteenth mares that were in vernal transition were accompanied until the first pre-ovulatory follicle was detected. At this moment, the ovulation was induced with 2,500 IU hCG (IV) and the mares were inseminated every other day until ovulation. Seven days after the ovulation, embryo recovery was performed and the progesterone concentration was determined. After detection of the first pre-ovulatory follicle of breeding season with > and = 25mm, it took 14.92 ± 10.80 days for the follicle to reach the pre-ovulatory size and 18.00 ± 11.08 days to ovulation. After administration of hCG, 11/13 mares ovulated in 48 hours. These follicles growth 2.19 ± 0.86 mm/ day on average. Nine of 13 mares (69.2%) produced embryos and ali were considered viable after morfological evaluation and fluorescence exams. The CL appeared competent producing 7.39 ± 2.11 ng/ml P4 on average, and responding to PGF2 alpha. According to these results the first ovulatory cycle of the year can be utilized to produce viable embryos.