Ovine Ovaries Research Articles

165 In vitro maturation of ovine and caprine oocytes during breeding and nonbreeding seasons

The current study investigated the in vitro meiotic competence of ovine and caprine oocytes that underwent nuclear maturation during the breeding and nonbreeding seasons. We hypothesised that maturation rates of ovine and caprine oocyte would be significantly lower during the nonbreeding season. Ovine (Katahdin crossbred) and caprine (mainly Spanish crossbred) ovaries were collected from a local abattoir in the southern United States. Age of the animals was not determined. Cumulus-oocyte complexes (COC) were harvested by slicing the ovaries and searching using a stereomicroscope. Oocytes with more than 3 layers of unexpanded cumulus cells and with evenly granulated cytoplasm were selected for in vitro maturation (IVM). A commercial bovine IVM media (IVF Bioscience, Falmouth, United Kingdom) was used throughout the study. After 24h of IVM, ovine and caprine oocytes were denuded and oocytes with an extruded polar body (meiotic metaphase II oocytes) were considered to have reached nuclear maturation. The seasons in this study were defined as follows: breeding season=September to April and nonbreeding season=May to July. The presence of corpus hemorrhagicum or corpus luteum in at least 70% of the ovaries indicated the breeding season for the animals. Proportions of oocytes undergoing nuclear maturation were analysed using a two-tailed Chi-squared test. Statistical significance was set at P ≤ 0.05. The ovine maturation rate was 59% (65/111) and 49% (254/519) and the caprine maturation rate was 70% (39/56) and 40% (64/162) during the breeding and nonbreeding seasons, respectively. These results show a significant difference in nuclear maturation for caprine oocytes (P<0.001) during the breeding and nonbreeding seasons; however, there was no significant difference in nuclear maturation for ovine oocytes (P=0.06) during the breeding and nonbreeding seasons. High environmental temperatures during the nonbreeding season may have had detrimental effects on oocyte nuclear maturation in caprine but not in ovine oocytes. Why oocytes from these 2 species differ on how they are adversely affected by season remains to be elucidated.

Molecular Cloning of the B4GALNT2 Gene and Its Single Nucleotide Polymorphisms Association with Litter Size in Small Tail Han Sheep.

Simple SummaryIn French Lacaune sheep, the B4GALNT2 (beta-1, 4-N-acetyl-galactosaminyl transferase 2) gene was considered as the potential gene for a FecL (mutation), which regulates the ovine ovulation rate. Three specific mutation sites linked with the FecL mutation have not been previously found in 11 sheep breeds. However, two mutations of g.36946470C > T and g.36933082C > T in the exon of B4GALNT2 were found to have had a significant effect on the litter size in the first parity for Small Tail Han (STH) Sheep (p < 0.05). B4GALNT2, which is mainly expressed in ovine ovary, also plays an important role in sheep reproduction. Furthermore, we discovered two transcription start sites (TSS) of B4GALNT2 in its 5′-flanking region in ovine granule cells in vitro.A new fecundity gene named the FecL (mutation), which regulates the ovulation rate, was discovered in French Lacaune sheep. The B4GALNT2 (beta-1, 4-N-acetyl-galactosaminyl transferase 2) gene was considered as the potential FecL mutation gene. This study explores whether the effect of the FecL mutation exists in other sheep breeds, and the features of the B4GALNT2 gene in terms of the molecular structure and its expression profile. Using Sanger sequencing, we found that high and low fecundity breeds from among 11 measured sheep breeds all had no variation in the three specific mutation sites, which were linked with the FecL mutation. However, two mutations of g.36946470C > T and g.36933082C > T in the exon of B4GALNT2 had a significant effect on litter size in the first parity for Small Tail Han (STH) Sheep (p < 0.05). Two transcription start sites (TSS) of B4GALNT2 in its 5′-flanking region were discovered in ovine granule cells in vitro, through the RACE (Rapid amplification of cDNA ends) method. Except for in the kidney and oviduct, no significant difference in expression levels had been found between STH sheep and Tan sheep breeds. The B4GALNT2 gene, as a candidate for FecL, may have a relationship with the differences in litter size in STH sheep. B4GALNT2 is mainly expressed in the ovine ovary, which also suggests that B4GALNT2 plays an important role in sheep reproduction.

Aneuploidy in dizygotic twin sheep detected using genome-wide single nucleotide polymorphism data from two commonly used commercial vendors

Early detection of karyotype abnormalities, including aneuploidy, could aid producers in identifying animals which, for example, would not be suitable candidate parents. Genome-wide genetic marker data in the form of single nucleotide polymorphisms (SNPs) are now being routinely generated on animals. The objective of the present study was to describe the statistics that could be generated from the allele intensity values from such SNP data to diagnose karyotype abnormalities; of particular interest was whether detection of aneuploidy was possible with both commonly used genotyping platforms in agricultural species, namely the Applied BiosystemsTM AxiomTM and the Illumina platform. The hypothesis was tested using a case study of a set of dizygotic X-chromosome monosomy 53,X sheep twins. Genome-wide SNP data were available from the Illumina platform (11 082 autosomal and 191 X-chromosome SNPs) on 1848 male and 8954 female sheep and available from the AxiomTM platform (11 128 autosomal and 68 X-chromosome SNPs) on 383 female sheep. Genotype allele intensity values, either as their original raw values or transformed to logarithm intensity ratio (LRR), were used to accurately diagnose two dizygotic (i.e. fraternal) twin 53,X sheep, both of which received their single X chromosome from their sire. This is the first reported case of 53,X dizygotic twins in any species. Relative to the X-chromosome SNP genotype mean allele intensity values of normal females, the mean allele intensity value of SNP genotypes on the X chromosome of the two females monosomic for the X chromosome was 7.45 to 12.4 standard deviations less, and were easily detectable using either the AxiomTM or Illumina genotype platform; the next lowest mean allele intensity value of a female was 4.71 or 3.3 standard deviations less than the population mean depending on the platform used. Both 53,X females could also be detected based on the genotype LRR although this was more easily detectable when comparing the mean LRR of the X chromosome of each female to the mean LRR of their respective autosomes. On autopsy, the ovaries of the two sheep were small for their age and evidence of prior ovulation was not appreciated. In both sheep, the density of primordial follicles in the ovarian cortex was lower than normally found in ovine ovaries and primary follicle development was not observed. Mammary gland development was very limited. Results substantiate previous studies in other species that aneuploidy can be readily detected using SNP genotype allele intensity values generally already available, and the approach proposed in the present study was agnostic to genotype platform.

Effects of a Sonic Hedgehog agonist on ovine oocyte maturation, epigenetic changes and development of parthenogenetic embryos

Purmorphamine is a Sonic Hedgehog agonist that plays an important role in activity regulation of receptors and transcription factors. This study was carried out to assess the effects of Purmorphamine on nuclear and cytoplasmic maturation, epigenetic changes of in vitro matured oocytes and development of parthenogenetic ovine embryos. Ovine ovaries were randomly collected from industrial slaughterhouse, and cumulus-oocyte complexes were cultured in media containing 250 or 500ng/ml Purmorphamine or without Purmorphamine as a control group. Then, after in vitro maturation (IVM), Hoechst stain, cell tracker blue and histone H4K12 antibody were used, respectively to assess the nuclear and cytoplasmic maturation, and histone acetylation rate of matured oocytes. Matured oocytes were activated and cultured to the blastocysts stage in order to determine the parthenogenetic embryo cleavage rate. Expression of Histone Deacetylase 1, 2 and 3 (Hdac1, Hdac2 and Hdac3) genes were evaluated in matured oocytes by quantitative Real Time PCR. Results showed that although the concentration of 500ng/ml Purmorphamine had no significant influence on the oocyte nuclear maturation, it led to an increase in oocyte cytoplasmic maturation. Also, no significant difference in the rates of cleavage and blastocysts per cleavage was observed (P>0.05) in the groups treated with Purmorphamine compared to the control. Quantitative PCR analysis indicated that in 500ng/ml Purmorphamine treatment, Hdac 2 and 3 transcripts decreased and the H4K12 acetylation increased significantly (P<0.05). Consequently, this study showed that 500ng/ml Purmorphamine can induce the cytoplasmic maturation of oocytes, and may possibly be a good addative for improvement of ovine oocyte cytoplasmic maturation.

Intrafollicular expression and potential regulatory role of cocaine- and amphetamine-regulated transcript in the ovine ovary

Follicular growth is regulated by a complex interaction of pituitary gonadotropins with local regulatory molecules. Previous studies demonstrated an important role for cocaine- and amphetamine-regulated transcript (CART) in regulation of granulosa cell estradiol production associated with dominant follicle selection in cattle. However, intraovarian expression and actions of CART in other species, including sheep, are not known. The objective of this study was to investigate the expression of CART in sheep follicles and determine the effects of CART on indices of ovine granulosa cell function linked to follicular development. Results demonstrated the expression of CART messenger RNA and prominent intraovarian localization of CART peptide in granulosa cells of sheep follicles. Granulosa cell CART messenger RNA was lower, but follicular fluid estradiol concentrations were higher in large (>5 mm) follicles vs smaller 3- to 5-mm follicles harvested from sheep ovaries of abattoir origin. CART treatment inhibited follicle stimulating hormone-induced estradiol production by cultured ovine granulosal cells and also blocked the follicle stimulating hormone-induced increase in granulosa cell numbers. Results demonstrate expression of CART in sheep follicular tissues and suggest potential biological actions of CART, which are inhibitory to ovine follicular growth and development.

Identification of novel and differentially expressed microRNAs in ovine ovary and testis tissues using Solexa sequencing and bioinformatics

MicroRNAs (miRNAs) are small, single stranded, non-coding RNA molecules, about 19–25 nucleotides in length, which regulate the development and functions of reproductive system of mammal. To discover novel miRNAs and identify the differential expression of them in ovine ovary and testis tissues, the study constructed two libraries by using next-generation sequencing technologies (Solexa high-throughput sequencing technique). As a result, 9 321 775 and 9 511 538 clean reads were obtained from the ovary and testis separately, which included 130 562 (90 genes of ovary) and 56 272 (85 genes of testis) of known miRNAs and 486 potential novel miRNAs reads. In this study, a total of 65 conserved miRNAs were significantly differentially expressed (P<0.01) between the two samples. Among them, 28 miRNAs were up-regulated and 3 miRNAs were down-regulated on ovary compared with testis. In addition, the known miRNAs with the highest expression level (5 miRNAs) and 30 novel miRNAs with the functions related to reproduction were validated using the real-time quantitative RT-PCR. Moreover, the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that differentially expressed miRNAs were involved in ovary and testis physiology, including signal transduction, gonad development, sex differentiation, gematogenesis, fertilization and embryo development. The results will be helpful to facilitate studies on the regulation of miRNAs during ruminant reproduction.

Extended ex vivo culture of fresh and cryopreserved whole sheep ovaries.

We describe an original perfusion system for the culture of whole ovine ovaries for up to 4 days. A total of 33 ovaries were divided into six groups: control (n=6), not perfused and fixed; Groups SM72 and SM72-FSH (n=6 each), perfused with a simple medium for 72h with or without FSH; Groups CM96 and CM96-FSH (n=6 each), perfused with a complex medium for 96h with or without FSH; Group CM96-FSH-cryo, (n=3) cryopreserved and perfused for 96h with Group CM96-FSH medium. Depending on the medium used, morphological parameters of cultured ovaries differed from fresh organs after 72 (SM72, SM72-FSH) or 96 (CM96, CM96-FSH) h of perfusion. Oestradiol and progesterone were secreted in all groups but FSH had an effect only on Group CM96-FSH, stimulating continued oestradiol secretion 10 times higher than in all other groups. Morphological parameters and hormone secretion of cryopreserved ovaries were not different from fresh controls. This method enables the culture of whole ovaries for up to 4 days, the time required in vivo for 0.5-mm follicles to grow to 2.2mm and then for these follicles to reach the ovulatory size of 4mm or more. It could be used as a research tool or to complement current techniques for preserving female fertility.

Evidence of a molecular clock in the ovine ovary and the influence of photoperiod

The influence of the central circadian clock on reproductive timing is well established. Much less is known about the role of peripheral oscillators such as those in the ovary. We investigated the influence of photoperiod and timing of the LH surge on expression of circadian clock genes and genes involved in steroidogenesis in ovine ovarian stroma. Seventy-two Suffolk cross ewes were divided into two groups, and their estrous cycles were synchronized. Progestagen sponge removal was staggered by 12 hours between the groups such that expected LH peak would occur midway through either the light or dark phase of the photoperiodic cycle. Four animals from each group were killed, and their ovaries were harvested beginning 36 hours after sponge removal, at 6-hour intervals for 48 hours. Blood was sampled every 3 hours for the period 24 to 48 hours after sponge removal to detect the LH surge. The interval to peak LH did not differ between the groups (36.2 ± 1.2 and 35.6 ± 1.1 hours, respectively). There was an interaction between group and the time of sponge removal on the expression of the core clock genes ARNTL, PER1, CRY1, CLOCK, and DBP (P < 0.01, P < 0.05, P < 0.01, P < 0.01, and P < 0.01, respectively). As no significant interaction between group and time of day was detected, the datasets were combined. Statistically significant rhythmic oscillation was observed for ARNTL, CLOCK, CRY1 (P < 0.01, respectively), PTGS2, DBP, PTGER2, and CYP17A1 (P < 0.05, respectively), confirming the existence of a time-sensitive functionality within the ovary, which may influence steroidogenesis and is independent of the ovulatory cycle.

Cryopreservation of whole ovine ovaries with pedicles as a model for human: parameters of perfusion with simultaneous saturations by cryoprotectants.

The aim of this research was to study the effectiveness of perfusion of intact ovine ovaries with different rates of perfusion and time-period elapsed between extraction of these ovaries and the beginning of perfusion. Ovaries were perfused through the arteria ovarica (ovarian arteries) with culture medium supplemented with 5% bovine calf serum, 6% dimethyl sulfoxide, 6% ethylene glycol, 0.15M sucrose, Indian ink, and 100 IU/mL heparin at room temperature (22 degrees C). In the first cycle of experiments, ovaries (n = 96) were perfused for 60 minutes just after extraction of ovaries at the following rates of perfusion (mL/hour): 150, 100, 75, 50, 25, 12.5, and 6.3. In the second cycle of experiments, ovaries (n = 26) were perfused at a rate of 25 mL/hour for 60 minutes after extraction of ovaries and their storage at room temperature for 2, 3, 4, and 5 hours, for groups 1, 2, 3, and, 4, respectively. Successful perfusion of blood vessels was detected visible by a blue coloration of the ovarian tissues. The first cycle of experiments showed that the optimal perfusion rates were 50 mL/hour and 25 mL/hour. In the second cycle of experiments, good perfusion of ovaries was established at a perfusion rate of 25 mL/hour for the ovaries of groups when 2 and 3 hours had elapsed after extraction. Effective perfusion of ovine intact ovaries with vascular pedicle was established using freezing medium at room temperature at the rate of perfusion of 25 mL/hour or 50 mL/hour. The ovaries must be perfused not later than 3 hours after the death of animals.

Identification of a novel miRNA from the ovine ovary by a combinatorial approach of bioinformatics and experiments.

MicroRNAs (miRNAs) are a class of short endogenous, single-stranded, non-coding small RNA molecules, about 19–25 nucleotides in length that regulate gene expression at the translation level and influence many physiological process, such apoptosis, metabolism, signal transduction, and occurrence and development of diseases. In this study, we constructed a library from the ovine luteal phase ovary by using next-generation sequencing technology (Solexa high-throughput sequencing technique) and identified 267 novel miRNAs by bioinformatics. One of the novel miRNAs (ovis_aries_ovary-m0033_3p), which expressed in the sheep ovary and testis, was confirmed by real time PCR and northern blot. Ovis_aries_ovary-m0033_3p was 21 nucleotides in length and located on chromosome 12, and it had 100% similarity to hsa-miR-214-3p, mmu-miR-214-3p, dre-miR-214and ssc-miR-214. Meanwhile, the pre-miRNA was 82 nucleotides in length and had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_ovary-m0033_3p had a function similar to hsa-miR-214-3p, which is involved in the fine regulation of cell survival, embryonic development, breeding activities and resistance to ovarian cancer, so we defined it as oar-miR-214-3p. These experimental results will enrich the miRNA database for ovis aries and provide the basis for researching the regulation mechanism of miRNA in relation to breeding activities of seasonal breeding animals.

Two methods of vitrification followed by in vitro culture of the ovine ovary: evaluation of the follicular development and ovarian extracellular matrix.

The aim of this study was to evaluate the influence of two vitrification techniques on the extra cellular matrix (ECM) and ovarian follicular development. The ovarian cortex was fragmented (9 mm(3)) and divided into six groups, viz. fresh control, cultured control, vitrified by the Ovarian Tissue Cryosystem (OTC) method, conventional solid surface vitrification (SSV) method, OTC/cultured and SSV/cultured. Follicles from all the fragments were analysed for morphology, development and viability. The ECM was evaluated based on the condition of collagen and reticular fibres and the immunolocalization of type I collagen and fibronectin. After 7 days of culture, the tissue vitrified by OTC revealed a higher percentage (p < 0.05) of morphologically normal (30.66%) and viable (60.00%) follicles when compared with those vitrified using the SSV technique (21.33% and 23.00%). In all the fragments cultured, regardless of the vitrification method, a significantly higher percentage of developing follicles was observed when compared with the non-cultured tissue. Analysis of the type I collagen showed increased immunostaining after the in vitro culture in the vitrified fragments. In conclusion, the OTC is better for preserving the follicular viability and morphology and maintaining the integrity of the extracellular matrix components of the ovine ovary.

Characterization and comparative profiling of ovarian microRNAs during ovine anestrus and the breeding season.

BackgroundSeasonal estrus is a critical limiting factor of animal fecundity, and it involves changes in both ovarian biology and hormone secretion in different seasons. Previous studies indicate that two classes of small RNAs (miRNAs and piRNAs) play important regulatory roles in ovarian biology. To understand the roles of small RNA-mediated post-transcriptional regulation in ovine seasonal estrus, the variation in expression patterns of ovarian small RNAs during anestrus and the breeding season were analyzed using Solexa sequencing technology. In addition, reproductive hormone levels were determined during ovine anestrus and the breeding season.ResultsA total of 483 miRNAs (including 97 known, 369 conserved and 17 predicated novel miRNAs), which belong to 183 different miRNA families, were identified in ovaries of Tan sheep and Small Tail Han (STH) sheep. Compared with the three stages of the breeding season, 25 shared significantly differentially expressed (including 19 up- and six down-regulated) miRNAs were identified in ovine anestrus. KEGG Pathway analysis revealed that the target genes for some of the differentially expressed miRNAs were involved in reproductive hormone related pathways (e.g. steroid biosynthesis, androgen and estrogen metabolism and GnRH signaling pathway) as well as follicular/luteal development related pathways. Moreover, the expression of the differentially expressed miRNAs and most of their target genes were negatively correlated in the above pathways. Furthermore, the levels of estrogen, progesterone and LH in ovine anestrus were significantly lower than those in the breeding season. Combining the results of pathway enrichment analysis, expression of target genes and hormone measurement, we suggest that these differentially expressed miRNAs in anestrus might participate in attenuation of ovarian activity by regulating the above pathways. Besides miRNAs, a large and unexpectedly diverse set of piRNAs were also identified.ConclusionsThe miRNA profiles of ovine ovaries in anestrus were presented for the first time. The identification and characterization of miRNAs that are differentially expressed between ovine anestrus and the breeding season will help understanding of the role of miRNAs in the regulation of seasonal estrus, and provides candidates for determining miRNAs which could be potentially used to regulate ovine seasonal estrus.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-899) contains supplementary material, which is available to authorized users.

Protein localization of epidermal growth factor in sheep ovaries and improvement of follicle survival and antrum formation in vitro.

The aims of this study were to characterize EGF protein expression in ovine ovaries and to verify the effect of EGF on the in vitro development of isolated pre-antral follicles. After collection, ovarian tissue was fixed for immunohistochemical analysis. Additional pairs of ovaries were collected, and secondary follicles were cultured for 18days in α-MEM(+) (control) alone or supplemented with EGF (1, 10 or 50ng/ml). The immunostaining for EGF was observed in oocytes from pre-antral and antral follicles, in granulosa cells of primary and secondary follicles, as well as in cumulus and mural cells of antral follicles. After 18days, the results showed that treatment with 50ng/ml EGF significantly increased the percentage of morphologically normal follicles compared with the control group (α-MEM(+) ) and significantly reduced the precocious extrusion of oocytes and increased the percentage of antral follicles compared with the control and 1ng/ml EGF. All the treatments induced a progressive and significant increase of the follicular diameter throughout the period of culture. However, there were no significant differences in follicular diameter or in the daily growth rate among treatments. In conclusion, this study demonstrated the presence of EGF in ovine ovaries. Moreover, 50ng/ml EGF increased the percentage of normal follicles and improved antrum formation in isolated ovine follicles after 18days of in vitro culture.

Immunohistochemical localization of fibroblast growth factor-2 in the sheep ovary and its effects on pre-antral follicle apoptosis and development in vitro.

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF-2 protein expression in ovine ovaries and to verify the effect of FGF-2 on the morphology, apoptosis and growth of ovine pre-antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7days in control medium (α-MEM(+) ) alone or supplemented with FGF-2 at different concentrations (1, 10, 50, 100 or 200ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF-2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10ng/ml FGF-2, and significantly higher than those observed in 50, 100 or 200ng/ml FGF-2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200ng/ml FGF-2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200ng/ml FGF-2 (p<0.05). However, the percentage of apoptotic cells only decreased (p<0.05) in ovarian tissues cultured in 1 or 10ng/ml FGF-2 compared with the control medium and other FGF-2 treatments. In conclusion, this study demonstrated the presence of FGF-2 in ovine ovaries. Furthermore, 10ng/ml FGF-2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.

In vitro development of ovine oocytes cultured in the presence of GDF9 at chemically defined or undefined maturation medium

The major factor that affects oocyte developmental competency is regulation of oocyte in vitro maturation (IVM). According to the literature, oocyte-secreted and exogenous growth differentiation factor 9 (GDF9) have little influence on ovine in vitro maturation. This result may have been masked by the presence of serum or other additive in the maturation medium. Thus, we aimed to compare the developmental competency of ovine oocyte cultured in the presence of GDF9 at chemically defined or undefined maturation medium.Abattoir-derived ovine ovaries were transported to the laboratory in saline. After aspiration of follicles, good qualities cumulus oocyte complexes (COCs) were cultured with and without serum in the presence of exogenous and endogenous GDF9. Then, morphological cumulus cell expansion, cleavage rate and blastocyst yield were evaluated between experimental groups.In general, the use of chemically defined maturation media leads to significantly lower cumulus expansion. Cumulus expansion index in the presence of endogenous GDF9 and serum was lower than its control whereas in the absence of serum, exogenous and endogenous GDF9 had little influence on cumulus expansion. In the presence of exogenous GDF9 and absence of serum, cleavage rate was lower than exogenous and endogenous GDF9 in the presence of serum. Blastocyst rates showed no statistically significant difference between groups. Factorial analysis of the effect of GDF9, serum and their interactions on cleavage and blastocyst rates revealed no statistically significant difference between groups.This study provides evidence that GDF9 was not predominant factor in ovine cumulus expansion and oocyte development. Moreover, our results suggest that GDF9 under certain conditions might have interacted with serum born factors.

118 EFFECTS OF EPIDERMAL GROWTH FACTOR ON IN VITRO SURVIVAL AND ANTRUM FORMATION OF ISOLATED OVINE PREANTRAL FOLLICLES

The aim of this study was to verify the beneficial effects of different concentrations of epidermal growth factor (EGF) on survival and antrum formation of isolated ovine preantral follicles cultured in vitro. Ovine ovaries (n = 50) were collected from a local slaughterhouse and secondary follicles (150–200 μm in diameter), without antral cavities, were mechanically isolated by microdissection using 26-gauge needles. After selection, the follicles were individually cultured in 100-μL droplets of culture medium at 39°C and 5% CO2 in air for 18 days. The basic control medium consisted of α-minimal essential medium (α-MEM) supplemented with BSA; insulin, transferring and selenium; glutamine; hypoxanthine; and ascorbic acid and then referred to as α-MEM+. For the experimental conditions, follicles were cultured in α-MEM+ alone (control) or in different concentrations of EGF (1, 10, or 50 ng mL–1). Every other day, 60 μL of the culture media was replaced with fresh media. The morphological aspects of all ovine follicles were assessed every 6 days using a precalibrated ocular micrometer in a stereomicroscope at 100× magnification. Only those follicles showing an intact basement membrane, with bright and homogeneous granulosa cells and an absence of morphological signs of degeneration, were classified as morphologically normal follicles. The rupture of the basement membrane was also observed and characterised as follicle extrusion. In addition, antral cavity formation was defined as the emergence of a visible translucent cavity within the granulosa cell layers. Data from morphologically normal follicles, extruded follicles, and antrum formation rate during in vitro culture were expressed as percentages and compared by the chi-squared test, and differences were considered significant when P &lt; 0.05. The results showed that the percentage of morphologically normal follicles decreased significantly throughout the culture periods in all the treatments, except in the 50 ng mL–1 EGF group, which maintained the percentage of normal follicles from Day 0 to 6. Considering the same culture period, 50 ng mL–1 EGF treatment significantly increased the percentage of morphologically normal follicles at Day 18 compared with the control group. Moreover, the addition of EGF to the culture medium, at 50 ng mL–1, significantly reduced the precocious extrusion of oocytes and increased the percentage of antrum formation compared with the control and 1 ng mL–1 EGF after 18 days of culture. Notably, there were no significant differences between 10 ng mL–1 EGF, control medium, and 1 ng mL–1 EGF treatments. In conclusion, this study demonstrated that the addition of EGF to the in vitro culture medium, at 50 ng mL–1, increased the proportion of morphologically normal follicles and antrum formation rate of isolated ovine preantral follicles. This work was supported by FACEPE (Process APQ-0705–5.05/10). L. P. Santos is a recipient of a grant from FACEPE (Brazil).

Whole Ovine Ovaries as a Model for Human: Perfusion with CryoprotectantsIn VivoandIn Vitro

These experiments were performed to test the perfusion of ovine as a model for human ovaries by cryoprotectants in vivo at high temperature when the permeability of capillaries is high and when blood is insensibly replaced by the solution of cryoprotectants. By our hypothetical supposition, ovaries could be saturated by cryoprotectants before their surgical removal. The objective was to examine the effectiveness of perfusion of ovine ovaries with vascular pedicle in vivo and in vitro. Arteria ovarica was cannuled and ovaries were perfused by Leibovitz L-15 medium + 100 IU/mL heparin + 5% bovine calf serum + 6% dimethyl sulfoxide + 6% ethylene glycol + 0.15 M sucrose + Indian ink in vivo and in vitro. In the first and second cycle of experiments, ovaries (n = 13 and n = 23) were perfused in vivo and in vitro, respectively, during 60 min with the rate of perfusion 50 mL/h (0.8 mL/min). It was established with in vivo perfusion that only about 10% of ovarian tissues were perfused due to an appearance of multiple anastomoses when the perfusion medium goes from arteria ovarica to arteria uterina without inflow into the ovaries. It was concluded that in vitro perfusion of ovine intact ovaries with vascular pedicle by freezing medium is more effective than this manipulation performed in vivo.

Exposure to chemical cocktails before or after conception – The effect of timing on ovarian development

Exposure of female fetuses to environmental chemicals (ECs) during pregnancy results in a disturbed ovarian adult phenotype. We investigated the influence of pre- and/or post-conception exposure to low-level mixtures of ECs on the structure and function of the fetal ovine ovary. We examined ovarian morphology, expression of oocyte and granulosa cell-specific genes and proteome. Female fetuses were collected at day 110 of gestation, from dams exposed continuously until, and after mating, by grazing in pastures treated with sewage sludge as a fertiliser (TT) or in control fields treated with inorganic fertiliser (CC). In addition, in a cross-over design, fetal ovaries were collected from dams maintained on sludge pastures up to the time of mating but then transferred to control pastures (TC) and, reciprocally, those transferred from control to treated pastures at mating (CT). On examination, the proportion of type 1a follicles (activating primordial follicles) was significantly lower in animals from the CT groups compared with CC and TT groups (P<0.05). Of the 23 ovarian gene transcripts studied, 14 were altered in the ovaries of exposed fetuses (CT, TC, and TT) relative to controls, with the largest number of changes observed in cross-exposure pattern groups (CT or TC). Continuous EC exposure (TT) produced fewer transcript alterations and only two genes (INHBA and GSN) presented differential profiles between CC and TT. Fetal ovarian proteome analysis (2-DE gels) showed, across all exposure groups, 86 differentially expressed protein spots compared to controls. Animals in the CT group exhibited the highest number (53) while TC and TT presented the same number of affected protein spots (42). Fetal ovarian proteins with altered expression included MVP (major vault protein) and several members of the heat-shock family (HSPA4L, HSP90AA1 and HSF1). The present findings indicate that continuous maternal EC exposure before and during gestation, are less deleterious for fetal ovarian development than a change in maternal EC exposure between pre and post-conception. The pathways by which the ovary responds to this chemical stress were common in TT, CT, TC exposed foetuses. In addition to the period of pregnancy, the pre-conception period appears also as crucial for conditioning long-term effects of EC exposure on ovarian development and primordial follicle reserve and hence future fertility.

English

This study was designed to identify the effects of the presence of corpus luteum (CL) on the meiotic competence of the ewe oocytes. In addition, due to the pivotalroles of oocyte recovery methods on efficiency of&nbsp;in vitro&nbsp;maturation and&nbsp;in vitrofertilization, this study considered the new oocyte recovery method &ldquo;Oocyte Recovery with Centrifuge&rdquo; (ORC); Ovine ovaries were obtained from local abattoir at Karaj, Iran. The ovaries were placed in 0.9% saline which was supplemented with penicillin/streptomycin in thermo flask at 37&deg;C and transported to the laboratory within 1 to 2 h of slaughter. The ovaries were assigned to two groups; group-1 with functional CL and group-2 without CL. The oocytes were recovered by means of aspiration pump or ORC. After oocyte recovery, they were cultured in TCM-199 for 24 h. The mean number of oocyte recovery per ovary in group 1 ovaries (1.8 &plusmn; 0.01 via aspiration and 3.84 &plusmn; 0.05 via ORC) was lower (P&lt;0.05) than group 2 ovaries (2.2 &plusmn; 0.00 via aspiration and 5.43 &plusmn; 0.01 via ORC). There were no significant differences (P&gt;0.05) between percentage of nuclear maturation in oocytes which were recovered from group 2 ovaries via aspiration and ORC (75.20 &plusmn; 0.00 vs. 74.95 &plusmn; 0.00, metaphase II; M-II) method. The nuclear maturation in oocytes which were obtained via ORC from group 2 ovaries was higher (P&lt;0.05) than group 1 ovaries (74.95 &plusmn; 0.00 vs.&nbsp;60.07 &plusmn; 0.00b). Nuclear maturation for oocytes obtained via ORC from group 1 ovaries was (P&lt;0.05) lower than oocytes obtained via aspiration (60.07 &plusmn; 0.00 vs. 74.70 &plusmn; 0.00). Result of the present study showed&nbsp;that the presence of CL on ovaries lead to decrease in the quality and the quantity of oocytes. ORC method increased the quantity and quality of recovered oocytes. &nbsp; Key words:&nbsp;Aspiration, corpus luteum,&nbsp;in vitro&nbsp;maturation (IVM), oocyte recovery.

205 GLUCOSE-6-PHOSPHATE DEHYDROGENAZE ACTIVITY IN OVINE OOCYTES IS ASSOCIATED WITH PRE-IMPLANTATION EMBRYONIC DEVELOPMENT IN VITRO

A large proportion of ovine oocytes fail to develop into viable embryos following maturation, fertilization, and culture in vitro. Accurate, fast, and noninvasive predictors of ovine oocyte quality are therefore in urgent need for oocyte selection before in vitro maturation (IVM). Recent studies have shown that oocyte competence can be predicted through the presence of the glucose-6-phosphate dehydrogenase (G6PD) enzyme, as indicated by brilliant cresyl blue (BCB), a dye that can be degraded by G6PD. Thus, oocytes that have completed their growth phase show decreased G6PD activity and exhibit cytoplasm with a blue colouration (BCB+), whereas growing oocytes are expected to have a high level of G6PD, which results in colourless cytoplasm (BCB–). The brilliant cresyl blue staining test, as a noninvasive intrinsic criterion, has been successfully used to identify the more competent oocytes in various species. Therefore, this study aimed to investigate whether BCB staining, as an indicator of G6PD activity, can be used to select developmentally competent ovine oocytes before IVM and thereby increase the efficiency of in vitro embryo production. Ovine ovaries were obtained from a local slaughterhouse and transported to the laboratory, where cumulus–oocyte complexes (COC) were recovered by slicing the ovaries. Only oocytes with one or more complete layers of unexpanded cumulus cells and a homogeneous cytoplasm were used. The COC were exposed to 26 mM BCB diluted in modified Dulbecco’s PBS for 90 min at 39°C in humidified air. After BCB exposure, the COC were examined under a stereomicroscope and divided into 2 groups: BCB+ (blue cytoplasm, low G6PD activity) and BCB– (colourless cytoplasm, high G6PD activity). Cumulus–oocyte complexes in the control group were incubated for IVM directly after selection, without exposure to BCB dye. After IVM, oocytes were subjected to IVF followed by embryo culture for 7 days (5% CO2, 39°C, humidified air). Results were analysed by a chi-square test, and P &lt; 0.05 was considered statistically significant. The proportion of oocytes that cleaved by Day 2 after insemination was significantly (P &lt; 0.05) higher for the control and BCB+ groups [67.3% (68/101) and 71.7% (81/113), respectively] than for the BCB– group [50.5% (46/91)]. Significant differences among groups were also observed on Day 7 after fertilization, when the embryos reached the blastocyst stage of development. The BCB+ group yielded a significantly (P &lt; 0.05) higher proportion of blastocysts [34.5% (39/113)] than both the control [20.8% (21/101)] and BCB– [4.3% (4/93)] groups. In addition, the blastocyst rate of development in the control group was significantly (P &lt; 0.05) higher than that for the BCB– group. In conclusion, results of this study show that selection of ovine oocytes based on G6PD activity through the BCB test can be used as an efficient predictor of in vitro embryonic developmental competence. This positive predictive parameter of oocyte quality may also be useful in increasing the efficiency of blastocyst production during in vitro embryo production procedures in the ovine.