Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids

Transcription

1 Human Reproduction vol.14 no.11 pp , 1999 Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids Se-Pill Park, Eun Young Kim, Deok Im Kim, Noh Hyung Park, Yu Seok Won, San Hyun Yoon, Kil Saeng Chung and Jin Ho Lim 1, To whom correspondence should be addressed This study demonstrates that higher survival of vitrifiedthawed bovine blastocysts can be obtained using electron microscope (EM) grids as embryo containers at freezing, rather than plastic straws. In-vitro produced day 7 bovine blastocysts after in-vitro fertilization (IVF) were vitrified on grids or in straws with EFS40 freezing solution and their survival after thawing was compared. Embryo survival was assessed as re-expanded and hatched rates at 4 and 48 h after thawing respectively. When the effects of exposure to vitrification solution and chilling injury from the freezing procedure were examined, embryo survival in the exposure group (4 h: 100, 48 h: 73.3%) was not different compared with that in the control group (100,84.4%). After vitrifica tion, the hatched rate of the EM grid group 48 h after thawing (67.8%) was significantly higher than that of the straw group (53.3%) (P < 0.05). Fast developing embryos (expanded blastocyst and early hatching blastocyst stage) showed better resistance to freezing than delayed ones (early blastocyst stage), irrespective of embryo containers (early: 4 h, 57.1 and 48 h, 4.4%; expanded: 84.7 and 60.6%; early hatching: 91.7 and 80.0%) ( P < 0.001). When using expanded and early hatching blastocysts, embryo survival rates in the vitrification-em grid group (67.8, 95.0% respectively) were significantly higher than that of the vitrification-straw group (53.0, 65.0%) at 48 h. Keywords: bovine blastocysts/in-vitro survival/em grid/plastic straw/vitrification Introduction The production of bovine embryos in vitro has become a routine procedure and thus successful cryopreservation methods are required for efficient utilization of these embryos (Leibo and Loskutoff, 1993). In the past decade, various new methods for embryo cryopreservation have been developed. Among these methods, vitrification has been widely used and is now regarded as a potential alternative to conservative slow freezing. In addition, to prevent the chilling injury during bovine 838 On the other hand, successful results from the vitrification of in-vitro derived bovine blastocysts have been demonstrated by many researchers (Kuwamaya et al., 199; Tachikawa et al., 1993; Mamoudzadeh et al., 1995). In a previous study (Park et al., 1998), we reported that higher survival of bovine in-vitro matured, fertilized and cultured (IVM/IVF/IVC) blastocysts can be obtained by a simple two-step vitrification method using straws and EFS40 freezing solution. On the basis of these results, we aimed in this study to find a better cryopreservation method for bovine IVM/IVF/IVC blastocysts. We examined the in-vitro survival rates of vitrified-warmed embryos from a new freezing method using EM grids, and compared our results with the survival rates from a previously established vitrification method using straws. Materials and methods Chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA) and media from GIBCO (Grand Island, NY, USA), unless otherwise stated. All of the manipulation procedures of oocytes, spermatozoa, freezing and warming were conducted at 5 C (room temperature). Production of bovine blastocysts The culture procedures employed in the production ofpreimplantation embryos from bovine follicular oocytes were as previously outlined (Park et al., 1999). Briefly, ovaries were obtained from a slaughter house and cumulus-oocyte complexes (COC) were aspirated from visible follicles (-6 mm in diameter). The COC were then washed with HEPES-buffered Tyrode's medium and cultured in maturation medium composed of TCM % fetal bovine serum (FBS) European Society of Human Reproduction and Embryology Downloaded from at Pennsylvania State University on March 4, 014 Maria Infertility Medical Institute, Sinseol-dong Dongdaemun-Gu, Seoul , Hanwoo Improvement Center, NLCF, Seosan, Chungnam , Maria Infertility Clinic, Sinseol-dong Dongdaemun-Gu, Seoul , ^Department of Animal Sciences, Kon-Kuk University, Mojin-dong, Kwangjin-gu, Seoul , Korea embryo cryopreservation, it has been suggested that rapid cooling may be better than slow cooling (Pollard and Leibo, 1994). Vitrification has potential advantages over conventional freezing in that it takes only a few seconds to cool embryos, and there is no extracellular crystallization, which is one of the major causes of cell injury (Rall and Fahy, 1985). It also offers lower osmotic and toxic effects and less severe chilling injury resulting from the rapid passage through the 'dangerous' temperature zone (Vajta et al., 1998). However, most vitrification methods use standard French mini-straws for holding the embryos during cooling, storage and thawing. Recently, to overcome the disadvantages of straws that have low cooling and warming rates, a few studies have been published. Among them, it has been reported (Martino et al., 1996) that an ultra-rapid freezing method using electron microscope (EM) grids was efficient for the cryopreservation of chilling sensitive bovine oocytes. However, until now, freezing studies using EM grids were limited to oocyte stages (Martino et al., 1996; Kim et al., 1998a,b).

2 30-45 s. Plastic 0.5 ml straws were loaded as follows. Briefly, a 4 cm length was filled with 0.5 M sucrose solution (prepared in m-dpbs containing 10% FBS) followed by a 1.5 cm air bubble, 0.5 cm EFS40, 0.5 cm air bubble, 0.5 cm EFS40, 0.5 cm air bubble, cm EFS40 and 1.5 cm air bubble. The remaining part of the straw was filled with 0.5 M sucrose. The straw was sealed with powder and heat after embryo loading. To prevent cracking of the straw, the first part of the straw filled with 0.5 M sucrose (4 cm) was slowly immersed into L N ; the remaining part of the straw was then plunged in. The average number of embryos loaded in each straw was about three to five. Table I. Comparison of characteristics of vitrification methods using two types of embryo container Specificity EM grids Straws Material Freezing step Prefreezing Thawing Dilution step Copper -step (1.5 min/30 s) Not needed Directly in warming solution -step (1.5 min/1.5 min) Plastic -step (3 min/30-45 s) Needed Air 5 s/water 15 s -step (5 min/5 min) Vitrification procedures In this study, two types of embryo containers (EM grids and straws) were used to cryopreserve the bovine blastocysts. Detailed information about two different freezing methods is given in Table I. In both vitrification methods, freezing was carried out in two steps. The freezing solution, EFS40, was prepared according to a previously described method (Kasai et al., 19), and consisted of 40% (v/v) ethylene glycol (EG), 18% (w/v) Ficoll (Ficoll 70, average MW: Da), 10.6% (w/v) sucrose and 10% FBS in modified Dulbecco's phosphate buffered saline (m-dpbs). To decrease embryo damage from sudden exposure in high concentrations ofcryoprotective agents, a pretreatment solution, m-dpbs (EG0) containing 0% EG and 10% FBS, was prepared. Vitrification using grid (V-G) The two-step freezing method was modified from that of Kim (Kim et al., 1998a,b). Briefly, in this method, 400 mesh copper EM grids (IGC 400; Pelco International, CA, USA) were used as a physical support to maximize cooling rates when the embryos were directly plunged into liquid nitrogen (LN). Before exposure to the vitrification solution, embryos (Figure 1A) were partially equilibrated in EG0 for 1.5 min. The embryos were then incubated in EFS40 (Figure 1B), loaded onto the EM grid (Figure 1C, D) and directly plunged in L N within 30 s. The mean number of blastocysts loaded on one grid was Vitrification using straws (V-S) Freezing was carried out as described (Park et al., 1998). In the first step, embryos were fully equilibrated in EG0 for 3 min. In the second step, embryos were incubated in EFS40, loaded into 0.5 ml French straws (IMV, L'Aigle, France) and vitrified in LN within Thawing procedures Grids Cryoprotectants in vitrified-thawed embryos were removed in two steps. After a few hours or days of storage in L N, embryos were warmed ultra-rapidly. EM grids stored in L N were directly transferred into 0.3 M sucrose solution (prepared in m-dpbs con taining 10% FBS) as soon as possible, and then quickly transferred into fresh 0.3 M sucrose and incubated for 1.5 min. Recovered embryos were transferred into m-dpbs containing 10% FBS. After another 1.5 min incubation, the embryos were washed with culture medium and co-cultured in cumulus cell monolayered drops (10 ml) of CR1 medium containing 10% FBS (Figure 1E). The cumulus cell drops were prepared with cumulus cells recovered from in-vitro matured bovine oocytes before IVF treatment. Straws Embryos were warmed rapidly by placing straws in air for 5 s and then in a 5 C water bath for s. The contents of each straw were emptied into 0.8 ml of 0.3 mol/l sucrose solution, and then recovered embryos were transferred into another 0.8 ml of 0.3 mol/l sucrose for 5 min. Embryos were then incubated in 0.8 ml ofm-dpbs containing 10% FBS for 5 min. After dilution, the embryos were cocultured in the same conditions as the grid embryos for 48 h. Assessment of embryo survival The post-thawing survival ofembryos was observed every 4 h under a microscope and embryos were judged as morphological survivors if they expanded into blastocysts within the first 4 h of culture, and hatched out totally within the next 48 h (Figure 1F). Experimental design To examine embryo damage from toxicity of the vitrification solution and to compare difference of chilling injury from the freezing procedure by embryo containers, bovine blastocysts were exposed to freezing solution and/or vitrified in LN respectively. In the exposure group, all treatments, including those before freezing and the warming procedures, were the same as used for the freezing group. To examine embryo survival in vitro after warming by embryo developmental stage, and to determine the effect of the two kinds of sample container on the embryo survival, day 7 blastocysts from each development stage were vitrified. Statistical analysis The significance of difference among treatment group in each experiment was compared with the c test using the Statistical Analysis System (SAS) Institute software package (SAS Institute Inc., 1985). Results Bovine embryo production in vitro From 0 replicates, 3318 bovine oocytes were subjected to in-vitro maturation and fertilization procedures. On day, 839 Downloaded from at Pennsylvania State University on March 4, 014 supplemented with 0. mmol/l sodium pyruvate, 1 mg/ml folliclestimulating hormone, 1 mg/ml oestradiol-17ß, and 5 mg/ml gentamycin sulphate at 39 C, 5% C O incubator. After incubation for -4 h in IVM medium, the COC were inseminated using highly motile spermatozoa recovered from frozen-thawed bull semen separated on a discontinuous Percoll column. Fertilization was assessed as cleavage rate (>-cell) after 44 ± h co-incubation with the spermatozoa. For in-vitro culture, cleaved embryos were cultured in CR1 (Rosenkrans et al., 1993) medium supplemented with 3 mg/ml fatty acid-free bovine serum albumin and then transferred into CR1 medium + 10% FBS at day 4 after in-vitro fertilization (IVF). For the study, blastocysts produced in vitro at day 7 after IVF were classified into early (i.e. the blastocoele is smaller than two-thirds of the whole embryo), expanded (i.e. the blastocoele is larger than twothirds of the whole embryo and the zona pellucida is thinning) and early hatching (i.e. the blastocoele is re-expanded to about four-fifths of the whole embryo after shrinkage which occurred with zona rupture) stages according to their developmental morphology (Kim et al., 1996) and they were divided into control, exposure and vitrified groups.

3 Figure 1. A series course of freezing and warming of bovine in-vitro matured, fertilized and cultured blastocysts using electron microscope (EM) grids (A-F). Bovine blastocysts (A) were equilibrated in EG0 for 3 min, exposed in EFS40 freezing solution (B) and were then loaded onto EM grids immediately before being plunged into LN (C, D) within 30 s. After warming, embryos were diluted in their cryoprotectants and then cultured in cumulus cell monolayered drops (E). Warmed blastocysts were hatched in vitro 48 h after culture (F). Original magnifications: A and B X150, C X40, D X60, E X100, F X80. Downloaded from at Pennsylvania State University on March 4, (88.0%) embryos had cleaved. The total number of embryos that developed to blastocysts at day 7 after IVF was 1503 (45.3%). When they were classified into early, expanded and beyond hatching blastocyst stages, 734 (.1%), 58 (17.5%) and 187 (5.6%) were produced respectively (Table II). For this study, day 7 blastocysts of morphologically good to excellent quality were selected from each replicate; thus 156 early blastocysts, 534 expanded blastocysts and 10 early hatching blastocysts were utilized according to each experimental purpose. In-vitro survival of exposed or vitrified bovine blastocysts The effects of exposure to vitrification solution and of chilling injury from the freezing procedure on in-vitro produced bovine blastocysts were examined. When embryo survival in vitro was assessed using re-expanded and hatched rates at 4 h and 48 h after thawing, as shown in Table III, the viability of bovine blastocysts was not decreased in spite of exposure to freezing solution (100.0, 73.3%) compared to the control group (100.0, 84.4%) respectively. However, their in-vitro survival rates at 4 h were significantly higher than those of the two 840

4 vitrified groups [vitrified in straws (V-S): 77.8; vitrified in grids (V-G): 87.8%] (P < 0.001). However, the embryo hatched rates in the V-G group (67.8%) at 48 h after warming were not significantly different from those in the exposure group only (73.3%), although there were significant differences from those in the control group (84.4%) (P < 0.01). Between two vitrified groups in particular, there were significant differences in hatched rates at 48 h after warming (V-S: 53.3; V-G: 67.8%) (P < 0.05). In-vitro survival of vitrified bovine blastocysts according developmental stage and embryo container The effects of embryo developmental stage (early, expanded and early hatching blastocysts) and embryo container (straw Table II. Development of bovine follicular oocytes at day 7 after IVF (expts = 0) No. of oocytes No. (%) of -cell Developed (%) to blastocysts at day 7 to or EM grid) used at freezing on the in-vitro survival of vitrified-thawed day 7 bovine blastocysts were examined. As shown in Table IV, fast developing embryos (expanded blastocyst or early hatching blastocyst stage) showed better resistance to cryopreservation than the delayed ones (early: blastocyst stage), irrespective of embryo containers (early: 57.1 and 4.4%; expanded: 84.7 and 60.6%; early hatching: 91.7 and 80.0%) (P < 0.001). In this study, among the blastocysts beyond hatching, early hatching stage embryos that had shrunk and then re-expanded were used as a comparison group. However, the survival rate of day 7 early hatching blastocysts was higher than that ofthe two other developmental stage embryos. In addition, when in-vitro survival of vitrified early hatching blastocysts according to embryo container was Total Early Expanded Hatching/hatched (88.0) 1503 (45.3) 734 (.1) 58 (17.5) 187 (5.6) Table III. In-vitro survival of exposed or vitrified bovine blastocysts in freezing solution EFS40 (expts = 6) Treatment No. of No. (%) of No. (%) of blastocysts surviving after thawing embryos embryos examined* recovered 4 h later 48 h later Control Exposed Vitrified Straw Grid (100) (100) (100) Expanded/re-expanded Hatching Hatched (100) a (100) a 70 (77.8) b 79 (87.8) b 80 (88.9) c 78 (86.7) c 61 (67.8) d 71 (78.9) c,d *Day 7 expanded blastocysts. Values within columns having different superscripts are significantly different (P < 0.001) a-b ; (P < 0.01) c-d ;(P < 0.05) d-e. 76 (84.4) c 66 (73.3) c,d 48 (53.3) e 61 (67.8) d Table IV. In-vitro survival of vitrified day 7 bovine blastocysts according to embryo developmental stage and embryo container Embryo stage Container No. of No. (%) of No. (%) of blastocysts surviving after thawing type embryos embryos examined recovered 4 h later 48 h later Downloaded from at Pennsylvania State University on March 4, 014 Early blastocyst Expanded blastocyst Re-expanded Hatching Hatched Straw (100) 4 (56.0) a 7 (36.0) a 17 (.7) a Grid (100) 47 (58.0) a 4 (9.6) a 1 (5.9) a Total (100) 89 (57.1)* 51 (3.7) * 38 (4.4) * Straw (94.8) 7 (86.7) b, t 59 (71.1) b 44 (53.0) b, t Grid (100) 7 (8.8) b, t 66 (75.9) b 59 (67.8) b, t Total (98.5) 144 (84.7) ** 15 (73.5) ** 103 (60.6) ** Early hatching blastocyst Straw (100) 51 (85.0) b, t 48 (80) b 39 (65.0) b Grid (100) 59 (98.3) b, t 59 (98.3) c 57 (95.0) c (100) 110 (91.7)** 107 (89.) *** 96 (80) *** Values within columns having different superscripts are significantly different (P < 0.001) a-c, *, **, ***; (P < 0.05)t or *. 841

5 compared, the survival rates of the V-G group (98.3, 95.0%) were significantly higher than those of the V-S group (85.0, 65.0%) at 4 h and 48 h post warming respectively ( P < 0.05, P < 0.001). Discussion Embryo cryopreservation using EM grids was originally designed for the vitrification of exceedingly chill-sensitive Drosophila embryos (Mazur et al., 199; Steponkus and Caldwell, 1993). As described in Table I, the V-G method is more simple and time saving than the V-S method. However, most recent attempts to improve the cryopreservation ofbovine embryos have been directed at the simplification of freezing/ thawing procedures. The vitrification method using EM grids for the cryopreservation of bovine oocytes was first introduced by the Martino group (Martino et al., 1996). In that study, it was demonstrated that the in-vitro survival of vitrified-thawed oocytes using EM grids was significantly higher than that of oocytes frozen in straws. These results may be caused by the characteristic of the grid, which has about 3-fold higher cooling rates, than those obtained with straws. It is known that the increased rate of cooling and thawing may considerably decrease the chilling injury of in-vitro produced bovine embryos. In addition, in our previous study (Kim et al., 1998a,b), we showed that the innate developmental capacity of bovine immature or mature oocytes vitrified and thawed using EM grids can be maintained. Although cryopreservation is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in-vivo or in-vitro produced) (Fahning and Garcia, 199). Generally, in-vitro produced embryos were much more sensitive to freezing than the in-vivo derived counterparts (Leibo and Loskutoff, 1993). However, recently reported improved survival rates after cryopreservation of in-vitro produced morulae and blastocysts are attributed more to the improved culture conditions than changes in the cryopreservation technology itself (Voelkel and Hu, 199; Massip et al., 1995). In our culture system using CR1 medium, 45.3% blastocysts from oocytes were developed at day 7 after IVF. Also, for this study, 810 day 7 healthy blastocysts were used. 84 In addition, cryoprotective agents were intimately related to embryo survival (Tachikawa et al., 1993). Since 19, EFS solution has been widely used to cryopreserve the various stage embryos of several species; it permeates the cell rapidly and has low toxicity. Using EFS freezing solution, we have tried to find a freezing method that covers a wide range of stages, from immature oocyte to hatched blastocyst, and thus appropriate concentrations of freezing solution and treatment methods according to each embryo stage were developed. In this study on the cryopreservation of bovine IVM/IVF/IVC blastocysts, the two-step vitrification method using EM grids and EFS40 freezing solution was efficient, particularly for day 7 expanded and early hatching blastocysts. References Fahning, M.L. and Garcia, M.A. (199) Status ofcryopreservation ofembryos from domestic animals. Cryobiology, 9, Kasai, M., Komi, H., Takakamo, A. et al. (19) A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J. Reprod. Fertil., 89, Kim, E.Y., Uhm, S.J., Kim, S.E. et al. (1996) ICM-trophectoderm cell numbers of bovine IVM/IVF/IVC blastocysts. Kor. J. Anim. Reprod., 0, Kim, E.Y., Kim, N.H., Yi, B.K. et al. (1998a) Developmental capacity of bovine follicular oocytes after ultra-rapid freezing by electron microscope grid I. Cryopreservation of bovine immature oocytes. Kor. J. Fert. Steril., 5, Kim, E.Y., Kim, N.H., Yi, B.K. et al. (1998b) Developmental capacity of bovine follicular oocytes after ultra-rapid freezing by electron microscope grid II. Cryopreservation of in vitro matured bovine oocytes. Kor. J. Ani. Reprod.,,1-9. Kuwamaya, M., Hamano, S. and Nagai, T. (199) Vitrification of bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro. J. Reprod. Fertil., 96, Leibo, S.P. and Loskutoff, N.M. (1993) Cryobiology of in vitro-derived bovine embryos. Theriogenology, 39, Mamoudzadeh, A.R., Van Soom, A., Bols, P. et al. (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, Martino, A., Songsasen, N. and Leibo, S.P. (1996) Developmental capacity of bovine oocytes cryopreserved after maturation in vitro and frozen-thawed bovine embryos derived from frozen mature oocytes. Theriogenology, 38, Massip, A., Mermillod, P. and Dinneys, A. (1995) Morphology and biochemistry of in-vitro produced bovine embryos: implications for their cryopreservation. Hum. Reprod., 10, Mazur, P., Cole, K.W., Hall, J.W. et al. (199) Cryobiological preservation of Drosophila embryos. Science, 58, Park, S.P., Kim, E.Y., Kim, D.I. et al. (1998) Systems for production ofcalves Downloaded from at Pennsylvania State University on March 4, 014 These data demonstrate that simpler and more efficient cryopreservation of bovine IVM/IVF/IVC blastocysts can be obtained by a vitrification method using EM grids (V-G). In a previous study (Park et al., 1998), we reported that in-vitro produced bovine blastocysts can be successfully cryopreserved by a simple two-step vitrification method using straws (V-S) and EFS40 freezing solution. However, in this study, the faster developed embryos in the V-G group tended to have a significantly higher survival in vitro compared with those in the V-S group after thawing. This difference was very high in early hatching blastocysts that re-expanded from a shrunken state caused by the osmotic difference between blastocoele fluid and culture medium following zona rupture. Almost all of the early hatching embryos vitrified using EM grids were hatched from their zonae 48 h after warming. This result indicated that hatching stage embryos were also efficiently utilized for future use. Of the expanded and early hatching blastocysts produced in vitro at day 7, using the V-G method, 67.8 and 95.0% hatched embryos were obtained 48 h after warming respectively. These embryo survival rates in vitro are reasonable compared to that of the Mamoudzadeh group (Mamoudzadeh et al., 1995) (68.8% hatched blastocysts/expanded blastocysts) observed 7 h after thawing. However, day 7 delayed blastocysts (early blastocyst) have been shown to be more sensitive to chilling injury than fast developing blastocysts (expanded blastocyst and early hatching blastocyst), irrespective of embryo con tainers. Their survival in vitro was significantly lower than that of the other two groups. However, to compare the survival between two container types, re-expansion of embryos in the V-G group seemed to be faster than that of the V-S group (data not shown).

6 from Hanwoo (Korean Native Cattle) IVM/IVF/IVC blastocyst I. Hanwoo IVM/IVF/IVC blastocyst cryopreserved by vitrification. Kor. J. Anim. Reprod.,, Park, S.P., Kim, E.Y., Yoon, S.H. et al. (1999) Enhanced hatching rate of bovine IVM/IVF/IVC blastocysts using a 1.48-m diode laser beam. J. Assist. Reprod. Genet., 16, Pollard, J.W. and Leibo, S.P. (1994) Chilling sensitivity of mammalian embryos. Theriogenology 41, Rall, W.F. and Fahy, G.M. (1985) Ice-free cryopreservation of mouse embryos at -196 C by vitrification. Nature, 313, Rosenkrans, C.F. Jr, Zeng, G.Q., McNamara, G.T. et al. (1993) Development of bovine embryos in vitro as affected by energy substrates. Biol. Reprod., 49, Steponkus, P.L. and Caldwell, S. (1993) An optimized procedure for the cryopreservation of Drosophila melanogaster embryos. Cryo-letters, 14, Tachikawa, S., Otoi, T., Kondo, S. et al. (1993) Successful vitrification of bovine blastocysts derived by in vitro maturation and fertilization. Mol. Reprod. Dev., 34, Vajta, G., Holm, P., Kuwayama, M. et al. (1998) Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev., 51, Voelkel, S.A. and Hu, Y.X. (199) Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient female. Theriogenology, 37, Received on April 3, 1999; accepted on August 1, 1999 Downloaded from at Pennsylvania State University on March 4,