1 Maria Infertility Medical Institute, 10311 Sinseol-dong Dongdaemun-Gu, Seoul 130110, 2 Hanwoo Improvement Center, NLCF, Seosan, Chungnam 356830, 3 Maria Infertility Clinic, Sinseol-dong Dongdaemun-Gu, Seoul 130110, 4 Department of Animal Sciences, Kon-Kuk University, Mojin-dong, Kwangjin-gu, Seoul 143701, Korea
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Abstract |
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Key words: bovine blastocysts/in-vitro survival/EM grid/plastic straw/vitrification
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Introduction |
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In addition, to prevent the chilling injury during bovine 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
).
On the other hand, successful results from the vitrification of in-vitro derived bovine blastocysts have been demonstrated by many researchers (Kuwamaya et al., 1992; 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 vitrifiedwarmed 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.
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Materials and methods |
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Production of bovine blastocysts
The culture procedures employed in the production of preimplantation embryos from bovine follicular oocytes were as previously outlined (Park et al., 1999). Briefly, ovaries were obtained from a slaughterhouse and cumulusoocyte complexes (COC) were aspirated from visible follicles (26 mm in diameter). The COC were then washed with HEPES-buffered Tyrode's medium and cultured in maturation medium composed of TCM199 + 10% fetal bovine serum (FBS) supplemented with 0.2 mmol/l sodium pyruvate, 1 µg/ml follicle-stimulating hormone, 1 µg/ml oestradiol-17ß, and 25 µg/ml gentamycin sulphate at 39°C, 5% CO2 incubator. After incubation for 2224 h in IVM medium, the COC were inseminated using highly motile spermatozoa recovered from frozenthawed bull semen separated on a discontinuous Percoll column. Fertilization was assessed as cleavage rate (
2-cell) after 44 ± 2 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 two-thirds 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.
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.
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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 (LN2). Before exposure to the vitrification solution, embryos (Figure 1A
) were partially equilibrated in EG20 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 LN2 within 30 s. The mean number of blastocysts loaded on one grid was 810.
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Thawing procedures
Grids
Cryoprotectants in vitrifiedthawed embryos were removed in two steps. After a few hours or days of storage in LN2, embryos were warmed ultra-rapidly. EM grids stored in LN2 were directly transferred into 0.3 M sucrose solution (prepared in m-DPBS containing 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 µl) 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 25°C water bath for 1015 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 0.8ml of m-DPBS containing 10% FBS for 5 min. After dilution, the embryos were co-cultured in the same conditions as the grid embryos for 48 h.
Assessment of embryo survival
The post-thawing survival of embryos was observed every 24 h under a microscope and embryos were judged as morphological survivors if they expanded into blastocysts within the first 24 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 LN2 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 2 test using the Statistical Analysis System (SAS) Institute software package (SAS Institute Inc., 1985).
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Results |
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Discussion |
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Embryo cryopreservation using EM grids was originally designed for the vitrification of exceedingly chill-sensitive Drosophila embryos (Mazur et al., 1992; 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 of bovine 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 vitrifiedthawed 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, 1992). 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, 1992
; 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. 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 72 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 containers. 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).
In addition, cryoprotective agents were intimately related to embryo survival (Tachikawa et al., 1993). Since 1990, 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.
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Notes |
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References |
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Submitted on April 23, 1999; accepted on August 12, 1999.