Ovarian morphometric characterization and in vitro maturation of oocytes obtained from buffalo (Bubalus bubalis) ovaries - partial results

Buffalo ovaries were collected from a slaughterhouse (Frigol, Brazil) and transported to the laboratory in saline solution at 36 degrees C. The ovaries were dissected to realize the evaluations (weight, length, width and height of the ovary; corpus luteum and dominant follicle diameters). The Cumulus-oocyte complexes (COCs) were recovered by aspiration of 2-8 mm follicles. Selected COCs were matured in TCM 199 supplemented with 10% fetal bovine serum, sodium pyruvate, LH, FSH, estradiol and gentamicin. In vitro maturation was carried out at 38.5 degrees C for 22-24 h and 34-36 h. For the evaluation of the nuclear maturation the oocytes were placed in TCM 199 medium added with type v hialuronidase where the granulosa cells were extracted. The denuded oocytes were transferred to 10 mu l of Hoescht 33342 and the chromosomic configuration was evaluated. The oocytes were classified according to meiosis stage in: Germinal Vesicle, Germinal Vesicle Breakdown, Metaphase I, Metaphase II and Degenerated. The means of weight, length, width and height of the ovary were 3.83 g, 2.27 cm, 1.08 cm and 1.56 cm, respectively. The means of corpus luteum and dominant follicle diameters were 1.40 cm and 7.77 mm. The proportion of oocytes that reached metaphase II stage was: 36.68%.

Use of oocyte transfer in a commercial breeding program for mares with reproductive abnormalities

In some mares with lesions of the reproductive tract, embryo collection and survival rates are low or collection of embryos is not feasible. For these mares, oocyte transfer has been proposed as a method to induce pregnancies. In this report, a method for oocyte transfer in mares and results of oocyte transfer performed over 2 breeding seasons, using mares with long histories of subfertility and various reproductive lesions, are described.Human chorionic gonadotropin or an implant containing a gonadotropin-releasing hormone analog was used to initiate follicular and oocyte maturation. Oocytes were collected by means of transvaginal ultrasound-guided follicular aspiration. Following follicular aspiration, cumulus oocyte complexes were evaluated for cumulus expansion and signs of atresia; immature oocytes were cultured in vitro to allow maturation. The recipient's ovary and uterine tube (oviduct) were exposed through a flank laparotomy with the horse standing, and the oocyte was slowly deposited within the oviduct. Oocyte transfer was attempted in 38 mares between 9 and 30 years old during 2 successive breeding seasons. All mares had a history of reproductive failure while in breeding and embryo transfer programs. Twenty pregnancies were induced. Fourteen of the pregnant mares delivered live foals. Results suggest that oocyte transfer can be a successful method for inducing pregnancy in subfertile mares in a commercial setting..

Effects of fetuin on zona pellucida hardening, fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment 1, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1 mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25 mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P < 0.05) were formed when FCS was used compared to PVA and 0.25 mg/ml of fetuin. In Experiment 11, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P < 0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P < 0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P < 0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes. (C) 2002 Published by Elsevier B.V. B.V.

Regulation and action of fibroblast growth factor 17 in bovine follicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Parthenogenetic development of domestic cat oocytes treated with ionomycin, cycloheximide, roscovitine and strontium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fertilizing Capacity of Frozen Epididymal Sperm Collected from Dogs

The collection of epididymal sperm may be a valuable tool for canine reproduction especially since it can enable collection of cells after death of a valuable dog. The aim of the present study was to evaluate the viability of epididymal sperm after freeze-thawing. Epididymides were obtained from four adult dogs by elective orchiectomy. The caudal portion of the epididymides and part of the deferential ducts were squeezed by means of an anatomic clamp into a Petri dish containing either 0.9% saline solution (Group 1) or Ringer solution without lactate (Group 2). Samples were centrifuged at 800 x g for 10 min, the supernatant was removed and the pellet was diluted in one step with a Tris/citric acid/OEP (Orvus Es Paste) extender containing 7% glycerol and subjected to semen freezing. Oocytes were obtained from canine ovaries, after ovariohysterectomy. Only oocytes that were approximately 100 mu m in diameter, with a dark ooplasm surrounded by three-or four-well formed cumulus cell layers were used for sperm testing. Frozen semen samples were thawed in a water bath at 70 degrees C for 8 s and analysed at room temperature for sperm motility and velocity. Oocytes were incubated with spermatozoa in humidified atmosphere containing 5% CO(2) at 38 degrees C for 18 h. Morphological and functional characteristics of spermatozoa were similar in both groups. However, the percentage of sperm cells bound to oocytes was significantly higher in Group 2 than in Group 1. This result suggests that the Ringer solution without lactate was a more suitable medium for collecting epididymal canine sperm than 0.9% saline.

Collection Rates and Morphology of Equine Oocytes Obtained from Immature Follicles after Treatment with Equine Pituitary Extract (EPE) and Human Chorionic Gonadotropin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Use of a Piezo Drill for Intracytoplasmic Sperm Injection into Cattle Oocytes Activated with Ionomycin Associated with Roscovitine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização estrutural do fator de crescimento de fibroblasto-10 (FGF-10) e seu papel na fisiologia folicular ovariana

Background: Interest in folliculogenesis has grown extensively in recent years. Nevertheless, several aspects of follicular activity are still poorly understood. Thus, in vitro culture of ovarian follicles using new substances has been established as a very viable model, enhancing the prospects for a better understanding of follicular activity. Among the family members of the fibroblast growth factor (FGFs), FGF-10 has received recent attention for its ability to regulate the development of ovarian follicles and oocyte maturation. Given the relevance of FGF-10 in the folliculogenesis process, this review aimed to describe the structural features, expression and the main biological effects of FGF-10 on the development of ovarian follicles in mammals.Review: Along this work, it was shown aspects related to structural characterization of FGF-10 and its receptors, as well as FGF-10 expression in different cell types, emphasizing its importance to follicular development. FGF-10 is a paracrine member of the family of FGFs, and is characterized by promoting biological responses via cell surface receptors (FGFRs) of tyrosine kinase-type. of these receptors, FGFR-1, FGFR-2 and FGFR-3 may undergo alternative transcriptional arrangements, enabling the formation of two isoforms (b and c) that have varying degrees of affinity for the various FGFs. Thus, seven FGFR proteins (FGFRs 1b, 1c, 2b, 2c, 3b, 3c and 4) with different binding specificities are generated from the four FGFR genes. The FGFRs transmit intracellular signals after binding with the ligand through the phosphorylation of tyrosine, which activates various transduction patterns in the cytoplasm. The signal transduction of FGF-10 may occur through three main pathways: protein of rat sarcoma (Ras)/MAPK, PLC gamma/Ca(2+) and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt), which are involved in the transmission of biological signals, leading to cellular proliferation and differentiation. FGF-10 mRNA expression was detected in the ovarian stroma, oocyte and theca cells of preantral and antral follicles. on the other hand, the expression of mRNA for FGF-10 receptors was found in, granulosa cells, theca cells, cumulus cells and oocytes. Although FGFs are widely distributed in different tissues and cell types, the importance and function of FGFs in the ovary are still poorly documented. FGF-10 has been shown to be an important mediator of mesenchymal and epithelial cell interactions during follicle development, promoting follicular survival, activation and growth. Besides the action on folliculogenesis, FGF-10 was recently identified as a growth factor able to improve oocyte competence. However, in antral follicles, the presence of FGF-10 is associated with increased follicular atresia, which matches its anti-estrogenic action.Discussion: From this review, we can conclude that FGF-10 is an important regulator of female reproduction. This growth factor acts in follicle survival, oocyte maturation, expansion of cumulus cells and proliferation of granulosa/theca cellsthrough direct and/or indirect actions in the control of folliculogenesis. Furthermore, FGF-10 seemed to have different effects throughout the follicular development. However, it is necessary to perform additional studies that may provide a better understanding about the importance of FGF-10 during folicullogenesis.

Angiotensin II, progesterone, and prostaglandins are sequential steps in the pathway to bovine oocyte nuclear maturation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Desenvolvimento das gônadas de Dermatobia hominis (Diptera: Cuterebridae)

O trabalho descreve o desenvolvimento das gônadas do berne (D. hominis) durante o período pupal. As pupas desenvolvidas de larvas com peso superior a 650 mg, deram imagos fêmeas, enquanto que as desenvolvidas daquelas pesando entre 500 e 650 mg deram macho, tendo havido um erro ao redor de 5%. Até o oitavo dia de pupação os testículos crescem mais que os ovários; a partir daí diminui o desenvolvimento, parando de crescer entre o vigésimo e vigésimo quinto dias. A espermatogênese inicia por volta do sétimo dia de pupa quando é grande o número de espermatócitos. No décimo dia alguns testículos apresentam considerável número de espermátides e os espermatozóides começam a aparecer por volta do vigésimo dia. A espermiogênese desenvolve-se sem interrupção e ao final da pupação quase toda loja testicular está repleta de espermatózóides. Os machos começam a nascer dois dias antes das fêmeas. Nessas, os ovaríolos aparecem formados por volta do oitavo dia de pupa; os folículos se individualizam por volta do vigésimo dia de pupa onde se distingue os trofócitos com núcleos politênicos e citoplasmas bem basófilos, enquanto o ovócito tem citoplasma mais acidófilo e núcleo com cromatina bastante frouxa. A vitelogênese tem início ao redor do vigésimo quinto dia de pupa e se completa ao nascimento da imago. A ligação das gônadas com suas respectivas estruturas somáticas acontece ao redor do décimo terceiro dia de pupação.

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Follicular somatic cell factors and follicle development

Considerable attention is currently paid to oocyte-derived secreted factors that act upon cumulus and granulosa cells. Also important for follicle development are somatic cell-derived secreted factors. This is illustrated by the ability of granulosa cell-derived Kit ligand (KITL) to promote primordial follicle activation, and the loss of follicle development that accompanies KITL gene disruption. This review summarises our current understanding of somatic cell factors during both preantral and antral follicle growth, involving not only signalling from granulosa cells to the oocyte, but also signalling between granulosa and theca cells. Principal granulosa cell-derived factors include activin, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Theca cells also secrete BMPs and FGFs. The interplay between these factors is equally important for follicle growth as the activity of oocyte-derived factors.

Expression and Function of Fibroblast Growth Factor 18 in the Ovarian Follicle in Cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Convective storm initiation in a moist tropical environment

Radar and satellite data from the Tropical Rainfall Measuring Mission-Large-Scale Biosphere-Atmosphere (TRMM-LBA) project have been examined to determine causes for convective storm initiation in the southwest Amazon region. The locations and times of storm initiation were based on the National Center for Atmospheric Research (NCAR) S-band dual-polarization Doppler radar (S-Pol). Both the radar and the Geostationary Operational Environmental Satellite-8 (GOES-8) visible data were used to identify cold pools produced by convective precipitation. These data along with high-resolution topographic data were used to determine possible convective storm triggering mechanisms. The terrain elevation varied from 100 to 600 m. Tropical forests cover the area with numerous clear-cut areas used for cattle grazing and farming. This paper presents the results from 5 February 1999. A total of 315 storms were initiated within 130 km of the S-Pol radar. This day was classified as a weak monsoon regime where convection developed in response to the diurnal cycle of solar heating. Scattered shallow cumulus during the morning developed into deep convection by early afternoon. Storm initiation began about 1100 LST and peaked around 1500-1600 LST. The causes of storm initiation were classified into four categories. The most common initiation mechanism was caused by forced lifting by a gust front (GF; 36%). Forcing by terrain (>300 m) without any other triggering mechanism accounted for 21% of the initiations and colliding GFs accounted for 16%. For the remaining 27% a triggering mechanism was not identified. Examination of all days during TRMM-LBA showed that this one detailed study day was representative of many days. A conceptual model of storm initiation and evolution is presented. The results of this study should have implications for other locations when synoptic-scale forcing mechanisms are at a minimum. These results should also have implications for very short-period forecasting techniques in any location where terrain, GFs, and colliding boundaries influence storm evolution.