Freezing bovine embryos with a portable liquid nitrogen freezer requiring no external seeding

One-hundred and twenty excellent morula to blastocyst stage bovine embryos were obtained nonsurgically from superovulated Holstein heifers. Completely portable, nonelectric (manual) liquid nitrogen (LN) freezers combined with simplified freezing curves utilizing self-seeding were compared to a programmable LN freezer (Planner-R204) following the conventional freezing rate for freezing embryos. Seeding was self induced in ampules at -6.8 degrees C and at -5.5 degrees C in straws in the manual freezers. Glycerol was used as the cryoprotectant at 1.5 M concentration. Post-thaw appearance, fluorscein diacetate testing (FDA), and growth after 12 and 24 h incubation were used as indicators of embryo viability. There were no significant differences between embryos frozen in the two types of freezers in terms of the viability tests used. Pregnancy rates resulting from transfer of embryos frozen in the two types of freezers will be determined in subsequent field trials. The manual LN freezers used in this study are capable of successfully freezing bovine embryos. The simplified nature of these freezers and the freezing procedures used with them greatly decreases the complexity and expense of freezing embryos.     

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.     

Freezing mammalian embryos: A review of the techniques

This article reviews the literature on freezing mammalian oocytes and embryos, with emphasis on embryos of domestic animals. Mammalian embryos must be stored in a quiescent state to retain viability for long periods. This has been accomplished by freezing and storing the embryos at ?196°C. To freeze embryos, a cryoprotectant like dimethyl sulfoxide (DMSO) or glycerol was required, slow cooling (0.1 to 2.0°C/min) and warming (1 to 50°C/min) rates were used, enucleation or seeding the freezing medium was a necessity, and stepwise addition and removal of the cryoprotectant at room temperature seemed to be beneficial. Using the above parameters embryos have been frozen and stored at ?196°C for several years and upon thawing and transfer to a suitable recipient, viable offspring have developed. Initially embryo viability was low after freezing-thawing, but with refinement of freezing-thawing techniques has increased sufficiently so that freezing embryos is no longer a laboratory technique, but is applicable to field use.     

In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycerol

The aim of the present study was to compare the survival rates of goat morulae and blastocysts after different freezing procedures. The viability of frozen-thawed embryos was assessed both in vivo and in vitro. Two cryoprotectants, ethylene glycol and glycerol, were used and three cryoprotectant removal procedures were compared: progressive dilution in 1.0, 0.5, 0.3 and 0 M of cryoprotectant in PBS; a similar progressive dilution with cryoprotectant in PBS plus 0.25 M of sucrose; or one-step transfer in PBS containing 0.25 M of sucrose. In vitro development of frozen-thawed blastocysts was always higher than that of frozen morulae irrespective of the cryoprotectant (52 129 = 40.3% vs 23 161 = 14.3% ; P< 0.001). In vivo, however, frozen-thawed morulae developed equally as well as blastocysts after an identical freezing-thawing protocol. Development both in vivo and in vitro showed ethylene glycol to be a better cryoprotectant than glycerol for goat embryos at both developmental stages (23 vs 0%, 45 vs 35% in vitro; 34.5 vs 21%, 35 vs 23% in vivo for morulae and blastocysts, respectively).     

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.     

Media alternatives for the collection, culture and freezing of mouse and cattle embryos

The replacement of biological products in media for the collection, culture and freezing of mammalian embryos was studied. To test the hypothesis that chemically defined surfactants can replace bovine serum albumin (BSA) or serum in embryo media, morula-stage mouse and cattle embryos were collected, cultured, and/or frozen in the surfactant compound, VF5. Collection efficiency of mouse and cattle embryos did not differ whether the medium contained serum or surfactant. In addition, morula-stage mouse and cattle embryos developed and hatched at similar rates in culture media containing either BSA or surfactant. Although the freeze/thaw survival and development in culture of bovine embryos was not significantly different in any of the media, there was a significantly lower hatching rate of mouse embryos frozen with serum or surfactant than with cryoprotectant alone or with cryoprotectant plus albumin-free serum. However, the absence of serum or surfactant in embryo freezing media resulted in embryo loss, presumably due to stickiness. The data suggest that serum can be replaced by a chemically defined surfactant in mouse and cattle embryo transfer systems for the collection, culturing and freezing of embryos. It is likely that the beneficial effects of serum are due to its surfactant properties.     

Quantification of embryo quality by respirometry

It is generally accepted that assessment of embryo metabolism, in particular oxygen consumption, may improve embryo selection by identifying the embryos with higher developmental competence. Several methods have been employed to measure embryonic oxygen consumption, but most of them were detrimental to subsequent embryo development. Recently, we have introduced the Nanorespirometer system, which is a non-invasive and highly sensitive technology developed for the individual measurement of embryonic respiration rates. This technology is able to perform single measurements at a fixed time or stage of embryonic development without adversely influencing embryo viability. Concomitantly, and based on the same principles, a second technology -- the Embryo Respirometer -- has been developed. The Embryo Respirometer allows the continuous measurement of individual respiration rates with simultaneous acquisition of digital images of each embryo, during the entire culture period (6-7 days). In this review, both technologies are described and their potential use as diagnostic tools for improving embryo selection in bovine and human following IVF treatments is discussed. Correlations between respiration rates of individual embryos and other parameters such as morphological quality, sex, stage of development, kinetics, diameter, expression of key metabolic genes and subsequent viability following embryo transfer are also examined. On the basis of the results obtained, it is postulated that assessment of embryonic respiration rates in association with other viability parameters allows for a more accurate embryo evaluation, both under clinical and research conditions.     

The effect of internal and external cryoprotectants on zebrafish (Danio rerio) embryos

The study investigated the effects of internal (DMSO, 1,2-propanediol, glycerol, ethylene glycol, methanol, N,N-dimethylacetamide) and external cryoprotectants (glucose, sucrose) on the viability and on morphometric parameters of zebrafish embryos. From the tested internal cryoprotectants, DMSO had the lowest toxicity, followed by 1,2-propanediol and glycerol. The external cryoprotectants were less toxic then the internal ones. Early ontogenetic stages were more sensible to cryoprotectant exposure than advanced stages. Two-step incubation procedures in increasing concentrations of internal and external cryoprotectants were superior to multiple-step exposure procedures. All tested vitrification solutions exceeded the tolerance limit of embryos. The tolerance of zebrafish embryos to cryoprotectants was highly variable in a concentration range causing approximately 50% embryo mortality. The width of the perivitelline space showed significant morphometrical changes due to cryoprotectant exposure. In the germinative tissue non-significant changes occurred. The yolk did not change morphometrically after exposure to internal cryoprotectants and showed no sign of dehydration after exposure to external cryoprotectants. Based on these results the study comes to the following conclusions: as yolk dehydration was impossible and as vitrification solutions were over the tolerance limit it seems unlikely that successful vitrification of zebrafish embryos can be achieved. Under these considerations slow freezing methods would be a better option as lower cryoprotectant concentrations can be used and embryos can be dehydrated during freezing.     

Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure

The study was designed to evaluate the efficiency of a modified (sealed) open pulled straw (mOPS) method for cryopreserving rabbit embryos by vitrification or rapid freezing. An additional objective was to determine whether the mOPS method could cause the vitrification of a cryoprotectant solution generally used in rapid freezing procedures. Two consecutive experiments of in vitro and in vivo viability were performed. In Experiment 1, the in vitro viability of rabbit embryos at the morula, compacted morula, early blastocyst and blastocyst stages was assessed after exposure to a mixture of 25% glycerol and 25% ethylene glycol (25GLY:25EG: vitrification solution) or 4.5 M (approximately 25% EG) ethylene glycol and 0.25 M sucrose (25EG:SUC: rapid freezing solution). Embryos were loaded into standard straws or mOPS and plunged directly into liquid nitrogen. The mOPS consisted of standard straws that were heat-pulled, leaving a wide opening for the cotton plug and a narrow one for loading embryos by capillarity. The embryos were aspirated into the mOPS in a column positioned between two columns of cryoprotectant solution separated by air bubbles. The mOPS were then sealed with polyvinyl-alcohol (PVA) sealing powder. The vitrification 25GLY:25EG solution became vitrified both in standard straws and mOPS, whereas the rapid freezing 25EG:SUC solution crystallized in standard straws, but vitrified in mOPS. The total number of embryos cryopreserved was 1695. Embryos cryopreserved after exposure to each solution in mOPS showed higher rates (88.2%) of survival immediately after thawing and removal of the cryoprotectant than those cryopreserved in 0.25 ml standard straws (78.8%; P < 0.0001). After culture, the developmental stage of the cryopreserved embryos significantly affected the rates of development to the expanded blastocyst stage. Regardless of the cryoprotectant used, lower rates of in vitro development were obtained when the embryos were cryopreserved at the morula stage, and higher rates achieved using embryos at blastocyst stages. Based on the results of Experiment 1, the second experiment was performed on blastocysts using the mOPS method. Experiment 2 was designed to evaluate the in vivo viability of cryopreserved rabbit blastocysts loaded into mOPS after exposure to 25GLY:25EG or 25EG:SUC. Embryos cryopreserved in mOPS and 25GLY:25EG solution gave rise to rates of live offspring (51.7%) not significantly different to those achieved using fresh embryos (58.5%). In conclusion, the modified (sealed) OPS method allows vitrification of the cryoprotectant solution at a lower concentration of cryoprotectants than that generally used in vitrification procedures. Rabbit blastocysts cryopreserved using a 25GLY:25EG solution in mOPS showed a similar rate of in vivo development after thawing to that shown by fresh embryos.     

Cryopreservation of bovine oocytes: is cryoloop vitrification the future to preserving the female gamete?

The cryoloop is a technique where a thin nylon loop is used to suspend a film of cryoprotectant containing the oocytes and directly immersing them in liquid nitrogen. 508 bovine oocytes were collected, of these 351 were cryopreserved by slow freezing using standard straws or a new vitrification method using our self-constructed cryoloops and the remainder were controls. After thawing, the oocytes were inseminated by ICSI or standard IVF. The cryoloop vitrification method yielded a survival rate of 90.5% and the slow freezing technique a rate of 54.4% (p < 0.0001). When ICSI was performed, cryopreservation by the cryoloop vitrification method resulted in very similar cleavage rate to controls (16.0% vs. 17.3%) but slow freezing produced a slightly lower rate (9.4%). Cleavage rates after IVF in fresh oocytes was higher than the cryopreservation groups (49.5% vs. 15.4% and 25.8%), whereas after ICSI the rates were similar in all groups (17.3% vs. 9.4% and 16%). It is concluded that the new cryoloop vitrification technique followed by ICSI produce good embryo formation results and they could hold the future for effective oocyte cryopreservation.     

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.     

Sheep embryo cryopreservation by vitrification and conventional freezing

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% propylene glycol and 25% glycerol. A group of embryos (simulated control) was processed without freezing to evaluate the toxicity of the vitrification solution. In Experiment II, embryos were exposed to a solution of PBS containing 10% glycerol and 0.25 M sucrose placed horizontally in a programmable freezer. Automatic seeding was applied at -7 degrees C in 2 positions on straws and cooled at -0.3 degrees C/min to -25 degrees C and then stored in liquid nitrogen. In vitro development rates of vitrified embryos were 12% (morulae) and 19% (blastocysts). Simulated embryos showed a higher rate of survival than embryos cryopreserved by vitrification (67 and 63%, morulae and blastocysts respectively). In conventional cooling, the blastocysts showed the highest viability percentage (67%) of all the experimental groups but these values decreased significantly in morulae (31%). Differences in temperature between straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Embryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the central point of this column (65 vs 21%). The damage of both cryopreservation methods on zona pellucida integrity (27 and 35% in vitrified and conventionally frozen embryos, respectively) had no effect on the in vitro survival.     

In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes

The aim of this study was to determine the influence of two ethylene glycol-based vitrification solutions on in vitro and in vivo survival after in-straw cryoprotectant dilution of vitrified in vitro-produced bovine embryos. Day-7 expanded blastocysts were selected according to diameter (> or = 180 microm) and osmotic characteristics and randomly assigned to one of three groups (i) VSa: vitrification in 40% EG+17.1% SUC+0.1% PVA; (ii) VSb: vitrification in 20% EG+20% DMSO; (iii) control: non-vitrified embryos. Vitrification was performed in hand-pulled glass micropipettes (GMP) and cryoprotectant dilution in 0.25 ml straws after warming in a plastic tube. Embryo viability was assessed by re-expansion and hatching rates after 72 h of IVC and by pregnancy rates after direct transfer of vitrified embryos. No differences in re-expansion rates were observed between vitrified groups after 24 h in culture (VSa=84.5%; VSb=94.8%). However, fewer VSa embryos (55.2%, P<0.05) hatched after 72 h than the VSb (75.8%) and control embryos (80.0%). To evaluate in vivo viability, vitrified embryos (VSa=20; VSb=21) were warmed under field conditions and individually transferred to synchronous recipients. Pregnancy rates (day 60) were similar between groups (VSa=20%; VSb=19%). Greater hatching rates occurred after 72 h of IVC for EG+DMSO than EG+SUC+PVA vitrification solutions. However, using a GMP vitrification container and in-tube warming, both solutions provided similar pregnancy rates after the in-straw cryoprotectant dilution and direct embryo transfer.     

Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification

Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.     

Study of cryopreservation of articular chondrocytes using the Taguchi method

This study evaluates the effect of control factors on cryopreservation of articular cartilage chondrocytes using the Taguchi method. Freeze-thaw experiments based on the L₈(2⁷) two-level orthogonal array of the Taguchi method are conducted, and ANOVA (analysis of variables) is adopted to determine the statistically significant control factors that affect the viability of the cell. Results show that the type of cryoprotectant, freezing rate, thawing rate, and concentration of cryoprotectant (listed in the order of influence) are the statistically significant control factors that affect the post-thaw viability. The end temperature and durations of the first and second stages of freezing do not affect the post-thaw viability. Within the ranges of the control factors studied in this work, the optimal test condition is found to be a freezing rate of 0.61 ± 0.03 °C/min, a thawing rate of 126.84 ± 5.57 °C/min, Me₂SO cryoprotectant, and a cryoprotectant concentration of 10% (v/v) for maximum cell viability. In addition, this study also explores the effect of cryopreservation on the expression of type II collagen using immunocytochemical staining and digital image processing. The results show that the ability of cryopreserved chondrocytes to express type II collagen is reduced within the first five days of monolayer culture.     

A comparative study of mouse embryo freeze-preservation including the examination of a thermoelectric freezing device

In Study 1 over 2000 4- to 8-cell mouse embryos were randomly pooled and assigned to 1 of 12 treatment groups. A 2 X 2 X 3 factorial design was used to analyze two types of cryoprotectant/post-thaw (PT) dilutions (dimethyl sulfoxide [Me2SO]/stepwise dilution versus glycerol/sucrose dilution), two storage containers (glass ampoules versus plastic straws), and three cooling treatments. Two commercial, controlled-rate freezing machines were examined, employing either nitrogen gas (Planer) or thermoelectric (Glacier) cooling. Embryos were cooled slowly (0.5 degrees C/min) to -35 or -80 degrees C and then cooled rapidly by transfer into liquid nitrogen (LN2). Thawed embryos were cultured for 24 hr after which developmental stage, post-thaw survival (PTS), embryo degeneration rate (EDR), quality grade (QG), and fluorescein diacetate viability grade (VG) were assessed. Overall, PTS and EDR were similar (P greater than 0.05) among the three freezing unit/plunge temperature treatments. Cumulative results of container and cryoprotectant/PT dilution treatments consistently demonstrated greater PTS, QG, and VG ratings and lower EDR values when embryos were frozen in ampoules using glycerol/sucrose dilution. Embryos treated with Me2SO/stepwise dilution were particularly sensitive to freezing damage when stored in plastic straws and plunged into LN2 at -35 degrees C. Study 2 was directed at determining whether Study 1 methods for diluting Me2SO-protected embryos markedly affected PTS rates. Post-thaw culture percentages were no different (P greater than 0.05) for four- to eight-cell Me2SO-treated embryos frozen in ampoules (using the forced-LN2 device), thawed, and diluted either conventionally in reduced concentrations of Me2SO or in the sucrose treatment normally accorded glycerolated embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

A Simple, Efficient Technique for Freezing Tetrahymena thermophila

ABSTRACT. We have developed a simple, efficient procedure for the long term freezing of Tetrahymena thermophila in liquid nitrogen. This technique yields excellent recovery of viable cells with all strains tested and does not require the use of a controlled rate low temperature freezer. To optimize the freezing technique, we have examined the effects of varying a number of parameters, including the physiological state of the cells prior to freezing, the time of exposure to cryoprotectant, and the rate of freezing and thawing. the frequency of viable cell recovery following freezing using this technique has been tested for a variety of different cell lines.     

Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos

Embryo cryopreservation is an important tool to preserve endangered species. As a cryoprotectant for mouse oocytes, antifreeze protein from Anatolica polita (ApAFP914) has demonstrated utility. In the present study, the effects of controlled slow freezing and vitrification methods on the survival rate of sheep oocytes fertilized in vitro after freezing-thawing were compared. Different ApAFP914 concentrations were added to the vitrification liquid for exploring the effect of antifreeze protein on the warmed embryos. The results showed that the survival and hatching rates of in vitro derived embryos were significantly higher than that of the slow freezing method. Furthermore, among the cryopreserved embryos at different developmental stages, the survival and hatching rates of the expanded blastocyst were significantly higher than those of the blastocysts, early blastocysts and morula. The survival and the hatching rates of the fast-growing embryos were both significantly higher than that of the slow-growing embryos. Additionally, treatment of ApAFP914 (5–30 μg/mL) did not increase the freezing efficiency of the 6–6.5 d embryos. However, addition of 10 μg/mL of ApAFP914 significantly increased the hatching rate of slow-growing embryos. In conclusion, our study suggests that the vitrification is better than the slow freezing method for the conservation of in vitro sheep embryos, and supplementation of ApAFP914 (10 μg/mL) significantly increased the hatching rate of slow-growing embryos after cryopreservation.     

Cryopreservation of Rainbow Trout (Oncorhynchus mykiss) Blastomeres: Influence of Embryo Stage on Postthaw Survival Rate

The cryopreservation of isolated fish blastomeres is likely to provide a valid alternative to embryo cryopreservation, the results of which are still discouraging. A repeatable technique for the cryopreservation of rainbow trout blastomeres has been established and the effect of embryonic developmental stage on freezing tolerance evaluated. Embryos at Ballard 6A, 6B, and 6C stages were dechorionated and left to dissociate in a Ca2+- and Mg2+-free medium. Cryoprotection was provided by step-wise addition of 1.4 M 1,2-propanediol. Cells were loaded into the middle of 250-μl straws and slowly frozen to -80 degreesC before being plunged into LN2. A low thawing rate was adopted, followed by step-wise removal of the cryoprotectant. Morphological evaluation was by microscopy and video recording. Metabolic activity and survival rate were determined by FDA and PI staining, by recovery of the ability to reassociate after 24 h culture in Leibovitz L15 + 2% Ultroser, and by measuring DNA synthesis in 6B cells by the method of BrdU incorporation. Survival rates were 53 +/- 9.3, 88 +/- 1.7, and 95 +/- 0.5% for stage 6A, 6B, and 6C cells, respectively. While 6A cells reassociated into clumps of cells, 6B and 6C cells formed holoblastic morulas in 24 h; proliferation of 6B cells was comparable to fresh control cells. The relationship between freezing tolerance and the physiological events occurring during early embryonic development is discussed in light of these results and conclusions are drawn that envisage the transfer of frozen-thawed blastomeres into recipient embryos. Copyright 1998 Academic Press.