Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes.

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.

Algriany O ，Bevers M ，Schoevers E ，Colenbrander B ，Dieleman S ... -  《THERIOGENOLOGY》

Effect of follicular fluid collected from various diameter follicles on the progression of nuclear maturation and developmental competence of pig oocytes.

Supplementing in vitro maturation medium with porcine follicular fluid (FF) improves maturation rate, male pronucleus formation, and monospermic fertilization of pig oocytes. This study examined, (1) if there are differences in FF derived from large follicles (LF, 5-6mm in diameter) and small follicles (SF, 3-4mm in diameter) on the effect of supplementing the maturation medium with FF on the progression of nuclear maturation, fertilization rate, and developmental competence of porcine oocytes; (2) whether the FF source influences the effect of the FF on the maturation medium on the survival rate and proliferation rate of cumulus cells (CCs) and the expansion of cumulus-oocyte-complexes (COCs); (3) whether the oocyte source (oocytes collected from LFs or SFs) influences the effect of FF on the progression of the nuclear maturation of oocytes; (4) whether the factors in the FF that affect the kinetics of nuclear maturation are proteins, and the range of the molecular weight of the FF factors. In experiment 1, adding FF from LFs (LFF) significantly accelerated nuclear maturation and improved the fertilization rate; the developmental ratio was comparable with those of adding FF from SFs (SFF). In experiment 2, adding LFF, but not SFF, improved the CC survival rate, although the FF source did not affect the proliferation rate. Expansion of COCs was greater with SFF than LFF. In experiment 3, LFF promoted nuclear maturation of oocytes collected from only LFs. There was a significant interaction between the FF source and the oocyte source in the effect on nuclear maturation stages at 36 h of maturation. In experiment 4, treatment of FF with heat or trypsin diminished the difference between the effect of LFF and SFF on the progression of nuclear maturation. In addition, the predominant effect of LFF compared to that of SFF on nuclear maturation was not affected by ultrafiltration of the FF with a 30-kDa filter, but was diminished by ultrafiltration with a 100-kDa filter. The present study suggests that some proteins present in LFF that range in molecular weight from 30 to 100 kDa improve the developmental competence of oocytes probably via progression of nuclear maturation and cumulus cells viability.

Ito M ，Iwata H ，Kitagawa M ，Kon Y ，Kuwayama T ，Monji Y ... -  《ANIMAL REPRODUCTION SCIENCE》

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.

Bijttebier J ，Van Soom A ，Meyer E ，Mateusen B ，Maes D ... -  《THERIOGENOLOGY》

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.

Suzuki M ，Misumi K ，Ozawa M ，Noguchi J ，Kaneko H ，Ohnuma K ，Fuchimoto D ，Onishi A ，Iwamoto M ，Saito N ，Nagai T ，Kikuchi K ... -  《THERIOGENOLOGY》

Effect of the removal of cumulus cells on the nuclear maturation, fertilization and development of porcine oocytes.

The present study was conducted to investigate the effects of attachment of cumulus cells to porcine oocytes during the process of maturation and fertilization on the nuclear maturation, fertilization and subsequent development after in vitro fertilization (IVF). In the first experiment, the cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 24 and 42 h after the onset of maturation culture and were then cultured until reaching 42 h of cultivation. In the second experiment, COCs were denuded as described in the first experiment, then fertilized and cultured for 7 days. As a control, cumulus cells were allowed to maintain attachment to the oocytes until the end of IVF. The proportion of oocytes reaching metaphase II significantly increased with the delay in the removal treatment of cumulus cells. The proportion of normal fertilization gradually increased with delay in the removal treatment of cumulus cells from COCs until the end of IVF. However, no significant difference in the proportion of normal fertilization was found between the 42-h and control groups. The removal treatment of cumulus cells in the 0- and 24-h group significantly (p < 0.05) decreased the proportion of cleaved embryos when compared with the control, and none of them developed to the blastocyst stage. The proportion of development to the blastocyst stage was significantly higher (p < 0.05) in the control group than in the 42-h group (18.1% vs 12.4%; p < 0.05). The present study indicates that the attachment of cumulus cells to the oocyte during maturation and fertilization is important to support oocyte nuclear maturation, fertilization and subsequent embryo development. Particularly, the attachment of cumulus cells to the oocyte during IVF promotes embryonic development.

Wongsrikeao P ，Kaneshige Y ，Ooki R ，Taniguchi M ，Agung B ，Nii M ，Otoi T ... -  《REPRODUCTION IN DOMESTIC ANIMALS》