Morphology Of Cumulus-oocyte Complexes Research Articles

Short- and Long-Term Storage of Non-Domesticated European Mouflon (Ovis aries musimon) Cumulus-Oocyte Complexes Recovered in Field Conditions.

Reproductive biotechnologies can be used as a supporting tool, through gamete conservation and in vitro embryo production, in the preservation of invaluable and irreplaceable animal genetic resources. In the present study, immature mouflon cumulus-oocyte complexes (COCs) collected from ovariectomized female ovaries underwent short- or long-term conservation (24 h maintained in Earle's/Hank's (EH) medium or vitrification) under field conditions and afterwards transported to the laboratory where they were cultured for in vitro maturation (IVM) and assessed for oocyte meiotic competence and bioenergetic-oxidative status. Utilization of both storage techniques led to COC morphology preservation, as well as cumulus expansion and oocyte meiotic resumption after the IVM procedure. Quantitative bioenergetic-oxidative parameters were reduced in vitrified oocytes compared with EH ones. Immature COC storage needs to be optimized in both domesticated and non-domesticated sheep as a part of the strategy to avoid the loss of valuable genotypes of these animal species.

Effect of follicular wave synchronization using a progesterone-releasing intravaginal device-based protocol on in vitro embryo production in Bos taurus cows subjected to 14-day intervals ovum pick-up

Follicular wave synchronization (FWS) before ovum pick-up (OPU) is one of the strategies used to improve the efficiency of in vitro embryo production (IVP). This study aimed to evaluate the effect of FWS on the total follicular number, cumulus-oocyte complex (COC) recovery, and in vitro embryo development in Angus cows (n = 33) subjected to OPU with 14-day intersession intervals. Additionally, it was also evaluated the presence of carryover effects given the short intersession interval used. The experiment was run as a 2-treatment (FWS vs. Control) x 2-period (1 vs. 2) crossover design. Animals in the FWS group received an intravaginal progesterone implant (1gr), estradiol benzoate (2 mg), and D-cloprostenol (150 μg) on day 0 and the OPU was performed on day 5. Control group animals did not receive any hormone treatment. The FWS increased the number of 6–10 mm follicles (P = 0.05), but it decreased the COC recovery rate (P < 0.01). The FWS did not affect the total or frozen embryo numbers (P = 0.49 and P = 0.17; respectively), but it increased the total blastocyst cell number (P < 0.01). A carryover effect was found on the total and < 6 mm follicles number (P = 0.10 and P < 0.01; respectively), and on the regular, atretic, viable, and total number of COC (P = 0.01, P = 0.08, P = 0.02 and P < 0.01; respectively). We concluded that the FWS increased the quality of embryos after OPU with 14-day intersession intervals in Angus cows and that this kind of OPU/IVP scheme enabled the existence of a carryover effect, especially on the follicle number and COC morphology.

200 Effect of duration of in vitro maturation and cumulus–oocyte complex morphology on in vitro embryo production in wood bison

200 Effect of duration of in vitro maturation and cumulus–oocyte complex morphology on in vitro embryo production in wood bison

Intercellular communication in the cumulus-oocyte complex during folliculogenesis: A review.

During folliculogenesis, the oocyte and surrounding cumulus cells form an ensemble called the cumulus-oocyte complex (COC). Due to their interdependence, research on the COC has been a hot issue in the past few decades. A growing body of literature has revealed that intercellular communication is critical in determining oocyte quality and ovulation. This review provides an update on the current knowledge of COC intercellular communication, morphology, and functions. Transzonal projections (TZPs) and gap junctions are the most described structures of the COC. They provide basic metabolic and nutrient support, and abundant molecules for signaling pathways and regulations. Oocyte-secreted factors (OSFs) such as growth differentiation factor 9 and bone morphogenetic protein 15 have been linked with follicular homeostasis, suggesting that the communications are bidirectional. Using advanced techniques, new evidence has highlighted the existence of other structures that participate in intercellular communication. Extracellular vesicles can carry transcripts and signaling molecules. Microvilli on the oocyte can induce the formation of TZPs and secrete OSFs. Cell membrane fusion between the oocyte and cumulus cells can lead to sharing of cytoplasm, in a way making the COC a true whole. These findings give us new insights into related reproductive diseases like polycystic ovary syndrome and primary ovarian insufficiency and how to improve the outcomes of assisted reproduction.

Ultrastructural changes in immature ovine cumulus-oocyte complexes vitrified in conventional and open pulled straws

This study aimed to evaluate the effect of different cryodevices [conventional straws (CS) and open pulled straws (OPS)] of vitrification on viability, morphology and ultrastructure of ovine immature oocytes. Ovaries of slaughtered ewes were transported from a local abattoir to our laboratory in 0.9 % normal saline at 25–30 °C. The visible follicles (1–6 mm in diameter) were sliced with a microblade and their cumulus-oocyte complexes (COCs) were released in Dulbecco’s phosphate buffer saline. Upon examination under a stereomicroscope, good quality oocytes (n = 266) were selected and randomly assigned into three groups: 1) Fresh oocytes (control, n = 78); 2) Oocytes vitrified using CS (n = 95) and 3) Oocytes vitrified using OPS (n = 93). The gross morphology of COCs was evaluated under a stereomicroscope whilst their viability was evaluated in Trypan blue stained smear. Moreover, glutaraldehyde-fixed COCs (n = 45) were processed for transmission electron microscopy. It was observed that the proportion of recovered normal ovine oocytes after vitrification in OPS (89.24 %) was significantly higher (P < 0.05) than those vitrified in CS (73.68 %). Meanwhile, the proportion of abnormal oocytes was greater (16.84 %) in CS than OPS (8.60 %). The proportion of viable ooplasm and viable cumulus oocytes was significantly (P < 0.05) higher in the fresh group (88.88 %) than OPS (69.11 %) and CS (54.54 %) groups with significant (P < 0.05) difference between the two vitrification groups. Notably, ultrastructural alterations of vesicles and lipid droplets in fresh oocytes were similar with OPS and lower than CS vitrified-warmed oocytes without significant difference between the two vitrified groups. In conclusion, OPS vitrified-warmed oocytes have less cryoinjuries and higher survivability in comparison with CS vitrified-warmed oocytes, although ovine immature oocytes were more vulnerable to cryopreservation by vitrification.

120 High variation in prediction of developmental competence of bovine oocytes based on visual examination

The quality of an oocyte is often represented by its morphological characteristics. Stringent selection of bovine cumulus–oocyte complexes (COCs) for invitro embryo production (IVP) is regularly based on parameters such as colour and granulation of the ooplasm, density of the surrounding cumulus cells, brightness of the zona pellucida, among other less common parameters. The link between developmental competence and morphology of the COC has been demonstrated in literature in group culture system. However, this has not been yet examined in an individual invitro production system. This study investigates the ability of IVP researchers to predict whether an oocyte will develop into a blastocyst at Day 8 of invitro culture in an individual production system. A set of 29 pictures of immature bovine COCs was presented in duplicates to eight bovine IVP researchers with different institutional backgrounds. The observers were asked if they would select each oocyte for further IVP processing, assuming that only the oocytes with the highest developmental potential can proceed, and thus a stringent selection is warranted. These 29 immature oocytes were matured, fertilized, and cultured individually invitro (using conventional methods) so that their ability to develop into a blastocyst at Day 8 post-fertilization was known. The ability to reach the blastocyst stage was stated as the gold standard and compared with the answers of the observers using kappa statistics and sensitivity (Se) and specificity (Sp) tests. Kappa (κ) for interobserver agreement was κ=0.01 (Se=50%, Sp=53%; positive predictive value=14%, negative predictive value=87%) with 50% accuracy of prediction. The intraobserver agreement was κ=0.69, with κ=1 referring to 100% agreement (Se=87%, Sp=83%), with an accuracy of 85%. The ability of trained embryologists to predict the fate of oocytes’ developmental competence was accurate for only half of the presented oocytes. Additionally, the proportion of false positive (1 − Sp) and false negative (1−Se) predictions was equally distributed. The low positive predictive value might be due to the lower development rates in individual culture systems versus group culture systems, whereas observers are mainly trained to select oocytes for group culture. This study demonstrated the subjective nature of the oocyte selection process that is based on visual examination of COC morphology because interobserver agreement was almost nonexistent. Interpretation of the morphology within an observer remains consequent, represented by the high intraobserver agreement. Consequently, there is a need to develop a new model for the objective determination of oocyte quality for individual IVP. The implementation of machine learning algorithms could objectively enhance oocyte selection and artificial reproductive technologies in extension.

Apoptosis in porcine cumulus-oocyte complexes: Relationship with their morphology and the developmental competence.

The aim of this study was to assess the presence and distribution of apoptosis in porcine cumulus-oocyte complexes (COCs) and its relations with COC morphology and developmental competence. The COCs were obtained from slaughterhouse ovaries, classified into A1 (top category), A2, B1, B2, C, and D based on their morphology. A1, A2, and B1 were matured and fertilized in vitro, and blastocyst rate was compared among them. Before and after in vitro maturation (IVM), annexin-V staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to assess early and late apoptosis, respectively. There was a significant increase in both annexin-V (+) oocytes and TUNEL (+) cumulus cells as morphology further deteriorated. There were no statistical differences regarding annexin-V (+) oocytes within immature and post-IVM COCs, but TUNEL (+) oocytes were only observed in post-IVM COCs. Early and late apoptosis was detected in cumulus cells of all categories of immature and post-IVM COCs. However, the difference was only significant for annexin-V (+). There were no significant differences in embryo development. Therefore, apoptosis increases as the morphological features of the immature COCs decrease. In conclusion, the selection of COCs from Categories A1, A2, and B1 may be used as a selection criterion for in vitro development.

In vitro-production of embryos using immature oocytes collected transvaginally from superstimulated wood bison (Bison bison athabascae)

Two experiments were done to test the hypothesis that morphologic characteristics of wood bison cumulus-oocyte complexes (COC) are reflective of the ability of the oocyte to develop to an advanced embryonic stage after in vitro maturation, fertilization and culture, and to determine the effect of prolonging the interval from the end of superstimulation treatment to oocyte collection (FSH starvation period). Experiments were done during the anovulatory season. In Experiment 1, ovarian superstimulation was induced in 10 bison with two doses of FSH given at 48 h intervals beginning at the time of follicular wave emergence. COC were collected 3 days (72 h) after the last dose of FSH by follicular aspiration and classified as compact, expanded or denuded. The COC were matured in vitro for 24 h before fertilization in vitro (Day 0). Embryo development was assessed on Days 3, 7 and 8. The blastocyst rate was 7/34, 2/10 and 0/3 in COC classified as compact, expanded and denuded, respectively; however, only compact COC resulted in embryos that reached the expanded blastocyst stage. In Experiment 2, COC were collected at either 3 or 4 days (72 or 96 h) after the last dose of FSH (n = 16 bison/group) to determine the effect of the duration of FSH starvation on oocyte competence. The COC were classified as compact good (>3 layers of cumulus cells), compact regular (1–3 layers of cumulus cells), expanded or denuded, and then matured, fertilized and cultured in vitro. Although follicles were larger (P < 0.05) in the 4-day FSH starvation group, there was no effect of starvation period on the distribution of COC morphology; overall, 112/194 (57.7%) were compact, 29/194 (26.3%) were expanded, 39/194 (20.1%) were denuded, and 14/194 (7.2%) were degenerated (P < 0.05). Similarly, there was no effect of starvation period on embryo development. Compact good COC had the highest cleavage (88%) and blastocyst rates (54%; P < 0.05), followed by compact regular COC at 73% and 25%, respectively. Expanded and denuded COC had low cleavage (40% vs. 59%, respectively) and blastocyst rates (5% vs. 8%, respectively). We conclude that morphologic characteristics of wood bison COC are reflective of the ability of the oocyte to develop into an embryo in vitro. Importantly, oocytes collected from superstimulated bison during the anovulatory season were competent to develop to the blastocyst stage following in vitro maturation, fertilization and culture.

Producción in vitro de embriones a partir de complejos cúmulos oocitos tipo II en bovinos Bos indicus

The morphological selection of cumulus-oocyte complexes (COCs) is animportant step for in vitro embryo production. It has been suggested thatCOC showing signs of atresia have the ability to generate embryos. Theobjective of this work was to evaluate the effect of COC morphology fromBos indicus animals with signs of early atresia versus no signs of atresiaon in vitro embryo production. COC were classified in: Type I (TI): homogeneousooplasm with ≥ 4 layers of compact cumulus cells (CC) and Type II(TII): granular ooplasm and ≥ 4 layers of CC slightly expanded. The COCwere matured in vitro for 24 hours in TCM199 medium and subsequentlyfertilized in vitro for 18 h. The suspected zygotes were cultured in vitro forseven days in modified SOFaa medium. Embryonic quality was determinedby blastomeric count following staining with Hoechst 33342. Student testwas used to determine statistical differences for cleavage, blastocyst rateand blastomeric counts between types of COC. The cleavage rate for TI (n= 220) and TII (n = 161) was 88 ± 4 % and 89 ± 8 % respectively (p&gt; 0.05);embryo development rate was 36 ± 7 % and 33 ± 8 % (p&gt;0.05) respectively.The blastomeric count for both groups was 101 and 104 cells for TI and TIIrespectively (n = 10), (p&gt;0.05). These results demonstrate that there is nodifference in the quantity and quality of embryos produced in vitro usingCOC type I or type II, suggesting that both types could be used for bovine invitro embryo production in Bos indicus cows.

Expression of cumulus-oocyte complex genes and embryonic development in goats subjected to progestogen-based estrus synchronization

The objective was to investigate the effect of short-term (7 days) and long-term (14 days) progesterone-based estrus synchronization on number of follicles, progesterone concentrations, cumulus-oocyte complex (COC) gene expression, and embryonic development in goats. Nulliparous Thai-native goats (n = 45) were randomly assigned to one of two estrus synchronization treatments. Goats were treated with intravaginal sponges containing 60-mg medroxyprogesterone acetate (MAP; Synchrogest esponjas, Spain) during 7 or 14 days (short-term or long-term protocol, respectively). Multiple follicular development was induced by intramuscularly injections of 300-IU eCG in both groups (1 day before sponge withdrawal). An ovariectomy was performed at 24 hours after sponge removal to evaluate number of follicle and collect oocyte for IVF. Oocyte quality (healthy or nonhealthy) was determined by morphology of COCs before IVM. Recovery of COCs and total cellular RNA isolation were applied to determine apoptosis-related gene expression. After IVF, embryos were evaluated during the eight-day culture as numbers of cleaved oocyte, morula, and blastocyst embryo. Total numbers of follicles and oocytes were similar for both treatments. Plasma progesterone concentrations were not different during MAP insertion period (P > 0.05). However, goats that received the short-term protocol had a greater number of 4 to 6-mm follicle, healthy oocytes, cleaved oocytes, and morula embryos than goats that received the long-term protocol (P < 0.01). In addition, the expression of B-cell lymphoma 2 messenger RNA was greater (P < 0.05) in COCs derived from the 7 days MAP-treated when compared to the 14 days MAP-treated goats. These data highlight that the 7-day progestin–based treatment may contribute to quality of oocytes and embryonic development in goats.

Characterization of gametes in two phyllostomid bat species: Artibeus jamaicensis and Sturnira lilium

Morphology of gametes is used to understand the physiological processes in reproduction among domestic and wild animals. These gametes are used in assisted reproductive technology (ART) and conservation programs. In the case of Artibeus jamaicensis and Sturnira lilium, few studies have been conducted related to these issues. The aim of this study was to describe the structure of spermatozoa, semen characteristics and also the morphology and quality of cumulus oocyte complexes (COC) of A. jamaicensis and S. lilium. Semen characteristics were: A. jamaicensis had a sperm concentration of 4.26×106sperm/ml, progressive motility of 34.55%; viability of 73.23%; head, tail and mid-piece abnormalities of 12.50%. Head length was 6.26μm, mid-piece 18.61μm and tail 70.92μm. S. lilium, had a sperm concentration of 5.15×106sperm/ml, progressive motility of 60.00%, viability of 83.82%; abnormalities in head, tail and mid-piece of 13.77%. Head length was 7.01μm, mid-piece 20.33μm and tail 70.50μm. On average 12.8 of right ovarian oocytes and 9.9 of left ovarian oocytes of A. jamaicensis were recovered. For S. lilium on average 10.7 oocytes from the right ovary and 10.9 oocytes from the left ovary were recovered, ranging in quality from excellent to poor. Sperm morphology and quality of COC were similar to those for other domestic and wild animals. Bat gametes can be used for the study of reproductive biology, in conservation programs and assisted reproductive technology (ART) among domestic and wild animals.

Multifactorial Analysis of the Follicular Environment is Predictive of Oocyte Morphology in Cattle

Numerous attempts have been recently made in the search for a reliable, fast and noninvasive assay for selection of oocytes suitable for in vitro embryo production. Potential markers have been described in the follicle such as follicular fluid (FF) or cumulus cells (CCs). However, the reported findings are contradictory, which may reflect the complexity of metabolism of the ovarian follicle. In the present experiment, a data set from individual follicles of known diameter was obtained: cumulus-oocyte complex (COC) morphology, fatty acid composition and glucose concentration in FF as well as apoptotic index in CCs. The obtained data was statistically analyzed either separately (univariate analysis) or simultaneously (multivariate analysis) to examine its predictive value in morphology assessment of bovine COCs. Although the univariate analysis yielded a complex relation system of the selected parameters, no clear outcome could be established. In multivariate analysis, the concentration of the four fatty acids (C16:0, C16:1, C18:1cis9, C22:5n3) and Δ9-desaturase (16) as well as elongase activities were selected as covariates. This allowed prediction of the morphology of a COC with an accuracy of 72%, which is the most interesting finding of the experiment. The present study indicates that the multifactorial model comprising of selected parameters related to the follicle appeared more effective in predicting the morphology of a bovine COC, which may improve the effectiveness of in vitro production systems.

217 TRANSCRIPT ABUNDANCE OF CATHEPSIN GENES IN CUMULUS CELLS AS A MARKER OF CATTLE OOCYTE QUALITY

Dynamics of follicular growth and atresia is closely connected with apoptosis. Cathepsins (CTS) are involved in diverse biological functions, whereas one member of this family, cathepsin B, plays a major regulatory role in the process of apoptosis. Oocyte quality is a complex trait shaped by the follicular components (e.g. cumulus cells, CC; follicular fluid, FF). A negative relationship between relative transcript abundance (RA) of CTSB, CTSS, and CTSZ genes in CC with the quality of corresponding oocytes was reported in cattle (Bettegowda et al. 2008). Fatty acid (FA) composition of the FF and mtDNA copy number in the oocyte are other markers of oocyte quality. Therefore, in this study, we analysed relations between selected traits of the 3 follicular components (FF, CC, oocyte) within the individual follicle with the focus on oocyte quality. The experiment was based on cumulus–oocyte-complexes (COC) and FF obtained from individual follicles of slaughterhouse ovaries. Each follicle was measured and assigned into 1 of 3 classes (small &lt;6 mm; medium 6 to 8 mm; large &gt;8 mm). The COC morphology (grades 1 to 4) was evaluated according to Stojkovic et al. (2001). The following analyses were performed: CC, mRNA abundance of CTSB, CTSS, CTSZ, and CTSK genes (real-time PCR, 100 replicates, ACTB as a reference gene); the oocyte, mtDNA copy number (real-time PCR, 93 replicates, COX1 gene); and FF and FA composition (gas chromatography). The following procedures were employed: total RNA isolation, mirVana Paris Kit (Ambion); total DNA isolation, High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN, USA); cDNA synthesis, Transcriptor High Fidelity cDNA Synthesis Kit (Roche); and the standard curve method, to analyse the qPCR data. For statistical analysis, the Kruskal-Wallis test as well as Spearman rank correlation were applied. The highest RA of CTSB gene was noted in CC from the grade 3 COC (P &lt; 0.05), whereas that of CTSK and CTSZ genes in CC from the grade 4 COC (P &lt; 0.01). Because grade 3 and 4 COC are not suitable for IVM, we assumed that high RA of CTS gene in CC may indicate reduced quality of the corresponding oocyte. Surprisingly, the highest RA for CTSB gene was observed in CC from the medium follicles (P &lt; 0.05). Significant (P &lt; 0.05) correlations were estimated between the following: RA of CTSB gene in CC and mtDNA copy number in the oocyte (r = 0.27), RA of CTSB gene in CC and C18.3 n-3 concentration in FF (r = 0.32), RA of CTSZ gene in CC and C18.3 n-3 concentration in FF (r = 0.37), as well as RA of CTSZ gene in CC and n-3 concentration in FF (r = 0.34). Although an increase in RA of the CTS genes in CC was accompanied by the inferior oocyte morphology, it was also correlated with higher mtDNA copy number in the oocyte and FA content in FF. The last 2 features have been previously attributed to oocytes of better quality, what contrasts with the high RA of the CTS genes. Thus, higher RA of CTS genes within CC may not mark the bovine oocyte of reduced quality. Funding–National Science Center, grant no. N N302 604438.

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.

Associations between free fatty acids, cumulus oocyte complex morphology and ovarian function during in vitro fertilization

To determine if follicular free fatty acid (FFA) levels are associated with cumulus oocyte complex (COC) morphology. Prospective cohort study. University in vitro fertilization (IVF) practice. A total of 102 women undergoing IVF. Measurement of FFAs in serum and ovarian follicular fluid. Total and specific follicular and serum FFA levels, correlations between follicular and serum FFAs, and associations between follicular FFA levels and markers of oocyte quality, including COC morphology. Predominant follicular fluid and serum FFAs were oleic, palmitic, linoleic, and stearic acids. Correlations between follicular and serum FFA concentrations were weak (r=0.252, 0.288, 0.236, 0.309, respectively for specific FFAs; r=0.212 for total FFAs). A receiver operating characteristic curve determined total follicular FFAs≥0.232 μmol/mL distinguished women with a lower versus higher percentage of COCs with favorable morphology. Women with elevated follicular FFAs (n=31) were more likely to have COCs with poor morphology than others (n=71; OR 3.3, 95% CI1.2-9.2). This relationship held after adjusting for potential confounders, including age, body mass index, endometriosis, and amount of gonadotropin administered (β=1.2; OR 3.4, 95% CI 1.1-10.4). Elevated follicular FFA levels are associated with poor COC morphology. Further work is needed to determine what factors influence follicular FFA levels and if these factors impact fertility.

Assessment of transcript and protein levels contributing to cell cycle control and gap junction connections in morphologically variable groups of porcine cumulus-oocyte complexes

Oocytes and somatic cumulus cells are connected by an extensive network of gap junctions. These connections contribute in a major way to oocyte maturation and developmental competence. Cumulus-oocyte complexes (COCs) were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The morphological classification of COCs is based on the number of cumulus cell layers and the degree of their compaction, as well as on cytoplasm composition (homogenous, heterogeneous). The obtained COCs were divided into four grades according to this classification system. By assessing the activity of glucose-6-phosphate dehydrogenase (G6PDH) using the brilliant cresyl blue (BCB) test, real-time quantitative PCR (RQ-PCR) reaction methods and confocal microscopic observations, we determined the transcript levels of connexins 43 and 45, cyclin dependent kinases (cdk5 and cdk5r), and cdk inhibitors 1 and 3 (p27kip1 and cdkn3) as well as cdk4 protein in morphologically different groups of porcine oocytes isolated from puberal gilts. To assess their nuclear status the completely denuded oocytes were subjected to DAPI staining. We found statistically increased cdkn3, cdk5 and connexin 45 mRNA levels in oocytes graded as I as compared to II, III, and IV. The cdkn1, cdk5r and connexin 43 transcript contents were higher only when comparing between oocytes graded as I, III and IV. The cdk4 protein in oocytes graded I and II is localized mainly in the zona pellucida, although in grade III COCs the expression of this protein is decreased and observed only in the cytoplasm. Grade IV COCs do not demonstrate a significant presence of cdk4 protein. With regards to nuclear maturation, the percentage of MII stage oocytes was significantly (P &amp;lt; 0.05) higher in grade I and II oocytes as compared to grade III and IV oocytes. Our results demonstrate for the first time that cdk4 protein localization and all of the investigated transcript levels are associated with COC morphology and may be related to further maturation ability as well as developmental competence of oocytes.

The morphology of porcine oocytes is associated with zona pellucida glycoprotein 3 and integrin beta 2 protein levels--

The morphology and quality of oocytes is set during oo- and folliculo-genesis, and is completed during final maturation. Early embryonic development is associated with the morphology of the cumulus-oocyte-complex (COC). However, current knowledge of the possible relationships between oocyte morphology and the level of proteins within the oocyte, which may reflect fertilization ability, is insufficient. Using western-blot analysis and confocal microscopic observation, we determined the levels of integrin beta-2 (integrin &amp;beta;2) and glycoprotein 3 (pZP3) protein levels in the porcine zona pellucida of four morphologically different types of oocytes, graded according to their cytoplasmic composition and cumulus structure. The level of integrin &amp;beta;2 protein was increased in grade I and II oocytes as compared to other grades (P &amp;lt; 0.05). Moreover, the level of pZP3 protein was 3&amp;ndash;4 fold higher in grade I oocytes (P &amp;lt; 0.01). We suggest that COC morphology may be associated with oocyte fertilization ability with respect to its sperm-oocyte interaction gene expression, which is the result of increased accumulation of specific proteins prior to fertilization in higher quality oocytes. Higher quality oocytes may also reflect better fertilization ability.

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had &gt;2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P &lt; 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P &lt; 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P &lt; 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P &lt; 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P &gt; 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P &lt; 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P &lt; 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

Effects of Season and Reproductive Phase on the Quality, Quantity and Developmental Competence of Oocytes Aspirated from Japanese Black Cows

The aim of the present study was to determine whether the season (hot and cool) and reproductive phase (pregnant and non-pregnant) of the cow affect follicular recruitment and oocyte development. Follicular oocytes were aspirated from Japanese black cows by the ovum pick-up (OPU) method, which was performed 2 to 6 times within 1.5 months in pregnant cows and 2 to 4 times within 2 months in non-pregnant cows, during the hot (July to September) and cool (October to November) seasons. After follicular aspiration, the number and morphology of cumulus-oocyte complexes (COCs) and the developmental competence of oocytes after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture were evaluated. The quality of aspirated COCs did not differ between the hot and cool seasons, irrespective of the reproductive phase of the donor cows. In the pregnant cows, the season did not affect follicular recruitment, early embryonic development or the quality of embryos. In the non-pregnant cows, however, the mean number of aspirated follicles and collected oocytes decreased during the hot season as compared with the cool season. When the data for the 2 seasons were combined to assess the effects of reproductive phase on oocyte development, the total proportions of cleavage, development into blastocysts and freezable embryos were higher for embryos obtained from pregnant cows (P<0.05) than those obtained from non-pregnant cows. In conclusion, the season did not have any apparent effects on the quality of aspirated COCs and the developmental competence of oocytes after IVM-IVF, but it may affect follicular recruitment in non-pregnant cows. Moreover, the reproductive phase may influence the developmental competence of the recovered oocytes.

Effect of maternal heat-stress on follicular growth and oocyte competence in Bos indicus cattle

The objective was to determine whether exposure of Gir (Bos indicus) cows to heat-stress (HS) causes immediate and delayed deleterious effect on follicular dynamics, hormonal profile and oocyte competence. The cows were kept in tie-stalls for an adaptive thermoneutral period of 28 days (Phase I, Days -28 to -1). In Phase II (Days 0-28) cows were randomly allocated into control (CG, n=5) and HS (HS, n=5) treatments. The HS cows were placed in an environmental chamber at 38 degrees C and 80% relative humidity (RH) during the day and 30 degrees C, 80% RH during the night for 28 days. The CG group was maintained in shaded tie-stalls (ambient temperature) for 28 days. During Phase III (Days 28-147) animals were placed in tie-stalls (Days 28-42) followed by pasture (Days 42-147) under thermoneutrality. In each phase, weekly ovum pick up (OPU) sessions were to evaluate follicular development, morphology of cumulus-oocyte complexes (COCs), and developmental competence after in vitro maturation, fertilization, and culture. Serum concentrations of progesterone (P(4)) and cortisol were evaluated by radioimmunoassay. Exposure of Gir cows to HS had no immediate effect on reproductive function, but exerted a delayed deleterious effect on ovarian follicular growth, hormone concentrations, and oocyte competence. Heat-stress increased the diameter of the first and second largest follicles from Days 28 to 49. Indeed, HS increased the number of >9 mm follicles (characterized as follicular codominance) during this phase. Cows exposed to HS had longer periods of non-cyclic activity (P(4)<1 ng/mL), as well as shorter estrous cycles. However, HS did not affect cortisol concentration as compared to CG. Although HS had no significant effect on cleavage rate, it reduced blastocyst development during Phase III. In conclusion, long-term exposure of B. indicus cattle to HS had a delayed deleterious effect on ovarian follicular dynamics and oocyte competence.