Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol

ELSEVIER

SURVIVAL OF IVF-DERIVED BOVINE EMBRYOS FROZEN IN GLYCEROL OR ETHYLENE GLYCOL J. F. Hasler, P.J. Hurtgen, Z.Q. Jin and J.E. Stokes Em Tran, Inc. Elizabethtown, PA 17022 USA Received for publication: September 20, 1996 Accepted: May 16, 1997

ABSTRACT Survival of IVF-derived bovine embryos of different ages and stages of development, produced in 2 different co-culture systems and frozen in 2 different cryoprotectants, was investigated. In vitro-derived bovine embryos (n=5,525) were utilized to study survival following exposure to ctyoprotectants and after freezing. Survival of the frozen embryos was based on blastocyst re-expansion 24 h and hatching 72 h after thawing. There was no difference in survival when embryos were exposed to either glycerol (Gly) or ethylene glycol (EG) for 10 or 40 min with the cryoprotectant diluted with or without freezing. In 2 of 3 experiments in which a comparison was possible, more blastocysts frozen in 1.4 M glycerol than in 1.5 M ethylene glycol survived. Addition of 0.25 M sucrose to 1.5 M ethylene glycol in the freezing solution did not improve embryo survivat. More blastocysts frozen on Day 7 of in vitro culture survived than those frozen on Day 6 or Day 6. On Days 6, 7 and 8, embryos in the most advanced stage of development survived better than those at less advanced stages. Post-thaw survival did not differ for embryos produced in co-culture with Buffalo Rat Liver (BRL) cells with either Menezo 82 Medium or Tissue Culture Medium 199 and frozen in 1.4 M glycerol. 0 1997 by Elsevier Science Inc. Key words: bovine embryos, IVF, cryopreservation, glycerol, ethylene glycol

INTRODUCTION Production of bovine embryos by in vitro procedures is now established as a commercial enterprise (12,18,32,37,46). Cryopreservation of in vitro-derived embryos by a variety of techniques with subsequent pregnancies after thawing and transfer has been reported by a number of research (1,10,33,36) and commercial organizations (12,16,32,49). However, survival rates of frozen in vitro-derived embryos, as measured either by post-thaw development in culture or by pregnancies following Acknowledgments This research was supported by Holland Genetics, Arnhem, The Netherlands. Semen was provided by Atlantic Breeders Cooperative, Lancaster, Pennsylvania. The authors thank S.P. Leibo, R.J. Mapletoft, T. Pantano and J.P. Parks for critically evaluating the manuscript. Theriogenology 46563-579. 0 1997 by Elsevier Science

1997 Inc.

009%691)(/97/$17 PII SOO93-691X(97)00274-4

00

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transfer, have been lower than those reported for in vivo-derived embryos. Survival of frozen-thawed in vitro-derived embryos has been reported to be affected by embryo age (4,5,12,22,24,38,43,49), stage of embryonic development (3,4,5,7,27,29,50), embryo quality (11 ,l4), cryoprotectant (25,39,41,43,44,48), pH of the freezing medium (13) freezing process (39,45,49) and the culture system in which the embryos are produced (31,33,35,47). Because of the large number of interactions involving factors such as culture system, embryo age and freezing system, comparisons among different published reports are contradictory. The objective of the present study was to investigate the survival of bovine embryos of various ages and stages produced in 2 different in vitro co-culture systems and frozen using 2 different cryoprotectants.

MATERIALS AND METHODS Cocyte Source Directly upon their collection from a slaughterhouse, ovaries were placed into a plastic bag, with no additional fluid added, and transported in an insulated box at 28 to 30°C. Upon arrival in the laboratory, the ovaries were rinsed with 28°C tap water and placed for 5 min into 28°C tap water containing 1% Nolvasan solution (Aveco Co., Inc., Fort Dodge, IA) and 1% 7X cleaning solution (ICN Biomedicals, Inc., Costa Mesa, CA), after which the ovaries were rinsed again with tap water and stored at ambient temperature (23 to 27°C) prior to aspiration. A 19 g, 3/4-inch needle attached to a Pioneer Pro-Pump (Pioneer Medical, Inc., Madison, CT) was used to aspirate the contentsof all follicies between 2 and 8 mm in diameter into a 50 ml centrifuge tube. The follicular aspirate was rinsed with PBS through an Em-Con filter (Immuno Systems, Spring Valley, WI). All oocytes with 3 or more compact layers of cumulus cells were utilized for maturation and were not graded on the basis of cytoplasmic appearance. Approximately 18,000 oocytes, aspirated from approximately 2,600 ovaries, were matured in vitro for this study. All culture procedures, including in vitro maturation, fertilization and culture were conducted in 4-well plates with no oil overlay (Nunclon, Kantrup, Denmark). Unless stated otherwise, all chemicals were obtained from Sigma Chemical Company (St. Louis, MO). In Vitro Maturation (IVM) Following aspiration, oocytes were rinsed 5 times in a modified Tyrode’s Medium (TAL-HEPES; Bio Whittaker, Inc., Walkersville, MD) and placed into maturation medium within 5 to 9 h after the ovaries had been collected at the slaughterhouse. Groups of 30 to 35 oocytes were matured for 24 h in 0.5 ml TCM-199 with Earle’s salts, supplemented with 2.2 g/L sodium bicarbonate (Gibco BRL, Grand Island, NY), 10% heat-inactivated fetal calf serum (FCS; Hyclone Laboratories, Logan, UT), 4 pg FSH and 6 pg LH (NOBL Laboratories, Sioux Center, IA). The oocytes were then incubated at 39°C in a humidified atmosphere of 5% CO2 in air.

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In Vitro Fertilization (IVF) At the end of IVM, oocytes were rinsed twice in TAL-HEPES and placed In 0.5 ml of fertilization medium, which consisted of modified Tyrode-Lactate-Pyruvate medium (TALP; 2).During the course of the experiment, semen from several bulls was used at concentrations ranging from 0.1 to 0.5 X 1061ml,depending on the sire. Frozen semen was thawed in a 35°C water bath and layered on a discontinuous gradient of Percoll (Sigma) in a 15ml centrifuge tube. The Percoll gradient was composed of 2 ml of 90% Percoll overlaid with 2 ml of 45% Percoll, both of which were prepared with Tyrode-Lactate (IL-HEPES; 26). After 30 min of centrifugation at 700 x g, the sperm pellet was recovered, resuspended in SP-TALP and the sperm concentration determined with a hemocytometer. At the time spermatozoa were added to the fertilization wells, 20 PM penicillamine, 10 /.IM hypotaurine and 1 PM epinephrine (PHE) and 2 ,ug/ml heparin were also added. After 18 h in IVF, oocytes were removed, rinsed twice in TALP, vortexed for 2 min to remove cumulus cells and then placed into in vitro culture. In Vitro Culture (IVC) Following vortexing, all oocytes were placed in a co-culture system consisting of 0.5 ml of either TCM-199 containing 10% FCS and 10 g/L BSA (A-4503, Sigma) or Menezo 82 medium (INRA, France) containing 10% FCS on a monolayer of BRL cells (American Type Culture Collection, Rockvillle, MD). The BRL were plated at a concentration of approximately 200,000, 100,000 or 50,000 cells at 24, 48 or 72 h before use. On the fourth day of IVC, the embryos were transferred to fresh co-culture wells. Incubation was conducted at a temperature of 39°C in a humidified atmosphere of 4% CO, in air. Embryo Freezing Groups of 10 to 20 embryos were frozen in 0.25 ml plastic straws (IMV International, Minneapolis, MN) containing a cryoprotectant solution composed of either 10% v/v (1.4 M) Gly or 1.5 M EG in Dulbecco’s PBS (Gibco BRL, Grand Island, NY) + 4 g/L BSA. Straws were seeded and maintained at -6” C for 15 min, cooled at -O.G”C/min to -32.5”C in the alcohol bath of a controlled-rate freezer (Bio-Cool, FTS Systems, Stone Ridge, NY) and then plunged into liquid nitrogen. Embryo Thawing Straws were thawed in air at ambient temperatures (23 to 27°C) for 10 set, then placed in a 35°C water bath until all ice melted. Embryos frozen in Gly were rehydrated by moving them sequentially for 5 min each into 0.94 M Gly + 0.3 M sucrose, 0.47 M Gly + 0.3 M sucrose and 0.3 M sucrose only, and then rinsed 3 times in PBS + 10% newborn calf serum (Irvine Scientific, Santa Ana, CA). Embryos frozen in EG were rehydrated by moving them through 5 rinses of PBS + 10% newborn calf

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serum. Approximately 1% of the embryos were not recovered at the time of thawing and were not included in the data analysis. Post Thaw Embryo Evaluation Following thawing and rehydration, all embryos were placed in co-culture consisting of 82 medium on a monolayer of BRL cells. Embryos were evaluated at 24, 48 and 72 h post thawing. Embryos that survived were recorded as either blastocysts that had expanded or hatched at 24 h or had hatched at 72 h. Chi-square analysis was used to compare differences. Experiment 1 The possible toxicity of cryoprotectants relative to the length of embryo exposure was examined. All early blastocysts, mid blastocysts and expanded blastocysts were removed on Day 7 from B2-BRL co-cultures and exposed to TALHEPES (controls), 1.4 M Gly or 1.5 M EG for 10 or 40 min. Embryos exposed to Gly and EG were diluted as previously described prior to being returned to co-culture. Seven replicates, ranging from 15 to 35 embryos per treatment group, were exposed to cryoprotectants and returned to co-culture for 72 h. Experiment 2 This experiment was an extension of Experiment 1. All early blastocysts, mid blastocysts and expanded blastocysts were removed on Day 7 from both B2-BRL and TCM199-BRL co-cultures and were frozen after 10 or 40 min of exposure to either 1.4 M Gly or 1.5 M EG. Five replicates, ranging from 9 to 40 embryos per treatment group, were exposed to cryoprotectant, frozen, thawed and cutlured. Experiment 3 To compare the efficacy of glycerol and ethylene glycol as cryoprotectants, all blastocysts at the early blastocyst, mid blastocyst and expanded blastocyst stages were selected from BBBRL co-cultures on Day 7 of IVC. Embryos were pooled without deliberate selection into 2 groups and frozen in either 1.4 M Gly or 1.5 M EG. Fifteen replicates, ranging from 13 to 88 embryos per treatment group, were frozen, thawed and cultured. Experiment 4 To determine if addition of sucrose to ethylene glycol influenced post-thaw embryo survival, all early blastocysts, mid blastocysts and expanded blastocysts were selected from BPBRL co-cultures on Day 7 of IVC. The embryos were divided into 2 groups and frozen in either 1.5 M EG or 1.5 M EG + 0.25 M sucrose. Ten replicates, ranging from 14 to 84 embryos per treatment group, were frozen, thawed and cultured.

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Experiment 5 To study the influence of the stage of development on post-thaw embryo survival, all Grade 1 early blastocysts, mid blastocysts and expanded blastocysts were selected from BP-BRL co-cultures on Day 7 of IVC and frozen separately by stage of development in 1.4 M Gly. Five replicates ranging from 10 to 61 embryos per treatment group were frozen, thawed and cultured. Experiment 6 This experiment was designed to determine embryo survival relative to possible interactions between embryo age and stage of development. All Grade 1 embryos of the following ages and developmental stages were selected from cultures on Day 6, 7 or 8, divided into 2 groups, and frozen in either 1.4 M Gly or 1.5 M EG: Day 6 - late morulae, EB; Day 7 - early blastocysts, mid blastocysts and expanded blastocysts; Day 8 - expanded blastocysts and hatched blastocysts. Five replicates ranging from 3 to 43 embryos per treatment group on each of 3 different days of development were frozen, thawed and cultured. Experiment 7 In this experiment we compared survival of different stages of embryos produced in 2 different culture systems. All early blastocysts, mid blastocysts and expanded blastocysts were removed on Day 7 from B2-BRL, and TCM-199-BRL cocultures that were run at the same time and frozen in 1.4 M Gly. Six replicates, ranging from 28 to 71 embryos per treatment group, were frozen, thawed and cultured. RESULTS Experiment 1 Survival rates did not decrease relative to controls following 24 h of culture when embryos were previously exposed to EG or Gly for periods of either 10 or 40 min. (Table 1). After 72 h of culture, there was a significantly higher hatching rate of embryos that had been exposed to EG for 40 min relative to controls. Experiment 2 Exposure of embryos prior to freezing either to Gly or EG for 40 min versus 10 min did not result in decreased post-thaw survival as judged by blastocysts at 24 h or hatched blastocysts at 72 h (Table 2). Within the category of cryoprotectant, there was no difference in the percentage of embryos surviving to blastocysts or hatched blastocysts, whether they were produced in 82 or TCM-199. However, when times of exposure were combined, fewer embryos frozen in EG hatched (3041444, 68.5%) compared to those frozen in Gly (3511455, 77.1%) (P
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Table 1. Influence of time of exposure to cryoprotectant (embryos not frozen) on the survival of Day 7 L/F-derived bovine blastocysts

Cryoprotectant

Exposure time

No. exposed

Blastocysts after 24 hours n

%

Hatched blastocysts after 72 hours n

(%)

Controls (Tyrode’s medium)

40 min

173

161

(93.1)

149

(86.1)a

Glycerol

IO min

167

161

(96.4)

152

(91.O)

40 min

171

160

(93.6)

146

(85.4)

10 min

172

164

(95.3)

156

(90.7)

40 min

180

171

(95.0)

168

(93.3)b

Ethylene Glycol

@Significantly different from Controls (PC 0.05)

Experiment 3 As shown by the data in Table 3, there was no difference between Gly and EG treatments in the percentage of blastocysts surviving 24 h after thawing. However, 72 h after thawing, a higher percentage of blastocysts frozen in Gly had hatched. of the blastocysts surviving after 24 h in culture, the rate of hatching after 72 h in culture was 87.9% for those frozen in Gly and 83.4% for EG. Experiment 4 As shown in Table 4, there was no difference in post-thaw survival rates of embryos frozen in 1.5 M EG versus those frozen in 1.5 M EG + 0.25 M sucrose. Experiment 5 The stage of embryonic development influenced post-thaw survival rates of Day 7 blastocysts frozen in Gly. As shown by the data in Table 5, both the survival rate and the hatching rate of blastocysts increased significantly as the degree of blastulation prior to freezing increased.

Embrvos cultured in B2

n (%)

Hatched blastocysts (72 hours)

n (%) n (%)

Blastocysts (24 hours)

Hatched blastocysts (72 hours)

(No. thawed and cultured)

Embrvos cultured in TCM-199

n (%)

Blastocysts (24 hours)

82 (78.8)

91 (87.5)

104

88 (72.7)

107 (88.4)

121

10 min

Crvoorotectant

86 (81.1)

100 (94.3)

106

95 (76.6)

111 (89.5)

124

65 (69.9)

82 (88.2)

93

80 (67.8)

102 (86.4)

ii8

76 (70.4)

93 (86.1)

108

83 (66.4)

110 (88.0)

125

40 min

Ethvlene alvcol

Crvoorotectant exoosure time 40 min 10 min

Glvcerol

Influence of times of exposure to cryoprotectant on the post-thaw survival of Day 7 IVF-derived bovine blastocysts produced in two different culture media

(No thawed and cultured )

Table 2.

5!

0‘ 9

B 2 0

3.

0

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Table 3. Post-thaw survival of Day 7 in vitro-derived bovine blastocysts frozen in either 1.4 M glycerol or 1.5 M ethylene glycol

Cryoprotectant

No. thawed

Blastocysts after 24 hours

Hatched blastocysts after 72 hours

n

(%)

n

W

Glycerol

543

428

(78.8)

376

(69.2)’

Ethylene glycol

588

440

(74.8)

367

(62.4)b

” Values in columns differ significantly (P-zO.02).

Table 4.

Post-thaw survival of Day 7 in vitro-derived bovine blastocysts frozen in either 1.5 M ethylene glycol or 1.5 M ethylene glycol plus 0.25 M sucrose

Cryoprotectant

No. thawed

Blastocysts after 24 hours

Hatched blastocvsts after 72 hours

n

%)

n

(%)

Ethylene glycol

385

301

(78.2)

242

(62.9)

Ethylene glycol + Sucrose

378

280

(74.1)

246

(65.1)

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Table 5.

571

Post-thaw survival of different stages of Day 7 in vitro-derived blastocysts blastocysts frozen in 1.4 M glycerol

Blastocvs staae (pre-fretzing)

No. thawed

Blastocvsts after 24 hours

Hatched blasfpUrsts after 72 hours

n

%

n

(%I)

Early

88

55

(62.5)’

44

(50.0)a

Mid

94

81

(86.2)’

64

(68.1)b

125

111

(88.8)c

105

(84.0)c

Expanded

abncValuesin columns differ significantly

(a vs c: PcO.001, a vs b and b vs c: P
Experiment 6 There were no differences between Gly and EG in the post-thaw survival rates of any of the age or stage categories of embryos. The post-thaw hatching rate for all age and stage categories of embryos combined after 72 h in culture was 61.3% for Gly (322/525) and 61 .O% for EG (319/523). As shown in Table 6, however, there were similar differences within each cryoprotectant in survival rates relative to both age and stage of embryos. Within the category of days, survival rates were higher for embryos at later stages of development than at earlier stages. Embryos at any given stage of development had highest survival rates when frozen at the earliest age the stage appeared. Experiment 7 After exposure to IVM and IVF, similar numbers of zygotes were placed in BRL co-culture with either 82 or TCM-199. On Day 7, more blastocysts had developed in co-culture with 82 (340/974 = 35%) than in TCM-199 (193/919 = 21%;Pc 0.001). After having been frozen in 1.4 M Gly, there were no differences in the post-thaw survival rates of the embryos derived from the two co-culture systems (Table 7).

68

88

MB

X6

63

82

57

20

28

21

n

(63.6) ’

(93.2)“’

(83.8)b

(48.8)’

(73.7)b

(20.6)*

%

Blastocysts after 24 hours

55

77

51

20

27

19

n

(55.6)af

(87.5)“’

(75.0)b

(48.8)”

(71.1)b,d

(18.6)a

%

Hatched blastocysts 72 hours

101

94

57

41

33

108

No. thawed

67

83

46

22

25

24

n

(66.3)’

(88.3)“’

(80.7)b

(53.7)”

(75.8)bBd

(22.2)’

(%)

Blastocysts after 24 hours

53

81

43

18

20

27

n

(52.5)”

(86.2)b*”

(75.4)b

(43.9)’

(60.6)b

(25.0)’

(%)

Hatched blastocysts after 72 hours

Ethvlene Glvcol

HB 89 __I ---73 (82.0)b 89 ____ ____ 77 (86.5)b ‘b’cWithin cryoprotectant and embryo age, values in columns differ significantly (a vs b,c:PcO.Ol and b vs c: PcO.05) d”“WRhin cryoprotectant and embryo stage, values in columns differ significantly (d vs e: PcO.05 and e vs f: P~O.001) M = morulae; EB = early blastocysts; MB = mid blastocysts; XB = expanded blastocysts; HB = hatched blastocysts.

99

41

Day 7 EB

XB

38

EB

Day8

102

No. thawed

Glvcerol

Post-thaw survival of different stages of in vitro-derived embryos frozen on Days 6,7 or 8 in 1.4 M glycerol versus 1.5 M ethylene glycol.

M

Day6

Age and stage at freezing

Table 6.

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Table 7.

Post-thaw survival of Day 7 in vitro-derived embryos produced in BPBRL or TCM-199-BRL co-cultures and frozen in 1.4 M glycerol

Culture Medium

No. Thawed

Blastocysts after 24 hours n

(%)

Hatched blastocysts after 72 hours n

W)

TCM-199

186

161

(86.6)

138,

(74.2)

B2

328

278

(84.8)

229

(69.8)

DISCUSSION In this study, both 1.4 M Gly and 1.5 M EG were found to be effective cryoprotectants for the freezing of n/F-derived bovine embryos, although comparisons of the efficacy of the 2 cryoprotectants were somewhat equivocal. In Experiment 3, equal numbers of Day 7 blastocysts frozen in Gly and EG survived as evidenced by reexpansion at 24 h post-thawing, whereas a slightly higher percentage (69 vs 62%) of embryos frozen in Gly hatched 72 h post thawing. By contrast, there was no difference between EG and Gly when embryos ranging from morulae to hatched blastocysts were separated by stage of development and frozen on Day 6,7 or 8 of IVC in Experiment 6. Yet, in Experiment 2, blastocysts produced in 2 different media and exposed to Gly for 10 or 40 min survived freezing better than those frozen in EG. Because the embryos frozen in Gly and EG were rehydrated differently, the comparison potentially involved more than just differences in cryoprotection. It was previously shown with early to expanded bovine IVF-derived blastocysts, that 1.5 M EG and 1.4 M Gly provided equal cryoprotection (48). Clearly, there are differences between in vivo and in vitro embryos involving, at the very least, membrane permeability (29) buoyant density, zonae enzymatic digestability and chilling sensitivity (17). However, when frozen in EG, both types of embryos survived direct dilution in isotonic solution (9,19,23,39,48), and pregnancies resulted when the embryos were transferred. These later studies have capitalized on the fact that ovine and bovine (40) and, in fact, equine (28) embryos are more permeable to EG than Gly. In contrast, direct rehydration of embryos frozen in Gly has not been successful (38,48), whereas survival of IVFderived embryos was good if 0.25 M sucrose was combined with the Gly (44). Research using rapidly permeating cryoprotectants such as EG has been driven, at least partially, by the advantages offered by direct post-thaw transfer of embryos, as described by Voekel and Hu (48). In commercial embryo transfer programs, large numbers of in vivo bovine embryos are now frozen in EG, thawed and transferred. However, there have been no reports of large-scale field trials directly comparing EG and G as cryoprotectants. Voekel and Hu (48) did not find a difference in a study

574

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involving small sample sizes. Looney et al. (19) reported a significantly lower pregnancy rate following direct transfer of more than 100 embryos frozen in EG compared with embryos frozen in Gly that were transferred following traditional rehydration. In contrast, both in vivo and in vitro-derived goat embryos survived better when frozen in EG than in Gly and then rehydrated by different methods (15). lt is questionable whether the results from our present study can be extended to in vivo embryos transferred under field conditions. Certainly, direct rehydration at ambient temperature in the laboratory does not duplicate the direct, intrauterine transfer of embryos. At the very least, however, it provides a starting point for the improvement of the cryopreservation of IVF embryos. In the present study, addition of 0.25 M sucrose to 1.5 M EG did not improve post-thaw survival of IVF-derived embryos. lt was previously shown that addition of 0.25 M sucrose to Gly allowed for direct rehydration with good survival following transfer (9,21), whereas there was no difference in pregnancy rates after the transfer of small numbers of in vivo-derived embryos frozen either in EG or EG + sucrose (9). In light of the non-permeating property of sucrose when added to cryoprotectants during freezing, further improvements might result from examining different combinations of sucrose concentrations and plunge temperatures and other cryoprotectants. In the present study, Day 7 expanded blastocysts frozen in Gly exhibited a higher post-thaw survival rate than blastocysts frozen at earlier stages of development. Similar results have been reported in other studies in which Gly was used as the cryoprotectant (3,7,11). Furthermore, it was reported that late-stage blastocysts survived vitrification better than at earlier stages (6,20). In studies in which the developmental stage of IVF-derived embryos was not clearly defined, Day 7 embryos survived freezing best in two studies (543) whereas in another study Day 6 embryos had the best survival rate (36). In one study, morulae and blastocysts survived equally well when frozen in Gly on either Day 6 or 7 of development; however, evaluation was performed immediately after thawing, without any post-thaw period of culture (24). Massip et al. (22) reported that expanded blastocysts frozen in Gly on either Day 6 or 7 of IVC survived significantly better than those frozen on Day 8. Since embryo age in the above study (22) was based on days in culture, one day should be added for comparison to embryo ages in our present study, which were based on days postinsemination. We previously reported that the transfer of frozen-thawed Day 7 IVFderived embryos, ranging from early to expanded blastocysts, resulted in a higher pregnancy than the transfer of Day 8 embryos (12). The transfer of fresh Day 7 expanded blastocysts also resulted in a higher pregnancy rate than other ages and stages of fresh embryos. Cell number of the embryos apparently does not account for this difference in viability, since the number of cells in expanded blastocysts collected on Days 7 and 8 in our laboratory was similar (unpublished data). Similar cell numbers were also reported in a vitrification study in which survival post-thaw for Day 8 expanded blastocysts was less than 50% that of Day 7 expanded blastocysts (34). Pollard and Leibo (29) reported that in a large number of bovine IVF-derived compact morulae (n=80), none survived conventional freezing in EG. In another study

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(30),they showed that although 65% of compact n/F-derived morulae survived chilling to 15”C, none survived when chilled to 10°C or below. Unfortunately, they did not specify the age of the embryos used in their experiments. In the present study, approximately 20% of late morulae removed from WC on Day 6 survived freezing in either Gly or EG. A surprisingly high pregnancy rate (45%) was reported for recipients into which 2 IVF-derived morulae frozen in 1.5 M Gly were transferred (27). The same authors also reported a 55% survival rate in culture for IVF-derived compact morulae frozen in Gly. In other studies, 6% (4) and 46% (50) of Day 6 morulae were reported to have survived freezing in Gly. This wide range of survival reported for IVF-derived morulae could be related to a number of factors such as cryoprotectant, culture system, embryo quality and embryo age. In the present study, there was no apparent advantage conferred to the postthaw survival of embryos co-cultured in 82 versus TCM-199. In addition, there was no difference in survival rate of morulae frozen in Gly or EG. In other studies, TCM-199 was used as the culture medium, both in the study in which the post-thaw survival of morulae was zero (29) and in studies with low to moderate survival rates (27,50). In the present study, there was a large difference in the survival of Day 6 late morulae (19 to 25%) compared with that of early blastocysts (61 to 71%). The presence of even a very small blastocoel cavity was correlated with dramatically increased post-thaw survival. In light of this, post-thaw survival rates in studies that combine early blastocysts and morulae in one category should be interpreted cautiously. The high post-thaw survival rate of hatched blastocysts frozen in either Gly or EG found in the present study has also been reported (7,8) for IVF-derived hatched blastocysts either frozen in Gly or vitrified. It is apparent that the zona pellucida is not necessary for mechanical protection during freezing. However, the viability of frozenthawed, IVF-derived, hatched bovine embryos has not been critically tested by transfer into recipients. Commercial embryo transfer practitioners often express concern regarding the possible toxicity of EG to bovine embryos. Holding the pre-freezing EG exposure time to less than 10 min is widely practiced in commercial embryo transfer. Takagi et al. (41) reported that when IVF-derived blastocysts were exposed to EG for 30 or 120 min. rehydrated and returned to culture, there was no difference in blastocyst expansion or hatching rates between the 2 exposure times. However, the hatching rates (30 min =40% and 120 min=22%) were well below the 81% hatching rate cited by the authors for control embryos. These authors also reported that various exposures of 10 to 120 min prior to freezing also did not result in differences in post-thaw hatching rates. In the present study, hatching rates after 10 or 40 min exposure, without freezing, were not lower than the hatching rate of control blastocysts that were not exposed to cryoprotectant. Likewise, there was no difference in survival relative to 10 or 40 min exposure time before freezing with either EG or Gly. Thus, it would appear that limiting EG exposure to a very brief duration prior to freezing for direct transfer is not necessary. Moreover, in another study, there was no difference in post-thaw survival of IVF-derived embryos exposed to the cryoprotectant solution containing 1.5 M EG for 1

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versus 30 min after thawing (42). It should be noted, however, that none of these experiments “mimicked” the rehydration conditions that an embryo encounters when transferred directly into the uterus. These experiments also utilized W-derived embryos, which may not be suitable models for in vivo-derived embryos with some of the cryoprotectants. In conclusion, bovine IVF-derived embryos co-cultured with BRL cells in 82 or TCM-199 survived freezing in 10% Gly equally well. Embryos frozen in 10% Gly survived slightly better than those frozen in 1.5 M EG in 2 of 3 experiments. Addition of 0.25 M sucrose to the EG did not improve embryo survival. A higher percentage of embryos frozen on Day 7 survived than those frozen on Day 6 or 8. On any given day of WC, embryos at the most advanced stage of development exhibited the highest survival rates. There was no difference from control embryos in survival, with or without freezing, when the embryos were exposed to either Gly or EG for 10 or 40 min.

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