Good Quality Oocytes Research Articles

Dynamic Morphology of the Human Ooplasm and Subsequent Embryonic Development Analyzed by Time-Lapse Cinematography

It has been reported that oocyte dysmorphisms, such as the appearance of a cytoplasmic vacuole, granularity, and deformed shape, are strongly associated with poor embryonic development. In this study, we analyzed the dynamics of cytoplasmic dysmorphisms of oocytes and zygotes using time-lapse cinematography (TLC) that we had originally developed. Mature oocytes (n=1,601) were classified into two groups: good quality oocytes (GQO), which indicated oocytes without any defects; and poor quality oocytes (PQO), oocytes with one or more defect. Of the 1601 oocytes, 74.1% were GQO and the remaining were PQO, which were evaluated by three senior embryologists. The rate of unfertilized oocytes was significantly lower in PQO. GQO also showed a higher development rate to good quality embryos than PQO. The time required for embryonic development in GQO was significantly less than that in PQO (P<0.01). In the zygotes (n=144), 11.4% showed vacuole-like phenomenon (VLP) in the cytoplasm. The VLP appeared in the peripheral cytoplasm after extrusion of the second polar body and moved synchronously as the mPN and fPN migrated toward the central cytoplasm. The VLP size gradually increased and remained after the first cleavage. The rate of multinucleated blastomere (MNB) formation was significantly higher in embryos with VLP than in those without VLP. The appearance of VLP was closely associated with aberrant embryonic development, particularly MNB formation. Our results suggest that the dynamic morphology of the cytoplasm in oocytes and zygotes influences subsequent embryonic development and viability, suggesting that the assessment of oocyte morphology could be a useful parameter in the prediction of embryonic development.

Different stimulation protocols for oocyte cryropreservation in oncological patients: a retrospective analysis of single university centre

Objective: To analyze the results obtain in cancer patients who receive the same controlled ovarian stimulation protocol, started in two different moments of the menstrual cycle, follicular or luteal phase. The stimulation is performed before cancer treatment in order to preserve fertility through oocytes cryopreservation.Study design: The study is a retrospective analysis about 25 cancer patients at our centre, Department of Reproductive Medicine of University of Pisa, in order to preserve their fertility before cancer treatment. Patients are divided into two groups depending on the menstrual cycle phase, follicular or luteal phase, at the moment of first examination. Standard stimulation protocol with gonadotropins is administered in the follicular group, whereas in the second group we use GnRH (gonadotropin-releasing hormone) antagonist before gonadotropins administration in order to have a rapid luteolysis. The outcome measures are the number of days needed before starting procedure, duration of stimulation, cumulative dosage of gonadotropins number of oocyte retrieved and percentage of mature oocytes.Results: Any difference showed between two groups based on days of stimulation, total amount of gonadotropins administered and the number of good mature quality oocytes was retrieved. The real difference is the number of days needed to start the procedure, lesser in the luteal group.Conclusions: This study suggests that oocytes can be obtained before cancer treatment, irrespective of menstrual cycle phase without compromising the efficacy of procedure. Moreover, starting ovarian stimulation anytime during menstrual cycle allows the patients to not postpone the beginning of cancer treatment. Different stimulation protocols, according to different kinds of disease, are available in order to obtain the maximum results without any complication for patients.

A microarray-based analysis of oocyte quality in the European clam Ruditapes decussatus

Abstract A microarray-based analysis was performed with the objective of describing genomic features of oocytes and looking for potential markers of oocyte quality in the economically important European clam, Ruditapes decussatus . Oocytes of 25 females from Ria de Aveiro, western coast of Portugal (40°42′N; 08°40′W) were selected for this study and oocyte quality was estimated by success of D-larval rate under controlled conditions, which appeared to vary from 0 to 95%. By genome-wide expression profiling with a DNA microarray, 526 probes appeared differentially expressed between two groups representing the largest and smallest value of D larval rates, named good (represented by a mean D-larval yield of 57 ± 22%) and poor (9 ± 5%) quality oocytes. Enrichment analysis showed “lysosome” (dre04142) as the single pathway represented in the enriched KEGG pathway terms, with 8 genes coding for putative cathepsins. Furthermore, differentially expressed genes involved in oocyte protection (DnaJ (Hsp40), Hsp70, cyclophilin B, PDI), maturation (Cam-PDE1C, PRDM9, G protein), sperm–egg interaction (PDI, G protein) and apoptosis (TNF) were also identified. From these, the apoptosis pathway was supposed to assume an important role in oocyte quality as the mRNA level of caspase 8 appeared also negatively correlated with the D-larval yield. Finally, a G-protein transcript was identified as more abundant in poor quality oocytes, which underlines the importance of release prophase I block and meiotic maturation in the oocytes of this species. This study provided new highly valuable information on genes specifically expressed by mature oocytes of R. decussatus , for further understanding of the mechanisms of early development in this species. Statement of relevance Understand the mechanisms of early development in the European clam R. decussatus .

Biodynamic imaging of live porcine oocytes, zygotes and blastocysts for viability assessment in assisted reproductive technologies.

The success of assisted reproductive technologies relies on accurate assessment of reproductive viability at successive stages of development for oocytes and embryos. The current scoring system used to select good-quality oocytes relies on morphologically observable traits and hence is indirect and subjective. Biodynamic imaging may provide an objective approach to oocyte and embryo assessment by measuring physiologically-relevant dynamics. Biodynamic imaging is a coherence-gated approach to 3D tissue imaging that uses digital holography to perform low-coherence speckle interferometry to capture dynamic light scattering from intracellular motions. The changes in intracellular activity during cumulus oocyte complex maturation, before and after in vitro fertilization, and the subsequent development of the zygote and blastocyst provide a new approach to the assessment of preimplant candidates.

Inhibition of cathepsin B activity reduces apoptosis by preventing cytochrome c release from mitochondria in porcine parthenotes.

Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05) enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria.

Is it time to consider patients suffering from endometriosis-related infertility as “novel candidates” for targeted peri-conceptional D-chiro inositol supplementation? Hypothesis, rationale and some considerations

Dear Editor, We read with great pleasure the interesting data reported by Piomboni et al. concerning several advantages patients affected by polycystic ovarian syndrome (PCOS) and undergoing controlled ovarian hyperstimulation (COH) with pre-treatment supplementation by D-chiro inositol and/or metformin treatment. [1] The demonstrat D-chiro inositol supplementation and metformin treatment were both shown to significantly improve the follicular fluid (FF) milieu by decreasing the oxidative damage (measured as free-SH groups levels) on FF proteins and, as a consequence, significantly increase the number of good quality MII oocytes. Although for many years it was known that the oxidative stress could have a negative effects on FF environment, recent evidence has suggested that good quality oocytes require a correct balance in FF pro-oxidative and anti-oxidative factors, [2] since an excess in pro-oxidative status is associated with poor quality oocytes, low fertilization and implantation rates while a deficit may be responsible for follicular atresia. [3, 4] Interestingly, several studies reported that women suffering from endometriosis frequently experience infertility and poor IVF outcomes (poor qualitative and quantitative ovarian response, low implantation rate), particularly when they have an imbalance in thiol-redox and oxidative status. [5–8] It is known that, frequently, abnormalities in oxidative status balance may reflect alterations in glucose and lipid metabolism. Similarly to PCOS patients, several studies noticed that patients suffering from endometriosis have abnormalities in both glucose and lipid metabolism. For example, McKinnon et al. found that in endometriotic lesions GLUT4 (glucose transporter-4) protein expression was enhanced, probably due to increase glucose availability in the growing lesions. [8] In contrast, Melo et al. report that patients affected by endometriosis frequently show “unfavourable” lipid profile (increase in low-density and reduction in high-density lipoprotein), potentially favouring the oxidative stress (via lipid peroxidation) typically increased in this cohort of women. [9–11] D-chiro inositol is a “dietary supplement” of Inositol, an essential component of the phospholipids that make up cellular membranes, detectable in every cell of the body. The available formulations for oral administration show good bioavailability, safe pharmacologic profiles and beneficial effects in improving spontaneous fertility (or success rate of ARTs) also after short-term treatment, particularly in patients affected by PCOS and anovulation. [11] Basically, oral formulations of D-chiro inositol appear to significantly reduce oxidative status as a result of its antioxidant properties that in turn lead to improvements in glucose metabolism (insulin-sensitizing effect mainly fulfilled by D-chiro inositol phosphoglycan via glycogen synthase activity) and a reduction in circulating levels of LDL and triglycerides in addition to BMI reduction. [12] The properties of D-chiro inositol in reducing the “unbalanced” oxidative status of PCOS patients without significant adverse effects prompts speculation as to whether patients suffering from endometriosis in association with hypo/infertility should be considered as “novel candidates” who may potentially benefit from this supplementation in a pre-conception period (or pre-treatment period when referred to ARTs). Our proposal warrants further investigation. Much effort is being made to improve the fertility of patients suffering from endometriosis, and more and more therapeutic strategies are targeted to oxidative stress reduction. [11, 12] In fact, several Authors [13] recently showed that pre-treatment administration of ultra-long GnRH agonist (1.8 mg s.c. every 28 days for three courses) was effective in balancing intra follicular oxidative stress (checked by 8-hydroxy-2-deoxyguanosine reduction and increase in melatonin levels) and in improving ARTs outcomes of women affected by endometriosis. Others [14] have proposed the use of statins for endometriosis targeted treatment. Despite the beneficial effects, potential adverse effects should not be underestimated. In the future both scientists and clinicians must join forces in seeking novel approaches to improve fertility in patients suffering from endometriosis reducing their “over” oxidative status. It is our opinion that the time has arrived to shift attention towards potential efficacy and safety of D-chiro inositol supplementation to balance the oxidative status of patients suffering from endometriosis, evaluating by appropriate studies the real cost-efficacy of peri-conceptional D-chiro inositol supplementation in this potential “novel candidate” cohort of patients.

307 MATURATION RATE AND GENE EXPRESSION ANALYSIS OF GOAT OOCYTES SELECTED BY FOLLICLE SIZE AND BRILLIANT CRESYL BLUE STAINING

Oocyte quality plays a critical role in determining the success of embryo development. Studies on cattle and goats indicate that oocytes derived from large follicles (LFO) have greater developmental competence than those derived from small follicles (SFO). Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase and is a commonly used noninvasive marker of oocyte competence. Studies in pigs, goats, cows, mice, and dogs showed that the maturation and blastocyst developmental rate of BCB+ oocytes is significantly higher than BCB– oocytes. The aim of this study was to evaluate the maturation rate of goat oocytes selected based on follicular size and BCB staining and compare their relative patterns of gene expression. Maturation rate and gene expression profile were expected to be different in these oocyte groups. Cumulus-oocyte complexes were recovered from abattoir-derived ovaries using a slicing technique. Eleven rounds of oocyte maturation and 4 rounds of BCB staining were carried out. During each replicate, oocytes from large (≥3 mm) and small (&lt;3 mm) follicles were collected separately from the same group of ovaries. Oocyte maturation rates were 54.3 ± 5.4% for LFO (n = 378) and only 33.5 ± 3.7% for SFO (n = 981; P &lt; 0.01). The BCB+ (n = 223) oocytes yielded a significantly higher maturation rate than the BCB– (n = 194) oocytes (56.1 ± 1.8 v. 20.6 ± 3.8%, respectively; P &lt; 0.001). Gene expression analysis was conducted on individual MII oocytes (21 oocytes per group). Specific target amplification was performed on a single oocyte directly by using the CellsDirect One-Step qRT–PCR Kit (Invitrogen). Quantitative real-time PCR was then performed using the 48.48 BioMark platform from Fluidigm. Forty two genes were selected from the following categories: growth factors, transcription factors, metabolism, pluripotency, cell cycle, apoptosis, and oocyte-specific genes. Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. The significance was assigned at P &lt; 0.05. The relative expression of CCNA2, CDK2, CCNB1, POU5F1, SOX2, EGF, FGF2, GDF9, ZP3, BCL2, GJA1, DDR1, PFKFB3, IGF2R, and GRB10 was significantly greater (P &lt; 0.05) in both LFO and BCB+ oocytes compared to SFO and BCB– oocytes, respectively. The proapoptotic gene BAX, the ACSL3 gene involved in fatty acid oxidation, and the growth factor IGF1 were expressed significantly higher (P &lt; 0.05) in SFO compared to LFO. By investigating these differentially expressed transcripts, we will better understand pathways involved in oocyte developmental competence and potentially use them as markers of oocyte quality. We expect that the ability to select oocytes of better quality based on BCB staining will improve outcomes of IVF and SCNT.

257 IN VITRO YAK EMBRYO PRODUCTION THROUGH CONVENTIONAL AND OVUM PICKUP METHODS

Yak is one of the most important economically useful animals for highlanders. The decline in the yak population demands effective measures for conservation and multiplication of elite germplasm. In vitro production of embryos and their cryopreservation and transfer to suitable recipients for production of elite calves may contribute to fulfill the objectives. The work was conducted at the National Research Center on Yak over a period of 3 years. The ovaries of slaughtered animals were used for collecting oocytes through aspiration of follicles followed by slicing of ovaries in the conventional method. Trials were conducted using 7 cyclic parous yaks for ultrasound-guided ovum pickup (OPU) at Nyukmadung farm (2700 m above mean sea level). The technique followed was similar to that in buffaloes with slight modification. Categories of oocytes classified A (2–3 layers of cumulus) and B (at least one layer of cumulus) obtained through the processes were subjected to in vitro maturation using standardized maturation medium (TCM-199 + 10% follicular fluid + sodium pyruvate + l-glutamine + 10% heat inactivated oestrus cow serum + pFSH + 17β oestradiol). The frozen-thawed yak sperm were capacitated using the swing-up method before their incubation with matured oocytes using BO medium. Oocytes matured for 24 h were washed 5 to 6 times with BO medium and then co-incubated with in vitro capacitated spermatozoa (0.1 to 0.25 million) for fertilization (8–10 oocytes per group) in 100-µL droplets of BO medium under mineral oil in 35-mm Petri dishes and placed in a CO2 incubator (5% CO2, 90% RH) at 38.5°C for 16 to 18 h. The presumed zygotes were washed several times in mCR2aa (modified Charles Rosenkrans) washing medium and then cultured in culture medium for 7 days on original beds of granulosa cells. The rates of maturation and fertilization of oocytes collected by conventional and OPU technique were comparable (Table 1). This may be attributed to greater numbers of good quality oocytes recovered in the conventional method. Embryos developed up to the stage of compact morula and blastocysts (24.66% through conventional and 22.73% through OPU) were cryopreserved using the vitrification method for further study. Thirteen embryos were transferred non-surgically to one each of 13 yak recipients; 5 became pregnant and only 1 recipient transferred with a cryopreserved-thawed embryo, developed through OPU, delivered one male calf, leading to the first successful production of an IVF yak calf in the world. The present findings are suggestive of using the OPU technique for in vitro embryo production, though resulting in lower numbers of transferable embryos (Table 1), because availability of ovaries for conventional IVF is a major constraint in yak. Table 1.Comparative in vitro yak embryo production rate with recovery of oocytes by conventional or ovum pickup (OPU) method

Effect of body condition and season on yield and quality ofin vitroproduced bovine embryos

The aim of our study was to examine the effects of cow's body condition score (BCS; scale 1-5) and season on the quality of bovine in vitro produced embryos. The proportion of good quality oocytes (Q1 and Q2) was higher (P < 0.05) in the BCS 2 (57.60%) and BCS 3 (60.90%) groups compared with the BCS 1 (43.60%) group. There were no statistical differences in embryo cleavage and blastocyst rate among the BCS groups. The highest total cell number (TCN, DAPI stain) of blastocysts (P < 0.05), recorded in BCS 1 (122.27 ± 6.90) in comparison with BCS 2 (101.8 ± 3.60) or BCS 3 (105.44 ± 3.70) groups, was related to higher dead cell (DCI, TUNEL) index in this group (7.07%) when compared with BCS 2 (6.54%) or BCS 3 (6.06%), respectively. The yield of good quality oocytes during spring was lower (P < 0.05) compared with the summer season. There were significant differences (P < 0.05) in maturation and cleavage rates between autumn and summer (73.42%, 76.2% vs. 85.0%, 41.8%, respectively). The highest (P < 0.01) blastocyst rate was noted during spring and summer months. Significant difference (P < 0.05) in the TCN among spring (99.38 ± 3.90), autumn (110.1 ± 4.58) or summer (108.96 ± 3.52) was observed. The highest proportion of embryos with the best (grade I) actin cytoskeleton (phalloidin-TRITC) quality was noted during the summer months. Our results indicate that body condition affects the initial quality of oocytes, but does not affect embryo cleavage, blastocyst rate and actin quality. This finding may suggest that development in vitro can mask the influence of BCS. The season affects yield and quality of blastocysts in the way that the autumn period is more favorable for embryo development.

Quality assessment of yak oocytes for in vitro production of yak embryos

Yak oocytes were isolated and morphologically classified into 3 categories, i.e. good, fair and poor quality oocytes according to the compactness of the cumulus cells, and were assigned for in vitro maturation, fertilization and embryonic development. Oocytes were retrieved by both aspiration and post-aspiration slicing technique, and the overall recovery rates of good, fair and poor quality oocytes were estimated at 46.55, 30.6 and 22.84% respectively. Maturation rates (M II) in good quality oocytes were significantly higher as compared to that in fair and poor quality oocytes. Total and normal fertilization rates were also higher in good quality oocytes than in fair and poor oocytes. Good quality oocytes led to embryonic development with significant numbers of transferrable embryos (i.e. morula and blastocyst); 60% of morula was further developed to blastocyst. Thus, it is concluded that the selection of good oocytes according to cumulus cells compactness is an important criterion for in vitro maturation, fertilization, and embryonic development in yaks.

Influence of corpus luteum and ovarian volume on the number and quality of bovine oocytes.

In order to evaluate whether ovarian volume, presence and diameter of the corpus luteum (CL) have effects on the number and quality of bovine recovered oocytes, 110 ovaries were obtained from the slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grades I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P < 0.05). Ovaries with CL showed greater numbers of good quality oocytes than ovaries without CL (P < 0.05). Further, presence of CL and its diameter positively influenced the probability of recovering good quality oocytes (P < 0.05). In conclusion, ovarian volume is not a good parameter itself to predict important ovarian characteristics; moreover, analysis of CL, its presence and diameter, may be a good tool to improve efficiency on in vitro embryo production programs.

Protein modification as oxidative stress marker in follicular fluid from women with polycystic ovary syndrome: the effect of inositol and metformin.

The purpose of this study was to evaluate the oxidative stress status (OS) of follicular fluid (FF) and the oocyte quality in women with polycystic ovary syndrome (PCOS) undergoing different ovarian stimulation protocols. FF samples were collected after gonadotropin administration in association or not with metformin or D-chiro-inositol (DCI). OS status was then evaluated by checking the follicular fluid protein oxidation profile after specific labeling of aminoacidic free-SH groups, and two-dimensional electrophoresis followed by qualitative and semiquantitative analysis. Oocyte quality was assessed by international morphological criteria. Our data indicated that both treatments, even if to different extent, recovered a significantly high level of free-SH groups in FF proteins of PCOS women clearly indicating a decrease of OS level with respect to that found in FF samples from gonadotropins alone treated women. A higher number of good quality MII oocytes was also observed in DCI (P < 0.05) or metformin (P < 0.05) study groups in comparison to untreated control group. A natural supplement and a drug both showed a statistically significant positive effect on follicular milieu by decreasing the oxidative damage on FF proteins, as well as in recovering good quality oocytes.

Influence of Dominant Follicle and Corpus luteum on Recovery of Good Quality Oocytes for In vitro Embryo Production in Cattle

The quality of oocytes is more critical for the success of bovine in vitro embryo production (IVP) than the conditions of in vitro culture of these structures. Despite knowledge about bovine follicular development, especially gonadotropin-dependent phase, be considerably wide, knowledge about effects of the estrous cycle phase, follicular or luteal, on recovery Mini -review Article

Hormonas esteroideas foliculares y su relación con los resultados globales del ciclo de estimulación ovárica

SUMMARY Objective: To relate the levels of follicular steroid hormones with the ovarian stimulation cycle and its overall results. Method: It was included patients ≤38 years old with sterility of male, tubaric or unknown origin who underwent a long protocol with GnRH agonists and rFSH. Samples were obtained from the first and second follicular aspiration of each ovary. A chemiluminescent immunoassay of estradiol, progesterone, testosterone and DHEAS was performed. Results: Figures of follicular DHEAS decreased as the days of treatment with GnRH agonists increased (p=0.0003) and levels of follicular testosterone and DHEAS increased along with the days of treatment with rFSH (p=0.03, p=0.03). In regard to the outcomes of the overall cycle it was found a negative correlation between follicular testosterone levels and the number of punctured complexes (r= -0.360; p=0.002) and between follicular testosterone and the number of D quality embryos (r= -0.233; p=0.047). The number of mature oocytes was lower in patients with higher levels of follicular testosterone (p=0.008). Follicular progesterone was higher in good quality oocytes as compared to those of no remarkable quality (p=0.006) and very poor quality (p=0.04). Conclusions: High levels of follicular testosterone were correlated with a fewer number of punctured complexes, mature oocytes and D quality embryos. Good oocyte quality was associated with higher follicular progesterone levels.

Gonadotrophins: The future.

The role of the IVF clinician is to make the ART treatment safe, patient-friendly, cost effective and at the same time offer good and high quality treatment. IVF protocols are a burden for women and are one of the potential reasons why women don’t return for subsequent cycles. Frequent injections may increase stress and also result in high error rates. Simple short treatment regimen with optimal recovery of good quality oocytes results in development of good quality embryos followed by SET in treatment and cryopreservation cycles are a less burden and result in related lesser discontinuation, side effects, treatment cycles in time and are more cost-effective.Development of FSH analogues with longer terminal t1/2 and slower absorption to peak serum levels will increase the efficiency, decrease the side effects and also is easy to administer. This makes it convenient for the patients increasing the compliance. A certain minimum LH concentration is necessary for adequate thecal cell function and subsequent oestradiol synthesis in the granulosa cells. Adjuvant r-HLH gives clinician's precise control over the dose of LH bioactivity administered to target the therapeutic window. New parenteral, transdermal, inhaled and oral fertility drugs and regimens are currently under research and development with the objective to further simplify treatment for ART.

Laparoscopic ovum pick up (LOPU) in goats: from hormonal treatment to oocyte possible destinations

Whether we are collecting oocytes from valuable donors as part of a program of in vitro embryo production (IVP) and/or to promote advances in emerging biotechnologies as cloning and transgenesis, the success rate depends on the development of methodological aspects of recovering oocytes. To succeed, sufficient number of good quality oocytes is the prerequisite for various reproductive techniques and laparoscopic ovum pick up (LOPU) is the recommended technique for obtaining them from live goats. However, the variability of the quantity and quality of the oocytes collected still limits the large-scale use of this technology. Under the current conditions, too large variability is reported with oocyte recovery rates ranging from 40 to 90%, and the number of harvested oocytes per female between 4 and 14, in different laboratories. This variability can depend on either intrinsic characteristic of the donors, such as breed, age or on aspects that we might be able to control, such as stimulation treatment, type of needle, aspiration pressure, among others. We believe that new investigations should contribute to significant improvement of LOPU yield. This review aims to report different factors influencing goat donor response for LOPU, presenting main steps for oocytes recovery as well as technical modifications proposed for improving LOPU efficiency. Furthermore, it is aimed to discuss about the potential use of goat oocytes after their recovery by LOPU and present overall results in goat IVP worldwide.

170 INFLUENCE OF OOCYTE QUALITY AND DIFFERENT MEDIA SUPPLEMENT ON IN VITRO MATURATION, CLEAVAGE, AND EMBRYO DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) OOCYTES

A series of 4 factorial-arranged experiments were conducted to study the effect of oocyte quality and different in vitro maturation (IVM) media supplements on IVM, cleavage, and embryo development of buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected at a local abattoir in a warm (32–35°C) saline (0.9% NaCl), and oocytes were aspirated using an 18-gauge needle. In experiment 1, oocytes (n = 320) were classified according to the number of cumulus cell layers and morphology of ooplasm as excellent, good, or fair. Oocytes were cultured for IVM, fertilization, and embryo culture (IVMFC) in TCM-199 + 10% FCS. In experiment 2, excellent quality oocytes (n = 237) were subjected to IVM in TCM-199 enriched with either 10% FCS or oestrous buffalo serum (EBS; 20–40 pg mL–1) and then fertilized using frozen–thawed buffalo semen capacitated in Bracket and Oliphant's (BO) medium containing heparin (0.02 mg mL–1) and sodium caffeine benzoate (3.89 mg mL–1). In experiment 3, oocytes (n = 290) were classified into 2 groups; Group 1, without gonadotropins, served as a control; Group 2, in which IVM medium was supplemented with 20 IU mL–1 equine chorionic gonadotropins (eCG). Experiment 4 was carried out to examine the suitable capacitating agent added to BO medium, either heparin or caffeine or both (n = 210 fertilized oocytes). In all experiments (multiple replicates), oocytes (2–6 mm in diameter) were kept at 39°C under 5% CO2 for IVMFC and examined several times for cleavage and embryo development (morula and blastocyst). Statistical analysis was carried out using Chi-squared test. Excellent and good quality oocytes produced a higher (P &lt; 0.05) maturation, cleavage, and morula development rates than poor quality oocytes (70% and 65% v. 33.3%), (50% and 46.2% v. 25%), and (42.9% and 33.3% v. 10%), respectively. Blastocyst production rate was also higher (P &lt; 0.05) for excellent compared with good quality oocytes (28.6% v. 16.7%, respectively). In experiment 2, the IVM and cleavage rates were significantly higher (P &lt; 0.05) in IVM medium plus 10% EBS than those cultured in 10% FCS (73% v. 45% and 50% v. 33.3%, respectively). In experiment 3, the addition of eCG to maturation medium increased (P &lt; 0.05) developmental competence of buffalo oocytes (IVMFC) compared with control medium (16% v. 4%). In experiment 4, the addition of heparin together with caffeine to BO medium produced significantly higher (P &lt; 0.05) cleavage and embryo developmental rates compared with heparin or caffeine alone (56.3% v. 33.3% and 35.7%, respectively; 22.2% v. 10% and 8%, respectively). In conclusion, excellent quality oocytes cultured in IVM medium supplemented with either EBS or eCG and fertilized with capacitated buffalo spermatozoa in BO medium enriched with heparin and caffeine progressively enhanced developmental competence of buffalo oocytes.

Endocrinological profile and follicular development in cyclic ewes subjected to repeated ovum pick-up

Blood concentrations of progesterone, FSH and oestradiol in Karagouniko ewes subjected to laparoscopic ovum pick-up (OPU) at specific stages of induced oestrous cycle, were measured. Twenty-four cyclic ewes were randomly allocated into four equal groups (A, B, C and D). Oestrus was synchronized with progestagen intravaginal sponges and detected by teaser rams (oestrus: day 0). In group A, during the induced oestrous cycle, OPU was performed on days 4, 9 and 14 (sessions A1, A2 and A3, respectively). In group B and group D, OPU was performed once, on day 9 and 14, respectively. In group C (controls), endoscopic observation of follicular population was performed three times, as in group A. Starting at sponge removal, progesterone was measured in blood samples collected on 22 daily occasions and oestradiol in samples collected on 27 occasions collected at various time-points starting 2h before to 24h after OPU. Follicular populations did not differ among A1, A2, A3 or between C1, C2, C3 and A1, A2, A3 or A1, B, D, respectively. Oocytes of better quality (category ‘1’ or ‘2’) were collected at A3 session compared with A1 (P<0.05). Progesterone concentration and oestrous cycle length did not differ among groups. Decreased oestradiol concentrations followed by FSH increase were recorded 3–5h after OPU. The results confirm the regulatory role of oestradiol on FSH secretion. The quality of collected oocytes was improved in subsequent pick-up sessions in the oestrous cycle. Moreover, OPU at specific stages of the luteal phase of the cycle, even when applied repeatedly, do not affect the normal oestrous cycle length of ewes.

Intracytoplasmic sperm injection improves in vitro embryo production from poor quality bovine oocytes

The objective was to use subzonal sperm injection (SUZI) to understand sperm penetration patterns and to use intracytoplasmic sperm injection (ICSI) to improve production of bovine embryos using poor quality gametes. In experiment 1, poor versus good quality oocytes were fertilized with sperm from two bulls, A and B, with poor and good sperm vigor, respectively. The blastocyst rate was higher for good versus poor quality oocytes (23.3% vs. 11.1%, P < 0.05), regardless of the bull used. There was no significant difference in blastocyst rate for bull A (low vigor) regardless of oocyte quality, and for bull B (high vigor), blastocyst rate was better for good versus poor quality oocytes (25.7% vs. 9.2%, P < 0.05). In experiment 2, poor quality oocytes were subjected to SUZI. The oocyte penetration rate was lower for bull A than for bull B (29.6% vs. 53.8%, P < 0.05), when SUZI was performed within 1 hour after sperm processing. However, when SUZI was performed 2 to 3 hours after sperm processing, penetrating capacity was similar between bulls, but for bull B, penetrating capacity significantly decreased after 3 hours of sperm processing. In an attempt to overcome sperm penetrating disorders, poor and good quality oocytes were subjected to ICSI (experiment 3). Irrespective of the bull or of the oocyte quality grade, there were no differences in cleavage or blastocyst rates. Both bulls had distinct IVF embryo production rates, which we inferred were because of particular individual sperm characteristics. In conclusion, ICSI was an effective means to achieve in vitro production of bovine embryos with gametes of variable quality.

Effect of oocyte quality and activation protocols on bovine embryo development following intracytoplasmic sperm injection

The purpose of this study was to investigate the effects of oocyte quality and activation protocols on the in vitro developmental competence of bovine embryos after intracytoplasmic sperm injection (ICSI). Bovine oocytes were grouped as being of excellent, good, and poor quality. All of the oocytes were activated using a calcium ionophore only, ethanol only, and 6-dimethylaminopurine (6-DMAP) following calcium ionophore. For the excellent quality oocytes, cleavage rates after ICSI were 70% in the Ca ionophore + 6-DMAP activation group, 47% in the ethanol activation group, 41.82% in the Ca ionophore group, and 30% in the nontreated control group. Cleavage rates for the good quality oocytes after activation with these different agents were 31%, 16.03%, 19.8%, and 12.9%, respectively, while the rates for the poor quality oocytes were lower (7%, 6%, 2%, and 1%, respectively). In conclusion, using Ca ionophore + 6-DMAP improves the in vitro developmental competence for bovine embryos after ICSI. Furthermore, the results for the excellent quality oocytes indicate the importance of the preselection of the oocytes and activation methods.