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Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)
Animal Reproduction Science 73 (2002) 151–158
Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP) Seong-Keun Cho a , Seong-Gyun Cho b , In-Hyu Bae b , Choong-Saeng Park a , Il-Keun Kong b,∗ a
Department of Animal Science, Gyeongsang National University, Chinju 660-701, South Korea b Department of Animal Science, Sunchon National University, Sunchon 540-742, South Korea
Received 26 June 2001; received in revised form 25 June 2002; accepted 25 June 2002
Abstract The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of in vitro-produced (IVP) bovine blastocysts and to compare the results with post-thaw survival rate of bovine blastocysts frozen in GMP with those frozen in open pulled straw (OPS) that have been previously investigated. The GMP vessel permitted higher freezing and warming rates than the OPS due to the higher heat conductivity of the glass and the lower mass of the solution that contained the embryos. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into either the OPS or GMP vessels and they immersed into LN2 within 20–25 s. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in a holding medium (HM: D-PBS supplemented with 5% FCS) and then in TCM-199 for 5 min in both cases. They was then cultured in TCM 199 supplemented with 10% FCS for 24 or 48 h. The rate of blastocyst re-expansion was significantly different between OPS (79.6%) and GMP (90.4%) methods. Neither was the hatching rate significantly different among OPS (51.8%), GMP (57.1%) methods and non-vitrified group (67.3%). Only the rate of post-thaw re-expanding of blastocysts loaded in narrow column was significantly higher than that of the wide column (83.3% versus 56.7%) (P < 0.05), although the GMP straw was loaded with three blastocysts per vessel.
∗ Correspondence author. Tel.: +82-61-750-3236; fax: +82-61-750-3208. E-mail address: [email protected] (I.-K. Kong).
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 2 ) 0 0 1 3 2 - X
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These results indicated that the GMP vessels provided high survival rates of bovine IVP blastocysts. The location of the embryos loaded into a narrow or wide portion was considered to be a limiting factor to the viability of bovine IVP embryos. © 2002 Elsevier Science B.V. All rights reserved. Keywords: GMP vitrification vessel; Cattle-reproductive technology; In vitro-produced blastocyst
1. Introduction Cryopreservation has allowed biological materials to be stored indefinitely without loss of functional activity and without genetic alteration (Whittingham, 1980; Mazur, 1984). The first successful cryopreservation of mammalian zygotes and embryos resulting in live births was achieved with mice (Whittingham et al., 1972). The first calf born from a frozen embryo was reported by Wilmut and Rowson (1973). Considerable progress has improved the methods for cryopreservation of in vivo-produced embryos (Fahning and Garcia, 1992; Niemann, 1991). With various new methods published (Niemann, 1991; Rall, 1992). Vitrification has been widely used and is now regarded as an alternative to transitional slow-rate freezing. There have been reports of successful embryo vitrification with various cryoprotectants in several species with the vitrification solution 3a solution (Rall and Wood, 1994; Dinnyes et al., 1995), the ethylene glycol/ ficoll/sucrose method (Kasai et al., 1990; Tachikawa et al., 1993), the ethylene glycol/polyvinyl pyrrolidone method (Leibo and Oda, 1993) and the ethylene glycol/dimethyl sulfoxide method (EDS: Vajta et al., 1998a). Vitrification is the solidification of the solution brought about but by elevation in viscosity during cooling not by crystalization. When embryos are cryopreserved by vitrification, ice crystal formation should be prevented by use of a high concentration of cryoprotectants and high cooling and warming rates. Other aspects of successful vitrification could include the use of biological cryoprotectants (Storey and Storey, 1990), equilibration at lower temperatures (Rall and Fahy, 1985) or shorter exposure time to the cryoprotectant at room temperature, and the use of the lowest concentration of cryoprotectant possible for a specific freezing protocol (Boutron, 1990). Acceleration of the speed of temperature changes may offer two advantages: it should permit use of lower concentrations of cryoprotectants with consequent reduction in toxicity and result in less severe chilling injury with the rapid passage through the “dangerous” temperature zone (Vajta et al., 1998a). The efficiency of vitrifying embryos has been markedly improved by increasing the speed of cooling and warming. Three techniques have been established for this purpose: direct immersion into liquid nitrogen (Landa and Tepla, 1990; Riha et al., 1991), using an electron microscopy grid to provide a protection (Martino et al., 1996) and the OPS method (Hurtt et al., 1999; Lewis et al., 1999; Vajta et al., 1998b). Vajta et al. (1998a) demonstrated that the OPS method can be raised with cooling and warming rates (over 20,000 C◦ /min) and decreasing toxic and osmotic damage. Although the OPS method is has been useful and easy, it has the disadvantage of lower heat conductivity and a larger volumes of the frozen sample than that of the GMP vessel from leading decreased freezing speed and possible damage to the embryo.
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To overcome this problem, we have investigated the use of a glass micropipette vessel for vitrification, which may offer even faster cooling and warming rates than the OPS vessel in both intact and vitrified blastocyst embryos.
2. Materials and methods 2.1. Reagents and culture media The inorganic salts were analytical grade from Mallinckrodt, Paris, Kentucky. Fetal bovine serum (FBS) and packaged media were from HyClone Laboratories, Logan, Utah. All of the other reagents were purchased from Sigma, St. Louis, Missouri, unless otherwise noted. “Embryo culture tested” grade was used whenever possible. 2.2. In vitro embryo production Bovine ovaries were obtained from a local abattoir. Cumulus-oocyte complexes were aspirated from 2 to 8 mm diameter antral follicles with an 18 gauge hypodermic needle, selected for an envelope of compact and complete cumulus cells, and then washed three times in HEPES-buffered Tyrode’s medium (Fissore et al., 1992) supplemented with 3 mg/ml BSA, 0.2 mM pyruvate, 100 IU/ml penicillin, 100 g/ml streptomycin (TL-HEPES). Oocytes were transferred into 500 l TCM 199 maturation medium containing 10% FBS, 0.5 g/ml bovine FSH, 5 g/ml bovine LH, 100 IU/ml penicillin, 100 g/ml streptomycin (M199) in 4-well culture dishes and cultured at 39 ◦ C in a humidified atmosphere of 5% CO2 and air for 24 h. Fertilization was initiated 23 h after onset of maturation. This was counted as day 0. Spermatozoa were prepared for IVF as previously described (Parrish et al., 1986). Cryopreserved semen was thawed in water at 37 ◦ C and transferred into 10 ml PBS for washing by centrifugation. The sperm was then capacitated with 400 l of 50 ng/ml heparin solution for 15 min at 39 ◦ C. The capacitated sperm were diluted with TL-FERT (Parrish et al., 1986) medium to approximately 1–2 × 106 sperms/ml in drops containing the oocytes. Embryos were cultured in hamster embryo culture medium-6 (HECM-6; McKieman et al., 1995) containing amino acids and 4 mg/ml BSA, 3 mg/ml PVA, 150 g/ml sodium citrate and/or 500 g/ml myo-inositol for 72 h after insemination. The cleaved embryos were counted after 3 days in culture. Those embryos that had cleaved beyond the 2-cell stage were cultured in TCM199 supplemented with 10% FCS on 30 mm dish with 50 l volume and approximately 30 embryos with paraffin oil overlay. Embryos that reached the expanded blastocyst stage at day 8 after insemination (initiation of insemination on day 0) were recovered on this day and randomly assigned to either vitrification or assisted hatching. 2.3. Making of OPS and GMP straw OPS straws were made with French mini-straws (0.25 ml, IMV, L’Aigle, France) at our laboratory as described by Vajta et al. (1998a). Briefly, French mini-straws were heat-softened over a hot plate, and pulled manually until the inner diameter and the wall thickness of the central part decreased from 1.7 mm to approximately 0.8 mm. The straws
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were cooled in air and then cut at the narrowest point with a razor blade. The GMP vessels were each constructed from a capillary glass pipette (outer/inner diameter (o.d./i.d.): 1.0/0.8 mm; Drummond Science Co., USA) by the method of Vajta et al. (1998a) with minor modifications. The capillary glass pipettes were pulled with a pipette puller (Narishige, Japan) until the o.d. of the central part decreased from 1.0 mm to approximately 0.3 mm. The GMP vessels were cooled in air and broken at the narrowest point after scribing with a diamond tip pen. If the volume loaded into 10 mm length of narrow column was calculated by 4/3 × r 3 × 10 (Takahashi and Kanagawa, 1990), the volume of OPS and GMP straws loaded into narrow column were 2.68 and 0.14 mm3 per straw, respectively. Every straw was sterilized by flushing with 70% ethanol and dried on a clean bench. 2.4. Vitrification procedure The vitrification solution consisted of VS1 {10% ethylene glycol (EG), 10% DMSO in HM} and VS2 {16.5% EG, 16.5% DMSO in HM}. The blastocysts collected were vitrified using EDS as reported previously (Vajta et al., 1998a). In brief, the embryos were first incubated in VS1 for 1 min, and then transferred within approximately 1–2 l VS1 solution into a 20 l droplet of VS2. Embryos were mixed quickly by pipetting with another drop containing approximately 1–2 l VS2 solution and embryos made using a 10 l automatic pipette. Loading and cooling were performed as described by Vajta et al. (1998a). The time between the contact of the embryos with the concentrated cryoprotectant solution and cooling did not exceed 25 s. In the GMP vitrification, the researcher has to carefully control the capillary reaction with a finger as the GMP was ultrasensitive to the capillary effect. The type of loading column containing embryos and vitrification solution was important to post-thaw survival of bovine IVP blastocysts. The loaded OPS or GMP straws were placed into LN2 first, almost horizontal for a few seconds, and then vertically immersed in the LN2 . Warming was performed by immersing the end of the straw containing the embryos into 1.0 ml of 0.25 M sucrose in HM prepared in a 4-well dish. After 1 min, the embryos were transferred into 1.0 ml of 0.15 M sucrose in HM for another 5 min, and then washed with HM twice for 5 min each time. The temperature of the media used for warming was held to approximately 35◦ C. Survival of cryopreserved embryos was determined by development to re-expanding or hatching blastocysts during in vitro culture for 24 or 48 h, respectively. The embryos were cultured in TCM199 supplemented with 10% FCS in humidified atmosphere of 5% CO2 at 39◦ C. 2.5. Statistics Data were analyzed by a General Linear Model technique (SAS, 1990). Statistical significance was established at the P < 0.05 level. 3. Results To determine the optimal vitrification vessels, the blastocysts were vitrified with in OPS or a GMP straw with VS2 vitrification solution. The post-thaw re-expanding rate
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Table 1 Effect of OPS or GMP vessels on post-thaw survival rates of vitrified IVP bovine blastocysts Vitrification vessels
OPS GMP Non-vitrified
No. of embryos developed to (%) Vitrified
Re-expanding
Hatching
54 63 49
43 (79.6) b 57 (90.4) a
28 (51.8) a 36 (57.1) a 33 (67.3) a
(a, b) Values with same letters in same column were not significantly different (P < 0.05). Table 2 Effect of type of loading column on post-thaw survival rate following GMP vitrification Types of loading column
Narrow column only Wide column
No. of embryos developed to (%) Loaded
Replicated
Re-expanding
30 30
10 10
25 (83.3) a 17 (56.7) b
Medium fills only the narrow column in the narrow column and then fills both narrow and wide in the wide column. (a, b) Values with different letters in column were significantly different (P < 0.05).
was significantly different between OPS and GMP vessels (79.6%: 43/54 versus 90.4%: 57/63), respectively (Table 1). The hatching rate was not significantly different among OPS, GMP and non-vitrified groups (51.8%: 28/54; 57.1%: 36/63; 67.3%: 33/49) (P > 0.05), respectively. These experiments were all conducted with three blastocyst loaded per straw. The blastocyst re-expansion rates were significantly different for the narrow and wide column types of column (83.3%: 25/30 versus 56.7%: 17/30, P < 0.05), although the embryos loaded per GMP vessels were limited to three blastocysts (Table 2).
4. Discussion The OPS and GMP vitrification vessels with VS2 vitrification solution were successfully for the cryopreservation of IVP bovine blastocysts. Re-expanding rates with OPS and GMP methods were different (79.6 and 90.4%; P < 0.05). Hatching rate was also not significantly different among OPS, GMP and non-vitrified group (51.8, 57.1 and 67.3%; P > 0.05). Although the GMP method was not significantly higher than the OPS method, it had the advantage of higher heat conductivity and a smaller volume of the frozen sample than that of OPS vessel. A problem with the OPS method was the straw floating in LN2 necessitating a cap to protect the floating of OPS vessel. The problem was overcome and cap is not necessary in GMP vessel because of higher density of the glass straw. However, there were no problem from a glass micropipette being more likely to break and resulting in loss of frozen embryos. In addition, the GMP straw may increase the freezing speed due to diminished pipette size and loading column, if only the narrow part of the pipette was filled. If this could increased, the post-thaw survival rates might be improved following vitrification of embryos of species that are far more difficult to cryopreserve.
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There is a limitation on how small a diameter can be formed with the OPS straw. They become fragile and often tear or break near the narrowest point, if OPS straw is pulled to under 0.8 mm o.d. However, the size of glass pipette can be decreased to approximately 0.3 mm o.d. and 0.26 mm i.d. with approximately 0.02 mm wall thickness. This would be three to four diameters larger than mouse embryos and approximately two diameters larger than cattle embryos. The weight of a GMP straw was also a little heavier rather than an OPS straw (0.098 g versus 0.070 g). When pulled as indicated, the volume of a GMP straw would be 19 times less than an OPS straw (0.14 mm3 versus 2.68 mm3 ), when the volume loaded was calculated for the 10 mm length of narrow column. Although the smaller size of a GMP straw could increase the freezing speed, it was more sensitive to the loading of embryos and vitrification solution by capillary reaction. Using the GMP method requires carefully loading of the embryo and vitrification solution by controlling capillarity with a finger. As shown in Table 2, the re-expansion rate in the narrow column was significantly higher than that of wide column (83.3% versus 56.7%; P < 0.05). The loading column is important to the viability of a vitrified blastocyst. Careful loading of the GMP straw might increase the speed of freezing or warming and decrease the loading volume and the damage of embryos. It is important in the GMP method to ensure that the embryos are loaded in the narrow column only. Loading more than three embryos in a GMP straw was detrimental to survival. Although the number of embryos loaded per straw may affect of type of loading column, the loading of fewer than three blastocysts per straw resulted in 83.3% survival rates in this study. The more embryos loaded per OPS vessel increased the probability of one being in the wide column. It is possible that increased numbers of embryos in the straw would space them outside of the narrowest portion of the fluid column resulting in slower freezing and cooling and decreased embryo survival. Kong et al. (2000) reported that vitrify unit less than four mouse embryos filled the narrow column; over six embryos increased the possibility of also filling the wide column. There was a high interaction between embryo numbers loaded per GMP vessels and type of loading column. If the number of the loaded embryos exceed four per GMP vessel, they may settle in the wide column, decreasing the freezing speed and viability of the vitrified embryos. They were limited to under three bovine blastocysts per GMP vessel in this study, because of better results with fewer than three embryos per GMP vessels. Vajta et al. (1998a) demonstrated that the OPS method with high cooling and warming rates (over −20,000 ◦ C/min) and short contact with concentrated cryoprotective additives before freezing (<30 s) offered the possibility to circumvent chilling injury and decreasing toxic and osmotic damage. The GMP method can achieve even more rapid freezing speed and heat conductivity, because of the capillary glass pipette. A disadvantage of both systems as well as the electron microscopy grid system is that there is the potential hazard of contamination as the embryo holding medium is directly in contact with liquid nitrogen (Tedder et al., 1995; Vajta et al., 1998b). To overcome the contamination problem, Vajta et al. (1998b) reported that the whole vitrification procedure should be performed in a laminar hood using filtered liquid nitrogen for cooling. The embryo-containing OPS would be then a placed into 0.5 ml plastic straw pre-cooled in the vapor of liquid nitrogen. At warming, the large straw would be kept vertical and only half-immersed in the liquid nitrogen, before the end was cut and then the OPS quickly removed and warmed directly in the culture medium.
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5. Conclusion The GMP method has been shown to increase the speed of freezing and warming, heat conductivity, post-thaw survival rates, and to decrease the loading volume and embryo damage by reducing straw size and the loading column.
Acknowledgements This research supported by Sunchon National University Non-directed Research Fund, 2000. References Boutron, P., 1990. Levo- and dextro-2,3 butanediol and their racemic mixture: very efficient solutes for vitrification. Cryobiology 27, 508–514. Dinnyes, A., Wallace, G.A., Rall, W.F., 1995. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods. Mol. Reprod. Dev. 40, 429–435. Fissore, R.A., Dobrinsky, J.R., Balise, J.J., Duby, R.T., Robl, J.M., 1992. Patterns of intracellular Ca2+ concentration in fertilized bovine eggs. Biol. Reprod. 47, 960–969. Fahning, M.L., Garcia, M.A., 1992. Status of cryopreservation of embryos from domestic animals. Cryobiology 29, 1–18. Hurtt, A.E., Squires, E.L., Seidel Jr., G.E., 1999. Vitrification of equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open pulled straws, ficoll and sucrose solution using open pulled straws. Theriogenology 51, 166 (Abstract). Kasai, M., Komi, J.H., Takakamo, A., Tsunoda, H., Sakurai, T., Machida, T., 1990. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution without appreciable loss of viability. J. Reprod. Fertil. 89, 91–97. Kong, I.K., Cho, S.G., Lee, S.I., Cho, S.K., 2000. Comparison of open pulled straw (OPS) versus open pulled capillary glass pipette (OPP) vitrification in mouse embryos. Theriogenology 53, 1817–1826. Landa, V., Tepla, O., 1990. Cryopreservation of mouse 8-cell embryos in microdrops. Folia Biol. (Praha) 36, 153–158. Leibo, S.P., Oda, K., 1993. High survival of mouse zygotes and embryos cooled rapidly or slowly in ethylene glycol plus polyvinylpyrrolidone. Cryobiol. Lett. 14, 133–144. Lewis, I.M., Lane, M.W., Vajta, G., 1999. Pregnancy rate following transfer of in vitro-produced bovine embryos vitrified by the open pulled straw (OPS) method. Theriogenology 51, 168 (Abstract). Martino, A., Songsasen, N., Leibo, S.P., 1996. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol. Reprod. 54, 1059–1069. Mazur, P., 1984. Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247, 125–142. McKieman, S.H., Clayton, M.K., Bavister, B.D., 1995. Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro. Mol. Reprod. Dev. 42, 188–199. Niemann, H., 1991. Cryopreservation of ova and embryos from livestock: current status and research needs. Theriogenology 35, 109–124. Parrish, J.J., Susko-Parrish, J.L., Leibfried-Rutledge, M.L., Crister, E.S., Eyestone, W.H., First, N.L., 1986. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 35, 547–555. Rall, W.F., Fahy, G.M., 1985. Ice-free cryopreservation of mouse embryos at −196 ◦ C by vitrification. Nature 313, 573–575. Rall, W.F., Wood, M.J., 1994. High in vitro and in vivo survival of day 3 mouse embryos vitrified or frozen in a non-toxic solution of glycerol and albumin. J. Reprod. Fertil. 101, 681–688. Rall, W.F., 1992. Cryopreservation of oocytes and embryos: methods and applications. Anim. Reprod. 28, 237–245.
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Riha, J., Landa, V., Kneissl, J., Matus, J., Jindra, J., Kloucek, Z., 1991. Vitrification of cattle embryos by direct dropping into liquid nitrogen and embryo survival after non-surgical transfer. Zivoc. Vir. 36, 113–120. SAS Institute Inc., 1990. The GLM procedure. SAS/STAT User’s Guide, Version 6, 4th Edition, Vol. 2, SAS Institute Inc., Cary, NC, USA, pp. 891–996. Storey, K.B., Storey, J.M., 1990. Frozen and alive. Sci. Am. 12, 92–97. 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. Takahashi, Y., Kanagawa, H., 1990. Effect of equilibration period on the viability of frozen-thawed mouse morula after rapid freezing. Mol. Reprod. Dev. 26, 105–110. Tedder, R.S., Zuckerman, M.A., Goldstone, A.H., Hawkins, A.E., Fielding, A., Briggs, E.M., Irwin, D., Blair, S., Gorman, A.M., Patterson, K.G., Linch, D.C., Heptonstall, J., Brink, N.S., 1995. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 346, 137–140. Vajta, G., Holm, P., Kuwayama, M., Booth, P.J., Jacobsen, H., Greve, T., Callesen, H., 1998a. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev. 51, 53–58. Vajta, G., Lewis, I.M., Kuwayama, M., Greve, T., Callesen, H., 1998b. Sterile application of the open pulled straw (OPS) vitrification method. Cryobiol. Lett. 19, 389–392. Whittingham, D.G., Leibo, S.P., Mazur, P., 1972. Survival of mouse embryos frozen to −196 and −269 ◦ C. Science 178, 411–414. Whittingham, D.G., 1980. Principles of embryo preservation. In: Ashwood-Smith, M.J., Farrant, J. (Eds.), Low Temperature Preservation in Medicine and Biology. Pitman Medical Ltd., UK, pp. 65–83. Wilmut, I., Rowson, L.E.A., 1973. Experiments on the low-temperature preservation of cow embryos. Vet. Rec. 92, 686–690.
Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP) Seong-Keun Cho a , Seong-Gyun Cho b , In-Hyu Bae b , Choong-Saeng Park a , Il-Keun Kong b,∗ a
Department of Animal Science, Gyeongsang National University, Chinju 660-701, South Korea b Department of Animal Science, Sunchon National University, Sunchon 540-742, South Korea
Received 26 June 2001; received in revised form 25 June 2002; accepted 25 June 2002
Abstract The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of in vitro-produced (IVP) bovine blastocysts and to compare the results with post-thaw survival rate of bovine blastocysts frozen in GMP with those frozen in open pulled straw (OPS) that have been previously investigated. The GMP vessel permitted higher freezing and warming rates than the OPS due to the higher heat conductivity of the glass and the lower mass of the solution that contained the embryos. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into either the OPS or GMP vessels and they immersed into LN2 within 20–25 s. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in a holding medium (HM: D-PBS supplemented with 5% FCS) and then in TCM-199 for 5 min in both cases. They was then cultured in TCM 199 supplemented with 10% FCS for 24 or 48 h. The rate of blastocyst re-expansion was significantly different between OPS (79.6%) and GMP (90.4%) methods. Neither was the hatching rate significantly different among OPS (51.8%), GMP (57.1%) methods and non-vitrified group (67.3%). Only the rate of post-thaw re-expanding of blastocysts loaded in narrow column was significantly higher than that of the wide column (83.3% versus 56.7%) (P < 0.05), although the GMP straw was loaded with three blastocysts per vessel.
∗ Correspondence author. Tel.: +82-61-750-3236; fax: +82-61-750-3208. E-mail address: [email protected] (I.-K. Kong).
0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 2 ) 0 0 1 3 2 - X
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S.-K. Cho et al. / Animal Reproduction Science 73 (2002) 151–158
These results indicated that the GMP vessels provided high survival rates of bovine IVP blastocysts. The location of the embryos loaded into a narrow or wide portion was considered to be a limiting factor to the viability of bovine IVP embryos. © 2002 Elsevier Science B.V. All rights reserved. Keywords: GMP vitrification vessel; Cattle-reproductive technology; In vitro-produced blastocyst
1. Introduction Cryopreservation has allowed biological materials to be stored indefinitely without loss of functional activity and without genetic alteration (Whittingham, 1980; Mazur, 1984). The first successful cryopreservation of mammalian zygotes and embryos resulting in live births was achieved with mice (Whittingham et al., 1972). The first calf born from a frozen embryo was reported by Wilmut and Rowson (1973). Considerable progress has improved the methods for cryopreservation of in vivo-produced embryos (Fahning and Garcia, 1992; Niemann, 1991). With various new methods published (Niemann, 1991; Rall, 1992). Vitrification has been widely used and is now regarded as an alternative to transitional slow-rate freezing. There have been reports of successful embryo vitrification with various cryoprotectants in several species with the vitrification solution 3a solution (Rall and Wood, 1994; Dinnyes et al., 1995), the ethylene glycol/ ficoll/sucrose method (Kasai et al., 1990; Tachikawa et al., 1993), the ethylene glycol/polyvinyl pyrrolidone method (Leibo and Oda, 1993) and the ethylene glycol/dimethyl sulfoxide method (EDS: Vajta et al., 1998a). Vitrification is the solidification of the solution brought about but by elevation in viscosity during cooling not by crystalization. When embryos are cryopreserved by vitrification, ice crystal formation should be prevented by use of a high concentration of cryoprotectants and high cooling and warming rates. Other aspects of successful vitrification could include the use of biological cryoprotectants (Storey and Storey, 1990), equilibration at lower temperatures (Rall and Fahy, 1985) or shorter exposure time to the cryoprotectant at room temperature, and the use of the lowest concentration of cryoprotectant possible for a specific freezing protocol (Boutron, 1990). Acceleration of the speed of temperature changes may offer two advantages: it should permit use of lower concentrations of cryoprotectants with consequent reduction in toxicity and result in less severe chilling injury with the rapid passage through the “dangerous” temperature zone (Vajta et al., 1998a). The efficiency of vitrifying embryos has been markedly improved by increasing the speed of cooling and warming. Three techniques have been established for this purpose: direct immersion into liquid nitrogen (Landa and Tepla, 1990; Riha et al., 1991), using an electron microscopy grid to provide a protection (Martino et al., 1996) and the OPS method (Hurtt et al., 1999; Lewis et al., 1999; Vajta et al., 1998b). Vajta et al. (1998a) demonstrated that the OPS method can be raised with cooling and warming rates (over 20,000 C◦ /min) and decreasing toxic and osmotic damage. Although the OPS method is has been useful and easy, it has the disadvantage of lower heat conductivity and a larger volumes of the frozen sample than that of the GMP vessel from leading decreased freezing speed and possible damage to the embryo.
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To overcome this problem, we have investigated the use of a glass micropipette vessel for vitrification, which may offer even faster cooling and warming rates than the OPS vessel in both intact and vitrified blastocyst embryos.
2. Materials and methods 2.1. Reagents and culture media The inorganic salts were analytical grade from Mallinckrodt, Paris, Kentucky. Fetal bovine serum (FBS) and packaged media were from HyClone Laboratories, Logan, Utah. All of the other reagents were purchased from Sigma, St. Louis, Missouri, unless otherwise noted. “Embryo culture tested” grade was used whenever possible. 2.2. In vitro embryo production Bovine ovaries were obtained from a local abattoir. Cumulus-oocyte complexes were aspirated from 2 to 8 mm diameter antral follicles with an 18 gauge hypodermic needle, selected for an envelope of compact and complete cumulus cells, and then washed three times in HEPES-buffered Tyrode’s medium (Fissore et al., 1992) supplemented with 3 mg/ml BSA, 0.2 mM pyruvate, 100 IU/ml penicillin, 100 g/ml streptomycin (TL-HEPES). Oocytes were transferred into 500 l TCM 199 maturation medium containing 10% FBS, 0.5 g/ml bovine FSH, 5 g/ml bovine LH, 100 IU/ml penicillin, 100 g/ml streptomycin (M199) in 4-well culture dishes and cultured at 39 ◦ C in a humidified atmosphere of 5% CO2 and air for 24 h. Fertilization was initiated 23 h after onset of maturation. This was counted as day 0. Spermatozoa were prepared for IVF as previously described (Parrish et al., 1986). Cryopreserved semen was thawed in water at 37 ◦ C and transferred into 10 ml PBS for washing by centrifugation. The sperm was then capacitated with 400 l of 50 ng/ml heparin solution for 15 min at 39 ◦ C. The capacitated sperm were diluted with TL-FERT (Parrish et al., 1986) medium to approximately 1–2 × 106 sperms/ml in drops containing the oocytes. Embryos were cultured in hamster embryo culture medium-6 (HECM-6; McKieman et al., 1995) containing amino acids and 4 mg/ml BSA, 3 mg/ml PVA, 150 g/ml sodium citrate and/or 500 g/ml myo-inositol for 72 h after insemination. The cleaved embryos were counted after 3 days in culture. Those embryos that had cleaved beyond the 2-cell stage were cultured in TCM199 supplemented with 10% FCS on 30 mm dish with 50 l volume and approximately 30 embryos with paraffin oil overlay. Embryos that reached the expanded blastocyst stage at day 8 after insemination (initiation of insemination on day 0) were recovered on this day and randomly assigned to either vitrification or assisted hatching. 2.3. Making of OPS and GMP straw OPS straws were made with French mini-straws (0.25 ml, IMV, L’Aigle, France) at our laboratory as described by Vajta et al. (1998a). Briefly, French mini-straws were heat-softened over a hot plate, and pulled manually until the inner diameter and the wall thickness of the central part decreased from 1.7 mm to approximately 0.8 mm. The straws
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were cooled in air and then cut at the narrowest point with a razor blade. The GMP vessels were each constructed from a capillary glass pipette (outer/inner diameter (o.d./i.d.): 1.0/0.8 mm; Drummond Science Co., USA) by the method of Vajta et al. (1998a) with minor modifications. The capillary glass pipettes were pulled with a pipette puller (Narishige, Japan) until the o.d. of the central part decreased from 1.0 mm to approximately 0.3 mm. The GMP vessels were cooled in air and broken at the narrowest point after scribing with a diamond tip pen. If the volume loaded into 10 mm length of narrow column was calculated by 4/3 × r 3 × 10 (Takahashi and Kanagawa, 1990), the volume of OPS and GMP straws loaded into narrow column were 2.68 and 0.14 mm3 per straw, respectively. Every straw was sterilized by flushing with 70% ethanol and dried on a clean bench. 2.4. Vitrification procedure The vitrification solution consisted of VS1 {10% ethylene glycol (EG), 10% DMSO in HM} and VS2 {16.5% EG, 16.5% DMSO in HM}. The blastocysts collected were vitrified using EDS as reported previously (Vajta et al., 1998a). In brief, the embryos were first incubated in VS1 for 1 min, and then transferred within approximately 1–2 l VS1 solution into a 20 l droplet of VS2. Embryos were mixed quickly by pipetting with another drop containing approximately 1–2 l VS2 solution and embryos made using a 10 l automatic pipette. Loading and cooling were performed as described by Vajta et al. (1998a). The time between the contact of the embryos with the concentrated cryoprotectant solution and cooling did not exceed 25 s. In the GMP vitrification, the researcher has to carefully control the capillary reaction with a finger as the GMP was ultrasensitive to the capillary effect. The type of loading column containing embryos and vitrification solution was important to post-thaw survival of bovine IVP blastocysts. The loaded OPS or GMP straws were placed into LN2 first, almost horizontal for a few seconds, and then vertically immersed in the LN2 . Warming was performed by immersing the end of the straw containing the embryos into 1.0 ml of 0.25 M sucrose in HM prepared in a 4-well dish. After 1 min, the embryos were transferred into 1.0 ml of 0.15 M sucrose in HM for another 5 min, and then washed with HM twice for 5 min each time. The temperature of the media used for warming was held to approximately 35◦ C. Survival of cryopreserved embryos was determined by development to re-expanding or hatching blastocysts during in vitro culture for 24 or 48 h, respectively. The embryos were cultured in TCM199 supplemented with 10% FCS in humidified atmosphere of 5% CO2 at 39◦ C. 2.5. Statistics Data were analyzed by a General Linear Model technique (SAS, 1990). Statistical significance was established at the P < 0.05 level. 3. Results To determine the optimal vitrification vessels, the blastocysts were vitrified with in OPS or a GMP straw with VS2 vitrification solution. The post-thaw re-expanding rate
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Table 1 Effect of OPS or GMP vessels on post-thaw survival rates of vitrified IVP bovine blastocysts Vitrification vessels
OPS GMP Non-vitrified
No. of embryos developed to (%) Vitrified
Re-expanding
Hatching
54 63 49
43 (79.6) b 57 (90.4) a
28 (51.8) a 36 (57.1) a 33 (67.3) a
(a, b) Values with same letters in same column were not significantly different (P < 0.05). Table 2 Effect of type of loading column on post-thaw survival rate following GMP vitrification Types of loading column
Narrow column only Wide column
No. of embryos developed to (%) Loaded
Replicated
Re-expanding
30 30
10 10
25 (83.3) a 17 (56.7) b
Medium fills only the narrow column in the narrow column and then fills both narrow and wide in the wide column. (a, b) Values with different letters in column were significantly different (P < 0.05).
was significantly different between OPS and GMP vessels (79.6%: 43/54 versus 90.4%: 57/63), respectively (Table 1). The hatching rate was not significantly different among OPS, GMP and non-vitrified groups (51.8%: 28/54; 57.1%: 36/63; 67.3%: 33/49) (P > 0.05), respectively. These experiments were all conducted with three blastocyst loaded per straw. The blastocyst re-expansion rates were significantly different for the narrow and wide column types of column (83.3%: 25/30 versus 56.7%: 17/30, P < 0.05), although the embryos loaded per GMP vessels were limited to three blastocysts (Table 2).
4. Discussion The OPS and GMP vitrification vessels with VS2 vitrification solution were successfully for the cryopreservation of IVP bovine blastocysts. Re-expanding rates with OPS and GMP methods were different (79.6 and 90.4%; P < 0.05). Hatching rate was also not significantly different among OPS, GMP and non-vitrified group (51.8, 57.1 and 67.3%; P > 0.05). Although the GMP method was not significantly higher than the OPS method, it had the advantage of higher heat conductivity and a smaller volume of the frozen sample than that of OPS vessel. A problem with the OPS method was the straw floating in LN2 necessitating a cap to protect the floating of OPS vessel. The problem was overcome and cap is not necessary in GMP vessel because of higher density of the glass straw. However, there were no problem from a glass micropipette being more likely to break and resulting in loss of frozen embryos. In addition, the GMP straw may increase the freezing speed due to diminished pipette size and loading column, if only the narrow part of the pipette was filled. If this could increased, the post-thaw survival rates might be improved following vitrification of embryos of species that are far more difficult to cryopreserve.
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There is a limitation on how small a diameter can be formed with the OPS straw. They become fragile and often tear or break near the narrowest point, if OPS straw is pulled to under 0.8 mm o.d. However, the size of glass pipette can be decreased to approximately 0.3 mm o.d. and 0.26 mm i.d. with approximately 0.02 mm wall thickness. This would be three to four diameters larger than mouse embryos and approximately two diameters larger than cattle embryos. The weight of a GMP straw was also a little heavier rather than an OPS straw (0.098 g versus 0.070 g). When pulled as indicated, the volume of a GMP straw would be 19 times less than an OPS straw (0.14 mm3 versus 2.68 mm3 ), when the volume loaded was calculated for the 10 mm length of narrow column. Although the smaller size of a GMP straw could increase the freezing speed, it was more sensitive to the loading of embryos and vitrification solution by capillary reaction. Using the GMP method requires carefully loading of the embryo and vitrification solution by controlling capillarity with a finger. As shown in Table 2, the re-expansion rate in the narrow column was significantly higher than that of wide column (83.3% versus 56.7%; P < 0.05). The loading column is important to the viability of a vitrified blastocyst. Careful loading of the GMP straw might increase the speed of freezing or warming and decrease the loading volume and the damage of embryos. It is important in the GMP method to ensure that the embryos are loaded in the narrow column only. Loading more than three embryos in a GMP straw was detrimental to survival. Although the number of embryos loaded per straw may affect of type of loading column, the loading of fewer than three blastocysts per straw resulted in 83.3% survival rates in this study. The more embryos loaded per OPS vessel increased the probability of one being in the wide column. It is possible that increased numbers of embryos in the straw would space them outside of the narrowest portion of the fluid column resulting in slower freezing and cooling and decreased embryo survival. Kong et al. (2000) reported that vitrify unit less than four mouse embryos filled the narrow column; over six embryos increased the possibility of also filling the wide column. There was a high interaction between embryo numbers loaded per GMP vessels and type of loading column. If the number of the loaded embryos exceed four per GMP vessel, they may settle in the wide column, decreasing the freezing speed and viability of the vitrified embryos. They were limited to under three bovine blastocysts per GMP vessel in this study, because of better results with fewer than three embryos per GMP vessels. Vajta et al. (1998a) demonstrated that the OPS method with high cooling and warming rates (over −20,000 ◦ C/min) and short contact with concentrated cryoprotective additives before freezing (<30 s) offered the possibility to circumvent chilling injury and decreasing toxic and osmotic damage. The GMP method can achieve even more rapid freezing speed and heat conductivity, because of the capillary glass pipette. A disadvantage of both systems as well as the electron microscopy grid system is that there is the potential hazard of contamination as the embryo holding medium is directly in contact with liquid nitrogen (Tedder et al., 1995; Vajta et al., 1998b). To overcome the contamination problem, Vajta et al. (1998b) reported that the whole vitrification procedure should be performed in a laminar hood using filtered liquid nitrogen for cooling. The embryo-containing OPS would be then a placed into 0.5 ml plastic straw pre-cooled in the vapor of liquid nitrogen. At warming, the large straw would be kept vertical and only half-immersed in the liquid nitrogen, before the end was cut and then the OPS quickly removed and warmed directly in the culture medium.
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5. Conclusion The GMP method has been shown to increase the speed of freezing and warming, heat conductivity, post-thaw survival rates, and to decrease the loading volume and embryo damage by reducing straw size and the loading column.
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