Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes

Immature oocytes were collected from immature female rats (60-65 g) 40 h after injection with 6 IU pregnant mare's serum gonadotropin (PMSG). Oocytes were matured cumulus-intact (CI) or cumulus-free (CF) in medium supplemented with 0.5% bovine serum albumin (BSA) or 5-20% serum for periods of up to 24 h. After assessment for nuclear maturation, the oocytes were exposed to epididymal sperm for fertilization in vitro. In vitro-matured and ovulated oocytes undergoing fertilization were transferred to unilaterally pregnant recipients for embryonic and fetal development. The presence of cumulus cells and serum shortened (by 2 h) the time required for polar body emission by in vitro-matured oocytes and also helped to increase significantly the penetrability of the oocytes by spermatozoa. A high proportion (45.6%) of fertilized oocytes showed evidence of abnormal fertilization following maturation in the absence of cumulus cells. Oocytes matured CI before fertilization were able to develop to viable fetuses (57.8%) in proportions similar to ovulated oocytes (55.0%) after in vitro fertilization. These findings indicate an essential role for cumulus cells in promoting normal cytoplasmic maturation of oocytes necessary for pronuclear formation and subsequent developmental capability.     

Influence of culture medium and protein supplementation on in vitro oocyte maturation and fertilization in the domestic cat

Domestic cat oocytes were cultured either in Waymouth MB 753/1 Medium (WAY) or in Eagle's Minimum Essential Medium (MEM) containing FSH, LH and estradiol-17beta and supplememted with one of the following: 5% fetal calf serum (FCS); 4 mg/ml bovine serum albumin (BSA); or 3 mg/ml polyvinylalcohol (PVA, a non-protein control). The oocytes were evaluated for: nuclear maturation after 48 hours of culture (in vitro maturation, IVM); fertilization and cleavage 24 to 30 hours postinsemination (in vitro fertilization, IVF); and early embryo development 48 hours postinsemination. Maturation rates were similar (P>0.05) for WAY + BSA (29.4%), MEM + BSA (46.7%) and MEM + PVA (43.3%), but were different (P<0.05) from the other treatments (range, WAY + FCS, 9.6% to WAY + PVA, 14.9%). Fertilization and cleavage rates were also similar (P>0.05) for WAY + BSA (51.4%, 30.5%), MEM + BSA (45.8%, 40.1%) and MEM + PVA (56.1%, 37.4%) and were greater (P<0.05) than all other treatments. These IVM/IVF oocytes were capable of culturing beyond 2-cells, with the highest proportion of 4- and 8- cell embryos forming in WAY and MEM media in the presence of BSA or in MEM medium containing PVA. In the domestic cat IVM/IVF system: both the type of culture medium and protein supplement influence the proportion of oocytes reaching Metaphase II; the type of protein supplement has a more significant (P<0.05) impact than medium on fertilization, cleavage and early embryo development; and nuclear maturation and fertilization in vitro can proceed in this species in the absence of supplementary protein.     

Pentoxifylline improves in vitro fertilization and subsequent development of bovine oocytes

The purpose of this study was to examine whether pentoxifylline improves in vitro fertilization and developmental rates of bovine oocytes. In the first experiment, we examined the effects on the fertilization rate of various concentrations of pentoxifylline (0-7.5 mM) combined with heparin (10 IU/mL). In the second experiment, we examined fertilization cleavage and blastocyst rates after frozen-thawed spermatozoa, obtained from four different bulls, were incubated with heparin (10 IU/mL) with or without caffeine (5 mM) or pentoxifylline (5 mM). In the first experiment, a significantly higher fertilization rate was obtained in heparin containing 5 mM pentoxifylline compared to that in heparin alone or in heparin containing 7.5 mM pentoxifylline (86% vs 60% vs 64%, respectively). The percentage of monospermy in 5 mM pentoxifylline (81%) was significantly higher than in heparin alone (57%). In the second experiment, the interactions among Bulls A, B, C, and D; between treatments (pentoxifylline-with-heparin, caffeine-with-heparin and heparin alone), and between bulls and treatments were analyzed for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts. Among bulls, there was a significant difference in the number of oocytes penetrated (P < 0.01), monospermic oocytes (P < 0.05), cleaved oocytes (P < 0.001), and blastocysts (P < 0.001). Between treatments, there was a significant difference in the number of oocytes penetrated (P < 0.001), monospermic oocytes (P < 0.01) and cleaved oocytes (P < 0.001). Interaction between bulls and treatments was observed for the number of oocytes penetrated (P < 0.05). Individually, for Bulls A, C and D, the numbers of oocytes penetrated and monospermic oocytes in pentoxifylline-with-heparin were significantly higher than in heparin alone. For Bull D, significantly higher results were obtained for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts in pentoxifylline-with-heparin compared to caffeine-with-heparin and heparin alone (P < 0.05). These results suggest that treating sperm with 5 mM pentoxifylline in combination with heparin is effective for bovine in vitro fertilization and it that this treatment is effective even for bulls that produce low fertilization and blastocysts after sperm treatment with caffeine-with-heparin or heparin alone.     

In vitro maturation, fertilization and culture to blastocysts of bovine oocytes in protein-free media

This study examined the role of protein supplementation at the various steps of the in vitro production of bovine embryos derived from two different morphological categories of COC. The basic medium was TCM 199 and was supplemented with hormones during maturation in vitro and either estrous cow serum (ECS), bovine serum albumin (BSA) at various concentrations or polyvinyl-alcohol (PVA). Fertilization in vitro was carried out using frozen-thawed semen or one bull in Fert-talp containing heparin, hypotaurin and epinephrine and either 6 mg/ml BSA or 1 mg/ml PVA. In vitro culture up to the blastocyst stage was performed in TCM 199 supplemented with either ECS, BSA or PVA. The first experiment investigated the influence of different medium-supplements (ECS, BSA or PVA) on nuclear maturation and revealed no significant differences among treatment groups nor between categories of COC (63.9% to 74.9% and 48.9% to 77.0%, respectively). The time course of in vitro fertilization was elucidated in Experiment 2 in medium supplemented with either protein or PVA during maturation and fertilization. Penetration was not affected (70.9% to 79.3% penetration 12 h after onset of oocyte-sperm-co-incubation), but formation of pronuclei was decreased (P < 0.05) 12 and 19 h after onset of oocyte-sperm-co-incubation and was retarded in medium supplemented with PVA (12 h: 63.8 vs 21.4 %; 19 h: 57.5 vs 20.8 %, respectively) while cleavage was not affected. In Experiment 3, six treatment groups were formed in which the two different morphological categories of cumulus-oocyte-complexes (COC) were incubated in basic medium supplemented with 1) ECS during maturation and embryo culture and BSA during fertilization; 2) PVA during maturation and embryo culture, fertilization medium with PVA; 3) PVA during maturation and embryo culture, fertilization medium with BSA; 4) BSA (1 mg/ml) during maturation, fertilization and embryo culture; 5) BSA (6 mg/ml) during maturation, fertilization and embryo culture; and 6) BSA (10 mg/ml) during maturation, fertilization and embryo culture. The rates of cleavage and the development to morulae or blastocysts did not differ (P > 0.05) among treatment groups and between both categories of COC and were showing a high degree of variability (cleavage 54.0% to 65.1% and 41.3% to 55.7%, respectively; morulae 25.3% to 53.0% and 26.0% to 51.2%, respectively; blastocysts 5.4% to 24.7% and 0.6% to 20.3%, respectively). Parthenogenetic activation only rarely occurred in medium containing PVA throughout all steps of in vitro production of bovine embryos (Experiment 4) and led to early cleavage stages (8%), but no development to morula- or blastocyst-stages was observed. It is concluded that 1) formation of pronuclei was retarded in medium lacking protein-supplementation, indicating that BSA is required for regular fertilization in vitro and 2) under our experimental conditions, protein-supplementation is not necessary for maturation and development up to the blastocyst stage in vitro.     

Influence of the collection technique of prepubertal goat oocytes on in vitro maturation and fertilization

Prepubertal goat ovaries obtained from a slaughterhouse were used to study the influence of the oocyte collection technique (dissection, aspiration and slicing) on the number of oocytes recovered and their capacity for maturation and fertilization in vitro. The oocytes were recovered using 3 techniques, were selected for culture and were classified according to the number of cumulus cell layers. The numbers of oocytes selected per ovary were 1.71, 1.27 and 6.05 for dissection, aspiration and slicing, respectively. The percentages of maturation obtained for slicing (56.9%) were lower than those obtained for dissection and aspiration (69.3 and 72.0%, respectively). The proportion of oocytes with the most cumulus cell layers (complete cumulus) was greatest for oocytes recovered by dissection, but this had no influence on their capacity for nuclear maturation. The total percentage of fertilization was similar for oocytes obtained by dissection and by slicing, but the latter yielded a lower percentage of normal fertilization (29.1 vs 18.2%). Of the oocytes obtained by slicing, no difference was observed in the fertilization rate between oocytes with a partial cumulus and a complete cumulus. The decrease in maturation time from 27 to 25.5 and 24 h did not improve the results for fertilization but caused a decrease in the percentage of nuclear maturation. In conclusion, the recovery of oocytes using the slicing technique yielded more oocytes per ovary than dissection or aspiration, although the in vitro fertilization capacity of oocytes obtained by the slicing method was lower than for oocytes obtained by dissection.     

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.     

Effects of in vitro culture of mouse fetal gonads on subsequent ovarian development in vivo and oocyte maturation in vitro

Under organ culture, female fetal gonads in mice cannot develop beyond the preantral follicle stage unless the follicles are individually isolated and cultured again. In this study, we investigated the effect of in vitro culture of female fetal gonads before transplantation on subsequent in vivo development. The gonads derived from female fetuses 12.5 days postcoitum were organ-cultured for 0, 7 and 14 days, and then were grafted underneath the kidney capsules of severe combined immunodeficient mice and recovered at 21, 14 and 7 days post-transplantation, respectively. The histological analysis of the grafts showed that the in vitro culture of the fetal gonads restricted follicular development to the antral follicle stage post-transplantation. In the grafts cultured for 14 days, particularly, no antral follicle was observed. However, the oocytes in these follicles had grown to around 65 µm in diameter and had competence to resume meiosis in vitro . When the fetal gonads were grafted after culture for 7 and 14 days, 13.0% and 6.8% of the oocytes progressed to the metaphase II stage, respectively. These data showed significant differences ( P  < 0.05) in comparison with the control group (25.3%). Our results indicate that the in vitro culture of female fetal gonads before transplantation affects the subsequent in vivo development of both follicular cells and oocytes, and in vitro oocyte maturation. However, this effect seems to be more severe in terms of follicular development when compared with oocyte growth and maturation.     

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKC alpha isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKC alpha isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.     

Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos

In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; P<0.05), total blastocysts (20.5+/-0.9% versus 15.3+/-1.3%; P<0.05), and hatching blastocysts (16.8+/-1.6% versus 12.0+/-1.5%; P<0.05). The greater survival in terms of hatching (78.6+/-7.0) following chilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.     

In vitro maturation and fertilization of goat oocytes

Goat oocytes were isolated from 3-5 mm diam. follicles. The oocytes with compact cumulus mass were matured and fertilized in vitro. Three different media, viz. modified Krebs-Ringer bicarbonate, Dulbecco's and Ham's F-12 with three different additives (bovine serum albumin, BSA; follicle stimulating hormone, FSH and fetal calf serum, FCS) were tested. The three basal media gave almost similar results with Ham's F-12 being slightly better. Addition of BSA (10 mg/ml) increased the rates of maturation and penetration. FSH + BSA (2.5 micrograms/ml + 10 mg/ml) further enhanced the rates while FCS (10%) proved to be even more effective. In modified Krebs-Ringer bicarbonate and Dulbecco media with additives FCS + BSA, around 60% oocytes matured to metaphase II of which 53% were penetrated by capacitated goat spermatozoa while in F-12 medium 70% reached metaphase II and 63% were penetrated. Ham's F-12 medium with additives FCS + BSA was slightly better for maturation and penetration of goat oocytes in comparison to two other media tested.     

Improvement on in vitro maturation, fertilization and development of minke whale (Balaenoptera acutorostrata) oocytes

The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P < 0.05) maturation rate (31.8%) than those in the medium without FWS (0%). Adding FWS to the maturation medium significantly (P < 0.01) improved the proportion of oocytes at Metaphase II (M-II): without FWS (7.9%), with 10% (19.4%) and 20% (21.4%) FWS. However, sexual maturity of whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P < 0.05) than did Grade A and B COCs cultured in the medium without FWS (0%). Neither Grade A nor B COCs cleaved when the medium was without FWS. The proportions of cleaved oocytes increased (P < 0.05) with FWS supplementation (6.9% and 8.1% for 1.0% FWS and 20% FWS, respectively). Grade A COC was significantly (P < 0.05) superior in its ability to cleave (14.5%) and develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA supplementation in fertilization medium was as effective as FWS supplementation for in vitro fertilization of matured oocytes. In vitro embryo production beyond the morula stage of minke whale oocytes could be possible, if Grade A COC was selected and cultured in the maturation medium supplemented with 10% or 20% FWS.     

Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells

The present study was conducted to determine the effects of cumulus cells and sodium pyruvate during in vitro maturation of bovine oocytes on maturation, fertilization, and subsequent development. Cumulus-enclosed oocytes (CEOs) and cumulus-denuded oocytes (CDOs) were cultured for 24 h in polyvinylpyrrolidone-Hepes-tissue culture medium 199 with or without sodium pyruvate. Oocytes were fertilized in vitro and then cultured in CR1aa for 10 days. Before in vitro fertilization, the glutathione (GSH) content of some oocytes was measured. Maturation and normal fertilization rates of CDOs cultured with sodium pyruvate and CEOs were higher than that of CDOs cultured without sodium pyruvate. The CEOs showed significantly higher rates of development to the blastocyst stage than CDOs. The GSH contents of oocytes significantly decreased in CDOs after maturation culture, but the GSH contents of oocytes in CEOs remained at the same level as oocytes before culture. These results indicate that sodium pyruvate promotes nuclear maturation of bovine CDOs and that a continuing presence of cumulus cells during maturation is important for subsequent development of zygotes to the blastocyst stage. However, blastocysts produced from CDOs in the presence of sodium pyruvate showed a developmental competence to be normal calves, but it is not known if CDOs cultured without sodium pyruvate also were capable of developing into calves.     

Effects of cysteamine, fsh and estradiol-17beta on in vitro maturation of porcine oocytes

Porcine cumulus-oocyte complexes (COCs) were cultured for 48 h with addition or absence of exogenous estradiol-17beta (E2; 1 microg/mL) in the maturation medium (mM199). The medium was supplemented with sodium pyruvate (0.1 mg/mL), 10% (v/v) FCS, various concentrations of FSH (0, 1 and 10 microg/mL) and with or without cysteamine (150 microM). When supplemented with E2, cysteamine enhanced the rates of germinal vesicle breakdown (GVBD) and maturation to metaphase-II (M-II) in COCs cultured in the medium with 0 and 1 microg/mL FSH (P<0.05). Among COCs cultured with FSH, oocytes cultured with 1 microg/mL FSH and E2 but without cysteamine showed the lowest rates of GVBD and M-II. The rates were, however, significantly increased when cysteamine was added to the same medium or by increasing FSH concentration to 10 microg/mL in the maturation medium. E2 significantly inhibited the rates of GVBD and M-II in COCs cultured without FSH and cysteamine (a group of oocytes with spontaneous maturation). When COCs were cultured in TCM 199 with 1 or 10 microg/mL FSH, with or without E2 (1 microg/mL) and fertilized in vitro, the rates of male pronucleus formation were not increased by increasing FSH concentration, but the addition of cysteamine to the maturation medium significantly enhanced the rates in the same FSH treatment. The results indicate that E2 inhibits spontaneous GVBD and maturation to M-II of porcine oocytes and that a low concentration of FSH (1 microg/mL) is not sufficient to induce full nuclear maturation, compared with 10 microg/mL FSH, but that it can complete nuclear maturation with cysteamine and E2. However, the cytoplasmic maturation is promoted only by the addition of cysteamine in the medium.     

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.     

In vitro growth, maturation, fertilization, and embryonic development of oocytes from porcine preantral follicles

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.     

In vitro maturation and fertilization of prepubertal goat oocytes

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

In vitro maturation and fertilization of follicular oocytes in cattle

This work aims towards developing research concerning the improvement of animal reproduction, embryo development and genetic engineering. In our laboratory, an attempt has been made to standardize in vitro conditions able to optimally support bovine oocyte maturation and fertilization in order to yield viable embryos. Ovaries from cows and heifers, obtained from local slaughter-house, were used for recovery of oocytes from antral follicles. Cumulus-oocyte complexes were statically cultured for 24h at 39 degrees C in medium TCM 199 supplemented with fetal calf serum inactivated, hormones, glucose and granulosa cells under a 5% CO2 and 95% humidity atmosphere. A first group of oocytes was used for fixing and staining procedure for evidence of in vitro maturation. After culture 69.4% (77/111) of oocytes reached full maturation showing cumulus expansion, first polar body extrusion and the 2nd metaphase plate. A 2nd group was used for in vitro fertilization. In vitro semen capacitation was obtained with swim-up system (8.9) with separation of high motility fraction in Talp Hepes medium. Oocytes and spermatozoa were coincubated for 18-20h in Talp medium at 39 degrees C with 5% CO2 and 95% humidity. At the end of culture stereoscope and microscope observations were made for evidence of fertilization. After IVF 67.4% (58/86) resulted fertilized. Most of them showed two pronuclei and residual sperm tail. In few cases oocytes with 1 pronucleus and the swollen sperm head or with syngamy or polyspermic were found. In these experiments high percentages of in vitro matured and in vitro fertilized oocytes have been obtained. These bovine zygotes can be considered an essential step to develop new technologies in cattle breeding.     

In vitro maturation of ovine oocytes in a portable incubator

Immature ovine oocytes were collected from ovaries obtained from an abattoir and assigned to one of three treatment groups for in vitro maturation. For Treatment 1 (T1), oocytes were matured in a conventional incubator, in tissue culture wells in an atmosphere of 5% CO(2) and air. Maturation medium consisted of bicarbonate buffered Tissue Culture Medium 199 (TCM199) supplemented with fetal calf serum (FCS), follicle stimulating hormone (FSH), luteinizing hormone (LH), and penicillin/streptomycin (pen/strep). For Treatment 2 (T2), oocytes were matured in a portable incubator, in plastic tubes containing the same medium as T1. The medium was equilibrated with 5% CO(2) and overlayed with oil. For Treatment 3 (T3) oocytes were matured in the portable incubator without CO(2) equilibration, in tubes containing HEPES buffered TCM 199 supplemented as in T1. After 24 hours at 39 degrees C, the percentage of oocytes undergoing normal nuclear maturation was 72.55, 68.14 and 66.96% for T1, T2 and T3, respectively (P >0.05). In a second experiment oocytes were matured in the 3 treatments described, then fertilized in vitro using frozen-thawed ram sperm. Fertilization rates were 44.09, 58.62 and 55.69% for T1, T2 and T3, respectively. T1 and T2 were significantly different (P < 0.05). For Experiment 3, oocytes matured and fertilized as described were cultured in drops of Modified Brinster's Mouse Ova Culture (MBMOC) containing bovine oviductal cells. These were incubated at 39 degrees C in an atmosphere of 5% CO(2) and air for 7 days. T1, T2 and T3 resulted in 20.26, 16.94 and 24.43% development to morulae, and 4.01, 3.06 and 1.85% development to blastocysts, respectively (P >0.05). The results of these experiments indicate that maturation, fertilization, and developmental rates of ovine oocytes matured in the portable incubator are similar to those of oocytes matured in a conventional incubator. This technique shows promise for transportation of oocytes to laboratories where abattoirs are not in close proximity, and holds promise for transportation of oocytes from non-domestic animals collected in the field or remote locations, to facilities capable of utilizing and preserving the gametes.     

Effects of carbon dioxide acidification of mouse oocytes before in vitro fertilization,culture, and transfer

The effects of varying pH levels on superovulated mouse oocytes before in vitro fertilization, culture, and transfer were investigated. Mouse oocytes acidified for 1 hour with 20% CO₂ (pH 6.9) exhibited a wide range of alterations. Five hours after insemination, 25% of these eggs showed different degrees of cytolysis and their perivitelline space contained numerous spermatozoa. At 12 hours normal-looking eggs from this group showed a higher proportion of polypronuclear zygotes than the control group (exposed, 51%; controls, 18%). Acidification thus markedly increased the permeability of the zona pellucida and/or interfered with the normal block to polyspermy. Fewer eggs achieved the two-cell stage among groups submitted to acidotic pH levels (6.9 to 6.6). In contrast, alkaline conditions (pH 7.8) did not reduce the proportion of two-cell embryos. However, only eggs maintained at pH 7.5 were capable of producing 80% of blastocysts. In other groups developmental blockage occurred mainly between stages 2 and 4. A significant reduction was found in the proportion of recipient females becoming pregnant after intrauterine transfer of blastocysts originating from CO₂-treated oocytes. This difference might be related to the existence of a large polyploid population among experimental eggs.