Effects of ovarian cortex cell co-culture during in vitro maturation on porcine oocytes maturation, fertilization and embryo development

The objective of the experiments was to evaluate the effects of porcine ovarian cortex cells (pOCCs) during in vitro maturation (IVM) of porcine oocytes on IVM of porcine oocytes, in vitro fertilization (IVF) parameters and subsequent embryo development. The pOCCs was cultured in the 500 microl TCM199 without hormone until the confluence, and then cultured in 500 microl TCM199 supplemented with hormone for 12 h before the oocytes added. Porcine oocytes were co-cultured with the pOCCs monolayers in the co-culture system for 44 h, following fertilized in the mTBM for 6 h. Finally, the presumptive zygotes were cultured for 144 h in the NCSU-23 supplemented with 0.4% BSA. The results showed that matured M II oocytes in the co-culture group were higher than that in the control group (P<0.05). Although penetration did not differ between the co-culture and control groups (P=0.481), polyspermy declined in the co-culture group (P<0.05), whereas male pronucleus (MPN) formation was improved in the co-culture group compared with the control group (P<0.05). More blastocysts developed in the co-culture group than that in the control group (P<0.05); however, the cleavage rates and the mean number cells per blastocyst showed no significant difference between the treated group and the control group (P=0.560 and 0.873, respectively). In conclusion, the presence of the pOCCs monolayers during IVM enhanced the maturation quality of the porcine oocytes, reduced the polyspermy, increased the percentages of MPN formation and blastocyst, but the blastocyst quality was not improved.     

Developmental ability of porcine oocytes matured and fertilized in vitro

The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42 71 ) and with monospermic penetration and male and female pronuclei (32%, 23 71 ) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18 71 and 8%, 6 71 , respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28 288 and 13%, 41 318 , respectively) were also higher (P < 0.05) than in controls (2%, 6 284 and 6%, 16 248 , respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9 288 in PFF-treated group and 2%, 6 284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3 100 ) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.     

Supplementation with vascular endothelial growth factor during in vitro maturation of porcine cumulus oocyte complexes and subsequent developmental competence after in vitro fertilization

The aim of the present study was to investigate whether the effects of vascular endothelial growth factor (VEGF) on porcine cumulus oocyte complexes (COCs) and subsequent blastocyst formation following in vitro fertilization are attributable to improved fertilization and cytoplasmic maturation. Porcine COCs were cultured for 42 h in TCM199 medium with 5 ng/mL human recombinant VEGF, and the resultant metaphase II oocytes were fertilized in vitro. COCs without VEGF supplementation served controls. Supplementation with VEGF during in vitro maturation (IVM) significantly (P < 0.05) improved the blastocyst formation rate and total cell number (46.7 ± 3.1% and 82.8 ± 6.7, respectively) compared with controls (32.5 ± 3.4% and 64.1 ± 5.6, respectively). On day 2, the percentage of four-cell stage embryos was significantly higher in the VEGF-matured group (49.1 ± 2.7%) than in the control (33.1 ± 5.8%), and the percentage of two-cell stage embryos was significantly higher in the control group (10.4 ± 1.4%) than in the VEGF-matured group (6.6 ± 0.9%). At 10 h after the onset of in vitro fertilization (IVF), oocytes with two pronuclei were considered as monospermically or normally fertilized, and oocytes with more than two pronuclei were considered as polyspermically fertilized. Monospermy was significantly higher in VEGF-matured oocytes (47.2 ± 4.3%) than in the control (20.0 ± 2.4%), and polyspermy and sperm penetration per oocyte were significantly higher in the control group (54.4 ± 3.8% and 2.3 ± 0.1, respectively) than in the VEGF-matured oocytes (43.9 ± 3.6% and 1.8 ± 0.1, respectively). Supplementation with VEGF during IVM significantly (P < 0.05) improved male pronuclear formation as compared with the control (91.1 ± 1.9 vs 74.4 ± 3.8%). Type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes (78.0%) than in the control (52.1%). These results suggest that VEGF supplementation during IVM enhanced the developmental potential of porcine IVF embryos through higher male pronuclear formation and higher monospermic fertilization rates as a consequence of improved cytoplasmic maturation.     

Effect of superoxide dismutase(SOD) on pronucleus formation of porcine oocytes fertilized in vitro

This study was undertaken to evaluate the effects of superoxide dismutase (SOD) on pronucleus formation in porcine oocytes fertilized in vitro by frozen-thawed spermatozoa. No differences were found in penetration rates when SOD was added to maturation or fertilization medium at any level tested in first and second experiments. Pronucleus formation rates were higher (P < 0.05) when SOD at 10 and 100 units was added to the maturation medium (46 and 53%, respectively) compared with the controls (26%). On the other hand, when the fertilization medium was supplemented with SOD at different concentrations (1, 10 and 100 units/ml), pronucleus formation rates (55, 52 and 50%) were significantly higher (P < 0.05) than in the control group. In third experiment, the oocytes were cultured in medium with (1 unit/ml) or without SOD for 8, 16, 24 and 32 h after insemination. The penetration rates had a tendency to increase as time of sperm-oocyte culture was prolonged. No significant differences, however, were observed in penetration rates between groups with and without SOD. On the other hand, the pronucleus formation rates were higher in medium with than without SOD at 8 (7 vs 0%), 16 (14 vs 3%), 24 (48 vs 16%; P < 0.01) and 32 h (49 vs 22%; P < 0.05). These findings demonstrate the advantage of culture with SOD on pronucleus formation in porcine oocytes penetrated by spermatozoa. However, SOD does not affect penetration rates and polyspermy.     

Ram-specific effects on in-vitro fertilization and cleavage of sheep oocytes matured in vitro

The present experiments were designed to identify possible male-specific effects on early embryonic development in vitro: Sheep oocytes were matured in vitro for 24-26 h and then fertilized in vitro using equal numbers of viable spermatozoa from 1 of 6 Clun Forest rams. At 15-18 h after insemination, oocytes were either fixed and examined for fertilization and polyspermy or further cultured in modified M 199 medium for 3 days in an oviduct epithelial co-culture system. There were significant differences in 5 separate trials between the rams with respect to the rate of fertilization, degree of polyspermy and cleavage rate after monospermic fertilization. The mean rate of fertilization varied from 89% in Ram B to 43% in Ram C while the percentage of polyspermic eggs varied from 5 to 34%. Both the absolute number of embryos cleaving to the 16-cell stage and the calculated percentage of monospermic eggs reaching the 16-cell stage differed markedly between groups of eggs fertilized by different rams. The results indicate that the development of sheep eggs in vitro is differentially affected by the ram from which the spermatozoa are collected.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

Embryotrophic effects of ethylenediaminetetraacetic acid and hemoglobin on in vitro porcine embryos development

This study investigated the embryotrophic effects of ethylenediaminetetraacetic acid (EDTA) and hemoglobin (Hb) on porcine preimplantation embryo development. Porcine embryos produced by in vitro maturation/fertilization were cultured for 6 days in modified North Carolina State University-23 medium (mNCSU-23) supplemented with EDTA and/or Hb. In Exp. 1, culturing porcine zygotes with 100 microM EDTA significantly increased cleavage frequencies (85.3%) at 48 h post insemination and the number of inner cell mass (ICM) (9.6+/-5.5) compared to the control (7.0+/-2.8). However, 100 microM EDTA did not improve blastocyst formation compared to 0, 1 or 10 microM EDTA. In Exp. 2, in vitro fertilized oocytes were cultured with 0, 1 or 10 microg/ml Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the cell numbers of blastocysts in 1 microg/ml Hb compared to 0 or 10 microg/ml Hb. In Exp. 3, culturing embryos with 100 microM EDTA+1 microg/ml Hb significantly improved frequencies of cleavage, blastocyst formation, and total cell numbers in blastocysts compared to the control. Moreover, 100 microM EDTA, 1 microg/ml Hb and their combination reduced reactive oxygen species (ROS) accumulation and decreased the incidence of apoptosis. In conclusion, the present study clearly demonstrated that the combining treatment of EDTA and Hb improved IVF porcine embryo development.     

The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.     

Zona drilling enhances fertilization by mouse caput epididymal sperm

Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500-1,000 sperm/oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 (11.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 (16.7%) oocytes, 50% of those fertilized developing to blastocyst (P = 0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.     

High oxygen tension during in vitro oocyte maturation improves in vitro development of porcine oocytes after fertilization

This study was conducted to evaluate the effect of oxygen tension during IVM and/or IVC on developmental competence of porcine follicular oocytes. Prospective, randomized experiments were designed, and oocytes were matured, inseminated and cultured in vitro in the designated condition. In experiment 1, either high (20%) or low (7%) oxygen tension was used for IVM. The high oxygen significantly improved blastocyst formation (23% versus 13%; P<0.01) after IVF than the low oxygen. Such treatment, however, did not significantly (P>0.05) improve the rates of nuclear maturation (89% in each treatment), sperm penetration (62-72%), monospermic fertilization (56-67%), pronuclear formation (90-96%), cleavage (49-53%) and blastocyst cell number (31-32 cells). In experiment 2, the combined effect of oxygen tension during IVM and IVC of embryos was evaluated by a 2 x 2 factorial arrangement. Again, the high oxygen tension during IVM supported blastocyst formation more efficiently (P<0.01) than the low oxygen, and this was independent of oxygen tension during IVC (26-28% versus 15-16%). In oocytes matured under the high oxygen, a tendency to increase blastomere number (P=0.0630) was found, when the low oxygen was used for IVC after insemination (39-45 cells/blastocyst). In conclusion, the use of high oxygen tension (20% maintained by exposure to 5% CO2 in air) for IVM of porcine oocytes promoted blastocyst formation in vitro.     

Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-alpha-glucoside during in vitro maturation.

The present study was conducted to examine the effect of ascorbic acid 2-O-alpha-glucoside (AA-2G), a stable ascorbate derivative, on the sustenance of cytoplasmic maturation responsible for subsequent developmental competence after in vitro fertilization of porcine oocytes. Cumulus-oocyte complexes were cultured for 44 h in North Carolina State University 37 medium supplemented with cysteine, gonadotropins, 10% (v:v) porcine follicular fluid, and 0-750 microM AA-2G. When oocytes were matured in the presence of 250 microM AA-2G, their ability to promote transformation of the sperm nucleus into the male pronucleus (MPN) was strongly enhanced after in vitro fertilization. Similarly, the presence of 25 microM beta-mercaptoethanol (ME) enhanced the degree of progression to MPN of penetrated sperm by associating with the increase in intracellular glutathione (GSH) content. Although the AA-2G treatment during oocyte maturation showed no influence on the GSH concentration, significantly higher levels of ascorbic acid (AsA) were detected in these oocytes than in those oocytes cultured without AA-2G (P < 0.05). The length of DNA migration encompassed by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase system, was not increased in the oocytes treated with AA-2G, whereas ME treatment could not block the DNA damage by ROS. These findings indicate that AA-2G in maturation medium can potentiate the cellular protection of oocytes against oxidative stress by continuously supplying AsA. The proportion of development to the blastocyst stage after in vitro insemination was significantly increased in oocytes matured with AA-2G (P < 0.05), and this proportion showed no difference in comparison with that of oocytes treated with ME. These findings suggest that a critical concentration of intracellular AsA, supplied by AA-2G during in vitro maturation, plays an important role in supporting the cytoplasmic maturation responsible for developmental competence after fertilization by prevention of oxidative stress against porcine oocytes.     

Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 microM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 microM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%). In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%). However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.     

Failure of male pronucleus formation is the major cause of lack of fertilization and embryo development in pig oocytes subjected to intracytoplasmic sperm injection

The objectives of this study were 1) to compare the efficiency of intracytoplasmic sperm injection (ICSI) with and without additional artificial stimulation using frozen-thawed sperm and in vitro-matured porcine oocytes and 2) to determine the nuclear anomalies of ICSI oocytes that failed to fertilize or develop. In experiments 1 and 2, we evaluated the effects of additional activation treatments, e.g., electrical stimulus, Ca ionophore (A23187), and/or cycloheximide, on fertilization and development of ICSI porcine oocytes. Significantly higher fertilization, cleavage, and blastocyst rates were obtained for oocytes treated with a combination of ICSI and electrical activation (EA) (P < 0.05) than for those treated with ICSI alone. However, different combinations of electrical and chemical activation treatments did not further improve the rates of fertilization, cleavage, and blastocyst development for ICSI embryos. To elucidate the association between sperm head decondensation and oocyte activation and to investigate the cause of embryonic development failure, in experiment 3 we evaluated the nuclear morphology of oocytes 16-20 h after ICSI. Nearly 100% of oocytes showed female pronucleus formation after ICSI regardless of activation treatment. However, failure of male pronucleus formation with intact or swelling sperm heads was observed in some ICSI embryos, suggesting that these embryos underwent cell division with the female pronucleus only. Artificial activation (EA and A23187) had a beneficial effect on embryonic development, sperm decondensation was independent of the resumption of meiosis, and the failure of formation of a male pronucleus was the major cause for fertilization failure in porcine ICSI embryos.     

Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 × 10⁸ cells/mL with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 ± 0.5 × 10⁴ to 575.0 ± 56.3 × 10⁴ cells/mL; mean ± SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 ± 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 ± 4.8%) than those at 7.5 mm (53.1 ± 6.0%) or further (41.9 ± 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 ± 0.040) than both standard IVF and transient IVF (0.222 ± 0.028 and 0.189 ± 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 ± 2.3%) than in either standard or transient IVF (22.6 ± 1.4 and 33.7 ± 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.     

In vitro production of pig embryos: comparisons of culture media and boars

The utilization of in vitro produced pig embryos for commercial production or research is dependent upon the development of improved methodology. Our objective was to establish a consistent in vitro embryo production (IVP) system and subsequently utilize the procedures to evaluate culture system components and boar effects. To summarize the IVP system, 403 inseminated oocytes from a total of 2243 were analyzed across 17 replicates for maturation and fertilization efficiency, while 1838 zygotes were cultured in 26 replicates for developmental data. Penetration, cleavage and blastocyst development rates were determined at 18, 44 and either 144 or 168 h post insemination, respectively. Monospermic penetration averaged 31.8+/-7.3% while polyspermy was 30.8+/-17.2%. Cleavage rate was 44.9+/-16.1%, with 21.8+/-7.5% of fertilized oocytes and 51.9+/-15.9% of cleaved embryos developing to blastocysts. For culture medium comparison, fertilized oocytes were cultured in either BECM-6, BECM-7, NCSU-23 or NCSU-23aa and supplemented on Day 5 post insemination (pi) with 10% FCS. These treatments resulted in 4.0, 4.9, 19.8 and 13.6% (+/-3.2%) blastocysts by Day 7 pi, with an average cell number of 44.4+/-9.0, 65.1+/-8.2, 61.3+/-4.5 and 64.4+/-4.8, respectively. These IVP procedures consistently produced zygotes from semen of several different boars, capable of forming blastocysts in vitro. Comparison of developmental rates among the boars indicated that this system is variable among boars but not strictly boar-dependent. Culture media comparisons suggest that NCSU-23 yielded a higher percentage of blastocysts than the other media in this IVP system.     

Effects of hexoses on in vitro oocyte maturation and embryo development in pigs

The objective was to determine the effects of supplementing hexoses in oocyte maturation and embryo culture medium on in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes and in vitro development of in vitro produced (IVP) porcine embryos. In the first experiment, oocytes were matured in vitro in modified North Carolina State University (NCSU)-37 medium, supplemented with hexoses (glucose, fructose or galactose) at various concentrations: 0 (control), 2.5, 5.5 and 10 mM. Supplementing the maturation medium with either glucose or fructose (5.5 mM) increased the percentages of oocytes that matured to metaphase II (79.4 and 70.2%, respectively), as compared with the control group (P < 0.05). However, supplementing galactose had no effects on meiotic maturation and fertilization. In the second experiment, cleaved embryos were collected 3 days after IVF of oocytes matured in the maturation medium supplemented with 5.5 mM of glucose; they were cultured for an additional 4 days in modified NCSU-37 medium, supplemented with 5.5mM of glucose, fructose or galactose. The incidence of blastocyst formation was higher (P < 0.05) in the glucose and fructose groups (18.6 and 18.2%, respectively) than in the galactose group and non-supplemented control group (12.9 and 9.2%). Moreover, fructose supplementation increased the total cell number/blastocyst (48.0 versus 37.6) and reduced the index of DNA-fragmented nucleus in the blastocysts (7.6% versus 11.8%), as compared with glucose supplementation (P < 0.05). In conclusion, fructose was a practical alternative to glucose for supporting IVM of porcine oocytes and fructose was superior to glucose for producing high-quality porcine embryos in vitro.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Development of in vitro-matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.     

Effects of hyaluronic acid on the development of 1- and 2-cell porcine embryos to the blastocyst stage in vitro

The purpose of this study was to evaluate the ability of hyaluronic acid to improve the development of 1- and 2-cell porcine embryos to the blastocyst stage in a simple medium. In Experiment 1, we confirmed the ability of Whitten's medium supplemented with 15 mg/ml BSA to support the development of porcine embryos to the blastocyst stage under our experimental conditions. Embryos collected from oviducts were cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in humidified air up to 6 d. After 2 d of culture, 82 and 78% of embryos reached the 4-cell stage or beyond in TCM199 supplemented with 10% fetal calf serum (FCS) and in Whitten's medium with BSA, respectively. However, no embryo developed to the morula stage in TCM199 after 6 d of culture. On the other hand, 26 and 15% of embryos developed to the morula and the blastocyst stage in Whitten's medium, respectively. In Experiment 2, we determined whether supplementation of hyaluronic acid in Whitten's medium would improve the development of porcine embryos to the blastocyst stage. After 6 d of culture, development of the embryos to the blastocyst stage was best supported in Whitten's medium with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid (70%). The proportion of degenerated embryos was lower in the presence than in the absence of hyaluronic acid. These results indicate that the supplementation of Whitten's medium with hyaluronic acid improves the development of 1- and 2-cell porcine embryos to the blastocyst stage.     

SD大鼠体外受精与早期胚胎体外培养体系的初步建立

目的改造IVF-30和HTFplus培养液,观察在改造后培养液里卵的受精和早期胚胎的发育状况,寻找适合SD大鼠体外受精和早期胚胎培养的实验体系。方法采用两种方法(超数排卵及体内成熟)采集成熟卵母细胞用于体外受精。选择两种商品化的培养液IVF-30(Vitrolife),HTFplus(Lifeglobal)。分别在以上的培养基中增加10、20、30mmol/L的NaCl,将培养液的渗透压提升至325~355mOsM之间,观察在改造前与改造后的培养液内SD大鼠卵母细胞受精率和囊胚发育率等指标。结果经改造的培养液HTFplus和IVF-30,体外受精率分别从13%、15%提升至70%、67%。改造后的IVF-30和HTFplus的2细胞,大于4细胞及囊胚的发育率分别为85%,74%,22%;96%,75%,32%。HTFplus,IVF-30经过改造后更适合于SD大鼠的体外受精及早期胚胎培养。与超排相比较更多的在自然周期的体内成熟卵母细胞(16%,28%)发育到了囊胚。结论成功建立了SD大鼠的体外受精和体外培养系统。