Competence Of Cumulus-oocyte Complexes Research Articles

Oocyte Quality, In Vitro Fertilization and Embryo Development of Alpaca Oocytes Collected by Ultrasound-Guided Follicular Aspiration or from Slaughterhouse Ovaries.

Simple SummaryWe compared the morphological quality and the in vitro developmental competence of cumulus–oocyte complexes (COCs) from live vs. slaughtered alpacas. COCs were recovered using: (i) manual aspiration in slaughterhouse ovaries (n = 15 females), or (ii) ovum pick-up (OPU) in live superstimulated alpacas (n = 13). COCs recovered were morphologically evaluated. Grade I–III COCs were in vitro matured and fertilized afterwards using fresh alpaca spermatozoa. Zygotes were in vitro cultured for seven days. The proportion of COCs recovered was similar between groups, but the mean number of COCs collected from individual ovaries was greater (p < 0.05) in slaughterhouse ovaries. A higher (p < 0.05) percentage of grades III and IV and a lower (p < 0.05) percentage of grade I COCs was obtained using OPU. Although, no significant difference in the percentage of cleavage and morula formation was observed between groups, the number of blastocysts, regarding cleavage and COCs collected, was higher (p < 0.007 and p < 0.0002, respectively) in the OPU group. However, the total number of blastocysts per female did not differ between groups. The recovery rate and morphological quality of COCs was significantly higher when follicles were manually aspirated from slaughterhouse alpaca ovaries; however, a greater developmental potential was observed in oocytes obtained from live alpaca.The morphological quality and the in vitro developmental competence of cumulus-oocyte complexes (COCs) collected from in vivo or slaughtered alpacas was compared. COCs were recovered from ovarian follicles using: (i) manual aspiration in ovaries of alpacas (n = 15) sacrificed at a local slaughterhouse, or (ii) transrectal ultrasound-guided follicular aspiration (or ovum-pick-up, OPU) in live alpacas (n = 13) 4 days after the administration of an ovarian superstimulation protocol (200 UI eCG). COCs recovered from both groups were morphologically evaluated and graded. Grade I to III COCs were in vitro matured for 26 h and in vitro fertilized afterwards for 20 h using fresh alpaca epididymal spermatozoa. Presumptive zygotes from both groups were in vitro cultured for 7 days. The proportion of COCs recovered over the total number of follicles punctured was similar between groups, but the mean number of COCs collected from individual ovaries was greater (p < 0.05) in slaughterhouse ovaries. A significantly higher (p < 0.05) percentage of low-quality COCs (grades III and IV) and a lower (p < 0.05) percentage of grade I COCs was obtained using OPU. The number of blastocysts, regarding cleavage and COCs collected, was higher (p < 0.007 and p < 0.0002 respectively) for COCs collected by OPU; however, the total number of blastocysts per female did not differ between groups. We can conclude that the recovery rate and morphological quality of COCs was significantly higher when follicles were manually aspirated from slaughterhouse alpaca ovaries; however, a statistically higher developmental potential was observed in oocytes collected by OPU from live alpaca donors.

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes.

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus–oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL+) and without (CCL-) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL+), contralateral (CL−), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL− cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL+ group; however, a higher proportion of embryos were obtained from the CCL− group. Besides, COCs from the CL+ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL− group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.

A multilevel analysis identifies the different relationships between amino acids and the competence of oocytes matured individually or in groups

High-protein diets contribute to an increase in urea follicular concentrations associated with decreased fertility. Urea has been shown to interfere with the epidermal growth factor (EGF)/EGFR system, which has been shown to have a beneficial effect during in vitro maturation (IVM) of oocytes. Of note, the number of cumulus-oocyte complexes (COCs) in the maturation medium can change the maturation and the developmental competence of COCs. Therefore, it was hypothesized that, the presence of urea and EGF may have a differential effect on the depletion/appearance of AAs and competence of COCs matured individually (I-IVM system) or in groups (G-IVM system). In the G-IVM system, COCs increased consumption (depletion) of AAs compared with other groups in the presence of high-level urea (40 mg/dl) + EGF (10 ng/ml). In the I-IVM system, the non-cleaved COCs depleted more AAs than the cleaved COCs, in particular in the presence of urea. The combination of urea and EGF increased the depletion of AAs in the G-IVM system. However, the EGF abrogated the urea-induced depletion of AAs by the I-IVM COCs. The use of N-acetyl-l-cysteine as an EGFR inhibitor canceled urea-induced depletion of AAs. This shows the inhibiting effect of urea over the EGF/EGFR system. In the presence of urea + EGF, COCs had a lower degree of developmental competence than control in both I- and G-IVM systems. Arginine had the best predictive power to identify highly competent COCs in the G-IVM system, while glutamine was the best predictor of the cleavage in the I-IVM system. In conclusion, this multi-level study shows that COCs matured individually or in groups may have different association with AAs metabolism. These findings provide new insights into the relationships between AA metabolism and the subsequent developmental competence of COCs.

Effect of Oocyte Quality Assessed by Brilliant Cresyl Blue (BCB) Staining on Cumulus Cell Expansion and Sonic Hedgehog Signaling in Porcine during In Vitro Maturation.

Brilliant cresyl blue (BCB) staining is used to select developmentally competent cumulus-oocyte complexes (COCs) for in vitro maturation (IVM). However, limited attention has been paid to what drives the higher developmental competence of BCB+ COCs. Sonic hedgehog signaling (SHH) is an important signaling pathway for ovarian follicular development and oocyte maturation. Therefore, this study investigated the effect of oocyte quality assessed by BCB staining on cumulus cell expansion, oocyte nuclear maturation, subsequent embryo development, apoptosis levels, and SHH signaling protein expression, in porcine COCs. After IVM, BCB+ COCs exhibited a significantly higher proportion of complete cumulus cell expansion and metaphase II rate in oocytes than BCB- COCs. After in vitro fertilization, the BCB+ group showed a significantly higher monospermy rate, fertilization efficiency, percentage of cleavage and blastocyst formation, with a higher total cell number and a lower apoptosis in blastocysts as compared with the BCB- group. Furthermore, significantly lower apoptosis levels and a higher expression of SHH-signaling proteins in COCs were observed, before and after IVM. In conclusion, high-quality oocytes had a greater potential to expand their surrounding cumulus cells with active SHH signaling and a lower apoptosis. This could provide COCs with a proper environment for maturation, thereby leading to a better subsequent embryo development.

Expression levels of FSHR, IGF1R, CYP11al and HSD3β in cumulus cells can predict in vitro developmental competence of bovine oocytes.

The efficiency of in vitro embryo production technologies would be improved by the development of suitable non-invasive biomarkers that allow the selection of good quality cumulus-oocyte complexes (COCs). The present study used whole, single oocyte culture to investigate whether the expression levels of follicle-stimulating hormone receptor (FSHR), insulin-like factor 1 receptor (IGF1R) and three steroidogenesis-related enzymes (CYP11al, CYP19al and HSD3β) in cumulus cells reflected the developmental competence of COCs. Cumulus cells were collected from single COCs before maturation culture and relative mRNA levels were assessed using real-time PCR. The analysis indicated that mRNAs for FSHR, IGF1R, CYP11al and HSD3β were present at higher levels in cumulus cells from COCs that failed to form blastocysts compared with cumulus cells from COCs that formed blastocysts. Moreover, FSHR and IGF1R mRNA levels were positively correlated with those of genes for steroidogenesis-related enzymes. In conclusion, poor developmental competence of COCs was related to higher expression of FSHR, IGF1R, CYP11al and HSD3β in cumulus cells, which may indicate the advanced differentiation of cumulus cells into granulosa cells.

Granulosa secreted factors improve the developmental competence of cumulus oocyte complexes from small antral follicles in sheep.

Oocyte in vitro maturation can be improved by mimicking the intra-follicular environment. Oocyte, cumulus cells, granulosa cells, and circulating factors act as meiotic regulators in follicles and maintain oocyte in the meiotic phase until oocyte becomes competent and ready to be ovulated. In a randomized experimental design, an ovine model was used to optimize the standard in vitro maturation media by Granulosa secreted factors. At first, the development capacity of oocyte derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles was determined. Differential gene expression of granulosa secreted factors and their receptors were compared between the cumulus cells of the two groups. Then, the best time and concentration for arresting oocytes at the germinal vesicle stage by natriuretic peptide type C (CNP) were determined by nuclear staining in both groups. Oocyte quality was further confirmed by calcein uptake and gene expression. The developmental competence of cumulus oocyte complexes derived from small size follicles that were cultured in the presence of CNP in combination with amphiregulin (AREG) and prostaglandin E2 (PGE2) for 24 h was determined. Finally, embryo quality was specified by assessing expressions of NANOG, SOX2, CDX2, OCT4, and TET1. The cumulus oocyte complexes derived from small size follicles had a lower capacity to form blastocyst in comparison with cumulus oocyte complexes derived from medium size follicles. Prostaglandin E receptor 2 and prostaglandin-endoperoxide synthase 2 had significantly lower expression in cumulus cells derived from small size follicles in comparison with cumulus cells derived from medium size follicles. Natriuretic peptide type C increased the percentage of cumulus oocyte complexes arresting at the germinal vesicle stage in both oocytes derived from medium and small follicles. Gap junction communication was also improved in the presence of natriuretic peptide type C. In oocytes derived from small size follicles; best blastocyst rates were achieved by sequential exposure of cumulus oocyte complexes in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. Increased SOX2 expression was observed in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)], while decreased OCT4 expression was observed in [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. It seems that the natriuretic peptide type C modulates meiotic progression, and oocyte development is probably mediated by amphiregulin and prostaglandin E2. These results may provide an alternative IVM method to optimize in vitro embryo production in sheep and subsequently for humans.

107 Follicular wave synchronization and FSH stimulation prior to ovum pickup for invitro embryo production

Invitro embryo production (IVP) has become a reliable alternative for genetic improvement in beef and dairy herds. Maximizing embryo yield and pregnancy per donor are key factors. The aim of this study was to compare ovum pickup (OPU) yields, developmental competence of cumulus-oocyte complexes (COCs), and pregnancy rates from Angus donors in a commercial IVP setting. Donors (&amp;gt;4-year-old pluriparous open dry cows) were handled under the same feeding and environmental conditions. Treatment groups were organised as follows: Group 1: no synchronization (SYNCH; n=5); Group 2: SYNCH with no superstimulation (SOV; n=5); Group 3: SYNCH + SOV (n=5) and OPU 36h after last FSH injection; Group 4: SYNCH + SOV (n=5) and OPU 48h after last FSH injection; and Group 5: SYNCH + SOV (n=5) and OPU 72h after last FSH injection. Follicular waves in groups 2, 3, 4, and 5 were synched by gonadotrophin-releasing hormone (GnRH), prostaglandin F2α (PGF), and controlled internal drug release (CIDR). No pre-synch was used. Injections of FSH (pFSH=180mg, Folltropin) were performed IM twice a day, for three days. A minimum of three replicates were performed for each donor. A Mindray DP30V equipped with a micro-convex transducer 5.0-8.5MHz probe, disposable 20-gauge needle, and a flow rate of 15mL min−1 were used for OPU. All visible follicles (Foll) were punctured and retrieved into a 50-mL 36°C warmed tube with media (phosphate-buffered saline, bovine serum albumin (BSA), and heparin). Viable oocytes were classified according to IETS guidelines. The COCs were matured in 100µL of M199 medium supplemented with ALA-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µgmL−1), epidermal growth factor (50ngmL−1), oFSH (50ngmL−1), bLH (3μgmL−1), cysteamine (0.1mM), and 10% fetal bovine serum (FBS) for 22 to 24h. Fertilization (Day 0) was carried out using highly fertile sires selected by discontinuous 40%/80% layers (PureSperm) and diluted to a final concentration of 1×106 spermmL−1. Matured oocytes were fertilized in 50µL of modified synthetic oviductal fluid (SOF) media supplemented with fructose (90µgmL−1), penicillamine (3µgmL−1), hypotaurine (11µgmL−1), and heparin (10µgmL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in 50-µL drops of SOF-BSA for 7 days. On Day 3.5, 2% of FBS was added. On Day 7, fresh transferable (grade 1 and 2, IETS standards) blastocysts were implanted into synchronized recipient cows. Around Day 30, ultrasound diagnosis was performed to determined pregnancy rate (PR). We used ANOVA for comparisons of mean values and X2 test for proportions, α=0.05 (Table 1). In conclusion, synchronization, FSH stimulation, and 48-h coasting before OPU in Angus cows increased the number of collected viable oocytes and embryo development rates. More transferrable embryos and higher rates of PR per OPU were obtained using 36- and 48-h coasting, respectively. Table 1.Follicles (Foll), viable oocytes, cleavage, blastocysts, and pregnancy rates (PR) in Angus cows after ovum pickup (OPU) or invitro embryo production (IVP) SYNCH1 FSH Coasting Foll/OPU Oocytes/OPU Viable oocytes Cleavage,% Embryos,% Embryos/OPU PR,% PR/OPU NO NO NO 10.8a 9.0a 8.9a 63a 25a 2.3a 45a 1.0a YES NO NO 10.1a 8.4a 8.1a 73b 27b 2.2a 51b 1.1a YES YES 36H 16.5b 13.8b 13.6b 70c 29b 4.0b 46a 1.8b YES YES 48H 16.1b 13.4b 13.3b 69c 28b 3.8c 52b 2.0b YES YES 72H 15.6b 13.0b 12.6b 64a 22c 2.8d 55b 1.6c a-dValues with different superscripts in the same column differ (P&amp;lt;0.05). 1SYNCH=synchronized.

Effect of cumulus cells of cumulus-oocyte complexes on in vitro maturation, embryonic developmental and expression pattern of apoptotic genes after in vitro fertilization in water buffalo (Bubalus bubalis)

In the present study, the potential of different grades of cumulus-oocyte complexes (COCs) for in vitro maturation (IVM) and embryonic development was assessed. Further, the association of the expression pattern of anti-apoptotic Mcl-1 and pro-apoptotic Bax genes in embryonic development was analyzed. Abattoir derived oocytes were graded into grade A and B based on surrounding cumulus rings. Out of 1050 ovaries, a total number of 770 and 1360, were of grade A and B COCs, respectively, were aspirated. After IVM, grade A COCs had a significantly higher number of polar bodies (92.04 ± 0.60%) as compared to grade B (85.88 ± 0.46%). On IVF and embryo culture, grade A COCs produced the significantly higher rate of cleavage and blastocyst (90.44 ± 0.71% and 41.55 ± 0.96%) as compared to grade B COCs (79.77 ± 0.76% and 30.44 ± 0.96%). The transcriptional analysis of apoptotic genes expression by Real-time PCR revealed a significantly higher expression of Mcl-1 gene in embryos of grade A as compared to grade B, whereas, the relative expression of Bax gene was down-regulated in grade A than grade B embryos. Thus it was concluded that the pattern of apoptotic genes expression in early-stage embryos can be used as a marker gene to predict the developmental competence of COCs.

189 EFFECT OF THE OVARIAN STIMULATION OF ANESTROUS CATS WITH eCG ON MORPHOLOGICAL QUALITY AND GENE EXPRESSION PROFILE OF CUMULUS-OOCYTE COMPLEXES

The objective of this research was to evaluate the effect of eCG on morphological quality and gene expression profile of cumulus-oocyte complexes (COC) recovered from anestrous cats. For this purpose, 3 experimental groups were made. Group 1 consisted of 11 oestrous cats (oestrous); Group 2 had 13 anestrous cats (anestrous); and Group 3 was made up of 11 anestrous cats treated with a single subcutaneous dose of 200 IU of eCG. In oestrous and anestrous groups the ovaries were obtained directly by ovariohysterectomy, whereas in the eCG group this was achieved 4 days after the dose injection. In all groups, each cat corresponded to an individual biological replicate, whereby the COC recovered from each cat were classified and processed separately for the experiments of gene expression analysis and in vitro maturation (IVM). The COC were collected by slicing of the ovaries and classified morphologically as grade I (excellent), grade II (good), grade III (fair), and grade IV (poor) quality. For gene expression analysis, pools of 8 to 10 grade I and II immature COC were made, resulting in 7 pools for each group. Quantitative RT-PCR was performed for gonadotrophin receptor genes (FSHR and LHCGR), FSH-induced genes (EGFR, EGR1, ESR2, and PTGS2), and genes related to oocyte competence (GDF9, BMP15, and GATM). The gene SDHA was used as the internal control. The total remaining proportion of grade I and II COC were used for IVM, and maturation rate was measured by visualisation of the first polar body. Statistical analysis was performed using the Kruskal–Wallis test. No differences were found in the total number of COC (mean ± standard deviation) recovered per cat among the oestrous (56.8 ± 20.5), anestrous (80.2 ± 35.2), and eCG groups (96.5 ± 62.0; P &gt; 0.05). With respect to morphological quality of COC, the eCG group had a higher proportion of grade I COC (33.6 ± 11.0%) than the oestrous and anestrous groups (16.5 ± 8.7 and 8.9 ± 6.0%, respectively; P &lt; 0.05). However, the anestrous group had a higher proportion of grade II COC (26.8 ± 6.4%) than the eCG group (21.1 ± 6.6%; P &lt; 0.05). On the other hand, the eCG group had a lower proportion of grade III and IV COC (45.3 ± 12.8%) than the anestrous group (64.3 ± 9.1%; P &lt; 0.05), without differences from the oestrous group (57.1 ± 12.0%; P &gt; 0.05). Concerning to gene expression analysis, COC from the eCG group had a higher relative expression of FSHR, LHCGR, and EGFR than COC from the oestrous and anestrous group (P &lt; 0.05). Furthermore, the COC from the eCG group had a higher relative expression of EGR1 than COC from the anestrous group and a higher expression of ESR2 than COC from the oestrous group (P &lt; 0.05). However, COC from the eCG group had a lower relative expression of GATM and PTGS2 than COC from the oestrous group and a lower expression of GDF9 and BMP15 than COC from the anestrous group (P &lt; 0.05). Although a higher number of oocytes with a first polar body could be seen in the eCG group after IVM, no significant differences in the maturation rate were found among the eCG (55.3 ± 13.2), oestrous (43.7 ± 11.1), and anestrous groups (47.9 ± 13.6; P &gt; 0.05). In conclusion, the treatment with eCG improved the morphological quality of COC recovered from anestrous cats, which agrees with an increased relative expression of FSHR, LHCGR, EGFR, EGR1, and ESR2 and might be related to an enhanced competence of COC.

A three-dimensional alginate system for in vitro culture of cumulus-denuded feline oocytes.

In the case of high valuable individuals with very precious genetic material, widening the genetic pool including gametes with poor morphological characteristics, as cumulus-denuded oocytes (CDOs), could be an option. To improve the in vitro culture of low-competence feline CDOs, an enriched three-dimensional (3D) system in association with competent cumulus-oocyte complexes (COCs) was developed. For this purpose, domestic cat CDOs were cultured with or without companion COCs in the 3D barium alginate microcapsules. The overall viability and the meiotic progression of feline CDOs cocultured with COCs or cultured separately in 3D or in 2D (traditional microdrops) system were compared. The 3D system was able to support viability and meiotic resumption of the feline oocytes, as well as the 2D microdrops. In 3D microcapsules, the presence of COCs resulted in a higher viability of CDOs (91.1%, p<.05), than that obtained without COCs or in 2D microdrops (71.2% and 67.3%, respectively), but the percentages of meiotic resumption were similar of those of CDOs cultured separately (55.4% vs. 40.4%, p>.05). It is notable that the presence of CDOs seemed to enhance the meiotic progression of the associated COCs. In conclusion, the 3D barium alginate microcapsules are a suitable system for feline oocytes in vitro culture, but more specific enriched conditions should be developed to improve the CDOs full competence in vitro.

Identification of molecular markers for oocyte competence in bovine cumulus cells.

Cumulus cells (CCs) have an important role during oocyte growth, competence acquisition, maturation, ovulation and fertilization. In an attempt to isolate potential biomarkers for bovine invitro fertilization, we identified genes differentially expressed in bovine CCs from oocytes with different competence statuses, through microarray analysis. The model of follicle size, in which competent cumulus-oocyte complexes (COCs) were recovered from bigger follicles (≥8.0mm in diameter) and less competent ones from smaller follicles (1-3mm), was used. We identified 4178 genes that were differentially expressed (P<0.05) in the two categories of CCs. The list was further enriched, through the use of a 2.5-fold change in gene expression as a cutoff value, to include 143 up-regulated and 80 down-regulated genes in CCs of competent COCs compared to incompetent COCs. These genes were screened according to their cellular roles, most of which were related to cell cycle, DNA repair, energy metabolism, metabolism of amino acids, cell signaling, meiosis, ovulation and inflammation. Three candidate genes up-regulated (FGF11, IGFBP4, SPRY1) and three down-regulated (ARHGAP22, COL18A1 and GPC4) in CCs from COCs of big follicles (≥8.1mm) were selected for qPCR analysis. The selected genes showed the same expression patterns by qPCR and microarray analysis. These genes may be potential genetic markers that predict oocyte competence in invitro fertilization routines.

Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenetic protein 15 and follicle stimulating hormone.

The developmental competence of cumulus oocyte complexes (COCs) can be increased during in vitro oocyte maturation with the addition of exogenous oocyte-secreted factors, such as bone morphogenetic protein 15 (BMP15), in combination with hormones. FSH and BMP15, for example, induce different metabolic profiles within COCs-namely, FSH increases glycolysis while BMP15 stimulates FAD and NAD(P)H accumulation within oocytes, without changing the redox ratio. The aim of this study was to investigate if this BMP15-induced NAD(P)H increase was due to de novo NADPH production. Cattle COCs were cultured with FSH and/or recombinant human BMP15, resulting in a significant decrease in glucose-6-phosphate dehydrogenase activity (P < 0.05). Inhibition of isocitrate dehydrogenase (IDH) during this process decreased NAD(P)H intensity threefold in BMP15-treated oocytes, suggesting that BMP15 stimulates IDH and NADPH production via the tricarboxylic acid cycle. As NADPH is a reducing agent, reduced glutathione (GSH), H2O2, and mitochondrial activity were also measured to assess the general redox status of the oocyte. FSH alone decreased GSH levels whereas the combination of BMP15 and FSH sustained higher levels. Expression of genes encoding glutathione-reducing enzymes were also lower in oocytes cultured in the presence of FSH alone. BMP15 supplementation further promoted mitochondrial localization patterns that are consistent with enhanced developmental competence. Metabolomics revealed significant consumption of glutamine and production of alanine by COCs matured with both FSH and BMP15 compared to the control (P < 0.05). Hence, BMP15 supplementation differentially modulates reductive metabolism and mitochondrial localization within the oocyte. In comparison, FSH-stimulation alone decreases the oocytes' ability to regulate cellular stress, and therefore utilizes other mechanisms to improve developmental competence.

In vitro fertilization and development of cumulus oocytes complexes collected by ultrasound-guided follicle aspiration in superstimulated llamas

The objective was to evaluate the developmental competence of cumulus-oocyte complexes (COC) collected by follicular aspiration in llamas treated with FSH or eCG. Llamas were assigned randomly to two groups (n = 16 per group) and treated, at the time of ovarian follicular wave emergence, with either: 1) 25 mg of FSH im, twice daily for 4 d; or 2) 1000 IU of eCG as a single i.m. dose. The start of gonadotropin treatment was considered Day 0. Both groups were given 5 mg of Armour Standard LH im on Day 6, and COC were collected by follicle aspiration on Day 7. Expanded COC collected from FSH- (n = 157) and eCG-treated llamas (n = 151) were fertilized in vitro using epididymal sperm, and presumptive zygotes were in vitro cultured in SOF medium for 8 d. The FSH and eCG treatment groups did not differ with respect to: the number of follicles ≥7 mm (16.0 ± 2.7 vs 14.0 ± 1.9, respectively; P = 0.5); the number of COC collected (11.5 ± 1.9 vs 9.7 ± 1.2; P = 0.4); the number of expanded COC (9.8 ± 1.4 vs 9.4 ± 1.2; P = 0.8); or the percentage of presumptive zygotes which developed into 2 to 8 cell stage embryos (65.3 vs 63.1), morulas (46.2 vs 42.5), or blastocysts (23.1 vs 20.5; P > 0.05). In conclusion, FSH and eCG treatments were equally effective for recovery of a high number of expanded COC which were used directly for in vitro fertilization. Furthermore, rate of embryo development was not significantly affected by the gonadotropin treatment used.

Zona pellucida glycoprotein 3 (pZP3) and integrin β2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test

Brilliant cresyl blues (BCB) staining test is a useful tool in assessing the competence of cumulus-oocyte-complexes (COCs) in several mammalian species. It is mostly used to select gametes after they are recovered from the ovary or before and after IVM to isolate those oocytes that reach developmental competency. However, there is evidence that double exposure to BCB test may lead to impaired fertilization or even have a toxic effect on cells. The aim of the present study was to investigate the expression pattern of sperm-egg interaction molecules in oocytes after single and double exposure to BCB test. Follicles were dissected from porcine ovaries after slaughter and aspirated COCs were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The BCB test was applied to COCs before and after IVM. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH; BCB test), real-time quantitative PCR reaction methods, western blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoprotein 3 (pZP3), and integrin beta 2 (ITGB2), as well as the levels of pZP3 and ITGB2 proteins. In the control group, assessment of the expression of the investigated genes was performed before and after IVM without BCB test. We observed a significantly higher level of pZP3 mRNA in oocytes after single exposure to BCB test compared to control before and after IVM (P < 0.001), and to double staining (P < 0.05). The level of ITGB2 mRNA was also increased in gametes after single exposure to BCB test as compared to control before and after IVM (P < 0.001, P < 0.01, respectively), and double staining (P < 0.05). Western blot analysis demonstrated a higher level of pZP3 protein in oocytes after single staining with BCB as compared to control both before and after IVM (P < 0.001, P < 0.05, respectively) and double staining (P < 0.05). Confocal microscopic observations have revealed the same pattern of increased level of pZP3 and ITGB2 expression after single exposure to BCB test. In both cases we detected specific cytoplasmic localization of both proteins. The ITGB2 protein has zona pellucida and membrane localization in control oocytes before IVM. After IVM and after single exposure to BCB, ITGB2 was also strongly detected in the cytoplasm. In both cases, after double exposure to BCB both proteins were detected only partially in the cytoplasm. Our results suggest that (i) single exposure to BCB increased the expression of sperm-oocyte interaction genes, (ii) double exposure to BCB leads to only partial expression of pZP3 and ITGB2 in oocyte cytoplasm, (iii) the BCB staining test itself may be a cause of specific pZP3 translocation from the zona pellucida to the cytoplasm, and that (iv) in vitro maturation of oocytes may increase ITGB2 expression and translocation from the zona pellucida to the cytoplasm.

315 USE OF PERIFOLLICULAR BLOOD FLOW TO PREDICT THE DEVELOPMENTAL COMPETENCE OF BOVINE CUMULUS-OOCYTE COMPLEXES COLLECTED DURING REPEATED OVUM PICKUP SESSIONS ONCE OR TWICE WEEKLY

On average, only 20% of the cumulus–oocyte complexes (COC) develop to the blastocyst stage (Merton et al. 2003 Theriogenology 59, 651–674). An increase in the blood supply to individual follicles appears to be associated with follicular growth rates, whereas a reduction seems to be closely related to follicular atresia (Acosta et al. 2003 Reproduction 125, 759–767). The purpose of this study was to determine whether qualitative perifollicular blood flow changes can be used to predict the developmental competence of COC collected during repeated ovum pickup (OPU) sessions once or twice weekly. Lactating Holstein cows (n = 20) were used as oocyte donors. After dominant follicle removal, OPU was performed twice (group 1, for 3 weeks) or once (group 2, for six weeks) weekly employing a 7.5-MHz transducer (GE 8C-RS) of an ultrasound scanner (GE Logiq Book). Follicle size and Doppler characteristics were recorded by transvaginal ultrasonography just before COC collection using color flow imaging. Owing for technical limitations for measurement of blood flow in small individual follicles, only the presence or absence of blood flow was assessed for each follicle. When a clearly visible blue or red spot (blood flow) was detected in the follicle wall, it was considered as a follicle with detectable blood flow. Follicles with or without detectable blood flow from each individual cow were aspirated separately. After morphological classification of COC, standard protocols for IVP were used for embryo production (Wrenzycki et al. 2001 Biol. Reprod. 65, 323–331). Cleavage and blastocyst rates were recorded at Day 3 and Day 8, respectively. In total, 464 (246 with and 218 without detectable blood flow) and 243 (125 with and 118 without detectable blood flow) follicles ≥3 mm were aspirated in group 1 and group 2, respectively. Morphology of the COC was similar in all groups. Developmental rates for COC stemming from follicles with or without detectable blood flow in group 1 did not show differences for cleavage rates, 54.0% (34/63) and 56.7% (45/81), and for blastocyst rates, 25.4% (16/63) and 22.2% (18/83), respectively. In group 2, the cleavage rates were also similar for COC originating from follicles with and without detectable blood flow, 54.3% (25/46) and 51.5% (34/66). However, developmental rates up to the blastocyst stage did show a significant difference, 23.9% (11/46) and 15.2% (10/66) for COC aspirated from follicles with or without detectable blood flow (P ≤ 0.05). These results show that using COC originating from follicles with detectable perifollicular blood flow collected once weekly may have a higher developmental competence compared to those from follicle without detectable blood flow. Within the detection limits of this study, differences in perifollicular blood flow during repeated OPU sessions once weekly were predictive of oocyte competence. Ruthe Research Farm, Germany, for providing the animals; Masterrind GmbH, Germany, for donation of the semen; and the HW Schaumann Stiftung for financial support.

193 DIFFERENTIAL GENE EXPRESSION IN CUMULUS CELLS OF DEVELOPMENTALLY COMPETENT V. CHALLENGED BOVINE OOCYTES

It is well established that exposure of cumulus–oocyte complexes (COC) to heat stress during the first 12 h of maturation reduces blastocyst development by 42 to 65%. Previous research supports the notion that some of the effects of heat stress on oocyte competence may be cumulus-mediated. To determine the extent to which this may occur, COC were matured at 38.5°C for 24 h (control) or 41°C for the first 12 h of maturation followed by 38.5°C for remaining 12 h (heat stress). A subset of COC underwent IVF with Percoll-prepared sperm and then was cultured in KSOM containing 0.5% BSA to assess developmental competence. Remaining oocytes were denuded. Cumulus cells, kept separate by treatment, were stored in lysis buffer at –80°C until RNA extraction. Total RNA from cumulus was amplified prior to hybridization to bovine Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA, USA; n = 8 pools per treatment collected on 8 different occasions; n = 16 chips). Following pre-processing using the MAS5.0 algorithm, microarray data were subjected to linear modeling and empirical Bayes analyses (Bioconductor, Limma package). False discovery rate was controlled using the Benjamini and Hochberg method, and differentially expressed genes were selected by an adjusted P-value (P &lt; 0.05). Functional annotation of selected genes was performed using NetAffx (Affymetrix Inc.) and Database for Annotation, Visualization and Integrated Discovery (DAVID; NIAID, NIH, Bethesda, MD, USA). Heat stress of COC reduced blastocyst development (27.2 v. 16.1% for control v. heat stress, respectively; SEM = 1.6; P &lt; 0.002). Approximately 66 and 65% of 24 000 possible genes were called present (i.e. expressed) in RNA from cumulus of competent (control) v. challenged (heat-stressed) oocytes, respectively. In cumulus from developmentally challenged COC, increased abundance of 42 genes (36 currently annotated) was noted. Use of DAVID demonstrated enrichment of genes important for electron transport and energy generation (NOS2A, MAOB, CYP11A1, HSD11B1L, LTB4DH). Further examination of gene ontology identified genes associated with mitochondrial function (SLC25A10, MAOB, CYP11A1), cell signaling (similar to JAK-3, FSHR, CYP11A1, WNT2B), cytoskeleton (ACTA1), antioxidant activity (GSTA1), and extracellular region (FMOD). In contrast, cumulus from developmentally competent COC had increased expression of 22 genes (20 currently annotated), of which 15% were related to protein binding (CAV1, MMP9, TGFB2) according to DAVID. Further analysis using gene ontology revealed genes associated with extracellular matrix formation (MMP9, MMP19, PCOLCE2) and neural tissue (METRNL). In summary, alterations in cumulus gene expression were associated with differences in developmental competence of oocytes. Additional research is necessary to examine the extent to which identified genes account for functional differences in oocyte competence. This research was supported in part by National Research Initiative Competitive Grant no. 2004-35203-14772 from the USDA Cooperative State Research, Education, and Extension Service.

313 EVALUATION OF COLOR DOPPLER ULTRASONOGRAPHY OF THE PERIFOLLICULAR BLOOD FLOW TO PREDICT THE DEVELOPMENTAL COMPETENCE OF BOVINE CUMULUS - OOCYTE COMPLEXES COLLECTED DURING REPEATED OVUM PICK-UP SESSIONS

The population of cumulus–oocyte complexes (COCs) used in OPU–IVP (ovum pick-up combined with in vitro production of embryos) is largely homogeneous due to repeated sessions resulting in the elimination of dominant and atretic follicles, especially when a 3–4 day interval is used. However, on average only 20% of the COCs develop to the blastocyst stage (Merton et al. 2003 Theriogenology 59, 651–674). Different blood flow changes within the follicle wall influence the fate of the follicles, and detectable blood flow and vasculature are associated with follicle viability. Furthermore, blood flow in follicles may be involved in not only selection of the dominant follicle but also early follicular development including follicular recruitment (Miyamoto et al. 2006 J. Reprod. Dev. 52, 153–160). However, no data are available regarding the quality of the COCs collected from follicles with or without blood flow. The purpose of this study was to determine whether qualitative perifollicular blood flow changes can be used to predict the developmental competence of COCs collected during repeated OPU sessions. Lactating Holstein cows were used as oocyte donors. After dominant follicle removal, OPU was performed twice weekly employing a 7.5-MHz transducer (GE 8C-RS) of an ultrasound scanner (GE Logiq Book). Follicle size and Doppler characteristics (color flow imaging) were recorded by transvaginal ultrasonography just before COC collection. Due to technical limitations for measurement of blood flow in small individual follicles, only the presence or absence of blood flow was assessed for each follicle. When a clearly visible blue or red spot (blood flow) was detected in the follicle wall, it was considered as a follicle with detectable blood flow. Follicles with or without detectable blood flow from each individual cow were aspirated separately. After morphological classification of COCs, standard protocols for IVP were used for embryo production. Cleavage and blastocyst rates were recorded at Day 3 and Day 8, respectively. In total, 464 (246 with and 218 without detectable blood flow) follicles e3 mm were aspirated. The percentage of follicles with detectable blood flow increased depending on follicle size (3 mm: 28.7, 4 mm: 48.4, 5 mm: 50.5, 6 mm: 62.5, 7 mm: 64.8, 8 mm: 71.4, and 9 mm: 76.9). Cleavage rates for COCs stemming from follicles with or without detectable blood flow did not show differences, 45.5% (35/77) and 56.7% (38/67), respectively. The rates of blastocyst formation were also similar in COCs originating from follicles with and without detectable blood flow, 16.9% (13/77) and 14.9% (10/67), respectively. These results show that perifollicular blood flow increases during early follicular growth. Within the detection limits of this study, differences in perifollicular blood flow during repeated OPU sessions twice weekly did not seem to be predictive of oocyte competence. We acknowledge the Ruthe Research Farm, Germany, for providing the animals, and Masterrind GmbH, Germany, for donating the semen.

Ultrasound image characteristics of ovarian follicles in relation to oocyte competence and follicular status in cattle

Assessment of the quality of the female gamete has become paramount for in vitro procedures. There is a need to identify reliable indicators of oocyte competence and develop a simple, non-invasive method to assess competence. The aim of this study was to investigate the relationships among ultrasonographic attributes of a follicle, its stage of development and the competence of the oocyte that it contains. We tested the hypotheses that follicular echotexture characteristics are related to: (1) the phase of development of the follicle, (2) the presence of the corpus luteum (CL) and/or the dominant follicle in the ovary, and (3) developmental competence of cumulus oocyte complexes (COC) from the same ovary. Crossbred beef cows ( n=143), age 4–14 years, were given a luteolytic dose of dinoprost to cause ovulation. Ultrasound-guided ablation of all follicles ≥4 mm was done 8 days later to induce new follicular wave emergence during a luteal phase. Ultrasonographic images of dominant follicles and the three largest subordinate follicles ( n=402 follicles; 84 cows) were acquired on Days 2, 3, 5 or 7 of the follicular wave (Day 0: wave emergence), i.e. growing, early-static, late static, and regressing phases of subordinate follicle development, respectively. From a subset of these animals ( n=33), ovaries were collected within 30 min of slaughter and COC from subordinate follicles ≥3 mm underwent in vitro maturation, fertilization and culture to the blastocyst stage. Image analysis revealed differences in echotexture between dominant and subordinate follicles among Days 2–7 of the follicular wave. Images of dominant and subordinate follicles at Day 7 of the wave displayed consistently lower grey-scale values ( P<0.05) in the peripheral antrum, follicular wall and perifollicular stroma than all other days. Follicle images displayed a consistent pattern of variation in echotexture among follicular phases. Data did not support the hypothesis of a local effect of the CL or dominant follicle on follicular echotexture. Echotexture values of the perifollicular stroma were lower in ovaries that did not produce embryos compared to ovaries that produced embryos. Our results showed that the changes in follicular image attributes are consistent with changes in follicular status. The sensitivity of the technique is not yet sufficient for use in a diagnostic setting, but results provide rationale for further development of image analysis as a tool for evaluating oocyte competence in situ.

Bovine cumulus-oocyte-complex-quality is reflected in sensitivity for α-amanitin, oocyte-diameter and developmental capacity

The aim of the present study was to find more parameters to define developmental competence of cumulus-oocyte-complexes (COCs). Bovine COCs were divided into five groups based on their morphology. In order of increasing level of atresia: COC-A had a bright, compact cumulus investment; COC-B1 also had a compact cumulus investment, but was darker than COC-A; the color of COC-B2 was comparable with COC-B1 but the corona radiata appeared to dissociate from the rest of the cumulus investment; the cumulus of COC-B3 was almost black and the corona radiata was almost completely dissociated from the rest of the cumulus investment; COC-C had a strongly expanded cumulus investment with dark spots of degenerated cells. An increasing level of atresia was accompanied by: (1) an increasing zona pellucida diameter (147.6, 150.8, 151.0, 154.3 and 155.1 μm, respectively, for COC-A, COC-B1, COC-B2, COC-B3 and COC-C); (2) an increasing oocyte diameter except for COC-C (120.9, 122.8, 122.8, 123.9 and 118.4 μm, respectively); (3) an increasing developmental competence except for COC-C (13.9, 14.7, 17.4, 19.1 and 11.5%, respectively, development to morula and blastocyst after in vitro embryo production (IVP)) and (4) by a increasing percentage of oocytes exhibiting germinal vesicle breakdown (GVBD) after 24 h culture with α-amanitin and FSH, except for COC-A (34.4, 68.6, 51.0, 22.0 and 4.8%, respectively, oocytes arrested in GV). In general, embryo-quality, expressed in nuclei-count, was significantly affected by COC-quality ( P<0.05) with B3>B1>B2>A>C. However, if the developmental stages were compared separately, this effect was less evident. It was found that, in absence of FSH, α-amanitin was unable to inhibit GVBD and that the success of GVBD inhibition was positively correlated to the amount of cumulus cells surrounding the oocyte. COCs that had been exposed to α-amanitin and FSH during maturation retained the ability to cleave after IVF, but were unable to develop any further.

Effect of ovarian phase and follicle quality on morphology and developmental capacity of the bovine cumulus-oocyte complex.

In order to study the effect of the follicular environment on the quality and developmental competence of cumulus-oocyte complexes (COC), COC were collected from nonatretic (NA), light atretic (LA), atretic (A), and heavily atretic (HA) follicles. Cumulus-oocyte complexes were also collected from early-luteal phase ovaries (EL), from late-luteal phase ovaries (LL), from follicular phase ovaries (F), and from ovaries from non-cyclic animals (NC). Each COC was assigned to one of three quality groups based on the appearance of the cumulus investment: 1) COC-A: compact and bright, 2) COC-B: less compact and dark, and 3) COC-C: strongly expanded cumulus with dark spots. There was a high correlation between follicle quality and the distribution of the COC over the three COC qualities. The COC-A were mainly but not exclusively derived from NA follicles, COC-B were mainly derived from all classes of atretic follicles, and COC-C nearly exclusively originated from HA follicles. The developmental capacity of COC-A and COC-B, which was measured by in vitro embryo production, was consistent over the follicle qualities, except for COC-B obtained from HA follicles. They showed lower development (10.6%, P < .05) compared with COC-B from the other follicle qualities (18.9, 18.7, and 19.8%, respectively, for COC-B from NA, A, and LA follicles). The COC-B from atretic follicles produced more blastocysts (19.8%) than COC-A (12.7%, P < .05). The overall percentage of produced embryos per follicle class seemed to increase with increasing signs of atresia, except if the COC were derived from HA follicles. This increased percentage of embryos was, however, not due to a better quality of COC, but to a higher percentage of COC-B coming from these follicles. The stage of the cycle had no effect on the distribution of the COC over the three COC qualities or on the developmental capacity of COC-A or COC-B, except for COC-A from EL ovaries, which produced more (P < .05) blastocysts than COC-A from the other luteal phases (12.5% vs approximately 8%). A follow-up study was performed trying to elucidate why COC-B possess a higher developmental potency than COC-A. The answer was sought in the oocyte maturation. At several time points during maturation, oocytes were evaluated for their nuclear stage. At all time points COC-B seemed to be a few hours ahead of COC-A, and after 24 h of maturation more COC-B had reached the metaphase-2 stage. This might mean that COC-A need a longer maturation period than COC-B.