Follicles Of Different Diameters Research Articles

Features of the functioning of Ovis Aries granulosa cells from follicles of different diameters

Features of the functioning of Ovis Aries granulosa cells from follicles of different diameters

Analysis of functional activity of granulosa cells of porcine ovarian follicles

Functioning of the granulosa cells of animal ovarian follicles (OF) determines the growth and development of the OF and the oocyte formed in it, which determines their use in modeling the composition of media for oocyte maturation in vitro in cell reproductive technologies. The purpose of the study is to determine the peculiarities of functional activity of granulosa cells in follicles of different diameters (< 3, 3–5, 5–8 mm) using complex testing of granulosa cell viability parameters (apoptosis level, production of reactive oxygen species, functional activity of mitochondria and lipidome) and hormonal status of follicle fluid (estradiol, testosterone, anti-Müllerian hormone level) in order to optimize the composition of the culture media for in vitro maturation of pig oocytes. The experiments found that the proportion of apoptotic cells in the OF increased as the diameter of the OF increased (by 11% in OFs 5–8 mm in diameter, by 4% in OFs < 3 mm in diameter). There was an increase in the number of high reactive oxygen species cells (by 10% in 5–8 mm diameter OF compared with < 3 mm diameter OF) and high mitochondrial activity (by 12% in 5–8 mm diameter OF compared with < 3 mm diameter OF). Correlations were found between the group of cells with high generation of reactive oxygen species and the level of apoptosis (0.500, p < 0.05), as well as with high levels of mitochondrial activity (0.500, p < 0.05). Estradiol concentration in follicular fluid increased as the OF grew (1.50 ng/ml in OF < 3 mm diameter and 2.09 ng/ml in OF 3–5 mm diameter, p < 0.01), being almost unchanged in OF 5–8 mm diameter (2.11 ng/ml), as was the testosterone concentration. Correlations were found between the proportion of the cells with high NileRed/LD FI (a marker of lipid droplet functional activity) and the levels of estradiol (0.998, p < 0.05), testosterone (0.500, p < 0.05), and anti-Müllerian hormone (–0.500, p < 0.05) in the dynamics of folliculogenesis, as well as between estradiol concentration and the proportion of cells with high mitochondrial activity (0.746, p < 0.05). Correlations were detected between the concentrations of estradiol and anti-Müllerian hormone, testosterone and anti-Müllerian hormone (–1.000, p < 0.05), between the level of apoptotic cells and the concentration of anti-Müllerian hormone in follicular fluid (–1.000, p < 0.05). The results of the monitoring of the viability and functional activity of porcine OF granulosa cells supplement the available data on the peculiarities of their functioning in the dynamics of folliculogenesis and can be used in modeling the systems of in vitro maturation of donor oocytes of Sus Scrofa Domesticus.

Features of the thyroid gland morphology of some representatives of the Cervidae family, living in the Altai

The authors studied the morphofunctional (anatomical and histological) features of the thyroid gland of dapple deer, maral, moose and roe deer. During the macroscopic examination of the organ, the number of lobes, the presence of an isthmus, the length, width and thickness of the lobes, and the weight of the gland were determined. During histological examination, morphometric parameters were evaluated, such as: diameter of the follicles, height of the thyrocytes, volume of the thyrocyte nuclei, and the Brown index was calculated. In all animals, the thyroid gland consisted of two lobes without an isthmus. It differs between species in the shape, size, and thickness of the lobes. The gland weight correlates with the size of the animals. Morphological differences between species consist in the heterogeneity of the location of follicles of different diameters in relation to each other. In the dapple deer, large-diameter follicles are located in the center of the organ, and smaller-diameter follicles are located both in the center and on the periphery. In roe deer, the gland follicles are almost identical in shape and size, that is, they are of the same type. The gland has a similar morphological structure in maral and moose. The colloid of the follicles has a homogeneous consistency, it is compacted and intensively stained with hematoxyl-eosin, resorption vesicles are in small quantities.

The features of proliferative processes in the thyroid gland of the Wistar rat’s offspring after intrauterine action of dexamethasone

The features of proliferative processes in the thyroid gland of the Wistar rat’s offspring after intrauterine action of dexamethasone were studied. Animals were divided into 3 groups: I – intact rats; II – control – animals, which on the 18th day of the dated pregnancy transuterine, transdermal, subcutaneously in the interscapular area was injected with 0.9 % saline in the amount of 0.05 ml; III – experimental group – animals, which during laparotomy by intrauterine, transdermal subcutaneous injection in the interscapular area was injected with a solution of dexamethasone at a dose of 0.05 ml at a dilution of 1:40 intrauterinely on the 18th day of pregnancy (Ukrainian patent No.112288). In the experimental subgroups used the allowable, generally accepted number of animals for statistical processing and obtaining reliable results – 6 animals. The thyroid gland with the tracheal area was removed on the 21st, 30th, 45th, 60th, 90th, 120th days of life. Immunohistochemical study was performed according to the protocol recommended for a particular antibody manufacturer. Monoclonal antibodies ki-67 (Ki-67), Fox-1 Antibody (A-12) were used to assess proliferative activity, the company Santa Cruz Biotechnology, Inc. (USA). The study found that the thyroid gland of rats of infantile period, which prenatally exposed to dexamethasone, is structurally represented by chaotically located follicles of different diameters with a predominance of large with desquamated cells in the lumen, and proliferative changes aimed at forming extrafollicular which is confirmed immunohistochemically by the presence of Ki-67-positive cells. Intracellularly, protein- synthesizing organelles of thyrocytes also proliferate, to which there is a clear cytoplasmic and nuclear reaction with Fox-1 antibodies. Dining the juvenile period, proliferative processes in the thyroid gland of animals of the experimental group are stabilized while maintaining the morphological structure of the hypofunctional type, and remain lower compared to the control and intact groups. Morphological signs of functional tension of the thyroid gland animals exposed prenatally to dexamethasone, which correlate with a decrease in proliferative activity, indicate a functional compensatory response of synthetic and hormone-producing function, but suppression of proliferative processes, despite the slight manifestations. The thyroid gland of morphological hypofunctional type after prenatal action of dexamethasone in young rats, indicates an adaptogenic compensatory response and morphofunctional immaturity of the organ during this period, which may be the basis for provoking the preservation of such morphogenetic factors under the influence of stressors. Keywords: thyroid gland, proliferation, dexamethasone, experiment, rats.

FSH promotes the proliferation of sheep granulosa cells by inhibiting the expression of TSP1

Thrombospondin (TSP1) plays an important role as an antiangiogenic factor in the reproductive system of female mammals. However, its expression and function in sheep are still unclear. In the present research, the Altay sheep (a native Chinese breed) was used to analyze the expression of TSP1 in the ovary and its potential function in granulosa cells. TSP1 was widely expressed in most tissues, as shown by qPCR. In the ovary, TSP1 mRNA expression decreased during follicular to luteal growth. The TSP1 protein was expressed in a wide variety of follicles of different diameters and localized to the cytoplasm and nucleus of granulosa cells. In in vitro studies, follicle-stimulating hormone (FSH) significantly inhibited the expression of TSP1 in sheep granulosa cells. Functionally, FSH- and TSP1-specific siRNAs can promote the proliferation of sheep granulosa cells. In contrast, TSP1 mimetic peptide, ABT510, offsets the proliferation of sheep granulosa cells. Different signaling pathway inhibitors all promoted FSH-inhibited TSP1 expression, but each inhibitor had different effects on TSP1. Among them, the PI3K and ERK pathway inhibitors significantly promoted TSP1 expression and inhibited the proliferation of sheep granulosa cells.

Reproductive biology, morphology, and morphometry of ovaries and oviducts of Trachemys scripta elegans in Brazilian Cerrado

ABSTRACT: Although Trachemys scripta elegans is an exotic species popular as a pet in Brazil, studies on reproductive biology and capacity are non-existent in the Brazilian Cerrado. This study analyzed ovarian and oviduct characteristics and the egg production capacity of T. scripta elegans grown in this biome. The findings will associate with the size of the specimens and the sexual maturity, aiming at comparisons with native and exotic populations, as well as interspecific and contributing to the understanding of its impact on the invaded ecosystems and the establishment of eradication programs. Thus, 39 females had evaluated the body biometry and the morphology and morphometry of the ovaries and oviducts. G2 (N=20): with Class I (>5-10mm) follicles, with Class I and Class II (>10-fold) follicles, 25mm) and G3 (N=9) with Class I, Class II and Class III (>25mm) follicles. Analysis of variance, Scott-Knott’s test, and Pearson’s correlation analysis showed that there was no significant difference between the groups in body biometry; in the mean gonadosomatic index and gonadal morphometry, only the width of the oviducts in the right antimer and the mass and width in the left antimer were higher in G3, the only one that presented eggs. There was positive and harmonic development between body mass, carapace, and plastron, and gonadal growth occurred concomitantly with body growth, indicating a higher reproductive potential and a positive relationship between the size of the litter and the female litter. The gonadosomatic index proved to be an excellent reproductive indicator, and the ovarian evaluation was a better indicator of sexual maturity than the maximum carapace length. Ovaries were irregular structures, without delimitation between the cortical and medullary regions and filled with vitelogenic follicles of different diameters, atresic follicles, and corpora lutea, which reflected the ovarian complexity of the species and the presence of follicular hierarchy. In the scarce stroma, two germinative beds were observed per ovary and the presence of gaps very close to the follicles and associated with the blood vessels. Analysis of gonadal tissue revealed three types of oocytes according to cytoplasmic characteristics: homogeneous, vesicular or vesicular in the cortex with apparent granules. Oviducts were functional and separated, joining only in the final portion to form the cloaca and subdivided into infundibulum, tuba, isthmus, uterus, and vagina. The structure of the uterine tube was composed of serosa, muscular and mucous, which was full of glands. The presence of eggs in the oviducts indicated that the specimens can reproduce in the Brazilian Cerrado. This study provides necessary and relevant information on the reproductive biology and capacity of T. scripta elegans in the Brazilian Cerrado and can contribute to the understanding of its impact on the invaded ecosystems and the establishment of eradication programs. The extraction of females with capacity can reduce the annual reproductive yield of the species and decrease its effect on local biodiversity.

Oxidative homeostasis in oocyte competence for in vitro embryo development.

The objective of this study was to evaluate the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes from follicles of different diameters and their relevance in the in vitro production of embryos (IVPE). Bovine ovaries were aspirated according to the diameter of the follicle [2-8 (general), 4-8 (large), and 2<4mm (small)]. The oocytes were evaluated for levels of ROS, GSH, in vitro maturation, and IVPE. Higher levels of ROS and GSH were observed (p<0.05) in oocytes of the large group (85.6±7.2 and 140.0±9.6) followed by those in the general (81.1±10.5 and 134.3±7.8) and small (73.5±10.1 and 125.0±10.6) groups. However, the proportion of ROS/GSH did not differ (p>0.05) between the general, large, and small groups. The maturation was higher (p<0.05) in the large group (87.8±3.0%) than in the small group (72.2±5.8%), but both were similar (p>0.05) to that in the general group (82.2±2.5%), whereas the IVPE of the large group (57.3±3.0%) was higher (p<0.05) than those in the general (44.7±4.4%) and small (34.0±4.0%) groups. We report that oocytes from large follicles are more competent for IVPE, whereas higher levels of ROS and GSH appear to be correlated with oocyte competence, as long as oxidative homeostasis is retained.

Rex Rabbit Somatic Cell Nuclear Transfer with In Vitro-Matured Oocytes.

Somatic cell nuclear transfer (SCNT) requires large numbers of matured oocytes. In vitro-matured (IVM) oocytes have been used in SCNT in many animals. We investigated the use of IVM oocytes in Rex rabbit SCNT using Rex rabbit ovaries obtained from a local abattoir. The meiotic ability of oocytes isolated from follicles of different diameters was studied. Rex rabbit SCNT was optimized for denucleation, activation, and donor cell synchronization. Rex rabbit oocytes grew to the largest diameter (110 μm) when the follicle diameter was 1.0 mm. Oocytes isolated from <0.5-mm follicles lacked the ability to resume meiosis. More than 90% of these oocytes remained in the germinal vesicle (GV) stage after in vitro culture (IVC) for 18 h. Oocytes isolated from >0.7-mm follicles acquired maturation ability. More than 90% of these oocytes matured after IVC for 18 h. The developmental potential of oocytes isolated from >1-mm follicles was greater than that of oocytes isolated from 0.7- to 1.0-mm follicles. The highest activation rates for IVM Rex rabbit oocytes were seen after treatment with 2.5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) and 5 μg/mL cycloheximide (CHX) for 1 h. Ionomycin induced the chromatin of IVM oocytes to protrude from the oocyte surface, promoting denucleation. Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were more suitable for Rex rabbit SCNT than skin fibroblast cells (SFCs) (blastocyst rate was 35.6 ± 2.2% and 38.0 ± 6.0% vs. 19.7 ± 3.1%). The best fusion condition was a 2DC interval for 1 sec, 1.6 kV/cm voltages, and 40 μsec duration in 0.28 M mannitol. In conclusion, the in vitro maturation of Rex rabbit oocytes and SCNT procedures were studied systematically and optimized in this study.

In vivo collection of follicular fluid and granulosa cells from individual follicles of different diameters in cattle by an adapted ovum pick-up system

BackgroundMost studies on granulosa cell (GC) function in cattle have been performed using GC and follicular fluid (FF) samples collected from slaughterhouse ovaries. Using this approach, the follicular developmental stage and functional status are unknown and indirectly inferred, limiting data interpretation. Ultrasound-guided follicle aspiration has previously been used to recover GC or FF samples, but this was mostly carried out in large follicles or pools of small follicles, without recording the efficiency of recovery. The present study was aimed at adapting and evaluating an ovum pick-up (OPU) system for the in vivo recovery of FF and GC from individual follicles of different diameters.MethodsIn the first trial, the losses of fluid inside the tubing system were calculated using a conventional or an adapted-OPU system. Blood plasma volumes equivalent to the amount of FF in follicles of different diameters were aspirated using a conventional OPU Teflon circuit. The OPU system was then adapted by connecting 0.25 mL straws to the circuit. A second trial evaluated the efficiency of FF recovery in vivo. Follicles ranging from 4.0 to 16.8 mm in diameter were aspirated individually using the conventional or adapted-OPU systems. A third trial assessed the in vivo recovery of GC and the subsequent amount of RNA obtained from the follicles of different diameters from Holstein and Gir cattle.ResultsIn Trial I, the plasma recovery efficiency was similar (P > 0.05) for the volumes expected for 12 and 10 mm follicles, but decreased (P < 0.05) for smaller follicles (45.7+/−4.0%, 12.4+/−4.3% and 0.0+/−0.0% for 8, 6, and 4 mm follicles, respectively). Using the adaptation, the losses intrinsic to the aspiration system were similar for all follicle diameters. In Trial II, the expected and recovered volumes of FF were correlated (r = 0.89) and the efficiency of recovery was similar among follicles <12 mm, while larger follicles had a progressive increase in FF losses that was not related to the tubing system. In Trial III, the number of GC and amount of RNA obtained were not affected (P > 0.05) by follicle size, but differed according to breed (615,054+/−58,122 vs 458,095+/−36,407 for Holstein and Gir, respectively; P < 0.05).ConclusionsThe adapted-OPU system can be successfully used for the in vivo collection of FF and GC from follicles of different diameters. This will enable further endocrine, cellular, and gene expression analyses.

184 INTERCELLULAR COUPLING AND CHROMATIN CONFIGURATION STATE IN HORSE OOCYTE - CUMULUS CELL COMPLEXES OF DIFFERENT ORIGINS

Fewer follicles and of variable size are found at any time on the mare ovary compared with other livestock species, also influenced by seasonal variation. This is reflected on the population of cumulus–oocyte complexes (COC) collected that is characterised by a high heterogeneity. Recovered immature oocytes presumably need different culture conditions. The main factors contributing to this high heterogeneity are the follicle diameter, the status of the cumulus oophorus, and the reproductive seasonality. Therefore, the aim of the present study was to assess the nuclear chromatin configuration and the oocytes–cumulus cell gap junction-mediated communication (GJC) functionality in COC of different origins because these parameters are indicative of the oocyte’s metabolic state and should be taken into account when designing IVM strategies. The COC with compact (Cp) or expanded (Ex) cumulus oophorus were collected from follicles of different diameters (&lt;1, 1–2, and &gt;2 cm) in October–November, January–February, and April–May. The GCJ functionality was assessed by Lucifer Yellow microinjection and chromatin configuration was evaluated by Hoechst and Lacmoid staining, after cumulus cells removal. Data were obtained from a total of 1003 oocytes and were analysed by chi-squared test. Overall, GJC functionality was impaired in the majority of Ex COC in each follicle category, even though a certain proportion of them had open GJC (% of Ex COC with open GJC was 39.7, 29.3, and 39.3 in &lt;1, 1–2, and &gt;2 cm follicles, respectively). Moreover, the proportion of Ex COC with open GJC did not differ significantly between periods (% of Ex COCs with open GJC in October–November, January–February, and April–May was, respectively, 43.3, 28.6, and 41.7 in &lt;1 cm follicles; 45.5, 19.3, and 26.47 in 1–2 cm follicles; 66.7, 50, and 16.7 in &gt;2 follicles). On the contrary, the majority of Cp COC from follicles &lt;1 and 1–2 cm, showed open GCJ in October–November and April–May, whereas they decreased significantly in January–February. This tendency was not maintained in Cp COC from follicles &gt;2 cm, where GJC functionality did not differ significantly between periods (% of Cp COC with open GJC in October–November, January–February, and April–May was, respectively, 74.4, 35.7, and 75 in &lt;1 cm follicles; 73.8, 42.1, and 67.7 in 1–2 cm follicles; 58.3, 58.3, and 68.6 in &gt;2-cm follicles). Chromatin configuration analysis revealed that the highest proportion (23.9%) of oocytes with fibrillar chromatin was found in Cp oocytes from &lt;1 cm follicles, whereas the proportion of oocytes with fibrillar chromatin ranged from 5.4 to 12.5% in the other groups. Moreover, the increase in follicle diameter was generally associated with an increase of chromatin condensation in Cp COC. Interestingly, the chromatin configuration distribution did not differ significantly among seasons. Our data could be useful in setting up new in vitro cultural strategies aimed to improve horse-assisted reproductive technology efficiency as well as in the understanding of horse oocyte biology. Funding: Grant no. 26096200 ‘Ex Ovo Omnia’ from Regione Sardegna &amp; Lombardia and ‘Dote Ricerca Applicata’ (VL and IT).

125 FOLLICULAR FLUID AND GRANULOSA CELL RECOVERY FROM BOVINE FOLLICLES OF DIFFERENT DIAMETERS USING AN ADAPTED TRANSVAGINAL-GUIDED FOLLICULAR ASPIRATION SYSTEM

Most studies of granulosa cell (GC) function have been performed in vitro, using follicular fluid (FF) and GC recovered from slaughterhouse ovaries. This approach does not consider the reproductive status and follicular developmental stage of the donor, limiting data interpretation and usefulness. The aim of this study was to evaluate the efficiency of an adapted ultrasound-guided transvaginal follicular aspiration (TVFA) procedure to recover FF and mural GC from live cows. A preliminary in vitro trial was performed to calculate fluid losses using a conventional TVFA circuit. A known volume of PBS, expected to be the volume of FF in follicles ranging from 4 to 12 mm in diameter, was aspirated using a 20 G needle connected to a Teflon circuit 80 cm long, with a 1.0-mm internal diameter, connected to a 1.5-mL tube. Losses of the expected volume of FF were 12.7 ± 1.1, 19.9 ± 2.5, 54.4 ± 4.0, 87.6 ± 4.3 and 100%, for follicles of 12, 10, 8, 6 and 4 mm in diameter (0.90, 0.52, 0.27, 0.11 and 0.03 mL, respectively). When an adapted system for small-volume recoveries was used, there was a decrease (P &lt; 0.05) in fluid losses for follicles of 8, 6 and 4 mm. An experiment was performed in vivo, using the adapted system, to evaluate FF and GC recovery from follicles of 4 and 5 (n = 16), 6 (n = 19), 7 (n = 7), 8 (n = 13), 9 (n = 15), 10 (n = 24), 11 (n = 11), 12 (n = 15), 13 (n = 13), 14 (n = 6) and 16 (n = 9) mm in diameter. Follicular wave emergence was synchronized with 2 mg of oestradiol benzoate and an intravaginal progesterone device and follicles that reached the desired diameter were aspirated using an ultrasound machine equipped with a 7.5-MHz probe and disposable 20 G needle. The recovered FF volume was measured and centrifuged at 600 × g for 10 min. The GC pellet was vortexed with 0.1% hyaluronidase (5 min) and washed twice in PBS and the number of cells was determined using a Neubauer chamber. Ribonucleic acid was extracted using an RNAeasy Microkit and quantified in NanoDrop. The efficiency of FF recovery was estimated by the difference between the recovered and expected volumes for each follicle diameter (4/3πr3), which were compared by ANOVA. From all the follicles aspirated, the recovery of FF and GC was not successful in 2 (1.3%). Overall, FF recovery efficiency was 84.7%. The recovered volume ranged from 0.03 to 3.80 mL and increased with follicular diameter (y = 0.011x2 – 0.012x + 0.043; R2 = 0.99). Losses of FF were significant (P &lt; 0.05) for follicles larger than 12 mm. The mean (±s.e.m.) number of mural GC recovered was 716,708 ± 68,536, providing 6.8 ± 0.7 samples of 100 000 cells with 14.8 ± 0.7 ng of RNA μL–1 for each punctured follicle. A high coefficient of variation (57.4%) was observed in cell recovery. There was no difference in the number of cells recovered from follicles of different diameters, but sample contamination with blood was more frequent (75%) in follicles larger than 10 mm. In conclusion, an adapted TVFA system can be used successfully on an individual basis and from follicles of different diameters for in vivo recovery of FF and GC for further endocrine and gene expression analyses. This research was supported by CNPq and Fapemig (CVZ APQ 02863/09).

Progesterone Production in Mares and Echographic Evaluation of the Corpora lutea Formed after Follicular Aspiration

Ultrasound-guided follicular aspiration was performed in 26 Criollo crossbred mares, followed by the evaluation of ultrasonographic images of the Corpus luteum (CL) that was formed after puncture of follicles of different diameters (Group 25-29 mm; Group 30-35 mm and Group >35 mm). Serum progesterone (P(4) ) concentrations were measured to determine CL function. The size of the CL was measured and the CL was classified based on the following echoscore: 1- anechoic tissue; 2- poorly defined luteal structure with low echogenicity; 3- echogenicity analogous to a luteal structure. The proportion of aspirated follicles that formed a functional CL (based on P(4) concentration) 8 days after aspiration was 57.1% (4/7; CL size 25-29 mm), 75.0% (6/8; CL size 30-35 mm) and 72.7% (8/11; CL size >35 mm), respectively (p > 0.05). The echographic scores of aspirated follicles (indicating the presence or absence of a CL) were consistent with serum P(4) concentrations (p < 0.0001). Of 26 aspirations, 18 resulted in luteal function confirmed by increased progesterone concentrations ([P(4) ] > 1.0 ng/ml); 17 of these mares (94.4%) had an echoscore (2-3) compatible with luteinization (p = 0.0372). Eight days after aspiration, serum [P(4) ] > 2.0 ng/ml was associated with high (p = 0.0056) CL echoscore (3) in 15 of 17 mares (88.2%). The echoscore used in this study was valuable as a screening test to detect the presence of a functional CL after aspiration. An echoscore of 3 served as a practical and efficient method to confirm luteinization.

344 FOLLICULAR FLUIDS COLLECTED FROM LARGE FOLLICLES IMPROVED THE PROGRESSION OF IN VITRO NUCLEAR MATURATION OF PIG OOCYTES

The present study examined the effects of supplementation of in vitro maturation medium with porcine follicular fluids (pFFs) on the progression of nuclear maturation, subsequent fertilization, and development of pig oocytes. The pFFs were collected from follicles of different diameters: small follicles (SFs, 3–4 mm in diameter) and large follicles (LFs, 5-6 mm). In experiment 1, oocytes were collected from follicles 3–6 mm in diameter. The oocytes were cultured in maturation medium (NCSU-37) supplemented (10% v/v) with pFF from SFs (SpFF) or LFs (LpFF); the kinetics of nuclear maturation, fertilization rate, and developmental competence of oocytes were evaluated. In experiment 2, oocytes were collected from SFs or LFs, and the effects of supplementation of SpFF and LpFF on the nuclear maturation of these oocytes were examined. In experiment 3, each pFF was denaturated by heat treatment (60�C for 30 min) or trypsin treatment (200 �g mL-1), and the effects of these pFFs on the nuclear maturation of pig oocytes were examined. In experiment 4, pFFs were ultrafiltered, and separated into fractions greater and less than 30 kDa; the effects of the separated pFFs on nuclear maturation were examined. We used Student&amp;apos;s t-test to examine the differences among the results; a P value less than 0.05 was considered to be significant. In experiment 1, the progression of nuclear maturation was significantly faster when the maturation medium was supplemented with LpFF than with SpFF (MI rates at 36 h after culture: LpFF, 20.0%, and SpFF, 35.0%; MII rates at 36 h after culture: LpFF, 33.3%, and SpFF, 20.0%). The rates of polyspermic fertilization were significantly lower in the maturation medium with LpFF than in that with SpFF (LpFF, 31.7%; SpFF, 66.7%). Moreover, the rates of monospermic fertilization (LpFF, 45.0%; SpFF, 21.7%) and blastocyst formation (LpFF, 15.2%; SpFF, 3.0%) were significantly greater in the medium supplemented with LpFF than with SpFF. In experiment 2, we observed that LpFF accelerated the progression of nuclear maturation of pig oocytes collected from LFs (MII rates at 36 h after culture: LpFF, 61.7%; SpFF, 35.5%), but not those collected from SFs (MII rates at 36 h after culture: LFs, 15.0%; SFs, 11.7%). In experiment 3, the accelerating effect of pFF on nuclear maturation was eliminated by heat (MII rates at 36 h after culture: LpFF, 30.0%; SpFF, 25.0%) or trypsin treatment (MII rates at 36 h after culture: LpFF, 20.0%; SpFF, 21.7%). In experiment 4, only LpFF with a molecular weight greater than 30 kDa had positive effects on the progression of nuclear maturation (MII rates at 36 h after culture with LpFF: &amp;gt;30 kDa, 30.0%; &amp;lt;30 kDa, 11.7%). The present study suggests that LpFF contains some proteins that improve the progression of nuclear maturation and the developmental competence of oocytes.

Annexin II mRNA expression in bovine oocytes during follicular development

We investigated the expression of calcium-dependent phospholipid binding protein annexin-II (Ann-II) messenger RNA (mRNA) during preantral follicle development and in oocytes from antral follicles of different diameters (< 3 mm, 5 to 8 mm and > 8 mm). The action of retinol on Ann-II mRNA expression in mature oocytes was also examined. Only oocytes from secondary preantral follicles expressed Ann-II mRNA and at the germinal vesicle stage expression by oocytes from follicles larger than 8 mm was significantly higher (p < 0.05) compared with oocytes from follicles smaller than 3 mm or between 5 and 8 mm. Ann-II mRNA expression by metaphase II oocytes from follicles larger than 8 mm was significantly higher (p < 0.05) than that from oocytes from follicles smaller than 3 mm, with oocytes from both these size-classes showing similar levels of Ann-II mRNA expression as oocytes recovered from 5-8 mm follicles. In the presence of retinol, Ann-II mRNA expression was higher than when retinol was absent (p < 0.05). Our data indicate that Ann-II mRNA expression is highest in competent oocytes and that retinol increases Ann-II mRNA and may be involved in the regulation of oocyte competence by decreasing the translation and/or degradation of Ann-II mRNA.

Fertility in the mare after repeated transvaginal ultrasound-guided aspirations

Ovum pick-up (OPU) by transvaginal ultrasound guided aspiration (TUGA) is a procedure applied in equine-assisted reproduction programs such as oocyte transfer and in vitro embryo production. Despite a large number of studies reporting that it is a repeatable and safe technique, little information is available about the effect of repeated punctures on fertility of mares. Moreover, even if flushing follicles improves the oocyte recovery rate, to our knowledge the efficiency of flushing estrous and diestrous follicles has not been evaluated. The aims of the present study were (1) evaluate if repeated TUGAs negatively effects fertility and (2) investigate the influence of flushing the follicular cavity (as compared to aspiration only-unflushed) on the recovery rate from follicles of different sizes and in different stages of the estrous cycle. Seventy-six TUGAs were carried out on 20 mares during the breeding season; 153 follicles were aspirated and 31 oocytes were recovered (20.3% per follicle; 40.8% per TUGA attempt). Of the 76 aspirations, 52 were carried out during estrus and 24 in diestrus. Flushing the follicular cavity significantly increased ( P < 0.01) the oocyte recovery rate from estrous follicles (13/28, 46.4% flushed versus 3/24, 12.5% aspirated only) but not ( P > 0.05) from diestrous follicles of different diameters (3/30, 10% flushed versus 2/36, 5.6% aspirated only for follicles <2 cm in diameter; 6/20, 30% flushed versus 4/15, 26.7% aspirated only for follicles ≥2 cm in diameter). Mares underwent ultrasonic examinations after every aspiration and no alteration was found with the exception of two mares in which the corpus luteum (CL) did not form following aspiration of estrous follicle. Of the 20 mares involved in this study, 10 were artificially inseminated with fresh semen from a single fertile stallion at the first spontaneous heat following the previous aspiration. Of the 10 inseminated mares, 7 were found to be pregnant 16, 30 and 50 days after artificial insemination (AI), indicating that repeated TUGAs did not adversely affect fertility.

Desenvolvimento embrionário de oócitos bovinos mantidos em fluido folicular bovino de folículos de diferentes diâmetros

Oocitos bovinos tem sido mantidos em fluido folicular como meio para transporte e para aumento de sua competencia, antes da maturacao. Oitocentos e oitenta e um (881) oocitos foram aspirados de foliculos de 2 a 8mm, de ovarios de abatedouro, para avaliar o efeito da manutencao de oocitos bovinos em fluido folicular bovino de foliculos de diferentes tamanhos sobre o desenvolvimento embrionario. Os oocitos foram distribuidos aleatoriamente em quatro tratamentos, com sete repeticoes cada. No grupo controle (n=217), os oocitos foram maturados por 24h em TCM-199 com Soro de Egua em Estro (SEE), piruvato e rFSH-h, em estufa a 39°C, com 5,00% de CO2 e umidade saturada. No tratamento FFpequeno (n=216), os oocitos foram mantidos por 6h em fluido folicular de foliculos de 3 a 5mm a 30°C e posteriormente maturados por 18h nas mesmas condicoes do grupo controle. Os oocitos dos tratamentos FFmedio (n=226) e FFgrande (n=222) foram mantidos em fluido folicular de foliculos com 5,1 a 8mm e foliculos maiores de 8,1mm, respectivamente e, apos, maturados por 18h. Apos a fecundacao por 18h, os zigotos foram cultivados por 8 dias em SOFaaci com 5,00% de SEE, em estufa a 39°C, em bolsas gaseificadas com 5,00%CO2, 5,00%O2 e 90,00%N2. Oocitos do grupo FFpequeno resultaram em menor (P 0,05) entre os grupos. O fluido folicular de foliculos medios e grandes pode ser utilizado para o manutencao de oocitos bovinos por 6h a 30°C, antes da maturacao por 18h.