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Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity
Animal Reproduction Science 52 Ž1998. 93–104
Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity I. Donnay a , Ph. Auquier a , S. Kaidi a , C. Carolan b, P. Lonergan b, P. Mermillod b, A. Massip a,) a
UniÕersite´ Catholique de LouÕain, Unite´ des Sciences Veterinaires, Place Croix du Sud 3, ´´ B-1348 LouÕain-la-NeuÕe, Belgium b INRA, Station de Physiologie de la Reproduction, F-37380 Nouzilly, France Accepted 4 June 1998
Abstract Methodological studies were undertaken to test the validity of a three-step vitrification procedure for bovine in vitro produced embryos using glycerol and ethylene glycol as cryoprotectants. Embryos were produced in a low-phosphate culture system Žmedium VT1 q 10% foetal calf serum. and vitrified at day 7 post-insemination either in a mixture of 25% glycerol—25% ethylene glycol or a mixture of 10% glycerol—40% ethylene glycol. In the first mixture 67% Ž n s 283. of blastocysts were re-expanded after 72 h of culture and 53% were hatched while in the second one Ž n s 65. only 5% survived. The mean number of cells of the surviving blastocysts was correlated with the rate of survival Ž R 2 s 0.47; P s 0.0024.. Embryo size Ždiameter - or ) to 180 mm. did not influence blastocyst survival or cell number, but hatching rate was higher for embryos ) 180 mm. Embryo survival, hatching rate and cell number 72 h post-warming were not affected by the mode of vitrification Ždirect plunging into nitrogen liquid or vitrification into nitrogen liquid vapour., the mode of preparation of the vitrification solutions Žmolar or molal basis. or by the concentration of galactose used as a diluent Ž0 to 0.85 M.. Only one calf was born after transfer of 22 vitrified blastocysts. These results confirm the apparent lack of correlation for cryopreserved embryos between in vitro survival or hatching and viability after transfer. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cattle-embryology; In vitro embryo production; Vitrification; Cryopreservation
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Corresponding author: Tel.: q32 10 47 37 56; fax: q32 10 47 37 17; e-mail: [email protected]
0378-4320r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 0 9 8 - 0
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1. Introduction In vitro produced ŽIVP. bovine embryos differ from their in vivo counterparts in many respects: for example, in vitro developmental capacity, morphology, resistance to freezing and survival after transfer Žfor reviews, see Greve et al., 1993; Gordon, 1994; Avery and Greve, 1995; Massip et al., 1995; Van Soom et al., 1996.. The improvement of IVP embryo cryopreservation is an important goal and recent data indicate that rapid, rather than slow cooling, may be beneficial for their survival ŽLeibo and Loskutoff, 1993; Mahmoudzadeh et al., 1994; Pollard and Leibo, 1994; Niemann, 1995.. IVP bovine embryos have been vitrified successfully, in terms of in vitro survival, by most of the methods used for in vivo embryos with or without modifications ŽTachikawa et al., 1993; Mahmoudzadeh et al., 1995; Massip et al., 1995; Vajta et al., 1995, 1996a.. The pregnancy rate after transfer of vitrified in vitro produced embryos is highly variable Ž23 to 50% of the transferred embryos. but the numbers of transfers in the published studies are usually low ŽKuwayama et al., 1992; Tachikawa et al., 1993; Wurth et al., 1994; Agca et al., 1994; Delval et al., 1996; Holm et al., 1996.. Agca et al. Ž1994. adapted the original vitrification method of Massip et al. Ž1986. to in vitro embryos and obtained pregnancies after transfer. They used a three-step equilibration procedure with glycerol and ethylene glycol as cryoprotectants and sucrose as diluent. They stressed the importance of time and temperature of equilibration in the previtrification solutions on in vitro survival and demonstrated that the best conditions were achieved when embryos were equilibrated for 5 min at 278C during the second step. We have used their new procedure, but with galactose as diluent instead of sucrose, to examine the effect of a number of variables on the outcome of vitrification: Ži. composition of the vitrification solution; Žii. blastocyst size; Žiii. preparation of the solutions on a molar vs. molal basis; Živ. mode of vitrification Žnitrogen liquid vapour vs. direct plunging into nitrogen liquid.; Žv. dilution medium after warming. Re-expansion and hatching rates of the vitrifiedrwarmed embryos were assessed during 72 h of co-culture on Buffalo Rat Liver cells, as well as the cell number of the surviving embryos. Transfers into recipient heifers were also performed to assess the viability of the vitrified embryos.
2. Materials and methods 2.1. Source of embryos Embryos were generated from abattoir ovaries. Oocytes were collected by aspiration of 3–6 mm follicles and washed four times in modified Dulbecco’s phosphate buffered saline ŽPBS. containing 0.3 mM sodium pyruvate ŽSigma, St. Louis, MO, USA., 0.4 mg bovine serum albumin mly1 ŽFraction V; Sigma. and 50 mg mly1 gentamicin ŽGibco, Paisley, UK.. Groups of 100 randomly selected oocytes were allocated to four well culture plates ŽNunc, Roskilde, Denmark. containing 500 ml maturation medium ŽTCM 199, Sigma M-4530. supplemented with 10% heat treated foetal calf serum ŽFCS-JRH Biosciences, Lenaxa, KS, USA. and 10 ng mly1 epidermal growth factor ŽSigma.. After
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24 h maturation at 398C under 5% CO 2 in a humidified atmosphere, oocytes were washed four times in PBS and transferred to 500 ml fertilization medium ŽTyrode’s medium with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mly1 fatty acid-free albumin fraction ŽSigma., 10 mg mly1 heparin–sodium salt w167 U mgy1 ; Calbiochem, San Diego, CA, USAx. and 50 mg mly1 gentamicin. Thawed semen was centrifuged on a Percoll ŽPharmacia, Uppsala, Sweden. discontinuous density gradient Ž45% and 90%. for 30 min at 700 = g. Spermatozoa were added to each well to a final concentration of 2 = 10 6 mly1 . Oocytes and spermatozoa were incubated together for 18 h. The same ejaculate from one bull was used throughout the experiments. Presumptive zygotes were vortexed for 2 min in PBS to remove cumulus cells, washed three times in PBS and twice in culture medium. Denuded zygotes were cultured in groups of about 30 in 30 ml droplets of medium VT1 ŽDonnay et al., 1996. under paraffin oil and 5% O 2 , 5% CO 2 , 90% N2 at 398C in humidified air. The composition of VT1 medium was 114 mM NaCl, 7.16 mM KCl, 25.07 mM NaHCO 3 , 0.49 mM MgCl 2 , 0.3 mM Na-pyruvate, 2.5 mM Ca-lactate, 1 mM glutamine, 3 mg mly1 BSA, 20 ml ly1 essential amino acids mixture ŽSigma. and 10 ml ly1 non-essential amino acids ŽSigma.. On day 2 post-insemination Žpi., 10% FCS was added. Blastocysts were collected on day 7 pi. 2.2. Vitrification procedure On day 7 pi, morphologically normal blastocysts according to the I.E.T.S. standard classification Žfrom small to expanded. were retrieved and pooled in a Petri dish containing either HEPES buffered culture medium ŽVT1 q 10% FCS q 5.96 g ly1 HEPES; Sigma. or embryo transfer freezing medium ŽGibco. with 20% FCS and no BSA ŽETF.. Those media were also used for the dilution of the cryoprotectants. Samples of 10 embryos were treated at a time, the remainder being held in the incubator. Unless otherwise indicated, treatment consisted in a three-step equilibration process at room temperature Ž25–328C.: step 1: 5 min in 10% glycerol Žvrv; AnalaR, Poole, UK.; step 2: 5 min in a mixture of 10% glycerol ŽGly. q 20% ethylene glycol ŽEg. ŽAnalaR.; and step 3: 30 s in a vitrification mixture of 25% Gly q 25% Eg. The blastocysts were equilibrated for 5 min in step 1; 5 min in step 2, then placed, for step 3, five at a time, in small drops of the vitrification solution, and aspirated, within 30 s, into 0.25 ml French straws ŽIMV, L’Aigle, France. containing 0.85 M d-galactose ŽSigma. dissolved in ETF medium. The straws were then plunged immediately into nitrogen liquid, unless otherwise indicated. 2.3. RecoÕery of embryos and assessment of surÕiÕal After storage from a few days to a few weeks, the straws were warmed for 10 s in water at 378C, unless otherwise indicated. The contents of each straw were expelled into a Petri dish and gently agitated to mix galactose with cryoprotectant. After 5 min, the embryos were transferred to holding medium for 5 min then washed in 199 mediumq 10% FCS and co-cultured in 50 ml drops of this medium on a confluent monolayer of Buffalo Rat Liver cells ŽBRL. at 398C in a humidified atmosphere of 5% CO 2 in air
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ŽKaidi et al., 1996.. The BRL cells ŽBRL 3A. were stored at y808C in TCM199 supplemented with 10% Žvrv. FCS and 10% Žvrv. DMSO ŽBDH.. They were thawed in a 378C water bath for 1 min and washed in 10 ml of TCM 199 q FCS. Fifty ml drops of cell suspension under paraffin oil containing 1 = 10 5 cells mly1 were prepared and cultured until confluency under 5% CO 2 in air. Confluency was achieved after 2 days. Embryo survival was defined as the percentage of vitrified embryos that had re-expanded after 72 h of culture but data were also available at 24 and 48 h. At the end of this period, expanded and hatched blastocysts were fixed and stained with Hoechst 33342 ŽCalbiochem. for determination of total cell number ŽPursel et al., 1985.. 2.4. Experimental design Each experiment was replicated three times. 2.4.1. Experiment 1: Effect of blastocyst size and composition of Õitrification solution Blastocysts recovered on day 7 pi were arbitrarily separated in two groups: - 180 mm and ) 180 mm diameter and vitrified as described above. The vitrification solutions were 25% Gly q 25% Eg or 10% Gly q 40% Eg, the aim being to test the effect of increasing Eg concentration, the Gly concentration remaining constant at 10%. Steps 1 and 2 and treatment after warming were identical in each case. 2.4.2. Experiment 2: Effects of mode of Õitrification and warming and of the composition of the physiological saline component (molar or molal basis) of the Õitrification solution Two modes of vitrification were tested: in the first one, the straws were placed for 2 min into nitrogen liquid vapour on a styrofoam boat then plunged into nitrogen liquid and warmed in water at 208C. In the second one, straws were plunged slowly into nitrogen liquid then warmed in water at 378C. This experiment also compared two ways of preparing the cryoprotectant solutions. In the first one Žmolal basis., cryoprotectants were diluted as in the other experiments in ETF medium with 20% FCS on a vrv basis; in the second one Žmolar basis., the salts concentrations in the culture medium ŽHEPES VT1. used as a diluent were adapted to maintain their initial concentration in the solution of cryoprotectants throughout the three-step equilibration process. 2.4.3. Experiment 3: Effect of galactose dilution of Õitrified embryos Straws were loaded in three ways. As previously described, air bubbles were positioned on each side of the cryoprotectant solution containing the embryos. In the first case, 0.85 M galactose was present on both sides of the bubbles, in the second case, holding medium was on one side with 0.85 M galactose on the other, and in the third case, holding medium was present on both sides. Dilution was carried out as described in the vitrification protocol. 2.4.4. Experiment 4: Embryo transfers After warming and dilution, embryos were transferred singly to day 7 recipients synchronized with a Norgestomet ear implant ŽCrestar, Intervet, France.. Recipients were Holstein or crossbreed Holstein heifers from the experimental herd at the INRA
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Fig. 1. Evolution of the rates of expansion and hatching after warming.
Fig. 2. Correlation between embryo survival Žrate of re-expansion after 72 h. and mean number of cells.
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Table 1 Effect of the size of day 7 in vitro blastocysts on in vitro survival and total cell number after 72 h of co-culture with BRL cells Blastocyst diameter
-180 mm )180 mm a
Results 72 h post-warming a N
Re-expanded mean %"SD
Hatched mean %"SD
Mean cell number"SEM
30 32
55"20 36"33
42"21b 100"33 c
137"21 Ž ns9. 150"18 Ž ns12.
Results of three replicates. Values with different superscripts within the same column are different ŽTwo-way ANOVA, P - 0.05..
bc
Physiology Station. Transfers were non-surgical and always carried out by the same individual. In a first series, 11 vitrified embryos produced in VT1 were transferred in autumn 1996 at the INRA station; a second series of 11 were transferred in spring and autumn 1997 on recipients of our herd on natural heat. 2.4.5. Statistical analysis One- or Two-way analysis of variance ŽANOVA. were performed to analyse the effect of treatments on all parameters. When data were proportions, an arcsinus square-root transformation was applied before analysis ŽDagnelie, 1986..
3. Results 3.1. Embryo production From a total of 12 replicate experiments, 3466 oocytes were fertilized and cultured for 6 days. On day 7 pi, the peak time of blastocyst appearance, 741 Ž21%. blastocysts were recovered and 536 Ž72%. were selected, of these 348 were used for methodological studies, and the remainder for transfers and other programmes. In the experiments using 25% Gly q 25% Eg, on average 67% Žout of 283. of blastocysts of all sizes survived the vitrification and 53% were hatched 72 h post-warming. The percentage of survival
Table 2 Effect of the mode of preparation of the cryoprotectant solutions and of the mode of vitrification on in vitro survival and mean cell number of in vitro day 7 bovine embryos after vitrification Preparation of the solutions
Mode of vitrification
Molal basis
Direct plunging in LN2 LN2 vapour Direct plunging in LN2 LN2 vapour
Molar basis
)
Results 72 h post-warming ) N Re-expansion Hatching mean %"SD mean %"SD
Mean cell number"SEM
29 30 29 29
154"21 Ž ns 23. 169"21 Ž ns 22. 191"20 Ž ns 24. 190"21 Ž ns 22.
90"10 83"5 93"8 87"11
72"20 56"10 80"20 80"20
Results are from three replicates. No significant difference was observed ŽTwo-way ANOVA, P ) 0.05..
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Table 3 Effect of the type and concentration of the diluent after warming on in vitro survival and mean cell number of vitrified day 7 in vitro bovine blastocysts ) Diluent
0.85 M galactose 0.42 M galactose Culture medium )
Results 72 h post-warming N
Re-expansion mean %"SD
Hatching mean %"SD
Mean cell number"SEM
34 35 35
73"14 55"10 53"19
63"16 34"8 44"14
127"17 Ž ns 21. 125"19 Ž ns18. 142"19 Ž ns17.
Results are from three replicates. No significant difference was observed ŽTwo-way ANOVA, P ) 0.05..
Žre-expansion. did not vary between 24, 48 or 72 h post-warming although the rate of hatching increased ŽFig. 1.. A significant correlation was found between the rate of embryo survival and the mean cell number of the surviving blastocysts 72 h post-warming Ž R 2 s 0.47, P s 0.0024, Fig. 2.. 3.1.1. Experiment 1: Effect of blastocyst size and composition of Õitrification solution With 10% Gly q 40% Eg Ž n s 65., embryo survival was very low: 5% vs. 45% for 25% Gly q 25% Eg, P - 0.05. Survival, hatching rate and mean cell numbers after vitrification in 25% Gly—25% Eg were not significantly different between the two classes of blastocyst size ŽTable 1.. 3.1.2. Experiment 2: Effects of mode of Õitrification and warming and of the composition of the physiological saline component (molar or molal basis) of the Õitrification solution No significant difference was found in the survival and hatching rates or mean cell numbers of blastocysts between the two modes of vitrification Ždirect plunging into nitrogen liquid or 2 min exposure to nitrogen liquid vapours. or of the preparation of the physiological solutions ŽTable 2.. 3.1.3. Experiment 3: Effect of galactose dilution of Õitrified embryos Survival parameters Žre-expansion and hatching rate. and blastocyst cell numbers were not significantly affected by the concentration of galactose used in the dilution medium ŽTable 3.. 3.1.4. Experiment 4: Embryo transfers In the first group of transfers, from the 11 transferred blastocysts produced in VT1 medium, none led to pregnancies but three animals had a delayed return to oestrus Žday 25 to day 27 post-oestrus.. In the second group, one calf is born and two recipients had a few days prolonged cycle.
4. Discussion The in vitro survival of in vitro produced bovine blastocysts after vitrification reported in this study Ž67% re-expansion and 53% hatching. was assessed after 72 h of
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culture post-warming. Similar rates of re-expansion were obtained after 24 or 48 h although the rate of hatching increased from 24 to 72 h ŽFig. 1.. Our mean survival rate is slightly lower than those of Kuwayama et al. Ž1992. Ž87% at 24 h in the best conditions., Dinnyes et al. Ž1995. Ž81% at 48 h., Mahmoudzadeh et al. Ž1995. Ž69 to 89% at 72 h. and Vajta et al. Ž1995. Ž84% at 24 h. who all used different methods for embryo production and vitrification. The variation we observed in the percentage of re-expanding embryos between experiments Ž45 to 86%. and in the mean cell number of blastocysts at 72 h post-warming Ž131 to 176., is likely to be due to variabilities in embryo quality ŽGreve et al., 1993.. Those two parameters are not independent as we found a significant correlation between the rate of embryo survival and the mean number of cells of surviving blastocysts ŽFig. 2.. Blastocysts failed to survive when vitrified in the mixture 10% Gly q 40% Eg. The first mixture Ž25% Gly q 25% Eg. was therefore adopted. Day 7 cattle embryos are more permeable to ethylene glycol than to glycerol ŽSzell ´ et al., 1989.: at high concentration, the intracellular concentration of ethylene glycol increases more rapidly, due to its low molecular weight, and could become toxic. However, higher survival rates have been obtained with 40% ethylene glycol in other studies ŽMahmoudzadeh et al., 1995; Ohboshi et al., 1997; Palasz et al., 1997.. In those studies, however, the vitrification solutions also contained macromolecules and saccharides that reduce intracellular concentration of ethylene glycol at room temperature. To avoid the detrimental effect of high concentrations of ethylene glycol, Vajta et al. Ž1996b. stressed the importance of working at 48C or, when working at room temperature, of exposing the embryos for a very short time only, a precaution adopted in our present studies. We had also carried out the equilibration steps at room temperature since Agca et al. Ž1994. showed that this temperature is more suitable when using a glycerolq ethylene glycol mixture, at least for the second step. Survival rate was not influenced by blastocyst size, whereas higher hatching rates were observed for the more expanded blastocysts. Similar results with vitrified in vitro produced bovine embryos were obtained by Vajta et al. Ž1996b., although it is generally recognised that advanced stage embryos Žexpanded blastocysts. survive cryopreservation better than those frozen earlier ŽPollard and Leibo, 1994; Han et al., 1994; Dinnyes et al., 1995; Leibo et al., 1996.. In our present work, blastocysts were all collected on the same day Žday 7 pi.. Preparation on a molar basis allows to maintain electrolyte isotonicity and it has been suggested that this method is preferable when the cryoprotectant concentration is greater than 4 M ŽMeryman and Douglas, 1982.. Rall Ž1987. recommended the molar basis method and Papis et al. Ž1993. reported that this method gives greater rabbit embryo viability post-vitrification than the molal Žvrv. method. However, in our study the preparation of solutions on a molar or molal basis did not seem to influence the results. Under our conditions, in vitro survival rates and mean cell numbers were similar irrespective of the mode of vitrification. Vitrification in vapour is thought to prevent sample fracture which can damage embryos or the zona pellucida ŽRall and Meyer, 1989; Ali, 1992; Kasai, 1996; Kasai et al., 1996.. Fractures are known to occur with solutions of high concentration cooled ultrarapidly by plunging abruptly into nitrogen liquid. By contrast, a solution of the same concentration rarely fractures when cooled in
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nitrogen vapour for 2 min followed by plunging into nitrogen liquid. This has been attributed to a uniformity of temperature in the sample during cooling on the boat. The recent data of Vajta et al. Ž1997a. indicate that the fracture of the zona pellucida could be due to pressure changes in the solutions induced by rapid shrinkage and expansion of air bubbles during the changes of temperature. Whatever the origin of fractures, the optimal conditions of cooling and warming need to be found for a given vitrification solution ŽKasai et al., 1996.. In our study, both modes Ždirect plunging into nitrogen liquid followed by warming at 378C or cooling for 2 min in nitrogen liquid vapour before plunging into nitrogen liquid followed by warming at 208C. were equally efficient. Survival was not significantly affected by the concentration of the diluent used, though the results were slightly better when the cryoprotectant was diluted in 0.85 M galactose. Similarly, Mahmoudzadeh et al. Ž1995., using various concentrations of sucrose as diluent Ž0 to 0.5 M., did not observe a significant difference on survival rates. We choose galactose as diluent because monosaccharides have much lower viscosity than disaccharides solutions of equivalent osmolalities and can be mixed more efficiently with cryoprotective agents ŽMcWilliams et al., 1995.. Our objective was to simplify the dilution procedure so that transfers could be performed directly after warming. To achieve this, the diluent was loaded into the straw and separated from the cryoprotectant containing the embryo by air bubbles. These bubbles have to be dislodged after warming to allow the contents of the straw to mix. This was performed by expelling the contents into a Petri dish with gentle mixing to mimic direct transfer. Direct in-straw rehydration of vitrified in vitro bovine embryos was reported by Vajta et al. Ž1995, 1996b. to give a good in vitro survival rate Ž) 80%. and various diluents with or without protein additives gave similar results. Direct transfer upon thawing of 40 in vivo day 7 bovine morulae and early blastocysts vitrified by the method of Rall Ž1992. in VS3a, resulted in a 43% pregnancy rate at day 60 ŽVan Wagtendonk-De Leeuw et al., 1995.. With in vitro embryos and the same vitrification procedure, 23% of 85 recipients became pregnant ŽWurth et al., 1994.. In this case, the diluent was 1 M sucrose in PBS. These data demonstrate that, despite high concentrations of cryoprotectants, in-straw dilution and therefore direct transfer are possible, so making the vitrification method attractive, particularly in developing countries. Transfer experiments are necessary to assess embryonic survival, especially for in vitro produced bovine embryos. For example, the number of ICM cells as well as the ratios ICMrtrophectoderm cells can vary without affecting in vitro survival and hatching ŽThompson et al., 1995; Van Soom et al., 1996., resulting in various developmental capacities of the embryos after transfer ŽHolm et al., 1994; Bavister, 1995.. With our production system one calf was obtained out of 22 transfers of vitrified embryos. Our disappointing results are unlikely to be only related to the quality of the embryos produced in our low-phosphate embryo culture system ŽVT1 q 10% FCS., as calves have been born following transfer of fresh Ž2r2. or frozen-thawed Ž5r15. embryos cultured in this medium. Moreover, results were also poor after transfer of blastocysts produced in SOF medium and vitrified with the same procedure Žtwo pregnancies out of 13 transferred embryos; P. Mermillod, personal communication.. The reasons for the low yields of pregnancy obtained with our vitrification procedure for bovine embryos
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are not easy to elucidate due to the possible interactions between culture conditions, vitrification and warming procedures. It should be noted that the same protocol of vitrification was successfully applied to ovine ŽMermillod et al., 1997. and caprine ŽA. de Souza, G. Baril, B. Leboeuf and P. Mermillod, personal communication. in vitro produced embryos with initial pregnancy rates of respectively 54% and 44%. Recent data ŽVajta et al., 1997b. indicate that even an efficient vitrification method causes profound but transient changes to blastocyst ultrastructure. Vitrified embryos can survive such damage but a long term effect on their capacity to develop after transfer cannot be excluded. The impact of subcellular injury may also vary with the culture system used to produce the embryos. Indeed, Agca et al. Ž1994, 1996., using the same vitrification method to the one we have adopted, reported 12 fetuses from 32 transferred embryos obtained in CR1aa. In conclusion, a three-step vitrification procedure using Eg and Gly as cryoprotectants leads to a good in vitro survival of IVP bovine blastocysts irrespective of their size. The in vitro survival of embryos seems not affected by the mode of vitrification, the mode of preparation of the vitrification solutions or by the concentration of galactose used as a diluent. This procedure is simpler than those generally used and could allow for the direct transfer of the embryos. However, the extremely poor number of pregnancies following transfer of those embryos needs further investigation. Our data also confirm the apparent lack of correlation between in vitro survival or hatching and viability after transfer already observed by Van Wagtendonk-De Leeuw et al. Ž1995..
Acknowledgements This research was supported by EU Biotechnology programme ŽBIO2-CT92-0067. and by the ‘Ministere wallonne de Belgique’ ŽMinistre G. ` de l’Agriculture de la Region ´ Lutgen.. We thank M.-A. Mauclet for typing the manuscript and J.-L. Touze´ for technical assistance in animal care and embryo transfers. We would like to thank Dr. H.J. Leese for critical reading of the manuscript.
References Agca, Y., Monson, R.L., Northey, D.L., Abas-Mazni, O., Rutledge, J.J., 1994. Post-thaw survival and pregnancy rates of in vitro produced bovine embryos after vitrification. Theriogenology 41, 154. Agca, Y., Monson, R.L., Northey, D.L., Schaefer, D.M., Rutledge, J.J., 1996. Post-thaw pregnancy rates comparison of vitrified and frozen in vitro produced bovine embryos. Theriogenology 45, 175. Ali, J., 1992. Factors affecting ultrarapid vitrification and cryopreservation of embryos. PhD Thesis, The John Curtin School of Medical Research, Australian National University, Canberra, Australia. Avery, B., Greve, T., 1995. Apparent Žab.normalities of in vitro produced bovine embryos. In: Enne, G., Greppi, G.F., Lauria, A. ŽEds.., Reproduction and Animal Breeding, Advances and Strategy. Elsevier, pp. 171–183. Bavister, B.D., 1995. Culture of preimplantation embryos: facts and artefacts. Human Reprod. Update 1, 91–148. Dagnelie, P., 1986. Les methodes de l’inference statistique. In: Theorie et Methodes Statistiques. Gembloux ´ ´ ´ ´ Agronomic Press, pp. 213–240.
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Delval, A., Ectors, F.J., Touati, K., Beckers, J.F., Ectors, F., 1996. Vitrification of bovine embryos produced in vitro: survival, hatching and pregnancy rates. Theriogenology 45, 178. Dinnyes, A., Carolan, C., Lonergan, P., Solti, L., Massip, A., Mermillod, P., 1995. In vitro survival of in vitro produced ŽIVP. bovine embryos frozen or vitrified by techniques suitable for direct transfer. Theriogenology 43, 197. Donnay, I., Van Langendonckt, A., Auquier, P., Massip, A., Dessy, F., 1996. A new phosphate-free simple medium for culture and co-culture of cattle embryos. J. Reprod. Fertil., Abstract series, 18, Abstract 97. Gordon, I., 1994. Laboratory Production of Cattle Embryos. CAB International, Wallingford. Greve, T., Avery, B., Callesen, H., 1993. Viability of in vivo and in vitro produced embryos. Reprod. Domestic Anim. 28, 164–169. Han, Y.M., Yamashina, H., Koyama, N., Kee, K.K., Fukui, Y., 1994. Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing. Theriogenology 42, 645–654. Holm, P., Smith, S., Callesen, H., 1994. Post-thaw in vitro viability of bovine blastocysts produced in vitro in different media and gas atmospheres. Report of the 10th scientific meeting of the European Embryo Transfer Association, Lyon, September 9 and 10, 182. Holm, P., Vajta, G., Greve, T., Callesen, H., 1996. Pregnancy rates with direct transfer of vitrified and in-straw rehydrated intact and biopsied in vitro produced bovine embryos. Report of the 12th scientific meeting of the European Embryo Transfer Association, Lyon, September 13 and 14, 138. Kaidi, S., Donnay, I., Bralion, V., Massip, A., Dessy, F., 1996. Comparison of two co-culture systems for the assessment of bovine blastocyst survival after cryopreservation. J. Reprod. Fertil., Abstract Series, 18, Abstract 98. Kasai, M., 1996. Simple and efficient methods for vitrification of mammalian embryos. Anim. Reprod. Science 42, 67–75. Kasai, M., Zhu, S.E., Pedro, P.B., Nakamura, K., Sakurai, T., Edashige, K., 1996. Fracture damage of embryos and its prevention during vitrification and warming. Cryobiology 33, 459–464. Kuwayama, M., Hamano, S., Nagai, T., 1992. Vitrification of bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro. J. Reprod. Fertil. 96, 187–193. Leibo, S., Loskutoff, N.M., 1993. Cryobiology of in vitro-derived bovine embryos. Theriogenology 39, 81–94. Leibo, S.P., Martino, A., Kobayashi, S., Pollard, J.W., 1996. Stage dependent sensitivity of oocytes and embryos to low temperature. Anim. Reprod. Science 42, 45–53. Mahmoudzadeh, A.R., Van Soom, A., Ysebaert, M.T., de Kruif, A., 1994. Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos. Theriogenology 42, 1389–1397. Mahmoudzadeh, A.R., Van Soom, A., Bols, P., Ysebaert, M.T., de Kruif, A., 1995. Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of developmental stage, two-step addition of cryoprotectant and sucrose dilution on embryonic survival. J. Reprod. Fertil. 103, 33–39. Massip, A., Van Der Zwalmen, P., Scheffen, B., Ectors, F., 1986. Pregnancies following transfer of cattle embryos preserved by vitrification. Cryo-Letters 7, 270–273. Massip, A., Mermillod, P., Dinnyes, A., 1995. Morphology and biochemistry of in vitro produced bovine embryos: implications for their cryopreservation. Human Reprod. 10, 3004–3011. McWilliams, R.B., Gibbons, W.E., Leibo, S.P., 1995. Osmotic and physiological responses of mouse zygotes and human oocytes to mono- and disaccharides. Human Reprod. 10, 1163–1171. Mermillod, P., Traldi, A.S., Guerin, Y., Poulin, N., Massip, A., Cognie, Y., 1997. Report of the 13th scientific meeting of the European Embryo Transfer Association, Lyon, September 12 and 13, 182. Meryman, H.T., Douglas, M.St.J., 1982. Isotonicity in the presence of penetrating cryoprotectants. Cryobiology 19, 565–569. Niemann, H., 1995. Advances in cryopreservation of bovine oocytes and embryos derived in vitro and in vivo. In: Enne, G., Greppi, G.F., Lauria, A. ŽEds.., Reproduction and Animal Breeding, Advances and Strategy. Elsevier, Amsterdam, pp. 117-128. Ohboshi, S., Etoh, T., Sakamoto, K., Fujihara, N., Yoshida, T., Tomogane, H., 1997. Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro. Theriogenology 47, 1237–1243. Palasz, A.T., Gustafsson, H., Rodriguez-Martinez, H., Gusta, L., Larsson, B., Mapletoft, R.J., 1997. Vitrifica-
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tion of bovine IVF blastocysts in an ethylene glycolrsucrose solution and heat-stable plant-extracted proteins. Theriogenology 47, 865–879. Papis, K., Fujikawa, S., Kojima, T., Oguri, N., 1993. Effect of the composition of vitrification media on survival of rabbit embryos. Cryobiology 30, 98–105. Pollard, J.W., Leibo, S.P., 1994. Chilling sensitivity of mammalian embryos. Theriogenology 41, 101–107. Pursel, V.G., Wall, R.J., Rexroad, C.E., Hammer, R.E., Brinster, R.L., 1985. A rapid whole-mount staining procedure for nuclei of mammalian embryo. Theriogenology 24, 687–691. Rall, W.F., 1987. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24, 387–402. Rall, W.F., 1992. Cryopreservation of oocytes and embryos: methods and applications. Anim. Reprod. Science 28, 237–245. Rall, W.F., Meyer, T.K., 1989. Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos. Theriogenology 31, 683–692. Szell, ´ A., Shelton, J.N., Szell, ´ K., 1989. Osmotic characteristics of sheep and cattle embryos. Cryobiology 26, 297–301. Tachikawa, S., Otoi, T., Kondo, S., Machida, T., Kasai, M., 1993. Successful vitrification of bovine blastocysts derived by in vitro maturation and fertilization. Mol. Reprod. Dev. 34, 266–271. Thompson, J.G., Gardner, D.K., Pugh, P.A., McMillan, W.H., Tervit, H.R., 1995. Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol. Reprod. 53, 1385–1391. Vajta, G., Holm, P., Greve, T., Callesen, H., 1995. Direct in-straw rehydration after thawing of vitrified in vitro produced bovine blastocysts. Vet. Rec. 137, 672. Vajta, G., Holm, P., Greve, T., Callesen, H., 1996a. Overall efficiency of in vitro embryo production and vitrification in cattle. Theriogenology 45, 683–689. Vajta, G., Holm, P., Greve, T., Callesen, H., 1996b. Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration. Anim. Reprod. Science 45, 191–200. Vajta, G., Booth, P.J., Holm, P., Greve, T., Callesen, H., 1997a. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw ŽOPS. method. Cryo-Letters 18, 191–195. Vajta, G., Hyttel, P., Callesen, H., 1997b. Morphological changes of in vitro produced bovine blastocysts after vitrification, in-straw direct rehydration and culture. Mol. Reprod. Dev., in press. Van Soom, A., Boerjan, M., Ysebaert, M.T., De Kruif, A., 1996. Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media. Mol. Reprod. Dev. 45, 171–182. Van Wagtendonk-De Leeuw, A.M., Den Daas, J.H.G., Kruip, T.H.A.M., Rall, W.F., 1995. Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos. Cryobiology 32, 157–167. Wurth, Y.A., Reinders, J.M.C., Rall, W.F., Kruip, T.H.A.M., 1994. Developmental potential of in vitro produced bovine embryos following cryopreservation and single-embryo transfer. Theriogenology 42, 1275–1284.
Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity I. Donnay a , Ph. Auquier a , S. Kaidi a , C. Carolan b, P. Lonergan b, P. Mermillod b, A. Massip a,) a
UniÕersite´ Catholique de LouÕain, Unite´ des Sciences Veterinaires, Place Croix du Sud 3, ´´ B-1348 LouÕain-la-NeuÕe, Belgium b INRA, Station de Physiologie de la Reproduction, F-37380 Nouzilly, France Accepted 4 June 1998
Abstract Methodological studies were undertaken to test the validity of a three-step vitrification procedure for bovine in vitro produced embryos using glycerol and ethylene glycol as cryoprotectants. Embryos were produced in a low-phosphate culture system Žmedium VT1 q 10% foetal calf serum. and vitrified at day 7 post-insemination either in a mixture of 25% glycerol—25% ethylene glycol or a mixture of 10% glycerol—40% ethylene glycol. In the first mixture 67% Ž n s 283. of blastocysts were re-expanded after 72 h of culture and 53% were hatched while in the second one Ž n s 65. only 5% survived. The mean number of cells of the surviving blastocysts was correlated with the rate of survival Ž R 2 s 0.47; P s 0.0024.. Embryo size Ždiameter - or ) to 180 mm. did not influence blastocyst survival or cell number, but hatching rate was higher for embryos ) 180 mm. Embryo survival, hatching rate and cell number 72 h post-warming were not affected by the mode of vitrification Ždirect plunging into nitrogen liquid or vitrification into nitrogen liquid vapour., the mode of preparation of the vitrification solutions Žmolar or molal basis. or by the concentration of galactose used as a diluent Ž0 to 0.85 M.. Only one calf was born after transfer of 22 vitrified blastocysts. These results confirm the apparent lack of correlation for cryopreserved embryos between in vitro survival or hatching and viability after transfer. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cattle-embryology; In vitro embryo production; Vitrification; Cryopreservation
)
Corresponding author: Tel.: q32 10 47 37 56; fax: q32 10 47 37 17; e-mail: [email protected]
0378-4320r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 0 9 8 - 0
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1. Introduction In vitro produced ŽIVP. bovine embryos differ from their in vivo counterparts in many respects: for example, in vitro developmental capacity, morphology, resistance to freezing and survival after transfer Žfor reviews, see Greve et al., 1993; Gordon, 1994; Avery and Greve, 1995; Massip et al., 1995; Van Soom et al., 1996.. The improvement of IVP embryo cryopreservation is an important goal and recent data indicate that rapid, rather than slow cooling, may be beneficial for their survival ŽLeibo and Loskutoff, 1993; Mahmoudzadeh et al., 1994; Pollard and Leibo, 1994; Niemann, 1995.. IVP bovine embryos have been vitrified successfully, in terms of in vitro survival, by most of the methods used for in vivo embryos with or without modifications ŽTachikawa et al., 1993; Mahmoudzadeh et al., 1995; Massip et al., 1995; Vajta et al., 1995, 1996a.. The pregnancy rate after transfer of vitrified in vitro produced embryos is highly variable Ž23 to 50% of the transferred embryos. but the numbers of transfers in the published studies are usually low ŽKuwayama et al., 1992; Tachikawa et al., 1993; Wurth et al., 1994; Agca et al., 1994; Delval et al., 1996; Holm et al., 1996.. Agca et al. Ž1994. adapted the original vitrification method of Massip et al. Ž1986. to in vitro embryos and obtained pregnancies after transfer. They used a three-step equilibration procedure with glycerol and ethylene glycol as cryoprotectants and sucrose as diluent. They stressed the importance of time and temperature of equilibration in the previtrification solutions on in vitro survival and demonstrated that the best conditions were achieved when embryos were equilibrated for 5 min at 278C during the second step. We have used their new procedure, but with galactose as diluent instead of sucrose, to examine the effect of a number of variables on the outcome of vitrification: Ži. composition of the vitrification solution; Žii. blastocyst size; Žiii. preparation of the solutions on a molar vs. molal basis; Živ. mode of vitrification Žnitrogen liquid vapour vs. direct plunging into nitrogen liquid.; Žv. dilution medium after warming. Re-expansion and hatching rates of the vitrifiedrwarmed embryos were assessed during 72 h of co-culture on Buffalo Rat Liver cells, as well as the cell number of the surviving embryos. Transfers into recipient heifers were also performed to assess the viability of the vitrified embryos.
2. Materials and methods 2.1. Source of embryos Embryos were generated from abattoir ovaries. Oocytes were collected by aspiration of 3–6 mm follicles and washed four times in modified Dulbecco’s phosphate buffered saline ŽPBS. containing 0.3 mM sodium pyruvate ŽSigma, St. Louis, MO, USA., 0.4 mg bovine serum albumin mly1 ŽFraction V; Sigma. and 50 mg mly1 gentamicin ŽGibco, Paisley, UK.. Groups of 100 randomly selected oocytes were allocated to four well culture plates ŽNunc, Roskilde, Denmark. containing 500 ml maturation medium ŽTCM 199, Sigma M-4530. supplemented with 10% heat treated foetal calf serum ŽFCS-JRH Biosciences, Lenaxa, KS, USA. and 10 ng mly1 epidermal growth factor ŽSigma.. After
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24 h maturation at 398C under 5% CO 2 in a humidified atmosphere, oocytes were washed four times in PBS and transferred to 500 ml fertilization medium ŽTyrode’s medium with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mly1 fatty acid-free albumin fraction ŽSigma., 10 mg mly1 heparin–sodium salt w167 U mgy1 ; Calbiochem, San Diego, CA, USAx. and 50 mg mly1 gentamicin. Thawed semen was centrifuged on a Percoll ŽPharmacia, Uppsala, Sweden. discontinuous density gradient Ž45% and 90%. for 30 min at 700 = g. Spermatozoa were added to each well to a final concentration of 2 = 10 6 mly1 . Oocytes and spermatozoa were incubated together for 18 h. The same ejaculate from one bull was used throughout the experiments. Presumptive zygotes were vortexed for 2 min in PBS to remove cumulus cells, washed three times in PBS and twice in culture medium. Denuded zygotes were cultured in groups of about 30 in 30 ml droplets of medium VT1 ŽDonnay et al., 1996. under paraffin oil and 5% O 2 , 5% CO 2 , 90% N2 at 398C in humidified air. The composition of VT1 medium was 114 mM NaCl, 7.16 mM KCl, 25.07 mM NaHCO 3 , 0.49 mM MgCl 2 , 0.3 mM Na-pyruvate, 2.5 mM Ca-lactate, 1 mM glutamine, 3 mg mly1 BSA, 20 ml ly1 essential amino acids mixture ŽSigma. and 10 ml ly1 non-essential amino acids ŽSigma.. On day 2 post-insemination Žpi., 10% FCS was added. Blastocysts were collected on day 7 pi. 2.2. Vitrification procedure On day 7 pi, morphologically normal blastocysts according to the I.E.T.S. standard classification Žfrom small to expanded. were retrieved and pooled in a Petri dish containing either HEPES buffered culture medium ŽVT1 q 10% FCS q 5.96 g ly1 HEPES; Sigma. or embryo transfer freezing medium ŽGibco. with 20% FCS and no BSA ŽETF.. Those media were also used for the dilution of the cryoprotectants. Samples of 10 embryos were treated at a time, the remainder being held in the incubator. Unless otherwise indicated, treatment consisted in a three-step equilibration process at room temperature Ž25–328C.: step 1: 5 min in 10% glycerol Žvrv; AnalaR, Poole, UK.; step 2: 5 min in a mixture of 10% glycerol ŽGly. q 20% ethylene glycol ŽEg. ŽAnalaR.; and step 3: 30 s in a vitrification mixture of 25% Gly q 25% Eg. The blastocysts were equilibrated for 5 min in step 1; 5 min in step 2, then placed, for step 3, five at a time, in small drops of the vitrification solution, and aspirated, within 30 s, into 0.25 ml French straws ŽIMV, L’Aigle, France. containing 0.85 M d-galactose ŽSigma. dissolved in ETF medium. The straws were then plunged immediately into nitrogen liquid, unless otherwise indicated. 2.3. RecoÕery of embryos and assessment of surÕiÕal After storage from a few days to a few weeks, the straws were warmed for 10 s in water at 378C, unless otherwise indicated. The contents of each straw were expelled into a Petri dish and gently agitated to mix galactose with cryoprotectant. After 5 min, the embryos were transferred to holding medium for 5 min then washed in 199 mediumq 10% FCS and co-cultured in 50 ml drops of this medium on a confluent monolayer of Buffalo Rat Liver cells ŽBRL. at 398C in a humidified atmosphere of 5% CO 2 in air
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ŽKaidi et al., 1996.. The BRL cells ŽBRL 3A. were stored at y808C in TCM199 supplemented with 10% Žvrv. FCS and 10% Žvrv. DMSO ŽBDH.. They were thawed in a 378C water bath for 1 min and washed in 10 ml of TCM 199 q FCS. Fifty ml drops of cell suspension under paraffin oil containing 1 = 10 5 cells mly1 were prepared and cultured until confluency under 5% CO 2 in air. Confluency was achieved after 2 days. Embryo survival was defined as the percentage of vitrified embryos that had re-expanded after 72 h of culture but data were also available at 24 and 48 h. At the end of this period, expanded and hatched blastocysts were fixed and stained with Hoechst 33342 ŽCalbiochem. for determination of total cell number ŽPursel et al., 1985.. 2.4. Experimental design Each experiment was replicated three times. 2.4.1. Experiment 1: Effect of blastocyst size and composition of Õitrification solution Blastocysts recovered on day 7 pi were arbitrarily separated in two groups: - 180 mm and ) 180 mm diameter and vitrified as described above. The vitrification solutions were 25% Gly q 25% Eg or 10% Gly q 40% Eg, the aim being to test the effect of increasing Eg concentration, the Gly concentration remaining constant at 10%. Steps 1 and 2 and treatment after warming were identical in each case. 2.4.2. Experiment 2: Effects of mode of Õitrification and warming and of the composition of the physiological saline component (molar or molal basis) of the Õitrification solution Two modes of vitrification were tested: in the first one, the straws were placed for 2 min into nitrogen liquid vapour on a styrofoam boat then plunged into nitrogen liquid and warmed in water at 208C. In the second one, straws were plunged slowly into nitrogen liquid then warmed in water at 378C. This experiment also compared two ways of preparing the cryoprotectant solutions. In the first one Žmolal basis., cryoprotectants were diluted as in the other experiments in ETF medium with 20% FCS on a vrv basis; in the second one Žmolar basis., the salts concentrations in the culture medium ŽHEPES VT1. used as a diluent were adapted to maintain their initial concentration in the solution of cryoprotectants throughout the three-step equilibration process. 2.4.3. Experiment 3: Effect of galactose dilution of Õitrified embryos Straws were loaded in three ways. As previously described, air bubbles were positioned on each side of the cryoprotectant solution containing the embryos. In the first case, 0.85 M galactose was present on both sides of the bubbles, in the second case, holding medium was on one side with 0.85 M galactose on the other, and in the third case, holding medium was present on both sides. Dilution was carried out as described in the vitrification protocol. 2.4.4. Experiment 4: Embryo transfers After warming and dilution, embryos were transferred singly to day 7 recipients synchronized with a Norgestomet ear implant ŽCrestar, Intervet, France.. Recipients were Holstein or crossbreed Holstein heifers from the experimental herd at the INRA
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Fig. 1. Evolution of the rates of expansion and hatching after warming.
Fig. 2. Correlation between embryo survival Žrate of re-expansion after 72 h. and mean number of cells.
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Table 1 Effect of the size of day 7 in vitro blastocysts on in vitro survival and total cell number after 72 h of co-culture with BRL cells Blastocyst diameter
-180 mm )180 mm a
Results 72 h post-warming a N
Re-expanded mean %"SD
Hatched mean %"SD
Mean cell number"SEM
30 32
55"20 36"33
42"21b 100"33 c
137"21 Ž ns9. 150"18 Ž ns12.
Results of three replicates. Values with different superscripts within the same column are different ŽTwo-way ANOVA, P - 0.05..
bc
Physiology Station. Transfers were non-surgical and always carried out by the same individual. In a first series, 11 vitrified embryos produced in VT1 were transferred in autumn 1996 at the INRA station; a second series of 11 were transferred in spring and autumn 1997 on recipients of our herd on natural heat. 2.4.5. Statistical analysis One- or Two-way analysis of variance ŽANOVA. were performed to analyse the effect of treatments on all parameters. When data were proportions, an arcsinus square-root transformation was applied before analysis ŽDagnelie, 1986..
3. Results 3.1. Embryo production From a total of 12 replicate experiments, 3466 oocytes were fertilized and cultured for 6 days. On day 7 pi, the peak time of blastocyst appearance, 741 Ž21%. blastocysts were recovered and 536 Ž72%. were selected, of these 348 were used for methodological studies, and the remainder for transfers and other programmes. In the experiments using 25% Gly q 25% Eg, on average 67% Žout of 283. of blastocysts of all sizes survived the vitrification and 53% were hatched 72 h post-warming. The percentage of survival
Table 2 Effect of the mode of preparation of the cryoprotectant solutions and of the mode of vitrification on in vitro survival and mean cell number of in vitro day 7 bovine embryos after vitrification Preparation of the solutions
Mode of vitrification
Molal basis
Direct plunging in LN2 LN2 vapour Direct plunging in LN2 LN2 vapour
Molar basis
)
Results 72 h post-warming ) N Re-expansion Hatching mean %"SD mean %"SD
Mean cell number"SEM
29 30 29 29
154"21 Ž ns 23. 169"21 Ž ns 22. 191"20 Ž ns 24. 190"21 Ž ns 22.
90"10 83"5 93"8 87"11
72"20 56"10 80"20 80"20
Results are from three replicates. No significant difference was observed ŽTwo-way ANOVA, P ) 0.05..
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Table 3 Effect of the type and concentration of the diluent after warming on in vitro survival and mean cell number of vitrified day 7 in vitro bovine blastocysts ) Diluent
0.85 M galactose 0.42 M galactose Culture medium )
Results 72 h post-warming N
Re-expansion mean %"SD
Hatching mean %"SD
Mean cell number"SEM
34 35 35
73"14 55"10 53"19
63"16 34"8 44"14
127"17 Ž ns 21. 125"19 Ž ns18. 142"19 Ž ns17.
Results are from three replicates. No significant difference was observed ŽTwo-way ANOVA, P ) 0.05..
Žre-expansion. did not vary between 24, 48 or 72 h post-warming although the rate of hatching increased ŽFig. 1.. A significant correlation was found between the rate of embryo survival and the mean cell number of the surviving blastocysts 72 h post-warming Ž R 2 s 0.47, P s 0.0024, Fig. 2.. 3.1.1. Experiment 1: Effect of blastocyst size and composition of Õitrification solution With 10% Gly q 40% Eg Ž n s 65., embryo survival was very low: 5% vs. 45% for 25% Gly q 25% Eg, P - 0.05. Survival, hatching rate and mean cell numbers after vitrification in 25% Gly—25% Eg were not significantly different between the two classes of blastocyst size ŽTable 1.. 3.1.2. Experiment 2: Effects of mode of Õitrification and warming and of the composition of the physiological saline component (molar or molal basis) of the Õitrification solution No significant difference was found in the survival and hatching rates or mean cell numbers of blastocysts between the two modes of vitrification Ždirect plunging into nitrogen liquid or 2 min exposure to nitrogen liquid vapours. or of the preparation of the physiological solutions ŽTable 2.. 3.1.3. Experiment 3: Effect of galactose dilution of Õitrified embryos Survival parameters Žre-expansion and hatching rate. and blastocyst cell numbers were not significantly affected by the concentration of galactose used in the dilution medium ŽTable 3.. 3.1.4. Experiment 4: Embryo transfers In the first group of transfers, from the 11 transferred blastocysts produced in VT1 medium, none led to pregnancies but three animals had a delayed return to oestrus Žday 25 to day 27 post-oestrus.. In the second group, one calf is born and two recipients had a few days prolonged cycle.
4. Discussion The in vitro survival of in vitro produced bovine blastocysts after vitrification reported in this study Ž67% re-expansion and 53% hatching. was assessed after 72 h of
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culture post-warming. Similar rates of re-expansion were obtained after 24 or 48 h although the rate of hatching increased from 24 to 72 h ŽFig. 1.. Our mean survival rate is slightly lower than those of Kuwayama et al. Ž1992. Ž87% at 24 h in the best conditions., Dinnyes et al. Ž1995. Ž81% at 48 h., Mahmoudzadeh et al. Ž1995. Ž69 to 89% at 72 h. and Vajta et al. Ž1995. Ž84% at 24 h. who all used different methods for embryo production and vitrification. The variation we observed in the percentage of re-expanding embryos between experiments Ž45 to 86%. and in the mean cell number of blastocysts at 72 h post-warming Ž131 to 176., is likely to be due to variabilities in embryo quality ŽGreve et al., 1993.. Those two parameters are not independent as we found a significant correlation between the rate of embryo survival and the mean number of cells of surviving blastocysts ŽFig. 2.. Blastocysts failed to survive when vitrified in the mixture 10% Gly q 40% Eg. The first mixture Ž25% Gly q 25% Eg. was therefore adopted. Day 7 cattle embryos are more permeable to ethylene glycol than to glycerol ŽSzell ´ et al., 1989.: at high concentration, the intracellular concentration of ethylene glycol increases more rapidly, due to its low molecular weight, and could become toxic. However, higher survival rates have been obtained with 40% ethylene glycol in other studies ŽMahmoudzadeh et al., 1995; Ohboshi et al., 1997; Palasz et al., 1997.. In those studies, however, the vitrification solutions also contained macromolecules and saccharides that reduce intracellular concentration of ethylene glycol at room temperature. To avoid the detrimental effect of high concentrations of ethylene glycol, Vajta et al. Ž1996b. stressed the importance of working at 48C or, when working at room temperature, of exposing the embryos for a very short time only, a precaution adopted in our present studies. We had also carried out the equilibration steps at room temperature since Agca et al. Ž1994. showed that this temperature is more suitable when using a glycerolq ethylene glycol mixture, at least for the second step. Survival rate was not influenced by blastocyst size, whereas higher hatching rates were observed for the more expanded blastocysts. Similar results with vitrified in vitro produced bovine embryos were obtained by Vajta et al. Ž1996b., although it is generally recognised that advanced stage embryos Žexpanded blastocysts. survive cryopreservation better than those frozen earlier ŽPollard and Leibo, 1994; Han et al., 1994; Dinnyes et al., 1995; Leibo et al., 1996.. In our present work, blastocysts were all collected on the same day Žday 7 pi.. Preparation on a molar basis allows to maintain electrolyte isotonicity and it has been suggested that this method is preferable when the cryoprotectant concentration is greater than 4 M ŽMeryman and Douglas, 1982.. Rall Ž1987. recommended the molar basis method and Papis et al. Ž1993. reported that this method gives greater rabbit embryo viability post-vitrification than the molal Žvrv. method. However, in our study the preparation of solutions on a molar or molal basis did not seem to influence the results. Under our conditions, in vitro survival rates and mean cell numbers were similar irrespective of the mode of vitrification. Vitrification in vapour is thought to prevent sample fracture which can damage embryos or the zona pellucida ŽRall and Meyer, 1989; Ali, 1992; Kasai, 1996; Kasai et al., 1996.. Fractures are known to occur with solutions of high concentration cooled ultrarapidly by plunging abruptly into nitrogen liquid. By contrast, a solution of the same concentration rarely fractures when cooled in
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nitrogen vapour for 2 min followed by plunging into nitrogen liquid. This has been attributed to a uniformity of temperature in the sample during cooling on the boat. The recent data of Vajta et al. Ž1997a. indicate that the fracture of the zona pellucida could be due to pressure changes in the solutions induced by rapid shrinkage and expansion of air bubbles during the changes of temperature. Whatever the origin of fractures, the optimal conditions of cooling and warming need to be found for a given vitrification solution ŽKasai et al., 1996.. In our study, both modes Ždirect plunging into nitrogen liquid followed by warming at 378C or cooling for 2 min in nitrogen liquid vapour before plunging into nitrogen liquid followed by warming at 208C. were equally efficient. Survival was not significantly affected by the concentration of the diluent used, though the results were slightly better when the cryoprotectant was diluted in 0.85 M galactose. Similarly, Mahmoudzadeh et al. Ž1995., using various concentrations of sucrose as diluent Ž0 to 0.5 M., did not observe a significant difference on survival rates. We choose galactose as diluent because monosaccharides have much lower viscosity than disaccharides solutions of equivalent osmolalities and can be mixed more efficiently with cryoprotective agents ŽMcWilliams et al., 1995.. Our objective was to simplify the dilution procedure so that transfers could be performed directly after warming. To achieve this, the diluent was loaded into the straw and separated from the cryoprotectant containing the embryo by air bubbles. These bubbles have to be dislodged after warming to allow the contents of the straw to mix. This was performed by expelling the contents into a Petri dish with gentle mixing to mimic direct transfer. Direct in-straw rehydration of vitrified in vitro bovine embryos was reported by Vajta et al. Ž1995, 1996b. to give a good in vitro survival rate Ž) 80%. and various diluents with or without protein additives gave similar results. Direct transfer upon thawing of 40 in vivo day 7 bovine morulae and early blastocysts vitrified by the method of Rall Ž1992. in VS3a, resulted in a 43% pregnancy rate at day 60 ŽVan Wagtendonk-De Leeuw et al., 1995.. With in vitro embryos and the same vitrification procedure, 23% of 85 recipients became pregnant ŽWurth et al., 1994.. In this case, the diluent was 1 M sucrose in PBS. These data demonstrate that, despite high concentrations of cryoprotectants, in-straw dilution and therefore direct transfer are possible, so making the vitrification method attractive, particularly in developing countries. Transfer experiments are necessary to assess embryonic survival, especially for in vitro produced bovine embryos. For example, the number of ICM cells as well as the ratios ICMrtrophectoderm cells can vary without affecting in vitro survival and hatching ŽThompson et al., 1995; Van Soom et al., 1996., resulting in various developmental capacities of the embryos after transfer ŽHolm et al., 1994; Bavister, 1995.. With our production system one calf was obtained out of 22 transfers of vitrified embryos. Our disappointing results are unlikely to be only related to the quality of the embryos produced in our low-phosphate embryo culture system ŽVT1 q 10% FCS., as calves have been born following transfer of fresh Ž2r2. or frozen-thawed Ž5r15. embryos cultured in this medium. Moreover, results were also poor after transfer of blastocysts produced in SOF medium and vitrified with the same procedure Žtwo pregnancies out of 13 transferred embryos; P. Mermillod, personal communication.. The reasons for the low yields of pregnancy obtained with our vitrification procedure for bovine embryos
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are not easy to elucidate due to the possible interactions between culture conditions, vitrification and warming procedures. It should be noted that the same protocol of vitrification was successfully applied to ovine ŽMermillod et al., 1997. and caprine ŽA. de Souza, G. Baril, B. Leboeuf and P. Mermillod, personal communication. in vitro produced embryos with initial pregnancy rates of respectively 54% and 44%. Recent data ŽVajta et al., 1997b. indicate that even an efficient vitrification method causes profound but transient changes to blastocyst ultrastructure. Vitrified embryos can survive such damage but a long term effect on their capacity to develop after transfer cannot be excluded. The impact of subcellular injury may also vary with the culture system used to produce the embryos. Indeed, Agca et al. Ž1994, 1996., using the same vitrification method to the one we have adopted, reported 12 fetuses from 32 transferred embryos obtained in CR1aa. In conclusion, a three-step vitrification procedure using Eg and Gly as cryoprotectants leads to a good in vitro survival of IVP bovine blastocysts irrespective of their size. The in vitro survival of embryos seems not affected by the mode of vitrification, the mode of preparation of the vitrification solutions or by the concentration of galactose used as a diluent. This procedure is simpler than those generally used and could allow for the direct transfer of the embryos. However, the extremely poor number of pregnancies following transfer of those embryos needs further investigation. Our data also confirm the apparent lack of correlation between in vitro survival or hatching and viability after transfer already observed by Van Wagtendonk-De Leeuw et al. Ž1995..
Acknowledgements This research was supported by EU Biotechnology programme ŽBIO2-CT92-0067. and by the ‘Ministere wallonne de Belgique’ ŽMinistre G. ` de l’Agriculture de la Region ´ Lutgen.. We thank M.-A. Mauclet for typing the manuscript and J.-L. Touze´ for technical assistance in animal care and embryo transfers. We would like to thank Dr. H.J. Leese for critical reading of the manuscript.
References Agca, Y., Monson, R.L., Northey, D.L., Abas-Mazni, O., Rutledge, J.J., 1994. Post-thaw survival and pregnancy rates of in vitro produced bovine embryos after vitrification. Theriogenology 41, 154. Agca, Y., Monson, R.L., Northey, D.L., Schaefer, D.M., Rutledge, J.J., 1996. Post-thaw pregnancy rates comparison of vitrified and frozen in vitro produced bovine embryos. Theriogenology 45, 175. Ali, J., 1992. Factors affecting ultrarapid vitrification and cryopreservation of embryos. PhD Thesis, The John Curtin School of Medical Research, Australian National University, Canberra, Australia. Avery, B., Greve, T., 1995. Apparent Žab.normalities of in vitro produced bovine embryos. In: Enne, G., Greppi, G.F., Lauria, A. ŽEds.., Reproduction and Animal Breeding, Advances and Strategy. Elsevier, pp. 171–183. Bavister, B.D., 1995. Culture of preimplantation embryos: facts and artefacts. Human Reprod. Update 1, 91–148. Dagnelie, P., 1986. Les methodes de l’inference statistique. In: Theorie et Methodes Statistiques. Gembloux ´ ´ ´ ´ Agronomic Press, pp. 213–240.
I. Donnay et al.r Animal Reproduction Science 52 (1998) 93–104
103
Delval, A., Ectors, F.J., Touati, K., Beckers, J.F., Ectors, F., 1996. Vitrification of bovine embryos produced in vitro: survival, hatching and pregnancy rates. Theriogenology 45, 178. Dinnyes, A., Carolan, C., Lonergan, P., Solti, L., Massip, A., Mermillod, P., 1995. In vitro survival of in vitro produced ŽIVP. bovine embryos frozen or vitrified by techniques suitable for direct transfer. Theriogenology 43, 197. Donnay, I., Van Langendonckt, A., Auquier, P., Massip, A., Dessy, F., 1996. A new phosphate-free simple medium for culture and co-culture of cattle embryos. J. Reprod. Fertil., Abstract series, 18, Abstract 97. Gordon, I., 1994. Laboratory Production of Cattle Embryos. CAB International, Wallingford. Greve, T., Avery, B., Callesen, H., 1993. Viability of in vivo and in vitro produced embryos. Reprod. Domestic Anim. 28, 164–169. Han, Y.M., Yamashina, H., Koyama, N., Kee, K.K., Fukui, Y., 1994. Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing. Theriogenology 42, 645–654. Holm, P., Smith, S., Callesen, H., 1994. Post-thaw in vitro viability of bovine blastocysts produced in vitro in different media and gas atmospheres. Report of the 10th scientific meeting of the European Embryo Transfer Association, Lyon, September 9 and 10, 182. Holm, P., Vajta, G., Greve, T., Callesen, H., 1996. Pregnancy rates with direct transfer of vitrified and in-straw rehydrated intact and biopsied in vitro produced bovine embryos. Report of the 12th scientific meeting of the European Embryo Transfer Association, Lyon, September 13 and 14, 138. Kaidi, S., Donnay, I., Bralion, V., Massip, A., Dessy, F., 1996. Comparison of two co-culture systems for the assessment of bovine blastocyst survival after cryopreservation. J. Reprod. Fertil., Abstract Series, 18, Abstract 98. Kasai, M., 1996. Simple and efficient methods for vitrification of mammalian embryos. Anim. Reprod. Science 42, 67–75. Kasai, M., Zhu, S.E., Pedro, P.B., Nakamura, K., Sakurai, T., Edashige, K., 1996. Fracture damage of embryos and its prevention during vitrification and warming. Cryobiology 33, 459–464. Kuwayama, M., Hamano, S., Nagai, T., 1992. Vitrification of bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro. J. Reprod. Fertil. 96, 187–193. Leibo, S., Loskutoff, N.M., 1993. Cryobiology of in vitro-derived bovine embryos. Theriogenology 39, 81–94. Leibo, S.P., Martino, A., Kobayashi, S., Pollard, J.W., 1996. Stage dependent sensitivity of oocytes and embryos to low temperature. Anim. Reprod. Science 42, 45–53. Mahmoudzadeh, A.R., Van Soom, A., Ysebaert, M.T., de Kruif, A., 1994. Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos. Theriogenology 42, 1389–1397. Mahmoudzadeh, A.R., Van Soom, A., Bols, P., Ysebaert, M.T., de Kruif, A., 1995. Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of developmental stage, two-step addition of cryoprotectant and sucrose dilution on embryonic survival. J. Reprod. Fertil. 103, 33–39. Massip, A., Van Der Zwalmen, P., Scheffen, B., Ectors, F., 1986. Pregnancies following transfer of cattle embryos preserved by vitrification. Cryo-Letters 7, 270–273. Massip, A., Mermillod, P., Dinnyes, A., 1995. Morphology and biochemistry of in vitro produced bovine embryos: implications for their cryopreservation. Human Reprod. 10, 3004–3011. McWilliams, R.B., Gibbons, W.E., Leibo, S.P., 1995. Osmotic and physiological responses of mouse zygotes and human oocytes to mono- and disaccharides. Human Reprod. 10, 1163–1171. Mermillod, P., Traldi, A.S., Guerin, Y., Poulin, N., Massip, A., Cognie, Y., 1997. Report of the 13th scientific meeting of the European Embryo Transfer Association, Lyon, September 12 and 13, 182. Meryman, H.T., Douglas, M.St.J., 1982. Isotonicity in the presence of penetrating cryoprotectants. Cryobiology 19, 565–569. Niemann, H., 1995. Advances in cryopreservation of bovine oocytes and embryos derived in vitro and in vivo. In: Enne, G., Greppi, G.F., Lauria, A. ŽEds.., Reproduction and Animal Breeding, Advances and Strategy. Elsevier, Amsterdam, pp. 117-128. Ohboshi, S., Etoh, T., Sakamoto, K., Fujihara, N., Yoshida, T., Tomogane, H., 1997. Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro. Theriogenology 47, 1237–1243. Palasz, A.T., Gustafsson, H., Rodriguez-Martinez, H., Gusta, L., Larsson, B., Mapletoft, R.J., 1997. Vitrifica-
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I. Donnay et al.r Animal Reproduction Science 52 (1998) 93–104
tion of bovine IVF blastocysts in an ethylene glycolrsucrose solution and heat-stable plant-extracted proteins. Theriogenology 47, 865–879. Papis, K., Fujikawa, S., Kojima, T., Oguri, N., 1993. Effect of the composition of vitrification media on survival of rabbit embryos. Cryobiology 30, 98–105. Pollard, J.W., Leibo, S.P., 1994. Chilling sensitivity of mammalian embryos. Theriogenology 41, 101–107. Pursel, V.G., Wall, R.J., Rexroad, C.E., Hammer, R.E., Brinster, R.L., 1985. A rapid whole-mount staining procedure for nuclei of mammalian embryo. Theriogenology 24, 687–691. Rall, W.F., 1987. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24, 387–402. Rall, W.F., 1992. Cryopreservation of oocytes and embryos: methods and applications. Anim. Reprod. Science 28, 237–245. Rall, W.F., Meyer, T.K., 1989. Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos. Theriogenology 31, 683–692. Szell, ´ A., Shelton, J.N., Szell, ´ K., 1989. Osmotic characteristics of sheep and cattle embryos. Cryobiology 26, 297–301. Tachikawa, S., Otoi, T., Kondo, S., Machida, T., Kasai, M., 1993. Successful vitrification of bovine blastocysts derived by in vitro maturation and fertilization. Mol. Reprod. Dev. 34, 266–271. Thompson, J.G., Gardner, D.K., Pugh, P.A., McMillan, W.H., Tervit, H.R., 1995. Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol. Reprod. 53, 1385–1391. Vajta, G., Holm, P., Greve, T., Callesen, H., 1995. Direct in-straw rehydration after thawing of vitrified in vitro produced bovine blastocysts. Vet. Rec. 137, 672. Vajta, G., Holm, P., Greve, T., Callesen, H., 1996a. Overall efficiency of in vitro embryo production and vitrification in cattle. Theriogenology 45, 683–689. Vajta, G., Holm, P., Greve, T., Callesen, H., 1996b. Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration. Anim. Reprod. Science 45, 191–200. Vajta, G., Booth, P.J., Holm, P., Greve, T., Callesen, H., 1997a. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw ŽOPS. method. Cryo-Letters 18, 191–195. Vajta, G., Hyttel, P., Callesen, H., 1997b. Morphological changes of in vitro produced bovine blastocysts after vitrification, in-straw direct rehydration and culture. Mol. Reprod. Dev., in press. Van Soom, A., Boerjan, M., Ysebaert, M.T., De Kruif, A., 1996. Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media. Mol. Reprod. Dev. 45, 171–182. Van Wagtendonk-De Leeuw, A.M., Den Daas, J.H.G., Kruip, T.H.A.M., Rall, W.F., 1995. Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos. Cryobiology 32, 157–167. Wurth, Y.A., Reinders, J.M.C., Rall, W.F., Kruip, T.H.A.M., 1994. Developmental potential of in vitro produced bovine embryos following cryopreservation and single-embryo transfer. Theriogenology 42, 1275–1284.