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Culture of early stage ovine embryos to blastocyst enhances survival rate after cryopreservation
Theriogenology 63 (2005) 2233–2242 www.journals.elsevierhealth.com/periodicals/the
Culture of early stage ovine embryos to blastocyst enhances survival rate after cryopreservation R.M. Garcia-Garcia*, A. Gonzalez-Bulnes, V. Dominguez, A. Veiga-Lopez, M.J. Cocero Departamento de Reproduccio´n Animal INIA, Avda Puerta de Hierro s/n, 28040 Madrid, Spain Received 1 July 2004; received in revised form 17 September 2004; accepted 7 October 2004
Abstract The current study assessed both the effects of in vitro culture and developmental stage of early stage in vivo produced ovine embryos on their ability to survive cryopreservation. Early stage embryos (n = 226) were recovered from the oviduct, at different days of the early luteal phase, at three different developmental stages: 2- to 4-cell, 5- to 8-cell and 9- to 12-cell. For each stage, half of the embryos were cultured to the blastocyst stage and frozen thereafter (CF), while the remainder was frozen just after recovery (EF). A third experimental group (BF; n = 43) included blastocysts obtained from the uterus and frozen immediately after recovery. Embryo viability post-thawing was determined by assessing their rate of development to the hatched blastocyst stage following in vitro culture. Culture negatively affected embryo viability, since survival rate was higher in blastocysts obtained from the uterus than in those from culture (83.7% versus 66.1%; P < 0.05); also the cryosurvival of cultured embryos was lower when the timing of blastocyst formation was extended (P < 0.01). However, survival following freezing–thawing of early developmental stages was significantly lower when compared to viability of their counterparts cultured to the blastocyst stage (23.1% versus 66.1%, P < 0.0001). In conclusion, our results indicate that, despite the deleterious effects of culture per se, the culture of early in vivo produced ovine embryos to the blastocyst stage prior to be frozen improves their survival after thawing. # 2004 Elsevier Inc. All rights reserved. Keywords: Cryosurvival; Developmental stage; Embryo culture; Freezing–thawing; Sheep
* Corresponding author. Present address: Departamento de Fisiologia Animal. UCM. Avda Puerta de Hierro s/n, 28040 Madrid, Spain. Tel.: +34 91 3943858; fax: +34 91 3943864. E-mail address: [email protected] (R.M. Garcia-Garcia). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.10.006
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1. Introduction In spite of the efforts towards the improvement of the techniques of in vitro embryo production (IVP), embryo developmental rates are still far from ideal [1,2], and embryos show poor quality and low cryotolerance [3,4]. Recently, it has been reported that cryosurvival of bovine blastocysts is influenced by the in vitro culture system in which they are produced [5,6], even when zygotes derived from oocytes of high developmental potential (e.g., in vivo matured/fertilized) are used [7]. However, there is a lack of conclusive data about effects of culture on embryo cryotolerance in most mammalian species [8]. Studies carried out in laboratory animals have led to conflicting results, with no effect [9] or deleterious effects [10] following culture periods of long duration [11]. Similarly, in the bovine, a low resistance to freezing–thawing procedures after in vitro culture of both in vivo derived zygotes [7] and morulae and blastocysts cultured for 24 h [12] has been reported. Thus, cryopreservation of embryos at an early stage of development would limit possible negative effects of in vitro culture [13], increasing developmental rates and alleviating logistical problems in other reproductive techniques, such as cloning [14,15]. Studies relating to the cryotolerance of early stage embryos are scarce, but the results obtained in cattle were low [15,16]. However, as chilling sensitivity is species-dependent [17], cryopreservation methods must be adapted for each species [18]. In the present study, sheep embryos were in vivo fertilized and recovered from the oviduct to avoid the influence of potentially suboptimal in vitro maturation and/or fertilization [7]. The objective was to determine the influence of in vitro culture and embryo developmental stage on the cryotolerance of in vivo derived ovine embryos. This would lead, in practice, to the development of better strategies for improving embryo cryosurvival.
2. Materials and methods 2.1. Study design A total of 269 in vivo produced ovine embryos were used to assess the possible influence of culture and developmental stage on survival after freezing–thawing procedures. Early stage embryos classified as good or excellent after flushing, on the basis of their morphology and synchrony between their stage and recovery time, were distributed in three experimental groups according to the stage of development: 2- to 4cell (n = 76), 5- to 8-cell (n = 73) and 9- to 12-cell (n = 77). For each stage, half of the embryos were randomly allocated in two groups. In the first, embryos were cultured in vitro until the blastocyst stage and frozen thereafter (group CF), while in the second group, early stage embryos were frozen just after recovery (EF). Additionally, a third group of blastocysts obtained in vivo from superovulated ewes were frozen immediately after recovery (BF), acting as controls (n = 43). After freezing–thawing, embryo viability was evaluated by assessing attainment of the blastocyst to the hatched blastocyst stage [19]. In agreement with previous studies [20,21], only blastocysts
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hatching from the zona pellucida within a maximum period of 3 days after thawing were classified as viable. Embryo quality was also assessed by counting the number of cells in the hatched blastocysts [22] after staining, and by the kinetics of appearance of hatched blastocysts post-thawing. Effect of culture on embryo cryosurvival was studied by assessment of hatching rates of embryos from groups CF and BF, whilst influence of embryo developmental stage was determined by comparison of hatching blastocyst rate from early stage embryos (EF) versus their counterparts cultured to the blastocyst stage (CF). 2.2. Embryo recovery The embryos were recovered from 4 to 7 years old Manchega ewes, maintained outdoors with access to indoor facilities, at the experimental farm of the INIA Animal Reproduction Department in Madrid, Spain, at a latitude of 408N. They were superovulated with the protocols commonly used in our laboratory [23]. Briefly, estrus was synchronized with the insertion of one intravaginal progestagen impregnated sponge (40 mg fluorogestone acetate, FGA, Chronogest1, Intervet International B.V., Boxmeer, The Netherlands) on Day 0, which was exchanged for a new sponge on Day 7 that was maintained until Day 14. Ewes were also given a single dose of 125 mg, i.m., cloprostenol (Estrumate, Mallinckrodt Vet GmbH, Friesoythe, Germany) at the time of the first FSH dose on Day 12. The superovulatory treatment consisted of eight decreasing doses (1.5 mL 3, 1.25 mL 2, and 1 mL 3) of FSH, i.m. (OvagenTM, ICP, Auckland, New Zealand) administered twice daily starting 60 h before sponge removal and finishing 24 h after progestagen withdrawal. Progestagen removal was coincident with the sixth FSH dose and estrus detection was performed with adult Manchega rams after 24 h. Mating was repeated at 36 and 48 h after sponge withdrawal. The embryos were obtained by surgical access to the genital tract, through prepubian laparotomy under general anaesthesia with xylazine (Rompun, Bayer Ag, Leverkusen, Germany, 6 mg i.m.) and ketamine (Imalge`ne 1000, Merial, Lyon, France, 130 mg i.m.). The ovarian response in terms of number of corpora lutea was determined by laparoscopy just before the laparotomy, thus avoiding surgery in non-responding females. Oviduct retrievals for early stages of development were performed at different intervals after sponge removal to obtain the different developmental stages required: on Day 4 to obtain 2- to 4- cell embryos, on Day 4.5 for 5- to 8-cell, and on Day 5 for 9- to 12-cell. Both oviducts were flushed with PBS (Dulbecco’s phosphate buffered saline) supplemented with 0.2% BSA (bovine serum albumin Fraction V, Sigma Chemical Co., St. Louis, MO, USA) and 10 mg mL 1 of gentamicin (Sigma) from a catheter fed through a blunt needle inserted at the uterotubal junction. The catheter was fixed with silk to avoid backflow of the flushing medium, towards another catheter without a needle introduced into the fimbria and also kept fixed with silk. Uterine recoveries for blastocysts were performed on Day 7 after sponge withdrawal, by flushing both uterine horns with PBS-BSA from a blunt needle inserted at the major curvature of the horn, kept fixed with forceps to avoid backflow of flushing medium, towards another catheter provided with a blunt needle and fixed with silk at the utero-tubal junction.
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2.3. Embryo freezing and thawing Excellent and good embryos categorized by morphologically criteria [24] were selected for conventional freezing with a two-step method with ethylene glycol (ETG) as cryoprotectant. All cryoprotectant solutions were prepared in PBSS (PBS plus 20% FCS, fetal calf serum, Sigma). First, embryos were equilibrated in 0.75 M ETG at 32 8C for 10 min and then placed for a further 10 min in 1.5 M ETG under the same conditions. Embryos were loaded in groups of two to four into 0.25 mL straws (IMV, L’Aigle, France) and placed in a middle column of cryoprotectant-PBSS separated by air bubbles from similar columns of cryoprotectant-PBSS. Afterwards, the straws were placed in a controlled-rate freezer (Lauda E300 Konigshofen, Germany). The slow freezing program carried out was similar to that described by Willadsen et al. [25]: 1 8C min 1 to 7 8C, seeding, 0.3 8C min 1 to 30 8C, and 0.1 8C min 1 to 35 8C. The straws were then plunged into liquid nitrogen and stored for 1 month to 1 year. Thawing of straws was performed by immersion in water at 32 8C for 30 s. Embryos were then transferred to a 0.25 M sucrose solution in PBSS for 10 min, and washed three times in PBSS for 5 min. 2.4. Embryo culture Culture of early stage embryos to the blastocyst stage in the CF group was carried out on ovine oviduct cell monolayers prepared 48 h before the start of culture. Briefly, oviducts collected in the abattoir were trimmed free from adjacent connective tissue and two small portions of the oviduct (1–1.5 cm) were selected (one close to the utero-tubal junction and another one close to the fimbria). Portions were longitudinally dissected and the luminal surface was scraped with a scalpel blade to collect epithelial cells. The cell suspension was aspirated and expelled several times to break up the sheets of cells into smaller clumps and after a centrifugation step, the concentrated pellet was re-suspended in culture medium TCM 199 supplemented with 10% FCS, 1% L-glutamine and antibiotic-antimycotic solution (all products from Sigma) at 37 8C. The cells were stained with 4% trypan blue to assess their viability and the concentration of viable cells was counted with a haemocytometer. Then, the concentration of the viable cells was adjusted to 5 105 mL 1 by dilution in a culture media, seeded into a four- well dish (NunclonTM, Nunc International, Roskilde, Denmark) in a volume of 500 mL per well, and cultured at 38.5 8C in a humidified atmosphere of 5% CO2. Embryos were examined with an inverted microscope (Nikon Eclipse TE300, Tokyo, Japan). Culture of thawed blastocysts to the hatched blastocyst stage was performed in TCM 199 supplemented with 10% FCS, 1% L-glutamine and antibiotic-antimycotic solution (all products from Sigma). Culture plates were maintained at 38.5 8C in a humidified environment of 5% CO2. For the estimation of cell number, blastocysts were air dried on a slide and fixed in ethanol for 24 h. They were then stained with Hoechst 33342 (10 mg/mL in 2.3% sodium citrate) and examined under an epifluorescent microscope.
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2.5. Statistical analysis The effects of the embryo stage and the length of culture on development and hatching rates were analysed using chi-squared test. Yates correction was applied when it was necessary. The log-linear model determined possible interactions between variables. Blastocyst total cell numbers are given as the mean S.E.M. and the statistical significance was accepted from P < 0.05.
3. Results 3.1. Effect of culture on embryo cryosurvival The survival rate after freezing–thawing (Table 1) was significantly higher in blastocysts produced in vivo (BF, 83.7%) than in those obtained following the in vitro culture of early stage embryos (CF, 66.1%, P < 0.05), the lowest survival being for 2- to 4cell embryos (56.0% versus 83.7%, P < 0.005). Although viability after freezing–thawing was different, the number of nuclei of hatched blastocysts was similar between CF and BF groups (196.8 0.3 versus 199.4 0.5, NS). The mean range of time elapsed to hatching of blastocysts after thawing was also similar (around 2–3 days) for both CF and BF groups and for the different developmental stages in CF group. Embryo survival after freezing–thawing in CF group was related to timing of blastocyst formation (P < 0.01). The kinetics of blastocyst appearance for early developmental stage embryos was also determined by the stage of the embryo at the beginning of culture (P < 0.0001). However, irrespective of the developmental stage, any delay in reaching the blastocyst stage was associated with a decrease in survival following freezing/thawing (Table 2). This effect was stronger in the 9- to 12-cell embryos; those embryos developing to blastocysts in 4 days showed higher developmental rates after thawing when compared with embryos going beyond 5 or 6 days (94.4% versus 56.3% versus 40.0%; P < 0.01). 3.2. Effect of embryo stage on cryosurvival Embryos cultured to the blastocyst stage and frozen thereafter (CF) had a significantly higher viability than their counterparts frozen at earlier cleavage stages (EF), (66.1% Table 1 Survival rate (%) of frozen-thawed blastocysts derived from culture of 2- to 4-cell, 5- to 8-cell and 9- to 12-cell embryos, and in vivo obtained ovine blastocysts Blastocysts derived from culture of 2- to 4-cell (n) Survival percentage after freezing–thawing
41 (56.0)a*
5- to 8-cell (n) 38 (71.0)
Different superscripts indicate significant differences (P < 0.05). differences (P < 0.005).
In vivo derived blastocysts (n)
39 (71.7)
118 (66.1)a a,b
9- to 12-cell (n)
43 (83.7)b a*,b
Different superscripts indicate significant
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Table 2 Influence of the kinetics of blastocyst appearance of cultured ovine embryos on their survival rate (%) after freezing–thawing Day of blastocysts appearance
Cryosurvival rate (%) Embryo stage at start the in vitro culture
Day Day Day Day
4 5 6 7
2- to 4-cell (n)
5- to 8-cell (n)
9- to 12-cell (n)
Overall (n)
4 2 30 5
3 (100.0) 16 (68.8) 19 (68.4) –
18 (94.4)a 16 (56.3)b 5 (40.0)b –
25 34 54 5
(25.0) (100.0) (63.3) (20.0)
(84.0) (64.7) (62.9) (20.0)
a,b
Values in the same row with different superscripts differ significantly (P < 0.01).
versus 23.1%; P < 0.0001), as shown in Table 3. Probability for reaching the hatched blastocyst stage after thawing of 2- to 4-cell embryos frozen just after recovery was 6.2 folds lower than of embryos obtained in the same stage but cultured to the blastocyst stage before to be frozen. Such differences increased to 5.8 folds for the group of embryos with 5to 8-cell and to 9.8 for those with 9- to 12-cell at retrieval.
4. Discussion The results of the present study show that the ability of blastocysts produced by in vitro culture of early stage embryos to withstand freezing–thawing is lower than that of blastocysts obtained in vivo and frozen without culture. Such decreases in cryosurvival rate were mainly observed in blastocysts derived by culture of the earliest stages of development, 2- to 4-cell embryos. These results clearly indicate a deleterious effect of culture, which was greater with a longer exposure to culture conditions, confirming that sheep embryos may have temporal sensitivities to the culture environment, in a similar way to cattle [26]. Culture in sequential media would be useful to comply with the requirements of earliest developmental stage embryos, enhancing their developmental competence, as is used in human medicine [27]. Table 3 Developmental rates of fresh ovine embryos to the blastocyst stage before freezing and development of early stages embryos either frozen just after recovery or frozen after in vitro culture to hatched blastocyst stage Treatment group
Embryo stage
Blastocyst rate in culture before freezing (%)
Survival post-thaw (%)
Total survival post-thaw (%)
Direct freezing
2- to 4-cell (n) 5- to 8-cell (n) 9- to 12-cell (n)
– – –
35 (17.1)a 35 (31.4)a 38 (21.1)a
108 (23.1)a
Freezing post-culture
2- to 4-cell (n) 5- to 8-cell (n) 9- to 12-cell (n)
61 (67.2)1 45 (84.4)2 43 (90.6)3
41 (56.0)b 38 (71.0)b 39 (71.7)b
118 (66.1)b
a,b Different superscripts indicate significant differences (P < 0.0001). 1,2Different superscripts indicate significant differences (P < 0.001). 1,3Different superscripts indicate significant differences (P < 0.0001).
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In the current study, the early stage embryos were able to reach the blastocyst stage during the in vitro culture, but failed to hatch from the zona pellucida after freezing– thawing. These findings could be related to the negative effects of culture on the patterns of expression of genes involved in the expansion and hatching of embryos, described by previous authors [28,29]. On the other hand, in our study, cryosurvival was affected by day of blastocyst appearance. Post-thawing hatching rates of embryos exhibiting a delay in reaching the blastocyst stage were reduced, confirming previous data that reported a better survival to cryopreservation of earlier appearing IVP blastocysts [30]. However, these discrepancies can be related to a possible influence of the initial quality of the embryos on timing of blastocyst formation. Such a relationship has been described in both in vivo [12,31] and in vitro produced embryos [12,30,32]. In current study, this effect was avoid or, at least, minimized as far as possible, by (1) using only those embryos strictly classified as excellent or good by morphology and chronology between age and recovery day and (2) avoiding any effect of possible alterations of developmental competence undetected by microscopic evaluation by randomly allocating the chosen embryos between the different groups of treatment. In our study, the timing of development into a hatched blastocyst after freezing– thawing, and cell number was similar between embryos cultured to the blastocyst stage and blastocysts obtained in vivo, suggesting a similar quality. This is in contrast to the findings of Rizos et al. [7], who reported a lower cryotolerance when in vivo produced bovine zygotes were in vitro cultured to the blastocyst stage. Possible causes for this difference may be related either to the use of different species (bovine versus ovine), different culture media (SOF versus oviductal cell coculture) or even different cryopreservation procedures (vitrification versus conventional freezing). Differences between bovine and ovine may confirm previous studies indicating some inherent species variations concerning cryotolerance [15,33]. Coculture with somatic cells has been reported to improve embryo quality [34–37] and cryoresistance [38,39]. Conversely, semidefined media, such as SOF supplemented with serum or albumin, has been reported to be effective for blastocyst production [34,40], but still affects intrinsic embryo quality in terms of cryosurvival [34]. Differences in cryotolerance between studies may be also related to the cryopreservation method [11]. We have used conventional freezing with ethylene glycol (ETG) due to our previous experience with sheep morulae and blastocysts indicating high survival rates [41]. The ETG offers low toxicity and high permeation into embryo cells [42], and is considered the most suitable cryoprotectant for preservation of embryos from all mammal species [15]. However, conservation procedure may be less important for these divergences between studies, since similar results have been described after transfer of either vitrified or conventional freezing ruminants embryos [15,16,43–45]. In spite of the deleterious effects of culture, current results indicate that culture of early developmental stages to the blastocyst stage prior to freezing procedure enhances viability after thawing. Possible effects derived from undetected alterations in developmental competence of early stage embryos may be disregarded. Embryos from each female were randomly allocated between freezing and culture groups after morphological microscopic evaluation, and the embryos cultured to the blastocyst stage before freezing were able to successfully overcome the so-called ‘‘developmental block’’ [46]. Our data are in agreement with previous studies indicating that developmental stage influences the
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cryotolerance of sheep embryos [47]; early developing stages would have a high sensitivity to freezing, a finding also described for cattle embryos [15]. This increase in cryosurvival rates with more advanced stages seems to be determined by the higher number and the smaller size of cells [38,39], which improve chilling tolerance when compared to early stage embryos with few and large cells [48]. These considerations, supporting the hypothesis that different embryo stages have different responses to the same cryopreservation procedure [16], point to the need to modify freezing protocols, adjusting them to the embryo stage used. These efforts may be even more important in IVF protocols, since IVP embryos show even poorer cryotolerance than their in vivo counterparts [20]. This is the trend in human medicine, although results remain controversial with different studies reporting higher [49], similar [50] and lower survival rates for cryopreserved blastocysts than for early stage embryos [51]. However, such differences may be caused by the inherent limitations in the selection of human embryos used for cryopreservation [52], due to the necessity to overcome a fertility problem, since variable results have been found even for blastocyst cryopreservation [50,52]. In conclusion, ovine embryo survival after freezing–thawing is enhanced by the culture of early developmental stages to the blastocyst stage prior to cryopreservation, despite the possible deleterious effects of culture conditions and the loss of some embryos during the period of culture. However, culture may also be considered a method of selection of developmentally competent embryos, as embryos exhibiting a delay in the time to reach the blastocyst stage will show higher chilling sensitivity after a freezing–thawing procedure.
Acknowledgments The authors wish to thank Dr. R. Calvo for the kind help with the statistical analysis of data. The valuable critical revision of this manuscript by Dr. C.J.H. Souza and by Dr. P. Lonergan is gratefully acknowledged. This work was supported by funds from the project INIA SC00-051-C3.1. AGB was supported by the programme Ramon y Cajal and AVL by an INIA research grant.
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[31] Gonzalez-Bulnes A, Garcia-Garcia RM, Castellanos V, Santiago-Moreno J, Ariznavarreta C, Dominguez V, et al. Influence of maternal environment on the number of transferable embryos obtained in response to superovulatory FSH treatments in ewes. Reprod Nutr Dev 2003;43:17–28. [32] Massip A, Mermillod P, Dinnyes A. Morphology and biochemistry of in vitro produced bovine embryos: implications for their cryopreservation. Hum Reprod 1995;10:3004–11. [33] Rizos D, Fair T, Papadopoulus S, Boland MP, Lonergan P. Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro. Mol Reprod Dev 2002;62:320–7. [34] Rizos D, Ward F, Boland MP, Lonergan P. Effect of culture system on the yield and quality of bovine blastocysts as assessed by survival after vitrification. Theriogenology 2001;56:1–16. [35] Kaufmann RA, Menezo Y, Hazout A, Nicollet B, DuMont M, Servy EJ. Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil Steril 1995;64:1125–9. [36] Menezo Y, Guerin P. The mammalian oviduct: biochemistry and physiology. Eur J Obstet Gynecol Reprod Biol 1997;73:99–104. [37] Xu JS, Cheung TM, Chan STH, Ho PC, Yeung WSB. Temporal effect of human oviductal cell and its derived embryotrophic factors on mouse embryo development. Biol Reprod 2001;65:1481–8. [38] Menezo Y, Nicollet B, Harbaut N, Andre D. Freezing cocultured human blastocysts. Fertil Steril 1992;58:977–80. [39] Wiemer KE, Cohen J, Tucker MJ, Godke RA. The application of coculture in assisted reproduction: 10 years of experience with human embryos. Hum Reprod 1998;13(Suppl 4):226–38. [40] Krisher RL, Lane M, Bavister BD. Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media. Biol Reprod 1999;60:1345–52. [41] Cocero MJ, Procureur R, De la Fuente J, Chupin D. Glycerol or ethylene glycol for embryo cryoprotection of deep frozen ewe embryos. Theriogenology 1988;29:238 [abstract]. [42] Szell A, Shelton JN, Szell K. Osmotic characteristics of sheep and cattle embryos. Cryobiology 1989;26:297–301. [43] Massip A, Mermillod P, Van Langendonckt A, Touze JL, Dessy F. Survival and viability of fresh and frozenthawed in vitro bovine blastocysts. Reprod Nutr Dev 1995;35:3–10. [44] Baril G, Traldi AL, Cognie Y, Leboeuf B, Beckers JF, Mermillod P. Successful direct transfer of vitrified sheep embryos. Theriogenology 2001;56:299–305. [45] Kaidi S, Bernard S, Lambert P, Massip A, Dessy F, Donnay I. Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro. Biol Reprod 2001;65:1127–34. [46] Wright Jr RW, Bondioli KR. Aspects of in vitro fertilization and embryo culture in domestic animals. J Anim Sci 1981;53:702–29. [47] Cocero MJ, Lopez Sebastian A, Barragan ML, Picazo RA. Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol. Cryobiology 1996;33:502–7. [48] Szell A, Shelton JN. Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour. J Reprod Fertil 1986;76:401–8. [49] Langley MT, Marek DM, Gardner DK, Doody KM, Doody KJ. Extended embryo culture in human assisted reproduction treatments. Hum Reprod 2001;16:902–8. [50] Rienzi L, Ubaldi F, Iacobelli M, Ferrero S, Minasi MG, Martinez F, et al. Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer. Hum Reprod 2002;17:1852–5. [51] Alper MM, Brinsden P, Fischer R, Wikland M. To blastocyst or not to blastocyst? That is the question Hum Reprod 2001;16:617–9. [52] Behr B, Gebhardt J, Lyon J, Milky AA. Factors relating to a successful cryopreserved blastocyst transfer program. Fertil Steril 2002;77:697–705.
Culture of early stage ovine embryos to blastocyst enhances survival rate after cryopreservation R.M. Garcia-Garcia*, A. Gonzalez-Bulnes, V. Dominguez, A. Veiga-Lopez, M.J. Cocero Departamento de Reproduccio´n Animal INIA, Avda Puerta de Hierro s/n, 28040 Madrid, Spain Received 1 July 2004; received in revised form 17 September 2004; accepted 7 October 2004
Abstract The current study assessed both the effects of in vitro culture and developmental stage of early stage in vivo produced ovine embryos on their ability to survive cryopreservation. Early stage embryos (n = 226) were recovered from the oviduct, at different days of the early luteal phase, at three different developmental stages: 2- to 4-cell, 5- to 8-cell and 9- to 12-cell. For each stage, half of the embryos were cultured to the blastocyst stage and frozen thereafter (CF), while the remainder was frozen just after recovery (EF). A third experimental group (BF; n = 43) included blastocysts obtained from the uterus and frozen immediately after recovery. Embryo viability post-thawing was determined by assessing their rate of development to the hatched blastocyst stage following in vitro culture. Culture negatively affected embryo viability, since survival rate was higher in blastocysts obtained from the uterus than in those from culture (83.7% versus 66.1%; P < 0.05); also the cryosurvival of cultured embryos was lower when the timing of blastocyst formation was extended (P < 0.01). However, survival following freezing–thawing of early developmental stages was significantly lower when compared to viability of their counterparts cultured to the blastocyst stage (23.1% versus 66.1%, P < 0.0001). In conclusion, our results indicate that, despite the deleterious effects of culture per se, the culture of early in vivo produced ovine embryos to the blastocyst stage prior to be frozen improves their survival after thawing. # 2004 Elsevier Inc. All rights reserved. Keywords: Cryosurvival; Developmental stage; Embryo culture; Freezing–thawing; Sheep
* Corresponding author. Present address: Departamento de Fisiologia Animal. UCM. Avda Puerta de Hierro s/n, 28040 Madrid, Spain. Tel.: +34 91 3943858; fax: +34 91 3943864. E-mail address: [email protected] (R.M. Garcia-Garcia). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.10.006
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1. Introduction In spite of the efforts towards the improvement of the techniques of in vitro embryo production (IVP), embryo developmental rates are still far from ideal [1,2], and embryos show poor quality and low cryotolerance [3,4]. Recently, it has been reported that cryosurvival of bovine blastocysts is influenced by the in vitro culture system in which they are produced [5,6], even when zygotes derived from oocytes of high developmental potential (e.g., in vivo matured/fertilized) are used [7]. However, there is a lack of conclusive data about effects of culture on embryo cryotolerance in most mammalian species [8]. Studies carried out in laboratory animals have led to conflicting results, with no effect [9] or deleterious effects [10] following culture periods of long duration [11]. Similarly, in the bovine, a low resistance to freezing–thawing procedures after in vitro culture of both in vivo derived zygotes [7] and morulae and blastocysts cultured for 24 h [12] has been reported. Thus, cryopreservation of embryos at an early stage of development would limit possible negative effects of in vitro culture [13], increasing developmental rates and alleviating logistical problems in other reproductive techniques, such as cloning [14,15]. Studies relating to the cryotolerance of early stage embryos are scarce, but the results obtained in cattle were low [15,16]. However, as chilling sensitivity is species-dependent [17], cryopreservation methods must be adapted for each species [18]. In the present study, sheep embryos were in vivo fertilized and recovered from the oviduct to avoid the influence of potentially suboptimal in vitro maturation and/or fertilization [7]. The objective was to determine the influence of in vitro culture and embryo developmental stage on the cryotolerance of in vivo derived ovine embryos. This would lead, in practice, to the development of better strategies for improving embryo cryosurvival.
2. Materials and methods 2.1. Study design A total of 269 in vivo produced ovine embryos were used to assess the possible influence of culture and developmental stage on survival after freezing–thawing procedures. Early stage embryos classified as good or excellent after flushing, on the basis of their morphology and synchrony between their stage and recovery time, were distributed in three experimental groups according to the stage of development: 2- to 4cell (n = 76), 5- to 8-cell (n = 73) and 9- to 12-cell (n = 77). For each stage, half of the embryos were randomly allocated in two groups. In the first, embryos were cultured in vitro until the blastocyst stage and frozen thereafter (group CF), while in the second group, early stage embryos were frozen just after recovery (EF). Additionally, a third group of blastocysts obtained in vivo from superovulated ewes were frozen immediately after recovery (BF), acting as controls (n = 43). After freezing–thawing, embryo viability was evaluated by assessing attainment of the blastocyst to the hatched blastocyst stage [19]. In agreement with previous studies [20,21], only blastocysts
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hatching from the zona pellucida within a maximum period of 3 days after thawing were classified as viable. Embryo quality was also assessed by counting the number of cells in the hatched blastocysts [22] after staining, and by the kinetics of appearance of hatched blastocysts post-thawing. Effect of culture on embryo cryosurvival was studied by assessment of hatching rates of embryos from groups CF and BF, whilst influence of embryo developmental stage was determined by comparison of hatching blastocyst rate from early stage embryos (EF) versus their counterparts cultured to the blastocyst stage (CF). 2.2. Embryo recovery The embryos were recovered from 4 to 7 years old Manchega ewes, maintained outdoors with access to indoor facilities, at the experimental farm of the INIA Animal Reproduction Department in Madrid, Spain, at a latitude of 408N. They were superovulated with the protocols commonly used in our laboratory [23]. Briefly, estrus was synchronized with the insertion of one intravaginal progestagen impregnated sponge (40 mg fluorogestone acetate, FGA, Chronogest1, Intervet International B.V., Boxmeer, The Netherlands) on Day 0, which was exchanged for a new sponge on Day 7 that was maintained until Day 14. Ewes were also given a single dose of 125 mg, i.m., cloprostenol (Estrumate, Mallinckrodt Vet GmbH, Friesoythe, Germany) at the time of the first FSH dose on Day 12. The superovulatory treatment consisted of eight decreasing doses (1.5 mL 3, 1.25 mL 2, and 1 mL 3) of FSH, i.m. (OvagenTM, ICP, Auckland, New Zealand) administered twice daily starting 60 h before sponge removal and finishing 24 h after progestagen withdrawal. Progestagen removal was coincident with the sixth FSH dose and estrus detection was performed with adult Manchega rams after 24 h. Mating was repeated at 36 and 48 h after sponge withdrawal. The embryos were obtained by surgical access to the genital tract, through prepubian laparotomy under general anaesthesia with xylazine (Rompun, Bayer Ag, Leverkusen, Germany, 6 mg i.m.) and ketamine (Imalge`ne 1000, Merial, Lyon, France, 130 mg i.m.). The ovarian response in terms of number of corpora lutea was determined by laparoscopy just before the laparotomy, thus avoiding surgery in non-responding females. Oviduct retrievals for early stages of development were performed at different intervals after sponge removal to obtain the different developmental stages required: on Day 4 to obtain 2- to 4- cell embryos, on Day 4.5 for 5- to 8-cell, and on Day 5 for 9- to 12-cell. Both oviducts were flushed with PBS (Dulbecco’s phosphate buffered saline) supplemented with 0.2% BSA (bovine serum albumin Fraction V, Sigma Chemical Co., St. Louis, MO, USA) and 10 mg mL 1 of gentamicin (Sigma) from a catheter fed through a blunt needle inserted at the uterotubal junction. The catheter was fixed with silk to avoid backflow of the flushing medium, towards another catheter without a needle introduced into the fimbria and also kept fixed with silk. Uterine recoveries for blastocysts were performed on Day 7 after sponge withdrawal, by flushing both uterine horns with PBS-BSA from a blunt needle inserted at the major curvature of the horn, kept fixed with forceps to avoid backflow of flushing medium, towards another catheter provided with a blunt needle and fixed with silk at the utero-tubal junction.
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2.3. Embryo freezing and thawing Excellent and good embryos categorized by morphologically criteria [24] were selected for conventional freezing with a two-step method with ethylene glycol (ETG) as cryoprotectant. All cryoprotectant solutions were prepared in PBSS (PBS plus 20% FCS, fetal calf serum, Sigma). First, embryos were equilibrated in 0.75 M ETG at 32 8C for 10 min and then placed for a further 10 min in 1.5 M ETG under the same conditions. Embryos were loaded in groups of two to four into 0.25 mL straws (IMV, L’Aigle, France) and placed in a middle column of cryoprotectant-PBSS separated by air bubbles from similar columns of cryoprotectant-PBSS. Afterwards, the straws were placed in a controlled-rate freezer (Lauda E300 Konigshofen, Germany). The slow freezing program carried out was similar to that described by Willadsen et al. [25]: 1 8C min 1 to 7 8C, seeding, 0.3 8C min 1 to 30 8C, and 0.1 8C min 1 to 35 8C. The straws were then plunged into liquid nitrogen and stored for 1 month to 1 year. Thawing of straws was performed by immersion in water at 32 8C for 30 s. Embryos were then transferred to a 0.25 M sucrose solution in PBSS for 10 min, and washed three times in PBSS for 5 min. 2.4. Embryo culture Culture of early stage embryos to the blastocyst stage in the CF group was carried out on ovine oviduct cell monolayers prepared 48 h before the start of culture. Briefly, oviducts collected in the abattoir were trimmed free from adjacent connective tissue and two small portions of the oviduct (1–1.5 cm) were selected (one close to the utero-tubal junction and another one close to the fimbria). Portions were longitudinally dissected and the luminal surface was scraped with a scalpel blade to collect epithelial cells. The cell suspension was aspirated and expelled several times to break up the sheets of cells into smaller clumps and after a centrifugation step, the concentrated pellet was re-suspended in culture medium TCM 199 supplemented with 10% FCS, 1% L-glutamine and antibiotic-antimycotic solution (all products from Sigma) at 37 8C. The cells were stained with 4% trypan blue to assess their viability and the concentration of viable cells was counted with a haemocytometer. Then, the concentration of the viable cells was adjusted to 5 105 mL 1 by dilution in a culture media, seeded into a four- well dish (NunclonTM, Nunc International, Roskilde, Denmark) in a volume of 500 mL per well, and cultured at 38.5 8C in a humidified atmosphere of 5% CO2. Embryos were examined with an inverted microscope (Nikon Eclipse TE300, Tokyo, Japan). Culture of thawed blastocysts to the hatched blastocyst stage was performed in TCM 199 supplemented with 10% FCS, 1% L-glutamine and antibiotic-antimycotic solution (all products from Sigma). Culture plates were maintained at 38.5 8C in a humidified environment of 5% CO2. For the estimation of cell number, blastocysts were air dried on a slide and fixed in ethanol for 24 h. They were then stained with Hoechst 33342 (10 mg/mL in 2.3% sodium citrate) and examined under an epifluorescent microscope.
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2.5. Statistical analysis The effects of the embryo stage and the length of culture on development and hatching rates were analysed using chi-squared test. Yates correction was applied when it was necessary. The log-linear model determined possible interactions between variables. Blastocyst total cell numbers are given as the mean S.E.M. and the statistical significance was accepted from P < 0.05.
3. Results 3.1. Effect of culture on embryo cryosurvival The survival rate after freezing–thawing (Table 1) was significantly higher in blastocysts produced in vivo (BF, 83.7%) than in those obtained following the in vitro culture of early stage embryos (CF, 66.1%, P < 0.05), the lowest survival being for 2- to 4cell embryos (56.0% versus 83.7%, P < 0.005). Although viability after freezing–thawing was different, the number of nuclei of hatched blastocysts was similar between CF and BF groups (196.8 0.3 versus 199.4 0.5, NS). The mean range of time elapsed to hatching of blastocysts after thawing was also similar (around 2–3 days) for both CF and BF groups and for the different developmental stages in CF group. Embryo survival after freezing–thawing in CF group was related to timing of blastocyst formation (P < 0.01). The kinetics of blastocyst appearance for early developmental stage embryos was also determined by the stage of the embryo at the beginning of culture (P < 0.0001). However, irrespective of the developmental stage, any delay in reaching the blastocyst stage was associated with a decrease in survival following freezing/thawing (Table 2). This effect was stronger in the 9- to 12-cell embryos; those embryos developing to blastocysts in 4 days showed higher developmental rates after thawing when compared with embryos going beyond 5 or 6 days (94.4% versus 56.3% versus 40.0%; P < 0.01). 3.2. Effect of embryo stage on cryosurvival Embryos cultured to the blastocyst stage and frozen thereafter (CF) had a significantly higher viability than their counterparts frozen at earlier cleavage stages (EF), (66.1% Table 1 Survival rate (%) of frozen-thawed blastocysts derived from culture of 2- to 4-cell, 5- to 8-cell and 9- to 12-cell embryos, and in vivo obtained ovine blastocysts Blastocysts derived from culture of 2- to 4-cell (n) Survival percentage after freezing–thawing
41 (56.0)a*
5- to 8-cell (n) 38 (71.0)
Different superscripts indicate significant differences (P < 0.05). differences (P < 0.005).
In vivo derived blastocysts (n)
39 (71.7)
118 (66.1)a a,b
9- to 12-cell (n)
43 (83.7)b a*,b
Different superscripts indicate significant
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Table 2 Influence of the kinetics of blastocyst appearance of cultured ovine embryos on their survival rate (%) after freezing–thawing Day of blastocysts appearance
Cryosurvival rate (%) Embryo stage at start the in vitro culture
Day Day Day Day
4 5 6 7
2- to 4-cell (n)
5- to 8-cell (n)
9- to 12-cell (n)
Overall (n)
4 2 30 5
3 (100.0) 16 (68.8) 19 (68.4) –
18 (94.4)a 16 (56.3)b 5 (40.0)b –
25 34 54 5
(25.0) (100.0) (63.3) (20.0)
(84.0) (64.7) (62.9) (20.0)
a,b
Values in the same row with different superscripts differ significantly (P < 0.01).
versus 23.1%; P < 0.0001), as shown in Table 3. Probability for reaching the hatched blastocyst stage after thawing of 2- to 4-cell embryos frozen just after recovery was 6.2 folds lower than of embryos obtained in the same stage but cultured to the blastocyst stage before to be frozen. Such differences increased to 5.8 folds for the group of embryos with 5to 8-cell and to 9.8 for those with 9- to 12-cell at retrieval.
4. Discussion The results of the present study show that the ability of blastocysts produced by in vitro culture of early stage embryos to withstand freezing–thawing is lower than that of blastocysts obtained in vivo and frozen without culture. Such decreases in cryosurvival rate were mainly observed in blastocysts derived by culture of the earliest stages of development, 2- to 4-cell embryos. These results clearly indicate a deleterious effect of culture, which was greater with a longer exposure to culture conditions, confirming that sheep embryos may have temporal sensitivities to the culture environment, in a similar way to cattle [26]. Culture in sequential media would be useful to comply with the requirements of earliest developmental stage embryos, enhancing their developmental competence, as is used in human medicine [27]. Table 3 Developmental rates of fresh ovine embryos to the blastocyst stage before freezing and development of early stages embryos either frozen just after recovery or frozen after in vitro culture to hatched blastocyst stage Treatment group
Embryo stage
Blastocyst rate in culture before freezing (%)
Survival post-thaw (%)
Total survival post-thaw (%)
Direct freezing
2- to 4-cell (n) 5- to 8-cell (n) 9- to 12-cell (n)
– – –
35 (17.1)a 35 (31.4)a 38 (21.1)a
108 (23.1)a
Freezing post-culture
2- to 4-cell (n) 5- to 8-cell (n) 9- to 12-cell (n)
61 (67.2)1 45 (84.4)2 43 (90.6)3
41 (56.0)b 38 (71.0)b 39 (71.7)b
118 (66.1)b
a,b Different superscripts indicate significant differences (P < 0.0001). 1,2Different superscripts indicate significant differences (P < 0.001). 1,3Different superscripts indicate significant differences (P < 0.0001).
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In the current study, the early stage embryos were able to reach the blastocyst stage during the in vitro culture, but failed to hatch from the zona pellucida after freezing– thawing. These findings could be related to the negative effects of culture on the patterns of expression of genes involved in the expansion and hatching of embryos, described by previous authors [28,29]. On the other hand, in our study, cryosurvival was affected by day of blastocyst appearance. Post-thawing hatching rates of embryos exhibiting a delay in reaching the blastocyst stage were reduced, confirming previous data that reported a better survival to cryopreservation of earlier appearing IVP blastocysts [30]. However, these discrepancies can be related to a possible influence of the initial quality of the embryos on timing of blastocyst formation. Such a relationship has been described in both in vivo [12,31] and in vitro produced embryos [12,30,32]. In current study, this effect was avoid or, at least, minimized as far as possible, by (1) using only those embryos strictly classified as excellent or good by morphology and chronology between age and recovery day and (2) avoiding any effect of possible alterations of developmental competence undetected by microscopic evaluation by randomly allocating the chosen embryos between the different groups of treatment. In our study, the timing of development into a hatched blastocyst after freezing– thawing, and cell number was similar between embryos cultured to the blastocyst stage and blastocysts obtained in vivo, suggesting a similar quality. This is in contrast to the findings of Rizos et al. [7], who reported a lower cryotolerance when in vivo produced bovine zygotes were in vitro cultured to the blastocyst stage. Possible causes for this difference may be related either to the use of different species (bovine versus ovine), different culture media (SOF versus oviductal cell coculture) or even different cryopreservation procedures (vitrification versus conventional freezing). Differences between bovine and ovine may confirm previous studies indicating some inherent species variations concerning cryotolerance [15,33]. Coculture with somatic cells has been reported to improve embryo quality [34–37] and cryoresistance [38,39]. Conversely, semidefined media, such as SOF supplemented with serum or albumin, has been reported to be effective for blastocyst production [34,40], but still affects intrinsic embryo quality in terms of cryosurvival [34]. Differences in cryotolerance between studies may be also related to the cryopreservation method [11]. We have used conventional freezing with ethylene glycol (ETG) due to our previous experience with sheep morulae and blastocysts indicating high survival rates [41]. The ETG offers low toxicity and high permeation into embryo cells [42], and is considered the most suitable cryoprotectant for preservation of embryos from all mammal species [15]. However, conservation procedure may be less important for these divergences between studies, since similar results have been described after transfer of either vitrified or conventional freezing ruminants embryos [15,16,43–45]. In spite of the deleterious effects of culture, current results indicate that culture of early developmental stages to the blastocyst stage prior to freezing procedure enhances viability after thawing. Possible effects derived from undetected alterations in developmental competence of early stage embryos may be disregarded. Embryos from each female were randomly allocated between freezing and culture groups after morphological microscopic evaluation, and the embryos cultured to the blastocyst stage before freezing were able to successfully overcome the so-called ‘‘developmental block’’ [46]. Our data are in agreement with previous studies indicating that developmental stage influences the
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cryotolerance of sheep embryos [47]; early developing stages would have a high sensitivity to freezing, a finding also described for cattle embryos [15]. This increase in cryosurvival rates with more advanced stages seems to be determined by the higher number and the smaller size of cells [38,39], which improve chilling tolerance when compared to early stage embryos with few and large cells [48]. These considerations, supporting the hypothesis that different embryo stages have different responses to the same cryopreservation procedure [16], point to the need to modify freezing protocols, adjusting them to the embryo stage used. These efforts may be even more important in IVF protocols, since IVP embryos show even poorer cryotolerance than their in vivo counterparts [20]. This is the trend in human medicine, although results remain controversial with different studies reporting higher [49], similar [50] and lower survival rates for cryopreserved blastocysts than for early stage embryos [51]. However, such differences may be caused by the inherent limitations in the selection of human embryos used for cryopreservation [52], due to the necessity to overcome a fertility problem, since variable results have been found even for blastocyst cryopreservation [50,52]. In conclusion, ovine embryo survival after freezing–thawing is enhanced by the culture of early developmental stages to the blastocyst stage prior to cryopreservation, despite the possible deleterious effects of culture conditions and the loss of some embryos during the period of culture. However, culture may also be considered a method of selection of developmentally competent embryos, as embryos exhibiting a delay in the time to reach the blastocyst stage will show higher chilling sensitivity after a freezing–thawing procedure.
Acknowledgments The authors wish to thank Dr. R. Calvo for the kind help with the statistical analysis of data. The valuable critical revision of this manuscript by Dr. C.J.H. Souza and by Dr. P. Lonergan is gratefully acknowledged. This work was supported by funds from the project INIA SC00-051-C3.1. AGB was supported by the programme Ramon y Cajal and AVL by an INIA research grant.
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