M.T. Paramio

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.

Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test

The aim of this study was to evaluate the usefulness of the brilliant cresyl blue (BCB) test in the selection of more competent heifer oocytes for in vitro embryo production (IVEP). IVEP from selected BCB heifer oocytes was compared to IVEP from morphologically selected heifer (control group) and cow oocytes. BCB staining determines the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with less activity in grown oocytes. Six hundred and fifty seven heifer cumulus-oocyte complexes (COC) were classified morphologically as Grade 1-3 and exposed to 26 microM of BCB and classified as: blue (or grown) oocytes (BCB+) or unstained oocytes or growing oocytes (BCB-). Grade 1-3 heifer oocytes showed significantly different percentages of BCB+ oocytes (78.6, 66.2, and 51.1%, respectively; P<0.05). The diameter of BCB+ oocytes was significantly higher than BCB- oocytes (152.6+/-5.8 microm and 147+/-5.9 microm, respectively; P<0.001). The percentage of BCB+ oocytes reaching the blastocyst stage was significantly higher than those of BCB- and control heifer oocytes (12.3, 1.6, and 5.2%, respectively; P<0.05), but lower than those of cow oocytes (30.0%; P<0.05). In conclusion, heifer oocytes selected by the BCB test (BCB+) are larger and more competent for IVEP than control heifer oocytes. However, fewer heifer oocytes selected using the BCB test develop to blastocyst stage compared to cow oocytes.

Meiotic competence of prepubertal goat oocytes.

The object of this work was to evaluate in vitro maturation of follicular oocytes from the ovaries of prepubertal goats obtained from the slaughterhouse. To obtain the oocytes, follicles were dissected and classified according to their diameters. In the first experiment, oocytes were matured in vitro with granulosa cells. No significant differences were detected in the percentages of maturation between adult and prepubertal goat oocytes recovered from follicles of 2.5 to 6.0 mm in diameter (81.82 vs 72.47%, respectively). The percentage of maturation increased to 88.0% in prepubertal goat oocytes from 3.0 to 6.0-mm follicles. In the second experiment, the percentage of maturation of prepubertal goat oocytes was greater after 27 than after 24 h. In the third experiment, the maturational capacity of prepubertal goat oocytes according to follicular diameter was evaluated. The percentages of maturation after 27 h of culture with no granulosa cells were 24.14, 56.60 and 74.78%, respectively, for follicles 1.0 to 1.9 mm, 2.0 to 2.9 mm, and 3.0 to 6.0 mm in diameter. As the follicular diameter increased, growth of the oocyte as well as a greater number of oocytes with more cumulus cell layers were observed. A correlation between the diameter of the oocyte and its competence to complete in vitro maturation was also observed. Oocytes with more cumulus cell layers showed only a slight superiority in their capacity for maturation in large-size follicles (3.0 to 6.0 mm), but the difference was not significant. In conclusion, oocytes from prepubertal goats complete their growth and reach meiotic competence in follicles larger than 3.0 mm. With these oocytes it is possible to obtain in vitro maturation results similar to those from adult goats.

Developmental capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats

The developmental competence of oocytes from prepubertal and adult goats was studied through in vitro maturation, fertilization and embryo culture up to the blastocyst stage. Oocytes were recovered from antral follicles of prepubertal and adult goat ovaries, with or without ovarian stimulation with exogenous FSH. The effect of different sources of granulosa cells during IVM on the developmental competence of prepubertal goat oocytes was also noted. Oocytes were matured for 27 h at 38.5 degrees C in 5% CO(2) in air in 50-microl microdrops in TCM199 supplemented with 20% estrus goat serum, FSH, LH and estradiol-17beta or in 2 ml of the same medium supplemented with granulosa cells. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation At 24 h post-insemination, the oocytes were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 10. Results show that the developmental ability of embryos from prepubertal goats after IVM and IVF is similar to those from adult goats. Treatment of the prepubertal and adult goats with FSH did not improve the developmental capacity of the resulting embryos. On studying the addition of different sources of granulosa cells to a maturation system of 2 ml of medium, a significantly positive effect of the cells from primed females was observed on the percentage of maturation, on embryo cleavage and on the percentage of embryos that overcame the in vitro developmental block from 8 to 16 cells.

EFFECT OF CULTURE MEDIA ON EMBRYO DEVELOPMENT FROM PREPUBERTAL GOAT IVM-IVF OOCYTES

Experiments were carried out to develop and improve in vitro culture systems for IVM-IVF prepubertal goat oocytes. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM-199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air. Matured oocytes were placed in drops of TALP- fert medium supplemented with hypotaurine (1 microgram/mL) and inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (69) but with 100 micrograms/mL heparin. At 24 h post insemination the ova were transferred to various in vitro culture media, and early embryo development was evaluated until Day 8 post insemination. Specifically, in the studies described here, we have compared the effects of (Experiment 1) co-culture systems using oviductal ephitelial cells (OEC) and cumulus cells (CC), both caprine and bovine; (Experiment 2) the presence of serum and/or OEC; (Experiment 3) 4 culture media (TCM199, Hams F10, CZB abd SOF) for co-culture with OEC; and (Experiment 4) conditioned medium with OEC. In Experiment 1, the percentage of morulae plus blastocysts was higher in culture with OEC, both caprine and bovine (15.1 and 14.8%, respectively) than with CC (4.1 and 6.7%, respectively). In Experiment 2, the OEC with EGS did not improve the percentage of morulae and blastocysts obtained with OEC alone (14.3 and 23.1% respectively). In Experiment 3, this percentage was higher using OEC with TCM-199 compared to CZB medium (21.3 and 12.3%, respectively) and in Experiment 4, the results were 3.7, 11.2 and 21.3% for TCM-199 without cells, Conditioned Medium and co-culture with OEC, respectively.

EFFECT OF SEMEN PREPARATION ON IVF OF PREPUBERTAL GOAT OOCYTES

The aim of these experiments was to study the effects of different methods of washing and selection of spermatozoa on the IVF of IVM oocytes from prepubertal goats. Fresh ejaculates from 3 males of proven fertility were processed according to the following treatments: 1) centrifugation in TALP, 2) centrifugation in sucrose-based Ficoll medium, 3) centrifugation in Percoll gradients at 40 and 80%, 4) by swim-up and 5) by dilution of spermatozoa (1:40) in (1:1) TALP. In all 5 treatments spermatozoa were incubated for 45 min with 100 microg/mL of heparin and then added to Fert-TALP. Oocytes were matured for 27 h in TCM-199 supplemented with 20% estrous goat serum (EGS), FSH, LH and estradiol-17beta. Spermatozoa (4x10(6) cells/mL) were coincubated with oocytes in 100 microL of Fert-TALP with hypotaurine for 24 h, after which the oocytes were transferred to a granulosa cells monolayer in TCM-199 plus 10% of EGS for 24 h (48 h post insemination). At 17 h post insemination a sample of sperm-exposed oocytes was taken and stained in lacmoid to observe sperm penetration and the formation of pronuclei. At 48 h post insemination the cleavage rate of oocytes was evaluated. Motility, viability and acrosome status of the spermatozoa were evaluated immediately after the mixing of the ejaculates, after washing and selection treatments, and after incubation with heparin and at 17 h post insemination. The different ejaculate treatments did not affect the penetration and cleavage rates of oocytes. At 48 h post insemination the cleavage rate was 46.9, 36.6 and 29.0% for dilution, Ficoll and swim-up preparations, respectively. Only the swim-up protocol improved sperm motility and viability compared with that of the initial semen sample and with the other sample treatments. At 17 h post insemination the semen parameters were the same for all sperm sample treatments.

Effect of in vitro and in vivo culture on embryo development from prepubertal goat IVM-IVF oocytes

The aim of this study was to analyze different culture systems on embryo development of prepubertal goat oocytes. We compare (i) the effect of the age of donor (goat) of oocytes on in vitro maturation, fertilization and subsequent embryo development, (ii) the effect of the origin of oviduct cells from coculture of prepubertal goat embryo development, and (iii) the effect of in vivo culture in rabbit oviducts for 1, 2 and 3 days on the development of prepubertal goat embryos produced in vitro. In Experiment 1, at 24 h post-insemination (hpi), oocytes from adult goats were allocated in TCM199 with oviduct cells from adult goats, and oocytes from prepubertal goats were randomly placed in drops with oviduct epithelial cells from adult (aOEC) or prepubertal (pOEC) goats. Cleavage rate and embryo development were evaluated at 48 hpi and after 7 days coculture, respectively. In Experiment 2, at 24 hpi, prepubertal oocytes were allocated in TCM 199 with pOEC. At 40-42 hpi, a group of embryos remained in the coculture (control group), and the rest were transferred to rabbit oviducts (three rabbits for replicate) for culturing in vivo for 24, 48 and 72 h. After these in vivo cultures, embryos were recovered, evaluated and placed in TCM199 with pOEC until Day 8 post-insemination. The maturation, fertilization and blastocyst rates did not differ significantly between oocytes obtained from adult and prepubertal goats. The percentage of blastocysts obtained from prepubertal goat embryos cocultured with aOEC or pOEC was also similar (12.1% versus 12.2%). The transfer of prepubertal goat embryos to rabbit oviducts for 1, 2 and 3 days did not improve the blastocyst rate compared to the control group (9.7, 10.9, 4.1 and 11.5%, respectively). In conclusion, in our conditions, there were no significant differences in embryo development between oocytes obtained from prepubertal and adult goats, and the embryo development from prepubertal goat oocytes were similar in the different culture systems compared.

Oocyte secreted factors improve embryo developmental competence of COCs from small follicles in prepubertal goats

Oocytes secrete soluble paracrine factors called Oocyte Secreted Factors (OSFs) which regulate the cumulus cell phenotype. Follicle populations in ovaries from prepubertal females have smaller diameters than their adult counterparts. Oocytes from small follicles are less competent than those from large follicles. The aim of this study was to investigate, in prepubertal goats, the effect of OSFs secreted by denuded oocytes (DOs) from small (<3 mm) or large (>or=3 mm) follicles during IVM on embryo development and the blastocyst quality of cumulus-oocyte complexes (COCs) from small follicles and to determine if GDF9 participates in this process. Treatment groups were: (A) COCs non selected by their follicle size (control group); (B) cumulus oocytes complexes from small follicles (SFCOCs), (C) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from small follicles (SFCOCs + SFDOs), and (D) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from large follicles (SFCOCs + LFDOs). The effect of the addition of kinase inhibitor SB-431542, which antagonizes GDF9, was tested in A, C, and D treatment groups. Co-cultured SFCOCs with SFDOs or LFDOs significantly augmented the blastocyst rate in comparison to SFCOCs alone (15.77%, 17.39% vs. 10.31%, respectively). Blastocysts from SFCOCs + LFDOs group showed higher rates of tetraploid nuclei than blastocysts from SFCOCs and the control group (14.43% vs. 5.45% and 5.24%, respectively; P < 0.05). However, we did not observe differences in the hatching rate, mean cell number or embryo cryotolerance (P > 0.05) between the four treatment groups. The addition of SB-431542 during IVM did not have any effect on blastocyst rate (P > 0.05). In conclusion, in prepubertal goats, COCs with a low embryo developmental competence as a consequence of follicle size can be improved by coculturing them with denuded oocytes from both small and large follicles. GDF9 does not seem play a role in this improvement.

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.

Effect of Hoechst 33342 staining on developmental competence of prepubertal goat oocytes.

This study was undertaken to evaluate the effects of Hoechst staining on nuclear maturation and fertilisation when used at different stages of in vitro maturation (IVM) in prepubertal goat oocytes. Oocytes were matured in TCM1999 supplemented with 10% fetal bovine serum, 10 microg LH/ml, 10 microg FSH/ml and 1 microM 17beta-estradiol for 27 h. Frozen-thawed sperm cells were prepared by centrifugation in a discontinuous Percoll gradient and resuspended in DMH medium with 20% steer serum. Oocytes were fertilised in DMH medium with 7.75 mM calcium lactate. During IVM oocytes were exposed to 0.5 microg/ml of Hoechst 33342 staining and to ultraviolet light for a mean time of 3 s at 0 h, 8 h, 15 h, 20 h and 27 h. The percentage of metaphase II oocytes decreased significantly when oocytes were stained with Hoechst dye at 0 h, 8 h and 15 h of IVM. There was a decrease in total fertilisation rate and normal fertilisation rate of Hoechest-stained oocytes, independently of the time of Hoechst staining. Hoechst staining produces a significant reduction in oocyte viability when it is used in the early stages of in vitro maturation.