A mouse embryo culture system for quality control testing of human in vitro fertilization and embryo transfer media and fetal cord sera

Swiss white mice were superovulated, mated, and sacrificed to recover two-cell embryos that were cultured in Ham's F-10 supplemented with 15% fetal serum. In 16 experiments, media enriched with fetal bovine serum (FBS) supported blastocyst development from 80% ± 19% (mean ± S.D.) of two-cell embryos. Culture media + FBS was the positive control when 74 batches of heat-inactivated human fetal cord serum (hFCS) were tested. Statistical analyses indicated two distinct populations: 49 hFCS promoted blastocyst formation and 25 hFCS grew fewer blastocysts. In five studies, 35/47 two-cell embryos recovered from mice oviducts in media + FBS and immediately incubated formed blastocysts (75% ± 10%). In six comparison studies where the recovered embryos stood at room temperature for 30 minutes before incubation, only 18/57 (29% ± 21%) became blastocysts. When the colony was housed for 1 week in rooms with Shell No Pest Strips as treatment for mites, only 11/125 two-cell embryos became blastocysts (9%). In contrast, animals housed in quarters decontaminated with chlorine bleach had reduced breeding efficiency and produced fewer two-cell embryos. We conclude that (1) Ham's F-10 + FBS is an excellent positive control to test new batches of hFCS; (2) hFCS that supports blastocyst formation from ≥75% of two-cell embryos is adequate for human use; (3) pesticide treatment of breeding colonies and cooling of murine embryos during harvest both impaired in vitro blastocyst development; and (4) chlorine bleach cleansing of animal quarters reduced the number of successful matings.     

In vitro fertilization and preimplantation embryo development of African green monkeys (Cercopithecus aethiops)

Ovaries of five adult female African green monkeys were stimulated by repeated administrations of equine chorionic gonadotrophin (eCG), followed by a single administration of human chorionic gonadotrophin (hCG). Oocytes were collected from enlarged follicles 28 h after hCG administration and incubated in vitro for 288 h. Oocytes that had extruded the first polar body were inseminated with spermatozoa that had been incubated for 4 to 6 h in medium with caffeine and dibutyryl cyclic AMP. Of these oocytes, 66% were fertilized and the incidence of polyspermy was 37%. Eighty-two percent of the fertilized eggs cleaved, with some developing into expanded blastocysts. Am. J. Primatol. 43:43–50, 1997. © 1997 Wiley-Liss, Inc.     

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

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

Effects of oxygen tension and humidity on the preimplantation development of mouse embryos produced by in vitro fertilization: analysis using a non-humidifying incubator with time-lapse cinematography

To examine the effects of oxygen tension and humidity on early embryonic development, the preimplantation development of mouse embryos produced by in vitro fertilization was assessed by time-lapse cinematography to evaluate morphokinetic development with higher precision. Zygotes were produced from spermatozoa and oocytes from ICR mice and cultured in KSOM under low or high oxygen tension in a non-humidified incubator with time-lapse cinematography (CCM-iBIS). The developmental rates of embryos to the 4-cell and blastocyst stages under lower oxygen tension in CCM-iBIS were significantly higher than those under higher oxygen tension in CCM-iBIS. Ninety-six hours after insemination, a large number of embryos cultured under low oxygen tension developed to the hatching blastocyst stage. Embryonic development was more synchronized under lower oxygen tension. Non-humidified cultures did not affect embryonic development. On average, mouse embryos cultured at lower oxygen tension reached 2-cell at 18 h, 3-cell at 39 h, 4-cell at 40 h, initiation of compaction at 58 h, morula at 69 h, and blastocyst at 82 h after insemination. In conclusion, lower oxygen tension better supports preimplantation development of mouse embryos fertilized in vitro, and non-humidified culture conditions do not influence the embryonic development in vitro.     

Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization

The present study examined the effect of epidermal growth factor (EGF) during in vitro maturation (IVM) and embryo culture on blastocyst development in the pig. In experiment 1, cumulus oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, hormonal supplements, and with or without EGF (0–40 ng/ml) for 20–22 hr. They then were cultured for an additional 20–22 hr without hormones. After maturation, cumulus-free oocytes were co-incubated with frozen-thawed spermatozoa for 5–6 hr. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 hr. In experiment 2, oocytes were matured in medium containing 10 ng/ml EGF, inseminated, and putative embryos were cultured in the presence of 0–40 ng/ml EGF. In experiment 3, oocytes were cultured in the presence of 0, 10 and 40 ng/ml EGF to examine the kinetics of meiotic maturation. In experiment 4, 2- to 4-cell and 8-cell to morula stage embryos derived from oocytes matured with 10 ng/ml EGF were transferred to the oviduct and uterus, respectively, of each of three recipient gilts (3 and 4 days post-estrus, respectively). The presence or absence of EGF during IVM did not affect cumulus expansion, nuclear maturation, fertilization parameters, or cleavage rate. However, compared to no addition (21%), presence of 1 (33%) and 10 ng/ml EGF (42%) during IVM increased (P < 0.01) the rate of blastocyst development in a concentration-dependent manner. Compared to 10 ng/ml EGF, higher concentrations (20 and 40 ng/ml) reduced (P < 0.01) blastocyst development in a concentration-dependent manner (35% and 24%, respectively). No difference was observed between no addition and 40 ng/ml EGF (22%). Compared to no addition and 10 ng/ml EGF, a significantly (P < 0.001) higher proportion (25% vs. 55%) of oocytes reached metaphase II stage 33 hr after IVM with 40 ng/ml EGF. However, no difference was observed at 44 hr. Transfer of embryos to six recipient gilts resulted in three pregnancies and birth of 18 piglets. The results show that EGF at certain concentrations in IVM medium can influence the developmental competence of oocytes. However, addition of EGF during the culture of pig embryos derived from oocytes matured in the presence of EGF is without effect. Birth of piglets provides evidence that embryos derived from oocytes matured in a medium containing EGF are viable. Mol. Reprod. Dev. 51:395–401, 1998. © 1998 Wiley-Liss, Inc.     

Birth of a Western Lowland gorilla (Gorilla gorilla gorilla) following in vitro fertilization and embryo transfer

A 21-year-old multiparous female exhibiting 31–41 day menstrual cycles was given hFSH (225 IU/day, Metrodin 75, from cycle day 3 through 9 (menses = day 1) and hCG (10,000 IU, Profasi, on day 10 to stimulate follicular development. At 35 h after hCG, under isoflurane (AErrane) anesthesia, follicles were aspirated by controlled suction under transvaginal ultrasound guidance. Metaphase II oocyctes (n = 11) were placed in modified human tubal fluid (mHTF, 100 μl) medium under oil at 37°C in humidified 5% CO₂. Frozen semen, collected by voluntary ejaculation, was thawed (70°C H₂O bath, 6 sec), diluted slowly, centrifuged, and resuspended in mHTF, and 160,000 motile spermatozoa/ml were added at 6 h after oocyte recovery. At 21 h postinsemination (p.i.) eight oocytes were at the two-cell stage, five were cryopreserved, and three were cultured to the six- to eight-cell stage in mHTF with granulosa cells before transcervical uterine transfer at 47 h p.i. using a Teflon catheter. Micronized progesterone (400 mg/d) was orally administered for 10 weeks posttransfer (p.t.). Ultrasound examination revealed a single fetus at 15 weeks p.t., and unassisted delivery of a live 1.37 kg female infant occurred at 29 weeks. Am. J. Primatol. 41:247–260, 1997. © 1997 Wiley-Liss, Inc.     

Chick embryo retina development in vitro: The effect of insulin

In this paper we study the development of chick embryo retina culturedin vitro and the effects exerted by insulin. Retinas were removed from 7-day embryos and cultured in serum-and hormone-free medium for 7 additional days. Under these conditions retinal cells survived and underwent cholinergic differentiation, as previously ascertained by Hausman et al. (Dev. Brain Res., 1991, 59: 31–37). However, a great retardation of development was noted compared to uncultured control, 14-day retina. In fact both wet weight and DNA and protein content increased much slower than in ovo and the tubulin content decreased below even the starting value. In addition, although after 7 days in culture retinal cells were organized in identifiable layers, nevertheless the typical organization equivalent to 14-day in ovo retina was absent. The addition of insulin in the medium markedly increased the wet weight of cultured retinas, their protein content and the level of tubulin pools, particularly that of non-assembled fraction. Nevertheless insulin did not modify DNA synthesis and did not induce the increment of both neuron specific enolase and actin. Morphological observations show that insulin markedly increased the number and the thickening of the fiber layers. These results, together with the facts that retina synthesizes and secretes insulin and possesses specific insulin receptors suggest that insulin can have autocrine or paracrine regulatory functions in retinal development by exerting a general effect on retinal growth and a more specific one on tubulin production.     

Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization

The incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P < 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.     

A preliminary report on the establishment of pregnancies in an in vitro fertilization (IVF) programme at the University of British Columbia (UBC)

Between July 1982 and November 1983, two pregnancies were established using in vitro fertilization and embryo transfer (IVF and ET) procedures with three different schedules to induce follicular maturation. All women were cycling normally and had inoperable or absent fallopian tubes. Of 83 oocytes aspirated from 24 patients (31 cycles), 75% were considered mature and 25% immature by the morphological characteristics of the oocytes and cumulus cells. Oocytes were preincubated for 6–24 hours, and after insemination, 60% cleaved to the two-to-four-cell stage. The superovulation induction schedule employing hMG administered according to the individually adjusted treatment scheme established two pregnancies. This schedule was considered the superior regimen, as it gave the highest proportion of mature oocytes (89%) which cleaved (78%). The pregnancy-attaining follicle showed a high progesterone:estradiol-17β ratio (P₄/E₂) in its microenvironment of aspirated follicular fluid, culture media of granulosa cells, and oocyte-cumulus complex. Our observations indicate a high P₄/E₂ ratio in the pregnancy-attaining follicle, and thereby reflect a further parameter in influencing maturation of the oocytes most likely to implant.     

Effect of estrus-associated glycoprotein and tissue inhibitor of metalloproteinase-1 secreted by oviduct cells on in vitro bovine embryo development

The effect of two glycoproteins (estrus-associated glycoprotein [EGP] and tissue inhibitor of metalloproteinase [TIMP-1]) secreted by bovine oviduct cells on in vitro bovine embryo development was assessed. A first set of experiments was conducted to determine whether the embryotrophic activity of the bovine oviduct-conditioned medium (BOCM) was correlated with the presence of EGP or TIMP-1. EGP and TIMP-1 were detected in BOCM, supporting the development of 22% zygotes to the blastocyst stage, as well as in BOCM yielding a low blastocyst rate (3–4% blastocysts). These glycoproteins do not seem to be necessary for bovine embryo development up to the blastocyst stage in our BOCM. In a second experiment, zygotes were cultured in modified synthetic oviduct fluid (mSOF) supplemented with different concentrations (0.5, 5, 50, and 500 μg/ml) of purified bovine EGP. In the third experiment, since purified bovine TIMP-1 was not available, zygotes were cultured in BOCM depleted of TIMP-1 by immunoprecipitation treatment. Adding EGP to mSOF, or removing TIMP-1 from BOCM, did not affect bovine embryo development up to the blastocyst stage, or mean number of cells per blastocyst after 8 days of culture. The results indicate that, under our culture conditions, EGP and TIMP-1 do not play an important role in sustaining bovine embryo development, and do not influence blastocyst quality, assessed in terms of total number of cells per embryo. Mol. Reprod. Dev. 46:527–534, 1997. © 1997 Wiley-Liss, Inc.     

Pregnant mares' serum gonadotropin source influences fertilization and fresh or thawed embryo development,but the effect is genotype specific

The influence of the source of pregnant mares' serum gonadotropin (PMSG) on the num ber, quality, and in vitro development of mouse embryos before and after freezing was evaluated among three genotypes: N:NIH(S), C57BL/6N, and C3H/HeN-MTV^?. Immature females were given PMSG from one of five commercial sources. Following col lection ( 116 hr later), embryos were evaluated for stage of development, and four-to eight-cell embryos were pooled within genotype and assigned to standardized fresh or freeze-thaw culture trials. Different PMSG sources stimulated the production of different num bers of total embryos (P < 0.05) but not necessarily more embryos suitable for freezing. Differences in embryo production among genotypes indicated that absolute embryo num bers using a single mouse genotype may not accurately reflect the potency of a specific gonadotropin source. The PMSG source also affected the ability of an embryo to survive in culture either immediately after collection or after frozen storage. The effect, however, was genotype specific, with some mouse strains being relatively insensitive to PMSG source, whereas gonadotropin source played a major role in determining in vitro viability in others. Development rates for freshly collected embryos differed, often inconsistently, from those of thawed embryos regardless of the PMSG source used, demonstrating that fresh embryo development cannot be used to estimate expected post-thaw survival. In vitro development of thawed embryos is influenced not only by genotype, but also the source of the gonadotropin used to promote follicular development and oocyte maturation. These findings may explain, in part, the wide variation in embryo viability and culture rates reported among laboratories and intraspecies animal populations.     

Comparison of strains and culture media used for mouse in vitro fertilization

Success rates of superovulation in response to gonadotropic hormone treatment and in vitro fertilization (ie, mitotic cleavage following insemination) of mouse eggs from outbred CD-1, hybrid CB6F_l, or hybrid B6CBAF₁, mice were compared using either a mouse inseminationmedium, modified Krebs-Ringer-bicarbonate (m-KRB), or a human insemination medium, Ham's F10 nutrient mixture. Inseminations were performed in either organ culture dishes or screw-top, flat-side tissue culture tubes. Mean superovulation rates (± SD) were 24.2 (5.1) for CD-1, 33.0 (5.8) for CB6F₁, and 16.3 (6.6) for B6CBAF₁ mice. For in vitro cleavage the best combination of mouse strain, insemination medium, and culture container was achieved using CB6F₁, mice, m-KRB medium, and culture tubes. However, Ham's medium used with either hybrid mouse strain was shown to be employable for fertilization of mouse eggs in vitro as a quality control assay and/or experimental model system for testing the human in vitro fertilization procedure.     

An essential role for functional telomeres in mouse germ cells during fertilization and early development

Late generations of telomerase-null (TR(-/-)) mice exhibit progressive defects in highly proliferative tissues and organs and decreased fertility, ultimately leading to sterility. To determine effects of telomerase deficiency on germ cells, we investigated the cleavage and preimplantation development of embryos derived from both in vivo and in vitro fertilization of TR(-/-) or wild-type (TR(+/+)) sperm with either TR(-/-) or TR(+/+) oocytes. Consistently, fertilization of TR(-/-) oocytes with either TR(+/+) or TR(-/-) sperm, and TR(-/-) sperm with TR(+/+) oocytes, resulted in aberrant cleavage and development, in contrast to the normal cleavage and development of TR(+/+) oocytes fertilized by TR(+/+) sperm. Many (>50%) of the fertilized TR(-/-) eggs developed only one pronucleus, coincident with increased incidence of cytofragmentation, in contrast to the normal formation of two pronuclei and equal cleavage of wild-type embryos. These results suggest that both TR(-/-) sperm and oocytes contribute to defective fertilization and cleavage. We further found that a subset (7-9%) of telomeres was undetectable at the ends of some metaphase I chromosomes from TR(-/-) spermatocytes and oocytes, indicating that meiotic germ cells lacking telomerase ultimately resulted in telomere shortening and loss. Dysfunction of meiotic telomeres may contribute to aberrant fertilization of gametes and lead to abnormal cleavage of embryos, implying an important role of functional telomeres for germ cells undergoing fertilization and early cleavage development.     

In vitro pollination fertilization of maize: influence of explant factors on kernel development

The influence of donor plant genotype, ear maturity, explant size, and the ratio of ovule-to-cob tissue on kernel development from in vitro pollinated ovules was examined. All genotypes evaluated in this study were capable of in vitro pollination/fertilization, however, significant differences were observed for the responses measured. Genotype means for complete kernel formation ranged from 1.5% to 25.4% with B73 exhibiting the highest response. Averaged over all genotypes, ear maturity effects were not significant, however, the genotype x ear maturity mean square was significant for swelling percentage. Explant size had a profound effect on in vitro kernel development. Averaged over all genotypes and ear maturities, 30-ovule explants resulted in more than twice as many ovules classified as complete kernels when compared to 10-ovule explants. Ovule-to-cob tissue ratio was also found to have highly significant effects on all three variables measured. An ovule-to-cob tissue ratio of 4:24 resulted in the highest percentages of swelling, embryos with incomplete embryos, and complete kernels.     

Sequential analysis of zona thickness during in vitro culture of human zygotes: Correlation with embryo quality,age, and implantation

Zona pellucida thickness was measured daily in zygotes and cleavage stage embryos. Measurements were performed on a Nikon inverted microscope equipped with Hoffman modulation optics, using an ocular micrometer. Zona thickness of each zygote/embryo was measured four times, the zygote/embryo was then “rolled over,” and four more measurements were repeated for a total of eight. The zygotes/embryos were photographed daily and the measurements repeated on the prints. Subsequently, the mean zona thickness for each stage was calculated. A total of 81 patients (mean age 33.8 ± 4.2) participated in the study. A total of 427 embryos were evaluated. Categorical data differences between groups were evaluated by ANOVA and multiple linear regression. For nominal data, the Kruskal-Wallis test was applied; when P < 0.05 the differences were considered to be significant. We found that the average zona thickness on day 1 of in vitro culture was 17.7 ± 0.14 μm; 16.3 ± 0.14 μm on day 2 and 14.9 ± 0.14 μm on day 3 (P < .0001). When the zona thickness was analyzed in relation to the number of blastomeres on day 3 of culture, there was a highly significant correlation with blastomere number (P < .0001). Similarly, there was a highly significant correlation with embryo grade (P < .005) and fragmentation (P < .001). The data were also analyzed for embryos transferred that resulted in a successful pregnancy, revealing that embryos in a pregnancy cycle had significantly thinner zonae pellucidae (P < .0001), when compared to embryos that were not transferred or from nonconceptual cycles. The average zona thickness also decreased with age, and was most apparent after 35 years. Changes in zona thickness correlated with the number of blastomeres, grade, fragmentation, age and were more evident in embryos transferred from cycles resulting in successful pregnancies. Therefore, zona pellucida measurements should be included in the overall assessment of embryo quality, since this information may be useful in the selection of optimal embryos for transfer. Mol. Reprod. Dev. 47:99–104, 1997. © 1997 Wiley-Liss, Inc.     

Sequential culture medium promotes the in vitro development of Macaca fascicularis embryos to blastocysts

In vitro production of blastocyst stage embryos from Macaca fascicularis (Mf) has not previously been demonstrated without cell support. Historical data indicates that a large proportion of Mf embryos arrest at the morula stage in nonsequential culture medium (NSM) lacking serum supplementation and/or cell support. Here we report the application of a sequential culture system supporting in vitro production of Mf blastocysts. Mf embryos produced by in vitro fertilization (IVF; n = 69) were subjected to in vitro culture without cell support in either a commercial sequential embryo culture medium (SM) or an NSM. At 24 hr post-insemination (PI) embryos generated from in vivo and in vitro matured oocytes and cultured in the NSM cleaved to two or more cells in significantly greater proportions (15/23; 65%) compared to embryos cultured in SM (14/46; 30%). However, by day 3 PI embryo development beyond eight cells was not different in NSM (9/23; 39%) compared to SM (25/46; 54%). At day 5 PI embryo development to the morula stage was slightly lower in NSM (8/23, 35%) compared to SM (21/46, 45%), and embryo degeneration was slightly higher in NSM (9/23, 39%) compared to SM (9/46, 20%). After 7-9 days of in vitro culture, embryo development to the blastocyst stage and embryo degeneration were significantly lower and higher, respectively, in NSM (0/23, 0%; and 23/23, 100%) compared to SM (9/46, 20%; and 26/46, 56%). In this study the sequential culture system was better able to support in vitro development of Mf embryos compared to nonsequential culture systems.     

Mammalian oocyte growth and development in vitro

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes from preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. © 1996 Wiley-Liss, Inc.     

Developmental capacity of rat embryos produced by in vivo or in vitro fertilization

This work compares the ability of rat zygotes fertilized in vitro or in vivo to develop into viable embryos. All oocytes were from adult cyclic females. After the first cleavage, the zygotes were transferred to oviducts of pseudopregnant recipients. Their fate was examined on day 13 at laparotomy and again on day 20. Ninety-five of 146 in vivo fertilized zygotes developed into normal sized 13-day fetuses and 72 (55%) to apparently normal near-term fetuses. Forty-six of 135 in vitro fertilized zygotes developed up to day 13, and 30 (24%) developed to term. It appears that the probability that in vitro fertilized rat zygotes will develop into viable embryos is about half the chance of in vivo fertilized zygotes. Since the two types of zygotes were morphologically identical, the morphological appearance of the two-cell stage is not an adequate criterion for judging developmental potential.