Theriogenology 42:25-35, 1994

CRYOPRESERVATION OF PORCINE EMBRYOS BY VITRIFICATION: A STUDY OF IN VITRO DEVELOPMENT

J.R. Dobrinsky and L.A. Johnson

Germplasm and Gamete Physiology Laboratory Agricultural Research Service

United States Department of Agriculture Beltsville, MD 20705

Received for publication: September 7, 1993 Accepted: May 2, 1994

ABSTRACT

Until recently, attempts to preserve porcine embryos have been unsuccessful. Vitrification has been developed as a method of cryopreserving mammalian embryos by avoiding ice crystal formation, assuring a cryopreserved glass state during storage in liquid nitrogen. Vitrification may be a useful method of overcoming the deleterious effects of chilling injury when pig embryos are cryopreserved using conventional slow freezing procedures. In this study, we applied vitrification procedures for rodent and/or bovine embryos to cryopreserve porcine embryos. Following warming, survival was defined as normal development of embryos in culture, namely the formation or re- expansion of the blastocoelic cavity. Experiment 1 tested the relative toxicity of 3 vitrification procedures on Day-5, 6 and 7 porcine embryos. Embryos equilibrated in vitrification solution (VS3a) continued to develop in vitro at rates comparable to that of untreated control embryos. Experiment 2 was designed to evaluate embryonic development following cryopreservation by vitrification in VS3a. Day-5 porcine embryos did not survive cryopreservation while Day-6 and Day-7 embryos survived and continued development in vitro. In Experiment 3, we evaluated a period of culture prior to vitrification and its effect on cryosurvivability of porcine embryos. A 3-h culture period prior to vitrification had no effect on cryosurvivability over that of freshly recovered, immediately vitrified embryos. These studies indicate, for the first time, that porcine embryos can be successfully cryopreserved by vitrification based on morphology and subsequent development in vitro. However, survival following cryopreservation appears to depend upon embryonic age or stage of development.

Key words: porcine embryo, vitrification, cryopreservation, cryoprotectant, toxicity, development

Acknowledgements The authors gratefblly acknowledge the expert technical assistance of Lori L. Schreier; animal care by Jim Piatt, Russ Lange and Del Parsons; and the helpful advice of Drs. V.G. Pursel, W.F. Rall and D.L. Garner.

Copyright 0 1994 Butterworth-Heinemann

26 Theriogenology

INTRODUCTION

An unexplained characteristic of porcine embryos is their high sensitivity to chilling injury when cooled to temperatures below 15’C. Polge et al. (23) and others (17,lS) first described the chilling sensitivity of porcine embryos. Recently, it has been shown that porcine embryos chilled below 15’C lyse within 12 h of being placed in culture after cooling (22). This phenomenon is not apparent in embryos from other domestic and laboratory species, such as cattle, sheep, rodents and rabbits. It is also interesting to note that porcine semen is extremely sensitive to cooling and cryopreservation, suggesting that this phenomenon may be species specific (9,24).

Classically, mammalian embryo preservation has almost always involved the freezing of embryos in a cryoprotectant suspension. Recently, reports have appeared in the literature claiming successfi~l cryopreservation of porcine embryos (10,12,19). These papers have reported of the use of slow cooling/conventional freezing of porcine embryos, two (10,12) which reported the birth of live offspring after frozen-thawed embryos were transferred to recipient females. These reports (12,19) indicated that porcine embryo survival was dependent upon embryonic age and/or stage of development and one indicated that 150 to 300 urn embryos developed more frequently after cryopreservation (19). A period of culture prior to cryopreservation, the presence of a protein in the freezing solution and breed differences also affected freeze-thaw survival (19). The success rate of these procedures was variable and their repeatability has not been confirmed. It is perplexing that porcine embryos are susceptible to chilling below 15’C, yet survive during slow cooling ( l°C/min) during conventional freezing.

Recently, vitrification has been utilized as a method of cryopreserving mammalian embryos. Vitrification (25) involves the rapid cooling of liquid medium in the absence of ice crystal formation. The solution forms an amorphous glass as a result of rapid cooling by direct submersion of the embryo in a plastic straw into liquid nitrogen (LN2). The glass retains the normal molecular/ionic distributions of a liquid but remains in an extremely viscous, supercooled form. In this state, the glass is devoid of all ice crystals, and embryos are not subjected to the physical damage that is associated with ice crystal formation. Rodent (2,8,25,26,28), rabbit (1) sheep (30) and bovine (3,6,7,14,26) embryos have been successfully cryopreserved by vitrification. It may be possible that the rapid cooling during vitrification could bypass or “out-race” chilling-induced cellular changes that take place during slow cooling while avoiding both intracellular and extracellular ice formation,. Some vitrification solutions have been shown to be extremely toxic to porcine embryos (4,5,34). The objective of this work was to determine the relative toxicity of vitrification solutions, the efficacy of vitrification procedures and age of embryo for cryopreserving porcine embryos and determine if a period of culture prior to vitrification affects in vitro survival rates.

MATERIALS AND METHODS

Embryo Production

All embryos in these studies were produced corn a pool of naturally cyclic, nonsuperovulated crossbred gilts that were 6 mo of age or older and weighed at least 100 kg at the time of use.

Theriogenology 27

Nonsuperovulated, mature gilts decrease variability in the embryonic pool and, thereby, standardize the embryonic stages per day ofrecovery. All gilts were artificially inseminated twice during the period of standing estrus (-24-h) with semen (pooled from at least 3 mature boars) that was extended in Beltsville TS (l3TS; 11) to a concentration of >SxlO’ spermatozoa (80 to 100 ml volume per insemination). Per replicate, 3 to 4 donors were used per day of recovery (Day 5, 6 or 7, Day O=estrus onset). Embryos were collected from excised reproductive tracts obtained daily during slaughter at the Beltsville abattoir The tracts were kept warm (37’C), and embryos were recovered by flushing the excised uterine horns with sterile modified Dulbecco’s phosphate buffered saline (mDPBS, Gibco 3 lo-404OAJ supplemented with 3 mg/ml BSA-V and 5.5 mM glucose) within 10 min after the tracts were dissected. Only late morulae/early blastocysts, expanded blastocysts, or hatched blastocysts recovered from donor females on Day 5, 6, or 7, respectively, from donor females, and graded excellent or good for developmental stage and appearance, were randomly allocated across all treatment groups and used in the experiments. Per replicate, 28 to 40 morphologically normal embryos were randomly allocated across a 3x4 (Experiment 1, 240 total embryos) factorial design, a 3x3 (Experiment 2,381 total embryos) factorial design, or a 2 treatment direct comparison (Experiment 3,46 total embryos). All experiments had a minimum of 3 replicates.

Embryo Cryopreservation

Vitrification procedures used were based on established protocols developed for cryopreserving murine and/or bovine embryos; solutions were composed of glyceroVpropylene glycol (UM-ABSI), ethylene glycol/BSA (EG) or glycerol/BSA (VS3a). Unless otherwise indicated, all embryos were processed at 25’C on a warming plate in room atmosphere.

UM-ABSl : As adapted from Massip et al. (14) and Dobrinsky et al. (6,7), embryos were incubated for 5 min in modified Hepes-buffered TALP (mHBT) and then placed into equilibration medium (10% glycerol, 20% propylene glycol in mHBT) for 7 to 8 min. After equilibration, embryos were washed in vitrification medium (25% glycerol, 25% propylene glycol in mHBT) for 30 set and either put into dilution medium (for noncryopreserved toxicity tested embryos, Experiment 1) or placed into prepared 0.25-ml straws (for cryopreservation, Experiments 2 and 3) containing 100% vitrification media. The straws were heat-sealed on both ends and immediately lowered into LN2 storage for l-4 mo. The vitrified straws were removed from LN2 and placed into a 25’C water bath for 10 set and the contents emptied into a plastic petri dish. All embryos (cryopreserved and non- cryopreserved) were located and immediately placed into 3 ml dilution media (0.3M sucrose in mHBT) and incubated for 10 min. Following dilution, embryos were rehydrated by transfer into 3 ml of mHBT for 15 min and then placed into culture.

EG: As adapted from Leibo and Oda (13) embryos were incubated for 5 min in 3 ml of mDPBS and then equilibrated for 5 min in 3 ml of mDPBS-6 (5.5mM glucose, 6Omg/ml BSA). Embryos were then placed into 3 ml of equilibration medium (2M EG, in mDPBS-6) for 5 mm. Following equilibration, the embryos were washed in vitrification medium (8M EG in mDPBS-6) for 30 set and either put into dilution medium (for non-cryopreserved toxicity tested embryos, Experiment 1) or placed into prepared 0.25~ml straws (for cryopreservation, Experiments 2 and 3) containing 100% vitrification media. Straws were heat-sealed and cooled rapidly in LN2. Atter the straws were removed from storage in LN2 and embryos were located as described above, they were

28 Theriogenology

placed into dilution medium (1M sucrose in mDPBS). All embryos (cryopreserved and non- cryopreserved) were held in 3 ml of dilution medium (10 min), rehydrated in 3 ml of mDPBS (15 min) and then placed into culture.

B: As adapted from Rail (27) embryos were washed in 3 ml of mDPBS for 5 min and then equilibrated in 3 ml of mDPBS-6 for 5 min. Embryos were then equilibrated 20 min in 25% VS3a. After equilibration, embryos were washed in 65% VS3a for 45 set, washed again in 100% VS3a (6.5M glycerol, 6Omg/ml BSA in mDPBS) for 1 min, and then put into dilution medium (Experiment 1) or placed into prepared 0.25-ml straws (for cryopreservation, Experiments 2 and 3) containing 100% VS3a. Straws were heat-sealed and placed into LN2 storage. After storage in LN2 straws were warmed and embryos were located as described above and then were placed into 3 ml of dilution medium (1M sucrose in mDPBS) for 3 min at 37’C. Embryos were then placed into 3 ml of dilution medium for 3 min at 25’C to continue dilution. After dilution, embryos were rehydrated in 3 ml of mDPBS for 15 min and then placed into culture.

Embryo Culture

Embryos that were cultured prior to cryopreservation were incubated for 3 h in Beltsville Embryo Culture Medium-3 (BECM-3, Table 1.) in NUNC 4-well tissue culture plates (520 embryos/well). After cryopreservation, all embryos were cultured in 1 ml of BECM3 supplemented with 10% heat-treated fetal bovine serum. All embryo cultures were incubated for 24 h at 38.7’C in a humidified 5% CO2 and air atmosphere.

Table 1. Composition of Beltsville Embryo Culture Medium-3 : BECM-3a

Compound mM gnb

NaCl 94.59 KC1 6.00 MgSO4.7H20 1.19 CaC12.2H20 1.71 NaHC03 25.07 Glucose 5.56 Glutamine 1.00 Na Lactate (60% syrup) 23.00 Na Pyruvate 0.33 BSA-V (Sigma A2153) 12 mg/ml MEM Amino Acids (Sigma M7145) 10 ml/l BME Amino Acids (Sigma B6766) 20 mlil Phenol Red <O.OOl g/l

5.54 0.45 0.29 0.25 2.11 1.00 0.15

3.35 ml/l 0.04 12.00

a -290 mOsm. b Except where indicated.

Theriogenology 29

Experiment 1

The toxicity associated with equilibration in each of the vitrification solutions was tested factorially as follows. Excellent and good quality embryos collected on Day-5, 6 and 7 were cultured for 3 to 4 h in BECM-3 and then equilibrated in 1 of the 3 vitrification solutions as described above. Immediately after equilibration, embryos were diluted and rehydrated without the cooling step of vitrification. Rehydrated embryos were transferred into mDPBS containing trypan blue at 25’C for 10 min. Those embryos excluding dye were cultured in BECM-3 to assess developmental competence. Survival was based on the total number of embryos treated and was defined as morulae that formed a blastocoelic cavity, blastocysts that re-expanded their blastocoelic cavity or hatched out of their zona pellucida, and hatched blastocysts that re-expanded their blastocoelic cavity during 24 h of culture. Differences in the rates of survival across treatment groups was analyzed using ANOVA-LSM probability determination (29).

Experiment 2

Based on the results from Experiment 1, excellent and good-quality Day-5, 6 and 7 embryos were cryopreserved by vitrification in VS3a medium. Embryos remained cryopreserved for at least 1 mo. At the time of recovery from storage in LN2, all embryos were diluted, rehydrated, stained with trypan blue and cultured as described above. Embryonic development after cryopreservation was compared to nontreated controls and noncryopreserved VS3a-treated (VS3a-TC) embryos and was evaluated as described above in Experiment 1. Blastocyst development was analyzed across treatments with ANOVA-LSM probability determination (29).

Experiment 3

This experiment was designed to ascertain the effect of a culture period prior to cryopreservation on the survivability of D7 porcine embryos. Embryos were vitrified in VS3a within 15 min after recovery from donor gilts or after 3 to 4 h culture in BECM-3. Embryos remained cryopreserved for at least 1 mo. At the time of recovery from storage in LN2, all embryos were diluted, rehydrated, stained with trypan blue and cultured as described above. Embryonic development after cryopreservation was evaluated as described above in Experiments 1 and 2. Blastocyst development was analyzed with Chi-square analysis (29).

RESULTS

Experiment 1

This experiment tested the relative toxicity of each vitrification system on noncryopreserved Day-5, 6 and 7 pig embryos (Table 2). Within embryo age, Day-5 embryos survived across all treatments, Day-6 embryos developed in EG and VS3a while VS3a was superior (p’O.05) for Day-7 embryos. In UM-ABSI, only Day-5 embryos developed at rates comparable to controls. After

30 Theriogenology

exposure to EG, a large proportion of Day-5 and Day-6 embryos developed. The VS3a solution was only slightly toxic to Day-6 embryos, while it had no effect on the development of Day-5 and Day-7 embryos. Therefore, VS3a was used as the vitrification solution in subsequent studies.

Table 2. Percentage of embryosa developing at 24 hours following exposure to different vitrification solutions

Vitrification Solutions Embryo

age Culture control UM-ABSl EG VS3a

Day 5 90bTd 6SbTd 7ObTd 70b7d

Day 6 1 oob,d 4ob,c,f 7+,d,e 7ob7”

Day 7 85b7d 2oc7e 40CJe 9ObTd

a n=20 per tre at m ent; at least 3 replicates/treatment. b$ Differences within columns: PcO.05; d,epf differences within rows: P<O.OS.

Experiment 2

This experiment evaluated embryonic development following cryopreservation in VS3a (Table 3). Day-6 and Day-7 embryos survived vitrification while Day-5 embryos did not. A high percentage of Culture Control and VS3a treatment control (VS3a-TC) embryos developed in vitro. The percentage of Day-6 embryos developing after exposure to VS3a solution was significantly lower than for their culture control counterparts. Vitrification of Day-6 and 7 embryos resulted in additional significant reductions in the percentage of embryos developing as compared with the VS3a treatment control embryos.

Table 3. Percentage of embryos developing after exposure to or cryopreservation in VS3a

Embryo age

Vitrification Treatments (embryos treated)

Culture control VS3a-TC VS3 a-LN2

Day 5 88(24)a7C 68(25)a2b3c o(59)aTd

Day6 1oo(33)“‘c S8(33)b>d 27(64)bpe

Day 7 86(29)a>C 83(30)a7C 39(84)byd

a,b Differences within columns:P<0.05; c,dye differences within rows:P<O.OS.

Theriogenology 31

Experiment 3

This experiment was designed to evaluate if a period of culture prior to cryopreservation at&ted the survivability of Day-7 embryos after cryopreservation. The percentages of embryos that continued to develop were similar (P>O. 10) for embryos vitrified immediately after recovery (7120, 35%) and embryos vitrified after 3 to 4 h in culture (1 l/26, 42%).

DISCUSSION

The success of freezing porcine embryos by conventional slow freezing procedures has been variable. Porcine embryos have been shown to be extremely sensitive to chilling below 15’C (2). Preliminary data from our laboratory indicated that slow cooling during the cryopreservation seemed to be detrimental toward embryo survival. During freezing, the embryo suspension must be cooled at -l°C/min from room temperature to seeding temperature, which is often found to be near -6’C. We designed these experiments to determine if vitrification may be especially suited for the cryopreservation of porcine embryos, because vitrification may bypass deleterious effects of chilling since embryos are cooled rapidly by direct submersion into LN2.

Some vitrification solutions have been shown to be extremely toxic to porcine embryos (4,5,34). Bovine embryos have been shown to undergo similar damage during vitrification as cortical f-a&n, cortical tubulin (20,21) and mitochondria (6) are especially sensitive to high levels of certain cryoprotectants. Vitrification requires high concentrations of cryoprotectants to achieve the vitreous state in liquid nitrogen, and these high levels can be toxic to cells. The relative toxicity of vitrification solutions and procedures that have been found to be successtbl in cryopreserving embryos of other species were tested with porcine embryos. Our results indicate that porcine embryos, dependent upon embryonic age and/or stage of development, can withstand the effects of certain cryoprotectant mixtures and protocols needed to achieve cryopreservation in a vitreous state. Certain cryoprotectants, after periods of equilibration, were detrimental to the development of embryos exposed to vitrification media but not cryopreserved. Embryos that did not survive appear to have alterations in the plasma membrane and/or cellular structures (Pigure la-d). Total disruption of the plasma membranes and lysis of individual blastomeres, as well as lack of blastocoelic cavity reformation during culture indicate that porcine embryos cannot tolerate certain cryoprotectants or cryoprotectant treatments as the present protocols prescribe and that membrane integrity and metabolic activity is jeopardized. Most Day-6 and Day-7 porcine embryos that survived the vitrification protocols had evidence of some cellular damage although development in vitro appeared to continue normally.

The procedure yielding the greatest in vitro development after cryoprotectant equilibration and subsequent dilution was used to determine survival of embryos after cryopreservation by vitrification. The VS3a solution provided the highest rates of survival following exposure to noncryopreserved porcine embryos. Our results indicate that although noncryopreserved Day-5 (late morulae/early blastocysts) embryos can withstand exposure to the VS3a protocol, none survived cryopreservation by vitrification. This data is consistent with the frozen Day-5 early blastocysts/blastocysts data of Nagashima et al. (16).

32 Theriogenology

Figure 1. a) Day-7 hatched blastocyst-stage porcine embryo. b) Developing Day-7 hatched blastocyst-stage porcine embryos 12 hours in culture following cryopreservation by vitrnication. c) Day-6 expanded blastocyst-stage porcine embryo. d) Developing Day-6 expanded blastocyst-stage porcine embryos 12 hours in culture following cryopreservation by vitrification.

It has been suggested (33) that the porcine embryo lacks tolerance to freezing primarily due to a suspected high lipid content of embryos during early stages of development. Nagashima et al. (19) report an undocumented observation that the size of neutral lipid droplets in the cells of porcine blastocysts decreased after hatching. They speculated that lipid turnover may be involved and that lipid may be important for hatching and normal development, thus survivability of porcine embryos may peak during the perihatching stage. Our data shows Day-5 embryos tolerate equilibration in all three vitrification solutions but none survive cooling in VS3a. Day-6 embryos include both blastocysts and expanded blastocysts, which in these studies can develop in vitro (27%) afler vitrification, approximately 50% of that seen in noncryopreserved treatment controls (VS3a-TC) and was not different to that of vitrified Day-7 embryos. Vitrified Day-7 hatched blastocysts survived vitrification and developed in vitro (39%) for at least 24 h. They also exhibit some cellular damage to the plasma membrane and/or the cytoskeleton. After vitrification, similar to Day-6 embryos, the Day-7 embryos develop at a rate approximately 50% of that found with noncryopreserved treatment controls and culture controls.

Theriogenology 33

Nagashima et al. (19) observed that porcine embryos L300um fail to develop following I&zing; optimum freezability was attained with embryo diameters between 150 to 300 urn in recently hatched blastocysts. They conclude that optimal freezability is attained immediately after hatching and that freezability decreases rapidly as the embryo increases in size. In our studies, all embryos were pooled by day of recovery from donors, and Day-7 embryo diameters ranged from 200 to 400um. Although we did not design our study to assess the effect of embryo size on cryosurvival, it appears that embryos that survived cryopreservation were not of any specific diameter.

A culture period prior to cryopreservation was tested to determine its effect on embryonic development following cryopreservation. Nagashima et al. (19) reported that a period of culture in BSA containing medium prior to cryopreservation enhanced in vitro development of porcine embryos after cryopreservation. Their work speculates the involvement of membrane fluidity as a site of potential damage to the cryopreserved embryo, and that a period of culture in bovine serum albumin- supplemented medium prior to cryopreservation might be favorable to change the membrane and give better support for cryosurvivability. The BSA-V is a protein source that contains high levels of lipid. Rall(26,27) has included high levels of BSA-V in VS3a, and our data suggest a beneficial effect in contrast to vitrification media with lower concentrations of BSA-V. In our study, however, we find no benefit of culturing embryos for 3 h prior to cryopreservation in BSA-V supplemented culture medium. This finding may be useful as a tool in the application of vitrification. Surgical embryo recovery from multiple donor females requires a great great deal of preparation and time. Embryo collection can take up to 45 min per donor, resulting in almost 3 h for up to 4 donors. If desired, it is advantageous to have embryos in culture so that they may be pooled for use at one time. The data presented in our manuscript suggest that a culture period of up to 3 h prior to vitrification causes no ill-effects on embryo survival after cryopreservation.

As in the pig, Drosonhila melanogaster embryos are extremely sensitive to subzero temperatures in the absence of ice (15). When the embryos are chilled during slow cooling (which is needed to remove intracellular water), death occurs rapidly. Mazur et al. (15) speculated that this chilling injury could result from the loss of synchrony of coupled reactions involved in early embryonic development. Steponkus et al. (32) have recently reported that Drosophila embryos can be cryopreserved by vitrification. As was suggested by Mazur et al. (15) vitrification may be able to “out-race” the deleterious effects that are evident during slow cooling. Rapid cooling during vitrification may enable porcine embryos to attain intracellular glass formation before changes in cellular structure and function take place. These studies indicate that porcine embryos can be successfully cryopreserved by vitrification. However, survival following cryopreservation seems to be dependent upon embryonic age or stage of development. Based on morphological evaluation of all vitrified embryos, there appears to be cellular damage following vitrification, and this cellular damage may be in part induced by the high levels of cryoprotectants needed to insure proper development of the vitrified state. Also, zona intact embryos can survive vitrification at rates similar to those of hatched blastocysts. This is a significant finding in terms of the future potential of porcine embryo transfer technology as the intact zona pellucida provides protection to the embryo from infection (3 1). Further testing is needed to determine the specific sites of cellular damage during cryopreservation and in vivo development will determine the long-term developmental competence of vitrified porcine embryos.

34 Theriogenology

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Theriogenology 35

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