- Email: [email protected]
Effect of ice nucleation by droplet of immobilized silver iodide on freezing of rabbit and bovine embryos
THERIOGENOLOGY
EFFECT
OF
ICE NUCLEATION ON FREEZING T.
BY DROPLET OF IMMOBILIZED SILVER OF RABBIT AND BOVINE EMBRYOS
Kojima,
T.
Soma
and
Department of Animal National Institute of Tsukuba-Norindanchi, P.O.Box Received
for
publication: Accepted:
N.
Oguri
Reproduction, Animal Industry 5, Ibaraki, 305,
December September
IODIDE
31,
19,
Japan
1987
1988
ABSTRACT Silver iodide was immobilized by applying the insoluble reacti.on between sodium alginate and calcium chloride. The immobilized silver iodide was immersed into a freezing solution ice nucleation. order to trigger Temperature in change postthaw in vitro development of during cooling and embryos in order to evaluate the effectiveness of the were examined silver iodide immobilized (AgI alginate-gel droplet) on embryo Samples containing the AgI alginate-gel droplets development. released the latent heat of fusion at a higher subzero temperature than samples without the AgI alginate-gel droplets. When the AgI alginate-gel droplet was added to the freezing solution of rabbit and bovine embryos, they were successfully preserved in liquid nitrogen. Key
words:
immobilized nucleation,
silver embryo
iodide, freezing
sodium
alginate,
ice
INTRODUCTION confirmed silver iodide It has been that can be induce effectively ice 'formation in extracellular used to solution in the process of slow cooling of mammalian embryos (1,Z). Using this procedure a high postthaw survival rate of obtained without employing conventional ice rabbit embryos was However, original silver iodide techniques. the seeding has some limitations in practical seeding method (192) in principle air For example, should be aspirated application. between the column of freezing solution into a straw and the the embryos column of silver iodide to containing prevent them from coming into contact. There is the possibility iodide could affect embryonic growth silver that some molecules of silver iodide came into deleteriously if the embryos at time of thawing, even though no with contact harmful effect on embryonic growth has been observed yet. A usable procedure of silver iodide needs to be developed to safe, We are grateful to Dr. A. Hanada for reviewing Acknowledgments: We also wish to thank Mr. Y. Hori, Mr. S. Ito the manuscript. of Nippon Oil & Fats Co. Ltd. for their and IMr. K. Hashimoto technical suggestions.
DECEMBER
1988 VOL.
30 NO. 6
1199
THERIOGENOLOGY
apply the silver iodide seeding reliably. In the present study, iodide was immobilized silver that it could be used more easily than in the original procedsuore (1,2). In the present study we examined the effect of ice nucleation by immobilized silver droplet) on iodide alginate-gel embryonic development. (AgI MATERIALS
AND
METHODS
Immobilization Silver iodidea was added to a 1 vel of 2% in distilled % water containing 2% w/v sodium alginate . The solution was mixed with a magnetic stirrer. Using a syringe and a 19-gauge obtuse the solution was added, drop by drop, to the distilled needle, 1OOmM calcium solution containing chloride dihydrate. water Immobilization of the silver iodide coincides with the reaction of sodium alginate and calcium chloride. Silver iodide was fixed gel of calcium alginate. Sodium alginate calcium in a and chloride are used to immobilize certain types of enzymes, yeasts of immobilized silver iodide (AgI and bacteria (3). The droplets were then removed from droplets) the calcium alginate-gel and washed in distilled chloride solution water. The AgI alginate-gel droplet was sterilized by autoclaving (121"C, 20 the droplets were preserved in sterilized distilled min) and water at 4'C until use. Observation
of
Temperature
Change
The freezing solution consisted of 10% v/v glycerolC in Dulbecco's phosphate buffered saline (PBS) supplemented with 20% strawe (0.5 ml), a glass ampoule fetal bovine serumd . A plastic (2 ml) and a polypropylene tubef (2 ml) were used as freezing changes, vessels to observe temperature especially those at the level. The quantity of latent heat releasing the freezing was 400 1-11 in the plastic straw, 600 ul in the solution used and 150 ul in the polypropylene tube. A ampoule and 1 ml without the AgI alginate-gel droplet or with freezing solution the substrate (calcium alginate) were employed in control only Approximately one droplet of silver iodide was put experiments. into each freezing vessel. Following cooling at 2'C/min, from a of 25°C to O"C, with a programmable liquid room temperature freezing machineg the respective samples nitrogen vapor were cooled at l"C/min until the latdnt heat of fusion occurred. change during cooling was monitored Temperature by a thermocouple connected to a thermocouple copper-constantan information The was recorded charts. recorder. on a Wako Chemical Industries, Ltd., Osaka, Japan. Sigma Chemical Co., St.Louis, MO, USA. b A-2033, Sigma Chemical Co., St.Louis, MO, USA. c G-9012, dGIBC0 Laboratories, Grand Island, MD, USA. e Fujihira Industries Ltd., Tokyo, Japan. f Corning Glass Works, Corning, NY, USA. Osaka Sanso Kogyo Inc., Osaka, Japan. g FFP-190,
1200
DECEMBER
1988 VOL.
30 NO. 6
THERIOGENOLOGY
of the curve was repeated three or four times except cooling in the case of the polypropylene tube. Formation of ice crystals monitored by the occurrence of the latent heat of fusion. was analyzed for statistical significance by Student's Data were t-test. Postthaw
In
Vitro
Viability
of
Rabbit
Embryos
Adult female Japanese White rabbits (Oryctolagus cuniculus ver domesticas) were superovulated by administration of follicle stimulating hormone (FSH)h and human chorionic gonadotrophin FSH was subcutaneously injected (0.5 mg/rabbit) twice per (hCG)=. day at 12-h intervals for 3 days. The hCG was intravenously injected (80 IU) 12 h after the last injection of FSH. Immediately after hCG administration, the rabbits were naturally mated with New Zealand White male adult rabbits. At 60 h after the injection of hCG, embryos at the morula stage were collected by flushing out the oviducts and the uteri with PBS containing 0.3% bovine serum albuminj (PBStBSA). The morphologically normal were pooled and washed twice embryos with PBStBSA at room The embryos were temperature. then put into freezing vessel glass ampoule and polypropylene tubes) containing (plastic straw, a freezing solution (11% dimethyl sulfoxidekin PBS supplemented 20% pooled heat-inactivated rabbit serum) with or without with of AgI alginate-gel, or into a solution with one one droplet droplet of calcium alginate (the substrate of immobilization). The quantities of the freezing solutions were 400~1 (straw), 600 and 1 ml and 150~1 (polypropylene tubes). The ul (glass ampoule), freezing vessels were filled with embryos and the freezing solution by the manner described earlier and were cooled using the programmable freezer. Following cooling at 1 or 2'C/min from the temperature of the cooling bath was room temperature to -7"C, held at -7°C for 10 min. Then, cooling continued at l"C/min to -30°C (4). When the temperature of a ~dummy sample -with -3O"C, the freezing vessels were immersed in thermccouple reached liquid nitrogen. Then, the frozen samples were transferred to 37'C water bath for thawing. The warming rate was 1200"C/min (straw), 450"C/min (glass ampoule), and 150"C/min (1 ml) and 350"C/min (150 ~1) in the case of polypropylene tube. After the dilution of dimethyl sulfoxide was performed in a thawir.g, single step at room temperature (4,5). The embryos recovered were washe<. in fresh PBStBSA. The morphology of the embryos was 40x magnification after the removal of dimethyl examined at with normal blastomeres and an intact aona sulfoxide. Embryos pellucida were regarded as transferable. After morphological embryos were assessment all recovered placed in 0.3 ml of Ham's F-10 '1 supplemented with antibiotics and 10% rabbit serum, with light oil covered paraffin and incubated at 37'C in h i j k 1
Denka Pharmaceutical Co. Ltd., Tokyo, Japan. ANTRIN, Puberogen, Sankyo Zoki Co. Ltd., Tokyo, Japan. Sigma Chemical Co., St.Louis, MO, USA. A-9647, Dojin Chemical Ltd., Kumamoto, Japan. GIBCO Laboratories, Grand Island, MD, USA.
DECEMBER
1988 VOL. 30 NO. 6
1201
THERIOGENOLOGY
a gas phase viability of into expanded
Postthaw
In
by the microdroplet of 5% CO2 in air WSS assessed by their embryos ability blastocysts after 48-h culture.
Vitro
Viability
of
Bovine
method. The to develop
Embryos
collected from Japanese Black cows and Bovine embryos were The embryo donors were synchronized heifers at our institute. superovulated FSH with prostaglandin and with F2," (3Omg) dosage In six injections). The donors were total (24mg inseminated with the frozen semen (one-unit straw) of a Japanese Black bull at 24-h after the onset of standing estrus. Embryos were collected from the donors on Day 7 or 8 (Day O=the onset of standing estrus) by a nonsurgical method using PBS+BSA. Isolated embryos were placed in a modified PBS solution supplemented with 20% heat-inactivated fetal bovine serum (PBltFBS) under paraffin oil at temperature until equilibration with a freezing room V/Y glycerol in PBltFBS). Embryos were graded for solution (10% stage of development and for quality, and ranged from compacted morulae to expanded blastocysts. All were of excellent or good The embryos were dehydrated with the freezing solution quality. within 5 h after collection. A one-step dehydration procedure was carried out at 37°C. Next, and it was the embryos were used, one of two groups as follows: A portion of the assigned to embryos was loaded individually into sterile plastic straws with freezing the solution and one droplet of 400 p1 of AgI Other embryos were- put individually into sterile alginate-gel. polypropylene tubes with 1 ml of the freezing solution and one droplet of AgI alginate-gel. Thirty minu~tes later, all dehydrated were placed in the cooling apparatus. The freezing embryos were cooled from room temperature to -7'C at l"C/min vessels for -10 min, then cooled to -35°C at 0.3"C/min. and held at -7°C At -35"C, the freezing vessels were plunged into liquid nitrogen. the frozen sainples were thawed in water at Following 1 h storage, The straw was immersed for 10 sec. The polypropylene tube 37OC. the was immersed and agitated in water bath until complete decrystallization took place. After thawing, embryo normality The thawed embryos were rehydrated by a one-step was assessed. The embryos were sucrose procedure. loaded into a 0.25-ml (lo-mm long) containing the freezing solution. plastic straw" This column was separated by an air bubble with 0.5 M sucroseoin PBltFBS of the length of 70 mm. The heat-sealed end of the straw was held and tapped softly until the air bubble rose to the top of the straw. Then, the straw was held in an upright position for room temperature. The transferred to 5 min at embryos were another 0.5 M sucrose solution in a petri dish and held for 5 min at room temperature before washing in PBl+FBS. another the rehydrated embryos were transferred to Ham's After washing, supplemented with antibiotics 10% FBS. The F-10 medium and m PANACELAN-Hi n A201, I.M.V., Sigma o S-9378,
1202
Daiichi L'Aigle, Chemical
Parmaceutical Ltd., Tokyo, France. Co., St. Louis, USA.
DECEMBER
Japan.
1988 VOL.
30 NO. 6
THERIOGENOLOGY
culture procedure was similar to that ,previously described for rabbit embryos. The viability of embryos was assessed by their into hatching or hatched ability to develop blastocysts. RESULTS
AND DISCUSSION
If AgI alginate-gel droplet plays a role in a triggering nucleation in an solution ice aqueous under supercooling then a sample containing the AgI alginate-gel would conditions, level at a releasf: the latent heat of fusion at a constant temperature slightly lower than the freezing point of the aqueous solution. Table 1 shows the clear differences in the latent temperature of solution containing heat-releasing the AgI it in each vessel used in the alginate-gel and that without present study. Comparing the average releasing temperature of the latent heat of the respective vessels with the droplet, the temperature of the straw was higher than those of the other vessels. There was a significant difference (PcO.1) between the straw and the glass ampoule. Cryomicroscopic clarified that the droplet of the AgI alginate-gel observation began to swell slightly at constant temperature and increase in brightness suggesting an ice formation in the droplet, and that a of ice germinated from the surface of the small projection after swelling (6). Theoretically, this immediately droplet always occurs at the same constant temperature in spite of kinds and materials used for the freezing vessels. The surface area the atmosphere of the cooling contacting chamber in the respec*:ive freezing vessels in the straw is larger than those of vessels. Therefore', after other the ice is nucleated the originally, ice grows more rapidly in the straw than in the other
Table
1. The differences in the releasing temperature of latent heat of ~fusion in freezing solution with and without droplet of silver iodide alginate-gel (10% glycerol in PBS+FBS)a
Straw
("C)
Ampoule
("C)
Fre~ezing vessel 400 ul
With silver iodide alginate-gel
-6.7+0.3*b (n=3>
-11.3+1.2 Without silver iodide alginate-gel (n=3)
600 ul
-9.3'0.9**' (n=4) -13.6+0.9 (n=3)
Polypropylene tube ("C) 1 ml 150 ul
$1)
$1)
-13 (n=l)
-13 (n=l)
Mean i: standard error. * P
DECEMBER 1988 VOL. 30 NO. 6
(n=3).
1203
with AgIb without .AgI with Arg
40 5 12 9
1 ml with AgI with Arg
150 1 with AgI with Arg
7 6
59 62 15
Frozen
12 ‘7
38 2
7 5
57 55 12
Recovered
NO.
embryos
10ca3) 0
0
34(89)
5(71) 0
53(93) a(151 0
Transferable
of
3(43)
0
5(13) 0
5( 9) I( 2) 3(25)
With ZP broken
(%ja
7(58) 0
12(32) 0
6Ca6) 0
52(91) 9(16) 7(58)
Developed
frozen by using the silver iodide alginate-gel
t Percentage of the number of embryos recovered. AgI means the silver iodide alginate-gel droplet. i Arg means calcium alginate. pseudoblastocysts.
Polypropylene tube
Glass with AgI ampoule without AgI
Straw
Freezing vessel
Table 2. Postthaw development of rabbit morulae droplet seeding method
THERIOGENOLOGY
vessels.
This is the reason for the higher releasing temperature latent heat in the straw compared with the other vessels. Table 2 shows postthaw in vitro survival data of rabbit morulae that were frozen using the AgI alginate-gel droplet seeding method. Comparing the survival rates of these embryos with those of the controls (the non silver iodide method) the effectiveness of the silver iodide was evident. The proportion of embryo development was especially high after thawing in straws. However, it was similar to the result of our earlier study (4) using a conventional seeding method. Although some rabbit morulae survived freezing under the present seeding method in the glass ampoule and in the polypropylene tube, the survival rate was relatively low. This low viability rate could be attributed to the 1o.d releasing temperature of latent heat (7) and the low thawing rate (8). In this sense, the plastic straw was superior ampoule or the to the glass polypropylene tube as a freezing cryopreservation of vessel for embryos under the conditions in the present study. presented The proportion of embryos with zonae pellucidae was very low after thawing, compared damaged our previous study with (4) in which a conventional seeding However, this point method was used. should be studied further. of
Table 3 shows postthaw in vitro development of bovine Day 7 or 8 embryos frozen by using the AgI alginate-gel droplet method. group was set up for the present Although no control experiment frf?ezing bovine embryos, it on was confirmed that the AgI alginate-gel droplet acted effectively to induce ice in the solution of bovine embryos. freezing The rate of postthaw survival (84%) was very high. Recently, it has been reported that survival postthaw in vitro was approximately 65% in embryos were ranked excellent or that good (9). Besides rabbit and bovine embryos, the seeding method by the alginate-gel AgI could be utilized in the freezing of other drople-: mammalian embryozs. AEter thawing, cracks of the .droplet of AgI alginate-gel were 'observed very seldom. Therefore, it was considered that
Table
3.
Postthaw development of using the seeding method
bovine Day 7 or 8 embryos frozen by AgI alginate-gel droplet.
No. Freezing
Frozen
Plastic
of
embryos
(%)a
vessel
straw
DECEMBER
of
Transferable
Cultured
Developed
22
19
15
19
16(84)
7
7
5
5
4(57)
Polypropylene tube a Percentage
Recovered
the
number
1988 VOL.
of
embryos
30 NO. 6
recovered.
1205
THERIOGENOLOGY
this could prevent method embryos from directly contacting with a molecule of silver iodide. There would be no possibility that silver iodide is introduced to the lumen of uterus of embryo-recipient animal with frozen-thawed embryos. Silver iodide is hardly soluble in water (the solubility = O.O56mg/L at 25°C). So, it is not considered that silver iodide in itself shows the deleterious effect to the embryo in an aqueous solution. The our previous study using the original silver iodide method has demonstrated that two normal calves were born by transferring the frozen-thawed embryos to recipients (2). When rabbit morulae were cultured of AgI alginate-gel in Ham's F-10 with one droplet medium, were developed all embryos normally to hatched blastocysts after fully expanding (our unpublished observation). Some recipients become pregnant after transfer of bovine embryos frozen by using the present seeding method (in preparation for publication). In conclusion, the present study was undertaken to improve the original silver iodide seeding method by immobilizing the iodide silver and thus preventing it from contaminating the embryos. The present study also supports the importance of ice inoculation in obtaining high postthaw embryonic survival rates in the slow freezing procedure.
REFERENCES 1.
Kojima, T., Soma, T. and Oguri, N. Effect of silver iodide as an ice inducer on viability of frozen-thawed rabbit (1986). morulae. Theriogenology 3:341-352
2.
Kojima, T., Soma, T. and Oguri, N. Application of ice induction method using silver iodide to cryopreservation 33.73-76 (1987). of bovine embryos. Jpn. J. Anim. Reprod. _.
3.
Nilsson, K., Birnbaum, S., Flygare, S., Linse, L., Schroder, U., Jeppsson, U., Larsson, P-O., Mosbach, K. and Brodelius, P. A general method for the immobilization of cells with preserved viability. Eur. J. Appl. Microbial. Biotechnol. -17:319-326 (1983).
4.
Kojima, T., Soma, T. and Oguri N. Effect of rapid addition and dilution of dimethyl sulfoxide and 37°C equilibration on viability of rabbit morulae thawed rapidly. Cryobiology %:247-255 (1987).
5.
Kojima, T., Soma, T. and Oguri, N. Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of (1985). frozen-thawed rabbit morulae. Cryobiology =:409-416
6.
Kojima, T., Oguri, N., Shimada, K. and Souzu, H. Cryomicroscopic observation of ice crystal germination initiated by silver iodide-alginate gel droplets in various solutions. Cryo-Lett. in press (1988).
1206
DECEMBER 1988 VOL. 30 NO. 6
THERIOGENOLOGY
7.
Whittingham, D. G. Some factors affecting embryo storage in laboratory animals. In: Elliott, K. and Whelan, .I., (eds). The Freezing of Mammalian Embryos. Ciba Foundation Elsevier/Excerpta Medica/NorthSymposium 52 (new series), Amsterdam, 1977, pp. 97-108. Holland,
8.
S. M. Factors affecting the survival of sheep Willadsen, embryos during deep-freezing and thawing. In: Elliott,K. and Whelan, J., (eds). The Freezing of Mammalian Embryos. Cit'a Foundation Symposium 52 (new series), Elsevier/Excerpta Medics/North-Holland, Amsterdam, 1977, pp. 175-189.
9.
R. S., Spire, Prather, M. F. and Schalles, of cryopreservation techniques for bovine Theriogenology 2:195-204 (1987).
DECEMBER
1988 VOL.
30 NO. 6
R. R. Evaluation embryos.
1207
EFFECT
OF
ICE NUCLEATION ON FREEZING T.
BY DROPLET OF IMMOBILIZED SILVER OF RABBIT AND BOVINE EMBRYOS
Kojima,
T.
Soma
and
Department of Animal National Institute of Tsukuba-Norindanchi, P.O.Box Received
for
publication: Accepted:
N.
Oguri
Reproduction, Animal Industry 5, Ibaraki, 305,
December September
IODIDE
31,
19,
Japan
1987
1988
ABSTRACT Silver iodide was immobilized by applying the insoluble reacti.on between sodium alginate and calcium chloride. The immobilized silver iodide was immersed into a freezing solution ice nucleation. order to trigger Temperature in change postthaw in vitro development of during cooling and embryos in order to evaluate the effectiveness of the were examined silver iodide immobilized (AgI alginate-gel droplet) on embryo Samples containing the AgI alginate-gel droplets development. released the latent heat of fusion at a higher subzero temperature than samples without the AgI alginate-gel droplets. When the AgI alginate-gel droplet was added to the freezing solution of rabbit and bovine embryos, they were successfully preserved in liquid nitrogen. Key
words:
immobilized nucleation,
silver embryo
iodide, freezing
sodium
alginate,
ice
INTRODUCTION confirmed silver iodide It has been that can be induce effectively ice 'formation in extracellular used to solution in the process of slow cooling of mammalian embryos (1,Z). Using this procedure a high postthaw survival rate of obtained without employing conventional ice rabbit embryos was However, original silver iodide techniques. the seeding has some limitations in practical seeding method (192) in principle air For example, should be aspirated application. between the column of freezing solution into a straw and the the embryos column of silver iodide to containing prevent them from coming into contact. There is the possibility iodide could affect embryonic growth silver that some molecules of silver iodide came into deleteriously if the embryos at time of thawing, even though no with contact harmful effect on embryonic growth has been observed yet. A usable procedure of silver iodide needs to be developed to safe, We are grateful to Dr. A. Hanada for reviewing Acknowledgments: We also wish to thank Mr. Y. Hori, Mr. S. Ito the manuscript. of Nippon Oil & Fats Co. Ltd. for their and IMr. K. Hashimoto technical suggestions.
DECEMBER
1988 VOL.
30 NO. 6
1199
THERIOGENOLOGY
apply the silver iodide seeding reliably. In the present study, iodide was immobilized silver that it could be used more easily than in the original procedsuore (1,2). In the present study we examined the effect of ice nucleation by immobilized silver droplet) on iodide alginate-gel embryonic development. (AgI MATERIALS
AND
METHODS
Immobilization Silver iodidea was added to a 1 vel of 2% in distilled % water containing 2% w/v sodium alginate . The solution was mixed with a magnetic stirrer. Using a syringe and a 19-gauge obtuse the solution was added, drop by drop, to the distilled needle, 1OOmM calcium solution containing chloride dihydrate. water Immobilization of the silver iodide coincides with the reaction of sodium alginate and calcium chloride. Silver iodide was fixed gel of calcium alginate. Sodium alginate calcium in a and chloride are used to immobilize certain types of enzymes, yeasts of immobilized silver iodide (AgI and bacteria (3). The droplets were then removed from droplets) the calcium alginate-gel and washed in distilled chloride solution water. The AgI alginate-gel droplet was sterilized by autoclaving (121"C, 20 the droplets were preserved in sterilized distilled min) and water at 4'C until use. Observation
of
Temperature
Change
The freezing solution consisted of 10% v/v glycerolC in Dulbecco's phosphate buffered saline (PBS) supplemented with 20% strawe (0.5 ml), a glass ampoule fetal bovine serumd . A plastic (2 ml) and a polypropylene tubef (2 ml) were used as freezing changes, vessels to observe temperature especially those at the level. The quantity of latent heat releasing the freezing was 400 1-11 in the plastic straw, 600 ul in the solution used and 150 ul in the polypropylene tube. A ampoule and 1 ml without the AgI alginate-gel droplet or with freezing solution the substrate (calcium alginate) were employed in control only Approximately one droplet of silver iodide was put experiments. into each freezing vessel. Following cooling at 2'C/min, from a of 25°C to O"C, with a programmable liquid room temperature freezing machineg the respective samples nitrogen vapor were cooled at l"C/min until the latdnt heat of fusion occurred. change during cooling was monitored Temperature by a thermocouple connected to a thermocouple copper-constantan information The was recorded charts. recorder. on a Wako Chemical Industries, Ltd., Osaka, Japan. Sigma Chemical Co., St.Louis, MO, USA. b A-2033, Sigma Chemical Co., St.Louis, MO, USA. c G-9012, dGIBC0 Laboratories, Grand Island, MD, USA. e Fujihira Industries Ltd., Tokyo, Japan. f Corning Glass Works, Corning, NY, USA. Osaka Sanso Kogyo Inc., Osaka, Japan. g FFP-190,
1200
DECEMBER
1988 VOL.
30 NO. 6
THERIOGENOLOGY
of the curve was repeated three or four times except cooling in the case of the polypropylene tube. Formation of ice crystals monitored by the occurrence of the latent heat of fusion. was analyzed for statistical significance by Student's Data were t-test. Postthaw
In
Vitro
Viability
of
Rabbit
Embryos
Adult female Japanese White rabbits (Oryctolagus cuniculus ver domesticas) were superovulated by administration of follicle stimulating hormone (FSH)h and human chorionic gonadotrophin FSH was subcutaneously injected (0.5 mg/rabbit) twice per (hCG)=. day at 12-h intervals for 3 days. The hCG was intravenously injected (80 IU) 12 h after the last injection of FSH. Immediately after hCG administration, the rabbits were naturally mated with New Zealand White male adult rabbits. At 60 h after the injection of hCG, embryos at the morula stage were collected by flushing out the oviducts and the uteri with PBS containing 0.3% bovine serum albuminj (PBStBSA). The morphologically normal were pooled and washed twice embryos with PBStBSA at room The embryos were temperature. then put into freezing vessel glass ampoule and polypropylene tubes) containing (plastic straw, a freezing solution (11% dimethyl sulfoxidekin PBS supplemented 20% pooled heat-inactivated rabbit serum) with or without with of AgI alginate-gel, or into a solution with one one droplet droplet of calcium alginate (the substrate of immobilization). The quantities of the freezing solutions were 400~1 (straw), 600 and 1 ml and 150~1 (polypropylene tubes). The ul (glass ampoule), freezing vessels were filled with embryos and the freezing solution by the manner described earlier and were cooled using the programmable freezer. Following cooling at 1 or 2'C/min from the temperature of the cooling bath was room temperature to -7"C, held at -7°C for 10 min. Then, cooling continued at l"C/min to -30°C (4). When the temperature of a ~dummy sample -with -3O"C, the freezing vessels were immersed in thermccouple reached liquid nitrogen. Then, the frozen samples were transferred to 37'C water bath for thawing. The warming rate was 1200"C/min (straw), 450"C/min (glass ampoule), and 150"C/min (1 ml) and 350"C/min (150 ~1) in the case of polypropylene tube. After the dilution of dimethyl sulfoxide was performed in a thawir.g, single step at room temperature (4,5). The embryos recovered were washe<. in fresh PBStBSA. The morphology of the embryos was 40x magnification after the removal of dimethyl examined at with normal blastomeres and an intact aona sulfoxide. Embryos pellucida were regarded as transferable. After morphological embryos were assessment all recovered placed in 0.3 ml of Ham's F-10 '1 supplemented with antibiotics and 10% rabbit serum, with light oil covered paraffin and incubated at 37'C in h i j k 1
Denka Pharmaceutical Co. Ltd., Tokyo, Japan. ANTRIN, Puberogen, Sankyo Zoki Co. Ltd., Tokyo, Japan. Sigma Chemical Co., St.Louis, MO, USA. A-9647, Dojin Chemical Ltd., Kumamoto, Japan. GIBCO Laboratories, Grand Island, MD, USA.
DECEMBER
1988 VOL. 30 NO. 6
1201
THERIOGENOLOGY
a gas phase viability of into expanded
Postthaw
In
by the microdroplet of 5% CO2 in air WSS assessed by their embryos ability blastocysts after 48-h culture.
Vitro
Viability
of
Bovine
method. The to develop
Embryos
collected from Japanese Black cows and Bovine embryos were The embryo donors were synchronized heifers at our institute. superovulated FSH with prostaglandin and with F2," (3Omg) dosage In six injections). The donors were total (24mg inseminated with the frozen semen (one-unit straw) of a Japanese Black bull at 24-h after the onset of standing estrus. Embryos were collected from the donors on Day 7 or 8 (Day O=the onset of standing estrus) by a nonsurgical method using PBS+BSA. Isolated embryos were placed in a modified PBS solution supplemented with 20% heat-inactivated fetal bovine serum (PBltFBS) under paraffin oil at temperature until equilibration with a freezing room V/Y glycerol in PBltFBS). Embryos were graded for solution (10% stage of development and for quality, and ranged from compacted morulae to expanded blastocysts. All were of excellent or good The embryos were dehydrated with the freezing solution quality. within 5 h after collection. A one-step dehydration procedure was carried out at 37°C. Next, and it was the embryos were used, one of two groups as follows: A portion of the assigned to embryos was loaded individually into sterile plastic straws with freezing the solution and one droplet of 400 p1 of AgI Other embryos were- put individually into sterile alginate-gel. polypropylene tubes with 1 ml of the freezing solution and one droplet of AgI alginate-gel. Thirty minu~tes later, all dehydrated were placed in the cooling apparatus. The freezing embryos were cooled from room temperature to -7'C at l"C/min vessels for -10 min, then cooled to -35°C at 0.3"C/min. and held at -7°C At -35"C, the freezing vessels were plunged into liquid nitrogen. the frozen sainples were thawed in water at Following 1 h storage, The straw was immersed for 10 sec. The polypropylene tube 37OC. the was immersed and agitated in water bath until complete decrystallization took place. After thawing, embryo normality The thawed embryos were rehydrated by a one-step was assessed. The embryos were sucrose procedure. loaded into a 0.25-ml (lo-mm long) containing the freezing solution. plastic straw" This column was separated by an air bubble with 0.5 M sucroseoin PBltFBS of the length of 70 mm. The heat-sealed end of the straw was held and tapped softly until the air bubble rose to the top of the straw. Then, the straw was held in an upright position for room temperature. The transferred to 5 min at embryos were another 0.5 M sucrose solution in a petri dish and held for 5 min at room temperature before washing in PBl+FBS. another the rehydrated embryos were transferred to Ham's After washing, supplemented with antibiotics 10% FBS. The F-10 medium and m PANACELAN-Hi n A201, I.M.V., Sigma o S-9378,
1202
Daiichi L'Aigle, Chemical
Parmaceutical Ltd., Tokyo, France. Co., St. Louis, USA.
DECEMBER
Japan.
1988 VOL.
30 NO. 6
THERIOGENOLOGY
culture procedure was similar to that ,previously described for rabbit embryos. The viability of embryos was assessed by their into hatching or hatched ability to develop blastocysts. RESULTS
AND DISCUSSION
If AgI alginate-gel droplet plays a role in a triggering nucleation in an solution ice aqueous under supercooling then a sample containing the AgI alginate-gel would conditions, level at a releasf: the latent heat of fusion at a constant temperature slightly lower than the freezing point of the aqueous solution. Table 1 shows the clear differences in the latent temperature of solution containing heat-releasing the AgI it in each vessel used in the alginate-gel and that without present study. Comparing the average releasing temperature of the latent heat of the respective vessels with the droplet, the temperature of the straw was higher than those of the other vessels. There was a significant difference (PcO.1) between the straw and the glass ampoule. Cryomicroscopic clarified that the droplet of the AgI alginate-gel observation began to swell slightly at constant temperature and increase in brightness suggesting an ice formation in the droplet, and that a of ice germinated from the surface of the small projection after swelling (6). Theoretically, this immediately droplet always occurs at the same constant temperature in spite of kinds and materials used for the freezing vessels. The surface area the atmosphere of the cooling contacting chamber in the respec*:ive freezing vessels in the straw is larger than those of vessels. Therefore', after other the ice is nucleated the originally, ice grows more rapidly in the straw than in the other
Table
1. The differences in the releasing temperature of latent heat of ~fusion in freezing solution with and without droplet of silver iodide alginate-gel (10% glycerol in PBS+FBS)a
Straw
("C)
Ampoule
("C)
Fre~ezing vessel 400 ul
With silver iodide alginate-gel
-6.7+0.3*b (n=3>
-11.3+1.2 Without silver iodide alginate-gel (n=3)
600 ul
-9.3'0.9**' (n=4) -13.6+0.9 (n=3)
Polypropylene tube ("C) 1 ml 150 ul
$1)
$1)
-13 (n=l)
-13 (n=l)
Mean i: standard error. * P
DECEMBER 1988 VOL. 30 NO. 6
(n=3).
1203
with AgIb without .AgI with Arg
40 5 12 9
1 ml with AgI with Arg
150 1 with AgI with Arg
7 6
59 62 15
Frozen
12 ‘7
38 2
7 5
57 55 12
Recovered
NO.
embryos
10ca3) 0
0
34(89)
5(71) 0
53(93) a(151 0
Transferable
of
3(43)
0
5(13) 0
5( 9) I( 2) 3(25)
With ZP broken
(%ja
7(58) 0
12(32) 0
6Ca6) 0
52(91) 9(16) 7(58)
Developed
frozen by using the silver iodide alginate-gel
t Percentage of the number of embryos recovered. AgI means the silver iodide alginate-gel droplet. i Arg means calcium alginate. pseudoblastocysts.
Polypropylene tube
Glass with AgI ampoule without AgI
Straw
Freezing vessel
Table 2. Postthaw development of rabbit morulae droplet seeding method
THERIOGENOLOGY
vessels.
This is the reason for the higher releasing temperature latent heat in the straw compared with the other vessels. Table 2 shows postthaw in vitro survival data of rabbit morulae that were frozen using the AgI alginate-gel droplet seeding method. Comparing the survival rates of these embryos with those of the controls (the non silver iodide method) the effectiveness of the silver iodide was evident. The proportion of embryo development was especially high after thawing in straws. However, it was similar to the result of our earlier study (4) using a conventional seeding method. Although some rabbit morulae survived freezing under the present seeding method in the glass ampoule and in the polypropylene tube, the survival rate was relatively low. This low viability rate could be attributed to the 1o.d releasing temperature of latent heat (7) and the low thawing rate (8). In this sense, the plastic straw was superior ampoule or the to the glass polypropylene tube as a freezing cryopreservation of vessel for embryos under the conditions in the present study. presented The proportion of embryos with zonae pellucidae was very low after thawing, compared damaged our previous study with (4) in which a conventional seeding However, this point method was used. should be studied further. of
Table 3 shows postthaw in vitro development of bovine Day 7 or 8 embryos frozen by using the AgI alginate-gel droplet method. group was set up for the present Although no control experiment frf?ezing bovine embryos, it on was confirmed that the AgI alginate-gel droplet acted effectively to induce ice in the solution of bovine embryos. freezing The rate of postthaw survival (84%) was very high. Recently, it has been reported that survival postthaw in vitro was approximately 65% in embryos were ranked excellent or that good (9). Besides rabbit and bovine embryos, the seeding method by the alginate-gel AgI could be utilized in the freezing of other drople-: mammalian embryozs. AEter thawing, cracks of the .droplet of AgI alginate-gel were 'observed very seldom. Therefore, it was considered that
Table
3.
Postthaw development of using the seeding method
bovine Day 7 or 8 embryos frozen by AgI alginate-gel droplet.
No. Freezing
Frozen
Plastic
of
embryos
(%)a
vessel
straw
DECEMBER
of
Transferable
Cultured
Developed
22
19
15
19
16(84)
7
7
5
5
4(57)
Polypropylene tube a Percentage
Recovered
the
number
1988 VOL.
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THERIOGENOLOGY
this could prevent method embryos from directly contacting with a molecule of silver iodide. There would be no possibility that silver iodide is introduced to the lumen of uterus of embryo-recipient animal with frozen-thawed embryos. Silver iodide is hardly soluble in water (the solubility = O.O56mg/L at 25°C). So, it is not considered that silver iodide in itself shows the deleterious effect to the embryo in an aqueous solution. The our previous study using the original silver iodide method has demonstrated that two normal calves were born by transferring the frozen-thawed embryos to recipients (2). When rabbit morulae were cultured of AgI alginate-gel in Ham's F-10 with one droplet medium, were developed all embryos normally to hatched blastocysts after fully expanding (our unpublished observation). Some recipients become pregnant after transfer of bovine embryos frozen by using the present seeding method (in preparation for publication). In conclusion, the present study was undertaken to improve the original silver iodide seeding method by immobilizing the iodide silver and thus preventing it from contaminating the embryos. The present study also supports the importance of ice inoculation in obtaining high postthaw embryonic survival rates in the slow freezing procedure.
REFERENCES 1.
Kojima, T., Soma, T. and Oguri, N. Effect of silver iodide as an ice inducer on viability of frozen-thawed rabbit (1986). morulae. Theriogenology 3:341-352
2.
Kojima, T., Soma, T. and Oguri, N. Application of ice induction method using silver iodide to cryopreservation 33.73-76 (1987). of bovine embryos. Jpn. J. Anim. Reprod. _.
3.
Nilsson, K., Birnbaum, S., Flygare, S., Linse, L., Schroder, U., Jeppsson, U., Larsson, P-O., Mosbach, K. and Brodelius, P. A general method for the immobilization of cells with preserved viability. Eur. J. Appl. Microbial. Biotechnol. -17:319-326 (1983).
4.
Kojima, T., Soma, T. and Oguri N. Effect of rapid addition and dilution of dimethyl sulfoxide and 37°C equilibration on viability of rabbit morulae thawed rapidly. Cryobiology %:247-255 (1987).
5.
Kojima, T., Soma, T. and Oguri, N. Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of (1985). frozen-thawed rabbit morulae. Cryobiology =:409-416
6.
Kojima, T., Oguri, N., Shimada, K. and Souzu, H. Cryomicroscopic observation of ice crystal germination initiated by silver iodide-alginate gel droplets in various solutions. Cryo-Lett. in press (1988).
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THERIOGENOLOGY
7.
Whittingham, D. G. Some factors affecting embryo storage in laboratory animals. In: Elliott, K. and Whelan, .I., (eds). The Freezing of Mammalian Embryos. Ciba Foundation Elsevier/Excerpta Medica/NorthSymposium 52 (new series), Amsterdam, 1977, pp. 97-108. Holland,
8.
S. M. Factors affecting the survival of sheep Willadsen, embryos during deep-freezing and thawing. In: Elliott,K. and Whelan, J., (eds). The Freezing of Mammalian Embryos. Cit'a Foundation Symposium 52 (new series), Elsevier/Excerpta Medics/North-Holland, Amsterdam, 1977, pp. 175-189.
9.
R. S., Spire, Prather, M. F. and Schalles, of cryopreservation techniques for bovine Theriogenology 2:195-204 (1987).
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R. R. Evaluation embryos.
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