Blastocyst development after intracytoplasmic sperm injection of equine oocytes vitrified at the germinal-vesicle stage

We evaluated the meiotic and developmental competence of GV-stage equine oocytes vitrified under different conditions. In a preliminary study, using dimethyl sulfoxide (D), ethylene glycol (EG) and sucrose (S) as cryoprotectants, the maturation rate was higher for cumulus-oocyte complexes (COCs) held overnight before vitrification (37%) than for those vitrified immediately (14%; P < 0.05). Thereafter, all COCs were held overnight before vitrification. In Experiment 1 we compared 1 min (1m) and 4 min (4m) exposure to vitrification and warming solutions; oocytes that subsequently matured were fertilized by ICSI. The maturation rate was similar between timing groups (29–36%), but was significantly lower than that for controls (73%). The 1m treatment yielded one blastocyst (11%), vs. 19% in controls. In Experiment 2, propylene glycol (PG) and trehalose (T) were also used. We compared two base solutions: M199 with 10% FBS (M199+), and 100% FBS; three cryoprotectant combinations: D-EG-S; PG-EG-S; and PG-EG-T; and two timings in vitrification solution: ∼30 s (30s) and 1 min (1m). The most effective treatment (FBS/PG-EG-T/30s) yielded 42% maturation, 80% cleavage and 1 blastocyst (10%), vs. 49%, 93% and 29%, respectively for controls (P > 0.1). In Experiment 3, we evaluated the toxicity of the M199/D-EG-S/1m and FBS/PG-EG-T/30s treatments, without actual vitrification. These treatments did not affect maturation but both significantly reduced blastocyst development (0% and 0%, vs. 21% for controls). This represents the second report of blastocyst development after vitrification of GV-stage equine oocytes, and presents the highest developmental competence yet achieved; however, more work is needed to increase the efficiency of this system.     

Viable rabbits derived from reconstructed oocytes by germinal vesicle transfer after intracytoplasmic sperm injection (ICSI)

Abnormal oocyte spindle due to the improper function of ooplasm is associated with female infertility of advanced maternal age. A possible way to overcome this problem is to transfer an oocyte germinal vesicle (GV) which contains genetic materials of a patient with a history of poor embryo development to the cytoplast from a donor oocyte. Here we demonstrate that GV transfer is feasible using a rabbit model. When the GVs were transferred to auto- or hetero-cytoplasts of GV stage oocytes, around 80% of the reconstructed oocytes could mature in vitro and 7.1-9.4% of the oocytes developed to blastocyst stage after intracytoplasmic sperm injection (ICSI). Transfer of 93 fertilized eggs reconstructed via GV transfer into six recipients resulted in two live offspring. Results of this experiment indicate that GV transfer can potentially become a new approach in treatment of infertility because of advanced maternal age.     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.     

Holding bovine oocytes in the absence of maturation inhibitors: kinetics of in vitro maturation and effect on blastocyst development after in vitro fertilization

Holding immature oocytes before maturation simplifies the transport of oocytes and aids in scheduling later manipulations. We examined the effect of holding bovine oocytes in the absence of meiotic inhibitors on their subsequent meiotic and developmental competence. Oocytes were matured immediately after recovery (control) or were held in a mixture of 40% TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts, and 20% FBS, at room temperature for 16 to 18h (EH-held) and then matured. Chromatin status was determined at 0, 10, 14, 18, and 22h of maturation culture. Oocytes were fertilized in vitro after either 18 or 22-24h maturation. The EH treatment maintained oocytes at the germinal vesicle stage (79.3%, vs. 87.7% for control oocytes at 0h; P>0.05). Upon culture, held oocytes matured more quickly than did control oocytes. The proportions of mature oocytes were not significantly different between groups at 18h (EH-held, 80.6% and control, 79.3%); however, after 22h significantly more EH-held than control oocytes had degenerated (24.1% vs. 4.5%, P<0.0001). Blastocyst development was similar between groups for oocytes fertilized after 18h maturation (EH-held, 29.6% and control, 27.8%). When oocytes were fertilized after 22-24h maturation, EH-held oocytes yielded lower blastocyst development than did control oocytes (16.5% vs. 29.3%, P<0.05). In conclusion, bovine oocytes may be effectively held in the EH treatment before maturation without adversely affecting meiotic or developmental competence. However, holding affects the kinetics of maturation and this must be taken into account when subsequent manipulations are performed.     

Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes

There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes were fixed immediately after collection to assess whether the collection method influenced the initial chromatin configuration of oocytes. In Experiment 2, in vitro maturation rates of oocytes recovered by aspiration or scraping were compared. In Experiment 3, oocytes were matured in vitro and submitted to intracytoplasmic sperm injection (ICSI). Initial chromatin configuration differed according to collection method in that there was a significantly higher prevalence of diffuse chromatin within the germinal vesicle in oocytes recovered by scraping than in oocytes recovered by aspiration (29/87, 33% and 28/166, 17%, respectively; P < 0.01). Maturation of oocytes to metaphase II did not significantly differ between scraped and aspirated oocytes (56/101, 55.4 % vs. 65/106, 61.4%, respectively). The overall pronucleus formation rate after ICSI of oocytes recovered by scraping was not significantly different than that of oocytes recovered by aspiration (50/99, 52.6% vs. 50/85, 68.5 %, respectively); however, the rate of abnormal fertilization was significantly higher for oocytes collected by aspiration (14/73, 19% vs. 6/94, 6%, respectively; P <0.05). These results demonstrate that the collection method affects the population of recovered oocytes and may contribute to differences in results observed among laboratories working with horse oocytes.     

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.     

Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection

Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2 g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to 9.1) or oocytes recovered (4.8 to 5.0) per mare per aspiration session. Of 314 oocytes recovered, 180 (57%) matured in culture. Thirty-six mature oocytes were subjected to ICSI; 33% formed blastocysts (63% per mare per aspiration session). In Experiment 2, the preovulatory follicle was aspirated every 14 d for three to four cycles. Prostaglandin F_2α was given on Days 6 and 7 after aspiration. A follicle ≥25 mm in diameter was present on Day 13, the day of deslorelin administration, in 23 of 24 cycles, and ovulatory response (granulosa expansion) was seen in 24 of 25 follicles aspirated. Blastocyst development after ICSI was 41% per injected oocyte, or an estimated 33% per mare per aspiration session. We concluded that both aspiration of immature follicles and aspiration of the preovulatory follicle can be performed effectively every 14 d without monitoring ovarian follicular growth. As performed in these separate experiments, aspiration of immature follicles provided more blastocysts per aspiration session.     

Configuration of maternal and paternal chromatin and pertaining microtubules in human oocytes failing to fertilize after intracytoplasmic sperm injection

The microtubules and chromosomes of 180 human oocytes failing to fertilize after intracytoplasmic sperm injection were observed in order to establish how sperm chromatin and sperm astral microtubule configuration is related to the phases of oocyte cell cycle, and to find the defects in those structures causing fertilization arrest. As many as 125 (69%) oocytes were arrested at metaphase II. In one-fourth of them, damages of the second meiotic spindle were noted. In their cytoplasm intact sperm were found in 38 (30%) cases, a swollen sperm head in 36 (29%) and prematurely condensed sperm chromosomes (G1-PCC)-a result of active mitosis promoting factor (MPF)-in 51 (41%) cases. G1-PCC were mostly (73%) surrounded by the bipolar paternal spindle instead of astral microtubules. A male pronucleus was never presented in metaphase II oocytes. In 19 (11%) oocytes, arrested at anaphase II, no intact sperm were found. As many as 9 (47%) oocytes contained sperm in G1-PCC form, which proves that anaphase II oocytes mostly retain active MPF, despite oocyte activation. As many as 78% of 36 monopronucleate oocytes contained sperm, with delay in the process of sperm nucleus decondensation. Sperm in G1-PCC form and a bipolar paternal spindle were never found in monopronucleate oocytes. From this we conclude that sperm that does not activate the oocyte may continue decondensing the chromatin, but the oocyte prevents male pronucleus formation before the female one, mostly by causing PCC in the sperm and by duplicating the sperm centrosome. Mol. Reprod. Dev. 55:197-204, 2000.     

Full-term development of golden hamster oocytes following intracytoplasmic sperm head injection

The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.     

Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been applied successfully in the treatment of male infertility in humans and in fertilization research in mice. However, the technique has had limited success in producing offspring in other species including the rabbit. The aim of this research was to test the in vitro and in vivo developmental of rabbit oocytes after ICSI. Sperm used for ICSI were collected from mature Dutch Belted buck and washed 2-3 times with PBS +0.1% polyvinyl alcohol (PVA) and then mixed with 10% polyvinyl pyrrolidone (PVP) prior to microinjection. Oocytes were collected from superovulated does 14-15 hr after hCG injection and were fertilized by microinjection of a single sperm into the ooplasm of each oocyte without additional activation treatment. After ICSI, the presumed zygotes were either cultured in KSOM +0.3% BSA for 4 days or transferred into oviducts of recipient does at the pronuclear or 2-cell stage. A high percentage of fertilization (78%, n = 114) and blastocyst development (39%) was obtained after ICSI. Control oocytes, receiving a sham injection, exhibited a lower activation rate (31%, n = 51) and were unable to develop to the blastocyst stage, suggesting that the blastocysts developed following ICSI were derived from successful fertilization rather than parthenogenetic development. A total of 113 embryos were transferred to six recipient does. Two recipients became pregnant and delivered seven live young. Our results demonstrated that rabbit oocytes can be successfully fertilized and activated by ICSI and can result in the birth of live offspring.     

Cysteamine or beta-mercaptoethanol added to a defined maturation medium improves blastocyst formation of porcine oocytes after intracytoplasmic sperm injection

The present study was carried out to investigate the effect of adding 100 microM cysteamine (Cys) or 100 microM beta-mercaptoethanol (beta-ME) to a defined maturation medium on in vitro maturation (IVM), and fertilization and developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The two control media for IVM culture were modified TCM199 containing 10% (v/v) porcine follicular fluid (pFF) or 0.05% (w/v) polyvinyl alcohol (PVA), and Cys or beta-ME was supplemented to the PVA-control medium. There was no significant difference in the proportions of in vitro matured oocytes among the four treatment groups (94.5-98.4%). The percentages of pronuclear formation (51.0-64.2%) after ICSI were also not significantly different among the four groups. The cleavage rate (72.8%) in the oocytes treated with Cys showed no significant difference compared with those of the two control media containing pFF (72.2%) or PVA (61.5%), but was higher (P<0.05) than that in the oocytes treated with beta-ME (56.3%). However, the rates of blastocyst formation of Cys (36.7%), beta-ME (27.1%) and pFF (31.4%) were higher (P<0.05) than that using the control medium containing PVA (15.6%). The mean cell number of blastocysts ranged from 42 to 52 among the four groups, without significant differences. In conclusion, the addition of Cys or beta-ME to a defined maturation medium enhanced blastocyst formation after ICSI, to a level similar to that achieved by adding pFF.     

Embryo development after vitrification of immature and in vitro-matured equine oocytes

Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata (MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the Cryotech® method. Warming medium was equilibrated either in 5% CO₂ or Air. IMM oocytes underwent in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO₂- than Air-equilibrated medium (86 ± 3 vs. 76.9 ± 4%, respectively). Maturation for all vitrified-warmed oocyte treatments (37 ± 6.5 to 45.9 ± 5.8%) was not different from control (50 ± 4.1%), except for MAT CR- CO₂ (20.3 ± 4.6%). Cleavage for MAT CR- CO₂ and Air groups was similar to control (67.7 ± 12.1, 71.4 ± 8.1, and 78 ± 5.3%, respectively). One blastocyst was produced (MAT CR + CO₂), representing the first equine blastocyst reported after vitrification of an in vitro-matured oocyte.     

Microtubule arrays in the cortex and near the germinal vesicle of immature starfish oocytes

An extensive array of long, crisscrossing microtubules has been discovered in the cortex of oocytes of the starfish Pisaster ochraceus. The microtubules were visualized in cortex preparations by indirect immunofluorescence microscopy using antibodies to tubulin. The cortical array of microtubules is present in all oocytes before and for about 30 min after the application of 1-methyladenine, the hormone that induces oocyte maturation. The presence of microtubules was confirmed by electron microscopy. The microtubules in this array are depolymerized when oocytes are treated with colchicine or nocodozole and are augmented when oocytes are treated with taxol. Dihydrocytochalasin B treatment of the oocytes causes the microtubules to aggregate, presumably by altering a microfilament network also found in the cortex. The distribution of microtubules was also explored in whole oocytes stained with antitubulin. One or two aster-like structures were observed adjacent to the germinal vesicle of each oocyte.     

Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.     

Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig

The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 °C for up to 48 h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Cζ (PLCζ) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCζ in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18 h at 24 °C (78%, 62%, and 35%, respectively) and for 48 h at 14 °C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCζ in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.     

Effects of vitrification cryoprotectant treatment and cooling method on the viability and development of buffalo oocytes after intracytoplasmic sperm injection

In vitro matured (IVM) buffalo oocytes at the metaphase of the second meiotic division (MII) were vitrified in 20% Me(2)SO: 20% EG (v/v) and 0.5M sucrose (VA), or 35% EG (v/v), 50mg/mL polyvinylpyrrolidone (PVP), and 0.4M trehalose (VB), either on cryotops or as 2μL microdrops. The viability was assessed after warming by fluorescein diacetate (FDA) staining and all surviving oocytes were subjected to ICSI and ethanol activation. All vitrified groups had similar recovery rates but both VA groups had significantly higher survival and pronuclear formation rates than either of the VB groups. Non treated control oocytes and non cryopreserved oocytes exposed to FDA had significantly higher survival, 2nd polar body extrusion, PN and blastocyst formation rates than any of the four vitrified groups (P<0.05). In conclusion The cryotop and microdrop methods are equally effective for buffalo oocyte vitrification, and although vitrification in VA solution yielded higher rates of survival and formation of 2 pronuclei than VB, the rate of blastocyst formation was comparable for both solutions. A detailed analysis of oocytes that extruded the second polar body after ICSI and activation revealed that only a minority (7-20% of the vitrified and 46-48% of the control oocytes) also had two pronuclei, indicating that normal activation is compromised by vitrification.     

Cytoskeleton and chromatin reorganization in horse oocytes following intracytoplasmic sperm injection: patterns associated with normal and defective fertilization

Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is accompanied by a series of integrated cytoskeleton-mediated events, failure of which results in developmental arrest.     

Blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection

This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoele and capsule. In vitro development was assessed in three culture systems. Culture of zygotes in modified Chatot, Ziomek, Bavister medium with BSA containing either 5.5 mM glucose for 7.5 days or 0.55 mM glucose for 3 days, followed by 3 mM glucose for 2 days, then 4.3 mM glucose for 2.5 days, did not result in blastocyst formation. Culture of zygotes in Dulbecco modified Eagle medium (DMEM)/F-12 with 10% fetal bovine serum with and without coculture with equine oviductal epithelial explants yielded 16% and 15% blastocyst development, respectively. Development to blastocyst was significantly lower in G1.3/2.3/BSA than in DMEM/F-12/BSA or in either medium with 10% added serum (2% vs. 18%, 18% or 20%; P < 0.05), suggesting that requirements for equine embryo development differ from those for other species. These results indicate that in vitro-matured equine oocytes are sufficiently competent to form 36% blastocysts in an optimal environment (in vivo). While we identified an in vitro culture system that provided repeatable blastocyst development without coculture, this yielded only half the rate of development achieved in vivo.