Number Of Good Quality Oocytes Research Articles

Vitamin and Carotenoid Intake and Outcomes of In Vitro Fertilization in Women Referring to an Italian Fertility Service: A Cross-Sectional Analysis of a Prospective Cohort Study

Nutrition may impact reproductive health and fertility potential. The role of dietary antioxidants in affecting conception and birth outcomes is a topic of emerging interest. This cross-sectional analysis from a prospective cohort study aims to explore the relationship between the intake of antioxidants, vitamins, and carotenoids and the outcomes of assisted reproduction techniques. Information on the socio-demographic characteristics, health histories, lifestyle habits, and diet information of subfertile couples referred to a fertility center was obtained. A total of 494 women were enrolled. According to the four IVF outcomes considered, 95% of women achieved good quality oocytes, 87% achieved embryo transfer, 32.0% achieved clinical pregnancies, and 24.5% achieved pregnancy at term. Associations were found between age and the number of good quality oocytes (p = 0.02). A moderate level of physical activity in the prior 5 years was associated with a better rate of achieving clinical pregnancy (p = 0.03). Smoking habits, alcohol intake, and caffeine consumption did not show associations with any outcome. No associations were found, even after accounting for potential confounders, with the intake of vitamins C, D, E, and α-carotene, β-carotene, beta-cryptoxanthin, lutein, and folate. Further research is needed to understand how antioxidant intake may have a role in modulating fertility.

Efficacy and safety of newly developed ganirelix acetate in infertile women for assisted reproductive technology: a prospective, randomised, controlled study

This study aimed to investigate the efficacy of Ganilever pre-filled syringe (PFS), a newly developed ganirelix acetate, for the inhibition of premature luteinising hormone (LH) surge in in vitro fertilisation (IVF). A prospective randomised controlled study was conducted (NCT03051087). A total of 236 women (Ganilever group: 114, Orgalutran group: 122) were finally analysed. The patients with LH of >10 mIU/mL on the day of human chorionic gonadotropin (hCG) injection were 0 (0.0%) and 3 (2.5%) in the Ganilever and Orgalutran groups, respectively (p= .25). The number of retrieved oocytes from two groups did not show any significant difference (12.0 ± 6.4 vs. 11.8 ± 6.3, p= .73). Furthermore, the two groups did not show significant differences in the number of good-quality oocytes and embryo, and the rate of fertilisation. Similar safety profiles were also observed. In conclusion, Ganilever PFS showed comparable IVF outcomes and safety profile in IVF, as compared to the Orgalutran. Impact Statement What is already known on this subject? Premature LH surge during controlled ovarian stimulation results in the induction of luteinisation of the immature follicles. Thus, gonadotrophin-releasing hormone (GnRH) antagonist protocol was suggested as an option for suppression of premature LH surge. Currently, one of GnRH antagonists being widely used is ganirelix acetate (Orgalutran®; Organon, Oss, The Netherlands). Ganilever pre-filled syringe (PFS) is a newly developed GnRH antagonist containing ganirelix acetate as an active ingredient. What do the results of this study add? Our study demonstrated that Ganilever PFS showed comparable IVF outcomes and patient safety profile in infertile women undergoing in IVF-ET, as compared to the Orgalutran. What are the implications of these findings for clinical practice and/or further research? The results of our study will provide another available GnRH antagonist to be used in patients with IVF.

P–169 Does increasing the time interval between Oocyte-Retrieval and Oocyte-Denudation improve the results in ICSI cycles ?

Abstract Study question What should be the optimal time interval which elapses between oocyte retrieval and denudation followed by ICSI , for optimal results in ART cycles ? Summary answer Our study suggests that an optimum interval between oocyte retrieval and oocyte denudation followed by ICSI, leads to better results in ART cycles. What is known already It is widely accepted that the best timing for OPU is 34–39 hours after ovulation trigger. Some studies suggest that preincubation time before ICSI can be beneficial when it comes to fertilization and pregnancy rates while late ICSI (fertilization) may have negative results due to oocyte ageing. Other studies claim that there is no significant difference in ART results when ICSI is performed between 2–6 hours post Oocyte-Retrieval (OR) . Few studies state that 1–3 hours of COC-culture prior to denudation and oocyte injection is better as far as fertilization , embryo quality and improved oocyte cytoplasmic maturity is concerned. Study design, size, duration RCT of 234 ICSI cycles was carried out between 2017–2019. Patients were divided into two groups-: A- Early denudation with ICSI and B- Late denudation with ICSI.Both the groups were comparable in terms of female age, number of oocytes, day of transfer, number of embryos transferred and embryo quality. Fresh or frozen embryos were transferred , which were always derived from the same stimulation cycle. Exclusion criteria were : Severe male factor / TESA / PESA. Participants/materials, setting, methods 234 ICSI cycles with similar ovarian stimulation protocols were analyzed as per time range between triggering, OPU, denudation and ICSI. Patients were divided into two groups: A- Early denudation (1–2 hours after OPU) with ICSI (1–2 hours after denudation) and B- Late denudation (4–6 hours after Oocyte-Retrieval ) with ICSI (1–2 hours after denudation).Primary outcomes were oocyte maturation and fertilization rates and secondary outcomes were clinical pregnancy rate and abortion rates. Main results and the role of chance In group B ( Late denudation and ICSI), the mean fertilization rate was 67% and the Clinical Pregnancy rate was 46%. This was better than the mean fertilization rate of 56% and clinical pregnancy rate of 39% observed in group A ( Early denudation and ICSI). However the difference was not statistically significant. Therefore, ideal maturation rates were observed when denudation ( followed by ICSI ) was delayed and done 4–6 hours after Oocyte-Retrieval. In ICSI cycles in ART , ovarian stimulation is used to induce the simultaneous growth of multiple follicles, followed by final maturation and ovulation triggering with exogenous hCG. or GnRH-Agonist or both. Generally, oocyte retrieval (OR) is performed 34 - 36h later. In addition, 2–4 hours in culture of the cumulus oocyte complexes (COC) prior to oocyte injection is believed beneficial for fertilization and embryo quality, probably due to improved oocyte cytoplasmic maturity. However, in large ART centers with high workloads, following such definite time intervals is frequently very difficult. Limitations, reasons for caution In large busy centers , maintaining meticulous time intervals is difficult . As our study numbers are small, larger multicentric trials are required in order to confirm our findings and to provide more robust data . This data cannot be applied to IVM, TESE / PESE and severe male-factor infertility. Wider implications of the findings: To achieve a successful fertilization, both nuclear and cytoplasmic maturity are required. Our Study indicates that a slight delay in denudation following Oocyte-Retrieval , will yield a higher number of good quality oocytes. A higher success rate can also be expected due to more number of embryos available for transfer. Trial registration number Not applicable

Association between exposure of light rare earth elements and outcomes of in vitro fertilization-embryo transfer in North China

The adverse health effects of rare earth elements (REEs) on reproductive health remain a subject of debate, and few clinical observations are available. This study investigated the association between light REEs (LREEs) exposure and the outcome of in vitro fertilization–embryo transfer (IVF-ET). We recruited a total of 305 women undergoing IVF-ET in Beijing City and Shandong Province of northern China. Their demographic information and lifestyle characteristics were collected using questionnaires at enrollment. Fasting blood samples were collected on the day before the IVF-ET treatment cycle began. Serum concentrations of the LREEs of concern were analyzed using inductively coupled plasma-mass spectrometry, and four LREEs were measured with a high detection rate, including lanthanum (La), cerium (Ce), praseodymium (Pr), and neodymium (Nd). We found that a higher serum La concentration was associated with a 30% increased likelihood of clinical pregnancy failure [relative risk (RR) = 1.30, 95% confidence interval (CI): 1.00–1.67] and a 230% increased likelihood of preclinical spontaneous abortion (RR = 3.30, 95% CI: 1.57–6.94). There was a negative correlation between serum La concentration and the number of good-quality oocytes. For the other LREEs, no statistically significant associations were observed. We concluded that a high serum La concentration may have an adverse effect on IVF-ET outcomes.

Estradiol benzoate treatment before ovum pick-up increases the number of good quality oocytes retrieved and improves the production of transferable embryos in Japanese Black cattle

The aim of this study was to evaluate the efficacy of treatment with estradiol benzoate (EB) at luteal phase prior to the ovum pick-up (OPU) during in vitro production of transferable embryos in Japanese Black cattle. A total of 15 cows were used as oocyte donors for OPU. Of those, four donors were randomly allocated (three times) into each of two treatment groups as a crossover study, and OPU session was carried out six times per one donor. Another eleven donors were used in a paired difference test by one crossover trial. Donors in the control group received no hormonal treatment; whereas, donors in the EB group received 1 mg of EB as a single injection. First, we observed dynamics of ovarian follicles and emergence of follicular wave after EB injection using transrectal ultrasonography. The number and proportion of medium-sized follicles with 4 to 6 mm in diameter increased gradually and achieved a peak at 72 and 96 hours after EB injection. The OPU was performed 88 hours after EB injection. The EB-treated donors had a higher proportion of follicles with 4 to 6 mm in diameters at the time of OPU. The stimulation with EB significantly increased the numbers of follicles aspirated, and the good quality cumulus-oocyte complexes per OPU. Furthermore, in the EB group, the percentage of transferable blastocysts was significantly greater than that in the control group (P<0.05). In conclusion, a single EB injection before OPU increases the number of medium-sized follicles and can produce more transferable embryos.

Clinical utility of newly developed highly purified human menopausal gonadotrophins: a randomized controlled trial

The aim of this study was to evaluate the safety and efficacy of IVF-M HP, a newly developed highly purified human menopausal gonadotrophin preparation, for ovarian stimulation in women with infertility undergoing IVF, intracytoplasmic sperm injection (IVF–ICSI) and embryo transfer using a GnRH antagonist protocol. This was a multicentre, randomized, active-controlled, parallel design, open-label, non-inferiority clinical study. Of the 112 patients randomized for treatment using the GnRH antagonist protocol, 111 were treated. No significant difference was found in the number of oocytes retrieved from the IVF-M HP and Menopur groups (13.1 ± 7.6 versus 10.3 ± 6.7, respectively). The lower limit of the one-sided 97.5% confidence interval for the difference between the groups was −0.25, i.e., greater than the pre-defined non-inferiority margin (−5). Therefore, the IVF-M HP treatment was considered non-inferior to Menopur. Furthermore, no significant difference was observed between the groups in the number of good-quality oocytes, leading follicles, good-quality embryos, or in fertilization, implantation, positive beta-HCG and clinical pregnancy rates. The safety analysis revealed that 40.4% and 35.2% in the IVF-M HP and Menopur groups, respectively, reported adverse events. In conclusion, IVF-M HP had comparable clinical efficacy and safety profiles to Menopur.

Antioxidants reduce oxidative stress in follicular fluid of aged women undergoing IVF

BackgroundThe status characterized by the imbalance between pro-oxidants and antioxidants molecules, defined as oxidative stress, has been suggested to be involved in the pathogenesis of subfertility in females. This study aims to evaluate the impact of a complete micronutrients supplementation on oxidative stress levels in follicular microenvironment as well as on in vitro fertilization (IVF) outcome.MethodsThis preliminary study was conducted between January 2014 and July 2015 at the Siena University Hospital Infertility Clinic. Serum and follicular fluid were collected from infertile women aged > 39 years who underwent two in vitro fertilization cycles: in the first cycle they were treated with GnRH-antagonist protocol and gonadotropins for controlled ovarian hyperstimulation, whereas in the second cycle ovarian stimulation protocol was associated to micronutrients supplementation, starting three months earlier. Protein oxidation levels and total antioxidant capacity in serum and in follicular fluid were evaluated in IVF cycles with or without micronutrients supplementation. Differences in IVF outcome parameters were statistically evaluated.ResultsTwo-dimensional electrophoresis analyses demonstrated that when patients assumed micronutrients before IVF cycles, follicular fluid and serum proteins were protected from oxidative damage. Comparable results were obtained when total antioxidant capacity was measured. Moreover, the mean number of good quality oocytes retrieved when patients received micronutrients supplementation was significantly increased.ConclusionThe additional treatment with micronutrients, starting three months before IVF cycles, protects the follicular microenvironment from oxidative stress, thus increasing the number of good quality oocytes recovered at the pick up.

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

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.

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.

Effect of aging on follicular function may be relieved by exogenous gonadotropin treatment in a sheep model

The current study investigated hormonal and ovarian changes during physiological reproductive aging in Sarda ewes. In a first experiment, follicular and corpus luteum dynamics were compared during an induced oestrus cycle in aged (12-14 years) and young adult ewes (4-5 years). Oestrus cycle characteristics did not differ between the two experimental groups. However, follicular function during the follicular phase showed significant alterations in aged ewes, as determined by a lack of dominance effect and by lower mean values of circulating oestradiol (E(2)) and inhibin levels, compared with young adult ewes. In a second experiment, differences in follicle growth, hormonal milieu and oocyte quality in response to exogenous FSH administration were assessed in aged and adult ewes. No differences were recorded in ovarian response to FSH treatment between young adult and aged ewes, as evaluated by ultrasonographic data and circulating concentrations of LH, E(2) and inhibin-A. Although the total number of recovered oocytes was similar in the two age groups, the number of good quality oocytes selected for IVM was significantly lower in aged ewes compared with adult ones. Thereafter, no differences were recorded in cleavage rates, total blastocyst output, embryo developmental kinetic and quality between aged and adult groups. In conclusion, this study demonstrated that reproductive aging in sheep is associated with impaired follicle functionality and an increase in the proportion of oocytes showing morphological abnormalities. However interestingly, oocyte developmental competence in vitro and embryo cryotolerance were not affected by the aging process, when only good quality oocytes were chosen.

Genetic Profile of SNP(s) and Ovulation Induction

Obtaining an adequate number of good quality oocytes while minimizing adverse drug reactions (ADRs) and cycle cancellation rates is considered the gold standard in controlled ovarian hyperstimulation (COH) for fertility treatment. Patients who undergo IVF/ICSI cycles tend to present with different responses to exogenous gonadotrophin administration. Research has shown that the secret probably lies in the various single nucleotide polymorhisms (SNPs) in their receptor genes. The decryption of human genome provided specialists with additional information in assessing and even predicting ovarian response to COH. In this context, the study of Pharmacogenomics, Pharmacogenetics and SNPs unravels as a promising field in optimizing fertility treatment. Several SNPs in FSH and estrogen receptor genes have been detected so far, but only three of them, one in FSH receptor and two in estrogen receptor genes have been associated with ovarian response to COH. It seems that the Asn/Ser variant of the FSH receptor functions more efficiently, while the Ser/Ser and Asn/Asn variants have a tendency to resist to FSH stimulation. With regards to estrogen receptor 1 (ESR1), the Pvull and the Xbal polymorphisms seem to be associated with differences in the response to ovarian stimulation, while the Rsal polymorphism in estrogen receptor 2 (ESR2) is currently under investigation. There exists evidence supporting the hypothesis that a set of genes, all related to the FSH hormone mechanism of action, may participate along with other factors to the control of ovarian response to FSH, thus a cautious interpretation of polymorphism detection results is considered mandatory. However, identifying potential genetic markers that could predict ovarian response and implementing them in routine screening tests for every woman entering an IVF/ICSI cycle, would be able to tailor fertility treatment to each patients needs thus maximizing the success rate and eliminating potential side-effects of fertility drugs.

In vitro oocyte fertilization and subsequent embryonic development after cryopreservation of bovine ovarian tissue, using an effective approach for oocyte collection

This study was undertaken to assess dissection/puncture combined technique for collecting large number of oocytes from bovine ovaries and to determine the effect of ovarian tissue cryopreservation on the oocytes capability to undergo in vitro maturation, fertilization and subsequent embryonic development. Ovaries ( n = 31) of slaughtered cows were cut into small fragments using a scalpel blade and the ovarian tissues were randomly assigned to cryopreserved by slow freezing and vitrification and non cryopreserved (fresh) groups. Oocytes were collected from non-atretic follicles from fresh and post-thawing ovarian tissue by the puncture method. The advantage of this technique appeared through morphologically good quality cumulus–oocyte complex (COC) recovery rate from fresh tissue (31.7 ± 2.0 oocytes/ovary). However, the cryopreservation affected the post thawing total and good quality COC recovery rates from slow freezing (26.6 ± 2.0 and 23.5 ± 2.3 oocytes/ovary, respectively) and vitrification groups (21.7 ± 1.1 and 17.6 ± 1.8 oocyte/ovary, respectively). The maturation rate resulted in significant differences between the fresh tissue (94.1 ± 1.1%) and the two cryopreservation groups. Moreover, this rate was significantly higher in the slow freezing group (80.1 ± 1.3%) than in the vitrification group (73.0 ± 1.9%). No statistical differences were observed in the cleavage and the embryonic developmental rates between fresh tissue group and cryopreservation groups. Furthermore the number of embryos produced per animal was statistically higher for fresh tissues than for slow freezing and the vitrification groups (34.4 ± 1.4, 27.8 ± 3.1 and 22.0 ± 0.7, respectively). In conclusion, dissection method followed by puncture of bovine ovaries greatly maximizes the number of good quality oocytes recovered, as well as the number of embryos obtained per animal. Ovarian tissue can be successfully cryopreserved by slow freezing and vitrification.

Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian buffaloes ( Bubalus bubalis)

The objective of this study was to determine the best combined hormonal treatment to utilize in order to obtain a high number of good quality in vivo and in vitro matured oocytes from prepuberal Mediterranean Italian buffalo calves ( Bubalus bubalis). Transvaginal ultrasound follicular aspiration was employed to recover oocytes from antral follicles. Fifteen barn housed buffalo calves, between 5 and 9 months of age were used in this study and randomly divided into control (Group A) and treated groups. A commercially available preparation of 2000 IU eCG was administered to animals in the treatment groups, followed by 2000 IU of hCG given either 12 h (Group B), or 24 h (Group C) before ovum pick up (OPU). From the time of administration of eCG treatments, the best timing for hCG administration before OPU was determined and integrated with the administration of 500 IU of FSH-LH in a decreasing dosage protocol over 4 days (Group D). Expanded cumulus oocyte complexes (COCs) recovered from all groups were immediately fixed for later aceto-orcein staining. All other COCs were processed for in vitro maturation using standard procedures and then fixed and stained for assessment of nuclear maturation. Collectively, hormonal stimulation did not increase the number of ovarian antral follicles available compared to the control group ( P>0.05), but did result in higher output of medium (Group B: 9.8±7.1; Group C: 3.4±6.7; Group D: 15.6±4.9 versus Group A: 1.6±2.2) and large follicles (Group B: 44.8±22.9; Group C: 8.7±6.1; Group D: 70.2±10 versus Group A: 6.1±6.3). Administration of hCG 12 h before follicle aspiration proved to be the best strategy to obtain high numbers of immature and mature oocytes from antral follicles ( P<0.05; Group B: 70.8±12 and Group D: 82±12.6 versus Group A: 43.6±13.9 and Group C: 27.2±13.9). A significantly higher number of expanded COCs was obtained from hormonally stimulated groups compared to the control group ( P<0.05; Group B: 28.7±16.8, Group C: 16.3±5.9 and Group D: 27.1±16.9 versus Group A: 6.2±6). A higher oocyte maturational competence ( P<0.05) was found in Groups A, B and D (80.8±7.9, 87.5±8.2, and 86.5±4.3, respectively) compared to Group C (60±26.2). In conclusion, in prepuberal buffalo calves combined gonadotrophin stimulation protocols yielded higher numbers of medium to large size follicles compared to a control group. A high number of good quality oocytes were recovered by transvaginal ultrasound follicle aspiration, and a high rate of metaphase II progression was reached after in vivo and in vitro maturation.

Factors affecting number of surface ovarian follicles and oocytes yield and quality in Egyptian buffaloes.

Three experiments were conducted to evaluate factors affecting number of surface ovarian follicles and oocytes yield and quality in buffalo. In Experiment 1, ovaries (n = 126) were collected in pairs from slaughtered anoestrus, early pregnant and cyclic buffaloes. Ovarian follicles (1-3, 4-9 and > or = 10 mm diameter) were counted, aspirated and oocytes were recovered and evaluated. In Experiment 2, ovaries were divided into 2 groups. Group 1, ovaries bearing a CL (n = 74) and Group 2 non-bearing CL (n = 74), ovarian follicles (2-8 mm) were counted, aspirated and oocytes evaluated. In Experiment 3, oocytes were recovered using aspiration or slicing methods. In all experiments, oocytes were classified into good, fair, poor and denuded. Results showed that the development of small and total ovarian follicles are continuous and independent in early pregnant or cyclic buffalo cows, however, it significantly decreased (P < 0.01) in the ovaries of anoestrus buffaloes. Number of medium and large size follicles was significantly increased (P < 0.01) in cyclic buffaloes on Days 10-16 and 17-22 of oestrous cycle, while large follicles was significantly decreased (P < 0.01) in the ovaries of pregnant buffaloes. A significantly higher (P < 0.01) percentage of poor and denuded oocytes were recovered from ovaries of anoestrus and pregnant buffalo. While, the highest (P < 0.01) percentage of good quality oocytes were recovered from ovaries of cyclic buffaloes on Days 1-3 and 10-16 of oestrous cycle, eliciting that the stage of oestrous cycle is affecting the quality of buffalo oocytes. In addition, the presence of a CL stimulates the development of a significantly higher (P < 0.01) number ovarian follicles which produced a significantly higher (P < 0.05) number of good quality oocytes. Slicing of buffalo ovaries produced a significantly higher number of fair, poor and denuded oocytes. In conclusion, number of ovarian follicles and yield and quality of oocytes were affected by the reproductive status, stage of the oestrous cycle, presence of a CL and the method of oocytes retrieval.

Live foals produced from sperm-injected oocytes derived from pregnant mares

Recently, in vitro fertilization (IVF) in the horse has met with less than anticipated results. Various problems associated with equine IVF include: (1) the inability to collect large numbers of good quality oocytes, (2) the alteration of the zona pellucida associated with in vitro maturation of equine oocytes, and (3) the improper preparation of equine sperm cells for IVF of these oocytes. Therefore, this study was conducted to achieve fertilization via sperm injection of equine oocytes and to produce live offspring from this IVF procedure. Oocytes were collected by transvaginal ultrasound-guided oocyte retrieval procedures from early pregnant mares of mixed breeds (day 14 to day 70 of pregnancy) and were matured in vitro and subjected to intracytoplasmic sperm injection (ICSI). Injected oocytes were then cultured for 48 hours in either TCM-199 or P-1 medium (glucose and phosphate-free medium) supplemented with 15% fetal bovine serum. Cleavage rates for embryos cultured in the two culture media were different (47% vs. 63% in TCM-199 and P-1, respectively). Also, four Grade 1 embryos were surgically transferred into the oviducts of four recipient mares (one embryo/mare) at 48 hours post-ICSI, with three pregnancies (75%) developing as ultrasonically demonstrated by the presence of an embryonic vesicle in the uterine body by day 16 post-ICSI. On June 23rd one live filly was born after 328 days of gestation and subsequently, a second healthy filly was born after 319 days of gestation. To our knowledge, this is the first report of live foals resulting from in vitro fertilization (via ICSI) of in vitro matured oocytes recovered from pregnant mares using an efficient, repeatable transvaginal ultrasound-guided procedure.

Factors promoting embryo implantation in in vitro fertilization and embryo transfer.

The low implantation rate after embryo transfer (ET) is the most important problem to be solved in in vitro fertilization and embryo transfer (IVF-ET). Various factors which may affect embryo implantation in the endometrium have been examined. In this study, the influence of the timing of luteinization on the establishment of pregnancy was investigated. Follicle stimulation was performed by luteinizing hormone-releasing hormone agonist, human menopausal gonadotropin and human chorionic gonadotropin (hCG). hCG was injected on the day when the mean diameter of two follicles exceeded 16 mm, and 36 h after the injection the oocyte was retrieved. Progesterone (P4) supplement was started on the day of ovum retrieval (50 mg), followed by 30 mg for 14 days. The patients were divided into three groups: (1) the pregnant patients (n = 20: group Pre); (2) the nonpregnant patients without bleeding during P4 injection (n = 35; group N), and (3) the nonpregnant patients with bleeding during P4 injection (n = 30; group B). The number of follicles that developed, endometrial thickness, the day of operation, the number of oocytes harvested, the number of good quality oocytes, the number of oocytes fertilized, the number of embryos transferred, estradiol (E2) and P4 levels, and the E2/P4 ratio were examined. The endometrial thickness of group Pre (10.9 +/- 0.6 mm; mean +/- SEM) on the day of hCG injection was significantly (p less than 0.05) greater than that of groups N (9.0 +/- 0.4 mm) and B (9.2 +/- 0.3 mm).(ABSTRACT TRUNCATED AT 250 WORDS)