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Biology of Reproduction 65, 1127-1134 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Effect of Conventional Controlled-Rate Freezing and Vitrification on Morphology and Metabolism of Bovine Blastocysts Produced In Vitro1

S. Kaidia, S. Bernarda, P. Lambertb, A. Massipa, F. Dessya, and I. Donnay2,a

a Université Catholique de Louvain, Unité des Sciences Vétérinaires b Institut de Statistiques, B-1348 Louvain-la-Neuve, Belgium

ABSTRACT

This study compares the effects of conventional controlled-rate freezing and vitrification on the morphology and metabolism of in vitro-produced bovine blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct fluid with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25% glycerol, 25% ethylene glycol. Cell alterations and in vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) cell number as well as glucose, pyruvate, and oxygen uptakes, and lactate release by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservation procedures, while alterations in nuclear staining were more frequent in vitrified embryos. After culture, similar survival and hatching rates were observed. Both procedures decreased cell number immediately after thawing and after 72 h. However, the number of TE cells was lower in frozen embryos than in vitrified ones. In relation to this, frozen blastocysts showed a decrease in glucose, pyruvate, and oxygen uptake, although those parameters were not altered in vitrified embryos. An increased glycolytic activity was also observed in frozen embryos, indicating a stress response to this procedure.

developmental biology, early development, in vitro fertilization

INTRODUCTION

In vitro-produced (IVP) bovine embryos are characterized by an increased chilling sensitivity and a lower freezability compared to their in vivo counterparts [1, 2]. After cryopreservation, the rate of re-expansion and hatching in vitro is high, while development after transfer to recipient animals remains low [3–6]. Loss of viability after cryopreservation and transfer is probably due to cellular damage or metabolic disturbances occurring during the procedure. The type and degree of such cryoinjuries are likely to depend on the method of cryopreservation.

Two major cryopreservation methods are used: conventional freezing and vitrification. They induce different injuries in bovine blastocysts. Intracellular and extracellular ice crystal formation occurring during freezing can induce damage to junctions between trophectoderm (TE) cells [7, 8], to membranes and cristae in mitochondria, to nuclear and plasma membranes [9], fractures of the zona pellucida, and cytoplasmic defects [10]. This leads to a decrease in total cell number, accumulation of cellular debris in the perivitelline space, and death of inner cell mass (ICM) cells [10, 11]. Vitrification is defined as the solidification of a solution brought about, not by crystallization, but by extreme elevation in viscosity during cooling [12]. During vitrification, no ice is formed but the osmotic changes are often more severe than during freezing [13] and the higher concentrations of cryoprotectants can increase their toxic effects on embryonic cells [13–15]. Damage to embryos after vitrification includes a decrease in numbers of microvilli in trophectoderm cells, loss of plasma membrane integrity, mitochondrial changes and swelling of the rough endoplasmic reticulum, formation of small vesicles, and distinct intramembrane particle aggregations in the plasma membranes [16–18]. Some of these changes are apparently reversible [16–18], but cell death also occurs, as suggested by the decrease in blastocyst cell number [18] and the accumulation of cellular debris in the perivitelline space and the blastocoele cavity [16].

Alterations in embryo metabolism after freezing have also been reported in cattle [19–21]. A general decrease in both nutrient uptake and utilization is observed after blastocyst freezing. Some results are consistent with a partial uncoupling of the inner mitochondrial membrane [22]. Embryos may be able to compensate by increasing glycolytic activity [19]. Vitrification appears to alter glycolytic metabolic profiles of bovine embryos [23].

Until now, however, direct comparison between freezing and vitrification has led to differing results depending on the method (cryoprotectants, number of steps). Most of the results concerned developmental rates. Uechi et al. [24] observed a decrease in implantation rate of blastocysts developed in vitro from 2-cell mouse embryos vitrified in a mixture of ethylene glycol, Ficoll, and trehalose by comparison with those developed from embryos frozen in a mixture of propanediol and sucrose. Rall and Wood [25] compared the effect of cooling rate (slow freezing or vitrification) in 6.5 mol glycerol on 8- to 12-cell mouse embryos and observed no difference in the rate of development in vitro and after transfer. Sommerfeld and Niemann [26] used the same approach on bovine blastocysts using ethylene glycol as sole cryoprotectant. Hatching rate in vitro after conventional freezing was similar to the control while it was lower after vitrification. Van Wagtendonk-De-Leeuw et al. [27] compared four methods of cryopreservation (two freezing and two vitrification procedures) and observed some differences that seemed unrelated to the rate of cooling. Mahmoudzadeh et al. [28] obtained better in vitro survival and hatching rates after vitrification of expanded IVP bovine blastocysts in a mixture of ethylene glycol, Ficoll, and sucrose than after conventional freezing in glycerol. Agca et al. [29] did not observe significant differences in pregnancy rates between IVP bovine blastocysts frozen in glycerol or vitrified in a mixture of glycerol and ethylene glycol.

Direct comparisons between the two procedures in terms of their effects on embryo morphology and metabolism are scarce. Concerning embryo metabolism, the only comparison was made by Uechi et al. [24] on 2-cell mouse embryos. In their study, glucose uptake was lower after vitrification than after freezing.

The present study aimed to compare directly the effects of conventional controlled-rate freezing and vitrification on the in vitro development, morphology, and metabolism of IVP bovine embryos. The following parameters were evaluated either immediately or 72 h after thawing, 1) in vitro re-expansion and hatching, 2) cell numbers and membrane alterations directly after thawing and after a culture period, 3) ICM and TE cell numbers, and 4) embryo metabolism by measuring uptakes of oxygen, glucose, and pyruvate and release of lactate.

MATERIALS AND METHODS

Embryo Production In Vitro

Cumulus-oocyte complexes (COCs) were obtained by aspiration of 2- to 6-mm follicles from abattoir ovaries. After four washes in tissue culture medium (TCM) 199-Hepes, they were matured in 500-µl of TCM 199 (Sigma Chemical Co., St. Louis, MO) containing 60 U/ml penicillin and 60 µg/ml streptomycin (Sigma), 10% heat-treated fetal calf serum (FCS; ICN Biosciences, Lenexa, KS), and 10 ng/ml mouse epidermal growth factor (Sigma) for 24 h at 39°C in an atmosphere of 5% CO2 in humidified air.

After maturation, the COCs were transferred into 500 µl of modified Tyrode medium supplemented with 6 mg/ml fatty-acid-free fraction V BSA (Sigma), 4 mg/ml sodium lactate (Sigma), 0.11 mg/ml sodium pyruvate, and 17 U/ml heparin (Calbiochem, San Diego, CA). Motile spermatozoa were separated on a Percoll (Pharmacia, Uppsala, Sweden) discontinuous density gradient (45–90%) and diluted to a final concentration of 2 x 106/ml. Oocytes and spermatozoa were incubated together for 18 h at 39°C in a humidified atmosphere of 5% CO2 in air.

After removal of cumulus cells by vortexing, presumptive zygotes were cultured in groups of about 20 in 20-µl droplets under oil of synthetic oviduct fluid (SOF) culture medium modified according to Holm et al. [30] and containing 0.34 mM of citrate; 2.77 mM of myoinositol, 4.2 mM of lactate, 0.73 mM of pyruvate, and 5% FCS but no glucose and no BSA. Culture dishes were incubated in an atmosphere of 5% O2, 5% CO2, and 90% N2 at 39°C for 6 days. The day of fertilization was considered as Day 0.

Cryopreservation Procedures

Controlled-rate freezing The freezing procedure was that described by Massip and Van Der Zwalmen [31] using glycerol and sucrose as cryoprotectants.

Blastocysts were collected on Day 7 postinsemination and pooled in embryo transfer freezing medium (ETF; Sigma). They were exposed for 10 min at room temperature to a mixture of 1.36 M glycerol (GLY) (v:v; AnalaR BDH Ltd., Poole, England) and 0.25 M sucrose (Sigma) in ETF supplemented with 20% FCS. Groups of five embryos were loaded in this mixture into 0.25-ml straws (ZA 482 IMV, L'Aigle, France) between two columns of 0.5 M sucrose in ETF separated by air bubbles. The straws were placed vertically into a freeze control machine (CL 863) and precooled at -7.5°C for 10 min (including seeding). Straws were then cooled at 0.3°C/min down to -25°C, following which they were immersed and stored in liquid nitrogen. For thawing, the straws were held 5 sec in air, then immersed for 10 sec in a water bath at 20°C. The contents of each straw were emptied in a petri dish and mixed by slight agitation for 10 min. Embryos were transferred to ETF medium for 5 min at room temperature and then cocultured on Buffalo rat liver (BRL) cells.

Vitrification The vitrification procedure used was described by Donnay et al. [32]. Blastocysts were collected on Day 7 postinsemination and pooled in ETF. The embryos were exposed at room temperature to increasing concentrations of cryoprotectants in three steps as follows: step 1, 10% GLY (v:v) for 5 min; step 2, 10% GLY + 20% ethylene glycol (EG) (v:v; AnalaR BDH) for 5 min; step 3, 25% GLY + 25% EG for 30 sec. Groups of five embryos were rapidly loaded into a 0.25-ml straw between two columns of a 0.85 M galactose solution in ETF (GAL) (Sigma) separated from the embryos by air bubbles. The straws were placed in liquid nitrogen vapors for 2 min before immersion. For thawing, the straws were immersed for 10 sec in a water bath at 20°C. The contents of each straw were emptied into a petri dish and mixed by slight agitation. After 5 min, the embryos were transferred to 0.42 M GAL solution for 5 min, then to ETF medium for a further 5 min. Then, they were cocultured on BRL cells.

Embryo Recovery and Assessment of Survival

Coculture on BRL cells Embryos were washed in culture medium (TCM 199 medium + 10% FCS) and cocultured in 50-µl drops of this medium on a confluent monolayer of BRL at 39°C in a humidified atmosphere of 5% CO2 in air, as described by Kaidi et al. [33].

Survival and hatching Re-expansion and hatching rates were defined as the percentage of blastocysts that had respectively re-expanded and hatched over the total number of treated blastocysts.

Fluorescent Staining of Embryonic Nuclei

Double staining A double-staining method was used to assess cell membrane alterations [18]. The two dyes were 1) propidium iodide (PI; Sigma), which is a nucleic acid marker excluded by intact cells; it can only enter cells with altered membrane integrity (red color following UV excitation); and 2) bisbenzimide (BIS, Hoechst 33342; Calbiochem), which enters all cells and binds both specifically and quantitatively to DNA (blue color following UV excitation). In cells with membrane alterations, the BIS fluorescence is quenched by the PI, which absorbs this energy and emits red fluorescence. Double staining is particularly suitable for assessing membrane lesions in TE cells. Indeed, PI will reach the ICM cells only if intercellular junctions between TE cells are damaged.

Blastocysts were incubated for 15 min at 39°C in Hepes-buffered TCM 199 medium supplemented with 10 µg/ml PI. They were fixed in iced 70% ethanol for 5 min and then transferred into ethanol containing 10 µg/ml BIS for 5 min at room temperature. Embryos were transferred to a drop of glycerol on a glass slide, covered with a coverslip, and examined by fluorescent microscopy. Although both blue and red nuclei could be visualized using the same excitation wavelengths and filter (excitation wavelengths at 330:380 nm and barrier filter at 420 nm), for counting PI-stained nuclei more precisely, the red nuclei were recounted after excitation at 510:560 nm, with a barrier filter at 590 nm.

Triple staining A triple-staining method was used to discriminate between cells with membrane alteration, cells with perturbed BIS staining, and intact cells [18]. Blastocysts were double stained as described above. After counting PI-stained and BIS-stained nuclei, 20-µl of PI (10 µg/ml) was injected under the coverslip, and the total PI-stained nuclei were counted (total cells). The difference between the numbers of nuclei counted after double staining and after triple staining was evaluated for each embryo to evaluate the number of cells with intact membranes showing alteration in BIS staining.

Differential staining Differential cell counting of ICM and TE was carried out by the method of Hardy et al. [34] modified by Van Soom et al. [35]. Briefly, trinitrobenzene-sulfonic acid (TNBS; Sigma) and rabbit anti-dinitrophenol-BSA (ICN Biomedicals NV, Asse-Relegem, Belgium) were used for labeling TE cells. Guinea pig complement (CL5000; Cedarlane Laboratories, Sanbio BV, Uden, Holland) was added for lysing TE cells. Staining was then performed using PI and BIS. ICM nuclei labeled with BIS appeared blue and TE (labeled with both BIS and PI) appeared red when examined by fluorescence microscopy.

Metabolic Measurements

Determination of glucose and pyruvate uptake and lactate release Pyruvate, D-glucose, and L-lactate concentrations were assessed in spent culture drops using microfluorescence assays as described by Leese and Barton [36] and Gardner and Leese [37]. The assays for measuring substrate concentrations are based on the generation or consumption of the reduced pyridine nucleotides NADH and NADPH. These nucleotides fluoresce when excited with light at 340 nm, whereas the oxidized forms, NAD+ and NADP+ do not. Fluorescence was measured using a photometer and photomultiplier attached to a Leitz fluorescence inverted microscope (DMIRB). Pyruvate and glucose uptake and lactate release by the embryos were calculated by comparing their concentrations in spent culture medium with those in embryo-free control drops incubated simultaneously. Assays were performed in triplicate.

Determination of oxygen uptake Oxygen consumption was determined using the technique of Houghton et al. [38], which involves pyrene, an oil-soluble fluorescent compound. A 5-µl graduated glass pipette (PCR micropipettes; Drummond, Broomall, PA) was loaded with 1 µl of 3 mg/ml pyrene (P2146, Sigma) in mineral oil (Sigma) followed by 2 µl Hepes-buffered SOF containing blastocysts. A control with 0% oxygen was prepared by overnight incubation of 1 mg/ml baker's yeast in a 60 mmol/L D-glucose solution. Fluorescence was measured using a photometer and photomultiplier attached to a Leitz fluorescence inverted microscope. The fluorescence of pyrene is quenched in proportion to oxygen concentration.

Experimental Design

Experiment 1—cell alterations and in vitro development In this experiment, assessment of cell alterations and total cell numbers immediately after cryopreservation and thawing, as well as re-expansion, hatching, and total cell numbers after 72 h of culture period were performed.

Day 7 expanded blastocysts were randomly allocated to three groups of eight blastocysts: 1) untreated blastocysts; 2) blastocysts frozen in 1.36 M GLY, 0.25 M sucrose; and 3) blastocysts vitrified in 25% GLY, 25% EG. After thawing, four embryos were immediately submitted to triple staining. The other four were cocultured on BRL cells for 72 h. Re-expansion and hatching were evaluated, as well as total cell numbers at the end of culture period. This experiment was replicated seven times (n = 168).

Experiment 2—differential effect on ICM and TE cells This experiment aimed to compare the effect of freezing and vitrification on the two cell lineages population of the blastocyst.

Day 7 expanded blastocysts were randomly allocated by groups of six blastocysts to one of the following treatments: 1) untreated blastocysts; 2) blastocysts frozen in 1.36 M GLY, 0.25 M sucrose; and 3) blastocysts vitrified in 25% GLY, 25% EG. After thawing and 48 h of coculture on BRL cells, hatched blastocysts were submitted to differential staining. This experiment was replicated seven times (n = 126).

Experiment 3—effect on carbohydrate metabolism This study was performed to compare carbohydrate metabolism before and after freezing or vitrification as well as to compare the effects of the two methods on embryo metabolism after cryopreservation.

Glucose and pyruvate uptakes, as well as lactate release were measured in the same Day 7 expanded blastocysts before and after cryopreservation. Before cryopreservation, three groups of five embryos were incubated for 3 h in 1-µl drops of SOF with 5% FCS and 1.5 mM glucose. Drops of medium without embryos were incubated in parallel. Afterward, embryos were recovered and spent culture drops were frozen at -80°C until analysis. Each group of embryos was then allocated to one of the following three treatments: 1) untreated, 2) frozen, and 3) vitrified. After treatment, embryos were cocultured for 18 h on BRL cells. Surviving embryos in each group were then incubated in 1-µl drops for 3 h. At the end of the incubation, embryos were recovered, and submitted to triple staining and spent culture drops were frozen. Pyruvate and glucose uptake and lactate release were evaluated microfluorometrically and expressed in pmol/embryo/h or in pmol/embryo intact cell/h. Glycolytic activity was evaluated as the ratio of lactate release (x2) on glucose uptake, assuming that 1 mol glucose produces 2 mol lactate. The whole experiment was replicated five times.

Experiment 4—effect on oxygen uptake Measurements of oxygen uptake were performed on Day 7 blastocysts (n = 66). After cryopreservation, thawing, and 18 h of coculture on BRL cells, groups of three to five surviving blastocysts were incubated in 2 µl Hepes-SOF for 3 h in order to measure oxygen consumption. After the measurement period, the embryos were recovered and triple stained. A computer-assisted mathematical model [38] was used to estimate the transfer of oxygen from the pyrene-oil phase toward the medium corresponding to the depletion of oxygen by the embryos. Results were expressed in nl/embryo/h or nl/embryo intact cell/h. This experiment was replicated five times.

Statistical Analysis

The effect and the comparison of treatments on the probabilities of re-expansion, and hatching rates, and cellular alterations were assessed using likelihood ratio tests associated with logistics regression models [39]. Differences between replicates were allowed in the considered models. Log-linear models (Poisson regression) [40] were used instead when the responses were unlimited counts such as the total number of nuclei.

Differences between treatment groups in the metabolism of glucose, pyruvate, and lactate and in oxygen consumption both before and after cryopreservation were tested using ANOVA models in which the data were the compared means weighted by the number, n, of cells generating them (which is equivalent to considering n observations of the corresponding mean). The levels of the classifying variables are the replicates and the compared treatments.

The metabolism (e.g., of glucose) before and after cryopreservation are compared using the mean quantities of metabolized glucose per number (Nb before and after treatment of alive embryos as data [weighted by 1/(Nb + 1/Na)] in an ANOVA model with the treatments as levels of classifying covariate.

RESULTS

Experiment 1—Cell Alterations and In Vitro Development

Immediately after thawing and double staining, membrane permeabilization (PI-stained nuclei) was observed in roughly one fifth of the cells (Table 1). The proportion was similar for frozen and vitrified embryos. After alcohol fixation and restaining of the same embryos with PI (triple staining), the total number of stained nuclei was lower in cryopreserved embryos by comparison with untreated ones (15–17% decrease by comparison with the controls), but no difference was observed between frozen and vitrified embryos. However some nuclei unstained after double staining were stained after triple staining. This difference resulted from the fact that some cells with intact membranes were not stained with BIS. The proportion of BIS-unstained nuclei was higher in vitrified embryos (19% versus 15%).


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  		TABLE 1. Cell alterations in frozen and vitrified Day 7 IVP bovine blastocysts directly after treatment or 72 h after coculture on BRL cells.*

After 72 h of coculture, a significant decrease in re-expansion and hatching rates was observed for frozen and vitrified blastocysts by comparison with the control (Fig. 1). Cryopreserved embryos also had fewer cells (Table 1). No difference in re-expansion and hatching rates was observed between the two procedures, while the decrease in cell number was greater in frozen embryos than in vitrified ones.



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  		FIG. 1. Re-expansion (A) and hatching (B) rates in frozen or vitrified Day 7 IVP bovine blastocysts after 72 h coculture on BRL cells. *Significantly different from the control (likelihood ratio test, P < 0.05). For more details, see Statistical Analysis

Experiment 2—Differential Effect on ICM and TE Cells

After 48 h of coculture, the numbers of ICM cells and TE cells of hatched cryopreserved blastocysts were decreased by comparison with untreated controls (Table 2). No difference was observed between frozen and vitrified embryos for the number of ICM cells (-24 versus -27%). However, the decrease in TE cells was greater in frozen blastocysts (-38%) than in vitrified ones (-20%). As a consequence, the ratio of ICM cells to total cells was increased, and the ratio of TE cells to total cells was decreased after freezing while they were not modified after vitrification.


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  		TABLE 2. Number of ICM and trophectoderm cells in frozen or vitrified Day 7 IVP bovine blastocysts 48 h after treatment and coculture on BRL cells.*

Experiment 3—Effect on Carbohydrate Metabolism

When results were expressed per embryo, lower glucose and pyruvate uptakes were observed after freezing by comparison with uptakes before freezing (Table 3). Vitrified and untreated blastocysts did not show differences in glucose and pyruvate uptakes before and after treatment. No change was detected in lactate release in any treatment.


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  		TABLE 3. Glucose and pyruvate uptake and lactate release of frozen or vitrified Day 7 IVP bovine blastocysts before and after treatment and coculture on BRL cells.*

Frozen-thawed embryos exhibited a decrease in glucose and pyruvate uptake as well as a significantly higher glycolytic activity by comparison with control and vitrified embryos. Lactate production was not affected but large variations were observed between replicates.

When the results are expressed per intact embryonic cell, however, differences between the treatments were no longer detected (Fig. 2, A–C).



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  		FIG. 2. Glucose (A) and pyruvate (B) uptake and lactate (C) release by frozen or vitrified Day 7 IVP bovine blastocysts re-expanded 18 h post-treatment. *Significantly different from the control (ANOVA, P < 0.05)

Experiment 4—Effect on Oxygen Uptake

A significant decrease in oxygen consumption by the embryos was observed after freezing but not after vitrification. This difference in oxygen uptake between frozen and vitrified embryos was not observed on an embryo cell scale (Fig. 3).



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  		FIG. 3. Oxygen uptake by frozen or vitrified Day 7 IVP bovine blastocysts re-expanded 18 h post-treatment. *Significantly different from the control (ANOVA, P < 0.05)

DISCUSSION

Comparison between conventional controlled-rate freezing and vitrification was performed for several parameters of bovine embryo morphology and metabolism. Similarities and differences were detected under our experimental conditions. Immediately after thawing, the proportion of embryo cells showing membrane alterations and a decrease in total cell number were similar in frozen and vitrified embryos (15–17%). Membrane alterations observed after freezing are likely to be due to ice crystal formation or osmotic shock, as described previously [7, 9, 10]. In vitrified embryos, such damage probably results from osmotic shock during the steps of equilibration and dilution of the cryoprotectants. Indeed, we observed similar alterations when embryos were exposed to the same cryoprotectants, without vitrification [18]. Damage to the plasma membrane after vitrification was also observed by Dobrinsky [15], Kuwayama et al. [13], Vajta et al. [16], and Ohboshi et al. [17]. Some of these alterations seem to be reversible, as most of the cells will recover after a few hours [16, 18]. However, as mentioned in the Materials and Methods, double staining and triple staining allowed for evaluating membrane lesions in the TE cells but probably not in ICM cells.

A decrease in total cell number (roughly 8%) was observed both in frozen and vitrified embryos directly after thawing. Cells probably degenerated quickly during the procedure, although we did not observe an increase in fragmented nuclei (data not shown). In cryopreserved embryos, some nuclei were not stained with BIS, although they were stained with PI after membrane permeabilization with alcohol. The different pattern of staining could be explained by the fact that BIS and PI show different DNA binding mechanisms. BIS shows a wide spectrum of sequence dependent DNA affinities and bind poly d(A-T) sequences [41, 42]. The conformation of the helix at the AT-rich site may affect its binding [43]. The PI dye is a DNA intercalating compound that binds to the double-stranded nucleic acids, with little or no sequence preference [44, 45]. This proportion of BIS-unstained nuclei was slightly higher in vitrified embryos (19%) than in frozen ones (15%). This is probably related to alterations in the fixation of the stain to DNA, maybe due to modifications in DNA structure [18].

After a 72-h culture period, survival and hatching rates were decreased in both frozen and vitrified embryos by comparison with untreated ones. Such a decrease has already been reported by several authors [6, 46, 32]. The decrease in hatching rate might be related to the decrease in cell number observed in cryopreserved embryos after 48 or 72 h of coculture by comparison with untreated ones [47]. It could be explained by necrosis and apoptosis of cells during the cryopreservation procedure and/or following the procedure as demonstrated by Baguisi et al. [48]. This decrease, however, could also be due to a slowing in embryonic cell proliferation after cryopreservation [49].

Frozen blastocysts had fewer cells than vitrified ones after several days of culture. This seems related mainly to a more marked decrease in TE cells after freezing. Indeed, while the decrease is similar in both cell lineages in vitrified embryos (20 and 24% for TE and ICM, respectively), frozen blastocysts exhibited a more marked decrease in TE cells (38% versus 27% in ICM) and thus a lower TE on total cell ratio. TE cells could thus be more sensitive to freezing than ICM cells. Indeed, the transport of water through the TE cells may increase their susceptibility to ice crystal formation characteristic of the freezing procedure [10]. This result is not in agreement with Renard [50] who observed as many degenerated cells in trophoblast as in ICM 12 h after freezing and thawing of bovine blastocysts, but his study was performed on in vivo-derived embryos. Iwasaki et al. [11] observed a decrease in total cell number and death of ICM cells in frozen-thawed in vitro bovine embryos. More recently, Takagi et al. [49] examined the rate of DNA synthesis by ICM in frozen-thawed bovine blastocysts. Their results suggested that the rates of proliferation of ICM cells in frozen-thawed bovine embryos tends to be lower than those of unfrozen embryos irrespective of the cryoprotectant used. In those two studies however, data on TE cells were not available.

In our study, we compared glucose and pyruvate uptake and lactate release both before and after cryopreservation and between the freezing procedures. The glycolytic activity was evaluated as the ratio of lactate release to glucose uptake, assuming that one glucose molecule produces two lactate molecules. It is generally accepted that bovine blastocysts utilize glucose via aerobic glycolysis and that the production of L-lactate accounts for almost all the glucose metabolized [51–54]. Pyruvate and oxygen uptakes were used to evaluate Krebs cycle and oxidative phosphorylation pathways. Results were expressed per embryo, as is usual in the literature, but we also expressed embryo metabolism at the cell level, as cells are the real metabolic units of any organism. Evaluation of metabolic activity was not performed directly after thawing, but after an overnight culture period, allowing discrimination between embryos that survived the procedure and embryos that did not. The metabolism of the latter embryos was not assessed, as the aim of our study was to evaluate the impact of cryopreservation on the quality of surviving embryos. This objective is thus different from the objectives of the study of Gardner et al. [22] and Khurana and Niemann [21] who performed metabolic studies to discriminate between viable and nonviable embryos.

On average, glucose uptake was lower in our study than in other studies on bovine blastocysts (3 ± 1.3 versus 14.6 ± 0.9 pmol/embryo/h [53]). This can be explained by the absence of added glucose in our culture medium up to Day 7 [30]. Lactate release was also on average reduced by comparison with data in the literature (19.7 ± 6.8 versus 37 ± 6 pmol/embryo/h for IVP bovine blastocysts, [23]) but to a lesser extent than glucose uptake. This means that the calculated glycolytic activity was high in all conditions (3.1–4.2 before treatment and 3.1–9.4 after treatment) and that only 27–69% of the released lactate could derive from exogenous glucose. Although comparisons between metabolic studies using different culture systems should be done cautiously, a high-calculated glycolytic activity is often considered as reflecting poor blastocyst quality [55]. The source for this additional lactate could be glycogenic reserves or exogenous pyruvate. Little is known about glycogen stores in ruminant embryos, but they are considered as low by comparison with mouse embryos [56, 57].

The values for pyruvate uptake obtained in our study were approximately half of those reported in other studies on bovine blastocysts (12 ± 7 versus 20.5 ± 2.4 pmol/embryo/h [53]). However, they were double those reported by Eckert et al. [23] (5.56 ± 0.71). This is probably due to differences in pyruvate concentrations in culture media between studies.

After freezing, uptake of glucose and pyruvate was reduced on a per embryo basis, while in control and vitrified embryos no significant change was observed. These results are in agreement with Partridge et al. [20], who demonstrated a decrease in glucose uptake by IVP frozen embryos. Gardner et al. [22] also reported reduced nutrient uptake and utilization by surviving bovine embryos after freezing and thawing. The decline in glucose uptake was explained by a decrease in glucose incorporation resulting from alteration in the membrane transport system. Indeed, Onohara et al. [58] observed that freezing procedures impair the metabolic functions, in particular the membrane transport system of mouse embryos. On the other hand, Uechi et al. [59] observed decreased glucose incorporation by mouse embryos after freezing, due to decreased expression of glucose transporter 1 (GLUT 1). Recently, Khurana and Niemann [21] showed that after freeze-thawing, the rate of CO2 production from glucose by IVP bovine embryos was reduced to 50% of their prefreeze levels while oxidation of glucose remained unchanged for in vivo embryos.

While we did not observe significant variations in lactate release between treatments, frozen embryos exhibited a threefold higher glycolytic activity than vitrified ones; this could be related to the stress induced by the freezing process and could reflect the poor quality of frozen-thawed embryos, as previously suggested by Lane and Gardner [55]. Although not detected in our study, an increase in lactate release after vitrification of bovine blastocysts was observed by Eckert et al. [23] using a different vitrification procedure.

Consumption of oxygen by frozen embryos was lower than by control and vitrified embryos. This disagrees with the increased substrate oxidation calculated in the study by Gardner et al. [22]. These authors concluded that freezing could lead to uncoupling of mitochondrial membranes resulting from damage to the inner membrane and that embryos could compensate by an increased glycolytic activity. However, those authors based their calculation on the amount of glucose and pyruvate uptake and lactate release and not on a direct evaluation of oxygen uptake.

When expressing our results on a per cell basis instead of on a per embryo basis, no difference between cryopreservation procedures could be detected for glucose, pyruvate, and oxygen uptake. This can be easily explained by the more marked decrease in cell number observed in frozen blastocysts. Indeed, it is logical that embryo metabolism correlates with cell number. Moreover, TE cells were more affected by the freezing procedure than by vitrification. Those cells are metabolically very active as blastocyst expansion is mediated through their activity [60]. This could increase the difference in oxidative metabolism between the two procedures of cryopreservation.

Both methods decreased survival and hatching rates to the same extent and induced similar cell alterations resulting probably from different mechanisms. These effects on embryo quality could account for a proportion of the embryo loss that occurs during implantation. Nevertheless, freezing affects more TE cells than vitrification, leading to a decrease in nutrient uptake by the embryos. Moreover, the stress response to cryopreservation measured by the glycolytic activity might be more important after freezing.

ACKNOWLEDGMENTS

We thank P. Lonergan for improving the manuscript. We also thank P. Bombaerts for technical assistance and M.A. Mauclet for typing the manuscript.

FOOTNOTES

First decision: 4 December 2000.

1 Part of this work was funded by Action de Recherche Concertée de la Direction générale de la Recherche scientifique, Communauté Française de Belgique (grant number: 96/01-196) and by European Union grant QLK3-CT-1999-00104 (Quality of Life). Back

2 Correspondence: I. Donnay, UCL, Unité des Sciences vétérinaires, Place croix du Sud, 3, 1348 Louvain-la-Neuve, Belgium. FAX: 32 10 47 37 17;donnay{at}vete.ucl.ac.be Back

Accepted: May 23, 2001.

Received: October 13, 2000.

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