Survival and apoptosis rates after vitrification in cryotopdevices of in vitro-produced calf and cow blastocystsat different developmental stages

Roser MoratoA,C, Dolors IzquierdoB, Maria Teresa ParamioB

and Teresa MogasA

ADepartament de Medicina i Cirurgia Animals, Facultat de Veterinaria,

Universitat Autonoma de Barcelona, Bellaterra 08193, Spain.BDepartament de Ciencia Animal i dels Aliments, Facultat de Veterinaria,

Universitat Autonoma de Barcelona, Bellaterra 08193, Spain.CCorresponding author. Email: roser.morato@uab.es

Abstract. Two experiments were designed to determine the ability of in vitro-cultured blastocysts at different stages of

development to survive the vitrification procedure using cryotop devices. Day 7 andDay 8 embryoswere classified as non-expanded, expanded or hatching and/or hatched blastocysts. In the first experiment, we examined the survival rate ofvitrified–warmed blastocysts after 3 h incubation in synthetic oviducal fluid (SOF) medium. In the second experiment,

vitrified–warmed blastocysts were evaluated using the terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) technique to detect nuclei with damaged DNA. In both experiments, resultsfor cow and calf blastocysts were compared. No differences in survival rates were observed after vitrification of Day 8

expanded (52.4%) and hatched (50%) cow blastocysts or Day 8 expanded (54.5%) and hatched (59.4%) calf blastocysts.When embryos were vitrified on Day 7, survival rates of 78.4% and 66.7% were observed after warming expanded andhatched cow blastocysts, respectively, compared with rates of 80% and 76.9%, respectively, for calf blastocysts. Lowestsurvival rates were recorded for non-expanded blastocysts (26%–54%) compared with the other developmental stages,

particularly those vitrified at Day 8 (�40%). The DNA integrity index obtained after vitrification–warming wascomparable to that for control fresh blastocysts, regardless of the length of embryo culture, the developmental stage ofthe embryo or the source of the oocytes. Our findings suggest that the cryotop vitrification method is particularly useful

for the cryopreservation of blastocysts presenting with a high degree of expansion (expanded or hatched blastocysts),particularly when vitrification is performed after 7 days of in vitro embryo culture.

Additional keywords: bovine, cryopreservation, embryo, prepubertal.

Introduction

The cryopreservation of in vitro-produced (IVP) bovineembryos is a prerequisite for their large-scale commercial use.The relatively low pregnancy rates achieved following the

transfer of thawed IVP embryos is a clear indication of theirlower quality compared with their in vivo-produced counter-parts. The cryopreservation of embryos at the blastocyst stage

allows better embryo selection, which serves to maximise theimplantation potential of subsequent embryo transfer andminimise the number of embryos being cryopreserved. Ofthe numerous factors that affect cryosurvival, it has been

reported that the age, developmental stage and quality of theembryo, as well as interactions between these factors, are key(Mahmoudzadeh et al. 1995; Carvalho et al. 1996; Saha et al.

1996; Pugh et al. 1998) for the freezing of IVP bovine embryos.Blastocyst stage at the time of vitrification could be a key factorinfluencing outcome parameters.

Blastocysts invariably represent an embryo that has exhib-

ited its developmental potential in vitro. Indeed, this selectionprocess is far more reliable than selection based on the mor-phology of earlier-stage embryos and makes the transfer of

blastocysts a better option. With recent advances in cryo-preservation protocols, the cryopreservation of blastocysts hasbecome a readily available and reliable tool with which to

preserve embryos that have shown the best in vitro develop-mental potential (Menezo 2004). However, the cryopreservationof blastocyst stage embryos is challenging because of theirinherent characteristics, such as: (1) irregular permeation of

the cryoprotectant into the blastocyst, largely because of itsmulticellular nature; (2) the zona pellucida (ZP) acting as aphysiological barrier to the permeation of the cryopreservation

medium; and (3) the presence of the blastocoele, which may beinadequately dehydrated during cryopreservation. The differentmethods developed for the cryopreservation of mammalian

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embryos have been designed to avoid the formation of intra-cellular ice, which causes cell damage. Vitrification is able to

solidify cells without crystallisation and has the added benefitsthat it allows flexibility and practicality, can be performedquickly (within a few minutes) and eliminates the need for

expensive and high-maintenance equipment (Liebermann andTucker 2004).

Blastocysts have been vitrified using a variety of different

carriers, such as electron microscopic grids, the cryotop, cryo-loops, hemistraws, open pulled straws and the cryotip. Thesecarriers have resulted in blastocyst survival rates of 69–97%(Mukaida et al. 2003; Kuwayama et al. 2005; Zech et al. 2005;

Lee et al. 2006). The technological aspects of vitrification,vitrification protocols and the type of carrier are likely to playa vital role in overall post-warming survival. The cryotop is

outstanding as a carrier for vitrification. The volume of fluidvolume is very small (o0.1mL) and direct exposure of theblastocysts to liquid nitrogen during the vitrification step may

improve survival rates due to the ultrarapid cooling rate, whichinduces transition to a glass-like state and thus reduces thepotential for cell damage.

Apoptosis is a cellular response to suboptimal conditions and

different kinds of stress that an embryo may encounter duringthe freeze–thaw cycle. Embryo viability and developmentalpotential have been related to the apoptotic index, calculated

as the number of damaged (i.e. terminal deoxyribonucleotidyltransferase-mediated dUTP–digoxigenin nick end-labelling(TUNEL)-positive cells) divided by the total number of blas-

tomeres. Thus, lower-grade embryos have a higher apoptoticindex. Assessment of DNA fragmentation using the TUNELassay is a reliable method for the detection of apoptosis in

embryos. However, there have been few reports on changes inthe apoptotic index of blastocysts in response to cryopreserva-tion or vitrification (Behr et al. 2002; Pomar et al. 2005).

Therefore, the aim of the present study was to examine the

capacity of Day 7 and Day 8 in vitro-cultured blastocysts fromboth cow and calf oocytes at different stages of developmentto survive vitrification–warming procedure using the cryotop

device. In addition, the study was designed to determine theeffects of the developmental stage or the time of in vitro cultureon cell death.

Materials and methods

Unless stated otherwise, all chemicals and reagents were pur-

chased from Sigma Chemical (St Louis, MO, USA).

Oocyte collection

Themethods used for the IVMof oocytes have been described in

detail elsewhere (Rizos et al. 2001). Briefly, ovaries from pre-pubertal calves (9–12 months of age) and cows (424 months ofage) were transported from a local abattoir to the laboratory in

phosphate-buffered saline (PBS) at 35–378C. Cumulus–oocytecomplexes (COCs) were obtained by aspirating follicles2–10mm in diameter. After three washes in modified PBS (PBS

supplemented with 36mgmL�1 Na-pyruvate, 50mgmL�1 gen-tamicin and 0.5mgmL�1 bovine serum albumin (BSA)), groupsof up to 50 COCs were placed in 500mL maturation medium in

four-well plates and cultured for 24 h at 38.58C in a 5% CO2

humidified air atmosphere. The maturation medium was com-

posed of TCM-199 supplementedwith 10% (v/v) fetal calf serum(FCS), 10 ngmL�1 epidermal growth factor and 50mgmL�1

gentamicin.

In vitro fertilisation

For IVF, COCs were washed four times in PBS and then infertilisation medium before being transferred, in groups of up to

50, to four-well plates containing 250mL fertilisation medium(Tyrode’s medium with 25mM sodium bicarbonate, 22mMNa-lactate, 1mM Na-pyruvate, 6mgmL�1 fatty acid-free BSAand 10mgmL�1 heparin–sodium salt; Calbiochem, Darmstadt,

Germany) in each well. Motile spermatozoa were obtainedby centrifuging frozen–thawed spermatozoa fromAsturian bulls(ASEAVA, Llanera, Asturias, Spain) for 8min at 700g at room

temperature (22–258C) on a discontinuous Percoll density gra-dient (2.5mL of 45% (v/v) Percoll over 2.5mL of 90% (v/v)Percoll; Pharmacia, Uppsala, Sweden). The pellet was washed

in HEPES-buffered Tyrode’s medium and centrifuged at 100gfor 5min. Spermatozoa were counted in a haemocytometer anddiluted in an appropriate volume of fertilisation medium to give

a final concentration of 2� 106 spermatozoa mL�1. A 250 mLaliquot of this suspension was then added to each fertilisationwell to obtain a final concentration of 1� 106 spermatozoamL�1. Plates were incubated for 22 h at 38.58C in a 5% CO2

humidified air atmosphere. Variations between individual bullswere reduced by mixing equal volumes of sperm samples fromtwo bulls for all experiments.

After coculture with spermatozoa for 22 h, presumptivezygotes were transferred to 25mL culture droplets of syntheticoviducal fluid (SOF)medium (Holm et al. 1999; 1 embryo permL)under mineral oil for 7 or 8 days at 38.58C in a 5% CO2, 5% O2

humidified atmosphere. Cleavage rates were recorded 48 h afterinsemination and the number of blastocysts was determined onDays 7 and 8 after insemination.

Blastocyst vitrification and thawing

Vitrification

Blastocysts were vitrified using the cryotop device andvitrification and warming solutions described by Kuwayamaet al. (2005). The cryotop consists of a narrow, thin strip of film(0.4mmwide, 20mm long and 0.1mm thick) attached to a hard

plastic handle. To protect the device from mechanical damageduring storage, a long plastic tube cap can be attached thatcovers the device (Kitazato Supply, Fujinomiya, Japan). The

holding medium (HM) used to formulate the vitrification–warming solutions was TCM-199 HEPES with 20% FCS. Allsteps were performed under a laminar flow hood heated to

38.58C using a stereomicroscope to visualise each step. Blas-tocysts were transferred into equilibration solution (ES) con-sisting of 7.5% ethylene glycol (EG) and 7.5% dimethyl

sulfoxide (DMSO) in HM for 10–15min. After an initialshrinkage, blastocysts regained their original volume; they werethen moved to the vitrification solution (VS) containing 15%EG, 15% DMSO and 0.5M sucrose dissolved in HM. After

incubation for 30–40 s, blastocysts (up to two) were loaded onto

1142 Reproduction, Fertility and Development R. Morato et al.

the cryotop, almost all the solution was removed to leave only athin layer covering the blastocyst and the sample was immersed

quickly into liquid nitrogen. Subsequently, the plastic cap wasattached to the cryotop. The entire process from immersion inVS to plunging into liquid nitrogen was completed within 1min.

The loaded devices were stored in liquid nitrogen.

Warming

All warming steps were performed at 38.58C. During warm-

ing, the protective cap was removed from the cryotop while itwas still submerged in liquid nitrogen. Next, the loaded cryotopwas plunged directly into the warming solution containing 1Msucrose dissolved in HM. After 1min, the recovered blastocysts

were placed in the dilution solution, which contained 0.5Msucrose dissolved in HM. Blastocysts were incubated in thedilution solution for 3min with gentle pipetting to facilitate

cryoprotectant diffusion out of the embryo. Subsequently,blastocysts were incubated in HM for 5min and rinsed againin HM for 1min before transfer to SOF culture medium and

incubation at 38.58C in a 5%CO2 and 5%O2 humidified atmo-sphere. The survival of vitrified blastocysts was determined asre-expansion rates after 3 h of recovery in SOF medium.

Embryo groups

After ensuring the quality of the embryos and before vitrifica-tion (Gomez et al. 2008), Day 7 and Day 8 blastocysts wereclassified according to the extent of blastocoele expansion into

one of three groups as follows: (1) non-expanded blastocysts, inwhich the blastocoele volume was less than one-half of the totalvolume of the blastocyst; (2) expanded blastocysts, in which the

blastocoele volumemore than one-half of the total volume of theblastocyst; and (3) hatched or hatching blastocysts, in which theexpanded blastocyst was without a ZP or had an opened ZP.

Embryo DNA fragmentation

Embryos were fixed in 4% paraformaldehyde in PBS for 1 h atroom temperature. After fixation, embryos were washed at least

three times in PBS containing 0.3%polyvinylpyrrolidone (PVP)and permeabilised in 0.5%Triton X-100 for 2min. The embryoswere then washed three times in PBS–PVP and incubated in theTUNEL reaction cocktail (In-situ Cell Death Detection System;

Roche Diagnostic, Indianapolis, IN, USA) at 378C for 1 h inthe dark. Positive and negative control samples were included in

each assay. Blastocysts exposed to DNase I for 15min at roomtemperature served as positive controls and blastocysts not

exposed to the terminal TdT enzyme served as negative controls.Embryoswerewashed thoroughly in PBS and finally transferredto the Hoechst 33342 staining solution (25mgmL�1) for 30min

at 378C in the dark. Finally, blastocysts weremounted on poly-L-lysine-treated slides, covered with a drop of mounting mediumand then a coverslip sealing the edges with nail polish, and

stored at �208C in the dark for confocal microscopy. Confocalimages were captured using a Leica TCS-SP2 laser scanningspectral confocal microscope (LeicaMicrosystems, Heidelberg,Germany). Excitation wavelengths were 364 nm for the Hoechst

stain and 488 nm for the fluorescein isothiocyanate-conjugatedTUNEL label. The fluorescence emitted from each of the twolabels was detected by two separate photomultiplier detectors

whose spectrophotometer slits were set for 400–490 and500–550 nm. Individual nuclei were scored as having eitherintact (TUNEL(�); red stain) or fragmented (TUNEL(þ); green

stain) DNA and counted. The apoptotic index was calculated asthe ratio of TUNEL(þ) cells/total number of nuclei, whereas theDNA integrity index was calculated as TUNEL(�) cells/totalnumber of nuclei.

Statistical analysis

Data were analysed using SAS version 8 (SAS Institute, Cary,NC, USA). Data from at least 13 replicates were collected.

The blastocyst cell counts in different experimental groupswere analysed using ANOVA (PROC GLM). Comparisons ofvitrified–warmed blastocyst survival rates between groups

were performed using the Chi-squared test. Survival data weretransformed to frequency percentages, whereas blastocysts cellcounts were expressed as absolute values. The level of statistical

significance was set at Po 0.05.

Results

Experiment 1: survival rates of vitrified–warmedblastocysts depending on developmental stageand time of in vitro culture

When results were analysed according to developmental stage(Table 1), a significant increase in survival rate was noted whencow expanded blastocysts were vitrified–warmed on Day 7 after

insemination compared with non-expanded blastocysts (78.4% v.

51.2%, respectively), whereas the survival rate of hatched

Table 1. Post-thaw survival rates after vitrification of blastocysts depending on development stage (non-expanded, expanded or hatched), source

of oocytes (cow or calf) and age (Day 7 or Day 8 after insemination)a,bValues within rows with different superscript letters differ significantly (Po 0.05); c,dvalues within columns with different superscript letters differ

significantly (Po 0.05)

Group Day No. vitrified–warmed

blastocysts

Non-expanded blastocysts Expanded blastocysts Hatched blastocysts

n Survival rate (n) n Survival rate (n) n Survival rate (n)

Cow (424 months) 7 127 84 51.2% (43)ac 37 78.4% (29)bc 6 66.7% (4)abcd

Calf (9–12 months) 7 76 33 54.5% (18)ac 30 80.0% (24)bc 13 76.9% (10)bd

Cow (424 months) 8 118 61 26.2% (16)ad 21 52.4% (11)bd 36 50.0% (18)bc

Calf (9–12 months) 8 84 30 40.0% (12)ac 22 54.5% (12)ad 32 59.4% (19)acd

Vitrification of bovine blastocysts Reproduction, Fertility and Development 1143

blastocysts (66.7%) did not significantly differ from either ofthese two groups. In the calf, significantly higher survival rates

were obtained after warming expanded and hatched blastocystscompared with non-expanded blastocysts (80% and 76.9% v.

54.5%, respectively). When blastocysts were vitrified–warmed

on Day 8 after insemination, significantly higher survival rateswhere observed for expanded and hatched blastocysts comparedwith non-expanded blastocysts (52.4% and 50% v. 26.2%,

respectively). Similar results were observed for non-expanded,expanded and hatched blastocysts from the calf (40%, 54.5% and59.4%, respectively), although there were no significant differ-ences between these groups.

When survival rates were compared depending on the time ofin vitro culture, significantly lower rates were observed whennon-expanded Day 8 cow blastocysts where vitrified–warmed

compared with non-expanded Day 7 cow or Day 7–8 day calfblastocysts. When expanded blastocysts were vitrified, signifi-cantly higher results were obtained for Day 7 calf and cow

blastocysts than for Day 8 calf and cow blastocysts. This sametendency was observed for hatched blastocysts, although thedifferences no longer reached statistical significance.

Experiment 2: cell death rates recorded in vitrified–warmedblastocysts according to developmental stage

Cell damage in vitrified–warmed bovine blastocysts wasassessed 3 h (Desai et al. 2008) after warming using the TUNEL

assay. Cell death rates were compared between calf and cowblastocysts vitrified at the non-expanded, expanded or hatchedstages and control blastocysts. Following vitrification, TUNEL

staining was conducted on both Day 7 calf (n¼ 23) and cow(n¼ 25) blastocysts, as well as on Day 8 calf (n¼ 22) and cow(n¼ 30) blastocysts (Table 2). After counting individual nuclei,DNA integrity and apoptotic indices were calculated for each

blastocyst. These results revealed a slight drop in the DNAintegrity index in response to the vitrification–warming proce-dure compared with fresh control embryos, although the dif-

ference did not reach statistical significance. No significantdifferences were observed between different developmentalstages for different durations of in vitro culture or between calf

or cow oocytes. Confocal microscopic images obtained afterTUNEL staining of the blastocysts are shown in Fig. 1.

Discussion

Several studies have reported differences in survival rates orhatching percentages after the cryopreservation of blastocystsobtained after different times of in vitro culture. However, some of

these studies addressed the use of slow freezing methods insteadof vitrification, emphasising that the duration of embryo cultureaffects the cryotolerance of the blastocysts produced (Hasler et al.1995, 1997; Cseh et al. 1996; Gustafsson et al. 2001; Havlicek

et al. 2009). Thus, Gustafsson et al. (2001) reported that blasto-cysts formed on Day 7 had an almost threefold greater chance ofsurviving freezing and thawing than embryos frozen on Days 8–9,

regardless of their developmental stage. In a more recent paper,Havlicek et al. (2009) recorded significantly higher survival andre-expansion rates of Day 7 embryos compared with Day 8 IVP

bovine embryos. Similar observations have been reported whenvitrification protocols have been used as the cryopreservationmethod (Mahmoudzadeh et al. 1995; Ohboshi et al. 1997; Saha

and Suzuki 1997). The findings of the present study are in agree-mentwith these previous observations because significantly highersurvival rates were recorded for vitrified–warmed Day 7 thanDay 8 blastocysts. In contrast, Dinnyes et al. (1999) have reported

that the survival of Day 7 and Day 8 blastocysts is similar.In our experiments, improved cryosurvival could be correlated

with a more advanced embryonic stage on a given day.When we

vitrified–warmed cow or calf embryos at different developmentalstages after 7 or 8 days of in vitro culture, higher survival rateswere observed for expanded blastocysts and those in the process

of hatching or those that had hatched completely comparedwith non-expanded blastocysts. In addition, similar results forthe DNA integrity index were obtained for vitrified–warmed

blastocysts and control fresh blastocysts. The fact that earlydeveloping embryos are better at surviving than later developingembryos has been highlighted in embryo transfer experiments inwhich frozen–thawed IVP bovine blastocysts cryopreserved on

the day of their appearance in culture rendered significantlyhigher pregnancy rates when Day 7 blastocysts were comparedwith Day 8 blastocysts (Hasler et al. 1995). The reasons for the

higher survival rates of more advanced stage embryos remain tobe determined. As shown for hatching and hatched pig embryos(Nagashima et al. 1992; Dobrinsky 1996), the characteristics of

expandedand hatching embryos that confer cryotolerance includemature junctional complexes among cells (Prather and First

Table 2. DNA integrity indices recorded for non-expanded, expanded and hatched blastocysts

The DNA integrity index was calculated as the number of terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling

(TUNEL)-negative blastomeres divided by the total number of blastomeres. Unless indicated otherwise, data are given as the mean� s.e.m.

Group Day No. vitrified

blastocysts

No. stained

blastocysts

DNA integrity index (n)

Non-expanded blastocysts Expanded blastocysts Hatched blastocysts

Fresh control

Cow 8 0 10 94.31� 2.12 (4) 97.78� 4.10 (3) 97.36� 6.13 (3)

Calf 8 0 14 92.13� 6.75 (5) 95.47� 4.25 (5) 94.64� 5.31 (4)

Cow (424 months) 7 127 25 92.22� 2.31 (12) 94.94� 3.09 (11) 98.87� 0.62 (2)

Calf (9–12 months) 7 76 23 88.42� 9.08 (12) 91.21� 7.57 (8) 91.57� 7.06 (3)

Cow (424 months) 8 118 30 94.74� 4.94 (8) 94.30� 4.55 (11) 92.30� 4.39 (11)

Calf (9–12 months) 8 84 22 86.65� 14.90 (11) 89.57� 6.87 (6) 91.62� 4.40 (5)

1144 Reproduction, Fertility and Development R. Morato et al.

(a)

Hoechst TUNEL-FITC Merged

(a�) (a�)

(b) (b�) (b�)

(c) (c�) (c�)

(d ) (d�) (d �)

Fig. 1. DNA staining of blastocysts showing DNA damage after vitrification–warming at different developmental stages. (a, a0, a00) Non-expanded (top) andexpanded (bottom) blastocysts on Day 7. (b, b0, b00) Hatching blastocysts on Day 7. (c, c0, c00) Expanded blastocyst on Day 7. (d, d0, d00) Hatched blastocysts onDay 8. All nuclei are stained red and those with DNA fragmentation have superimposed green staining. Individual nuclei were labelled as having either intact

(red) or fragmented (green) DNA and counted. The apoptotic index was calculated as the ratio of the number of green nuclei/total number of nuclei. Confocal

images were collected using a Leica TCS-SP2 laser scanning spectral confocal microscope (Leica Microsystems, Heidelberg, Germany). Samples were

excited at 364 nm for the Hoechst stain and 488 nm for the fluorescein isothiocyanate-conjugated terminal deoxyribonucleotidyl transferase-mediated dUTP–

digoxigenin nick end-labelling (TUNEL) stain.

Vitrification of bovine blastocysts Reproduction, Fertility and Development 1145

1993), small trophectoderm cells with an epithelial-like structureand a blastocoele containing a large amount of liquid. In the

present study, we observed a more pronounced and consistentdecrease in the volume of the blastocoele when expanded andhatching and/or hatched blastocysts were vitrified, suggesting a

more marked dehydration achieving better embryo protection,possibly due to the release of more water from the blastocoelecavity. Conversely,when the volume of the blastocoele increases,

theZPbecomes thinner andpossiblymorepermeable than inearlyblastocysts. In contrast with our findings, it has been reported thathuman expanded blastocysts exhibit relatively lower survivalrates after vitrification compared with early blastocysts (Cho

et al. 2002; Vanderzwalmen et al. 2002). These authors attributedthis reduced survival to greater mechanical damage suffered byexpanded than early blastocysts due to greater ice crystal forma-

tion in the blastocoele.Embryo transfer experiments are needed toconfirm that the improved in vitro cryosurvival of expanded andhatched blastocysts will indeed lead to higher pregnancy rates.

Hasler et al. (1995), who studied a large number of transfers usingfresh Day 7 and Day 8 IVP bovine embryos, reported that withinthe different age and grade categories, embryo stage failed tomodify pregnancy rate. In contrast, the transfer of large numbers

of bovine in vivo-produced embryos, independent of age andgrade, resulted in higher pregnancy rates when early and mid-blastocysts were used rather than morulae and expanded or

hatched blastocysts (Hasler et al. 1997).The survival rates achieved using hatching or hatched blas-

tocysts demonstrate that an intact ZP is not necessary for success-

ful vitrification.Our results are similar to those obtained by othersin studies in which biopsied embryos were slow frozen (Niemannet al. 1987; Schmidtet al. 1992;Gustafsson etal. 1994)orvitrified

(Agca et al. 1995) and may have practical implications for sexdetermination or for embryos produced by nuclear transfer (Galanet al. 2003; Hiraoka et al. 2004; Zech et al. 2005). The possibilityof vitrifying hatching or hatched blastocysts may be considered,

especially after preimplantation genetic diagnosis (PGD). Oneoptionwould be tocryopreservePGDembryos after extending theculture of the biopsied embryos to the blastocyst stage before

vitrification.Having established the survival of the vitrified blastocysts,

we assessed their integrity by determining the apoptotic index.

The apoptotic index of a blastocyst is an important factor that hasbeen negatively correlated with embryo developmental potential(Makarevich and Markkula 2002; Spanos et al. 2002). Aftercryopreservation, blastocysts undergo cell reorganisation, parti-

cularly of the cytoskeleton, which may be depolymerised by thepresence of cryoprotectant agents or disrupted by the cryopre-servation procedure itself (Dobrinsky 1996). In addition, cell

membranes are susceptible to different forms of damage duringcryopreservation (Wolfe and Bryant 1999; Acker and McGann2001) and the extent of this damage could affect embryo survival.

Although our results revealed no differences in apoptosis rates infresh andvitrified blastocysts, it shouldbenoted thatweexaminedDNA fragmentation 3 h after warming. This warming period was

perhaps not enough to identify the extent of damage caused by thevitrification–warming procedure.

We detected no significant differences between cow andcalf embryos when expanded or hatched blastocysts were

vitrified–warmed on Day 7 or 8 after insemination. The onlydifference detected was a higher survival rate when Day 8 calf

blastocysts where vitrified compared with Day 8 cow blasto-cysts. As far as we are aware, no prior study has compared thecryotolerance of adult and prepubertal blastocysts. Khatir et al.

(1998) observed no significant differences in total cell numbers,inner cell mass or trophectoderm cell numbers between calf andcow embryos. As reported by different authors and discussed

in this paper, improved cryosurvival has been linked to moreadvanced embryonic stages on a given culture day. In the presentstudy, calf blastocysts vitrified on Day 8 showed better survivalrates than cow blastocysts. This finding may be explained by a

slower in vitro development of calf blastocysts or improvedembryo quality on Day 8 compared with cow blastocysts. Whatdoes seem to be clear is that the age of the blastocyst and the time

of its formation are both important factors for the vitrificationof IVP bovine embryos. More specifically, our findings indicatethat the cryotop technique seems to be particularly useful for

blastocysts with a high degree of expansion (expanded andhatched blastocysts), particularly those blastocysts vitrified andwarmed on Day 7.

Acknowledgements

This study was supported by grants from the Spanish Ministry of Education

and Science (project no. AGL2007–60227/GAN) and from the Universitat

Autonoma de Barcelona (grant no. EME2004–25). The authors are grateful

to ASEAVA (Llanera, Asturias, Spain) for supplying doses of frozen bull

spermatozoa. The authors also acknowledge the Servei de Microscopia of

the Universitat Autonoma de Barcelona for technical assistance and stan-

dardisation of the confocal imaging procedure.

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Manuscript received 16 January 2010, accepted 20 March 2010

http://www.publish.csiro.au/journals/rfd

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