Coral larvae conservation: physiology and reproduction.

Coral species throughout the world's oceans are facing severe environmental pressures. We are interested in conserving coral larvae by means of cryopreservation, but little is known about their cellular physiology or cryobiology. These experiments examined cryoprotectant toxicity, dry weight, water and cryoprotectant permeability using cold and radiolabeled glycerol, spontaneous ice nucleation temperatures, chilling sensitivity, and settlement of coral larvae. Our two test species of coral larvae, Pocillopora damicornis (lace coral), and Fungia scutaria (mushroom coral) demonstrated a wide tolerance to cryoprotectants. Computer-aided morphometry determined that F. scutaria larvae were smaller than P. damicornis larvae. The average dry weight for P. damicornis was 24.5%, while that for F. scutaria was 17%, yielding osmotically inactive volumes (V(b)) of 0.22 and 0.15, respectively. The larvae from both species demonstrated radiolabeled glycerol uptake over time, suggesting they were permeable to the glycerol. Parameter fitting of the F. scutaria larvae data yielded a water permeability 2 microm/min/atm and a cryoprotectant permeability = 2.3 x 10(-4) cm/min while modeling indicated that glycerol reached 90% of final concentration in the larvae within 25 min. The spontaneous ice nucleation temperature for F. scutaria larvae in filtered seawater was -37.8+/-1.4 degrees C. However, when F. scutaria larvae were chilled from room temperature to -11 degrees C at various rates, they exhibited 100% mortality. When instantly cooled from room temperature to test temperatures, they showed damage below 10 degrees C. These data suggest that they are sensitive to both the rate of chilling and the absolute temperature, and indicate that vitrification may be the only means to successfully cryopreserve these organisms. Without prior cryopreservation, both species of coral settled under laboratory conditions.

Factors affecting the efficiency of embryo cryopreservation and rederivation of rat and mouse models.

The efficiency of embryo banking for rat and mouse models of human disease and normal biological processes depends on the ease of obtaining embryos. Authors report on the effect of genotype on embryo production and rederivation. In an effort to establish banks of cryopreserved embryos, they provide two databases for comparing banking efficiency: one that contains the embryo collection results from approximately 11,000 rat embryo donors (111 models) and another that contains the embryo collection results from 4,023 mouse embryo donors (57 induced mutant models). The genotype of donor females affected the efficiency of embryo collection in two ways. First, the proportion of females yielding embryos varied markedly among genotypes (rats: 16-100 %, mean =71 %; mice: 24-95 %, mean =65 %). Second, the mean number of embryos recovered from females yielding embryos varied considerably (rats: 4-10.6, mean =7.8; mice 5.3-32.2, mean =13.7). Genotype also affected the efficiency of rederivation of banked rat and mouse embryos models by embryo transfer. For rats, thawed embryos (n =684) from 33 genotypes were transferred into 66 recipient females (pregnancy rate, 78 %). The average rate of developing live newborns for individual rat genotypes was 30 % with a range of 10 to 58 %. For mice, thawed embryos (n =2,064) from 59 genotypes were transferred into 119 pseudopregnant females (pregnancy rate: 76 %). The average rate of development of individual mouse genotypes was 33 % with a range of 11 to 53 %. This analysis demonstrates that genotype is an important consideration when planning embryo banking programs.

Prolonged mating in prairie voles (Microtus ochrogaster) increases likelihood of ovulation and embryo number.

Prairie voles are induced ovulators that mate frequently in brief bouts over a period of approximately 24 h. We examined 1) impact of mating duration on ovulation and embryo number, 2) incidence of fertilization, 3) temporal pattern of embryo development, 4) embryo progression through the reproductive tract over time, and 5) embryo development in culture. Mating was videotaped to determine first copulation, and the ovaries were examined and the reproductive tracts flushed at 6, 8, 10, 12, 16, 20, and 24 h and 2, 3, and 4 days after first copulation. The number of mature follicles and fresh corpora lutea and the number and developmental stage of embryos were quantified. One, two-, and four-cell embryos were cultured in Whitten's medium. Mature follicles were present at the earliest time examined (6 h). Thirty-eight percent of females that had been paired for < 12 h after the first copulation ovulated, whereas all females paired >/= 12 h after the first copulation ovulated. Virtually all (> 99%) oocytes recovered from females paired for >/= 12 h after first copulation were fertilized. Pairing time after first copulation and mean copulation-bout duration were significant (p < 0.05) determinants of embryo number. Embryos entered the uterine horns and implanted on Days 3 and 4, respectively, after first copulation (Day 0). Embryos cultured in vitro underwent approximately one cell division per day, a rate similar to that in vivo. We conclude that prairie voles ovulate reliably after pairing for >/= 12 h, although some females showed exceptional sensitivity not predicted by the variables quantified. Prolonged mating for longer than 12 h increased the total embryos produced. This mechanism likely has adaptive significance for increasing offspring number.

Superovulation of rabbits with FSH alters in vivo development of vitrified morulae.

Morulae were flushed from the oviducts and uteri of New Zealand White (NZW) rabbits superovulated with either 6 (3 d) or 8 (4 d) injections of FSH and from non-superovulated controls. The percentages of embryos recovered from 4 d (100%, n = 8) donors was significantly higher (P < 0.01) than that of 3 d (76%, n = 16) and control (87%, n = 22) donors. Overall, fertilization rates were significantly lower for the 3 d embryos (P < 0.01). Most (86 to 90%) morulae were morphologically suitable for vitrification in an ethylene glycol-based solution. Following storage in liquid nitrogen, morulae were rapidly thawed and transferred to the uteri of pseudopregnant recipients. The total number of kits born for the 3 d, 4 d, and control groups was 40, 61 and 48, respectively. The percentage of live kits from morulae transferred was significantly lower for the 3 d (20%, n = 201) than either the 4 d (36%, n = 169; P < 0.01) or the control (31%, n = 157; P < 0.05) group. The mean number of kits born/recipient for the 3 d (2.4 +/- 2.9), 4 d (4.7 +/- 3.5), and control (3.0 +/- 2.2) protocols did not differ (P > 0.05). The estimated overall efficiency of producing kits based on normal morulae collected for control and 4 d groups, however, was nearly two-fold that for females given 6 FSH treatments. We conclude that the 4 d FSH superovulation regimen enhances the efficiency of rabbit reproductive biotechnology after embryo cryopreservation. These findings have important implications for rabbit colony management using embryo cryopreservation.

Water distribution and permeability of zebrafish embryos, Brachydanio rerio.

Teleost embryos have not been successfully cryopreserved. To formulate successful cryopreservation protocols, the distribution and cellular permeability to water must be understood. In this paper, the zebrafish (Brachydanio rerio) was used as a model for basic studies of the distribution to permeability to water. These embryos are a complex multi-compartmental system composed of two membrane-limited compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells (each surrounded by a plasma membrane). Due to the complexity of this system, a variety of techniques, including magnetic resonance microscopy and electron spin resonance, was used to measure the water in these compartments. Cellular water was distributed unequally in each compartment. At the 6-somite stage, the percent water (V/V) was distributed as follows: total in embryo = 74%, total in yolk = 42%, and total in blastoderm = 82%. A one-compartment model was used to analyze kinetic, osmotic shrinkage data and determine a phenomenological water permeability parameter, Lp, assuming intracellular isosmotic compartments of either 40 or 300 mosm. This analysis revealed that the membrane permeability changed (P < 0.05) during development. During the 75% epiboly to 3-somite stage, the mean membrane permeability remained constant (Lp = 0.022 +/- 0.002 micron x min-1atm-1 [mean +/- S.E.M.] assuming isosmotic is 40 mosm or Lp = 0.049 +/- 0.008 micron x min-1atm-1 assuming isosmotic is 300 mosm). However, at the 6-somite stage, Lp increased twofold (Lp = 0.040 +/- 0.004 micron x min-1atm-1 assuming isosmotic is 40 mosm or Lp = 0.100 +/- 0.017 micron x min-1atm-1 assuming isosmotic is 300 mosm). Therefore, the low permeability of the zebrafish embryo coupled with its large size (and consequent low area to volume ratio) led to a very slow osmotic response that should be considered before formulating cryopreservation protocols.

Chill sensitivity and cryoprotectant permeability of dechorionated zebrafish embryos, Brachydanio rerio.

The zebrafish (Brachydanio rerio) was used as a model for basic studies of the chilling sensitivity, permeability and toxicity of cryoprotectants. In both intact and dechorionated embryos, early-stage embryos (1.25, 1.5, 1.75, and 2 h) were more susceptible (P < 0.05) to chilling injury at 0 degrees C than late-stage embryos (50, 75, and 100% epiboly and three-somite stage). Moreover, enzymatic removal of the chorion did not alter (P > 0.05) this pattern of sensitivity to chilling. Eight-hour zebrafish embryos tolerated short-term exposures to temperatures ranging from 4 to 23 degrees C for 3.5 h with no detrimental developmental effects. The permeability of dechorionated embryos to cryoprotectants was examined by measuring the kinetics of volumetric change at various developmental stages (16 cells to six somites or ca. 1.25 to 14 h postfertilization) at 28.5 degrees C. The dechorionated zebrafish embryo is composed of two complex cellular compartments (i.e., a large yolk and the developing blastoderm). From 40 to 100% epiboly, the volumes of yolk and blastoderm remained constant, ca. 82 and 18%, respectively. However, these volumes changed rapidly after epiboly. For example, at the six-somite stage, the yolk composed 61% of the total volume, whereas the blastoderm composed 39%. When three- and six-somite embryos were placed in 1.5 and 2.0 M cryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurement of volume changes indicated no permeation of the cryoprotectants. However, some permeation was observed for six-somite embryos immersed in a 2.0 M methanol solution, but not for 3-somite embryos. For up to 30 min at room temperature, these cryoprotectant solutions were toxic to zebrafish embryos; however, 1.5 M glycerol and ethylene glycol solutions were. We conclude that the complex nature of the zebrafish embryo reduces the effectiveness and predictive value of light microscopical measurements for cryoprotectant permeability studies.

Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution.

We designed and conducted a field trial to obtain accurate pregnancy rates of Day 7 bovine embryos after vitrification in PB1 containing 6.5 M glycerol and 6% BSA (w/v) and one-step dilution in 1 M sucrose compared with controlled slow freezing in 1.5 M glycerol and three-step dilution. Embryos were collected from superovulated donor cows, and Grade 1 and 2 morulae and blastocysts were randomly assigned to each cryopreservation treatment group. Dutch farmers were solicited to participate in the field trial by an advertisement that offered cryopreserved embryos at subsidized cost. Within a period of 11 wk, one of six technicians visited 150 farms. Standard nonsurgical methods were used to transfer a total of 728 cryopreserved embryos. Recipient cows, mainly multiparous and of various breeds, the so-called "bottom-end" of the national herd, received embryos either 6, 7 or 8 d after standing estrus during natural estrous cycles. We compiled a database on 22 factors that may influence establishment of pregnancy in order to check randomization of each factor over cryopreservation treatment groups and embryo transfer technicians and to perform the statistical tests. Overall pregnancy rates were 44.5% (n = 393) for vitrified embryos and 45.1% (n = 335) for slowly frozen embryos. Pregnancy rates were not significantly different (ANOVA, P = 0.79 or Chi- square analysis, P = 0.88). The registered data confirm that all factors were randomly distributed over cryopreservation methods and technicians. Technician was not a significant source of variation in pregnancy rate (analysis of variance, P = 0.79). Although three technicians performed better with the one-step procedure and the other three performed better using the three-step method, the interaction between the technician and cryopreservation method was not significant (Tukey's test for nonadditivity, P = 0.13). Our results indicate that 1) vitrification and one-step dilution can be successfully used in the field without significant reduction in the pregnancy rate and 2) the pregnancy rate obtained using the "bottom-end" of the herd is satisfactory for practical application.

Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos.

Day 7 bovine morulae and early blastocysts were randomly assigned to one of four cryopreservation methods: (i) a modified conventional controlled slow freezing and stepwise dilution after thawing; and three methods which enable direct transfer of the embryo into the recipient upon thawing: (ii) conventional controlled slow freezing and a modification of a one-step procedure, (iii) vitrification with 6.5 M glycerol plus 6% BSA (w/v), and (iv) vitrification with 25% glycerol (v/v) and 25% propanediol (v/v). In a comparative in vitro study, the percentage of grade 1 and 2 embryos developing into expanded blastocysts in culture for cryopreservation methods 1-4 were, respectively, 53% (29/55), 33% (20/61), 44% (26/59), and 51% (17/33). Method 2 yielded a significantly lower survival rate than methods 1 (P < 0.1) and 4 (P < 0.05) and was excluded from a subsequent test of in vivo development. Pregnancy rates (Day 60) after transfer of embryos cryopreserved by methods 1, 3, and 4 were, respectively, 59% (20/34), 43% (17/40), and 24% (5/21). Method 4 yielded a significantly lower pregnancy rate than method 1 (P < 0.05). Method 3, however, did not yield a statistically different pregnancy rate (P > 0.1) when compared to method 1. Method 3 has considerable promise in providing a successful method for the cryopreservation of bovine embryos that (i) reduces the time required for equilibration and cooling, (ii) provides for simple and rapid one-step dilution of cryoprotectant after thawing, and (iii) enables more embryos to be thawed and transferred per unit time.

Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods.

We examined possible genotype effects on the survival of 8- to 16-cell mouse embryos isolated from four inbred strains (C57BL/6N, BALB/cAnN, DBA/2N, and C3H/HeN), a outbred stock (ICR), and various crosses after cryopreservation by vitrification or conventional slow freezing using glycerol solutions. The rates of in vitro development of C57BL/6N, BALB/cAnN, C3H/HeN, and ICR embryos to expanded blastocysts ranged from 86% to 94% after slow freezing and 85% to 97% after vitrification. The cryopreservation method did not significantly influence in vitro embryo survival after thawing (P > 0.05). Although genotype significantly influenced the in vitro survival of embryos (P = 0.008), this presumably resulted from an increased difficulty in assessing the quality grade of C3H/HeN embryos prior to cryopreservation. The rates in vivo development of C57BL/6N, BALB/cAnN, C3H/HeN, DBA/2N, and ICR embryos to normal day 18-19 fetuses ranged from 19% to 64% after slow freezing and from 18% to 63% after vitrification. The in vivo development of cryopreserved embryos was significantly influenced by cryopreservation method and genotype (P = 0.01 and P = 0.001, respectively). Vitrification yielded significantly higher rates of in vivo development than that after slow freezing (P > 0.05). In vivo development rates of DBA/2N and ICR female X B6D2F1 male embryos after cryopreservation were significantly higher than that of embryos from BALB/cAnN and C3H/HeN mice (P < 0.05). These results indicate that parental genotype exerts little or no effect on the ability of embryos to develop in vitro after vitrification or slow freezing. Differences in the ability of cryopreserved embryos to develop normally in vivo may reflect inherent genotype related differences in their post-implantation developmental potential and not their sensitivity to cryoinjury.

High in vitro and in vivo survival of day 3 mouse embryos vitrified or frozen in a non-toxic solution of glycerol and albumin.

A vitrification solution consisting of 6.5 mol glycerol l-1 and 6% (w/v) BSA in a modified Dulbecco's PBS (designated solution VS3a) was examined for the cryopreservation of 8-12-cell mouse embryos. Solution VS3a vitrified when cooled to -196 degrees C at rates of 10-2500 degrees C min-1 and vitrified suspensions did not crystallize when warmed at 200 or 2000 degrees C min-1. However, slow cooling at 5 degrees C min-1 or slow warming at 20 degrees C min-1 resulted in visible crystallization of solution VS3a. Embryos were equilibrated in solution VS3a in three steps at room temperature and placed into a 0.25 ml plastic straw in a way that permitted in-straw dilution with 1 mol sucrose l-1. Embryos equilibrated in solution VS3a and diluted immediately exhibited high rates of development in vitro to blastocysts (> 90%) if the total time of exposure to 100% solution VS3a did not exceed 5 min. Embryos exhibited high rates of development in vitro (75-97%) when equilibrated in 100% solution VS3a for 1 min and then cryopreserved using all combinations of three rates of cooling (5200 or 2500 degrees C min-1) and three rates of warming (20,000 or 2000 degrees C min-1). Although embryo suspensions visibly crystallized during slow cooling at 5 degrees C min-1, the rate of cooling was not a significant source of variance (P > 0.26). However, the rate of warming was found to have a small but significant effect on embryo survival (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Successful intrauterine insemination of Eld's deer (Cervus eldi thamin) with frozen-thawed spermatozoa.

This study tested the efficacy of assisted reproduction (synchronization of oestrus and intrauterine artificial insemination (AI)) in contributing to the captive propagation of an endangered species, the Eld's deer (Cervus eldi thamin). Semen was collected from males preselected on the basis of under-represented genotype. Motility of spermatozoa after thawing from ejaculates diluted with BF5F extender (8% glycerol), frozen on dry ice in 0.5 ml straws and stored in liquid nitrogen was 60-70%. Intravaginal progesterone-releasing devices (controlled internal drug release, CIDR-type G) were inserted into 20 adult Eld's deer hinds for 14 days. In all hinds, semen (7.5-10 x 10(6) motile spermatozoa per uterine horn) was deposited by laparoscopy performed 70 h after removal of the CIDR device. Ovarian activity, before and after AI, was monitored by analysing pregnanediol-3 alpha-glucuronide (PdG) concentrations in voided urine collected three to seven times per week. During the period of CIDR device insertion, urinary PdG profiles were equal to, or above, normal luteal phase concentrations in all hinds. Within 48 h of device withdrawal, PdG concentrations returned to baseline values in 17 of the 20 females, and the onset of behavioural oestrus occurred at this time in 12 hinds. On the basis of sustained increases in urinary PdG, 9 of the 20 hinds were diagnosed as pregnant by 90 days after AI, all of which delivered offspring after a mean gestation of 241.1 days (range, 235-245). Seven singletons (two females, five males) were born alive and survived, and one singleton and one set of twins were stillborn (three females).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos.

Two studies were conducted to evaluate the influence of cryoprotectant, cooling rate, container and cryopreservation procedure on the post-thaw viability of sheep embryos. In Study 1, late morula- to blastocyst-stage embryos were exposed to 1 of 10 cryoprotectant (1.5 M, glycerol vs propylene glycol)-plunge temperature treatments. Embryos were placed in glass ampules and cooled at 1 degrees C/min to -5 degrees C, seeded and further cooled at 0.3 degrees C/min to -15, -20, -25, -30 and -35 degrees C before rapid cooling by direct placement in liquid nitrogen (LN(2)). Post-thaw embryo viability was improved (P<0.01) when embryos were cooled to at least -30 degrees C before LN(2) plunging. Although there were no overt differences in embryo viability between cryoprotectant treatments (each resulted in live offspring after embryo transfer), there was a lower (P<0.01) incidence of zona pellucida damage using propylene glycol (4%) compared to glycerol (40%). In Study 2, embryos were equilibrated in 1.5 M propylene glycol or glycerol or a vitrification solution (VS3a). Embryos treated in propylene glycol or glycerol were divided into ampule or one-step((R)) straw treatments, cooled to -6 degrees C at 1 degrees C/min, seeded, cooled at 0.5 degrees C/min to -35 degrees C, held for 15 minutes and then transferred to LN(2). Embryos vitrified in the highly concentrated VS3a (6.5 M glycerol + 6% bovine serum albumin) were transferred from room air to LN(2) vapor, and then stored in LN(2). Propylene glycol- and glycerol-treated embryos in straws experienced lower (P<0.05) degeneration rates (27%) and yielded more (P<0.05) hatched blastocysts (73 and 60%, respectively) at 48 hours of culture and more (P<0.05) trophoblastic outgrowths (67 and 53%, respectively) after 1 week than vitrified embryos (47, 40 and 20%, respectively). In vitro development rate for VS3a-treated embryos was similar (P>0.10) to that of ampule controls, which had fewer (P<0.05) expanded blastocysts compared to similar straw treatments. Live offspring were produced from embryos cryopreserved by each straw treatment (propylene glycol, 3 of 7; glycerol, 1 of 7; VS3a, 2 of 7). In summary, freeze-preservation of sheep embryos was more effective in one-step straws than glass ampules and propylene glycol tended to be the optimum cryoprotectant. Furthermore, these findings demonstrate, for the first time, the biological competence of sheep embryos cryopreserved using the simple and rapid procedure of vitrification.

Deuterium oxide, dimethylsulfoxide and heat shock confer protection against hydrostatic pressure damage in yeast.

Deuterium oxide, dimethylsulfoxide (Me2SO) and heat shock treatment were all significantly effective at baro-injury as measured by plating efficiency after decompression. The content of unfreezable cell water was observed to increase during heat treatment, and this increase was associated with increase in viability.

Cryopreservation of Drosophila melanogaster embryos.

There is an urgent need to preserve the ever-increasing number (greater than 30,000) of different genetic strains of D. melanogaster that are maintained in national and international stock centres and in the laboratories of individual investigators. In all cases, the stocks are maintained as adult populations and require transfer to fresh medium every two to four weeks. This is not only costly in terms of materials, labour and space, but unique strains are vulnerable to accidental loss, contamination, and changes in genotype that can occur during continuous culture through mutation, genetic drift or selection. Although cryopreservation of Drosophila germ-plasm would be an enormous advantage, many attempts using conventional procedures have been unsuccessful. D. melanogaster embryos are refractory to conventional cryopreservation procedures because of the contravening conditions required to minimize mortality resulting from both intracellular ice formation and chilling injury at subzero temperatures. To overcome these obstacles, we have developed a vitrification procedure that precludes intracellular ice formation so that the embryos can be cooled and warmed at ultra-rapid rates to minimize chilling injury, and have recovered viable embryos following storage in liquid nitrogen. In a series of 53 experiments, a total of 3,711 larvae emerged from 17,280 eggs that were cooled in liquid nitrogen (18.4 +/- 8.8%). Further, using a subset from this population, approximately 3% of the surviving larvae (24/800) developed into adults. These adults were fertile and produced an F1 generation.

Prenatal diagnosis of sex in bovine fetuses by amniocentesis.

The sex of 1, 056 bovine fetuses was diagnosed by cytogenetic analysis of amniotic cells collected surgically from cows made pregnant as a result of embryo transfer. The fetuses ranged in gestational age from 7 to 22 wk at the time of amniocentesis. Amniotic cells were cultured for 5 to 30 d to obtain a sufficient number of cells for cytogenetic analysis. The growth rate of 819 samples was examined in detail. On average, amniotic cells from pregnancies that were 7 or 8 wks old required about 13 d to reach a cell concentration sufficient for analysis, whereas those from pregnancies that were 10 wk old or more required only 10 d or less to reach that concentration. Final disposition of 325 pregnancies subjected to amniocentesis was unequivocally confirmed. Of these, the sex of 302 fetuses (93%) was determined with a male: female ratio of 51:49. The gender of 213 of 220 fetuses (96.8%) was correctly dignosed, as verified by examination of either 33 intentionally induced abortuses or of 187 live calves. Thirteen percent (29) of 216 pregnancies that were allowed to proceed to term ended in spontaneous abortion, a rate only about 5% higher than that reported to occur normally in embryo transfer pregnancies. The remaining 53 pregnancies were induced to abort, but it was not possible to recover and verify the sex of those fetuses. The capability to diagnose fetal sex in utero yielded other useful information as well. For example, the sex ratio of 1,056 fetuses during development from 7 to 15+ wk of gestation was found to be the same (51:49) as it was at calving. Finally, amniocentesis to determine prenatal sex provides time to alter the sex ratio of a calf crop. It may ultimately prove valuable as an adjunct to genetic engineering of cattle to identify presumptive transgenic calves in utero.

Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos.

Although fracture damage to the zonae pellucidae and blastomeres is frequently observed after the cryopreservation of mammalian embryos, little is known of the mechanism by which this occurs. The incidence of damage to zonae was measured when bovine ova with normal zonae were frozen in straws or glass test tubes by standard embryo cryopreservation procedures that yield high rates of survival. Ova were examined for zona damage after warming by procedures that ought to produce little or no thermal stress (slow warming in 20 degrees C air) or high levels of stress (rapid warming in liquid baths). Ova frozen in straws exhibited no zona damage after slow warming at 150 degrees C/min in air (n = 206). However, the incidence of zona damage increased when the straws were warmed rapidly in 20 degrees C (n = 157) or 36 degrees C (n = 159) water (17 and 24%, respectively). Ova in straws warmed rapidly in nonaqueous liquids (ethylene glycol, or silicone oil) exhibited lower rates of zona damage (2 to 5%). Ova frozen in glass tubes exhibited a much higher incidence of zona damage than those frozen in straws, regardless of the warming conditions. Thus, 30% of 114 ova exhibited damage when tubes were warmed slowly at 25 degrees C/min in air, while 54% of 98 ova showed zona damage when tubes were warmed rapidly at 500 degrees C/min in 36 degrees C water. These results are consistent with the view that zona damage is associated with thermally-induced fracturing of the suspension during rapid changes of temperature.

Pregnancy resulting from cryopreserved human embryos using a one-step in situ dilution procedure.

In vitro fertilization and embryo transfer require the use of hormonal manipulation and surgery that may reduce the receptivity of the patient's uterus during the stimulated cycle. Cryopreservation of human embryos eliminates the need for immediate transfer, permitting them to be stored until they can be transferred during subsequent unstimulated cycles. Embryo cryopreservation is an established procedure in the breeding of laboratory and domestic animals, but has only recently been applied to humans. We report on a pregnancy using a simple cryopreservation procedure that permits embryos to be diluted out of the cryoprotectant solution without removing them from the plastic straw in which they were cryopreserved.

Factors affecting the survival of mouse embryos cryopreserved by vitrification.

Preimplantation stage mouse embryos have been used to examine the response of a simple multicellular system to cryopreservation by the complete vitrification of the suspension. Successful vitrification requires the use of a solution of cryoprotectants that is sufficiently concentrated to supercool and solidify into a glass at practicable cooling rates. Factors that influence the survival of embryos include the concentration and composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution. High rates of survival are obtained when embryos are dehydrated prior to vitrification in solutions composed of saline plus multimolar concentrations of either mixtures of permeating cryoprotectants (e.g. dimethyl sulphoxide-acetamide-propylene glycol) or single permeating cryoprotectants (propylene glycol or glycerol). Full permeation of cryoprotectants into the cells is not necessary and may lead to chemical toxicity and osmotic injury. Partial permeation and osmotic shrinkage concentrates the endogenous cytoplasmic macromolecules and greatly increases the likelihood of intracellular vitrification. Vitrification is a practical approach for embryo cryopreservation and offers new opportunities to examine fundamental aspects of cryoprotection and cryoinjury in the absence of freezing.

Development of mouse embryos cryopreserved by vitrification.

Eight-cell mouse embryos were cryopreserved by vitrification in a concentrated solution of dimethylsulphoxide, acetamide, propylene glycol and polyethylene glycol. This solution (designated VS1) does not crystallize when cooled to subzero temperatures but instead forms a glassy transparent solid. Embryos were exposed in three steps to a stock VS1 solution or a saline solution containing 90% of the cryoprotectants in the stock VS1 (90% VS1) and then the suspensions were vitrified by rapid cooling in liquid nitrogen. Of 568 embryos vitrified in 90% VS1, 80% developed in vitro and 98 normal fetuses or young (17% of the total) were produced after transfer to pseudopregnant recipients. By contrast, 22% of 153 embryos vitrified in the stock VS1 developed in vitro, but only one normal fetus was obtained after transfer. These results demonstrate that normal fetuses and young can be produced from embryos cryopreserved by the simple and rapid method of vitrification.

Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification.

The failure of complex mammalian organs, such as the kidney, to function following freezing to low temperatures is thought to be due largely to mechanical disruption of the intercellular architecture by the formation of extracellular ice. Classical approaches to the avoidance of ice formation through the imposition of ultra-rapid cooling and warming rates or by gradual depression of the equilibrium freezing point during cooling to -80 degrees C have not been adequate. An alternative approach relies on the ability of highly concentrated aqueous solutions of cryoprotective agents to supercool to very low temperatures. At sufficiently low temperatures, these solutions become so viscous that they solidify without the formation of ice, a process termed vitrification. When embryo suspensions are cryopreserved using conventional procedures, this supercooling behaviour allows intracellular vitrification, even in the presence of extracellular ice. We have therefore used mouse embryos to examine the feasibility of obtaining high survival following vitrification of both the intra- and extracellular solutions and report here that in properly controlled conditions embryos seem to survive in high proportions after cryopreservation in the absence of ice.