The effect of cumulus oophorus on the fertilization in vitro of mature oocytes of cattle

The effect of the cumulus oophorus on fertilisation was investigated by 478 in vitro maturated bovine oocytes. Culturing revealed that the amount of defects on the pellucid zone and in ooplasm of cumulus-free oocytes was higher than that in oocytes cultured in the cumulus-oocyte complex (38 percent or 77/203 versus eleven percent or 30/275). Insemination of the cumulus-oocyte complex (COC) seemed to increase the fertilisation capability of bovine oocytes (63 percent or 70/111), though its penetration rate was only 45 percent (111/245) and was thus below that of cumulus-free oocytes (55 percent or 69/126). Use of COC is recommended as basic material for in vitro maturation and fertilisation.     

Selected factors influencing the in vitro maturation of oocytes of cattle]

Studies were conducted into hormonal additives to medium and culturing time and their effects on in vitro maturation of bovine oocytes. The best maturation results were ensured in vitro by substitution of TCM-199 with FSH, HCG, and 17 beta-oestradiol: The stage of fertilisation capability (telophase I or metaphase II) was reached by 86% of all oocytes (115 of 134). The rate of maturation was worse with significance in FSH-free medium (62% or 101 of 163), and the amount of degenerated oocytes was twice as high (18%). Maturation in hormone-substituted medium for 28 to 30 hours is recommended under the condition that immature oocytes were cultured from juvenile antral follicles (2--5 mm in diameter).     

From the germinal cells to the newborn animal: the transmission of genes and life through the generations

The technology of reproduction progressed considerably during the last decade, leading to a certain availability of in vitro methods for fertilisation, oocyte maturation and embryo culture. The most spectacular manipulations are cloning and transgenesis. This review focuses on the early appearance of germinal cell precursors and the long-standing fate of gametes in mammals. The evident complexity and long-term programming of events in gametes and early embryos explain part of the difficulties encountered during the development of in vitro and in vivo methods such as multiple ovulation and embryo transfer (MOET), oestrus synchronisation, ovulation induction, superovulation, in vitro maturation and fertilisation, cryopreservation, transgenesis, nuclear transfer and cloning) and the occurrence of unexpected alterations of development, e.g. embryonic or fetal mortality, large-weight newborn syndrome and other dysregulations in imprinting or DNA transmission.     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响。结果显示,添加颗粒细胞传代接种第2天收集的20%颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P<0.05);然而单层颗粒细胞却显著抑制水牛卵母细胞体外成熟和早期胚胎发育(P<0.05)。结果表明,与单层颗粒细胞共培养相比,与颗粒细胞条件液共培养更有利于水牛卵母细胞体外成熟和早期胚胎发育。本研究为水牛卵母细胞体外成熟培养体系的完善提供一定理论依据。     

Results of endocrinologic studies in culture media after coculture of bovine oocytes with granulosa cells

The steroid hormones progesterone (P.), and testosterone (T.) were radio-immunologically determined in 108 medium samples, following co-culturing of bovine oocytes with granulosa cells. P. and T. values recorded from a control group were lower with significance than those recorded from co-culturing groups, that is 72 +/- 21 ng/ml and 264 +/- 84 pg/ml as compared to 208 +/- 138 ng/ml and 2,168 +/- 1,595 pg/ml in the oocyte plus fresh granulosa cell co-culturing group as well as 364 +/- 215 ng/ml and 825 +/- 233 pg/ml in the oocyte plus pre-incubated granulosa cell co-culturing group. These rises were accompanied by decline in maturation rate, increase in oocyte degeneration, and rises in the rates of fertilisation and segmentation.     

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.     

Application of in vitro fertilisation techniques to obtain calves from valuable cows after slaughter.

The ovaries of two infertile cows of high breeding value were recovered after slaughter, and a total of 222 oocytes were obtained. Of these, 156 were classified as of good or fair quality and were subjected to in vitro maturation, in vitro fertilisation (using frozen semen from three bulls of high breeding value) and in vitro culture procedures. After eight days, 27 embryos were obtained, of which 13 were transferred fresh, and 14 were frozen. Three recipients of fresh embryos became pregnant; two calved and one aborted at four months. One of eight recipients of frozen-thawed embryos became pregnant but aborted at three months.     

Development of vitrified matured cattle oocytes after thawing and culture in vitro

Bovine oocytes were partly denuded either at the beginning (t0) or six hours (t6) after the beginning of maturation and vitrified by the open pulled straw method at the end of the maturation process. After warming and fertilisation, their development in vitro and in vivo was assessed. The rates of production of blastocysts achieved in vitro were 3.4 per cent for the t0 group and 0.9 per cent for the t6 group compared with 40.4 per cent for the control oocytes. After transfer at the blastocyst stage pregnancies have been established in the three groups. Some of these pregnancies originated from vitrified oocytes which were further vitrified at the blastocyst stage before being transferred into synchronised recipients.     

Seasonal variations in the developmental competence of bovine oocytes matured in vitro

Ovaries were collected over a period of two years from heifers slaughtered at under 30 months of age and used to harvest 1757 oocytes. After in vitro maturation, fertilisation and culture, the proportions of oocytes and cleaved embryos that developed to blastocysts were significantly higher (P<0.01) in the autumn, from September to November, than in the spring, from March to May. In contrast, embryo development, as assessed by oocytes that developed to eight or more cells and blastocysts, was lowest (P<0.01) in the spring. These results were consistent during the two-year study, indicating a seasonal fluctuation in oocyte competence.     

Effect of open pulled straw (OPS) vitrification on the fertilisation rate and developmental competence of porcine oocytes

Freezing technologies are very important to preserve gametes and embryos of animals with a good pedigree or those having high genetic value. The aim of this work was to compare immature and in vitro matured porcine oocytes regarding their morphology and ability to be fertilised after vitrification by the open pulled straw (OPS) method. In four experiments 830 oocytes were examined. To investigate the effect of cumulus cells on oocyte survival after OPS vitrification, both denuded and cumulus-enclosed oocytes were vitrified at the germinal vesicle (GV) stage, then after vitrification they were matured in vitro. Besides, in vitro matured oocytes surrounded with a cumulus and those without a cumulus were also vitrified. The survival of oocytes was evaluated by their morphology. After in vitro fertilisation the rates of oocytes penetrated by spermatozoa were compared. Our results suggest that the vitrification/warming procedure is the most effective in cumulus-enclosed oocytes (22.35 +/- 1.75%). There was no difference between the order of maturation and vitrification in cumulus-enclosed oocytes, which suggests the importance of cumulus cells in protecting the viability of oocytes during cryopreservation.     

A new method for bovine embryo production: a potential alternative to superovulation

Eight cows were used to study the feasibility of transvaginal ultrasound-guided puncture of follicles as a method for the collection of immature oocytes for embryo production in vitro. In six trials at intervals of seven days, 104 oocytes were collected. After in vitro maturation and fertilisation the 104 oocytes were transferred to the oviducts of sheep. Six days later, 75 oocytes were recovered by flushing the oviducts. Twenty-four per cent of the recovered oocytes/embryos had developed into transferable and viable morulae and, or, blastocysts. The data show that this non-surgical and repeated collection of immature oocytes can be used successfully for the in vitro production of bovine embryos. The procedure may produce yields of embryos comparable to those obtainable by conventional superovulation procedures.     

Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

Background: The in vivo concentration of bicarbonate(HCO_3~-), one of the essential sperm capacitating effectors,varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO_3~-concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased.Results: Once exposed to the capacitation medium, the intracellular pH(pH_i) of spermatozoa increased immediately even at low concentrations of HCO_3~-, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation(pPKAs). Although with a significant delay, 15 mmol/L of HCO_3~-stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation(Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation(IVF)system based on the optimization of HCO_3~-concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes(8.6% in the standard system vs. 33.9%).Conclusions: Optimising HCO_3~-concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO_3~-in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine.     

The influence of follicular factors on the in vitro maturation of bovine oocytes]

Immature oocytes from antral follicles of cattle were tested for the effect of follicular factors on maturation. In vitro maturation was accomplished by use of follicular fluid from small (2--5 mm) and large (above 15 mm) follicles and by addition to the medium of a granulose factor (GF) which had been isolated from the surface of granulosa cells. The parent material, with 84% (72/86) of oocytes at the germinal vesicle stage (GV-S) at the beginning of culturing, could be rated immature. 46% of all oocytes (41/89) had reached telophase I or metaphase II (full maturation) after 24 hours of maturation in hormone-free control medium (TCM 199 + 10% of foetal calf serum). 36% of oocytes (53/84), on the other hand, stayed between GV breakdown (GVBD) and anaphase I (incipient maturation). Full maturation was reached by as little as 14%. GF and follicular fluid from small antral follicles were found to inhibit GVBD in the oocytes. 59% (36/61) or 48% (61/127) of oocytes were blocked at GV stage. Positive determination of maturation inhibiting action of the above follicular components may provide a chance for their target-oriented use in control of the maturation process. The pool of immature oocytes of the ovaries, under such circumstances, might be more systematically utilised for in vitro manipulations.     

Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro

The fertilization and cleavage of bovine oocytes matured by intra- or extra-follicular methods were investigated. Oocytes were fertilized in vitro or in the rabbit oviduct and cleavage was assessed after in vitro culture of in vitro fertilized oocytes and after in vivo culture (rabbit oviducts) of xenogenously fertilized oocytes. The effect of fertilization with fresh-diluted or frozen-thawed semen were also examined. The intra-follicular method did not increase the nuclear maturation rate as compared with the extra-follicular method (57.9 and 52.7%, respectively). However, the proportions of in vitro fertilized eggs (54.8%) and of cleaved eggs (two to eight cells; 34.6%) in the rabbit oviduct for 48 h after xenogenous fertilization were higher (P less than .025) in the intra-follicular oocytes than those of the extra-follicular oocytes (37.1 and 21.3%, respectively). It was also found that the use of fresh-diluted semen resulted in more cleaved eggs from the rabbit oviduct than the use of frozen-thawed semen (43.4 and 23.3% in the intra-follicular oocytes, P less than .025; 31.0 and 7.8% in the extra-follicular oocytes, P less than .05), while the appearance of cleaved eggs following in vitro fertilization was extremely low (0 to 6.6%). The present results demonstrated that the intra-follicular culture method of bovine oocytes provided a physiological environment for cytoplasmic maturation leading to higher fertilizability and development than the conventional in vitro culture of extra-follicular oocytes.     

Influence of cysteine in conjunction with growth factors on the development of in vitro-produced bovine embryos

Cysteine supplementation to in vitro maturation (IVM) media of bovine oocytes increases cellular glutathione production. Beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mm cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/ml); EGF (10 ng/ml); and IGF-I + EGF (100 + 10 ng/ml). Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the maturation media (12 h C IGF-I + EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I + EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in embryos was assessed. No treatment effect was observed on the expression of these genes. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.     

Effect of the in vitro maturation medium on equine oocytes: comparison of follicular fluid and oestrous mare serum

The present study evaluated the effect of supplementing the medium used to mature equine oocytes in vitro with oestrous mare serum (EMS) or horse follicular fluid (HFF). To this end, 144 ovaries were obtained from mares aged 16-21 months and transported to the laboratory in Dulbecco's phosphate buffered saline (D-PBS) at 30 degrees C. Oocytes were harvested from the ovaries by slicing, and then selected for in vitro maturation (IVM) according to the number of cumulus cell layers and the characteristics of the cytoplasm. The selected oocytes were washed three times in TCM199 medium plus HEPES (TCM-199H) or in the same medium plus glutamine (TCM-199G), then matured in vitro in six study groups established according to the in vitro maturation (IVM) treatment to see possible interactions between HEPES and glutamine on other supplements: Ten percent EMS was added to two of these media (TCM-199H+EMS and TCM-199G+EMS) and 10% HFF was added to the media in two other groups (TCM-199H+HFF and TCM-199G+HFF). IVM was performed at 38.5 degrees C for 40 h in a controlled atmosphere (5% CO2, 95% relative humidity). The findings indicate that the presence of EMS or HFF in the TCM-199H medium gives rise to the best results in terms of the proportions of oocytes reaching maturity (37.7% and 36.8%, respectively). The values obtained with EMS and HFF were statistically similar to each other but differed from the other treatments. The media containing glutamine led to the highest proportions of degenerated oocytes.     

Birth of calves after in vitro fertilisation using laparoscopy and rabbit oviduct incubation of zygotes

The infertility of many cows could be treated by in vitro fertilisation. In the present study laparoscopy was utilised to recover the in vivo matured oocytes from the ovary of a standing donor. After the capacitation of fresh semen with high ionic strength medium and in vitro fertilisation, a rabbit oviduct was employed as an incubator for four to five days, in order to obtain sufficiently aged embryos to be transferred to the uterus of a recipient. Using surgical or non-surgical transfer six calves were obtained.     

In vitro fertilisation of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hCG injection

The goal of the present study was to find out the best interval after hCG injection in PMSG primed prepuberal gilts for retrieval of in vivo matured oocytes for in vitro fertilisation (IVF). Altogether 66 gilts were superovulated with 1500 IU PMSG and 500 IU hCG 72 h later. Ovum pick up was performed endoscopically 24, 28, 32 or 36 h after hCG and a total of 869 cumulus-oocyte-complexes (COCs) were aspirated from 1400 follicles. COCs were tested for quality, and an aliquot was immediately fixed and stained to determine meiotic configuration. The remaining COCs were fertilised in vitro using frozen-thawed epididymal semen. Quality and developmental stage of embryos were tested after IVF, and the number of nuclei was counted. At 24 to 32 h after hCG only few oocytes have entered the second meiotic cycle (18 to 25% vs. 58% at 36 h, p < 0.05). The overall cleavage rate was significantly influenced by insufficient maturation rate at the early collection times (14% at 24 h vs. 49% at 36 h). Additionally, when oocytes were collected 24 to 32 h vs. 36 h the cleavage rate based on mature oocytes was lower (26 vs. 62%, p < 0.05). Once embryonic development has been initiated, the further in vitro development to blastocyst stages did not differ between groups. However, the number of cells was lower at collection times 24 to 32 h as compared to 36 h after hCG (12 to 15 cells vs. 22 cells, p < 0.05). The results indicate that the time of COC collection affects the in vitro developmental competence up to the blastocyst stage and should not be performed earlier than 36 h after hCG treatment.     

The effect of nerve growth factor on nuclear progression of porcine oocytes during in vitro maturation and embryo development

The present study examined the effect of nerve growth factor (NGF) on in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development of porcine oocytes. Cumulus-oocyte complexes were cultured with or without 1.0 ng/ml NGF for 40 h. After IVF, they were cultured in vitro for 6 days. After 10 and 20 h of IVM, there was no difference in nuclear status between the NGF-treated and control oocytes. Significant differences were detected in nuclear progression of oocytes matured in the presence or absence of NGF at 30 h of culture. A higher proportion of NGF-treated oocytes were at M-II stage compared to the control. Nevertheless, at the end of the 40-h IVM period, there was no difference in the proportion of M-II stage oocytes between the NGF-treated and control groups. NGF in IVM medium did not influence the developmental competence of putative embryos. Most embryos remained at the 2- to 4-cell stage; however, a significant amount of embryos reached the morula stage both in the NGF and the control groups. These results suggest that NGF during IVM accelerates nuclear progression of porcine oocytes by enhancing the post-diakinetic events of meiosis.     

Effect of low oxygen tension atmosphere and maturation media supplementation on nuclear maturation, cortical granules migration and sperm penetration in swine in vitro fertilization

The aim of this study was to evaluate the efficiency of low oxygen tension (5% CO(2) , 5% O(2) and 90% N(2) ) on in vitro oocyte maturation using defined media (0.1% polyvinyl alcohol - PVA) or 10% porcine follicular fluid (PFF)-supplemented media. To achieve this goal, oocytes were evaluated regarding cortical granules (GCs) migration, nuclear maturation and sperm penetration. Oocytes were in vitro matured under different conditions: 5% or 20% O(2) atmosphere and 0.1% PVA- or 10% PFF-supplemented media and evaluated at 0 and 44 h of maturation. To evaluate the migration of CGs and nuclear maturation, by confocal microscopy, oocytes were incubated with 100 μg of FITC-PNA/ml and 10 μg/ml of propidium iodide. To address sperm penetration, after maturation, in vitro fertilization for 6 h and in vitro culture for 18 h, zygotes were incubated with 10 mg/ml Hoechst 33342. Pronuclei and polar bodies were quantified using an epifluorescence microscope. Atmosphere conditions did not affect the CGs migration, but media supplementation did. Oocytes matured in 10% PFF media had a higher percentage of CGs in the oocyte periphery than oocytes matured in PVA-supplemented media. However, this fact did not have effect on in vitro sperm penetration levels. No effect of atmosphere conditions and media supplementation was observed on the rates of metaphase II oocytes. Therefore, the use of low oxygen tension in association with PVA maturation media does not improve the in vitro maturation system of porcine oocytes, because its use did not improve nuclear maturation, CGs migration and zygotes monospermic rates.