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Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium
Animal Reproduction Science 103 (2008) 239–248
Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium Kwang Taek Lim a,b , Goo Jang a , Kyung Hee Ko a , Won Wou Lee a,b , Hee Jung Park a , Joung Joo Kim a , Sung Keun Kang a , Byeong Chun Lee a,∗ a
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, San56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, South Korea b Embyro Research Center, Seoul Dairy Corporation, Gyeonggi 476-851, South Korea Received 29 July 2006; received in revised form 12 December 2006; accepted 18 December 2006 Available online 8 January 2007
Abstract The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozenthawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5 M ethylene glycol (EG), 0.1 M sucrose, 10% fetal bovine serum (FBS) in Dulbecco’s phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24 h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1 M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1 M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P < 0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1 M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P < 0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or
∗
Corresponding author. Tel.: +82 2 880 1247; fax: +82 2 884 1902. E-mail address: [email protected] (B.C. Lee).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.12.020
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BSA supplements at 24 h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P < 0.05), and 72 h (70.4% versus 49.1 and 46 4%; P < 0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P < 0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P < 0.05) and Day 60 (45.1%; P < 0.05) of gestation, and calving rate (43.7%; P < 0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer. © 2007 Elsevier B.V. All rights reserved. Keywords: Bovine; Embryo; Cryopreservation; Choline; Albumax
1. Introduction Cryopreservation is now an effective and established way of storing embryos from a number of mammalian species, notably those of cattle (Fuku et al., 1992), laboratory mice (Whittingham, 1977) and humans (Chen, 1986). Furthermoer, improvement of the cryopreservation protocols has significantly contributed to the success of bovine embryo storage (Fahning and Garcia, 1992). Research concerning cryopreservation techniques has included studies on the type and concentration of cryoprotectants, cooling and freezing rates, seeding and plunging temperatures, thawing temperatures and rates, and methods of cryoprotectant removal (Fahning and Garcia, 1992). The culture milieu for IVP embryos not only influences their development, but also exerts an effect on embryo survival following cryopreservation (Nedambale et al., 2004). Improving the in vitro culture conditions and modifying cryopreservation technology is an effective approach for increasing survival rates of cryopreserved embryos (Cho et al., 2002; Imai et al., 2002). In conventional methods of cryopreservation, FBS and BSA were widely used in the freezing medium as extracellular cryoprotectants (Gordon, 1994). Although embryo developmental competence, total cell numbers and survival rates were improved after freezing and thawing using undefined culture media containing FBS or BSA, such media might increase the incidence of large offspring syndrome (Young et al., 1998; Sinclair et al., 1999, 2000). In terms of international trade of cattle embryos between various countries, it may be increasingly necessary to replace FBS or BSA in the freezing medium by defined macromolecules to minimize any risk of disease transmission (Le Tallec et al., 2001; Hasler, 2003; Stringfellow et al., 2004). Up until now, D-PBS has been widely used as the base medium containing cryoprotectants, and sodium salts are major components of this medium. Sodium ions contribute significantly to the solution effects during cooling and re-warming of embryos. High intracellular sodium concentrations that may result from freezing are incompatible with normal cell function. Moreover, sodium toxicity has been specifically suggested to be a major factor in cryopreservation-related cell damage. In mice, substituting choline for sodium was beneficial for cryopreservation of unfertilized eggs (Stachecki et al., 1998a,b). Unlike the sodium ion, choline is thought not to cross the cell membrane and therefore would not be expected to contribute to the intracellular solute load. A previous study also suggested a possible membrane-protective role for choline (Toner et al., 1993). The objective of this study was to improve the survival of IVP embryos derived from defined culture medium by modifying the freezing medium with lipid-rich BSA (Albumax® I) and choline.
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2. Materials and methods 2.1. Oocyte collection and in vitro maturation Bovine ovaries were collected from a local slaughterhouse, placed into saline at 35 ◦ C and transported to the laboratory within 2 h. Cumulus-oocyte complexes (COCs) from follicles 2 to 8 mm in diameter were aspirated using an 18 gauge needle attached to a 10 mL syringe. The COCs with evenly granulated cytoplasm and enclosed by more than three layers of compact cumulus cells were selected, and washed three times in Hepes-buffered tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 2 mM NaHCO3 (Sigma–Aldrich Corp.), and 1% penicillin–streptomycin (v/v). For maturation, COCs were cultured in four-well dishes (30–40 oocytes per well, Falcon, Becton-Dickinson Ltd, Plymouth, UK) for 22 h in 450 L TCM199 supplemented with 10% FBS, 10 ng/mL epidermal growth factor (EGF), 0.005 IU/mL FSH (Antrin, Teikoku, Japan), and 1 g 17-estradiol (Sigma–Aldrich Corp.) at 39 ◦ C in a humidified atmosphere of 5% CO2 . 2.2. In vitro fertilization (IVF) and culture Motile spermatozoa were selected using the swim-up technique (Parrish et al., 1986). At 22 h of IVM, COCs were inseminated with 1 × 106 spermatozoa/mL for 18 h in 50 L/well of Tyrode’s Albumin-Lactate-Pyruvate (TALP)-IVF medium on a 4-well plate. Groups of six or seven zygotes were cultured in 25 L microdrops of two-step defined culture medium (Jang et al., 2005) overlaid with mineral oil (Sigma–Aldrich Corp.) for 7 days at 39 ◦ C in an atmosphere of 5% O2 , 5% CO2 and 90% N2 . Cleavage and blastocyst formation were examined at 2 and 7 days of culture, respectively. 2.3. In vivo embryo collection For production of in vivo blastocyst (multiple ovulation and embryo transfer, MOET), clinically healthy Holstein–Friesian donor cows were selected on the basis of cyclicity. Donors were superovulated by i.m. administration of a total of 400 mg Folltropin-V (Bioniche Animal Health, Canada) reconstituted in 20 mL sterile diluents and given twice daily in a series of declining doses as 2 mL (3.5 + 3.0 + 2.0 + 1.5) over a 4-day period commencing on Day 10 of the cycle. Estrus was induced by i.m. administration of 25 mg prostaglandin F2␣ on the sixth and seventh of FSH treatment. Estrus detection was performed twice daily beginning 24 h after the first prostaglandin F2␣ injection. Donor cows were artificially inseminated 12 and 24 h after first standing estrus with semen from a proven Holstein sire. Embryos used in this study were recovered Days 7.5 or 8 of the cycle (Day 0: first standing estrus). 2.4. Standard cryopreservation protocol At Day 7 after IVF, in vitro or in vivo blastocysts were equilibrated in Dulbecco’s phosphate buffered saline (D-PBS; Invitrogen, Carlsbad, CA) supplemented with 1.5 M ethylene glycol (EG), 0.1 M sucrose and 10% FBS. The embryos were loaded individually into 0.25 mL plastic straws. The straws were transferred into a controlled-rate freezer (FHK, Tokyo, Japan) equilibrated for 10 min at −7 ◦ C during which time seeding was initiated. Freezing was accomplished using a cooling rate of −0.3 ◦ C/min from −7 to −35 ◦ C, and then straws were plunged into liquid nitrogen
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Fig. 1. Morphology of in vitro produced bovine embryos after freezing–thawing at 0 h (A), 24 h (B), 48 h (C), and 72 h (D). The embryos were cryopreserved in medium in which sodium chloride had been replaced with choline chloride and supplemented with 0.4% lipid-rich BSA (Albumax I) instead of 10% FBS.
for storage. Frozen straws were subsequently warmed for 6–10 s at air temperature, followed by 15 s at 37 ◦ C. The thawed embryos were washed three times each for 4 min in Hepes buffered TCM 199 and further cultured in defined culture medium. After 24 and 48 h, viability of thawed embryo was evaluated by blastocyst expansion or hatching (Fig. 1). Preliminary studies in this laboratory showed that embryos frozen using this protocol had survival rates of 74.6% at 24 h and 53.3% at 48 h post-thaw using the standard protocol. 2.5. Embryo transfer (ET) After thawing, one or two blastocysts in Hepes-buffered TCM-199 supplemented with 2 mM NaHCO3 (Sigma–Aldrich Corp.) and 1% penicillin–streptomycin (v/v) were transferred to the uterine horn of each recipient cow by a transcervical method on Day 7 (estrus = 0 day). In order to determine embryo survival and pregnancy, cows were examined by rectal palpation 35 days after post-ET. Pregnant cows were monitored by rectal palpation at regular intervals thereafter. 2.6. Experimental designs 2.6.1. Experiment 1: effect of sucrose and xylose on embryo survival This experiment was to evaluate the influence of different sugars, xylose (monosaccharides) and sucrose (disaccharide) in the freezing medium. At Day 7, blastocysts derived from IVF were randomly allocated to three freezing protocols: D-PBS containing 1.5 M EG and 10% FBS, supplemented with (1) 0.1 M sucrose; (2) 0.1 M xylose; (3) none. Survival was defined as expansion, or partial or complete hatching during successive culture periods (24, 48 and 72 h).
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2.6.2. Experiment 2: effect of altering sodium concentration in the cryopreservation medium The effect of reducing the sodium concentration in the freezing medium on embryo cryopreservation was examined. Sodium-based freezing medium, which was D-PBS containing 1.5 M EG, 10% FBS and 0.1 M sucrose, was modified by substituting NaCl with an equal percentage of choline chloride to yield solutions containing either 0%, 50%, 75% or 100% replacement of sodium chloride. It should be noted that while in 100% choline chloride all the NaCl was replaced with choline chloride (medium named “CJ1”) some sodium ions were still present in the medium because of the inclusion of Na2 HPO4 and serum (Stachecki et al., 1998a); however, the amount of sodium present in the medium derived from these sources was very small. Blastocysts were frozen using these media and after thawing they were analyzed for their ability to develop to the expanding, hatching or hatched stages. 2.6.3. Experiment 3: effect of lipid-rich bovine serum albumin on frozen-thawed embryos Experiment 3 examined the effect of protein type (FBS, BSA, high lipid content Albumax® 1 (Cat. No. GIBCO 11020-021. Invitrogen, Carlsbad, CA)) in the freezing medium on post-thaw survival of embryos. The base freezing medium used (with choline chloride completely substituted for sodium chloride, containing 1.5 M EG and 0.1 M sucrose) was supplemented with either 10% FBS, 0.4% BSA or 0.4% Albumax® I. 2.6.4. Experiment 4: calving rates following transfer of embryos derived using the improved cryopreservation protocol In vitro produced embryos were frozen using a conventional method or the improved cryopreservation medium derived from Experiments 1 to 3. To evaluate the survival rates of frozen-thawed embryo as an in vivo study, thawed in vitro or in vivo preimplantational stage embryos were transferred into recipient cows. After ET, pregnancy was monitored at Days 35 and 60 by rectal palpation. 2.7. Statistical analysis Each experiment was replicated at least nine times and embryos were randomly allocated to each treatment group. Values in each variate were subjected to ANOVA in a generalized linear model (PROC-GLM) of statistical analysis system (SAS). When the model effect was significant in each parameter, each value after the treatment was compared by the least squares method. Significant difference among treatments was determined where the P-value was less than 0.05. 3. Results 3.1. Experiment 1: effect of sucrose and xylose on freezing–thawing of embryos Supplementation of the cryopreservation medium with 0.1 M sucrose resulted in higher postthaw survival rates of in vitro produced embryos at 24 h (71.3% versus 53.5 and 51.7%; P < 0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1 M xylose or no supplement, respectively (Table 1). 3.2. Experiment 2: effect of altering sodium concentration in the cryopreservation medium An increase in embryo survival was observed when NaCl was replaced by choline chloride (Table 2). When D-PBS in the freezing medium was completely replaced by CJ1, embryo survival
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Table 1 Influence of adding sucrose or xylose to the freezing medium (1.5 M ethylene glycol, 10% FBS) on the cryopreservation of in vitro produced preimplantation bovine embryos* Treatment
Number of embryos thawed
0.1 M sucrose 0.1 M xylose None a,b *
Number (%) of embryos surviving after 24 h (71.3)a
94 86 87
67 46 (53.5)b 45 (51.7)b
48 h
72 h
48 (51.1) 39 (45.3) 35 (40.2)
32 (34.0) 21 (24.4) 20 (23.0)
Values with different letters in the same column are significantly different (P < 0.05). All in vitro blastocysts were produced in chemically defined medium containing PVA (Jang et al., 2005).
Table 2 Effect of replacing sodium chloride with choline chloride in the freezing medium on the cryopreservation of in vitro produced bovine embryos* Composition ratio of freezing medium D-PBS (%)
CJ1 (%)**
0 25 50 100
100 75 50 0
Number of embryos thawed*
Number (%) of embryos surviving after 24 h
48 h
72 h
89 86 79 81
77 (86.5)a 73 (84.9)ab 60 (75.9)ab 59 (72.8)b
59 (66.3) 56 (65.1) 44 (55.6) 43 (53.1)
40 (44.9)c 39 (45.3)c 25 (31.6)cd 23 (28.4)d
a–d
Values with different letters in the same column are significantly different (P < 0.05). All in vitro blastocysts were produced in chemically defined medium containing PVA (Jang et al., 2005). ** The freezing medium which completely substituted choline chloride for sodium chloride was designated CJ1 (Stachecki et al., 1998a). Both freezing media contained 1.5 M EG, 0.1 M sucrose, and 10% FBS. *
at 24 and 72 h showed a significant difference (72.8% versus 86.5%, and 28.4% versus 44.9%, respectively). However, at 48 h the differences were not statistically significant. 3.3. Experiment 3: effect of lipid-rich bovine serum albumin on frozen-thawed embryos Survival was assessed by protein type (FBS, BSA and Albumax® I) during freezing and thawing. The addition of 10% FBS or 0.4% BSA significantly reduced survival compared with that of using 0.4% Albumax® I supplementation at 48 and 72 h (76.9%, 70.9% versus 87.2%, and 49.1%, 46%, 4% versus 70.4%, respectively). However, at 24 h, survival of thawed embryos was not affected by protein type during freezing. 3.4. Experiment 4: calving rates following transfer of embryos cryopreserved using the improved protocol Improved freezing conditions from Experiments 2 to 4 were beneficial for the survival of IVP embryos, compared to the conventional protocol (Table 4). Although pregnancy rates of in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax was not statistically different at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%), calving rates resulted in higher (22.6% versus 10.0%; P < 0.05) than those frozen in the standard medium. However, in vivo produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (50.7%; P < 0.05) and Day 60 (45.1%; P < 0.05) of gestation, and calving rate (43.7%; P < 0.05).
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4. Discussion In embryo cryopreservation, BSA or serum, which may contain a wide range of undefined proteins, is usually added to freezing solutions (Grilli et al., 1980; Schiewe et al., 1991; Ali and Shelton, 1993a,b; Szell and Windsor, 1994). It would appear that biological proteins have a beneficial role in embryo cryopreservation. However, culturing IVP embryos in media containing BSA or serum increased the incidence of large offspring syndrome (Young et al., 1998; Sinclair et al., 2000) and the risk of disease transmission (Wrathall, 1995, 1997; Wrathall et al., 2006). Without BSA and serum, our previous research suggested that developmental competence of embryos produced in defined culture media containing PVA was not inferior to the competence of those produced in undefined media and the former exhibited better pregnancy rates (Jang et al., 2005). There was a need to improve the preservation of IVP embryos derived from chemically defined media. It is well known that cryopreservation stresses embryos, and that IVP embryos are much more susceptible to freezing-induced damage than in vivo generated embryos. The results reported here demonstrated that improving the freezing medium by substituting choline for sodium chloride and adding 0.1 M sucrose and 0.4% Albumax® 1 enhances the quality of bovine embryos cultured in chemically defined medium in terms of embryo survival after freezing–thawing and calving rates compared to conventional freezing medium. However, the results in terms of calving rates were still not as good as with in vivo produced embryos that were cryopreserved using the same improved freezing medium. Literature reveals many different methods for the cryopreservation of embryos that have been developed in many laboratories around the world. Several cryoprotectants are being employed in various concentrations and occasionally in combination with one another. Cryoprotectants generally fall into two categories: intracellular and extracellular. Of the intracellular cryoprotectants, which are of rather low molecular weight, glycerol and ethylene glycol have been the most commonly used in freezing cattle embryos. The extracellular cryoprotectants are larger molecules, such as sugars and proteins. Non-penetrating molecules such as sucrose enable the embryo to reach equilibrium, in terms of its isotonic volume, without undergoing cell distention, and prevent swelling while the absence of cryoprotectants in the extracellular solution permits rapid efflux of the intracellular solute (Finn, 1979; Gordon, 1994). This study showed significantly higher survival rates in freezing medium containing 0.1 M sucrose than for any other group. No beneficial effects of using xylose (a monosaccharide) versus sucrose (a disaccharide) on embryo survival were observed (Table 1). Under physiological conditions, sodium ions diffuse into the cell slowly but do accumulate unless they are removed. But the excess is removed by sodium pumps, an activity that accounts for a substantial proportion of the cell’s total energy expenditure (Wolfe, 1993). During freezing, the rise in the extracellular solute concentration brought about by ice formation favors not only the flow of water out of the cell but also an increase in diffusion of sodium into the cell. At the same time, the sodium pumps may become increasingly disabled as the temperature decreases. It is therefore likely that the intracellular sodium load will have increased by the time the frozen cell is transferred to liquid nitrogen. If so, this situation will still exist immediately after thawing and could lead to post-thaw damage. Substituting choline chloride for sodium chloride in the freezing medium would help overcome this problem. Previous studies (Stachecki et al., 1998a) report that the high concentrations of sodium chloride in conventional freezing media is detrimental to cells and show that choline is a promising replacement for sodium chloride. Here, for the first time, we introduced the use of choline chloride to bovine embryo freezing. As in earlier studies, we hypothesize that reducing or virtually eliminating sodium chloride may allow oocytes, embryos
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Table 3 Effect of macromolecules (FBS, BSA or Albumax® I) in CJ1* medium on the cryopreservation of in vitro produced bovine embryos** Macromolecule
FBS 10% BSA 0.4% Albumax® 0.4%
Number of embryos thaweda 108 110 125
Number (%) of embryos surviving after 24 h
48 h
72 h (76.9)a
98 (90.7) 96 (87.3) 115 (92.0)
83 78 (70.9)a 109 (87.2)b
53 (49.1)c 51 (46.4)c 88 (70.4)d
a–d
Values with different letters in the same column are significantly different (P < 0.05). The freezing medium which completely substituted choline chloride for sodium chloride was designated CJ1 (Stachecki et al., 1998a). ** All in vitro blastocyst were produced in chemically defined medium containing PVA (Jang et al., 2005). *
and other cells to be frozen more efficiently. Replacing all of the freezing medium D-PBS with CJ1 containing choline chloride showed significantly higher post-thaw rates of blastocyst expansion or hatching than those in containing various proportions of D-PBS. Little is known about the mechanisms involved in cryoprotection by extracellular cryoprotectants, such as BSA, FBS and hyaluronic acid. It has been suggested by Leibo (1988) that BSA protects cell membranes in the immediate post-thaw phase and its most valuable role may be in stabilizing cell membranes in some way at this time. Aoyagi et al. (1996) reported that freezing medium supplemented with high lipid BSA increases pregnancy rates. In the present study, in order to further increase post-thaw embryo survival following replacement of sodium with choline, effects of various macromolecules (FBS, BSA and Albumax® I) was investigated. As shown in Table 3, freezing medium containing 0.4%, Albumax® I showed significantly higher survival up to expanding, hatching or hatched blastocyst at 48 or 72 h compared to BSA or FBS. This may be because Albumax® I, which has high lipid content, may help to stabilize the cell membranes during freezing and/or thawing of embryos. According to this study, the increase in post-thaw embryo survival in vitro with improved freezing medium was also reflected in higher calving rates compared to conventional methods (Table 4). In conclusion, this study showed that modifying the cryopreservation method for bovine embryos by adding 0.1 M sucrose and 0.4% Albumax® I and replacing sodium chloride with choline chloride enhanced the survival of embryos in vitro and allows post-thaw embryos to more efficiently develop into term calves. Table 4 Pregnancy and calving rates following transfer of one or two in vitro or in vivo produced bovine embryos after freezing–thawing* Freezing condition (1.5 M EG, 0.1 M sucrose) In vitro In vitro In vivo a–g
10% FBS in D-PBS Albumax® I 0.4% in CJ1** Albumax® I 0.4% in CJ1
Number of embryo transfersa 70 605 71
Number (%) of pregnancies Day 35 (22.9)a
16 193 (31.9)a 36 (50.7)b
Day 60 10 (14.3)c 146 (24.1)c 32 (45.1)d
Number (%) of calved 7 (10.0)e 137 (22.6)f 31 (43.7)g
Values with different letters in the same column are significantly different (P < 0.05). All in vitro blastocysts were produced in chemically defined medium containing PVA (Jang et al., 2005). ** The freezing medium which completely substituted choline chloride for sodium chloride was designated CJ1 (Stachecki et al., 1998a). *
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Acknowledgments This study was supported by grants from the Korea Ministry of Science and Technology, and Biogreen 21-1000520030100000. We thank Dr. Barry D. Bavister, University of New Orleans, for his great editing of the manuscript. The authors are grateful for a graduate fellowship provided by the Ministry of Education, through the BK21 program for Veterinary Science, Seoul National University. References Ali, J., Shelton, J.N., 1993a. Design of vitrification solutions for the cryopreservation of embryos. J. Reprod. Fertil. 99, 471–477. Ali, J., Shelton, J.N., 1993b. Successful vitrification of day-6 sheep embryos. J. Reprod. Fertil. 99, 65–70. Aoyagi, Y., Konishi, M., Takedomi, T., Itakura, H., Itoh, T., Yazawa, S., 1996. Effect of lipid-rich bovine serum albumin on direct transfer of frozen-thawed bovine embryos. Theriogenology 45, 165. Chen, C., 1986. Pregnancy after human oocyte cryopreservation. Lancet 1, 884–886. Cho, S.R., Cho, S.K., Lee, S.L., Lee, H.J., Choe, S.Y., Rho, G.J., 2002. Enhanced cryosurvival of bovine blastocysts produced in vitro in serum-free medium. J. Assist. Reprod. Genet. 19, 487–492. Fahning, M.L., Garcia, M.A., 1992. Status of cryopreservation of embryos from domestic animals. Cryobiology 29, 1–18. Finn, C.A., 1979. Oxford reviews of reproductive biology. In: Watson, P.F. (Ed.), The Preservation of Semen in Mammals. Clarendon Press/Oxford University Press, Oxford, New York, pp. 283–351. Fuku, E., Kojima, T., Shioya, Y., Marcus, G.J., Downey, B.R., 1992. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology 29, 485–492. Gordon, I., 1994. Laboratory Production of Cattle Embryos. CAB International, Wallingford, UK, pp. 245–371. Grilli, G., Porcellini, A., Lucarelli, G., 1980. Role of serum on cryopreservation and subsequent viability of mouse bone marrow hemopoietic stem cells. Cryobiology 17, 516–520. Hasler, J.F., 2003. The current status and future of commercial embryo transfer in cattle. Anim. Reprod. Sci. 79, 245–264. Imai, K., Matoba, S., Dochi, O., Shimohira, I., 2002. Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo. J. Vet. Med. Sci. 64, 887–891. Jang, G., Lim, K.T., Lee, W.W., Kim, J.I., Ko, K.H., Park, H.J., Kim, J.J., Kang, S.K., Lee, B.C., Hwang, W.S., 2005. Improved in vitro embryo development and increased efficiency in producing viable calves with the optimized defined two steps culture media. In: Proceedings of the 38th Annual Meeting Society for the Study of Reproduction, 151. Le Tallec, B., Ponsart, C., Marquant-Le Guienne, B., Guerin, B., 2001. Risks of transmissible diseases in relation to embryo transfer. Reprod. Nutr. Dev. 41, 439–450. Leibo, S.P., 1988. Cryopreservation. In: Proceedings of the Eleventh International Congress on Animal Reproduction and Artificial Insemination, vol. 5, Dublin, pp. 370–377. Nedambale, T.L., Dinnyes, A., Groen, W., Dobrinsky, J.R., Tian, X.C., Yang, X., 2004. Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification. Theriogenology 62, 437–449. Parrish, J.J., Susko-Parrish, J.L., Leibfried-Rutledge, M.L., Critser, E.S., Eyestone, W.H., First, N.L., 1986. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25, 591–600. Schiewe, M.C., Rall, W.F., Stuart, L.D., Wildt, D.E., 1991. Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology 36, 279–293. Sinclair, K.D., McEvoy, T.G., Maxfield, E.K., Maltin, C.A., Young, L.E., Wilmut, I., Broadbent, P.J., Robinson, J.J., 1999. Aberrant fetal growth and development after in vitro culture of sheep zygotes. J. Reprod. Fertil. 116, 177–186. Sinclair, K.D., Young, L.E., Wilmut, I., McEvoy, T.G., 2000. In-utero overgrowth in ruminants following embryo culture: lessons from mice and a warning to men. Hum. Reprod. 15 (Suppl. 5), 68–86. Stachecki, J.J., Cohen, J., Willadsen, S., 1998a. Detrimental effects of sodium during mouse oocyte cryopreservation. Biol. Reprod. 59, 395–400. Stachecki, J.J., Cohen, J., Willadsen, S.M., 1998b. Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 37, 346–354. Stringfellow, D.A., Givens, M.D., Waldrop, J.G., 2004. Biosecurity issues associated with current and emerging embryo technologies. Reprod. Fertil. Dev. 16, 93–102. Szell, A.Z., Windsor, D.P., 1994. Survival of vitrified sheep embryos in vitro and in vivo. Theriogenology 42, 881–889.
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Toner, M., Cravalho, E.G., Stachecki, J., Fitzgerald, T., Tompkins, R.G., Yarmush, M.L., Armant, D.R., 1993. Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential. Biophys. J. 64, 1908–1921. Whittingham, D.G., 1977. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at −196 ◦ C. J. Reprod. Fertil. 49, 89–94. Wolfe, S.L., 1993. Molecular and Cellular Biology. Wadsworth Publishing Company, Belmont, CA. Wrathall, A.E., 1995. Embryo transfer and disease transmission in livestock: a review of recent research. Theriogenology 43, 81–88. Wrathall, A.E., 1997. Risks of transmitting scrapie and bovine spongiform encephalopathy by semen and embryos. Rev. Sci. Technol. 16, 240–264. Wrathall, A.E., Simmons, H.A., Van Soom, A., 2006. Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilisation with virus-infected semen. Theriogenology 65, 247–274. Young, L.E., Sinclair, K.D., Wilmut, I., 1998. Large offspring syndrome in cattle and sheep. Rev. Reprod. 3, 155–163.
Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium Kwang Taek Lim a,b , Goo Jang a , Kyung Hee Ko a , Won Wou Lee a,b , Hee Jung Park a , Joung Joo Kim a , Sung Keun Kang a , Byeong Chun Lee a,∗ a
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, San56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, South Korea b Embyro Research Center, Seoul Dairy Corporation, Gyeonggi 476-851, South Korea Received 29 July 2006; received in revised form 12 December 2006; accepted 18 December 2006 Available online 8 January 2007
Abstract The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozenthawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5 M ethylene glycol (EG), 0.1 M sucrose, 10% fetal bovine serum (FBS) in Dulbecco’s phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24 h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1 M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1 M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P < 0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1 M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P < 0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or
∗
Corresponding author. Tel.: +82 2 880 1247; fax: +82 2 884 1902. E-mail address: [email protected] (B.C. Lee).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.12.020
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BSA supplements at 24 h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P < 0.05), and 72 h (70.4% versus 49.1 and 46 4%; P < 0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P < 0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P < 0.05) and Day 60 (45.1%; P < 0.05) of gestation, and calving rate (43.7%; P < 0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer. © 2007 Elsevier B.V. All rights reserved. Keywords: Bovine; Embryo; Cryopreservation; Choline; Albumax
1. Introduction Cryopreservation is now an effective and established way of storing embryos from a number of mammalian species, notably those of cattle (Fuku et al., 1992), laboratory mice (Whittingham, 1977) and humans (Chen, 1986). Furthermoer, improvement of the cryopreservation protocols has significantly contributed to the success of bovine embryo storage (Fahning and Garcia, 1992). Research concerning cryopreservation techniques has included studies on the type and concentration of cryoprotectants, cooling and freezing rates, seeding and plunging temperatures, thawing temperatures and rates, and methods of cryoprotectant removal (Fahning and Garcia, 1992). The culture milieu for IVP embryos not only influences their development, but also exerts an effect on embryo survival following cryopreservation (Nedambale et al., 2004). Improving the in vitro culture conditions and modifying cryopreservation technology is an effective approach for increasing survival rates of cryopreserved embryos (Cho et al., 2002; Imai et al., 2002). In conventional methods of cryopreservation, FBS and BSA were widely used in the freezing medium as extracellular cryoprotectants (Gordon, 1994). Although embryo developmental competence, total cell numbers and survival rates were improved after freezing and thawing using undefined culture media containing FBS or BSA, such media might increase the incidence of large offspring syndrome (Young et al., 1998; Sinclair et al., 1999, 2000). In terms of international trade of cattle embryos between various countries, it may be increasingly necessary to replace FBS or BSA in the freezing medium by defined macromolecules to minimize any risk of disease transmission (Le Tallec et al., 2001; Hasler, 2003; Stringfellow et al., 2004). Up until now, D-PBS has been widely used as the base medium containing cryoprotectants, and sodium salts are major components of this medium. Sodium ions contribute significantly to the solution effects during cooling and re-warming of embryos. High intracellular sodium concentrations that may result from freezing are incompatible with normal cell function. Moreover, sodium toxicity has been specifically suggested to be a major factor in cryopreservation-related cell damage. In mice, substituting choline for sodium was beneficial for cryopreservation of unfertilized eggs (Stachecki et al., 1998a,b). Unlike the sodium ion, choline is thought not to cross the cell membrane and therefore would not be expected to contribute to the intracellular solute load. A previous study also suggested a possible membrane-protective role for choline (Toner et al., 1993). The objective of this study was to improve the survival of IVP embryos derived from defined culture medium by modifying the freezing medium with lipid-rich BSA (Albumax® I) and choline.
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2. Materials and methods 2.1. Oocyte collection and in vitro maturation Bovine ovaries were collected from a local slaughterhouse, placed into saline at 35 ◦ C and transported to the laboratory within 2 h. Cumulus-oocyte complexes (COCs) from follicles 2 to 8 mm in diameter were aspirated using an 18 gauge needle attached to a 10 mL syringe. The COCs with evenly granulated cytoplasm and enclosed by more than three layers of compact cumulus cells were selected, and washed three times in Hepes-buffered tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 2 mM NaHCO3 (Sigma–Aldrich Corp.), and 1% penicillin–streptomycin (v/v). For maturation, COCs were cultured in four-well dishes (30–40 oocytes per well, Falcon, Becton-Dickinson Ltd, Plymouth, UK) for 22 h in 450 L TCM199 supplemented with 10% FBS, 10 ng/mL epidermal growth factor (EGF), 0.005 IU/mL FSH (Antrin, Teikoku, Japan), and 1 g 17-estradiol (Sigma–Aldrich Corp.) at 39 ◦ C in a humidified atmosphere of 5% CO2 . 2.2. In vitro fertilization (IVF) and culture Motile spermatozoa were selected using the swim-up technique (Parrish et al., 1986). At 22 h of IVM, COCs were inseminated with 1 × 106 spermatozoa/mL for 18 h in 50 L/well of Tyrode’s Albumin-Lactate-Pyruvate (TALP)-IVF medium on a 4-well plate. Groups of six or seven zygotes were cultured in 25 L microdrops of two-step defined culture medium (Jang et al., 2005) overlaid with mineral oil (Sigma–Aldrich Corp.) for 7 days at 39 ◦ C in an atmosphere of 5% O2 , 5% CO2 and 90% N2 . Cleavage and blastocyst formation were examined at 2 and 7 days of culture, respectively. 2.3. In vivo embryo collection For production of in vivo blastocyst (multiple ovulation and embryo transfer, MOET), clinically healthy Holstein–Friesian donor cows were selected on the basis of cyclicity. Donors were superovulated by i.m. administration of a total of 400 mg Folltropin-V (Bioniche Animal Health, Canada) reconstituted in 20 mL sterile diluents and given twice daily in a series of declining doses as 2 mL (3.5 + 3.0 + 2.0 + 1.5) over a 4-day period commencing on Day 10 of the cycle. Estrus was induced by i.m. administration of 25 mg prostaglandin F2␣ on the sixth and seventh of FSH treatment. Estrus detection was performed twice daily beginning 24 h after the first prostaglandin F2␣ injection. Donor cows were artificially inseminated 12 and 24 h after first standing estrus with semen from a proven Holstein sire. Embryos used in this study were recovered Days 7.5 or 8 of the cycle (Day 0: first standing estrus). 2.4. Standard cryopreservation protocol At Day 7 after IVF, in vitro or in vivo blastocysts were equilibrated in Dulbecco’s phosphate buffered saline (D-PBS; Invitrogen, Carlsbad, CA) supplemented with 1.5 M ethylene glycol (EG), 0.1 M sucrose and 10% FBS. The embryos were loaded individually into 0.25 mL plastic straws. The straws were transferred into a controlled-rate freezer (FHK, Tokyo, Japan) equilibrated for 10 min at −7 ◦ C during which time seeding was initiated. Freezing was accomplished using a cooling rate of −0.3 ◦ C/min from −7 to −35 ◦ C, and then straws were plunged into liquid nitrogen
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Fig. 1. Morphology of in vitro produced bovine embryos after freezing–thawing at 0 h (A), 24 h (B), 48 h (C), and 72 h (D). The embryos were cryopreserved in medium in which sodium chloride had been replaced with choline chloride and supplemented with 0.4% lipid-rich BSA (Albumax I) instead of 10% FBS.
for storage. Frozen straws were subsequently warmed for 6–10 s at air temperature, followed by 15 s at 37 ◦ C. The thawed embryos were washed three times each for 4 min in Hepes buffered TCM 199 and further cultured in defined culture medium. After 24 and 48 h, viability of thawed embryo was evaluated by blastocyst expansion or hatching (Fig. 1). Preliminary studies in this laboratory showed that embryos frozen using this protocol had survival rates of 74.6% at 24 h and 53.3% at 48 h post-thaw using the standard protocol. 2.5. Embryo transfer (ET) After thawing, one or two blastocysts in Hepes-buffered TCM-199 supplemented with 2 mM NaHCO3 (Sigma–Aldrich Corp.) and 1% penicillin–streptomycin (v/v) were transferred to the uterine horn of each recipient cow by a transcervical method on Day 7 (estrus = 0 day). In order to determine embryo survival and pregnancy, cows were examined by rectal palpation 35 days after post-ET. Pregnant cows were monitored by rectal palpation at regular intervals thereafter. 2.6. Experimental designs 2.6.1. Experiment 1: effect of sucrose and xylose on embryo survival This experiment was to evaluate the influence of different sugars, xylose (monosaccharides) and sucrose (disaccharide) in the freezing medium. At Day 7, blastocysts derived from IVF were randomly allocated to three freezing protocols: D-PBS containing 1.5 M EG and 10% FBS, supplemented with (1) 0.1 M sucrose; (2) 0.1 M xylose; (3) none. Survival was defined as expansion, or partial or complete hatching during successive culture periods (24, 48 and 72 h).
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2.6.2. Experiment 2: effect of altering sodium concentration in the cryopreservation medium The effect of reducing the sodium concentration in the freezing medium on embryo cryopreservation was examined. Sodium-based freezing medium, which was D-PBS containing 1.5 M EG, 10% FBS and 0.1 M sucrose, was modified by substituting NaCl with an equal percentage of choline chloride to yield solutions containing either 0%, 50%, 75% or 100% replacement of sodium chloride. It should be noted that while in 100% choline chloride all the NaCl was replaced with choline chloride (medium named “CJ1”) some sodium ions were still present in the medium because of the inclusion of Na2 HPO4 and serum (Stachecki et al., 1998a); however, the amount of sodium present in the medium derived from these sources was very small. Blastocysts were frozen using these media and after thawing they were analyzed for their ability to develop to the expanding, hatching or hatched stages. 2.6.3. Experiment 3: effect of lipid-rich bovine serum albumin on frozen-thawed embryos Experiment 3 examined the effect of protein type (FBS, BSA, high lipid content Albumax® 1 (Cat. No. GIBCO 11020-021. Invitrogen, Carlsbad, CA)) in the freezing medium on post-thaw survival of embryos. The base freezing medium used (with choline chloride completely substituted for sodium chloride, containing 1.5 M EG and 0.1 M sucrose) was supplemented with either 10% FBS, 0.4% BSA or 0.4% Albumax® I. 2.6.4. Experiment 4: calving rates following transfer of embryos derived using the improved cryopreservation protocol In vitro produced embryos were frozen using a conventional method or the improved cryopreservation medium derived from Experiments 1 to 3. To evaluate the survival rates of frozen-thawed embryo as an in vivo study, thawed in vitro or in vivo preimplantational stage embryos were transferred into recipient cows. After ET, pregnancy was monitored at Days 35 and 60 by rectal palpation. 2.7. Statistical analysis Each experiment was replicated at least nine times and embryos were randomly allocated to each treatment group. Values in each variate were subjected to ANOVA in a generalized linear model (PROC-GLM) of statistical analysis system (SAS). When the model effect was significant in each parameter, each value after the treatment was compared by the least squares method. Significant difference among treatments was determined where the P-value was less than 0.05. 3. Results 3.1. Experiment 1: effect of sucrose and xylose on freezing–thawing of embryos Supplementation of the cryopreservation medium with 0.1 M sucrose resulted in higher postthaw survival rates of in vitro produced embryos at 24 h (71.3% versus 53.5 and 51.7%; P < 0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1 M xylose or no supplement, respectively (Table 1). 3.2. Experiment 2: effect of altering sodium concentration in the cryopreservation medium An increase in embryo survival was observed when NaCl was replaced by choline chloride (Table 2). When D-PBS in the freezing medium was completely replaced by CJ1, embryo survival
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Table 1 Influence of adding sucrose or xylose to the freezing medium (1.5 M ethylene glycol, 10% FBS) on the cryopreservation of in vitro produced preimplantation bovine embryos* Treatment
Number of embryos thawed
0.1 M sucrose 0.1 M xylose None a,b *
Number (%) of embryos surviving after 24 h (71.3)a
94 86 87
67 46 (53.5)b 45 (51.7)b
48 h
72 h
48 (51.1) 39 (45.3) 35 (40.2)
32 (34.0) 21 (24.4) 20 (23.0)
Values with different letters in the same column are significantly different (P < 0.05). All in vitro blastocysts were produced in chemically defined medium containing PVA (Jang et al., 2005).
Table 2 Effect of replacing sodium chloride with choline chloride in the freezing medium on the cryopreservation of in vitro produced bovine embryos* Composition ratio of freezing medium D-PBS (%)
CJ1 (%)**
0 25 50 100
100 75 50 0
Number of embryos thawed*
Number (%) of embryos surviving after 24 h
48 h
72 h
89 86 79 81
77 (86.5)a 73 (84.9)ab 60 (75.9)ab 59 (72.8)b
59 (66.3) 56 (65.1) 44 (55.6) 43 (53.1)
40 (44.9)c 39 (45.3)c 25 (31.6)cd 23 (28.4)d
a–d
Values with different letters in the same column are significantly different (P < 0.05). All in vitro blastocysts were produced in chemically defined medium containing PVA (Jang et al., 2005). ** The freezing medium which completely substituted choline chloride for sodium chloride was designated CJ1 (Stachecki et al., 1998a). Both freezing media contained 1.5 M EG, 0.1 M sucrose, and 10% FBS. *
at 24 and 72 h showed a significant difference (72.8% versus 86.5%, and 28.4% versus 44.9%, respectively). However, at 48 h the differences were not statistically significant. 3.3. Experiment 3: effect of lipid-rich bovine serum albumin on frozen-thawed embryos Survival was assessed by protein type (FBS, BSA and Albumax® I) during freezing and thawing. The addition of 10% FBS or 0.4% BSA significantly reduced survival compared with that of using 0.4% Albumax® I supplementation at 48 and 72 h (76.9%, 70.9% versus 87.2%, and 49.1%, 46%, 4% versus 70.4%, respectively). However, at 24 h, survival of thawed embryos was not affected by protein type during freezing. 3.4. Experiment 4: calving rates following transfer of embryos cryopreserved using the improved protocol Improved freezing conditions from Experiments 2 to 4 were beneficial for the survival of IVP embryos, compared to the conventional protocol (Table 4). Although pregnancy rates of in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax was not statistically different at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%), calving rates resulted in higher (22.6% versus 10.0%; P < 0.05) than those frozen in the standard medium. However, in vivo produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (50.7%; P < 0.05) and Day 60 (45.1%; P < 0.05) of gestation, and calving rate (43.7%; P < 0.05).
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4. Discussion In embryo cryopreservation, BSA or serum, which may contain a wide range of undefined proteins, is usually added to freezing solutions (Grilli et al., 1980; Schiewe et al., 1991; Ali and Shelton, 1993a,b; Szell and Windsor, 1994). It would appear that biological proteins have a beneficial role in embryo cryopreservation. However, culturing IVP embryos in media containing BSA or serum increased the incidence of large offspring syndrome (Young et al., 1998; Sinclair et al., 2000) and the risk of disease transmission (Wrathall, 1995, 1997; Wrathall et al., 2006). Without BSA and serum, our previous research suggested that developmental competence of embryos produced in defined culture media containing PVA was not inferior to the competence of those produced in undefined media and the former exhibited better pregnancy rates (Jang et al., 2005). There was a need to improve the preservation of IVP embryos derived from chemically defined media. It is well known that cryopreservation stresses embryos, and that IVP embryos are much more susceptible to freezing-induced damage than in vivo generated embryos. The results reported here demonstrated that improving the freezing medium by substituting choline for sodium chloride and adding 0.1 M sucrose and 0.4% Albumax® 1 enhances the quality of bovine embryos cultured in chemically defined medium in terms of embryo survival after freezing–thawing and calving rates compared to conventional freezing medium. However, the results in terms of calving rates were still not as good as with in vivo produced embryos that were cryopreserved using the same improved freezing medium. Literature reveals many different methods for the cryopreservation of embryos that have been developed in many laboratories around the world. Several cryoprotectants are being employed in various concentrations and occasionally in combination with one another. Cryoprotectants generally fall into two categories: intracellular and extracellular. Of the intracellular cryoprotectants, which are of rather low molecular weight, glycerol and ethylene glycol have been the most commonly used in freezing cattle embryos. The extracellular cryoprotectants are larger molecules, such as sugars and proteins. Non-penetrating molecules such as sucrose enable the embryo to reach equilibrium, in terms of its isotonic volume, without undergoing cell distention, and prevent swelling while the absence of cryoprotectants in the extracellular solution permits rapid efflux of the intracellular solute (Finn, 1979; Gordon, 1994). This study showed significantly higher survival rates in freezing medium containing 0.1 M sucrose than for any other group. No beneficial effects of using xylose (a monosaccharide) versus sucrose (a disaccharide) on embryo survival were observed (Table 1). Under physiological conditions, sodium ions diffuse into the cell slowly but do accumulate unless they are removed. But the excess is removed by sodium pumps, an activity that accounts for a substantial proportion of the cell’s total energy expenditure (Wolfe, 1993). During freezing, the rise in the extracellular solute concentration brought about by ice formation favors not only the flow of water out of the cell but also an increase in diffusion of sodium into the cell. At the same time, the sodium pumps may become increasingly disabled as the temperature decreases. It is therefore likely that the intracellular sodium load will have increased by the time the frozen cell is transferred to liquid nitrogen. If so, this situation will still exist immediately after thawing and could lead to post-thaw damage. Substituting choline chloride for sodium chloride in the freezing medium would help overcome this problem. Previous studies (Stachecki et al., 1998a) report that the high concentrations of sodium chloride in conventional freezing media is detrimental to cells and show that choline is a promising replacement for sodium chloride. Here, for the first time, we introduced the use of choline chloride to bovine embryo freezing. As in earlier studies, we hypothesize that reducing or virtually eliminating sodium chloride may allow oocytes, embryos
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Table 3 Effect of macromolecules (FBS, BSA or Albumax® I) in CJ1* medium on the cryopreservation of in vitro produced bovine embryos** Macromolecule
FBS 10% BSA 0.4% Albumax® 0.4%
Number of embryos thaweda 108 110 125
Number (%) of embryos surviving after 24 h
48 h
72 h (76.9)a
98 (90.7) 96 (87.3) 115 (92.0)
83 78 (70.9)a 109 (87.2)b
53 (49.1)c 51 (46.4)c 88 (70.4)d
a–d
Values with different letters in the same column are significantly different (P < 0.05). The freezing medium which completely substituted choline chloride for sodium chloride was designated CJ1 (Stachecki et al., 1998a). ** All in vitro blastocyst were produced in chemically defined medium containing PVA (Jang et al., 2005). *
and other cells to be frozen more efficiently. Replacing all of the freezing medium D-PBS with CJ1 containing choline chloride showed significantly higher post-thaw rates of blastocyst expansion or hatching than those in containing various proportions of D-PBS. Little is known about the mechanisms involved in cryoprotection by extracellular cryoprotectants, such as BSA, FBS and hyaluronic acid. It has been suggested by Leibo (1988) that BSA protects cell membranes in the immediate post-thaw phase and its most valuable role may be in stabilizing cell membranes in some way at this time. Aoyagi et al. (1996) reported that freezing medium supplemented with high lipid BSA increases pregnancy rates. In the present study, in order to further increase post-thaw embryo survival following replacement of sodium with choline, effects of various macromolecules (FBS, BSA and Albumax® I) was investigated. As shown in Table 3, freezing medium containing 0.4%, Albumax® I showed significantly higher survival up to expanding, hatching or hatched blastocyst at 48 or 72 h compared to BSA or FBS. This may be because Albumax® I, which has high lipid content, may help to stabilize the cell membranes during freezing and/or thawing of embryos. According to this study, the increase in post-thaw embryo survival in vitro with improved freezing medium was also reflected in higher calving rates compared to conventional methods (Table 4). In conclusion, this study showed that modifying the cryopreservation method for bovine embryos by adding 0.1 M sucrose and 0.4% Albumax® I and replacing sodium chloride with choline chloride enhanced the survival of embryos in vitro and allows post-thaw embryos to more efficiently develop into term calves. Table 4 Pregnancy and calving rates following transfer of one or two in vitro or in vivo produced bovine embryos after freezing–thawing* Freezing condition (1.5 M EG, 0.1 M sucrose) In vitro In vitro In vivo a–g
10% FBS in D-PBS Albumax® I 0.4% in CJ1** Albumax® I 0.4% in CJ1
Number of embryo transfersa 70 605 71
Number (%) of pregnancies Day 35 (22.9)a
16 193 (31.9)a 36 (50.7)b
Day 60 10 (14.3)c 146 (24.1)c 32 (45.1)d
Number (%) of calved 7 (10.0)e 137 (22.6)f 31 (43.7)g
Values with different letters in the same column are significantly different (P < 0.05). All in vitro blastocysts were produced in chemically defined medium containing PVA (Jang et al., 2005). ** The freezing medium which completely substituted choline chloride for sodium chloride was designated CJ1 (Stachecki et al., 1998a). *
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Acknowledgments This study was supported by grants from the Korea Ministry of Science and Technology, and Biogreen 21-1000520030100000. We thank Dr. Barry D. Bavister, University of New Orleans, for his great editing of the manuscript. The authors are grateful for a graduate fellowship provided by the Ministry of Education, through the BK21 program for Veterinary Science, Seoul National University. References Ali, J., Shelton, J.N., 1993a. Design of vitrification solutions for the cryopreservation of embryos. J. Reprod. Fertil. 99, 471–477. Ali, J., Shelton, J.N., 1993b. Successful vitrification of day-6 sheep embryos. J. Reprod. Fertil. 99, 65–70. Aoyagi, Y., Konishi, M., Takedomi, T., Itakura, H., Itoh, T., Yazawa, S., 1996. Effect of lipid-rich bovine serum albumin on direct transfer of frozen-thawed bovine embryos. Theriogenology 45, 165. Chen, C., 1986. Pregnancy after human oocyte cryopreservation. Lancet 1, 884–886. Cho, S.R., Cho, S.K., Lee, S.L., Lee, H.J., Choe, S.Y., Rho, G.J., 2002. Enhanced cryosurvival of bovine blastocysts produced in vitro in serum-free medium. J. Assist. Reprod. Genet. 19, 487–492. Fahning, M.L., Garcia, M.A., 1992. Status of cryopreservation of embryos from domestic animals. Cryobiology 29, 1–18. Finn, C.A., 1979. Oxford reviews of reproductive biology. In: Watson, P.F. (Ed.), The Preservation of Semen in Mammals. Clarendon Press/Oxford University Press, Oxford, New York, pp. 283–351. Fuku, E., Kojima, T., Shioya, Y., Marcus, G.J., Downey, B.R., 1992. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology 29, 485–492. Gordon, I., 1994. Laboratory Production of Cattle Embryos. CAB International, Wallingford, UK, pp. 245–371. Grilli, G., Porcellini, A., Lucarelli, G., 1980. Role of serum on cryopreservation and subsequent viability of mouse bone marrow hemopoietic stem cells. Cryobiology 17, 516–520. Hasler, J.F., 2003. The current status and future of commercial embryo transfer in cattle. Anim. Reprod. Sci. 79, 245–264. Imai, K., Matoba, S., Dochi, O., Shimohira, I., 2002. Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo. J. Vet. Med. Sci. 64, 887–891. Jang, G., Lim, K.T., Lee, W.W., Kim, J.I., Ko, K.H., Park, H.J., Kim, J.J., Kang, S.K., Lee, B.C., Hwang, W.S., 2005. Improved in vitro embryo development and increased efficiency in producing viable calves with the optimized defined two steps culture media. In: Proceedings of the 38th Annual Meeting Society for the Study of Reproduction, 151. Le Tallec, B., Ponsart, C., Marquant-Le Guienne, B., Guerin, B., 2001. Risks of transmissible diseases in relation to embryo transfer. Reprod. Nutr. Dev. 41, 439–450. Leibo, S.P., 1988. Cryopreservation. In: Proceedings of the Eleventh International Congress on Animal Reproduction and Artificial Insemination, vol. 5, Dublin, pp. 370–377. Nedambale, T.L., Dinnyes, A., Groen, W., Dobrinsky, J.R., Tian, X.C., Yang, X., 2004. Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification. Theriogenology 62, 437–449. Parrish, J.J., Susko-Parrish, J.L., Leibfried-Rutledge, M.L., Critser, E.S., Eyestone, W.H., First, N.L., 1986. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25, 591–600. Schiewe, M.C., Rall, W.F., Stuart, L.D., Wildt, D.E., 1991. Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology 36, 279–293. Sinclair, K.D., McEvoy, T.G., Maxfield, E.K., Maltin, C.A., Young, L.E., Wilmut, I., Broadbent, P.J., Robinson, J.J., 1999. Aberrant fetal growth and development after in vitro culture of sheep zygotes. J. Reprod. Fertil. 116, 177–186. Sinclair, K.D., Young, L.E., Wilmut, I., McEvoy, T.G., 2000. In-utero overgrowth in ruminants following embryo culture: lessons from mice and a warning to men. Hum. Reprod. 15 (Suppl. 5), 68–86. Stachecki, J.J., Cohen, J., Willadsen, S., 1998a. Detrimental effects of sodium during mouse oocyte cryopreservation. Biol. Reprod. 59, 395–400. Stachecki, J.J., Cohen, J., Willadsen, S.M., 1998b. Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 37, 346–354. Stringfellow, D.A., Givens, M.D., Waldrop, J.G., 2004. Biosecurity issues associated with current and emerging embryo technologies. Reprod. Fertil. Dev. 16, 93–102. Szell, A.Z., Windsor, D.P., 1994. Survival of vitrified sheep embryos in vitro and in vivo. Theriogenology 42, 881–889.
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