Developmental competence of pig oocytes matured and fertilized in vitro

Pig follicles 3 to 6 mm in diameter were everted and matured for 44 h. The oocytes were then collected and exposed to capacitated boar sperm purified by centrifugation in a two step (65 and 70%) Percoll gradient. Of 110 ova fixed 14 h after in vitro fertilization, 78% were penetrated and 47% were monospermic. Next, 681 oocytes were cultured in vitro for 44 h after in vitro fertilization and the 266 embryos which had reached the two- to four-cell stage were transferred into the oviducts of 12 synchronized recipient gilts. Four days later, 211 embryos (79%) were recovered by uterine flushing. 40.7% of these were at the blastocyst stage, and 20% were at the morula stage. In a final experiment, four out of eight gilts which had received 40 to 50 two- to four-cell embryos, were diagnosed pregnant 30 and 37 d after in vitro fertilization. One sow farrowed nine live piglets and one stillborn, two pregnancies were in progress, while one sow returned to estrus 47 d after in vitro fertilization. These results demonstrate that pig oocytes matured and fertilized in vitro can develop to the blastocyst stage and establish a normal pregnancy resulting in the birth of live piglets.     

Effect of hyaluronic acid on the development of porcine 1-cell embryos produced by a conventional or new in vitro maturation/fertilization system

In pigs, it is difficult to produce normal fertilized embryos from immature oocytes in vitro. However, a new maturation/fertilization system in which the percentage of normal fertilized embryos is comparatively high has been developed recently. In the present study, porcine 1-cell embryos were produced both by a conventional and a new system and then cultured in NCSU-23 supplemented with hyaluronic acid at various concentrations. In the conventional system, the percentage of oocytes with monospermic penetration and 1 male pronucleus and 1 female pronucleus was only 6%. At 144 h after insemination, the percentage (5%) of embryos developing to the blastocyst stage in medium supplemented with 0.5 mg/mL hyaluronic acid was significantly (P<0.05) higher than that (2%) in medium without hyaluronic acid. When oocytes were matured and inseminated using the new system, monospermic penetration and the formation of 1 male and 1 female pronucleus were observed in 69% of the penetrated oocytes. However, blastocyst formation (8 to 14%) at 144 h after insemination was not affected by the concentration (0 to 1.0 mg/mL) of hyaluronic acid. These results indicate that the effect of hyaluronic acid on the development of in vitro-produced porcine embryos varies with the conditions of oocyte maturation and fertilization.     

Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum

Recently, piglets have been obtained from in vitro-produced blastocysts by using in vitro maturation systems in which oocytes have been matured in North Carolina State University (NCSU) solution supplemented with porcine follicular fluid (PFF). However, PFF is not available commercially. To prepare PFF from the ovaries required time and effort and there is substantial variation in quality among batches. Furthermore, PFF is considered a potential source of infectious agents. We evaluated another commercially available potential protein source, fetal bovine serum (FBS), for in vitro maturation, to produce embryos and piglets. Cumulus-oocyte complexes were matured in NCSU-37 with PFF or with one of four batches of FBS. The proportions of oocytes with expanded cumulus cells were lower in all FBS batch groups (P < 0.05, 15-41%) than that in the PFF group (74%). The proportions of oocytes that matured were also lower in all FBS batch groups (P < 0.05, 26-41%) than in the PFF group (73%), irrespective of cumulus expansion. However, the proportions of oocytes that underwent germinal vesicle breakdown were almost the same in all groups (76-96%). After in vitro fertilization, the rate of sperm penetration into matured oocytes was higher in the PFF group (P < 0.05, 63%) than in one batch of FBS (22%) and removal of the compacted cumulus cells after maturation did not affect fertilization status (21%). Subsequent in vitro embryo culture of the PFF and FBS groups for 6 day resulted in similar rates of blastocyst formation (17 and 19%, respectively) and similar numbers of cells per blastocyst (43 and 46 cells, respectively). When blastocysts obtained from oocytes matured with FBS were transferred into two recipients, one became pregnant and farrowed seven piglets. Transfer of blastocysts obtained from oocytes matured with PFF into two other recipients resulted in one pregnancy and production of four piglets. These data suggested that porcine in vitro maturation in NCSU-37 supplemented with FBS reduced the maturational ability of oocytes, but once oocytes have matured, they have the same ability to develop to term after in vitro fertilization and embryo transfer as those matured with PFF.     

Development potential of bovine oocytes matured in vitro or in vivo

Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.     

Pregnancy resulting from cattle oocytes matured and fertilized in vitro

Follicular oocytes (n = 81) collected from cattle at a local slaughterhouse were matured and fertilized in vitro. Of 27 ova 19 (70%) were penetrated by spermatozoa and 40/54 (74%) inseminated ova transferred surgically to the oviducts of a synchronized heifer were recovered by non-surgical flushing of the uterine horns 6 days later. Of the 40 ova 15 (38%) were at the morula, early blastocyst or diminutive morula stages. Culture in vitro sustained further development of all embryos and 9 were expanding or expanded blastocysts. One pregnancy resulted from non-surgical transfer of 2 blastocysts. The results demonstrate that immature oocytes from cattle can be matured and fertilized in vitro, subsequently develop to the blastocyst stage, and develop into a normal pregnancy after non-surgical transfer.     

The difference in embryo quality between Belclare and Suffolk ewes is not due to differences in oocyte quality

We have previously reported that the percentage of fertilized oocytes which reached the blastocyst stage by Day 6 after AI with frozen-thawed semen was higher for Belclare (94%) than Suffolk (59%) ewes. This may reflect differences in the timing of fertilization (Experiment 1) or differences in oocyte quality (Experiments 2 and 3). In Experiment 1, oocytes recovered from slaughterhouse ovaries were matured in vitro for 18, 20, 24, 28 or 30 h prior to fertilization and were then cultured in vitro. In Experiment 2, Belclare (n = 69) and Suffolk (n = 71) ewes were laparoscopically inseminated using frozen-thawed semen. Presumptive zygotes were recovered between 23 and 47 h post-insemination and cultured in vitro (grouped by breed). In Experiment 3, immature oocytes from Suffolk and Belclare ewes, were matured, fertilized and cultured in vitro (grouped by breed). Cleavage rate and blastocyst development was assessed. There was no effect of time of fertilization on cleavage rate, however, a lower proportion of cleaved oocytes reached the blastocyst stage after insemination at 30h compared to 24 h (P < 0.001). Ewe breed did not affect cleavage rate of oocytes matured and fertilized in vivo (41+/-9.6 and 47+/-10.1) or in vitro (47+/-9.4 and 52+/-9.4) for Belclare and Suffolk ewes, respectively (P > 0.05; %+/-S.E.). Likewise, ewe breed had no effect on the percentage (+/-S.E.) of cleaved oocytes developing to the blastocyst stage for in vivo (29+/-7.2 and 25+/-7.9) or in vitro matured and fertilized oocytes (29+/-6.1 and 36+/-5.9) from Belclare and Suffolk ewes, respectively (P>0.05). Based on this study oocyte quality does not differ between the breeds and in addition a 4h difference in the timing of fertilization, reflective of the breed difference in the timing of the LH surge in vivo, would not affect early embryo development.     

Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro

Developmental ability of porcine ova matured in porcine follicular fluid (pFF) with FSH in vitro and fertilized in vitro was examined by culturing in BMOC-2. Forty-eight hours after insemination, 35.6% of ova cleaved normally, and this rate was significantly higher (13.0%) than that of the ova matured in a modified Krebs-Ringer bicarbonate solution. Twenty-four percent (29 120 ) of ova matured in pFF with FSH developed to the four-cell stage and two of them developed to the eight-cell stage 66 h after insemination. Most cleaved embryos stopped developing at the four-cell stage and neither the morula nor blastocyst stage was observed throughout the culture period as reported in the in vivo matured ova. In culture at 37 degrees C, the appearance of two-cell and four-cell embryos was delayed from that of in vivo embryos, but their development was significantly accelerated by culturing at 39 degrees C. These results show that pFF is an excellent maturation medium for porcine oocytes, and the developmental capacity of the ova matured in pFF seems to be similar to that of in vivo matured ova. Culturing at 39 degrees C was found to be more suit-able for the development of ova than 37 degrees C.     

Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells.

Control ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty-one (38%) reached morula/blastocyst stage. Twenty-one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty-one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.     

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.     

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.     

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.     

Maturation and fertilization of porcine oocytes in vitro

Four experiments were conducted to study 1) factors affecting porcine oocyte maturation in culture medium and 2) a new method for oocyte maturation outside the porcine body. In Experiment 1, five groups of oocytes were cultured in m-TCM199 or m-KRB medium for 24 to 28, 32 to 36 or 40 to 42 hours and then were fertilized in vitro. The cleavage rate (two to four-cell stage) of oocytes cultured for 32 to 36 hours was significantly higher than those of the other oocytes. The results indicate that a suitable culture period for the in vitro maturation of porcine oocytes is 32 to 36 hours. In Experiment 2, four groups of oocytes were cultured in m-KRB or m-KRB supplemented with PFF, PMSG or FSH for in vitro maturation, and the cleavage rates of oocytes were 7.94, 22.56, 30.23 and 23.26%, respectively, after in vitro fertilization. The results show that porcine follicular fluid (PFF) and gonadotrophins added to the culture medium promote porcine oocyte maturation in vitro. In Experiment 3, oocytes were cultured in m-KRB or m-TCM199, supplemented with both gonadotrophin and pocine folliclar fluid for maturation in vitro. After fertilization in vitro, the cleavage rates of oocytes were 26.32 and 27.93% for the two media. The results indicate that the difference between m-KRB and m-TCM199 was insignificant when the media were used to culture porcine oocytes. But there was a significant difference when PFF and gonadotrophins were added to the basic media. In Experiment 4, porcine oocytes were transferred into the reproductive tracts of other animals for maturation. After 34 to 36 hours, the oocytes were collected and fertilized in vitro. The cleavage rates of oocytes were 10.42, 28.45, 3.33 and 36.36%, respectively, for the oocytes matured in mouse uterine horns, rat uterine horns, rat oviducts or rabbit oviducts. The results show that porcine oocytes can be matured in the reproductive tracts of other animals.     

Birth of normal calves resulting from bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro up to the blastocyst stage

Bovine oocytes matured in vitro were fertilized in high proportions (92% of matured oocytes) by sperm capacitated with Ca ionophore A23187. Eight percent of inseminated oocytes that were denuded 96 h after insemination developed to the morula stage when cultured for 6-120 h after insemination with cumulus cells from the original oocytes. Inseminated oocytes denuded 96 h after insemination developed to the blastocyst stage when cultured with or without cumulus cells or in the conditioned medium from 96 h to 168-216 h after insemination (9.0%, 8.1%, and 6.8% of inseminated oocytes respectively). Six frozen-thawed blastocysts were transferred nonsurgically to 3 recipients (2 embryos/recipient). Two of the 3 recipients became pregnant, with one delivering live twins at term. Seven fresh blastocysts were transferred nonsurgically to 6 recipients (1-2 embryos/recipient). Three of the 6 recipients became pregnant, with 2 delivering live calves.     

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology,fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.     

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.     

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.     

Meiotic arrest maintained by cAMP during the initiation of maturation enhances meiotic potential and developmental competence and reduces polyspermy of IVM/IVF porcine oocytes

We investigated effects of invasive adenylate cyclase (iAC), 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) on porcine oocyte in vitro maturation (IVM), in vitro fertilisation (IVF) and subsequent embryonic development. Porcine oocytes were collected in Hepes-buffered NCSU-37 supplemented with or without 0.1 microg/ml iAC and 0.5 mM IBMX. IVM was performed in a modified NCSU-37 supplemented with or without 1 mM dbcAMP for 22 h and then without dbcAMP for an additional 24 h. After IVF, oocytes were cultured in vitro for 6 days. After 12 h of IVM, no difference in nuclear status was observed irrespective of supplementation with these chemicals during collection and IVM. At 22 h, most (95%) of the oocytes cultured with dbcAMP remained at the germinal vesicle (GV) stage, whereas 44.3% of the oocytes cultured without dbcAMP underwent GV breakdown. At 36 h, oocytes cultured with dbcAMP had progressed to prometaphase I or metaphase I (MI) (32.6% and 49.3%, respectively), whereas non-treated oocytes had progressed further to anaphase I, telophase I or metaphase II (MII) (13.6%, 14.3% and 38.0%, respectively). At 46 h, the rate of matured oocytes at MII was higher in oocytes cultured with dbcAMP (81%) than without dbcAMP (57%), while the proportion of oocytes arrested at MI was lower when cultured with dbcAMP (15%) than without dbcAMP (31%). The rate of monospermic fertilisation was higher when oocytes were cultured with dbcAMP (21%) than without dbcAMP (9%), with no difference in total penetration rates (58% and 52%, respectively). The blastocyst rate was higher in oocytes cultured with dbcAMP (32%) than without dbcAMP (19%). These results suggest that a change in intracellular level of cAMP during oocyte collection does not affect maturational and developmental competence of porcine oocytes and that synchronisation of meiotic maturation using dbcAMP enhances the meiotic potential of oocytes by promoting the MI to MII transition and results in high developmental competence by monospermic fertilisation.     

Cloned piglets born after nuclear transplantation of embryonic blastomeres into porcine oocytes matured in vivo

Nuclear transplantation in the pig is more difficult than in other domestic animals and only one embryonic nuclear transplantation (NT) pig has been born to date. In this study, reconstituted porcine embryos were produced by electrofusion of blastomeres from in vivo four-cell embryos to enucleated in vivo or in vitro matured (IVM) oocytes. Nuclear transfer using cumulus cells as nuclear donors was also conducted. When blastomeres were used as donors, the electrofusion rate was significantly higher in oocytes matured in vivo (91.5%) than in those matured in vitro (66.1%) (p < 0.01). After fusion, the NT embryos reconstituted from in vivo matured oocytes developed to blastocysts at a rate of 10.3% after culture in rabbit oviducts for up to 5 days, while only 5.9% of the NT embryos reconstructed from in vitro matured oocytes developed to blastocyst stage. Electrofusion rate of cumulus cell nuclei with enucleated IVM oocytes was lower (47.6%) and only 1.5% (2/136) of the reconstituted eggs developed in vitro to morula stage, and 1.9% developed to blastocysts when cultured in the ligated rabbit oviducts. Transfer of 94 embryos reconstructed by blastomere NT with in vivo matured oocytes to five synchronous recipients resulted in the birth of two cloned piglets. No piglet was born following transfer to two recipients of embryos (n = 39) derived from NT with in vitro matured oocytes. The results demonstrate that in vivo matured oocytes are better recipients than those matured in vitro for pig cloning.     

Effect of low temperatures on in-vitro matured bovine oocytes

This research concerned effects of cooling in vitro matured bovine oocytes on subsequent fertilization and development in vitro. Oocytes were maintained at 39 degrees C (control), 20 degrees C, 10 degrees C or 0 degree C for 5, 10, or 20 min, then fertilized and cultured in vitro for 7 d. The proportion of fertilized oocytes that cleaved and developed to the morula/blastocyst stage was compared between different treatments. Duration of exposure had no effect on the results. Fertilization rate was higher (P < 0.05) for oocytes maintained at 39 degrees C (73.2%) than for oocytes cooled at 20 degrees C (58.6%), 10 degrees C (47.3%), or 0 degree C (36.9%). Cleavage rates were 58.3, 45.3, 15.7 and 7.0% for 39 degrees C, 20 degrees C, 10 degrees C and 0 degree C, respectively (P < 0.05). The lowest development rate to the blastocyst stage was obtained with oocytes cooled to 10 degrees C (0.0%) or 0 degree C (0.9%), followed by 20 degrees C (7.1%) and 39 degrees C (16.5%; P < 0.05). In a second experiment, the zona pellucida was removed after cooling but prior to fertilization (zona-free) from a portion of the in vitro- matured bovine oocytes in each treatment. When sperm penetration rates of zona-free oocytes were compared (percentage of oocytes exhibiting > or = 2 pronuclei), there was no difference (P > 0.05) between oocytes cooled at 0 degree C (59.7%) or 10 degrees C (67.9%). However, penetration rates in these 2 groups were lower (P < 0.05) when compared to zona-free oocytes cooled at 20 degrees C (83.1%) or those maintained at 39 degrees C (83.1%). Zona-free oocytes had higher penetration rates (P < 0.05) when cooled at 0 degree C (59.7%) or 10 degrees C (67.9%) than zona-intact oocytes cooled at 0 degree C (37.3%) or 10 degrees C (47.2%). However, there was no difference in the penetration rate when zona-free and zona-intact oocytes were cooled at 20 degrees C or maintained at 39 degrees C. These data demonstrate that cooling in vitro-matured bovine oocytes decreases the percentage of oocytes that undergo fertilization and subsequently develop in vitro. Moreover, at least part of the decrease in fertilization following oocyte cooling is due to effects on the zona pellucida.     

Antioxidant requirements for bovine oocytes varies during in vitro maturation,fertilization and development

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.