The quality of porcine oocytes is affected by sexual maturity of the donor gilt

Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (≥ 120 μm), more BCB+ oocytes and more high quality (both BCB+ and ≥ 120 μm) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported.     

Gilts and sows produce similar rate of diploid oocytes in vitro whereas the incidence of aneuploidy differs significantly

Oocytes derived from prepubertal gilts show reduced developmental competence when compared to oocytes collected from adult sows. Therefore, the aim of the study was to investigate whether gilts (4-5 months old) and adult sows (average age 3.5 years) of the same breed (Polish Landrace x Polish Large White crossbred) differ with regard to the rate of chromosomally unbalanced oocytes after IVM. COCs derived from individual pairs of slaughterhouse ovaries were matured in vitro and analyzed cytogenetically by conventional staining (Giemsa) and FISH methods (probes corresponding to centromeric regions of pig chromosomes 1 and 10). Altogether, 72 females (31 sows, 41 gilts) and 430 secondary oocytes (194 and 236 oocytes of sows and gilts, respectively) were investigated. Cytogenetic analysis revealed diploid (Giemsa, FISH) and aneuploid (FISH) spreads. The incidence of diploid oocytes was similar for sows (26.0%) and gilts (24.5%) whereas the rate of aneuploid oocytes (nullisomic/disomic) was eight times higher in gilts (10.8%) than in sows (1.3%). Diploid and aneuploid oocytes were observed in 64% of investigated females. Pig chromosome 10 was more frequently disomic/nullisomic compared to chromosome 1 suggesting, that like in human, small porcine chromosomes are often involved in the nondisjunction process. In conclusion, chromosomal imbalance significantly contributes to in vitro embryo production in the pig, since over 60% of females produced diploid or aneuploid gametes. The significantly higher rate of aneuploidy among oocytes derived from gilt ovaries may contribute to the reduced developmental competence of gametes collected from nonmature female pigs.     

Selection of prepubertal goat oocytes using the brilliant cresyl blue test

Brilliant cresyl blue stain allows us to determine the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with decreased activity in oocytes that have finished their growth phase. The objective of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth, in order to select competent prepubertal goat oocytes for in vitro embryo production. Oocytes were exposed to BCB diluted in PBS and were classified according to their cytoplasm coloration: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocytes without a blue cytoplasm or growing oocytes (BCB-). After exposure to different BCB concentrations, we evaluated in vitro maturation (IVM), in vitro fertilization (IVF) and embryo development parameters. We defined matured oocytes as those oocytes that reached the metaphase II (MII) stage after being cultured for 27 h. Oocytes showing two pronuclei at 20 h post-insemination were classified as normally fertilized oocytes. We assessed embryo development 8 days post-insemination and recorded the percentage of total embryos, morale and blastocysts. The mean percentage of BCB+ oocytes was 29.4%. Mean diameter of BCB+ oocytes (136.6+/-6.3 microm) was higher (P < 0.001) than that of BCB- oocytes (125.5+/-10.2 microm). The percentage of BCB+ oocytes reaching the MII stage (81.4%) was higher (P < 0.05) than that of BCB- (52.5%) and control oocytes (72.4%). Normal fertilization rate of BCB+ oocytes was also higher (23.5%) than that of BCB- (8.2%; P < 0.0001) and control oocytes (11.9%; P < 0.05). The percentages of total embryos undergoing development to >8-cell and the morula plus blastocyst stages were higher (P < 0.05) in the group of BCB+ (41.3 and 12.0%, respectively) than in BCB- oocytes (21.3 and 3.6%, respectively). In conclusion, the BCB test is a useful way to select more competent prepubertal goat oocytes for in vitro embryo production.     

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly (P<0.01) larger than NCL ovaries (width: 22.3+/-0.9 mm versus 15.9+/-0.4 mm, length: 33.2+/-1 mm versus 24.1+/-0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1+/-1.8 mm versus 46.8+/-2.2 mm), they had a similar number of follicles 3-8mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5+/-0.1 mm versus 3.3+/-0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159+/-1.3 microm versus 146.1+/-1.5 microm, zona free: 124.7+/-1.8 microm versus 113.1+/-1.6 microm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6+/-3.2% versus 92.4+/-3.2%), cleavage (88.4+/-11% versus 90.7+/-2.6%) and blastocyst formation (21.0+/-3.7% versus 23.7+/-5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.     

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation

The aim of this study was to assess the effect of oocyte selection using the brilliant cresyl blue (BCB) test plus the addition of cysteamine to the in vitro maturation (IVM) medium to improve the in vitro embryo development of prepubertal goat oocytes. The oocytes were exposed to 26 microM BCB and classified according to their cytoplasm coloration: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The oocytes were matured in a conventional IVM medium supplemented with cysteamine 100 microM. The control group consisted of oocytes not exposed to BCB and matured without cysteamine. The IVM-oocytes were inseminated and cultured in synthetic oviductal fluid (SOF) for 7 days. The normal fertilisation rate (oocytes showing 2 pronuclei and 1 sperm tail) of BCB+ oocytes (40%) was higher than those of BCB- (21%) and control oocytes (22%). The percentage of morulae plus blastocysts was higher (P < 0.05) in the BCB+ group than in the BCB- group (23.8 vs. 5.1%, respectively). In conclusion, the integration of the BCB test and the addition of cysteamine in the protocol of in vitro embryo production from prepubertal goat oocytes has improved the developmental rates of embryo development.     

Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test

Our previous studies have shown that the addition of 100 mircroM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB-). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 microM, 200 microM or 400 microM cysteamine. In Experiment 2, oocytes were matured with 400 microM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 microM and 100 microM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 microM and 200 microM cysteamine showed higher normal fertilization and embryo development rates than BCB- oocytes. Oocytes matured with 400 microM cysteamine did not present these differences between BCB+ and BCB- oocytes. In Experiment 2, the addition of 50 microM and 100 microM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB- oocytes (34.23% and 29.04%, respectively, P < 0.05). In conclusion, the addition of 400 microM cysteamine to the IVM improved normal fertilization and embryo development of BCB- oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.     

Cell coupling and maturation-promoting factor activity in in vitro-matured prepubertal and adult sheep oocytes.

We examined some differences between prepubertal and adult ovine oocytes; in particular we analyzed the functional status of the cumulus-oocyte complex, protein synthesis during in vitro maturation, and because no information is available on prepubertal and adult sheep, maturation-promoting factor (MPF) fluctuations throughout meiotic progression both in prepubertal and adult sheep oocytes. After 24 h of maturation, percentages of MII oocytes were similar between prepubertal and adult animals. Electron microscopy examinations showed that prepubertal oocytes had fewer transzonal projections than adult oocytes. Methionine uptake was significantly lower in prepubertal cumulus-enclosed oocytes examined through meiotic progression. On the contrary, denuded prepubertal oocytes showed a higher methionine incorporation in the first 4 h of incubation compared with adult oocytes. We also found some differences in MPF activity between prepubertal and adult oocytes at MII stage. In fact, prepubertal MII oocytes had a significantly lower level of MPF activity than adult oocytes did and, after fusion with germinal vesicle oocytes, they were unable to induce nuclear breakdown and chromosome condensation 1-2 h post-fusion, whereas adult MII oocytes could induce these processes. Our findings show that the lesser competence of prepubertal oocytes could be due to morphological anomalies and alterations in physiological activity and that oocytes do not reach full developmental competence until puberty.     

Cytogenetic analysis of caprine 2- to 4-cell embryos produced in vitro

Prepubertal goat in vitro matured/in vitro fertilised oocytes produce only a small percentage of blastocysts. The present study examines the incidence of chromosomal anomalies in 2- to 4-cell embryos in vitro produced (IVP) from prepubertal oocytes fertilised with the semen of two males. Cumulus-oocyte complexes were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% heat inactivated Donor Bovine Serum (DBS), 10 microg/ml FSH + 10 microg/ml LH + 1 microg/ml 17beta-oestradiol for 27 h at 38.5 degrees C in 5% CO2 in air. IVM oocytes were inseminated with the sperm from two males prepared using the swim-up and heparin-capacitation procedures. At 24 h postinsemination (hpi) the oocytes were transferred to 100 microl drops of SOF medium for a further 24 h. At 17 hpi a sample of oocytes was stained with lacmoid to evaluate the nuclear stage after fertilisation. The cleavage rate was determined at 24, 36 and 48 hpi and chromosome slides were prepared according to the gradual-fixation technique and stained with Leishman. A total of 1070 2- to 4-cell embryos from prepubertal goat oocytes were studied, but it was only possible to analyse 241 cytogenetically. Of these, 40% exhibited a normal diploid chromosome complement, 59% were haploid and 1% were triploid. There were significant differences between the two males in sperm oocyte penetration and oocyte cleavage but no differences were found in chromosomal anomalies. In conclusion, the low number of embryos karyotyped and the high number of haploid embryos found in this study suggested a high incidence of abnormal fertilised embryos and deficient cytoplasmic maturation of the oocyte which inhibits sperm head decondensation.     

No single way to explain cytoplasmic maturation of oocytes from prepubertal and cyclic gilts

The objective of this study was to evaluate selected aspects of cytoplasmic maturation in oocytes from prepubertal and cyclic crossbred gilts before and after in vitro maturation. For this purpose, cortical granule redistribution, mitochondrial DNA content and mitochondria translocation were analyzed. Moreover, for the first time the fatty acid profiles in follicular fluid (FF) of both gilt categories was evaluated. The nuclear maturation (the percentage of metaphase II oocytes was 83% in prepubertal gilts compared with 87% in cyclic gilts), cortical granule relocation from the cortex to peripheral ooplasm (98.7% vs. 98.8% of oocytes, respectively) and mitochondrial DNA content (227 543 vs. 206 660, respectively) was not affected by sexual maturity of the donor gilt. However, the redistribution of active mitochondria during in vitro maturation was observed only in the oocytes of cyclic gilts. With regard to FF analysis, saturated, unsaturated, and monounsaturated fatty acids were significantly more abundant in the FF of prepubertal females. In particular, stearic (C18:0) and palmitic (C16:0) fatty acids had significantly higher concentrations in the FF of prepubertal gilts. In conclusion, although the oocytes of prepubertal gilts matured in vitro at a rate similar to those of cyclic gilts, they differed with respect to the selected factors attributed to cytoplasmic maturation. We suggest that the higher content of particular fatty acids, which is known to have a negative influence on oocyte maturation, as well as impaired mitochondria redistribution are factors limiting the maturation potential of oocytes from prepubertal gilts.     

Effect of the addition of glutathione and glucose to the culture medium on embryo development of IVM-IVF prepubertal goat oocytes

Our previous studies have shown that larger and more competent oocytes can be selected using the brilliant cresyl blue (BCB) test. The objective of this study was to assess, in BCB-selected oocytes, the effect on the embryo development of prepubertal goat oocytes of the addition to in vitro culture (IVC) medium of either glutathione (GSH) alone or GSH in combination with glucose. Oocytes were exposed to 26 mM BCB and were classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocyteswithout blue cytoplasm or growing oocytes (BCB-). Oocytes were matured in TCM-199 with 100 microM cysteamine. Presumptive zygotes were cultured in synthetic oviductal fluid (SOF) in the presence or absence of 1 mM glutathione (experiment 1) for 7 days (8 days post-insemination, p.i.). In experiment 2 we tested the addition to culture of 2.78 mM glucose at day 5 p.i. BCB+ oocytes showed higher percentages of nuclear maturation than the BCB- and control groups (82.6%, 55.7% and 74.7%, respectively). The percentage of polyspermic oocytes was higher in BCB- than BCB+ oocytes. Supplementation of SOF medium with 1 mM GSH did not affect embryo development but the percentage of total embryos developed after culture was higher in BCB+ oocytes than in BCB- oocytes independently of the GSH supplementation. Glucose, alone or with GSH, added at 5 days p.i. did not affect embryo development. In conclusion, prepubertal goat oocytes were unable to develop beyond the 8-cell stage embryo under the culture conditions in this study.     

Chromosome abnormalities in secondary pig oocytes matured in vitro

Abnormalities of chromosome segregation during in vitro maturation of oocytes cause failure of in vitro fertilization. Oocytes collected from pig ovaries after slaughter were matured in vitro (IVM) for 30-48 h. In total, 1144 secondary oocytes were studied cytogenetically. An unreduced (diploid) chromosome set was identified in 146 spreads (12.8 %). A higher proportion of diploidy was noticed in secondary oocytes matured for 40 h and longer (15.0 %) than in the groups matured for 30 and 36 h (9.0 %). Among 998 secondary oocytes with the reduced chromosome number, 612 could be analyzed in detail. Hypohaploidy (n=19-1) was identified in 22 cells (3.59 %) and a hyperhaploid (n=19+1) set of chromosomes was identified in 15 cells (2.45 %). The rate of aneuploidy, estimated by doubling the rate of hyperhaploidy was 4.9 %. It was also found that aneuploid spreads occurred more frequently in the group of oocytes matured for 40 h and longer. Small acrocentrics were mostly found as an extra chromosome in the hyperhaploid spreads. Our study indicates that to avoid an excess of chromosomally abnormal secondary oocytes, IVM duration of pig oocytes should not exceed 40 h.     

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.     

Elimination of X-ray-induced chromosomal aberrations in the progeny of female mice

Female NMRI mice were irradiated with various doses of X-rays and induced chromosome aberrations were scored in MII oocytes (Dosage: 0.222, 0.666, 2 and 6 Gy). After irradiation with 2 Gy, early zygotes were examined in the 2-cell stage; additional dominant lethals were counted and surviving embryos were examined after 13.5 days of pregnancy. 87.2% of the MII oocytes showed structural chromosomal aberrations after irradiation with 2 Gy. Surviving embryos, however, failed to show any increase in the aberration rate. This result points to (almost) complete elimination of genetically damaged oocytes and zygotes already before birth. In addition to the structural aberrations, aneuploidies were induced. Most of them, however, were hypoploidies. Hence, the study confirmed the well-known susceptibility of oocytes around the time of fertilization for induced chromosome loss. Induced hyperploidies, however, were very rare. Evidence for induction of meiotic non-disjunction was weak. In surviving embryos, no increase in numerical aberrations, either hypoploid or hyperploid was discovered. The significance of these data for the prediction of chromosomal damage due to to ionizing radiation in humans is discussed. Recent risk estimates of UNSCEAR and other agencies represent very cautious upper levels.     

M-phase promoting factor (MPF) and mitogen activated protein kinases (MAPK) activities of domestic cat oocytes matured in vitro and in vivo

This work was undertaken in order to examine M-phase promoting factor (MPF) and mitogen-activated protein kinases (MAPK) activities during meiotic progression of cat oocytes cultured in two different media for two different incubation times and preovulatory cat oocytes that reached MII in vivo. Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 or SOF for 24 h and 40 h. In vivo matured oocytes were recovered by follicular aspiration from ovaries of domestic cats ovariectomized 24 h to 26 h after hormonal treatment. Results showed that the kinetic of MPF and MAPK activity was similar during meiotic progression of cat oocytes matured in TCM 199 and SOF. After 24 h of incubation, MII oocytes had significantly (p < 0.001) higher MPF and MAPK levels than MII oocytes cultured for 40 h in both culture media. MPF and MAPK activity was significantly (p < 0.01) lower in the oocytes matured in vitro than in those matured in vivo. This study provides evidence that the two different maturation media did not determine differences in MPF and MAPK fluctuations and levels during meiotic progression of cat oocytes and that the time of maturation influenced the level of the two kinases. Moreover, it shows that MPF and MPK activity is higher in in vivo matured oocytes than in in vitro matured oocytes, suggesting a possible incomplete cytoplasmic maturation after culture.     

The ploidy of in vitro matured bovine oocytes is related to the diameter

The aim of the present study was to investigate a possible relationship between bovine oocyte diameter and the ploidy after maturation in vitro. The cumulus-oocyte-complexes (COCs) were collected by slicing slaughterhouse ovaries and were matured in vitro in standard conditions. Oocytes were collected separately from each ovary and then processed in groups according to their origin. After maturation, the inside zona pellucida diameter of each cell was measured and cytogenetic slides were made. Four size categories were distinguished: <110, 110-115, 115-120 and >120 microm. Altogether, 600 oocytes derived from single ovaries of 50 donors were measured and cytogenetically analyzed. The diploid chromosome number (2MII) was found for 8.4% of oocytes (36/427) and for 44% of donors (22/50). The observed number of 2MII cells varied between 1 and 6 per donor. The size of secondary oocytes with unreduced chromosome numbers was significantly smaller (P < 0.01) than the haploid ones. We conclude that bigger oocytes underwent normal meiotic division, whereas their smaller counterparts tended to follow an abnormal path of maturation.     

Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured-in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed sermatozoa. Oocytes and presumptive zygotes were treated with anti-alpha-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 microg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.     

In vitro maturation and fertilization of prepubertal goat oocytes

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.     

Frequency of aneuploidy in in vitro-matured MII oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds as determined by dual-color fluorescent in situ hybridization

The current study was undertaken to investigate the aneuploidy rates in in vitro-matured meiosis II (MII) oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds by using dual-color fluorescent in situ hybridization (FISH). A total of 159 and 144 in vitro-matured MII oocytes of the Italian Friesian and Italian Brown breeds, respectively, were obtained according to the standard methods and analyzed by FISH using “Xcen” and “5” chromosome-specific painting probes, produced by chromosome microdissection and Degenerate Oligonucleotide Primer- Polymerase Chain Reaction (DOP-PCR). Oocytes with unreduced chromosome number were 10.1% and 16.7% in the two breeds, respectively. To avoid bias due to possible artifacts, the aneuploidy rates were determined by analyzing only oocytes with the corresponding polar bodies. In the Italian Friesian, 100 of 143 (69.9%) secondary MII oocytes showed clear MII plates with corresponding first polar bodies and were scored for aneuploidy detection; one oocyte was “nullisomic” for chromosome X (1.0%) and one “disomic” for chromosome 5 (1.0%). In the Italian Brown, 100 of 120 (83.3%) MII oocytes with corresponding first polar bodies were analyzed; one oocyte was nullisomic (1.0%) and one was disomic (1.0%), both for chromosome 5. Totally, 303 oocytes were analyzed, 40 of which showed an unreduced chromosome complement (13.2%); of 200 MII oocytes with the corresponding first polar bodies, the aneuploidy rate (nullisomy + disomy) for the two chromosomes scored was 2%. Assuming that each chromosome is equally involved in aneuploidy, it results that in cattle oocytes matured in vitro, at least 30% of the oocytes (1 × 30 haploid chromosomes) should be aneuploid. Premature separation of sister chromatids (PSSC) was also observed in 2% of the oocytes in the Italian Friesian breed involving chromosome 5 and in 1% of the Italian Brown breed involving the X chromosome. Estimation of the “baseline” level of aneuploidy in the in vitro-matured oocytes of the various domestic animal species and breeds is, to our opinion, a useful reference for improving the in vitro production of embryos as well as for monitoring future trends of the reproductive health of the species/breeds engaged in zootechnical productions, especially in relation to management errors and environmental hazards.     

Effect of vitrification solutions and cooling upon in vitro matured prepubertal ovine oocytes

The vitrification procedure effects on molecular and cytoskeletal components and on developmental ability of in vitro matured prepubertal ovine oocytes were evaluated. MII oocytes were divided into three groups: (1) vitrified in cryoloops (VTR); (2) exposed to vitrification solutions and rehydrated without being plunged into liquid nitrogen (EXP); (3) without further treatment as a control (CTR). Two hours after treatment, membrane integrity, assessed by propidium iodide/Hoechst staining, was lower in VTR and EXP than in CTR (70.6%, 88.5% and 95.2%, respectively). Cleavage rate after fertilization was statistically different among all groups (21.4%, 45.4% and 82.8% for VTR, EXP and CTR groups respectively; P<0.01). Blastocyst rate in VTR (0.0%) and EXP (2.8%) groups was lower (P<0.01) than in CTR (22.8%). Maturation promoting factor activity was lower (P<0.01) in VTR and EXP groups compared with CTR at both 0 h (82.2%, 83.6% and 100%, respectively) and 2 h (60% and 53.9% and 100%, respectively) after warming. Immediately after warming VTR and EXP oocytes showed a lower rate of normal spindle and chromosome configuration compared to CTR (59.1%, 48.0% and 83.3%, respectively; P<0.01). After 2 h of culture in standard conditions the percentage of oocytes with normal spindle and chromosome organization decreased in both VTR and EXP groups compared to CTR (36.4%, 42.8% versus 87.5%, respectively). In conclusion the exposition to the tested cryoprotectant solution and the vitrification in cryoloops modified cytoskeletal components and alter biochemical pathways that compromise the developmental capacity of prepubertal in vitro matured ovine oocytes.