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Cryobiology38, 290–300 (1999)Article ID cryo.1999.2172, available online at http://www.idealibrary.com on

Cryopreservation of Cattle Oocytes: Effects of Meiotic Stage,Cycloheximide Treatment, and Vitrification Procedure

Francois Le Gal1 and Alban MassipUnite des Sciences Ve´terinaires, Universite´ Catholique de Louvain,

Place Croix du Sud, 3, 1348 Louvain-la-Neuve, Belgium

Different parameters likely to influence the survival of bovine oocytes after a vitrification procedure were evaluatoocyte meiotic stage, cycloheximide treatment at the beginning or the end of maturation, and three vitrificatprocedures using conventional straws, open pulled straws (OPS), or microdrops. For each procedure a mixtucryoprotectants (25% ethylene glycol and 25% glycerol) was used. After the oocytes were warmed and subjectein vitro maturation and fertilization, the number that developed into blastocysts was determined. Results showcryoprotectant exposure reduced embryo development and that cycloheximide treatment had no beneficial effeoocytes vitrified in conventional straws. Among the three vitrification procedures, only the OPS method yieldblastocysts (approximately 3% of vitrified oocytes) irrespective of their initial meiotic stage. This result highlights thmajor influence of the cooling rate in an oocyte vitrification protocol.© 1999 Academic Press

Key Words:bovine; oocyte; maturation; vitrification; cycloheximide.

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Effective means of cryopreservation for ctle oocytes would offer many advantages,most important being the preservation amaintenance of genetic resources by the elishment of banks for the cattle breeding indtry. It would also allow the facilitation of planning in thein vitro production of embryos fromlaughtered cow ovaries for experimental poses and the use of oocytes from the sonor for testing the semen of several differulls at the same time (sibling test).However, despite considerable research

he recent years, limited progress has been mn the field of bovine oocyte cryopreservatnd survival of oocytes in terms of developmo the blastocyst stage still remains lomounting to about 3% of oocytes treated (cy 21). Only a few calves are born after transf blastocysts derived from frozen or vitrifiocytes (7, 9, 24, 30). The reasons forxtremely low yield must be found in the uniq

eatures of this cell that influence its cryobeh

Received September 16, 1998; accepted February 2,1 To whom correspondence should be addresse

resent address: Department of Reproductive Biollinik fu r Andrologie und Gyna¨kologie, Universita¨t Zurich,interthurerstrasse 268, CH-8057 Zurich, Switzerla

ax: (41) 1 635 89 03. E-mail: [email protected]

2900011-2240/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

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ior. The oocyte is a large cell with a hivolume–surface ratio, surrounded by a zonalucida (ZP) and several layers of granulosa cforming the cumulus–oocyte complex (COMany specific problems have been describedoocytes at different meiotic stages of matution. These include problems associated wthe cryopreservation of ovulated oocytesspindle disorganization (20), loss or clumpof microtubules resulting in some scatteringchromosomes (26), increased polyploidy attilization (2, 4, 8), and a decrease in fertilizat(8, 38). Freezing of immature oocytes at thestage might circumvent these problems becgenetic material is contained within the ctours of a nuclear envelope but very low svival rates have been reported for cattle (18,30) and pig oocytes (5). This could be explaiby the fact that the cells immediately adjacenthe oocyte (the corona radiata) have long cplasmic extensions which penetrate the ZPterminate in bulbous swellings closely assated with the oocyte membrane (10, 32).presence of these processes and of gap juncplays an important role in the metabolic coeration between oocyte and cumulus cellsing the growth phase and final maturation ofoocyte. All those features could explain why

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291BOVINE OOCYTE VITRIFICATION

success of oocyte cryopreservation in mostcies at these two maturation stages is veryHowever, rodents are an exception as goodsults were reported for mouse and hamstercytes (39). This could be explained by diffences in the susceptibility to cooling of tcytoskeletal system of those oocytes (37) othe fact that they are maturedin vivo.

Oocytes collected from a given batchslaughtered cattle ovaries originate from anerogenous follicular population. Even thoumost of these oocytes are arrested at thestage, they have not the same degree of cotence and they proceed through meiosis atferent rates (40) according to their GV staSo, in order to allow them to acquire develmental competence it was suggested by Loganet al. (19) to inhibit meiosis resumption b

sing reversible inhibitors such as cycloheide.Based on the results achieved to date, o

possibilities were investigated such as crpreservation at various stages of meiosis oruse of new vitrification procedures. Limet al.(18) showed that the meiotic stage affects ssequent development of cooled bovine oocyWhen oocytes were cooled at the GVBD (gminal vesicle breakdown) stage they cleaand reached the blastocyst stage in higherportions than those cooled at the GV or mphase II stages (3) and vitrification of oocyafter 12 h of maturation was superior compato other groups at 6-h intervals (11).

The low success rates reported using contional slow freezing methods with oocytes athe recent advances in cryopreservation ofbryos of Drosophila (22, 29) which are versensitive to chilling injury have lead to tadaptation of similar methods for mammaloocytes. They involve using extremely racooling rates. The simplest way to do this isplunge the sample directly in liquid nitrogeLanda and Tepla (13) attempted to vitrmouse eight-cell embryos in microdropscryoprotectant solutions (expelled into a pfloating on liquid nitrogen), thus allowingvery fast drop of temperature between rotemperature and that of liquid nitrogen. Ho

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ever, this procedure is not easy to carry oupractice. More recently, Martinoet al. (21) re-ported high survival and development of bovoocytes after ultrarapid cooling on electroncroscope grids in a film of cryoprotectant. Onagain, practical use is not easy. Finally, Vajtetal. (33) designed the OPS (open pulled strmethod in which 0.25-ml straws are heat puto half of both their diameter and their wthickness so that oocytes or embryos couldloaded by capillary effect in a minimum volumof cryoprotectant and immersed directly in LN2.

In this paper, we describe experimentswhich the effects of oocyte meiotic stage,influence of a treatment with cycloheximiprior to vitrification, and three different vitrification procedures (i.e., vitrification in strawsOPS, and in microdrops) were evaluatedsurvival and development of cattle oocytes.

MATERIAL AND METHODS

Maturation, Fertilization, and Culture

COCs were obtained by aspiration of 2-6-mm follicles from abattoir ovaries. After fowashes in modified PBS (supplemented wpyruvate and BSA, free fatty acid, SigmA8806) they were matured in TCM 199 cotaining penicillin (60 iu ml21) and streptomyci(60 mg ml21), 10% heat-treated fetal calf seru(FCS, ICN Flow, 2910149), and 10 ng/ml EG(epidermal growth factor, Sigma E4127)24 h in an atmosphere of 5% CO2 in air withmaximum humidity.

After maturation, COCs were washed fotimes in PBS and transferred into modifiedrode’s medium supplemented with fatty-acfree BSA (6 mg ml21), sodium lactate (4 mml21), sodium pyruvate (0.11 mg ml21), andheparin (10mg ml21). They were coincubatefor 18 h with Percoll-selected semen from oejaculate of a Belgian Blue Bull (23 106 sper-matozoa ml21 final concentration) in a humidfied atmosphere of 5% CO2 in air.

Presumptive zygotes were stripped of culus cells by gentle vortexing and cultured unparaffin oil in 30-ml droplets (one embryo/ml) ofVT1, a modified SOF without phosphate (6)

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292 LE GAL AND MASSIP

a humidified atmosphere of 5% CO2, 5% O2,and 90% N2. At 48 h after insemination (timecleavage evaluation), FCS was added to yiefinal concentration of 10%, and 1 vol of frecomplete culture medium was added 72 h laBlastocyst number was recorded 210 h possemination (day 8).

Vitrification Procedure with Glycerol (GLY)and Ethylene Glycol (EG) in Straw atDifferent Stages of the Maturation Proces

At different steps of their maturation proce(0, 17, and 24 h), COCs were successivexposed to PBSS solutions (20% FCS supmented PBS solution) containing 10% GLYmin), 10% GLY1 20% EG (5 min), and 25%

LY 1 25% EG for 1.25 to 1.5 min befoither dilution of the cryoprotectants (“exposocytes”) or loading into a 0.25-ml straw (72 per straw) and plunging directly into liquitrogen (“vitrified oocytes”). In both cases, tryoprotectant dilution procedure was the samin each of a PBSS solutions (3 ml) conta

ng 1.7, 0.85, and 0.4 M galactose, respectivnd three baths (10 min total) in PBSS.rocedures were performed at room temp

ure (25–27°C).

ycloheximide Treatment

COCs were matured in cycloheximide-caining medium (5mg ml21 in TCM 199 supplemented with FCS and EGF) either fromonset of maturation until the 18th hour to schronize the oocyte population (19) or from18th hour until the 42nd hour for inducing ocytes to exit from metaphase I–metaphastransition and enter an interphasic stage (The duration of this second cycloheximtreatment was chosen for practical convenieIn those experiments, total culture time w42 h.

Vitrification Procedure with GLY and EG inMicrodrops

COCs were exposed to PBSS cryoprotectcontaining solutions as described previouexcept that they remained only 30 s invitrification solution. Then, COCs were as

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rated into a yellow pipette tip and expelled i20-ml drop onto a chilled metal plate (2196°C).This plate was the oval lid (83 6 cm) of acandy box floating on liquid nitrogen in a Srofoam box. After 1 min, drops were transferto liquid nitrogen-filled cryotubes and storeda liquid nitrogen tank. Drops were devitrifiedusing chilled forceps for handling them froliquid nitrogen to PBSS galactose-containsolutions. The cryoprotectant dilution procedwas the same as above.

Vitrification Procedure with GLY and EG inOpen Pulled Straw

This protocol was adapted from that of Vaet al. (33). COCs were successively washePBSS containing 10% GLY for 1 min, 10GLY 1 10% EG for 1 min, and 20% GLY120% EG1 0.3 M galactose for 45–60 s duriwhich COCs were loaded into pulled stra(half of the original diameter, 7 to 12 COstraw). Straws were plunged directly into liqunitrogen. After storage in liquid nitrogen, strawere warmed in air for 3 s and then the pulleend was plunged into PBSS containing gatose and the COCs were expelled. Cryoptectant was removed by steps of decreaconcentrations of galactose in PBSS (0.85,0.2 M; 3 min/bath) and three final washesPBSS (7 min total).

Assessment of Maturation and FertilizationStages

After maturation and fertilization, a sampof oocytes and presumptive zygotes wasnuded of cumulus cells by vortexing, and thchromosomal status was assessed using arescent DNA-binding dye (Hoechst 33342).

Statistical Analysis

Maturation, fertilization, cleavage, and bltocyst rates were calculated from the ininumber of oocytes collected and matured.rates were compared by ANOVA after Arcstransformation (28), and differences ofP ,0.05 were considered as significant.

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RESULTS

In-Straw Vitrification of Oocytes at DifferentStages of the Maturation Process

Table 1 summarizes the results obtaiwhen oocytes were exposed to cryoprotector vitrified in straws at different stages of tmaturation process. Thein vitro maturation rat

f oocytes exposed to cryoprotectants beaturation (65.4%) or after 17 h of maturat

79.5%) was not significantly different frohat of unexposed control oocytes (71.3%).

When oocytes were vitrified before matuion, the maturation rate dropped to 19.8bout a one-third of them (25/67, unreporthowed signs of degeneration (clumpinghromosomes, granular aspect of cytoplaig. 1A). When oocytes were vitrified after 17f maturation (metaphase I–metaphase II tition), warmed, and then further maturedh, 41.7% reached the metaphase II stage

ignificantly less than controls) and 20% (9/ere degenerated.The fertilization rate for control oocytes w

1.6% (12.8% polyspermy) and was not sig

Survival and Development of Oocytes Exposed or V(Calculated from the Initial Num

Maturationstage andtreatment

Oocytestreated

(replicates)Maturation

(%)a Poly (%

Immaturecontrols

449 (6) 72/96 (71.3)a 11/83 (1

Immatureexposed

323 (5) 87/144 (65.4)a 6/31 (1

Immaturevitrified

245 (3) 11/67 (19.8)b 2/32 (6

17-h maturedexposed

202 (2) 35/44 (79.5)a 9/39 (2

17-h maturedvitrified

206 (2) 19/45 (41.7)a,b 13/43 (3

24-h maturedexposed

153 (2) Not done 12/48

24-h maturedvitrified

142 (2) Not done 4/43

Note.Values within columns with different superscripleast).

a Percentages are means of replicate percentages.

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icantly affected by exposure to vitrification slutions, irrespective of the meiotic stage. Hoever, a relative increase in polyspermy (fr12.8 to 15.7, 23.4, and 30% for controls, unmtured, 17-h matured, and 24-h matured expooocytes, respectively) was noticed.

Fertilization rates for oocytes vitrified befoduring, or after maturation were not signcantly lower than those for controls. The ratepolyspermy was much higher for oocytes vified after 17 h than before or after maturat(30.5 vs 6.7 and 7.4%, respectively).

Cleavage rates for oocytes exposed to cprotectants before, during, or after maturawere lower (but not significantly) than thosecontrols. However, this rate for oocytes expoto cryoprotectants after maturation was notnificantly different from that of controls. Cleaage rates for vitrified oocytes were lower thfor controls and did not differ irrespectivetheir initial meiotic stage at the time of vitrication (16.3, 17.6, and 21.8 vs 51.1% for vified oocytes and controls, respectively).

Overall blastocyst rate for controls w

fied in Straws at Different Stages of the Maturation Prof Oocytes Collected and Matured)

tilizationCleavage 48 hpi

(%)a

Blastocyst 210hpi (%)aTotal (%)a

) 59/83 (71.6) 132/270 (51.1)a 57/270 (21.8)a

) 16/31 (41.2) 56/148 (36.6)a,b 20/148 (13.9)a,b

12/32 (38.3) 23/146 (16.3)b 0/146 (0)c

) 27/39 (69.4) 45/119 (37.5)a,b 8/119 (6.7)a,b,c

) 29/43 (67.9) 21/118 (17.6)a,b 1/118 (1)b,c

) 31/48 (71.2) 52/105 (52)a,b 5/105 (4.3)a,b,c

) 15/43 (36.7) 23/99 (21.8)a,b 0/99 (0)b,c

are significantly different (ANOVA, Scheffe test,P , 0.05 a

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FIG. 1. Effects of vitrification and cycloheximide treatment onin vitro maturation of bovine oocytes. At theend of maturation, oocytes were denuded from cumulus cells, fixed in ethanol, and assessed for maturation sby using fluorescent DNA-binding dye (Hoechst 33342). (A) Degenerated oocyte after in straw vitrification

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21.8% (i.e., nearly 22 blastocysts of 100 oocwhich were induced to mature after recoveThis rate was higher than that for exposedvitrified oocytes (not significantly vs exposoocytes, significantly vs vitrified oocytes). Nblastocysts were obtained from oocytes vitribefore or after maturation, and only 1 (1%) wobtained from oocytes vitrified after 17 hmaturation.

Effect of Cycloheximide Treatment

On the maturation process.First of all, weevaluated the effects of using cycloheximidethe maturation process (Table 2). When cyheximide was added at the onset of the mattion process for 18 h, it was effective in blocing meiosis resumption as no metaphase II soocyte (0/77) was observed after 18 h of inbation. Most of these oocytes (70/775 90.9%)were still at the GV or GVBD stage (Fig. 1B

the GV stage: clumping of chromosomes and gOocyte treated with cycloheximide from the onOocyte treated with cycloheximide from the onsOocyte treated with cycloheximide from the onsmaturated for 24 h: metaphase II stage. The firtreated with cycloheximide from the onset to thefor 24 h: metaphase I stage. (F) Oocyte treamaturation: nucleus stage. The oocyte has emitta round shape structure is decondensed. Barsm

Effect of a Cycloheximide (CH

TreatmentOocytes treated

(replicates)Degenerated

(%)GV

Controlse 161 (9) 11 (6.9)CHX t0–18

f 77 (3) 70CHX t0–18 then

without t18–42g 162 (7) 18 (11.1)

CHX t18–42g 119 (5) 26 (21.8)

a Germinal vesicle and germinal vesicle breakdownb pro-Metaphase I stage.c Metaphase I stage.d Metaphase II stage.e Fixed att24.f Fixed att18.g Fixed att42.

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the remainder being blocked at prometapha(Fig. 1C). Extensive washing after 18 h ofcubation in the presence of the drug allowmeiosis to resume. After an additional 2incubation in drug-free medium, 105/162564.8% of the oocytes reached the metaphastage (Fig. 1D) whereas 35/1625 21.6%reached only the metaphase I stage (Fig. 1

When cycloheximide was added after 18 hmaturation, 22.7% showed decondensed cmatin (nucleus reformation, Fig. 1F) and 36.were still at the metaphase II stage whe41.1% (21.8 1 19.3) were degeneratedhowed only partial maturation.On cryopreservation.In these experiment

ocytes were treated with cycloheximide, eitrom the onset of maturation to 18 h (and thitrified, warmed, andin vitro matured for 24 hr from 18 to 42 h of maturation before beitrified. Two vitrification systems were use

ular aspect of the cytoplasm are particularly evident.to the 18th hour of maturation: GV–GVBD stage. (C

o the 18th hour of maturation: prometaphase I stage.o the 18th hour of maturation and then rinsed and furtholar body (pb) is facing the metaphase plate. (E) Ooc

th hour of maturation and then rinsed and further maturwith cycloheximide from the 18th to the 42nd hourthe first polar body (pb) and the chromatin contained wi

Treatment on Oocyte Maturation

BDa

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(%) MI c (%) MII d (%)Nucleus

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7 (4.3) 35 (21.7) 108 (67.1)0.9) 7 (9.1)

4 (2.5) 35 (21.6) 105 (64.8)23 (19.3) 43 (36.2) 27 (22

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one with conventional straws and the otherwith microdrops. Table 3 shows that matution, fertilization, and cleavage rates for oocythat had been vitrified and then warmed wlower than for controls, whatever the vitrifiction procedure. Moreover, the maturation,tilization, cleavage, and blastocyst rates wnot significantly different between the two vrification protocols irrespective of the cycloheimide treatment (except at the level of fertiliztion rate for oocytes vitrified aftercycloheximide treatment from 18 to 42 h (P 50.03).

Effects of Cycloheximide Treatment and Type of V(Calculated from the Initial Num

TreatmentVitrification

protocolOocytes treated

(replicates)Maturation

(%)a

Controls 467 (6) 52/106 (49.9)a

CHX t0–18

vitrif t1–18

Straws 252 (3) 8/60 (15.2)b

CHX t18–42

vitrif t42

Straws 104 (2) Not done

CHX t0–18

vitrif t18

Microdrops 210 (2) 9/28 (32.1)a,b

CHX t18–42

vitrif t42

Microdrops 71 (2) Not done



TABSurvival and Development of Immature and Mat

(Calculated from the Initial Num

Maturationstage

Vitrificationprotocol

Oocytes treated(replicates)

Maturation(%)a

Immature Not vitrified(controls)

658 (7) 55/119 (45.5)

Immature OPS 317 (5) 19/73 (27.5)24-h matured OPS 195 (4) Not doneImmature Microdrops 136 (2) 4/25 (16.2)24-h matured Microdrops 62 (2) Not done



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The OPS Protocol

Maturation rates for immature oocytes vified either by the OPS method (27.5%) ormicrodrops (16.2%) were lower than thosecontrol oocytes (45.5%, Table 4). Overall ftilization rates were also lower for immatuand mature oocytes vitrified in OPS or micdrops compared with controls (29.2, 25.820.9 and 43.7% vs 55.6%, respectively). It wparticularly evident when oocytes were vitrifibefore maturation: polyspermy was nil for bunmatured oocytes vitrified in OPS or mic

cation on Survival and Development of Vitrified Oocytof Oocytes Collected and Matured)

Fertilization Cleavageat 48 hpi

(%)a

Blastocystat 210 hpi

(%)aly (%)a Total (%)a

1 (17.6)a 54/91 (55.6)a 84/270 (31.5)a 42/270 (15.5)a

8 (4.4)a,b 22/68 (26)a 15/124 (12.3)a,b 0/124 (0)b

/61 (9.6)a,b 17/61 (28.4)a 2/43 (4.7)b 0/43 (0)b

4 (6.1)a,b 24/94 (22.3)a,b 13/88 (14.5)a,b 0/88 (0)b

/24 (0)b 0/24 (0)b 0/47 (0)b 0/47 (0)b


4Oocytes Vitrified in Open Pulled Straws or Microdropof Oocytes Collected and Matured)

Fertilization Cleavageat 48 hpi

(%)a

Blastocystat 210 hpi

(%)aoly (%)a Total (%)a

/114 (18.2)a 65/114 (55.6) 155/425 (35.5)a 77/425 (17.6)a

0/40 (0)b 12/40 (29.2) 48/204 (22.2)a 7/204 (3.3)b

9/51 (16.4)a,c 15/51 (25.8) 14/144 (10.6)a,b 3/144 (1.8)b

0/29 (0)b,c 6/29 (20.9) 6/82 (8.2)a,b 0/82 (0)b

5/16 (31.2)a 7/16 (43.7) 0/46 (0)b 0/46 (0)b


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drops compared with 18.2% for controls. Cleage rates were very low for vitrified oocy(10.6% for 24-h matured vitrified in OPS, 8.2for GV stage vitrified in microdrops, 0% f24-h matured vitrified in microdrops), excefor GV stage oocytes vitrified in OPS (22.2%Blastocyst rates were nil for oocytes vitrifiedmicrodrops and low for oocytes vitrified in OP(3.3 and 1.8% for GV stage and maturedcytes, respectively) compared with contr(17.6%).

DISCUSSION

In the present studies, we investigated sevfactors for their consequences on survivaldevelopment of vitrified bovine oocytes. In tfirst experiment, oocytes were exposed to vfication solutions or vitrified in straws at diffeent stages of their maturation process. No btocysts were obtained from oocytes vitrifieither before (GV stage) or after (metaphasstage)in vitro maturation and only one blasyst was produced from oocytes vitrified a7 h of maturation (metaphase I–metaphas

ransition). The detrimental effect of the vitriation procedure is due to cumulative effectxposure to vitrification solutions (as blastocates for exposed oocytes were lower than thor controls) and to vitrification per se (as blocyst rates for vitrified oocytes were signantly lower than those for exposed oocytehe same meiotic stage). The effect of expoo vitrification solution appears at the timerst cleavage. It thus seems that this vitrificatolution effect (cytotoxicity) is delayed, as oerved with immature goat oocytes (14). Deental effects of the vitrification procedure

ame more evident with the length of cultuhis confirms the results of GV mouse frozocytes (36).In the second experiment we first confirm

hat cycloheximide is able to block oocyte msis resumption as has already been publi

or cattle (19, 27) and other farm animal specsheep, 23; pig, 12; goat, 15). This effecotally reversible in cattle (19; this study) bnly partly in the goat (15).When the drug was added at metaph

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–metaphase II transition, only 22.7% of inthasic stages were obtained (i.e., reformationucleus, Table 2). The time point of 18 h a

he onset of maturation was chosen for praconvenience while 15–16 h would have brobably more accurate (according to 27). Nrtheless, we would expect to obtain more

erphasic stages with or without extrusion ofrst polar body when cycloheximide wresent from 18 to 42 h. It thus seemsovine oocytes are much more resistant to

ering an interphasic stage (27; this study) toat oocytes (15).When oocytes were treated with cycloheide from the onset of maturation to the 1our before vitrification, the completion of mration after warming was low: 15.2% wtraws and 32.1% with microdrops (Tablehen those oocytes were further submitte

n IVF procedure, 26 and 22.3% were fertilizespectively. This result is surprising becaertilization rates were calculated from the iial number of oocytes collected and maturne can explain this intriguing result by the f

hat oocyte samples which were fixed atained for evaluation of the maturation rere those left on the bench (in the maturaell) while the others were immediately rinsnd inseminated. This explanation agrees

he fact that maturation rates for controls wlso lower than their respective fertilizatiates (Tables 1, 3, and 4). Another possxplanation could be that at the end ofaturation period, many oocytes did not colete meiosis and were still at the metaphatage.When the cycloheximide treatment was

lied from 18 to 42 h of maturation, survivnd developmental rates after devitrificatere lower than those obtained for oocy

reated with cycloheximide at the onset of mration. However, if we compare Table 1 wables 3 and 4, cycloheximide treatment dot seem to have any beneficial effect prio

he vitrification procedure on maturation, ferzation, cleavage, and blastocyst rates forytes either vitrified in straws or in microdropIn the last experiment (Table 4), vitrificati

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procedures with a very rapid drop of tempeture, i.e., microdrops and OPS, were used.first one did not result in any blastocysts. Wthe OPS protocol higher blastocyst rates wobtained compared with their respective coterparts vitrified with conventional straws204 vs 0/146 and 3/144 vs 0/99 for immatand 24-h matured oocytes, respectively). Thdifferences could be evidence of the importaof the cooling rate between the time durwhich the oocytes were exposed to the cryotectant and their being plunged into liquidtrogen. In both cases, the cryoprotectant stion was nearly the same (25% EG1 25% GLYor vitrification in straws vs 20% EG1 20%LY 1 0.3 M galactose for vitrification iPS). This difference in the survival and devpment could also be explained by the fact

n the procedure of vitrification, the total timrom the beginning of the CPA exposurelunging of the straws into liquid nitrogen w5–90 s whereas with the OPS protocol,

ime was only 45–60 s. This latter durationonger than the one recommended (35) tonto account

(i) that glycerol is a less permeant cryopectant than DMSO or EG (17, 31)

(ii) that cumulus size was not reducedechanical pipetting,(iii) that permeability changes during ma

ation process, immature oocytes being lesseable to cryprotectant than mature ones6), and(iv) that the equilibration with the cryopr

ectant solution was done at room temperan this study (25–27°C) instead of 39°C (35

However, as the oocytes collapsed and beo reexpand during that exposure time, onessume that their internal cryoprotectant centration was already high (16).Since this study was performed, Vajtaet al.

34) reported very high rates of developmenitrified oocytes to blastocyst (up to 25%any reasons could explain such differenetween Vajtaet al.’s and our results: origin oiological material (dairy vs beef cows), wo

ng temperature (heated stages at 39°C vs r

-e

e-

ee

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s

e

r-,

e

nn-

f

s

m

emperature), cryoprotectants used (20% E10% Me2SO in 0.5 M sucrose-containing ho

ing medium vs 20% EG1 20% GLY in 0.3 Mgalactose-containing holding medium), reduvs unreduced size of the cumulus, and culturSOF medium vs VT1 medium. From all thodifferences in the whole procedure, one cantell what those are which influenced the mthe blastocyst production rate after warming

Martino et al. (21) using a cryoprotectaolution of 5.5 M EG1 1 M sucrose and a tot

exposure time of 30 s obtained 15% blastocwhen oocyte vitrification was done at very hcooling rate on electron microscope grwhereas blastocyst rates of less than 1% wobtained when the vitrification was made i0.25-ml conventional straws. They hypotsized that the extreme chilling sensitivitymature bovine oocytes could explain sucdifference in developmental rates. This chillsensitivity is assumed to be within115 and215°C, a temperature range for which therno evidence to suggest that cryoprotectantsprevent chilling damage. Vajtaet al. (34) re-ported that with OPS cooling rate is 7 totimes more rapid than with conventional stra

From the present data, we can concludethe key to success in a bovine oocyte vitrifition program would then reside in the use oprocedure that would circumvent the chillisensitivity of these cells.

ACKNOWLEDGMENTS

This work was supported by grant from the EuropCommission (Grant CEE/TMR ERB4001dt950900)F.L.G. The technical assistance of Mrs. Vale´rie Labrique fooocyte recovery and IVF procedures is greatly acknedged. The authors thank Mrs. Vale´rie Majerus and MrPierre Tilquin for assistance with statistical analysis.

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cryopreservation of cattle oocytes: effects of meiotic stage, cycloheximide treatment, and...

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