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Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived
Theriogenology 62 (2004) 481–493
Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived M. Dattenaa,*, C. Accardoa, S. Pilichia, V. Isachenkob, L. Maraa, B. Chessac, P. Cappaia a
Istituto Zootecnico e Caseario per la Sardegna, 07040 Olmedo, Sassari, Italy b Department of Gynecology, Endocrinology and Reproductive Medicine, University of Bonn, 53127 Bonn, Germany c Facolta` di Medicina Veterinaria, Facolta` di Medicina Veterinaria, Universita` di Sassari, 07100 Sassari, Italy
Received 18 June 2003; received in revised form 30 September 2003; accepted 25 October 2003
Abstract We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G þ 20% ethylene glycol (EG), then for 30 s in 25% G þ 25% EG (glycerol group), or for 3 min in 10% EG þ 10% dimethyl sulphoxide (DMSO), then for 30 s in 20% EG þ 20% DMSO þ 0:3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 ðTCM-199Þ þ 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of
* Corresponding author. Tel.: þ39-079-387268; fax: þ39-079-389450. E-mail address: [email protected] (M. Dattena).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.10.010
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IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols. # 2003 Elsevier Inc. All rights reserved. Keywords: Sheep embryos; In vitro produced; In vivo derived; Vitrification protocols; Lambing
1. Introduction Controlled slow freezing continues to be the most widely used technique of cryopreservation for in vivo and in vitro produced embryos. However, in the last decade the vitrification technique, a cryopreservation system involving the addition of higher concentrations of cryoprotectants and very rapid cooling [1], has been tested in different species with good results [2–8]. Many different vitrification protocols have been used to cryopreserve in vivo and in vitro produced embryos in the last 15 years. They differ in many ways, including type and concentration of cryoprotectant, number of equilibration steps, type of cryopreservation device used, time of exposure and number of dilution steps at warming [6,9–15], and it is widely believed that each of these factors can affect the results. The requirements of a good cryoprotectant are permeability and low toxicity. The most widely used vitrification cryoprotectants are glycerol, ethylene glycol, propylene glycol and dimethyl sulphoxide, employed in different combinations and concentrations, sometimes supplemented with non-permeable solutes such as sucrose, threalose etc. [1,6,13, 16–21]. The equilibration procedures can differ among the protocols either in the number of steps (one to three) or time of exposure (5–10 min), while exposure to the vitrification solution requires less than 1 min [6–8,20–24]. All of the above mentioned procedures together with the choice and concentration of cryoprotectant aim to achieve optimal intracellular dehydration and thereby avoid ice crystal formation. Glass ampules were originally used to store frozen cattle embryos [25]. In the 1980’s, the plastic straw became the container of choice for vitrification and storage of embryos [1,16]. A recent modification, the open pulled straw (OPS), reduces the volume of vitrification solution and allows direct contact between the embryo and liquid nitrogen, thus enhancing the cooling rate [6,15,26]. In fact, it is well known that minimum drop size and a rapid cooling rate are very important factors to improve vitrification results [6,27–29]. After warming, embryos are usually placed in a hypertonic solution to remove the permeating cryoprotectants before transferring them to an isotonic culture medium. Many authors report the use of one or more dilution steps with different concentrations of sucrose, which controls the degree of swelling during cryoprotectant removal [20,30,31]. However, the possibility of direct post-warming rehydration in an isotonic solution has been described [20,32,33]. This paper summarizes results obtained in our laboratory over the past several years, comparing five vitrification protocols carried out on in vitro produced and in vivo derived embryos. When it was possible, type of cryoprotectants (glycerol and DMSO), devices
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(French straw and open pulled straw), numbers of dilution-steps and type of diluent at warming were compared.
2. Materials and methods Except where otherwise indicated, all chemicals were obtained from Sigma Chemicals Company (St. Louis, MO, USA). 2.1. In vitro embryo production Ovaries were collected immediately after slaughter and transported to the laboratory in saline at approximately 35 8C within 1–2 h. Oocytes were obtained by aspiration of follicles using a 20-gauge needle fitted with a 2 ml syringe. Follicular oocytes covered by at least 2 layers of granulosa cells and with an evenly granulated cytoplasm were selected for IVM. The maturation medium was bicarbonate-buffered TCM-199 (osmolarity adjusted to 275 mOsm), containing 2 mM of glutamine, 10% FBS, 5 mg/ml follicle stimulating hormone (FSH) (Ovagen, ICP, Auckland, New Zealand), 5 mg/ml luteinizing hormone (LH), 1 mg/ml estradiol, 0.3 mM sodium pyruvate and 100 mM cysteamine. The oocytes were incubated in 400 ml of medium in four-well dishes (Nunc, Nunclon, Denmark) covered with mineral oil. In vitro maturation conditions were 5% CO2 in humidified air at 39 8C for 24 h. Following maturation, the oocytes were partially denuded of granulosa cells by gently pipetting in Hepes-TCM-199 (H-TCM-199) with 300 IU/ml of hyaluronidase. Fresh semen from a Sarda breed ram of proven fertility was used throughout the experiment. Collected ejaculate was kept at room temperature for up to 2 h, then washed in bicarbonatebuffered synthetic oviduct fluid (SOF) [34] that was enriched with 20% (v/v) heat inactivated estrous sheep serum. The washed ejaculate was centrifuged twice at 200 g for 5 min and directly added to the fertilization medium. Fertilization was conducted in 50 ml drops with 1 106 sperm/ml and a maximum of 15 oocytes per drop, at 39 8C with 5% CO2 in humidified air for 20 h. The presumptive zygotes were allocated to 20 ml-culture drops (4–5 embryos/drop), consisting of SOF supplemented with 1% (v/v) Basal Medium Eagle (BME)–essential amino acids, 1% (v/v) minimum essential medium (MEM)–non-essential amino acids, 1 mM glutamine and 8 mg/ml fatty acid-free bovine serum albumine (BSA). Embryos were incubated in an atmosphere of 7% O2, 5% CO2, 88% N2 at 39 8C at 100% humidity. Charcoal stripped FBS was added to the medium on the third and fifth day of culture (where day 0 was the day of fertilization). Culture was continued until 6–7 days post fertilization at which time the expanded blastocysts (n ¼ 225) were divided in five different vitrification protocols as follows: (A) Glycerol þ French straw þ three dilution-steps (n ¼ 68) þ transfer after blastocysts re-expansion (n ¼ 46); (B) Glycerol þ French straw þ one dilution-step þ direct transfer after warming (n ¼ 42); (C) Glycerol þ OPS þ one dilution-step þ direct transfer after warming (n ¼ 58);
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(D) DMSO þ 0:3 M sucrose þ OPS þ one dilution-step þ direct transfer after warming (n ¼ 36); (E) DMSO þ 0:75 M sucrose þ OPS þ direct dilution þ direct transfer after warming (n ¼ 21). 2.2. In vivo derived embryos The estrous cycles of Sarda ewes were synchronized by insertion of intravaginal sponges containing 40 mg fluorogestone acetate (Intervet, NL) for 14 days. The day before removal of the sponges, the ewes were superovulated with 16 mg porcine follicle stimulating hormone (p-FSH; Pluset, Calier, Italy) administered intramuscularly every 12 h in four decreasing doses (6, 5, 3 and 2 mg). The ewes were inseminated 48 h after sponge removal by intrauterine insemination with fresh semen from a fertile ram. Embryos were collected from the donors under general anaesthesia (Pentothal sodium, Gellini, Italy) 7 days after the start of estrus. Each uterine horn was flushed using a Foley catheter as described by Tervit and Havick [35]. The collection medium was HTCM-199 supplemented with 4 mg/ ml BSA. The embryos were classified according to their stage of development. All the 197 expanded blastocysts obtained in several collections were divided into four groups and subjected to one of the following vitrification protocols: (F) Glycerol þ French straw þ three dilution-steps (n ¼ 62) þ transfer after blastocysts re-expansion (n ¼ 52); (G) Glycerol þ OPS þ three dilution-steps (n ¼ 50) þ transfer after re-expansion (n ¼ 40); (H) Glycerol þ OPS þ one dilution-step (n ¼ 42) þ transfer after re-expansion (n ¼ 32); (I) DMSO 0.3 M sucrose þ OPS þ one dilution-step þ direct transfer after warming (n ¼ 43). 2.3. Vitrification procedures The vitrification procedures employed throughout this experiment were based on the methods originally designed by Yang [13] and by Vajta [6] for cow embryos and, after some modifications, used in our laboratory for preservation of in vivo derived and in vitro produced sheep embryos. The same operator made all the manipulations. Briefly, all vitrification solutions were prepared using phosphate-buffered saline (PBS) supplemented with 0.5 mM sodium pyruvate, 3.3 mM glucose and 20% FBS. The glycerol group of expanded ovine blastocysts, 168 IVP and 154 IVD, were exposed to the vitrification solution at room temperature (23 8C) according to the following procedure: 10% G for 5 min; 10% G plus 20% EG for 5 min followed by 25% G and 25% EG for 30 s. One hundred and ten IVP and 62 IVD embryos were loaded into the centres of 0.25 ml plastic insemination straws (IVM, L’Aigle, France) using a fine glass capillary pipette. Embryos were separated by two air bubbles from two surrounding drops of 0.5 M sucrose solution (90 ml) each. After heat-sealing, the straws were directly plunged into liquid nitrogen (LN2). The other 58 IVP and 92 IVD blastocysts were loaded into OPS. The DMSO þ 0:3 M sucrose group of 36 IVP and 43 IVD blastocysts were vitrified at room temperature (23 8C) as follows: 10% EG þ 10% DMSO for 3 min, followed by
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20% EG þ 20% DMSO þ 0:3 M S for 30 s. These blastocysts were loaded into OPS and directly plunged into LN2. A group of 21 IVP embryos were vitrified into OPS as above described, but the vitrification solution contained 0.75 M of sucrose (DMSO 0.75 group). 2.4. Warming and dilution All the embryos of glycerol and DMSO groups were diluted in one or three step dilutions. One hundred and ten IVP and 62 IVD embryos vitrified into the French straws were warmed by holding the straws for 6 s in air and then dipped into a 37 8C water bath for at least 15 s. The contents of each straw were emptied into a Petri dish containing the warming solution, and stirred gently to facilitate mixing between the two solutions. Sixtyeight IVP and 62 IVD embryos in the three dilution step group were picked up with a glass capillary pipette and serially transferred into 0.50, 0.25 and 0.125 M sucrose solutions at room temperature (23 8C) for 3 min each to allow removal of intracellular cryoprotectant. Then, they were held for 24 h in H-TCM 199 enriched with 20% FCS in humidified air containing 5% CO2 at 39 8C to permit re-expansion of the blastocoele. Another 42 IVP embryos were warmed as described above and diluted in a single dilution with 0.5 M sucrose for 5 min at room temperature before transfer. The 94 IVP and 135 IVD embryos vitrified into OPS were warmed by placing the straw into a Falcon tube with 8 ml of 0.5 M sucrose solution at 37 8C for 6 s and diluted as described above in either a one- or three-step dilution. They were then either directly transferred into synchronized ewes or cultured for 24 h before transfer. Twenty-one IVP embryos in the DMSO 0.75 group were directly warmed in H-TCM199 with 20% FCS and without sucrose at 37 8C, and transferred into synchronized recipient ewes (direct dilution group). 2.5. Transplantation of IVP and IVD vitrified embryos When re-expansion was performed after warming (protocol A for IVP and protocols F–H for IVD) only the re-expanded blastocysts were transferred. When only warming was performed (protocols B–E for IVP and protocol I for IVD) all the embryos were directly transferred in pairs into synchronized recipient ewes 7 days after the onset of natural estrous. Pregnancies were confirmed by ultrasonography at 40 days and were allowed to go to term. 2.6. Statistical analysis This study was designed to compare different overall vitrification protocols on in vitro produced (protocols A–E) and in vivo derived (protocols F–I). However, our analysis does include comparison of groups that differ in only one variable: cryoprotectant (C versus D, H versus I), device (B versus C, F versus G) and number of dilution steps at warming (A versus B, G versus H) (Tables 1 and 2). Comparisons among protocols were performed using the Chi-square test (SAS/STAT User’s Guide, 6.03 edition, SAS). Statistical significance was denoted as P < 0:05.
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Protocol
Cryoprotectant
Device
No. of dilution step at warming
Re-expansion (%)
Blastocysts transferred
Pregnancy (40 days) (%)
Lambs born/vitrified embryos (%)
Lambs born/transferred embryos (%)
A B C D E
G þ EG G þ EG G þ EG DMSO þ EG þ 0.3 M sucrose DMSO þ EG þ 0.75 M sucrose
French straws French straws OPS OPS OPS
3 1 1 1 1
46/68 (67.6) – – – –
46 42 58 36 21
12/24 12/21 15/29 9/18 5/10
10/68 10/42 13/58 7/36 5/21
10/46 10/42 13/58 7/36 5/21
No statistically significant differences among groups were found.
(Sucrose) (Sucrose) (Sucrose) (Sucrose) (20% FCS)
(50.0) (57.1) (51.7) (50.0) (50.0)
(14.7) (23.8) (22.4) (19.4) (23.8)
(21.7) (23.8) (22.4) (19.4) (23.8)
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Table 1 Re-expansion viability of IVP embryos and cryopreserved with five different vitrification protocols
Protocol
Cryoprotectant
Device
No. of dilution step at warming
Re-expansion
Blastocysts transferred
Pregnancy (40 d) (%)
Lambs born/vitrified embryos (%)
Lambs born/transferred embryos (%)
F G H I
G þ EG G þ EG G þ EG DMSO þ EG þ 0.3 M sucrose
French straws OPS OPS OPS
3 3 1 1
52/62 (83.8) 40/50 (80.0) 32/42 (76.1) –
52 40 32 43
19/27 14/20 12/16 15/21
39/62 30/50 24/42 26/43
39/52 30/40 24/32 26/43
No statistically significant differences among groups were found.
(Sucrose) (Sucrose) (Sucrose) (Sucrose)
(70.3) (70.0) (75.0) (71.4)
(62.9) (60.0) (57.1) (60.1)
(75.0) (75.0) (75.1) (60.1)
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Table 2 Re-expansion and viability of IVD embryos cryopreserved with four different vitrification protocols
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3. Results 3.1. Viability of vitrified IVP embryos No significant differences in either number of pregnancies at day 40 or lambing rate were found among vitrification protocols A–E (Table 1). 3.1.1. Cryoprotectants No significant differences were found either in the pregnancy (51.7% versus 50.0%) or lambing rate (22.4% versus 19.4%) when glycerol and DMSO were compared (C versus D) (Table 1). 3.1.2. Devices No significant differences were found in the pregnancy (57.1% versus 51.7%) or lambing rate (23.8% versus 22.4%) when French straws and OPS were compared (B versus C) (Table 1). 3.1.3. Warming 3.1.3.1. Number of dilution-steps. Three or one dilution-steps at warming did not significantly modify the pregnancy (50.0% versus 57.1%) or lambing rate (14.7% vs 23.8%: lambs born/vitrified embryos; 21.7% versus 23.8%: lambs born/transferred embryos.) (A versus B) (Table 1). 3.1.3.2. Diluent. The DMSO 0.75 group, warmed in a one-dilution step without sucrose, did not significantly differ in either pregnancy (50.0 versus 50.0%) or lambing rate (23.8% versus 19.4%) when compared with the DMSO 0.3 group (D versus E) (Table 1). 3.2. Viability of vitrified IVD embryos No significant differences were found either in pregnancy or lambing rates when comparisons among vitrification protocols F–I were tested (Table 2). 3.2.1. Cryoprotectants No significant differences were found in either pregnancy (75.0% versus 71.4%) or lambing rate (57.1% vs 60.1%: lambs born/vitrified embryos; 75.1% versus 60.1%: lambs born/transferred embryos.) when glycerol and DMSO were used (H versus I) (Table 2). 3.2.2. Devices When French straws and OPS were compared, no significant differences were found in the re-expansion (83.8% versus 80.0%), pregnancy (70.3% versus 70.0%) and lambing rate (62.9% vs 60.0%: lambs born/vitrified embryos; 75.0% versus 75.0%: lambs born/transferred embryos.) (F versus G) (Table 2).
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3.2.3. Number of dilution-steps at warming The use of three or one dilution-steps at warming did not significantly modify the reexpansion (80.0% versus 76.1%), pregnancy (70.0% versus 75.0%) and lambing rate (60.0% vs 57.1%: lambs born/vitrified embryos; 75.0% versus 75.1%: lambs born/ transferred embryos.) (G versus H) (Table 2).
4. Discussion The results demonstrate that the use of the tested vitrification protocols has no significant effects on the number of pregnancies or birth rates of either IVP or IVD sheep embryos. The lower response at lambing of IVP embryos if compared with IVD embryos, arises from fundamental differences in cellular structures, with the IVP embryos being the more sensitive to freezing as reported by Leibo and Loskutoff [36]. These workers describe the differences in buoyant density between in vivo and in vitro embryos due to the different ratio of lipids and proteins. This and other conditions significantly affect sensitivity to freezing of in vitro produced embryos [37,38]. Nevertheless, when fresh in vitro produced embryos are directly transferred into recipient ewes the lambing rate is higher than that obtained with the vitrified ones [39,40]. One of the most important factors to be considered in the choice of a cryoprotectant is its toxicity, which is related to its permeability. Greater permeability can result in higher chemical toxicity for embryos [41,42]. For this reason, the addition of glycerol (low permeability) has been suggested in a mixture of highly permeable cryoprotectants [9]. The use of highly permeable cryoprotectants (ethylene glycol or DMSO), singly or as part of a mixture, has been suggested to reduce the osmotic damage at warming and to avoid the need for multiple dilution-steps before the embryo transfer [32,42]. In our case, the use of a highly permeable cryoprotectant together with one of low permeability (EG þ G) or the use of two highly permeable cryoprotectants (EG þ DMSO) did not affect embryo viability. Exposure time to the cryoprotectants also affects toxicity: short periods of embryo exposure to the equilibration solution are relatively non-toxic [8,20,43–46]. Our results support this assumption: whether the maximum time of exposure to the equilibration solution was 10 min (glycerol group) or 3 min (DMSO group), it had no effect on lambing rates either on IVP or IVD embryos. Using traditional 0.25 ml insemination straws, the maximum cooling rate is approximately 2500 8C/min, which allows embryos to pass through certain critical temperature zones quickly and decreases chilling injuries [10]. Several new techniques have recently been developed to increase the cooling and the warming rates of vitrification technique. These include the electron microscopic grid, the open pulled straw and the cryoloop [6,27,29]. All these devices allow a cooling rate approximately tenfold faster than those achieved in standard straws, increasing the likelihood of success when cryopreserving oocytes, early and late embryos and other kinds of biological material particularly sensitive to low temperature. However, the major limit to the application of these types of vitrification techniques is the direct contact between the medium containing the embryos and the liquid nitrogen, which may introduce infection. In the last few years, there has been
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much discussions around the need to avoid contact between the embryos and liquid nitrogen, and the sterile application of the OPS has been proposed [47]. However, in our experience direct transfer of embryos vitrified in OPS into non-sterile liquid nitrogen has not been a problem. In any case, the trials reported here show that the use of the traditional French straw versus the OPS did not affect the pregnancy or the lambing rate, either in IVP or IVD embryos. The last variable considered in this study is the number of dilution-steps used at warming to remove the cryoprotectant. It is well known that the sucrose solution controls the degree of swelling during cryoprotectant removal by counteracting the high intracellular osmolarity of the cryoprotectant-permeated cells. As the cryoprotectant leaves the cells, the embryos will lose water and shrink [16,48–50]. Thereafter in isosmotic medium the cells regain their original volume. Many papers report the use of different concentrations of sucrose and different numbers of steps during dilution [31,51–53]. Our studies found no difference in embryo viability whether one or three dilution steps were employed. Direct rehydration presumes a very short exposure to a high concentration of nonpermeable solutes in the vitrification solution containing permeable cryoprotectants. This increases the dehydration of the embryos before they are plunged into liquid nitrogen, reducing the formation of intracellular ice. Good cell dehydration can be ensured by high concentrations of non-permeable compounds, such as sucrose or threalose; this may reduce toxicity by causing the embryos to shrink rapidly thereby reducing the amount of permeable cryoprotectants taken into the cell [16,17,50,54]. Consequently, after warming, the use of solutions without sucrose has no effect on embryo survival [20]: in fact, embryos cryopreserved as described above contain a minimal amount of cryoprotectant and therefore do not need a hyperosmotic extra-cellular environment to draw the cryoprotectants out of the cells. Thus, the optimal protocol for rapid cryopreservation with direct rehydration can be seen as a compromise between a maximal binding of cellular water by cryoprotectants that permeate the cell with a minimal quantity of these cryoprotectants remaining in the cytoplasm after warming [55]. In summary, our studies demonstrate that acceptable pregnancy and lambing rates of vitrified and warmed sheep embryos can be achieved using any of the tested protocols, despite the few number of observations occurred in some of the groups tested. On the other hand, the remarkable difference between IVP and IVD results, although not discussed in this study, leave us to presume that these outcomes could be affected by the quality of the embryos. In fact, several studies have reported that improved survival rates after cryopreservation of in vitro produced morulae and blastocysts are related more to the improved culture conditions than to changes in the cryopreservation technology itself [6,56–58].
Acknowledgements The authors thank Dr. D. Gillette and Dr. R. Prince for critical suggestions and revision of the manuscript; Mr. G. Camoglio and Mr. A. Pintadu for expert management of the animals and Mr. F. Chessa for assistance with experimental procedures.
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References [1] Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at 196 8C by vitrification. Nature 1985;313:573–5. [2] Massip A, Van Der Zwalmen P, Scheffen B, Ectors F. Pregnancies following transfer of cattle embryos preserved by vitrification. Cryoletters 1986;7:270–3. [3] Yuswiati E, Holtz W. Successful transfer of vitrified goat embryos. Theriogenology 1990;34:629–32. [4] Yoshino J, Kojima T, Shimizu M, Tomizuka T. Cryopreservation of porcine blastocysts by vitrification. Cryobiology 1993;30:413–22. [5] Van Wagtendonk-de Leeuw AM, Den Daas JHG, Kruip THAM, Rall WF. Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos. Cryobiology 1995;32:157–67. [6] Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51:53–8. [7] Dattena M, Ptak G, Loi P, Cappai P. Lambing rate following transfer after vitrification of in vitro and in vivo produced ovine embryos. Theriogenology 2000;53:252 [abstract]. [8] Isachenko V, Folch J, Isachenko E, Nawroth F, Krikokharchenko A, Vajta G, et al. Double vitrification of rat embryos at different developmental stages using an identical protocol. Theriogenology 2003;60: 445–52. [9] Massip A, Van Der Zwalmen P, Ectors F. Recent progress in cryopreservation of cattle embryos. Theriogenology 1987;27:69–79. [10] Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987;24:387–402. [11] Sze´ ll A, Zhang J, Hudson R. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at 180 8C. Reprod Fertil Dev 1990;2:613–8. [12] Schiewe MC, Rall WF, Stuart LD, Wildt DE. Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology 1991;36:279–93. [13] Yang NS, Lu KH, Gordon I, Polge C. Vitrification of bovine blastocysts produced in vitro. Theriogenology 1992;37:326 [abstract]. [14] Alı` J, Shelton JN. Vitrification of preimplantation stages of mouse embryos. J Reprod Fertil 1993;98: 459–65. [15] Dattena M, Ptak G, Loi P, Cappai P. Vitrification of in vivo derived (IVD) ovine embryos in two different devices: open pulled-straws and French mini-straws. Proc ICAR 2000, Sandnes-Norway: 66 [abstract]. [16] Massip A, Van Der Zwalmen P, Scheffen B, Ectors F. Some significant steps in the cryopreservation of mammalian embryos with a note on a vitrification procedure. Anim Reprod Sci 1989;19:117–29. [17] Kasai M, Komi JH, Takakamo A, Tsudera H, Sakurai T, Machida T. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990;89:91–7. [18] Alı` J, Shelton JN. Successful vitrification of day 6 sheep embryos. J Reprod Fertil 1993;99:65–70. [19] Vicente JS, Garcı`a-Xime´ nez F. Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae. Theriogenology 1994;42:1205–15. [20] Isachenko V, Isachenko EF, Ostashko FI, Grishchenko VI. Ultra rapid freezing of rat embryos with rapid dilution of permeable cryoprotectants. Cryobiology 1997;34:157–64. [21] Martinez AG, Matkovic M. Cryopreservation of ovine embryos: slow freezing and vitrification. Theriogenology 1998;49:1039–49. [22] Mahmoudzadeh AR, Van Soom A, Van Vlaenderen I, De Kruif A. A comparative study of the effect of one-step addition of different vitrification solutions on in vitro survival of vitrified bovine embryos. Theriogenology 1993;39:1291–302. [23] Massip A, Mermillod P, Van Langendonckt A, Touze JL, Dessy F. Survival and viability of fresh and frozen-thawed in vitro bovine blastocysts. Reprod Nutr Dev 1995;35:3–10. [24] Van Wagtendonk-de Leeuw AM, Den Daas JHG, Rall WF. Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution. Theriogenology 1997;48:1071–84.
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[25] Massip A, Van der Zwalmen P, Ectors F, De Coster R, D’Ieteren G, Hanzen C. Deep freezing of cattle embryos in glass ampules or French straws. Theriogenology 1979;12:79–84. [26] Isachenko V, Alabart JL, Dattena M, Nawroth F, Cappai P, Isachenko E, et al. New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos. Theriogenology 2003;59:1209–18. [27] Martino A, Pollard JW, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev 1996;45:503–12. [28] Arav A, Zeron Y. Vitrification of bovine oocytes using modified minimum drop size technique (MDS) is effected by the composition and the concentration of the vitrification solution and by the cooling conditions. Theriogenology 1997;47:341 [abstract]. [29] Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification of mammalian oocytes and embryos. Nature Biotechnol 1999;17:1234–6. [30] Sze´ ll A, Shelton JN. Sucrose dilution from mouse embryos frozen rapidly in liquid nitrogen vapour. J Reprod Fertil 1986;76:401–8. [31] Kobayashi K, Nagashima H, Yamakawa H, Kato Y, Ogawa S. The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method. Theriogenology 1990;33:777–88. [32] Vajta G, Holm P, Greve T, Callesen H. Direct in-straw rehydration after thawing of vitrified in vitro produced bovine blastocysts. Vet Rec 1995;23/30:672. [33] Vicente JS, Garcı`a-Xime´ nez F. Direct transfer of vitrified rabbit embryos. Theriogenology 1996;45:811–5. [34] Tervit HR, Whittingh DG, Rowson LEA. Successful culture of sheep and cattle ova. J Reprod Fertil 1972;30:493–7. [35] Tervit HR, Havik PG. A modified technique for flushing ova from the sheep uterus. NZ Vet J 1976;24: 138–40. [36] Leibo SP, Loskutoff NM. Cryobiology of in vitro derived bovine embryos. Theriogenology 1993;39:81–94. [37] Massip A, Mermillod P, Dinnye´ s A. Morfology and biochemistry of in vitro produced bovine embryos: implications for their cryopreservation. Hum Reprod 1995;10:3004–11. [38] Pollard JW, Leibo SP. Chilling sensitivity of mammalian embryos. Theriogenology 1994;41:101–6. [39] Dattena M, Ptak G, Loi P, Cappai P. Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts. Theriogenology 2000;53:1511–9. [40] Accardo C, Dattena M, Pilichi S, Mara L, Chessa B, Cappai P. Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability. Anim Reprod Sci 2004;81: 77–86. [41] Van der Zwalmen P, Touati K, Ectors FJ, Massip A, Beckers JF, Ectors F. Vitrification of bovine blastocysts. Theryogenology 1989;31:270 [abstract]. [42] Voelkel SA, Hu YX. Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females. Theriogenology 1992;37:686–97. [43] Li R, Trounson A. Rapid freezing of the mouse blastocyst: effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen. Reprod Fertil Dev 1991;3:175–83. [44] Takagi M, Otoi T, Suzuki T. Survival rate of frozen-thawed bovine IVM/IVF embryos in relation to postthaw exposure time in two cryprotectants. Cryobiology 1993;30:466–9. [45] Gutie´ rrez A, Artiga CG, Garde J, Pintado B. Effect of equilibration period and cryoprotectant in quick freezing of mouse morulae. Theriogenology 1993;39:229. [46] Agca Y, Monson RL, Northey DL, Abas Mazni O, Schaefer DM, Rutledge JJ. Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation length. Theriogenology 1998;50:147–62. [47] Vajta G, Lewis IM, Kuwayama M, Greve T, Callesen H. Sterile application of the Open Pulled Straw (OPS) vitrification method. Cryoletters 1998;19:389–92. [48] Leibo SP. A one-step method for direct non-surgical transfer of frozen-thawed bovine embryos. Theriogenology 1984;21:767–90. [49] Mazur P, Schneider U. Osmotic response of preimplantation mouse and bovine embryos and their cryobiological implications. Cell Byophis 1986;8:259–85. [50] Sze´ ll A, Shelton JN. Role of equilibration before rapid freezing of mouse embryos. J Reprod Fertil 1986; 78:699–703.
M. Dattena et al. / Theriogenology 62 (2004) 481–493
493
[51] Seshagiri PB, Bavister BD. Glucose inhibits development of hamster 8-cells embryos in vitro. Biol Reprod 1989;40:599–606. [52] Dobrinsky JR, Stice SL, Phillips PE, Duby RT, Robl JM. Development of IVM/IVF bovine embryos following vitrification dilution treatments. Theriogenology 1992;37:202 [abstract]. [53] Kobayashi K, Takei M, Kano M, Tomita M, Leibo SP. Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants. Cryobiology 1998;36:20–31. [54] Saha S, Otoi T, Takagi M, Boediono A, Sumantri C, Suzuki T. Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, threalose and polyvinylpyrrolidone. Cryobiology 1996;33:291–9. [55] Isachenko V, Alabart JL, Isachenko EF, Bezugly ND, Michelmann HW. Ultra rapid freezing and storage of rat embryos in an electric refrigerator at 130 8C without liquid cryo-agents, with ultra-short exposure in the freezing medium and direct re-hydration after thawing. Cryoletters 2000;21:13–8. [56] Greve T, Avery B, Callesen H. Viability of in vivo and in vitro produced bovine embryos. Reprod Domestic Anim 1993;28:164–9. [57] Shamsuddin MB, Rodriguez-Martinez H. Fine structure of bovine blastocysts developed either in serumfree medium or in conventional co-culture with oviduct epithelial cells. J Vet Med 1994;41:307–16. [58] Dobrinsky JR, Pursel VG, Long CR, Johnson LA. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod 2000;62:564–70.
Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived M. Dattenaa,*, C. Accardoa, S. Pilichia, V. Isachenkob, L. Maraa, B. Chessac, P. Cappaia a
Istituto Zootecnico e Caseario per la Sardegna, 07040 Olmedo, Sassari, Italy b Department of Gynecology, Endocrinology and Reproductive Medicine, University of Bonn, 53127 Bonn, Germany c Facolta` di Medicina Veterinaria, Facolta` di Medicina Veterinaria, Universita` di Sassari, 07100 Sassari, Italy
Received 18 June 2003; received in revised form 30 September 2003; accepted 25 October 2003
Abstract We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G þ 20% ethylene glycol (EG), then for 30 s in 25% G þ 25% EG (glycerol group), or for 3 min in 10% EG þ 10% dimethyl sulphoxide (DMSO), then for 30 s in 20% EG þ 20% DMSO þ 0:3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 ðTCM-199Þ þ 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of
* Corresponding author. Tel.: þ39-079-387268; fax: þ39-079-389450. E-mail address: [email protected] (M. Dattena).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.10.010
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IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols. # 2003 Elsevier Inc. All rights reserved. Keywords: Sheep embryos; In vitro produced; In vivo derived; Vitrification protocols; Lambing
1. Introduction Controlled slow freezing continues to be the most widely used technique of cryopreservation for in vivo and in vitro produced embryos. However, in the last decade the vitrification technique, a cryopreservation system involving the addition of higher concentrations of cryoprotectants and very rapid cooling [1], has been tested in different species with good results [2–8]. Many different vitrification protocols have been used to cryopreserve in vivo and in vitro produced embryos in the last 15 years. They differ in many ways, including type and concentration of cryoprotectant, number of equilibration steps, type of cryopreservation device used, time of exposure and number of dilution steps at warming [6,9–15], and it is widely believed that each of these factors can affect the results. The requirements of a good cryoprotectant are permeability and low toxicity. The most widely used vitrification cryoprotectants are glycerol, ethylene glycol, propylene glycol and dimethyl sulphoxide, employed in different combinations and concentrations, sometimes supplemented with non-permeable solutes such as sucrose, threalose etc. [1,6,13, 16–21]. The equilibration procedures can differ among the protocols either in the number of steps (one to three) or time of exposure (5–10 min), while exposure to the vitrification solution requires less than 1 min [6–8,20–24]. All of the above mentioned procedures together with the choice and concentration of cryoprotectant aim to achieve optimal intracellular dehydration and thereby avoid ice crystal formation. Glass ampules were originally used to store frozen cattle embryos [25]. In the 1980’s, the plastic straw became the container of choice for vitrification and storage of embryos [1,16]. A recent modification, the open pulled straw (OPS), reduces the volume of vitrification solution and allows direct contact between the embryo and liquid nitrogen, thus enhancing the cooling rate [6,15,26]. In fact, it is well known that minimum drop size and a rapid cooling rate are very important factors to improve vitrification results [6,27–29]. After warming, embryos are usually placed in a hypertonic solution to remove the permeating cryoprotectants before transferring them to an isotonic culture medium. Many authors report the use of one or more dilution steps with different concentrations of sucrose, which controls the degree of swelling during cryoprotectant removal [20,30,31]. However, the possibility of direct post-warming rehydration in an isotonic solution has been described [20,32,33]. This paper summarizes results obtained in our laboratory over the past several years, comparing five vitrification protocols carried out on in vitro produced and in vivo derived embryos. When it was possible, type of cryoprotectants (glycerol and DMSO), devices
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(French straw and open pulled straw), numbers of dilution-steps and type of diluent at warming were compared.
2. Materials and methods Except where otherwise indicated, all chemicals were obtained from Sigma Chemicals Company (St. Louis, MO, USA). 2.1. In vitro embryo production Ovaries were collected immediately after slaughter and transported to the laboratory in saline at approximately 35 8C within 1–2 h. Oocytes were obtained by aspiration of follicles using a 20-gauge needle fitted with a 2 ml syringe. Follicular oocytes covered by at least 2 layers of granulosa cells and with an evenly granulated cytoplasm were selected for IVM. The maturation medium was bicarbonate-buffered TCM-199 (osmolarity adjusted to 275 mOsm), containing 2 mM of glutamine, 10% FBS, 5 mg/ml follicle stimulating hormone (FSH) (Ovagen, ICP, Auckland, New Zealand), 5 mg/ml luteinizing hormone (LH), 1 mg/ml estradiol, 0.3 mM sodium pyruvate and 100 mM cysteamine. The oocytes were incubated in 400 ml of medium in four-well dishes (Nunc, Nunclon, Denmark) covered with mineral oil. In vitro maturation conditions were 5% CO2 in humidified air at 39 8C for 24 h. Following maturation, the oocytes were partially denuded of granulosa cells by gently pipetting in Hepes-TCM-199 (H-TCM-199) with 300 IU/ml of hyaluronidase. Fresh semen from a Sarda breed ram of proven fertility was used throughout the experiment. Collected ejaculate was kept at room temperature for up to 2 h, then washed in bicarbonatebuffered synthetic oviduct fluid (SOF) [34] that was enriched with 20% (v/v) heat inactivated estrous sheep serum. The washed ejaculate was centrifuged twice at 200 g for 5 min and directly added to the fertilization medium. Fertilization was conducted in 50 ml drops with 1 106 sperm/ml and a maximum of 15 oocytes per drop, at 39 8C with 5% CO2 in humidified air for 20 h. The presumptive zygotes were allocated to 20 ml-culture drops (4–5 embryos/drop), consisting of SOF supplemented with 1% (v/v) Basal Medium Eagle (BME)–essential amino acids, 1% (v/v) minimum essential medium (MEM)–non-essential amino acids, 1 mM glutamine and 8 mg/ml fatty acid-free bovine serum albumine (BSA). Embryos were incubated in an atmosphere of 7% O2, 5% CO2, 88% N2 at 39 8C at 100% humidity. Charcoal stripped FBS was added to the medium on the third and fifth day of culture (where day 0 was the day of fertilization). Culture was continued until 6–7 days post fertilization at which time the expanded blastocysts (n ¼ 225) were divided in five different vitrification protocols as follows: (A) Glycerol þ French straw þ three dilution-steps (n ¼ 68) þ transfer after blastocysts re-expansion (n ¼ 46); (B) Glycerol þ French straw þ one dilution-step þ direct transfer after warming (n ¼ 42); (C) Glycerol þ OPS þ one dilution-step þ direct transfer after warming (n ¼ 58);
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(D) DMSO þ 0:3 M sucrose þ OPS þ one dilution-step þ direct transfer after warming (n ¼ 36); (E) DMSO þ 0:75 M sucrose þ OPS þ direct dilution þ direct transfer after warming (n ¼ 21). 2.2. In vivo derived embryos The estrous cycles of Sarda ewes were synchronized by insertion of intravaginal sponges containing 40 mg fluorogestone acetate (Intervet, NL) for 14 days. The day before removal of the sponges, the ewes were superovulated with 16 mg porcine follicle stimulating hormone (p-FSH; Pluset, Calier, Italy) administered intramuscularly every 12 h in four decreasing doses (6, 5, 3 and 2 mg). The ewes were inseminated 48 h after sponge removal by intrauterine insemination with fresh semen from a fertile ram. Embryos were collected from the donors under general anaesthesia (Pentothal sodium, Gellini, Italy) 7 days after the start of estrus. Each uterine horn was flushed using a Foley catheter as described by Tervit and Havick [35]. The collection medium was HTCM-199 supplemented with 4 mg/ ml BSA. The embryos were classified according to their stage of development. All the 197 expanded blastocysts obtained in several collections were divided into four groups and subjected to one of the following vitrification protocols: (F) Glycerol þ French straw þ three dilution-steps (n ¼ 62) þ transfer after blastocysts re-expansion (n ¼ 52); (G) Glycerol þ OPS þ three dilution-steps (n ¼ 50) þ transfer after re-expansion (n ¼ 40); (H) Glycerol þ OPS þ one dilution-step (n ¼ 42) þ transfer after re-expansion (n ¼ 32); (I) DMSO 0.3 M sucrose þ OPS þ one dilution-step þ direct transfer after warming (n ¼ 43). 2.3. Vitrification procedures The vitrification procedures employed throughout this experiment were based on the methods originally designed by Yang [13] and by Vajta [6] for cow embryos and, after some modifications, used in our laboratory for preservation of in vivo derived and in vitro produced sheep embryos. The same operator made all the manipulations. Briefly, all vitrification solutions were prepared using phosphate-buffered saline (PBS) supplemented with 0.5 mM sodium pyruvate, 3.3 mM glucose and 20% FBS. The glycerol group of expanded ovine blastocysts, 168 IVP and 154 IVD, were exposed to the vitrification solution at room temperature (23 8C) according to the following procedure: 10% G for 5 min; 10% G plus 20% EG for 5 min followed by 25% G and 25% EG for 30 s. One hundred and ten IVP and 62 IVD embryos were loaded into the centres of 0.25 ml plastic insemination straws (IVM, L’Aigle, France) using a fine glass capillary pipette. Embryos were separated by two air bubbles from two surrounding drops of 0.5 M sucrose solution (90 ml) each. After heat-sealing, the straws were directly plunged into liquid nitrogen (LN2). The other 58 IVP and 92 IVD blastocysts were loaded into OPS. The DMSO þ 0:3 M sucrose group of 36 IVP and 43 IVD blastocysts were vitrified at room temperature (23 8C) as follows: 10% EG þ 10% DMSO for 3 min, followed by
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20% EG þ 20% DMSO þ 0:3 M S for 30 s. These blastocysts were loaded into OPS and directly plunged into LN2. A group of 21 IVP embryos were vitrified into OPS as above described, but the vitrification solution contained 0.75 M of sucrose (DMSO 0.75 group). 2.4. Warming and dilution All the embryos of glycerol and DMSO groups were diluted in one or three step dilutions. One hundred and ten IVP and 62 IVD embryos vitrified into the French straws were warmed by holding the straws for 6 s in air and then dipped into a 37 8C water bath for at least 15 s. The contents of each straw were emptied into a Petri dish containing the warming solution, and stirred gently to facilitate mixing between the two solutions. Sixtyeight IVP and 62 IVD embryos in the three dilution step group were picked up with a glass capillary pipette and serially transferred into 0.50, 0.25 and 0.125 M sucrose solutions at room temperature (23 8C) for 3 min each to allow removal of intracellular cryoprotectant. Then, they were held for 24 h in H-TCM 199 enriched with 20% FCS in humidified air containing 5% CO2 at 39 8C to permit re-expansion of the blastocoele. Another 42 IVP embryos were warmed as described above and diluted in a single dilution with 0.5 M sucrose for 5 min at room temperature before transfer. The 94 IVP and 135 IVD embryos vitrified into OPS were warmed by placing the straw into a Falcon tube with 8 ml of 0.5 M sucrose solution at 37 8C for 6 s and diluted as described above in either a one- or three-step dilution. They were then either directly transferred into synchronized ewes or cultured for 24 h before transfer. Twenty-one IVP embryos in the DMSO 0.75 group were directly warmed in H-TCM199 with 20% FCS and without sucrose at 37 8C, and transferred into synchronized recipient ewes (direct dilution group). 2.5. Transplantation of IVP and IVD vitrified embryos When re-expansion was performed after warming (protocol A for IVP and protocols F–H for IVD) only the re-expanded blastocysts were transferred. When only warming was performed (protocols B–E for IVP and protocol I for IVD) all the embryos were directly transferred in pairs into synchronized recipient ewes 7 days after the onset of natural estrous. Pregnancies were confirmed by ultrasonography at 40 days and were allowed to go to term. 2.6. Statistical analysis This study was designed to compare different overall vitrification protocols on in vitro produced (protocols A–E) and in vivo derived (protocols F–I). However, our analysis does include comparison of groups that differ in only one variable: cryoprotectant (C versus D, H versus I), device (B versus C, F versus G) and number of dilution steps at warming (A versus B, G versus H) (Tables 1 and 2). Comparisons among protocols were performed using the Chi-square test (SAS/STAT User’s Guide, 6.03 edition, SAS). Statistical significance was denoted as P < 0:05.
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Protocol
Cryoprotectant
Device
No. of dilution step at warming
Re-expansion (%)
Blastocysts transferred
Pregnancy (40 days) (%)
Lambs born/vitrified embryos (%)
Lambs born/transferred embryos (%)
A B C D E
G þ EG G þ EG G þ EG DMSO þ EG þ 0.3 M sucrose DMSO þ EG þ 0.75 M sucrose
French straws French straws OPS OPS OPS
3 1 1 1 1
46/68 (67.6) – – – –
46 42 58 36 21
12/24 12/21 15/29 9/18 5/10
10/68 10/42 13/58 7/36 5/21
10/46 10/42 13/58 7/36 5/21
No statistically significant differences among groups were found.
(Sucrose) (Sucrose) (Sucrose) (Sucrose) (20% FCS)
(50.0) (57.1) (51.7) (50.0) (50.0)
(14.7) (23.8) (22.4) (19.4) (23.8)
(21.7) (23.8) (22.4) (19.4) (23.8)
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Table 1 Re-expansion viability of IVP embryos and cryopreserved with five different vitrification protocols
Protocol
Cryoprotectant
Device
No. of dilution step at warming
Re-expansion
Blastocysts transferred
Pregnancy (40 d) (%)
Lambs born/vitrified embryos (%)
Lambs born/transferred embryos (%)
F G H I
G þ EG G þ EG G þ EG DMSO þ EG þ 0.3 M sucrose
French straws OPS OPS OPS
3 3 1 1
52/62 (83.8) 40/50 (80.0) 32/42 (76.1) –
52 40 32 43
19/27 14/20 12/16 15/21
39/62 30/50 24/42 26/43
39/52 30/40 24/32 26/43
No statistically significant differences among groups were found.
(Sucrose) (Sucrose) (Sucrose) (Sucrose)
(70.3) (70.0) (75.0) (71.4)
(62.9) (60.0) (57.1) (60.1)
(75.0) (75.0) (75.1) (60.1)
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Table 2 Re-expansion and viability of IVD embryos cryopreserved with four different vitrification protocols
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3. Results 3.1. Viability of vitrified IVP embryos No significant differences in either number of pregnancies at day 40 or lambing rate were found among vitrification protocols A–E (Table 1). 3.1.1. Cryoprotectants No significant differences were found either in the pregnancy (51.7% versus 50.0%) or lambing rate (22.4% versus 19.4%) when glycerol and DMSO were compared (C versus D) (Table 1). 3.1.2. Devices No significant differences were found in the pregnancy (57.1% versus 51.7%) or lambing rate (23.8% versus 22.4%) when French straws and OPS were compared (B versus C) (Table 1). 3.1.3. Warming 3.1.3.1. Number of dilution-steps. Three or one dilution-steps at warming did not significantly modify the pregnancy (50.0% versus 57.1%) or lambing rate (14.7% vs 23.8%: lambs born/vitrified embryos; 21.7% versus 23.8%: lambs born/transferred embryos.) (A versus B) (Table 1). 3.1.3.2. Diluent. The DMSO 0.75 group, warmed in a one-dilution step without sucrose, did not significantly differ in either pregnancy (50.0 versus 50.0%) or lambing rate (23.8% versus 19.4%) when compared with the DMSO 0.3 group (D versus E) (Table 1). 3.2. Viability of vitrified IVD embryos No significant differences were found either in pregnancy or lambing rates when comparisons among vitrification protocols F–I were tested (Table 2). 3.2.1. Cryoprotectants No significant differences were found in either pregnancy (75.0% versus 71.4%) or lambing rate (57.1% vs 60.1%: lambs born/vitrified embryos; 75.1% versus 60.1%: lambs born/transferred embryos.) when glycerol and DMSO were used (H versus I) (Table 2). 3.2.2. Devices When French straws and OPS were compared, no significant differences were found in the re-expansion (83.8% versus 80.0%), pregnancy (70.3% versus 70.0%) and lambing rate (62.9% vs 60.0%: lambs born/vitrified embryos; 75.0% versus 75.0%: lambs born/transferred embryos.) (F versus G) (Table 2).
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3.2.3. Number of dilution-steps at warming The use of three or one dilution-steps at warming did not significantly modify the reexpansion (80.0% versus 76.1%), pregnancy (70.0% versus 75.0%) and lambing rate (60.0% vs 57.1%: lambs born/vitrified embryos; 75.0% versus 75.1%: lambs born/ transferred embryos.) (G versus H) (Table 2).
4. Discussion The results demonstrate that the use of the tested vitrification protocols has no significant effects on the number of pregnancies or birth rates of either IVP or IVD sheep embryos. The lower response at lambing of IVP embryos if compared with IVD embryos, arises from fundamental differences in cellular structures, with the IVP embryos being the more sensitive to freezing as reported by Leibo and Loskutoff [36]. These workers describe the differences in buoyant density between in vivo and in vitro embryos due to the different ratio of lipids and proteins. This and other conditions significantly affect sensitivity to freezing of in vitro produced embryos [37,38]. Nevertheless, when fresh in vitro produced embryos are directly transferred into recipient ewes the lambing rate is higher than that obtained with the vitrified ones [39,40]. One of the most important factors to be considered in the choice of a cryoprotectant is its toxicity, which is related to its permeability. Greater permeability can result in higher chemical toxicity for embryos [41,42]. For this reason, the addition of glycerol (low permeability) has been suggested in a mixture of highly permeable cryoprotectants [9]. The use of highly permeable cryoprotectants (ethylene glycol or DMSO), singly or as part of a mixture, has been suggested to reduce the osmotic damage at warming and to avoid the need for multiple dilution-steps before the embryo transfer [32,42]. In our case, the use of a highly permeable cryoprotectant together with one of low permeability (EG þ G) or the use of two highly permeable cryoprotectants (EG þ DMSO) did not affect embryo viability. Exposure time to the cryoprotectants also affects toxicity: short periods of embryo exposure to the equilibration solution are relatively non-toxic [8,20,43–46]. Our results support this assumption: whether the maximum time of exposure to the equilibration solution was 10 min (glycerol group) or 3 min (DMSO group), it had no effect on lambing rates either on IVP or IVD embryos. Using traditional 0.25 ml insemination straws, the maximum cooling rate is approximately 2500 8C/min, which allows embryos to pass through certain critical temperature zones quickly and decreases chilling injuries [10]. Several new techniques have recently been developed to increase the cooling and the warming rates of vitrification technique. These include the electron microscopic grid, the open pulled straw and the cryoloop [6,27,29]. All these devices allow a cooling rate approximately tenfold faster than those achieved in standard straws, increasing the likelihood of success when cryopreserving oocytes, early and late embryos and other kinds of biological material particularly sensitive to low temperature. However, the major limit to the application of these types of vitrification techniques is the direct contact between the medium containing the embryos and the liquid nitrogen, which may introduce infection. In the last few years, there has been
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much discussions around the need to avoid contact between the embryos and liquid nitrogen, and the sterile application of the OPS has been proposed [47]. However, in our experience direct transfer of embryos vitrified in OPS into non-sterile liquid nitrogen has not been a problem. In any case, the trials reported here show that the use of the traditional French straw versus the OPS did not affect the pregnancy or the lambing rate, either in IVP or IVD embryos. The last variable considered in this study is the number of dilution-steps used at warming to remove the cryoprotectant. It is well known that the sucrose solution controls the degree of swelling during cryoprotectant removal by counteracting the high intracellular osmolarity of the cryoprotectant-permeated cells. As the cryoprotectant leaves the cells, the embryos will lose water and shrink [16,48–50]. Thereafter in isosmotic medium the cells regain their original volume. Many papers report the use of different concentrations of sucrose and different numbers of steps during dilution [31,51–53]. Our studies found no difference in embryo viability whether one or three dilution steps were employed. Direct rehydration presumes a very short exposure to a high concentration of nonpermeable solutes in the vitrification solution containing permeable cryoprotectants. This increases the dehydration of the embryos before they are plunged into liquid nitrogen, reducing the formation of intracellular ice. Good cell dehydration can be ensured by high concentrations of non-permeable compounds, such as sucrose or threalose; this may reduce toxicity by causing the embryos to shrink rapidly thereby reducing the amount of permeable cryoprotectants taken into the cell [16,17,50,54]. Consequently, after warming, the use of solutions without sucrose has no effect on embryo survival [20]: in fact, embryos cryopreserved as described above contain a minimal amount of cryoprotectant and therefore do not need a hyperosmotic extra-cellular environment to draw the cryoprotectants out of the cells. Thus, the optimal protocol for rapid cryopreservation with direct rehydration can be seen as a compromise between a maximal binding of cellular water by cryoprotectants that permeate the cell with a minimal quantity of these cryoprotectants remaining in the cytoplasm after warming [55]. In summary, our studies demonstrate that acceptable pregnancy and lambing rates of vitrified and warmed sheep embryos can be achieved using any of the tested protocols, despite the few number of observations occurred in some of the groups tested. On the other hand, the remarkable difference between IVP and IVD results, although not discussed in this study, leave us to presume that these outcomes could be affected by the quality of the embryos. In fact, several studies have reported that improved survival rates after cryopreservation of in vitro produced morulae and blastocysts are related more to the improved culture conditions than to changes in the cryopreservation technology itself [6,56–58].
Acknowledgements The authors thank Dr. D. Gillette and Dr. R. Prince for critical suggestions and revision of the manuscript; Mr. G. Camoglio and Mr. A. Pintadu for expert management of the animals and Mr. F. Chessa for assistance with experimental procedures.
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References [1] Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at 196 8C by vitrification. Nature 1985;313:573–5. [2] Massip A, Van Der Zwalmen P, Scheffen B, Ectors F. Pregnancies following transfer of cattle embryos preserved by vitrification. Cryoletters 1986;7:270–3. [3] Yuswiati E, Holtz W. Successful transfer of vitrified goat embryos. Theriogenology 1990;34:629–32. [4] Yoshino J, Kojima T, Shimizu M, Tomizuka T. Cryopreservation of porcine blastocysts by vitrification. Cryobiology 1993;30:413–22. [5] Van Wagtendonk-de Leeuw AM, Den Daas JHG, Kruip THAM, Rall WF. Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos. Cryobiology 1995;32:157–67. [6] Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51:53–8. [7] Dattena M, Ptak G, Loi P, Cappai P. Lambing rate following transfer after vitrification of in vitro and in vivo produced ovine embryos. Theriogenology 2000;53:252 [abstract]. [8] Isachenko V, Folch J, Isachenko E, Nawroth F, Krikokharchenko A, Vajta G, et al. Double vitrification of rat embryos at different developmental stages using an identical protocol. Theriogenology 2003;60: 445–52. [9] Massip A, Van Der Zwalmen P, Ectors F. Recent progress in cryopreservation of cattle embryos. Theriogenology 1987;27:69–79. [10] Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987;24:387–402. [11] Sze´ ll A, Zhang J, Hudson R. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at 180 8C. Reprod Fertil Dev 1990;2:613–8. [12] Schiewe MC, Rall WF, Stuart LD, Wildt DE. Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology 1991;36:279–93. [13] Yang NS, Lu KH, Gordon I, Polge C. Vitrification of bovine blastocysts produced in vitro. Theriogenology 1992;37:326 [abstract]. [14] Alı` J, Shelton JN. Vitrification of preimplantation stages of mouse embryos. J Reprod Fertil 1993;98: 459–65. [15] Dattena M, Ptak G, Loi P, Cappai P. Vitrification of in vivo derived (IVD) ovine embryos in two different devices: open pulled-straws and French mini-straws. Proc ICAR 2000, Sandnes-Norway: 66 [abstract]. [16] Massip A, Van Der Zwalmen P, Scheffen B, Ectors F. Some significant steps in the cryopreservation of mammalian embryos with a note on a vitrification procedure. Anim Reprod Sci 1989;19:117–29. [17] Kasai M, Komi JH, Takakamo A, Tsudera H, Sakurai T, Machida T. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990;89:91–7. [18] Alı` J, Shelton JN. Successful vitrification of day 6 sheep embryos. J Reprod Fertil 1993;99:65–70. [19] Vicente JS, Garcı`a-Xime´ nez F. Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae. Theriogenology 1994;42:1205–15. [20] Isachenko V, Isachenko EF, Ostashko FI, Grishchenko VI. Ultra rapid freezing of rat embryos with rapid dilution of permeable cryoprotectants. Cryobiology 1997;34:157–64. [21] Martinez AG, Matkovic M. Cryopreservation of ovine embryos: slow freezing and vitrification. Theriogenology 1998;49:1039–49. [22] Mahmoudzadeh AR, Van Soom A, Van Vlaenderen I, De Kruif A. A comparative study of the effect of one-step addition of different vitrification solutions on in vitro survival of vitrified bovine embryos. Theriogenology 1993;39:1291–302. [23] Massip A, Mermillod P, Van Langendonckt A, Touze JL, Dessy F. Survival and viability of fresh and frozen-thawed in vitro bovine blastocysts. Reprod Nutr Dev 1995;35:3–10. [24] Van Wagtendonk-de Leeuw AM, Den Daas JHG, Rall WF. Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution. Theriogenology 1997;48:1071–84.
492
M. Dattena et al. / Theriogenology 62 (2004) 481–493
[25] Massip A, Van der Zwalmen P, Ectors F, De Coster R, D’Ieteren G, Hanzen C. Deep freezing of cattle embryos in glass ampules or French straws. Theriogenology 1979;12:79–84. [26] Isachenko V, Alabart JL, Dattena M, Nawroth F, Cappai P, Isachenko E, et al. New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos. Theriogenology 2003;59:1209–18. [27] Martino A, Pollard JW, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev 1996;45:503–12. [28] Arav A, Zeron Y. Vitrification of bovine oocytes using modified minimum drop size technique (MDS) is effected by the composition and the concentration of the vitrification solution and by the cooling conditions. Theriogenology 1997;47:341 [abstract]. [29] Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification of mammalian oocytes and embryos. Nature Biotechnol 1999;17:1234–6. [30] Sze´ ll A, Shelton JN. Sucrose dilution from mouse embryos frozen rapidly in liquid nitrogen vapour. J Reprod Fertil 1986;76:401–8. [31] Kobayashi K, Nagashima H, Yamakawa H, Kato Y, Ogawa S. The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method. Theriogenology 1990;33:777–88. [32] Vajta G, Holm P, Greve T, Callesen H. Direct in-straw rehydration after thawing of vitrified in vitro produced bovine blastocysts. Vet Rec 1995;23/30:672. [33] Vicente JS, Garcı`a-Xime´ nez F. Direct transfer of vitrified rabbit embryos. Theriogenology 1996;45:811–5. [34] Tervit HR, Whittingh DG, Rowson LEA. Successful culture of sheep and cattle ova. J Reprod Fertil 1972;30:493–7. [35] Tervit HR, Havik PG. A modified technique for flushing ova from the sheep uterus. NZ Vet J 1976;24: 138–40. [36] Leibo SP, Loskutoff NM. Cryobiology of in vitro derived bovine embryos. Theriogenology 1993;39:81–94. [37] Massip A, Mermillod P, Dinnye´ s A. Morfology and biochemistry of in vitro produced bovine embryos: implications for their cryopreservation. Hum Reprod 1995;10:3004–11. [38] Pollard JW, Leibo SP. Chilling sensitivity of mammalian embryos. Theriogenology 1994;41:101–6. [39] Dattena M, Ptak G, Loi P, Cappai P. Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts. Theriogenology 2000;53:1511–9. [40] Accardo C, Dattena M, Pilichi S, Mara L, Chessa B, Cappai P. Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability. Anim Reprod Sci 2004;81: 77–86. [41] Van der Zwalmen P, Touati K, Ectors FJ, Massip A, Beckers JF, Ectors F. Vitrification of bovine blastocysts. Theryogenology 1989;31:270 [abstract]. [42] Voelkel SA, Hu YX. Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females. Theriogenology 1992;37:686–97. [43] Li R, Trounson A. Rapid freezing of the mouse blastocyst: effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen. Reprod Fertil Dev 1991;3:175–83. [44] Takagi M, Otoi T, Suzuki T. Survival rate of frozen-thawed bovine IVM/IVF embryos in relation to postthaw exposure time in two cryprotectants. Cryobiology 1993;30:466–9. [45] Gutie´ rrez A, Artiga CG, Garde J, Pintado B. Effect of equilibration period and cryoprotectant in quick freezing of mouse morulae. Theriogenology 1993;39:229. [46] Agca Y, Monson RL, Northey DL, Abas Mazni O, Schaefer DM, Rutledge JJ. Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation length. Theriogenology 1998;50:147–62. [47] Vajta G, Lewis IM, Kuwayama M, Greve T, Callesen H. Sterile application of the Open Pulled Straw (OPS) vitrification method. Cryoletters 1998;19:389–92. [48] Leibo SP. A one-step method for direct non-surgical transfer of frozen-thawed bovine embryos. Theriogenology 1984;21:767–90. [49] Mazur P, Schneider U. Osmotic response of preimplantation mouse and bovine embryos and their cryobiological implications. Cell Byophis 1986;8:259–85. [50] Sze´ ll A, Shelton JN. Role of equilibration before rapid freezing of mouse embryos. J Reprod Fertil 1986; 78:699–703.
M. Dattena et al. / Theriogenology 62 (2004) 481–493
493
[51] Seshagiri PB, Bavister BD. Glucose inhibits development of hamster 8-cells embryos in vitro. Biol Reprod 1989;40:599–606. [52] Dobrinsky JR, Stice SL, Phillips PE, Duby RT, Robl JM. Development of IVM/IVF bovine embryos following vitrification dilution treatments. Theriogenology 1992;37:202 [abstract]. [53] Kobayashi K, Takei M, Kano M, Tomita M, Leibo SP. Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants. Cryobiology 1998;36:20–31. [54] Saha S, Otoi T, Takagi M, Boediono A, Sumantri C, Suzuki T. Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, threalose and polyvinylpyrrolidone. Cryobiology 1996;33:291–9. [55] Isachenko V, Alabart JL, Isachenko EF, Bezugly ND, Michelmann HW. Ultra rapid freezing and storage of rat embryos in an electric refrigerator at 130 8C without liquid cryo-agents, with ultra-short exposure in the freezing medium and direct re-hydration after thawing. Cryoletters 2000;21:13–8. [56] Greve T, Avery B, Callesen H. Viability of in vivo and in vitro produced bovine embryos. Reprod Domestic Anim 1993;28:164–9. [57] Shamsuddin MB, Rodriguez-Martinez H. Fine structure of bovine blastocysts developed either in serumfree medium or in conventional co-culture with oviduct epithelial cells. J Vet Med 1994;41:307–16. [58] Dobrinsky JR, Pursel VG, Long CR, Johnson LA. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod 2000;62:564–70.