Transitional changes in arachidonic acid metabolism by bovine embryos at different developmental stages

In order to determine the profile of arachidonic acid (AA) metabolites synthesized by bovine embryos during early developmental stages, embryos collected from superovulated beef cattle (days 6 through 17) were incubated with AA and its metabolites were analyzed by high performance liquid chromatography and radioimmunoassay (RIA). Embryos harvested and cultured before day 12 of the estrous cycle metabolized AA primarily to prostaglandin E2 (PGE2), whereas, those harvested on day 13 of the cycle metabolized AA to both PGE2 and PGF2 alpha. Furthermore, embryos collected after day 15 of the cycle metabolized AA to PGI2 in addition to PGE2 and PGF2 alpha. In view of the luteotropic properties that have been attributed to PGE2 and the vasodilatory effect of PGI2, this transitional change in prostaglandin synthesis during early stages of embryonic development may be a part of the mechanism by which the embryo exerts a luteotropic effect leading to maternal recognition of pregnancy and by which the conceptus begins preparing for subsequent implantation.     

Glucose requirement at different developmental stages of in vitro fertilized bovine embryos cultured in semi-defined medium

Three experiments were conducted in which 2-cell bovine embryos were prepared from oocytes, obtained from abattoir ovaries, by in-vitro maturation for 22 to 24 hours, followed by exposure to spermatozoa for 8 hours and culture for 40 hours within the cumulus. The cumulus cells were then removed, and the cleaved embryos were cultured for a further 120 hours or longer, in the presence or absence of glucose, pyruvate and lactate. Very few embryos developed in the complete absence of energy substrates. Lactate and pyruvate, alone or combined, supported development to the 8-cell stage, but pyruvate was required to support development to the morula stage (Experiment 1). When present throughout culture or when added at 48 or 96 hours postinsemination, 5.56 mM glucose was detrimental to development (Experiments 1 and 2). However, when added at 120 hours postinsemination, 5.56 mM glucose improved development to the blastocyst and expanded blastocyst stages, compared with no glucose or 11.12 mM glucose (Experiment 3).     

Effect of yolk redistribution at different developmental stages on survivorship of zebrafish embryos

Survival of zebrafish Danio rerio embryos subjected to yolk redistribution during early development (cleavage to segmentation) was found to be dependent on the stage of manipulation and the quantity of redistributed yolk. The findings point to the presence and importance of yolk intrinsic organization during early development.     

Differential cleavage rates and sex determination in bovine embryos

The purpose of this study was to determine whether a correlation between embryonic cleavage rates and sex ratios in dairy cattle could be substantiated. Embryos were collected at Day 7 after estrus following superovulation and artificial insemination. Embryos from flushings where at least two different developmental stages within the same flushing could be identified were examined. A total of 476 such embryos was transferred to recipients and 110 calves were born. The sex effect was only demonstrable in flushings yielding embryos of at least three different developmental stages. The sex ratios from this group were 32%, 58% and 62% in the slow, intermediate and fast developing groups of embryos, respectively. The deviations were not significant (P = 0.084). Only 23% of the flushings provided embryos that fell into this cathegory, which limits the use of differential cleavage rates as a sexing method, unless additional embryo culture is introduced to the technique.     

Two-dimensional protein maps of xenopus eggs and embryos at different developmental stages.

Protein expression during the early development of Xenopus has been followed by 2D-polyacrylamide gel electrophoresis (PAGE). The analysis of two-dimensional maps of eggs and embryos at different stages of development has allowed the separation of more than 2000 spots. Identification of numerous polypeptides was obtained in four different ways: (1) immuno-blotting; (2) amino terminal sequence after blotting on to PVDF membranes; (3) comigration; and (4) assignment in comparison with proteins separated by 2D techniques on reference maps such as human liver, red blood cells, plasma and cerebrospinal fluid reported in the Swiss 2D-PAGE Data Base. The maps presented in this report are a step toward the study of the protein expression in Xenopus eggs and embryos and may be a powerful working tool since Xenopus embryos are popular models for the study of development.     

Influence of physiological concentrations of androgens on the developmental competence and sex ratio of in vitro produced bovine embryos

This study was designed to determine if the addition of androgens at ovarian follicular fluid (FF) concentrations to oocyte maturation media would alter the development and sex ratio of bovine embryos. To maximize hormone bioavailability, oil was removed and glass culture dishes were used during in vitro maturation (IVM) phase; this modified system was then used in the present experiment along with the standard IVM system utilizing plastic containers and incubation under oil. Ethanol (0.2%) was the vector for steroid hormone delivery. Oocytes were incubated for 22 h in the presence of two doses (“low” and “high”) of androstenedione (A₄) or testosterone (T); the doses were based on the concentrations of both androgens in preovulatory bovine follicles (A₄: 337.5 and 562.5 ng/ml; T: 22.2 and 42.6 ng/ml). The results of hormone assays indicated that bioavailability of steroid hormones remained relatively constant, regardless of the IVM system used. The plasticware with the addition of T resulted in significantly higher cleavage rates (80.0 ± 2.1%) than any other combination of treatments (plasticware × A₄: 71.5 ± 2.6%; glassware × T: 71.2 ± 1.9%; and glassware × A₄: 71.4 ± 2.4%). The blastocyst formation rate for the plasticware × T treatment (39.7 ± 2.5%) was significantly greater than for all other combinations (glassware × T: 28.7 ± 2.2%; glassware × A₄: 24.0 ± 2.8%; and plasticware × A₄: 19.8 ± 3.0%) and the low dose of T (37.1 ± 2.5%) resulted in higher (p < 0.05) blastocyst formation rates than all other treatments (T high dose: 29.2 ± 2.5%; A₄ high dose: 27.1 ± 2.9%; and A₄ low dose: 20.2 ± 3.0%). The proportion of male embryos was greater (p < 0.05) in plastic than glass dishes in the low-dose A₄ group (59.1 ± 8.7% vs. 38.2 ± 5.5%, plasticware vs. glassware, respectively) and it tended to be greater (p < 0.08) in the control groups and high-dose A₄ group, but not in the T groups. There was a moderate positive correlation between blastocyst formation rates across all treatment and control groups, and the percentage of male bovine embryos (r = 0.38, p < 0.05). In summary, specific combinations of androgen and glassware/plasticware treatments did alter early bovine embryo development and sex ratio. The addition of T to IVM media increased the cleavage and blastocyst formation rates in plasticware and may be employed to improve the efficiency of the standard in vitro embryo production systems. Androstenedione appeared to enhance whereas testosterone nullified the deviation in sex ratio (pro-femaleness) associated with the use of glass IVM dishes.     

Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol

Survival of IVF-derived bovine embryos of different ages and stages of development, produced in 2 different co-culture systems and frozen in 2 different cryoprotectants, was investigated. In vitro-derived bovine embryos (n = 5,525) were utilized to study survival following exposure to cryoprotectants and after freezing. Survival of the frozen embryos was based on blastocyst re-expansion 24 h and hatching 72 h after thawing. There was no difference in survival when embryos were exposed to either glycerol (Gly) or ethylene glycol (EG) for 10 or 40 min with the cryoprotectant diluted with or without freezing. In 2 of 3 experiments in which a comparison was possible, more blastocysts frozen in 1.4 M glycerol than in 1.5 M ethylene glycol survived. Addition of 0.25 M sucrose to 1.5 M ethylene glycol in the freezing solution did not improve embryo survival. More blastocysts frozen on Day 7 of in vitro culture survived than those frozen on Day 6 or Day 8. On Days 6, 7 and 8, embryos in the most advanced stage of development survived better than those at less advanced stages. Post-thaw survival did not differ for embryos produced in co-culture with Buffalo Rat Liver (BRL) cells with either Menezo B2 Medium or Tissue Culture Medium 199 and frozen in 1.4 M glycerol.     

Pregnancy rates relative to recipient plasma progesterone levels on the day of nonsurgical transfer of frozen/thawed bovine embryos

A total of 71 synchronized dairy heifers (Holstein Friesian x German Black Pied) were used as recipients of seven-day old frozen/thawed bovine embryos. Plasma progesterone concentrations and corpus luteum quality on the day of nonsurgical transfer (= day 7) were determined and related to pregnancy rates or estrus intervals in nonpregnant recipients. A total of 32 recipients (45.1 %) maintained pregnancy; 39 recipients (54.9 %) did not. No significant differences could be detected between progesterone levels in recipients that remained pregnant (3.14 +/- 0.24 ng/ml; x +/- SEM ) and those that did not maintain pregnancy (3.23 +/- 0.28 ng/ml). Optimal progesterone levels were between 2 and 5 ng/ml coinciding with a pregnancy rate of 51.1 % (24 47 ). Pregnancy rates apparently were decreased when progesterone levels were below 2 ng/ml (35.3 %; 6 17 ) or above 5 ng/ml (28.6 %; 2 7 ). Hence, optimal progesterone levels were identical to those for freshly collected embryos reported previously by Remsen et al. (1). Bovine corpus luteum quality graded by rectal palpation was related to some extent to progesterone levels but not to pregnancy rates. Out of 39 nonpregnant recipients seven animals (17.9 %) with a mean plasma progesterone level of 3.76 +/- 0.72 ng/ml showed an extended estrus interval of more than 55 days, probably indicating early embryonic mortality. Progesterone levels did not significantly differ between nonpregnant recipients with estrus intervals of various length. Plasma progesterone levels at the time of transfer are of limited diagnostic value for screening recipients prior to transfer of frozen/thawed embryos.     

Effect of lipid polarization by centrifugation at different developmental stages on post-thaw survival of bovine in vitro produced 16-cell embryos

The developmental rate to the blastocyst stage of frozen-thawed bovine in vitro produced embryos at stages earlier than Day 6 morula is not sufficiently high for practical utilization. The present study was undertaken to determine the effect of polarization of lipid droplets in the cytoplasm of bovine in vitro produced embryos from zygotes to the 8-cell stage, by centrifugation without following micromanipulation, on the survival rate of Day 4 16-cell embryos. After centrifugation at 15,500 x g in medium containing cytochalasin D, embryos were cultured to the 16-cell stage, classified as either mostly or partially delipidated by degree of lipid droplet removal, and then frozen. Embryos centrifuged at the 2-cell stage developed to the 16-cell stage similarly to those centrifuged at the 8-cell stage. The developmental rate to blastocysts after freezing of the mostly delipidated 16-cell embryos centrifuged at the 2-cell stage was higher than that of those centrifuged at the zygote stage, those that were partially delipidated at the 2-cell stage, and those that were not centrifuged. The results demonstrate that polarization of lipid droplets at the 2-cell stage by centrifugation without micromanipulation improved the survival rate of mostly delipidated 16-cell embryos after freezing.     

Vitrification of rat embryos at various developmental stages

The effect of developmental stage on the survival of cryopreserved rat embryos was examined. Wistar rat embryos at various developmental stages were vitrified by a 1-step method with EFS40, an ethylene glycol-based solution, or by a 2-step method with EFS20 and EFS40. After warming, the survival of the embryos was assessed by their morphology, their ability to develop to blastocysts (or expanded blastocysts for blastocysts) in culture, or their ability to develop to term after transfer. Most (91-100%) of the embryos recovered after vitrification were morphologically normal in all developmental stages. However, the developmental ability of 1-cell embryos was quite low; exposing them to EFS40 for just 0.5 min decreased the in vitro survival rate from 76 to 9%. The survival rates of 2-cell embryos and blastocysts, both in vitro and in vivo, were significantly higher with a 2-step vitrification process than with a 1-step vitrification process. Very high in vitro survival rates (94-100%) were obtained in 4- to 8-cell embryos and morulae in the 1-step method. Although survival rates in vivo of 4-cell (40%) and 8-cell (4%) embryos vitrified by the 1-step method were comparatively low, the values were similar to those obtained in non-vitrified fresh embryos. When morulae vitrified by the 1-step method were transferred to recipients, the in vivo survival rate (61%) was high, and not significantly different from that of fresh embryos (70%). These results show that rat embryos at the 2-cell to blastocyst stages can be vitrified with EFS40, and that the morula stage is the most feasible stage for embryo cryopreservation in this species.     

Double vitrification of rat embryos at different developmental stages using an identical protocol

The aim of the present investigation was to test the effectiveness of a method of vitrifying rat embryos at different stages of development (from early morula to expanding blastocyst) in a double vitrification procedure. Wistar rat embryos were vitrified and warmed in super-fine open-pulled straws (SOPS). Before being plunged into liquid nitrogen, the embryos were exposed to 40% ethylene glycol+0.75 M sucrose in TCM-199+20% fetal calf serum (FCS) for 20s at 38 degrees C. Subsequent warming and direct rehydration of the embryos was conducted in culture medium (TCM-199+20% FCS) at 38 degrees C. Early morula stage (7-10 blastomeres) embryos (n=358) were vitrified, warmed and cultured in vitro (EM group). Batches of these embryos were then cryopreserved again (revitrified) at the early blastocyst (EB group, n=87), blastocyst (B group, n=93) or expanding blastocyst stage (ExpB group, n=73). After the first (EM group) and repeated (EB, B, and ExpB groups) vitrification procedures, developmental rates of 81, 83, 34 and 76%, respectively were achieved (for EM-EB-ExpB P>0.1; for EM, EB, ExpB-B P<0.005). Our data demonstrate the possibility of using the described identical protocol for the SOPS vitrification of rat early morulae, early blastocysts and expanding blastocysts. The low survival rate of blastocysts subjected to double vitrification requires further investigation.     

Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures

In this study, three different vitrification systems (open pulled straw: OPS; superfine open pulled straw: SOPS; and Vit-Master technology using SOPS: Vit-Master-SOPS) were compared in order to investigate the influence of cooling rate on in vitro development of vitrified/warmed porcine morulae, early blastocysts, or expanded blastocysts. Embryos were obtained surgically on Day 6 of the estrous cycle (D0 = onset of estrus) from weaned crossbred sows, classified and pooled according their developmental stage. A subset of embryos from each developmental stage was cultured to evaluate the in vitro development of fresh embryos; the remaining embryos were randomly allocated to each vitrification system. After vitrification and warming, embryos were cultured in vitro for 96 h in TCM199 with 10% fetal calf serum at 39 degrees C, in 5% CO(2) in humidified air. During the culture period, embryos were morphologically evaluated for their developmental progression. The developmental stage of embryos at collection affected the survival and hatching rates of vitrified/warmed embryos (P < 0.001). The vitrification system or the interaction of vitrification system and developmental stage had no effect on these parameters (P > 0.05). Vitrified expanded blastocysts showed the best development in vitro (P < 0.001), with survival and hatching rates similar to those of fresh expanded blastocysts. The hatching rate of fresh morula or early blastocyst stage embryos was higher than their vitrified counterparts. In conclusion, under our experimental conditions, cooling rates greater than 20,000 degrees C/min, as occurs when SOPS or Vit-Master-SOPS systems are used, do not enhance the efficiency of in vitro development of vitrified porcine embryos.     

Hyperglycemia-induced apoptosis affects sex ratio of bovine and murine preimplantation embryos

The effect of glucose in the medium used during in vitro culture on both cell death by apoptosis and the sex ratio of bovine blastocysts derived from in vitro-matured and in vitro-fertilized oocytes was evaluated. Oocytes were matured, inseminated, and cultured in vitro in mSOF medium with 10% FCS with or without glucose supplementation. Exposure to high concentrations of glucose (10, 20, and 30 mM) during bovine embryo development in vitro from zygote to blastocyst resulted in a decrease in the number of cells per embryo and an increase in the frequency of apoptotic cells. A significantly higher proportion of females was found among those embryos that developed under hyperglycemic conditions in vitro. Moreover, both murine and bovine blastocysts incubated for 6 hr in 20 mM glucose had a significantly higher number of apoptotic cells in comparison to control. In this study, we also determined whether blastocyst production of the X-linked inhibitor of apoptosis protein (XIAP) differs between the sexes. Our results show that female bovine blastocysts produce significantly higher amounts of XIAP mRNA than males and this could be crucial in explaining the higher proportion of female blastocysts observed following in vitro culture under hyperglycemic conditions which induce apoptosis. Moreover, a higher proportion of female murine blastocysts cultured under hyperglycemic conditions were implanted in the uterus (65.3 of implantations from embryos cultured with 20 mM of glucose are females vs. 49% in control). This mechanism provides an explanation for the significant reduction of male children born to diabetic mothers.     

Survival of frozen-thawed sheep embryos cryopreserved at cleavage stages

This study evaluated the effect of freezing-thawing procedures on the viability of sheep embryos cryopreserved at various developmental stages. The survival rates of frozen-thawed embryos were compared with non-frozen counterparts. Embryos were recovered from the oviduct and uterus, at different days of the early luteal phase, and were classified at six different developmental stages: 2- to 4-cell (n = 72), 5- to 8-cell (n = 73), 9- to 12-cell (n = 70), early morulae (n = 42), morulae (n = 41), and blastocyst (n = 70). For each early cleavage stage and blastocysts, approximately half of the embryos, were frozen immediately by slow freezing with an ethylene glycol-based solution. The remaining embryos were cultured to the hatched blastocyst stage. All morulae and compact morulae were frozen after recovery with the same protocol. Cryoprotectants were removed using 1M sucrose solution, and then warmed the embryos were cultured to the hatched stage in a standardized in vitro culture. Embryo developmental stage had a significant effect on the ability to hatch following freezing (P<0.0001). The cryotolerance of the embryos fitted a regression (r2 = 0.908), increasing linearly from 2- to 4-cell embryos (17.1%) to morula stage (46.3%) and in a quadratic regression from the morula to the blastocyst stage (83.7%). Frozen early cleavage stage embryos had a significantly lower viability than their fresh counterparts (23.1 vs 83.1%; P<0.0001), with a similar rate of viability between fresh or frozen blastocysts (92.5 vs 83.7%). In conclusion, early sheep embryos are very sensitive to freezing per se and the survival rates following conventional freezing improve as embryo developmental stage progresses.     

Equilibrium vitrification of mouse embryos at various developmental stages

Previously, we developed a new method by which 2‐cell mouse embryos can be vitrified in liquid nitrogen in a near‐equilibrium state, and then kept at ?80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight‐cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol‐based solutions, named EFSc because of their composition of ethylene glycol (30–40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM‐70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at ?80°C. When 8‐cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at ?80°C, the survival rate was high even after 4 days in storage and remained high after re‐cooling in liquid nitrogen. On the other hand, the survival of vitrified‐expanded blastocysts kept at ?80°C was low. Therefore, 8‐cell embryos and morulae can be vitrified in a near‐equilibrium state using the same method as for 2‐cell embryos. A high proportion of C57BL/6J embryos at the 2‐cell, 8‐cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re‐cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near‐equilibrium vitrification method, which is effective for 2‐cell mouse embryos, is also effective for embryos at the 8‐cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice. Mol. Reprod. Dev. 79: 785–794, 2012. © 2012 Wiley Periodicals, Inc.     

Pregnancy rates after transfer of frozen bovine embryos: a field trial

An efficient and practical technique for bovine embryo cryopreservation is a fundamental issue in the widespread use of embryo transfer. The present study shows results obtained in field experiments. In the first experiment, two slow-freezing methods using glycerol and a one-step method using ethylene glycol were compared: glycerol added in two steps (5 and then 10%), glycerol added in one step (10%) and 1.5 M ethylene glycol with direct transfer. The three methods were equally effective; pregnancy rates of 40.4, 39.1 and 45.4%, respectively were achieved. In the second experiment, using 1.5 M ethylene glycol with direct transfer, 20 and 5 min of equilibration of the cryoprotectant were tested. There were no observed significant differences in pregnancy rates. In the third experiment, ethylene glycol and propylene glycol were combined with three sucrose concentrations (0, 0.1 or 0.3 M) in a one-step method. It was observed that ethylene glycol and 0.1 M sucrose yielded the highest pregnancy rate, not differing from fresh controls. Similar pregnancy rates were noted after using multiple-step or one-step methods, but the one-step method is preferable due to its simplicity and applicability to field conditions.     

The effect of glucosamine concentration on the development and sex ratio of bovine embryos

Glucosamine is a component of hyaluronic acid and an alternative substrate to glucose for the extracellular matrix synthesis of COCs. Its addition to an IVM medium reduces the glucose consumption of bovine COCs. Glucosamine is also metabolized to UDP-N-acetyl glucosamine (UDP-GlcNAc) via the hexosamine biosynthesis pathway and is utilized for O-linked glycosylation by the X-linked enzyme, O-linked GlcNAc transferase (OGT). Moreover, the inactivation of the second X chromosome in female embryos is influential in producing the sex ratio bias observed in vitro when embryos are cultured in the presence of glucose above 2.5mM. Accordingly, the aim of this study is to examine whether the presence of glucosamine during maturation or embryo culture causes a sex ratio bias in bovine blastocysts. Glucosamine was added to the medium in three different embryo developmental periods: in vitro maturation, the one-cell to eight-cell stage (before the maternal-zygotic transition, MZT), and the eight-cell to blastocyst stage (after MZT). When glucosamine was added during in vitro maturation, the developmental competence of oocytes was severely compromised. However, the sex ratio of embryos was not influenced. When glucosamine was added to embryo culture medium during development from one-cell to eight-cell stage (before MZT), it affected neither the development nor the sex ratio of bovine embryos. Finally, when glucosamine was added after MZT, the development rate of embryos was severely decreased, and the sex ratio was skewed toward males. Moreover, an inhibitor of OGT, benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (BADGP), negated the effect of glucosamine on the sex ratio when it was added to embryo culture medium from the eight-cell to blastocyst stage (after MZT). These results suggest that, like glucose, the supplementation of glucosamine into the medium skewed the sex ratio to males and that OGT, an X-linked enzyme, was involved in this phenomenon. Moreover, this effect of glucosamine was limited only to when it was present in the embryo culture medium after MZT.     

Survival of bovine embryos stored at 4 degrees C

These experiments were designed to test the efficacy of storing bovine embryos at 4 degrees C. Of particular interest were the age of embryo at which maximum post-storage survival could be achieved and longevity at 4 degrees C. A greater proportion of day 8 blastocysts developed in vitro at 37 degrees C following refrigeration for 48 hr than did embryos collected 2, 4 or 6 days after estrus (P<0.01). Survival of blastocysts stored at 4 degrees C for 48 hr was similar to that of nonstored blastocysts. In a subsequent experiment, day 8 blastocysts were recovered nonsurgically and assigned to one of the following treatments: (a) immediate transfer; (b) culture at 37 degrees C; or (c) storage at 4 degrees C for 1, 2, 3 or 5 days. Post-storage viability was assessed by either development in culture at 37 degrees C or embryo survival following nonsurgical transfer to synchronized recipients. In vitro survival of nonstored embryos and embryos stored 1 day did not differ. Survival decreased after storage for 2 days (P<0.10) or longer (P<0.05). Similar results were observed for survival after transfer, but embryo viability decreased even more rapidly with increasing duration of storage. In vitro survival was approximately 50% for blastocysts stored for 3 and 5 days, but few pregnancies resulted from transfer of embryos stored for these periods. In another experiment survival after transfer of blastocysts stored at 4 degrees C for up to 2 days was similar to that of nonstored blastocysts.     

Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryos: Effect on developmental potential

To evaluate the effect of karyoplast-cytoplast ratio on the development of nuclear transfer embryos, karyoplasts from day 4, day 5, and day 6 embryos were transferred to oocytes enucleated with different volumes of cytoplasm: Type 1, removal of a small volume of cytoplasm equivalent to the first polar body, Type 2, removal of a volume of cytoplasm approximately equal to the volume of the respective karyoplast, and Type 3, removal of half of the oocyte volume. In addition, the effect of experimental reduction of karyoplast cytoplasm was investigated in day 4 and day 5 karyoplasts. Intact day 4 karyoplasts fused to Type 3 cytoplasts did not support development to blastocysts, whereas these karyoplasts yielded blastocysts in combination with Type 1 (7%) and Type 2 cytoplasts (12%). After experimental reduction of cytoplasmic volume in day 4 karyoplasts, blastocysts (10%) were also obtained after fusion with Type 3 cytoplasts, probably due to reduction of cytoplasmic chimerism. With day 5 karyoplasts, blastocyst rate was higher in combination with Type 2 (34%) than with Type 1 (19%) and Type 3 cytoplasts (16%; P < 0.05). The use of day 6 intact karyoplasts resulted in a significantly (P < 0.05) higher proportion of blastocysts when fused with Type 2 (38%) or Type 1 cytoplasts (34%) than with Type 3 cytoplasts (16%). These results suggest that enucleation of oocytes with a volume similar to that of the respective karyoplast creates better conditions for cell cycle interactions with all types of karyoplasts than enucleation with minimal or large volume of cytoplasm. Mol. Reprod. Dev. 48:332–338, 1997. © 1997 Wiley-Liss, Inc.