Deep freezing of sheep embryos.

Sheep embryos, collected 1-8 days after oestrus, were placed in Dulbecco's phosphate-buffered saline medium (PBS). After treatment, the viability of the embryos was tested by temporary transfer to ligated rabbit oviducts. In Exp. 1, Days 5-8 embryos survived for at least 15 min at 0 degrees C in the presence of 1-5 M-DMSO. In Exp. 2, 12/14 Days 5-8 embryos survived after being frozen in 1-5 M-DMSO at 0-3 degrees C/min to temperatures ranging between-15 degrees and -60 degrees C and then thawed at 12 degrees C/min. In Exp. 3, Days 5-8 embryos were frozen in 1-5 M-DMSO at 0-3 degrees C/min to below-65 degrees C before being transferred to liquid nitrogen (-196 degrees C), and stored for 12 hr to 1 month. The embryos were thawed at 3 degrees C/min, 12 degrees C/MIN or 360 degrees C/min and, after transfer to rabbit oviducts, 0/4, 10/36 and 1/4, respectively, developed normally. The 11 embryos which were considered normal when recovered from the rabbit oviducts plus 1 slightly retarded embryo were transferred to 7 recipient ewes. Four ewes subsequently lambed, producing 5 lambs. In addition, 8 embryos were transferred to 4 ewes directly after thawing. Three of these ewes subsequently lambed, producing 3 lambs.     

Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of frozen-thawed rabbit morulae

The aim of the present study was to examine effects of altering thawing conditions and procedure of addition and dilution of Me2SO on the viability of frozen-thawed rabbit morulae. Five hundred and sixty two rabbit morulae were cooled from room temperature to -80 degrees C at 1 degree C/min in the presence of 1.5 M dimethyl sulfoxide (Me2SO) using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, cooled rapidly, and stored in liquid nitrogen. When Me2SO was added in a single step, the frozen embryos were thawed in ambient air at 40 degrees C/min and Me2SO was diluted in a single step, 99 of 107 (93%) embryos cultured for 48 hr and 12 of 32 (38%) embryos transferred to 6 recipients developed to expanding blastocysts and viable fetuses, respectively. When Me2SO was added in a single step and the frozen embryos were thawed at the same rate and transferred directly without removal of Me2SO to culture media or oviducts of 8 recipients, 67 of 75 (89%) embryos cultured and 12 of 40 (30%) embryos transferred developed to expanding blastocysts and viable fetuses, respectively. There were no significant differences between these survival rates and survival rates obtained by conventional method, i.e., frozen embryos were thawed at 4 degrees C/min by interrupted slow method and Me2SO was added and diluted in a stepwise manner.     

A simplified method for freezing mouse embryos

Mouse oviducts containing eight-cell embryos were frozen to ?196 °C in 1.45 m DMSO. The cooling rate was 0.3 °C/min and thawing occurred at 3 °C/min. Dilution of DMSO took place either before or after flushing of the thawed oviducts. The yield of intact embryos was higher in the second group.In one particular series involving 21 donor mice (natural ovulation) 88 recovered embryos were transferred to the oviducts of recently mated pseudopregnant mice without prior in vitro culture to the blastocyst stage. Fifty-five live young were born.It is concluded that the freezing of embryos in the oviduct is a reliable method for establishing an embryo bank. Handling and collection of isolated embryos is not required and a large amount of material can be frozen at once. In vitro culturing of embryos is not required immediately after thawing in order to obtain a high yield of live young.     

Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa

Spermatozoa from cauda epididymis of mature mice were suspended in preservation solution (Dulbecco's PBS containing raffinose in combination with glycerol, DMSO or skim milk as freezing protective agents). The suspension was frozen by the dry ice-alcohol method and preserved for 1-120 days in liquid nitrogen (-196 degrees C). Highest sperm viability after thawing was obtained with a combination of 10% raffinose and 5% glycerol or with a combination of 10% raffinose and 10% DMSO. These frozen thawed sperm were found to have fertilizing capacity when used for in vitro fertilization. The 2-cell embryos obtained through the above procedures developed into normal pups at a high rate when transferred into the oviducts of pseudopregnant female mice.     

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.     

Fast freezing of cow embryos in French straws with an automatic program

Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2 degrees C/min from seeding to -25 degrees C before being plunged into liquid nitrogen directly or after 10 min at -25 degrees C. Cooling rate from 20 degrees C to -5 degrees C was 9 degrees C/min. Seeding was induced automatically at -5 degrees C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20 degrees C (group I) or at 37 degrees C (group II). Survival was assessed by culture in vitro and by transfer. In group I, 35.7% were degenerated after thawing (compared to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42.8%) and hatching rate after 96h culture was quite similar (33.3% vs 34.4%). In group II, four pregnancies were obtained from 10 embryos transferred. Time at -25 degrees C did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos.     

Two-step freezing of two-cell rabbit embryos after partial dehydration at room temperature

The effect of rapid freezing and thawing on the survival of 2-cell rabbit embryos was examined. When embryos in 2.2 M-propanediol were directly plunged from room temperature to liquid nitrogen some of them survived after thawing (8%) but only if they had been pretreated by exposure to an impermeable solute, sucrose, that makes the blastomeres shrink osmotically before cooling. High survival (77-88%) in vitro was obtained when pretreated embryos were first held at -30 degrees C for 30-240 min before immersion into liquid nitrogen. Transfer of such frozen-thawed embryos gave a survival rate to live young similar to that obtained with controls (26% and 32% respectively). DMSO was less effective than propanediol; only 2 out of 38 sucrose-pretreated frozen-thawed embryos developed in vitro. The present work shows that a combination of partial dehydration of blastomeres at room temperature with their permeation by a cryoprotective agent offers a simple method for successful rapid freezing and thawing of rabbit embryos.     

Survival of mouse embryos after being frozen in glycerol-sucrose mixture

Random bred female albino mice (6-8 weeks old) were used as a source of embryos. 8- to 16 cell embryos were dehydrated in glycerol-sucrose mixture in 0.25 ml straws at room temperature. Straws were cooled at the rate of 5 degrees C/min to -7 degrees C. Seeding was induced by touching the out side of the straw at -7 degrees C. Straws were further cooled at 0.5 degree C/min down to -35 degrees C and then plunged into liquid N2. Thawing of straws was done by direct transfer into water at 35 degrees C. Frozen-thawed embryos were cultured in a CO2 incubator maintained at 39 degrees C. Out 190 embryos (8-16 cell) initially frozen, 169 (88.94%) were recovered on thawing. 158 (93.5%) out of 169 were apparently normal and used for culture. 75 (47.46%) developed to morulae/early blastocysts and 72 (45.56%) to expanded blastocysts on 24 and 48 hr culture respectively. In conclusion, the incorporation of sucrose in the freezing medium at a concentration of 0.25 M has led us to propose a freezing, thawing and transfer method without dilution of glycerol. The technique being quite simple is worth trying in farm animals where importance of this technique in non-surgical transfer of frozen-thawed embryos will be a boon.     

High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1-2 propanediol as cryoprotectant

A method for obtaining a high survival rate of frozen-thawed mouse embryos is presented. Eight-cell mouse embryos were frozen inside small plastic straws in the presence of 1-2 propanediol and stored at -196 C. After thawing, the embryos were diluted for only 5 min in a 1.0 M sucrose solution to remove the 1-2 propanediol from the cells. At high rate of thawing (is equivalent to 2500 C/min) more than 88% of the embryos survived in vitro to the blastocyst stage provided that the dilution of propanediol was performed rapidly during thawing. At a lower rate of thawing (is equivalent to 300 C/min), survival tended to be higher (94.7%) when dilution was done 5 min after thawing. When the frozen-thawed embryos were transferred to the oviducts of day 1 pseudopregnant recipients either directly after the dilution of 1-2 propanediol or after 24 or 48 hr of culture, a high proportion of them (65.9%) develop normally to viable fetuses.     

Survival of 16-celled and morula stage rabbit embryos frozen to -196 degrees C,.

Preimplantation stage (16-celled and morula) rabbit embryos were successfully frozen to -196 degrees C. The cooling rate (from a room temperature to 0 degrees C), the presence of the mucin layer surrounding embryos, the ice-seeding treatment and the thawing procedure were examined to determine their effects on the survival of the frozen embryos of Japanese white, New Zealand white and Dutch-Belted rabbits. A high proportion (51%; 16-celled, 69%; morula) of Dutch-Belted rabbit embryos developed in vitro, when they were frozen to -196 degrees C, applying the ice-seeding at -4 degrees C in the presence of 12.5% DMSO, after being cooled to 0 degrees C at the rate of 7-9 degrees C/min, and were diluted by a stepwise addition of 4 different strength PBS on thawing. The highest rate of in vitro development (81%; Japanese white, 75%; New Zealand white, 82%; Dutch Belted embryos) was obtained when the morula stage embryos were frozen to -196 degrees C applying seeding at -4 degrees C after being cooled to 0 degrees C at the rate of 1 degrees C/2.5 min and were diluted, on thawing, by stepwise addition of 6, 3 and 1% DMSO solution and a culture medium. No great difference was found in the survival rate between the embryos covered with the mucin layer and those which had not the coat. All the embryos frozen without applying seeding treatment failed to develop in vitro after being thawed and diluted. Nine out of 27 does each of which received 6 reimplantations of the embryos frozen-thawed became pregnant and were found to be carrying 37 normal fetuses on the 12th day of pregnancy.     

The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus

Embryos were collected at the 4-10-cell stage from the oviducts (Day 4; Day 1 = ovulation) or as morulae (Day 7) from the uterus of marmosets and frozen in 1.5 M-DMSO (Days 4 and 7) or 1.0 M-glycerol (Day 4 only), using a slow freezing and thawing technique. Of 22 Day-4 embryos frozen in DMSO, 18 were recovered and 16 of these were transferred to 10 synchronized recipients; 7 recipients became pregnant compared with all 7 control recipients receiving 10 unfrozen embryos. Fifteen frozen-thawed morulae were transferred to 9 Day-6 recipients; the pregnancy rate (55.6%) was lower than for control embryos (85.7%). Embryos frozen in glycerol suffered severe osmotic stress during glycerol addition and removal. Of 8 recipients, 3 (37.5%) became pregnant but only one fetus was carried to term. These results on embryo collection, freezing and transfer in the marmoset have important implications for developing improved methods for freezing human embryos and the breeding of endangered primates.     

Evaluation of cryopreservation techniques for bovine embryos

A total of 228 embryos was nonsurgically collected from superovulated cows and dehydrated in dimethyl sulfoxide (DMSO) or glycerol by a three-step procedure or a (T.I.T.) timed interval titration procedure. Embryos were loaded in straws, frozen by cooling to -6.0 degrees C at 1.0 degrees C/min, and seeded, followed by cooling to -30 degrees C at 0.3 degrees C/min and to -38 degrees C at 0.1 degrees C/min. At this time the straws were plunged into liquid nitrogen at -195 degrees C. Embryos were thawed in a 27 degrees C or 37 degrees C water bath and rehydrated by a six-step, three-step (sucrose) or one-step (sucrose) procedure. This yielded a 2 x 2 x 2 x 3 factorial treatment structure. Survival was based on development after 12 h in in vitro culture. The only significant single factor affecting survival was the initial quality grade of the embryo. Grades 1 and 2 embryos survived more often than Grade 3 embryos (P < 0.05). Using DMSO as the cryoprotectant resulted in better scores for the post dehydration to post thawing interval (P = 0.02). For both intervals, post dehydration to post thawing and post thawing to post rehydration, the previous quality grade was significant in determining the subsequent quality grade (P < 0.01). At each step of the freeze-thaw process, the embryos became progressively less morphologically intact.     

Two-step freezing of cattle blastocysts with dimethylsulfoxyde (DMSO) or glycerol

Sixty five cattle blastocysts were frozen by the so-called two-step freezing method: The samples were seeded at -7 degrees C and then directly brought at -30 degrees C for 30 minutes before being taken into liquid nitrogen. Results in terms of survival rates at thawing and after short term cultures were compared to two controlled linear cooling rate procedures (i.e. 0.3 degrees C/min and 1.3 degrees C/min). The results demonstrate that: 1) two-step freezing yielded approximately the same survival rate as the two others techniques and 2) Glycerol yielded better survival rates than DMSO treatments (56 vs 31% after 24 hours in culture).     

A new procedure for the cryopreservation of equine embryos

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).     

Development and viability of frozen-thawed cattle embryos

Embryos recovered 7 to 8 days after estrus were frozen from -7 to -30 degrees C at 0.3 degrees C/min, from -30 to -33 degrees C at 0.1 degrees C/min, and then plunged into liquid nitrogen. They were thawed in a 25 degrees C waterbath. In a preliminary study, 15 of 18 embryos continued to develop during the 24-hour culture post-thaw in either Ham's F-10 or modified Dulbecco's phosphate buffered saline (PBS). In the main study, 5 of 20 embryos developed to 60-day pregnancies when embryos were transferred within 5 hours after thawing. The incidence of extended estrous cycles (pregnancy or presumed embryonic mortality) was 10 of 14, when the zona pellucida was intact after thawing, and 0 of 6, when it was ruptured or absent (P<.05). Embryos cultured in PBS tended to develop more readily than those in Ham's F-10 (15 of 20 vs 9 of 20, respectively, P reverse similar.1). Quality of the embryos, at recovery from the donor and after thawing, affected development in culture (19 of 27 embryos excellent at recovery developed vs 5 of 13 poor to very good, P reverse similar.1; 23 of 33 embryos good to excellent after thawing developed vs 1 of 7 poor, P<.05). The proportion of pyknotic nuclei in embryos which were cultured ranged from 18 to 100%. The pregnancy rate from embryos which were cultured was low (2 of 20). Thirty percent of frozen and thawed embryos had damaged zonae pellucidae. The study showed that: the pregnancy rate from frozen embryos was approximately half that achieved with unfrozen embryos; culturing embryos for 24 hours before transfer was not beneficial; the PBS culture system appears to be the system of choice for assessing embryo viability in vitro .     

Effect of rapid addition and dilution of dimethyl sulfoxide and 37 degrees C equilibration on viability of rabbit morulae thawed rapidly

The aim of the present study was to examine the effects of various conditions of addition and dilution of dimethyl sulfoxide (Me2SO) and 37 degrees C equilibration, and also the effects of freezing in the solution which was prepared in advance and stored in plastic straws at -20 degrees C on the viability of rabbit morulae thawed rapidly. The embryos were cooled from room temperature to -30 degrees C at 1 degree C/min in the presence of 1.5 M Me2SO using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, then cooled rapidly, and stored in liquid nitrogen. The frozen straws were thawed rapidly (greater than 1000 degrees C/min). When Me2SO was added in a single step, equilibrated with embryos at 37 degrees C for 15 min and diluted out in a single step, a very high survival was obtained: transferable/recovered, 90%: developed/recovered, 96%. When embryos were pipetted into 1.5 M Me2SO that was prepared in advance, stocked in straws at -20 degrees C, and cooled, the proportions of transferable and developed embryos were equivalent to those of embryos frozen in the solution that was prepared immediately before use.     

Liquid nitrogen vapour freezing of mouse embryos

Mouse morulae were frozen rapidly to -196 degrees C in the presence of glycerol by a two-step procedure; the embryos were transferred directly from -7 degrees C after seeding into liquid nitrogen vapour at -170 to -180 degrees C and then into liquid nitrogen 10-15 min later. Suitable conditions for the survival of embryos frozen with liquid nitrogen vapour were found to be: 2 M-glycerol, 2 M-propylene glycol, 2 M-ethylene glycol; 5-30 min equilibration time at 0 degrees C; 3-60 min holding time in liquid nitrogen vapour; dilution of glycerol with sucrose out of the frozen-thawed embryos; morula and early blastocyst stage embryos. Relatively high survival rates (69-74%) were obtained after rapid freezing by liquid nitrogen vapour. Morulae frozen in this fashion, cultured and transferred to recipients developed into normal young.     

Effect of warming rate on mouse embryos frozen and thawed in glycerol

Mouse embryos (8-cell) fully equilibrated in 1.5 M-glycerol were cooled slowly (0.5 degrees C/min) to temperatures between - 7.5 and - 80 degrees C before rapid cooling and storage in liquid nitrogen (-196 degrees C). Some embryos survived rapid warming (approximately 500 degrees C/min) irrespective of the temperature at which slow cooling was terminated. However, the highest levels of survival of rapidly warmed embryos were observed when slow cooling was terminated between -25 and -80 degrees C (74-86%). In contrast, high survival (75-86%) was obtained after slow warming (approximately 2 degrees C/min) only when slow cooling was continued to -55 degrees C or below before transfer into liquid N2. Injury to embryos cooled slowly to -30 degrees C and then rapidly to -196 degrees C occurred only when slow warming (approximately 2 degrees C/min) was continued to -60 degrees C or above. Parallel cryomicroscopical observations indicated that embryos became dehydrated during slow cooling to -30 degrees C and did not freeze intracellularly during subsequent rapid cooling (approximately 250 degrees C/min) to -150 degrees C. During slow warming (2 degrees C/min), however, intracellular ice appeared at a temperature between -70 and -65 degrees C and melted when warming was continued to -30 degrees C. Intracellular freezing was not observed during rapid warming (250 degrees C/min) or during slow warming when slow cooling had been continued to -65 degrees C. These results indicate that glycerol provides superior or equal protection when compared to dimethyl sulphoxide against the deleterious effects of freezing and thawing.     

Two step freezing of cow embryos in 1.4 M glycerol

Day 6 1 2 -7 1 2 cow embryos were frozen in 1.4 M glycerol in PBS, at 0.3 degrees C/min to -30 (group I), -35 (group II), and -40 degrees C (group III) before being plunged into liquid nitrogen. They were subsequently thawed by direct transfer to water at 37 degrees C. In Experiment I, embryos frozen and thawed were cultured in vitro, 12 out of 19 embryos (63%) survived and there were no significant (p > 0.05) differences in survival rates among the three freezing groups. In Experiment II, 29 embryos frozen to -30 or -35 degrees C were transferred non-surgically to heifers on day 7. Seventeen of 29 recipients (59%) were pregnant at day 60. Five embryos frozen to -35 degrees C resulted in 5 pregnancies (100%) after thawing and surgical transfer.     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes.