Energy Status Characteristics of Porcine Oocytes During In Vitro Maturation is Influenced by Their Meiotic Competence.

The characteristics of energy status in porcine oocytes as related to their meiotic competence and in vitro maturation were studied. Cycling pubertal gilts in the early luteal to early follicular phases of the ovarian cycle were used as oocyte donors. The oocytes recovered from medium (MF) or small follicles (SF) were considered meiotically more or less competent, respectively. A half of oocytes from each category was matured by the standard protocol. The oocytes were examined before or after maturation by confocal microscopy, a bioluminescent cell assay and Western blotting. Four experiments, each in triplicate, were performed to assess both SF and MF oocytes in terms of metabolic units formed by mitochondria and lipids, ATP and lipid consumption and lipid droplets with adipose differentiation-related protein (ADRP) expression. The proportion of oocytes with metabolic units, the mean ATP content and the number of lipid droplets per oocyte, and the relative number of lipid droplets with ADRP expression were significantly higher in the MF compared to SF oocytes before maturation. On the other hand, after maturation, there was an increase in the proportion of oocytes with metabolic units and the relative number of lipid droplets with ADRP expression in the SF compared to MF oocytes. In conclusion, specific differences in energy characteristics between porcine oocytes with different meiotic competence were found. Meiotically more competent oocytes are more advanced in terms of energy reserves before maturation, while meiotically less competent oocytes are more active in replenishing energy stores during maturation.

Mitochondrial patterns in bovine oocytes with different meiotic competence related to their in vitro maturation.

This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.

Characteristics of bovine oocytes with different meiotic competence in terms of their mitochondrial status and expression of nuclear-encoded factors.

The present study was designed to characterize bovine oocytes with different meiotic competence and atresia levels in terms of their mitochondrial status. Oocyte subpopulations were recovered either from medium (MF) or small (SF) follicles and categorized as healthy, light-atretic and mid-atretic according to oocyte morphology. Mitochondrial activity, morphology and distribution, adenosine triphosphate (ATP) content and expression of mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1) were assessed before (GV) and after (MII) maturation. The data were related to follicular size regardless of or with regard to oocyte atresia. Regardless of atresia, the MF subpopulation showed a significantly higher mitochondrial activity and frequency of oocytes with granulated mitochondria at GV and clustered mitochondria at MII than the SF subpopulation. With regard to atresia, mitochondrial activity decreased from healthy to mid-atretic oocytes in both MF and SF subpopulations at GV, but in the SF subpopulation at MII, the mitochondrial activity and frequency of oocytes with clustered mitochondria were significantly higher in light-atretic than in healthy oocytes. The light-atretic oocytes also produced more ATP than healthy ones in both SF and MF subpopulations. However, a significantly higher relative abundance of mRNA TFAM was found in SF than MF subpopulations at GV, and this difference remained in mid-atretic oocytes at MII. It can be concluded that meiotic competence and atresia level influence mitochondrial status of immature bovine oocytes. After maturation, healthy oocytes from medium follicles and light-atretic oocytes from small follicles were more developed in terms of mitochondrial status than the other oocytes.

Utilization of the growth phase of the first follicular wave for bovine oocyte collection improves blastocyst production.

Characteristics of the follicle population and oocyte developmental competence at selected stages of follicular development were studied in cows with the aim to increase embryo production derived from oocytes collected by transvaginal aspiration. In Experiment 1, the growth phase before dominant follicle selection and the low dominant phase during dominant follicle regression were compared. Twenty-four cyclic Holstein cows, 4 to 6 yr of age, were divided into 2 groups. Animals were synchronized using two injections of prostaglandin F2alpha at 11 d intervals, and onset of estrus was determined (Day 0). Using ultrasonography, all follicles were counted and classified. Oocytes were aspirated once on Days I through 3 (Group 1, n=5) or Days 15 and 16 (Group 2, n=3) of the estrus cycle. The experiment was carried out in 3 replicates. In Experiment 2, the growth phase of the first follicular wave before dominant follicle selection was characterized in detail. Twelve cows of the same breed and age were divided into 3 groups. Their first estrus was synchronized as in Experiment 1, and each following estrus was induced using one injection of prostaglandin F2alpha administered 4 to 6 d after each aspiration performed. The ovaries were examined, and oocytes were collected repeatedly (total of 5 times per cow) on Days 1 (Group 3, n=4), 2 (Group 4, n=4) or 3 (Group 5, n=4) after estrus at 10 d intervals during a 40 d period. Viable oocytes were matured, fertilized and cultured using the standard methods. In Experiment 1, the mean numbers (+/-SD) of all follicles and of recovered and viable oocytes per donor were higher in Group 1 than in Group 2, but only the mean numbers (+/-SD) of larger follicles and recovered oocytes were statistically significant (8.0 +/- 0.6 and 6.2 +/- 0.6.vs. 3.3 +/- 0.5 and 2.8 +/- 0.2; P< 0.05). In Experiment 2, the percentage of larger follicles out of all visible follicles and the mean numbers (+/-SD) of larger follicles per donor were significantly higher (P<0.05) in Groups 4 (75.7 and 9.1 +/- 2.7) and 5 (66.3 and 8.5 +/- 2.9) when compared to Group 3 (27.9 and 3.8 +/- 0.8). The development rate of fertilized oocytes was significantly higher (P<0.05) in Groups 4 (27.8) and 5 (27.5) than in Group 3 (12.8). It can be concluded that it is possible to improve the efficiency of transvaginal aspiration and in vitro embryo production by utilization of the growth phase of the first follicular wave before dominant follicle selection.

Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production.

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.

Developmental competence of bovine embryos derived from oocytes collected at various stages of the estrous cycle.

The developmental competence of bovine oocytes collected from donors at various stages of the estrous cycle and fertilized in vitro was investigated by comparing the yields of embryos obtained from oocytes isolated from the ovaries of cows slaughtered on estrous cycle Days 7 and 14, 8 and 15, 9 and 16 and on Days 19, 20 and 2. The percentages of oocytes that developed into blastocysts at Day 8 after exposure to spermatozoa were: 11.9 vs 20.0; 13.2 vs 30.5; 20.8 vs 29.8; and 11.7, 4.4 and 16.9, respectively. A significantly higher proportion of oocytes developed into blastocysts following isolation on cycle Days 14 to 16 (24.3 %) than following recovery on Days 7 to 9 (13.0 %; P < 0.05), Days 19 to 20 (6.6 %; P < 0.05) or Day 2 (16.9 %; P < 0.05). Embryo development was also faster in oocytes isolated at the end of the luteal phase (Days 14 to 16). These results demonstrate that the stage of the estrous cycle may influence the developmental potential of oocytes and in vitro embryo production.

A potential relationship between the 16;20 and 14;20 Robertsonian translocations and low in vitro embryo development.

A possible effect of 16;20 and 14;20 Robertsonian translocations on the development of bovine oocytes matured and fertilized in vitro was assessed on the basis of embryo yield and blastocyst formation. Oocytes fertilized with semen from 2 bulls (A and B), which were heterozygous for these translocations, showed a significantly lower cleavage rate (36.3 +/- 2.25%; 39.8 +/- 3.88%) and percentage of blastocysts (3.7 +/- 1.24%; 3.2 +/- 1.20%) than those fertilized with semen from a bull (C) with a normal karyotype (control, 58.1 +/- 2.14%; 20.1 +/- 1.92%; P < 0.01). There was also a difference in the rate of further blastocyst development between the tested bulls and the control. The rates of expanded blastocysts were 6.6 and 11.1% for Bulls A and B, respectively, and 38.7% for the control bull on Day 7; while on Day 8 these values were 41.7 and 55.5% vs 76.0%. These results demonstrated that in the bulls carrying the 16;20 and 14;20 translocations, in vitro preimplantation embryo development was reduced, probably due to genetically unbalanced spermatozoa.

Frequency of aneuploidy in pig oocytes matured in vitro and of the corresponding first polar bodies detected by fluorescent in situ hybridization.

The objectives of this study were to develop a two-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in gilt oocytes using chromosome-specific DNA probes, and to establish baseline frequencies of aneuploidy in pig oocytes matured in vitro. The ovaries were collected from gilts at the local slaughterhouse. Immature oocytes were isolated by slicing the cortex of the ovaries. The oocytes were matured in microplate wells using TCM-199 medium supplemented with 10% estrous cow serum, sodium pyruvate, antibiotics, and gonadotrophins. After 44 h of maturation the oocytes were incubated with hyaluronidase and the cumulus cells were removed by vortexing. Single oocytes were transferred into 1 microL drops of a lysing buffer (0.01 N HCl/0.1% Tween 20) on clean microscopic slides. Two-color FISH was performed using probes specific for Chromosomes 1 and 10. The probe for Chromosome 1 was labeled with Cy3-dUTP and a probe labeled with fluorescein-11-dUTP was used for Chromosome 10. Only oocytes in which a complementary first polar body was found were confirmed as aneuploid. The final assessment of aneuploidy was based on results of 1189 haploid oocytes. Thirty-four (3%) of the examined oocytes were aneuploid. Disomy of Chromosome 1 and Chromosome 10 was found in 12 of 34 and 8 of 34 of the aneuploid oocytes, respectively. Nullisomy of Chromosome 1 and Chromosome 10 was found in 8 of 34 and 6 of 34 of the aneuploid oocytes. No significant differences were found in the frequencies of disomies and nullisomies of oocytes or in the frequencies of aneuploidies of Chromosomes 1 and 10. The frequency of aneuploid oocytes determined by FISH seems to be higher than that determined by conventional methods in other laboratories.

[Decontamination of continual cell lines spontaneously infected with mycoplasmas]. / Dekontaminace kontinuálních bunecných linií spontánne infikovaných mykoplazmaty.

The continual cell lines of bovine kidneys MDBK and AUBEK, and porcine kidneys RPD and IBRS, spontaneously infected with Mycoplasma arginini and Acholeplasma laidlawii, were decontaminated by the method of selective elimination. Two elimination procedures were modified to be used for the decontamination: one based on the reduction of infection by the light treatment of the cultures, the other based on the selection of mycoplasma-free cell population through cell clonation. On the basis of a long-continued control of the cell clones a methodical procedure of the preparation of mycoplasma-free cell lines was worked out.

[Electron microscopy of bovine leukosis viruses (BLV) in a fetal lamb spleen cell line]. / Elektronová mikroskopie viru bovinní leukózy (BLV) v bunecné linii fetální jehnecí sleziny.

The ultra-thin section method, the method of negative staining, and immunoelectron microscopy were used for detecting BLV and for determining its morphological characteristics in the FLS continual cell line used as a virus antigen producer for the ELISA test. Particles of C type, about 110 nm in size, having a structure corresponding to BLV, were detected in the FLS cells on the ultra-thin sections. The viruses were located extracellularly, in cytoplasmic vacuoles, and in different stages of maturation by budding from cell plasma membrane. BLV presence was also demonstrated by immunoelectron microscopy.

[Spontaneous infection of cell lines with mycoplasmas and their detection using the fluorescence method]. / Spontánní infekce bunecných linií mykoplazmaty a její detekce fluorescencní metodou.

The continual cellular lines of bovine kidneys MDBK and AUBEK and of monkey kidneys VERO were infected spontaneously in the course of long-range cultivation by the mycoplasmas M. arginini and A. laidlawii. The mycoplasmic infection reduced significantly the growth activity of the cells and influenced negatively their morphology. The method of an indirect proof of mycoplasmas by bisbenzimid Hoechst suited well to demonstrate the infection; it had been modified to a simple and rapid test. The starting infection could be demonstrated already at the time when the amount of infected cells in the culture was relatively low.

[Detection of enzootic bovine leukemia virus using the syncytium test]. / Prükaz viru enzootické leukózy skotu (BLV) syncytiálním testem (SyT).

BLV detection by the syncytial test was performed in 27 heifers experimentally and naturally infected by the enzootic bovine leukosis virus (BLV). The presence of BLV was demonstrated in 94.7% of the animals. The bovine foetal spleen cells (FBS) were found to be suitable for the syncytial test. Positive animals not reacting to infection by the production of anti-BLV antibodies were identified during the syncytial-test investigation. The importance of this finding for the programme of controlling enzootic bovine leukosis on farms is discussed. As suggested by the results, temporary occurrence of anti-BLV antibodies followed by their disappearance can be observed together with a negative result of the syncytial test in some circumstances. The discussion deals with the problems of the determination of anti-BLV antibodies in milk, and/or milk secretion, by the ELISA method.

[Experimental and natural infection with the enzootic leukosis virus of cattle]. / Experimentální a prirozená infekce virem enzootické leukózy skotu (BLV).

A trial was performed with heifers at the age of six to seven months. The animals were experimentally infected with the lymphocytes of a virus-productive donor. Infection was produced in all the nine cases, as demonstrated by means of the positive syncytial test. As indicated by the results of the trial, the antibodies to the enzootic bovine leucosis virus (BLV) were produced soon after experimental infection. A high sensitivity of the serum-neutralization test and the ELISA method was demonstrated in this connection: by these methods, the antibodies were identified already two to three weeks after experimental infection whereas by the immunodiffusion test they could be detected only after five weeks. Twenty-four animals were exposed to natural contact infection. Within 270 days of the trial, the disease after contact was recorded only in one heifer out of the four that were in close contact with the experimentally infected animals. In this case, as compared with experimental infection, the antibodies were produced much later--after 85 to 93 days. Leucosis was recorded in none of the remaining animals. The reasons why such a favourable result was obtained were the thorough disinfection of the stables after blood collections and the strict observance of the aseptic conditions. The results of experimental infection in three cows were identical with those obtained in young cattle. In the experimentally infected dairy cows, antibodies in milk were determined by the ELISA method. As found, in milk the antibodies to BLV appear two to three weeks later than they do in serum. The ELISA method of BLV antibody detection can be used for the identification of infected animals in herds where enzootic bovine leucosis occurs.

[Detection of PI-3 viruses, BVD-MD viruses and parvoviruses in bovine fetuses after experimental intrauterine infections]. / Detekce viru PI-3, BVD-MD a parvoviru, persistujicích v bovinních fétech po experimentální intrauterinní infekci.

Growing organ cultures prepared from foetuses experimentally infected in utero by the viruses PI-3 and BVD-MD or bovine parvovirus proved to be suitable in tests with the re-isolation of these viruses 7, 14, 21, 35, 42 and 70 days after infection. The attempts at demonstrating them by the traditional method of the inoculation of the primary cell culture of foetal kidneys were successful only at the re-isolation of the PI-3 virus seven days after infection. The PI-3 virus without cytopathic effect was demonstrated in the primary cell culture prepared from the spleen, lungs, kidneys and testes of foetuses delivered seven days from infection. The cytopathic effect manifested itself after cell transplantation in the first passage when further multiplication of the virus occurred.

[Multiplication of the IBR, PI-3 and BVD-MD viruses in cell and organ cultures of bovine fetuses after experimental intrauterine infections]. / Multiplikace viru IBR, PI-3 a BVD-MD v bunecných a orgánových kulturách pripravených z hovezích fétu po experimentální intrauterinní infekci.

The intrauterine infection of four- to nine-month-old bovine foetuses with the PI-3, BVD-MD viruses, performed 7 to 72 days prior to their delivery, did not exert any significant influence upon the susceptibility of primary cell cultures from foetal organs and tissues to further viral infection in vitro. The BVD-MD and IBR viruses multiplied in the primary cell cultures from the organs of a foetus infected with the PI-3 virus seven days before delivery even in the presence of endogenous PI-3 virus. Persisting infection with the PI-3 virus also failed to influence the susceptibility of foetal organ cultures to infection with the IBR and PI-3 viruses in vitro. The IBR virus and endogenous PI-3 virus multiplied simultaneously to high titres in the organ cultures of thymus and lungs whereas in the organ cultures of kidneys, spleen and testes the multiplication of endogenous PI-3 virus was suppressed.

[Chromosome Y microdeletions in Czech men with severe reproductive disorders]. / Mikrodelécie chromozómu Y u ceských muzov s t'azkou poruchou reprodukcie.

BACKGROUND: The Y chromosome microdeletions belong to the frequent genetical causes of male infertility. The aim of our study was to introduce reliable molecular genetic diagnosis of Y chromosomal microdeletions and to determine the prevalence of Y chromosomal microdeletions in Czech males with serious reproductive disorders. METHODS AND RESULTS: The Y chromosome microdeletions were screened in 198 Czech men with serious reproductive disorders with decreased sperm count. The Y chromosome microdeletions were disclosed in 8/198 (4.0%) examined males. The AZFc deletion type was revealed in 62.5% (5/8) and the combined AZFc + b microdeletion in 37.5% (3/8) of cases. Neither isolated AZFb nor AZFa microdeletion were found in any subject of the investigated group. CONCLUSIONS: Incidence of individual types of Y chromosomal microdeletions in Czech males with serious reproductive disorders was assessed. The standardised molecular genetic diagnosis of Y chromosomal microdeletions was introduced into the practice.

[Medical genetics in reproductive medicine]. / Vyuzití lékarské genetiky v reprodukcní medicíne.

Reproductive genetics (RG) is another new field of medical genetics, integrated with reproductive medicine, assisted reproduction and developmental genetic. RG is closely linked to the perioconceptional prevention, perinatology, ultrasound and biochemical screening in the end of the first and beginning of the second trimesters. RG is based on the system of specialized genetic counseling, clinical cytogenetics, molecular cytogenetics and molecular genetics to provide prefertilization, preimplantation and classical prenatal diagnosis in the Ist to IIIrd trimesters. Thus, RG is part of the fetal medicine and therapy. The six years experience with RG is summarized. A system of the specialized health care, organized, if possible in one integrated center of RG and reproductive medicine (RM) is presented. Reproductive medicine provides all necessary clinical gynecological and andrological surveillance, with assisted reproduction and further obstetrical ultrasound examinations, including nuchal translucency measurements and 2D, 3D ultrasound, echocardiography examinations, if indicated, as well as the invasive method of prenatal diagnosis and perinatology care. Specialized genetic counseling and cytogenetic analysis, if indicated, should be offered to all partners with reproductive disorders as well as to oocyte donors. Chromosome anomalies are disclosed in 6% of men with abnormal sperm analysis as well as in women with severe reproductive disorders. In males with severe oligo, azoospermia, the sperm aneuploidy analysis by molecular cytogenetic methods is recommended. Advised is also the molecular genetic detection of Y chromosome microdeletions, which is detected in 9% of our azoospermic men with deletions in AZFb region. CFTR gene mutations and intron 8 and 10 polymorphism examination is provided not only in men with obstructive azoospermia (CBAVD), but also if severe oligospermy with less than 1 x 10(6) sperm/ml is detected. Molecular genetic analysis of thrombophilic mutations of factor II., V. (Leiden) and MTHFR gene in unexplained recurrent abortions and in cases with unsuccessful IVF is part of the diagnostic strategy. The population frequencies of carriers of mutations of factor II. (2.3%), factor V.-Leiden (5.7%) and MTHFR gene (38%) were determined. The laser biopsy of the first polar body and of blastomeres was introduced for FISH analysis of chromosome aneuploidies. Quantitative fluorescent PCR (QFPCR) detection is used for testing of the most frequent delta F508 CFTR gene mutation and the most frequent aneuploidies of chromosome 13, 18, 21, X and Y. QFPCR was successfully tested for male fetal sex examination from partially purified fetal cells in the maternal blood. The first trimester ultrasound and biochemical screening is recommended to all successful pregnancies after different IVF methods. If borderline levels of first trimester biochemical screening of PAPP-A protein and beta hCG are detected without pathological ultrasound findings, classical triple test of biochemical screening in 16th week of gestation is recommended. If pathological results of ultrasound and biochemical screening are disclosed, invasive prenatal genetic diagnosis is indicated as well as in pregnancies after ICSL, if there is not any obstetrical contraindication.

Association of the transcription profile of bovine oocytes and embryos with developmental potential.

Although improvements in culture system have enhanced in vitro embryo production, success rates are still not adequate. The reasons for developmental arrest of a part of in vitro produced embryos are unknown, but are connected in part with low cytoplasmic competence of oocytes. The immaturity of cytoplasm can negatively influence fertilization efficiency and subsequent progression through embryonic genome activation (EGA), which are necessary steps in further pre-implantation development. A large number of studies have compared mRNA abundance among oocytes with different developmental competence with the aim to find markers of the normal embryo development. The amount of mitochondrial DNA (mtDNA) and mRNA for mitochondrial transcriptional factors directing oxidative phosphorylation belongs to such promising markers. Nevertheless, recently published studies revealed that the mammalian embryo is able to compensate for a reduced level of mtDNA in oocyte during subsequent pre-implantation development. The search for other molecular markers is in progress. Characterization of oocyte and embryonic mRNA expression patterns during the pre-implantation period, and their relationship to the successful in vitro and in vivo development will be essential for defining the optimized culture conditions or the nuclear transfer protocols. Microarrays technology enables us to reveal the differentially expressed genes during EGA, and to compare the expression profile of in vivo and in vitro produced embryos. Recent evidence indicates that the depletion of the pool of stored maternal mRNAs is critical for subsequent embryo development. All these experiments gradually offer a list of possible candidates for quality and developmental competence markers for mammalian oocytes and pre-implantation embryos.

Efficiency of in vitro fertilization is influenced by the meiotic competence of porcine oocytes and time of their maturation.

The effect of meiotic competence of oocytes and time of their maturation on the efficiency of fertilization was studied in pigs. Cycling gilts with synchronized estrous cycles were used as oocyte donors. To obtain oocytes with different meiotic competence, oocytes were recovered separately from small and medium follicles in the early, middle and late luteal or early follicular phase. They were matured for 40 h, 43 h or 47 h and fertilized by spermatozoa of a proven boar. The penetration and monospermy rates, and total efficiency of fertilization were assessed. The same data were related to the follicle size, with or without regard to the phase, and to the maturation time. Regardless of the phase and the time of maturation, the monospermy rate and total efficiency of fertilization were significantly lower for the small follicle-derived oocytes than for the medium follicle-derived oocytes (38.5±10.4% vs 63.1±7.0% and 24.7±6.3% vs 42.5±3.8%). With regard to the phase, in the small follicle-derived oocytes, the monospermy rate increased significantly (P<0.05) from the early luteal to the late luteal phase (from 25.4±2.4% to 46.4±3.9%) and remained unchanged in the early follicular phase. A similar tendency was observed in the total efficiency of fertilization. No differences were found in either of these parameters in medium follicle-derived oocytes in the late luteal and early follicular phase. With regard to the time of maturation, the total efficiency of fertilization was significantly higher (P<0.05) in the small follicle-derived oocytes matured for 47 h than in those matured for 40 h (27.7±7.4% vs. 20.5±6.1%) and in the medium follicle-derived oocytes matured for 40 h as compared with those matured for 47 h (47.1±1.9% vs. 32.7±1.1%). With regard to the phase and the time of maturation, the differences were significant only in the late luteal and early follicular phases. It can be concluded that greater meiotic competence of porcine oocytes positively influences monospermy rate and total efficiency of fertilization process. However adequate time of maturation is an important factor for oocytes with different meiotic competence to improve the IVF procedure.