Defined media for vitrification,warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos

The purpose of this study was to assess the viability (rates of re-expanding and hatching in vitro), of in vitro derived ovine blastocysts using vitrification and warming/rehydration media containing fetal calf serum (20% FCS) or polyvinyl alcohol (0.1% PVA), and the incorporation of labelled methionine in protein synthesised during the first 4h after cryopreservation. In experiment 1, after 60 h culture in TCM-199 supplemented with 10% FCS, the hatching rates of blastocysts that had been vitrified, warmed, and rehydrated in media containing only PVA (p/p) were significantly (P<0.05) lower than those vitrified in medium containing PVA with warming and rehydration in medium containing FCS (p/s). Blastocysts that were vitrified in medium containing FCS and warmed and rehydrated in medium with PVA (s/p) had hatching rates that were significantly lower (P<0.01) than those vitrified, warmed, and rehydrated in media with only FCS (s/s). After warming, the number of dead cells in the p/p group was significantly (P<0.05) lower than in all other groups. In experiment 2, the [35S]methionine uptake by embryonic cells of the s/p group was significantly (P<0.01) higher than in other groups. The incorporation of labelled methionine into newly synthesised proteins was significantly lower in the p/p group (P<0.01) than in all other groups. No differences in the newly synthesised proteins were observed between groups. In conclusion, these results suggest that it is possible to replace serum with defined macromolecules in vitrification and warming/rehydration media for in vitro derived ovine blastocysts but this leads to a decrease in viability and a reduction in protein synthesis after warming.     

The effect of dialysis of extended ram semen prior to freezing on post-thaw survival and fertility

The effect of dialysis on extended ram semen prior to cryopreservation was studied. Techniques were developed to improve post-thaw recovery of dialyzed semen and a fertility trial was used to evaluate the viability of dialyzed and frozen semen. Dialysis prior to freezing was shown to increase post-thaw recovery of motile cells and percentage of cells passing through a Sephadex filter. Freezing semen in pellets on dry ice was superior to freezing in French straws. Pellets were thawed in an aluminum thaw block at 42 to 45 degrees C before insemination of progestagen-PMSG synchronized ewes. Double inseminations were made at 12-hr intervals. Natural service of synchronized ewes was also made at 12-hr intervals as a control. There was no significant difference (P greater than 0.05) in fertility between naturally serviced ewes (44.4%) and ewes inseminated with frozen semen (44.7%).     

Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro

The objective of this study was to evaluate the in vitro development of frozen-thawed bovine embryos held at room temperature or refrigerated for 2, 6 or 12 h prior to freezing. After recovery, embryos were randomly assigned to be placed in holding media for 2 h (n=131), 6 h (n=136) or 12h (n=133) prior to freezing. Approximately one-half of the embryos were refrigerated (5 degrees C; n=203) while the remaining half were held at room temperature (22 degrees C; n = 197) until freezing. Embryos were frozen in 10% ethylene glycol and stored in liquid nitrogen. After thawing, embryos were cultured for 72 h in Ham's F-10 media supplemented with 4% fetal bovine serum. Embryos were evaluated for quality and stage of development prior to freezing and after culture. At the end of culture, it was determined if each embryo had developed beyond the stage recorded prior to freezing and if the embryo had hatched from the zona pellucida. The percentage of embryos that developed during culture was greater (P < 0.001) for Grade 1 (81%) than for either Grade 2 (65%) or Grade 3 (48%) embryos. Likewise, a greater proportion (P < 0.001) of Grade 1 embryos developed to hatched blastocysts (60%) than either Grade 2 (40%) or Grade 3 (24%) embryos. The holding temperature from collection to freezing did not influence embryo development, regardless of the interval from embryo collection to freezing. The proportion of embryos that developed to expanded blastocysts and hatched was greater (P < 0.005) for embryos held 2 h prior to freezing (64%) than for embryos held for 12 h (33%). Hatching rate of embryos held 6 h prior to freezing (54%) tended (P < 0.08) to be lower than the hatching percentage for embryos held for 2 h. Thus, post-thaw embryonic development was impaired the longer embryos were held prior to freezing and temperature during the interval from collection to freezing did not affect post-thaw development.     

Effect of glucose on embryo quality and post-thaw viability of in-vitro-produced bovine embryos

The present study was carried out to evaluate the effect of glucose absence during the first 24 h of culture on blastocyst quality and survival after freezing and thawing. In Experiment 1, IVM/TVF bovine zygotes from a slaughterhouse were cultured for 24 h in SOFm, either in the absence or in the presence of 1.5 mM glucose and then further cultured for 7 d in SOFm with 1.5 mM glucose. Absence of glucose during the first 24 h of culture increased (P < 0.001) the percentage of embryos that developed to the morula and blastocyst stages. In Experiment 2, presumptive zygotes were incubated for 24 h in the absence of glucose and were then cultured for 7 d in the presence of 1.5, 3 or 5 mM glucose. There were no differences in the percentages of embryos developing to morula or blastocyst stages at 1.5 or 3 mM glucose, whereas the 5 mM concentration appeared to be detrimental (P < 0.001). Blastocysts from Experiments 1 and 2 were assessed for freezing resistance by means of the ability of frozen-thawed embryos to re-expand their blastocoelic cavity and hatch after culture for 72 h in vitro. For Grade 1 and 2 blastocysts, the post-freezing survival rate was unaffected when glucose was omitted during the first 24 h of culture, provided that the glucose was subsequently maintained between 1.5 and 3 mM. At 5 mM glucose, blastocoelic re-expansion was inhibited (P < 0.03). Addition of 1.5 or 3 mM glucose to the culture medium following 24 h of culture without glucose did not affect embryo cell number, whereas 5 mM significantly decreased it (P < 0.01). These results indicate that the first 24 h of culture without glucose do not affect embryo quality or post-thaw viability, but an increase in blastocyst yield was observed. After 24 h of culture addition of glucose in the range 1.5 to 3 mM was beneficial, while as higher concentrations decreased the efficacy of this in vitro production technique.     

Cryopreservation of ovine embryos: slow freezing and vitrification

Different methods for the cryopreservation of ovine embryos were evaluated in vitro (survival upon culture in vitro) and in vivo (pregnancy and lambing rates after transfer in field conditions). In the first 2 experiments, slow freezing conditions were evaluated. When glycerol and ethylene glycol were compared, no differences in the overall pregnancy rate were found (40.2 vs 51.3%), but better results were obtained with ethylene glycol than with glycerol in morulae (29.7 vs 59.4%, P < 0.05). In the second experiment, 2 methods of removing ethylene glycol were compared: a 1-step procedure using 0.5-M sucrose and a 3-step process for decreasing ethylene glycol concentration. There were no differences in the overall pregnancy rate (48.0 vs 48.0%) between the 2 methods. The last series of experiments were designed to compare 2 vitrification solutions: propylene glycol--glycerol (PG) and ethylene glycol--Ficoll 70--sucrose (EFS). There were no differences between the 2 vitrification solutions, based on the overall pregnancy rate (28.1 vs 40.0%). The vitrification technique and specially with EFS solution has resulted in good pregnancy rates. The EFS solution was particularly efficacious with morulae (55.5% pregnancy). These results demonstrate that vitrification with EFS can be used successfully for the cryopreservation of ovine embryos.     

Effects of melatonin and undernutrition on the viability of ovine embryos during anestrus and the breeding season

This study examined the effects of melatonin and level of nutrition on embryo yield during anestrous and breeding season. Adult Rasa Aragonesa ewes were assigned randomly to one of the four treatment groups in two experiments using a 2x2x2 factorial design. Individuals were treated (+MEL) or not treated (-MEL) with a subcutaneous implant of melatonin for 42d (Melovine, CEVA) and fed 1.5 (control, C) or 0.5 (low, L) times the daily maintenance requirements for 20d. Ewes were mated at oestrus (Day=0) and embryos were recovered on Day 5. Level of nutrition and melatonin supplements did not have a significant effect on ovulation rate or the number of recovered ova per ewe in the Reproductive Season (RS) and the Anestrous Season (AS). During the RS, undernutrition reduced the number of viable embryos per ewe (C: 1.1+/-0.2; L: 0.6+/-0.2; P<0.05); however, the number of viable embryos per ewe in the L+MEL group (0.2+/-0.15) was significantly lower than it was in the L, C+MEL and C groups (0.9+/-0.3, 1.2+/-0.3, 1.0+/-0.4, respectively; P<0.05). In the AS, nutrition did not have a significant effect on the number of viable embryos per ewe, although melatonin supplements might have improved rates slightly. Embryo viability rate (% viable embryos/embryos recovered) was unaffected by melatonin supplements or level of nutrition in the RS and the AS. Season had a strong effect on the number of viable embryos per functional corpus luteum among ewes in the L+MEL group, only (RS: 0.2+/-0.1; AS: 0.6+/-0.2; P<0.05). In conclusion, undernutrition impaired the viability of sheep embryos in the RS, particularly among ewes that were given melatonin supplements subcutaneously, but melatonin appeared to improve embryo quality in the AS, which suggests that the mechanisms involved in the interactive effects of melatonin and nutrition on embryo development are influenced by season.     

Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3 M trehalose or liposome solution, then cooled with slow (0.95 ± 0.02 °C/min), medium (73 ± 3 °C/min) and fast (265 ± 12 °C/min) cooling rates and storage in liquid nitrogen, followed by a 37 °C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66% ± 5% vs 29% ± 4%) compared to control RBCs (c-RBC, p = 0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p = 0.039 and p = 0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p = 0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p = 0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.     

Impact of freezing/thawing procedures on the post-thaw viability of cryopreserved human saphenous vein conduits

BACKGROUND: Cryopreserved human blood vessels are important tools in reconstructive surgery. However, patency of frozen/thawed conduits depends largely on the freezing/thawing procedures employed. METHODS: Changes in tone were recorded on rings from human saphenous vein (SV) and used to quantify the degree of cryoinjury after different periods of exposure at room temperature to the cryomedium (Krebs-Henseleit solution containing 1.8M dimethyl sulfoxide and 0.1M sucrose) and after different cooling speeds and thawing rates following storage at -196 degrees C. RESULTS: Without freezing, exposure of SV to the cryomedium for up to 240 min did not modify contractile responses to noradrenaline (NA). Pre-freezing exposure to the cryomedium for 10-120 min attenuated significantly post-thaw maximal contractile responses to NA, endothelin-1 (ET-1) and potassium chloride (KCl) by 30-44%. Exposure for 240 min attenuated post-thaw contractile responses to all tested agents markedly by 62-67%. Optimal post-thaw contractile activity was obtained with SV frozen at about -1.2 degrees C/min and thawed slowly at about 15 degrees C/min. In these SV maximal contractile responses to NA, ET-1 and KCl amounted to 66%, 70% and 60% of that produced by unfrozen controls. Following cryostorage of veins for up to 10 years the responsiveness of vascular smooth muscle to NA was well maintained. CONCLUSION: Cryopreservation allows long-term banking of viable human SV with only minor loss in contractility.     

Effect of freezing and thawing rates on the post-thaw viability of boar spermatozoa frozen in FlatPacks and Maxi-straws

The effects of different freezing and thawing rates on the post-thaw motility and membrane integrity of boar spermatozoa, processed as split samples in Maxi-straws or flat PET-plastic packages (FlatPack) were studied. A programmable freezing device was used to obtain freezing rates of either 20, 50 or 80 degrees C/min. Thawing of the samples was performed in a bath of circulating water; for 40s at 50 degrees C or 27s at 70 degrees C for Maxi-straws and 23s at 35 degrees C, 13s at 50 degrees C or 8s at 70 degrees C for the FlatPacks. Sperm motility was assessed both visually and with a computer assisted semen analysis (CASA) apparatus, while plasma membrane integrity was assessed using the fluorescent probes Calcein AM and ethidium homodimer-1. Temperature changes during freezing and thawing were monitored in both forms of packaging. Values for motile spermatozoa, sperm velocity and lateral head displacement variables were significantly (p<0.05) higher for samples frozen in FlatPacks than in Maxi-straws, with superior results at higher thawing rates. Freezing at 50 degrees C/min yielded better motility than 20 or 80 degrees C/min, although the effect was rather small. Neither freezing rate nor thawing rate had any effect on membrane integrity (p>0.05). A significant boar effect was seen for several parameters. The most striking difference in temperature courses between containers was a 4-5-fold lowering of the thawing rate, between -20 and 0 degrees C, in the center of the Maxi-straw, compared with the FlatPack. This is apparently due to the insulating effect of the thawed water in the periphery of the Maxi-straw. The improvement in sperm motility seen when using the FlatPack appears to be related to the rapid thawing throughout the sample, which decreases the risk of cell damage due to recrystallization during thawing. Since sperm motility patterns have been reported to be correlated with fertility both in vitro and in vivo it is speculated that the use of the FlatPack might improve the results when using frozen-thawed boar spermatozoa for artificial insemination.     

Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying

AIMS: The effects of protective agents, rehydration media and freezing temperature on the viabilities of Lactobacillus brevis and Oenococcus oeni H-2 when subjected to freeze-drying were investigated. METHODS AND RESULTS: Several protectants and rehydration media were tested to improve the survival after freeze-drying. The cells were also frozen at -65 and -20 degrees C to check the effect of freezing temperature on the viability. CONCLUSIONS: The best protectant and rehydration medium to obtain the highest viability after freeze-drying varied with the species of bacteria. Yeast extract (4.0%) and sodium glutamate (2.5% ) gave maximum viability of L. brevis and O. oeni (67.8% and 53.6% respectively). The highest survival of L. brevis and O. oeni were obtained when rehydrated with 10% sucrose and MGY medium respectively. When the bacterial cells were frozen quickly (-65 degrees C) than slowly (-20 degrees C), L. brevis and O. oeni both showed increased viability after freeze-drying. SIGNIFICANCE AND IMPACT OF THE STUDY: The viabilities of L. brevis and O. oeni after freeze-drying were shown to be strain specific and dependent on protective agents, rehydration media and freezing temperature.     

Effect of post-thaw cell rehydration at 4 degrees C on survival of frozen and vitrified IVP-derived bovine embryos.

In vitro-produced bovine embryos (IVP) were either frozen in 10% glycerol in a phosphate-buffered saline solution (PBS) using conventional slow freezing or vitrified in 25% glycerol and 25% ethylene glycol in PBS. The results of viability and hatching rates were compared between frozen and vitrified embryos after thawing and dilution using one of three different protocols: (A) a three-step dilution procedure, (B) a one-step dilution procedure or (C) a procedure in which embryos were kept in situ inside the straw at 4 degrees C for 10 min during a one-step dilution procedure. No significant differences in embryo survival were found among protocols A, B and C for frozen embryos and between protocols A and B for vitrified embryos. Viability and hatching rates of vitrified embryos thawed and diluted by protocol C (73 and 62%) were significantly enhanced (P < 0.05) in comparison to those obtained with protocol A (55 and 41.6%) or protocol B (54.5 and 35.3%). These results indicate that for vitrified IVP bovine embryos, direct in-straw rehydration at 4 degrees C for 10 min improves embryo survival and it could be a practical procedure for use under field conditions where there is sometimes a longer interval between thawing and transfer.     

Influence of ovine oviducal amino acid concentrations and an ovine oestrus-associated glycoprotein on development and viability of bovine embryos

This study examined the effects of incorporating an ovine oviducal oestrus-associated glycoprotein (oEGP) and amino acids, at the concentrations present in the ovine oviduct around the time of oestrus, on in vitro production and subsequent viability of bovine embryos. The first experiment compared the influence of ovine oviducal concentrations of amino acids with MEM and BME amino acids. There was no treatment effect on cleavage rate (74.9% vs. 75.5%), but there was a higher (P < 0.05) blastocyst yield (30.4 vs. 25.2) and a shorter time (P < 0.05) to blastocyst formation (7.16 ± 0.64 vs. 7.27 ± 0.56 days) following use of oviducal concentrations of amino acids. Experiment 2 examined the influence of oEGP in combination with each of the amino acid treatments. oEGP had no effect on cleavage or blastocyst yield within amino acid treatments. Day of blastocyst formation significantly influenced nuclei numbers (P < 0.001) with higher numbers being obtained on day 7 than on either day 6 or day 8. There was also a significant (P < 0.01) interaction between day of blastocyst formation and amino acid treatment on blastocyst nuclei numbers. The third experiment studied the effects of the amino acid treatments on embryo viability. There was no effect of amino acid treatment of embryos on pregnancy rates (34.5 vs. 44.4%) following transfer of days 6 and 7 blastocysts to synchronized recipients. oEGP did not influence any of the parameters of bovine embryo development that were measured, suggesting that effects of this protein observed on ovine embryos are species specific. It is concluded that ovine oviducal amino acid concentrations are beneficial to blastocyst development in vitro but do not have any further beneficial effect following transfer of blastocysts to recipients. Mol. Reprod. Dev. 47:164–169, 1997. © 1997 Wiley-Liss, Inc.     

Effects of pre- and post-thaw thermal insults on viability characteristics of cryopreserved bovine semen

The magnitude of damage to the viability of cryopreserved bovine spermatozoa by pre- and post-thaw thermal insults was compared. Semen collected by artificial vagina from 5 Holstein bulls was diluted in egg yolk-citrate-7% glycerol extender (EYCG) and cryopreserved in 0.5 mL French straws at a sperm concentration of 40 to 60 x 10(6) cells/mL. In Experiment 1, straws were subjected to 22, 5 or -18 degrees C static air temperature for a duration of 1, 2, 3, 4 or 5 min before or after thawing in a 37 degrees C water bath for 1 min. Control straws were thawed in a 37 degrees C water bath for 1 min without further thermal insult. In Experiment 2, straws were thawed for 1 min in a 37 (control), 20 or 5 degrees C water bath, or were loaded into an insemination gun and plunged into a 37 degrees C water bath for 3 min. In both experiments, straws were returned to a 37 degrees C water bath for incubation prior to viability analysis. Viability evaluations, conducted in triplicate, included the percentage of motile spermatozoa at 1 min and at 3 h post thermal insult and the percentage of intact acrosomal membranes at 3 h post thermal insult. In both experiments, acrosomal integrity was more sensitive than motility to thermal insult. In Experiment 1, a significant interaction was observed between timing of thermal insult (pre- or post-thaw), static air temperature and duration of straw exposure. At 22 and 5 degrees C, thermal insults applied before thawing significantly (P<0.05) reduced acrosomal integrity at > or = 2 and > or = 4 min of exposure, respectively. However, post-thaw exposure to 22 and 5 degrees C for up to 5 min had no effect on any of the sperm viability parameters evaluated. In contrast, at -18 degrees C static air temperature, post-thaw exposure for > or = 3 min decreased acrosomal integrity (P<0.05), while 5 min of pre-thaw exposure was required for alteration of acrosomal integrity. In Experiment 2, each alternative thawing method resulted in significantly (P<0.05) lower incubated acrosomal integrity relative to the controls. These findings suggest that bovine spermatozoa cryopreserved in EYCG extender are more sensitive to pre-thaw than post-thaw thermal insults and that acrosomal integrity following 3-h incubation at 37 degrees C is superior to motility evaluations for detection of damage to sperm viability due to thermal insult.     

Human leukemia inhibitory factor improves the viability of cultured ovine embryos.

Embryos were collected from ewes on Day 6 after estrus (Day 0 = estrus), placed in M2 culture medium, and assigned to 1 of 4 treatment groups. Some embryos were transferred to recipient ewes on Day 6 of their estrous cycle either in pairs (group 1) or singularly (group 2) within 3 h of collection. The remaining embryos were individually cultured for 48 h in an atmosphere of 5% CO2 in humidified air in either synthetic oviduct fluid (SOF) medium (group 3) or SOF containing 1,000 U/ml of recombinant human leukemia inhibitory factor (hLIF) (SOF + hLIF: group 4). These embryos were then transferred to recipient ewes on Day 8 of their estrous cycle. The addition of hLIF to culture medium significantly improved the development of the embryos compared with control embryos prior to transfer (blastocysts hatching from the zona pellucida: group 3 = 16% vs. group 4 = 64%, p less than 0.05; those degenerative: group 3 = 27% vs. group 4 = 9%, p less than 0.05) and the subsequent pregnancy rates of the recipient ewes, receiving a single embryo, at Day 70 of pregnancy (group 3 = 16% vs. group 4 = 50%, p less than 0.05). The pregnancy rate of ewes given embryos cultured for 48 h in SOF + hLIF prior to transfer (50%; group 4) was similar to the group 2 ewes receiving a single embryo soon after collection (52%), but the pregnancy rate for both groups was significantly lower than that for the group 1 ewes receiving two embryos soon after collection (89%: 53% twins, 36% singles; p less than 0.05).     

Effects of kinetin supplementation on the post-thaw motility,viability, and structural integrity of dog sperm

Oxidative stress is one of the major issues associated with cryopreservation because it causes a marked reduction in the post-thaw quality of semen. This study investigated the ability of kinetin to preserve the structural and functional integrity of dog sperm during cryopreservation. Pooled ejaculates were divided into 5 equal aliquots, diluted with buffer 2 supplemented with different concentrations of kinetin (0, 25, 50, 100, and 200 μM), and finally cryopreserved. The optimal concentration of kinetin was 50 μM based on the significantly improved (P < 0.05) motion characteristics and viability of post-thaw sperm samples. Moreover, kinetin-supplemented samples exhibited significantly higher (P < 0.05) sperm counts with the intact plasma membrane, normal acrosomes, mitochondria, and chromatin than control. The beneficial effects of kinetin were also reflected by the significant increase in the expression levels of anti-apoptotic (B-cell lymphoma, BCL2) and protamine-related genes (protamine 2, PRM2; protamine 3, PRM3), and decrease in the expression of pro-apoptotic (BCL2-associated X, BAX) and mitochondrial reactive oxygen species-modulating genes (ROS modulator 1, ROMO1) in kinetin-supplemented sperm samples than in control. The results demonstrated that supplementation of buffer 2 with 50 μM kinetin is ideal for reducing the magnitude of oxidative damage during semen cryoprocessing and improving the post-thaw quality of dog semen.     

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows?. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.     

Influence of extender, freezing rate, and thawing rate on post-thaw motility, viability and morphology of coyote (Canis latrans) spermatozoa

The objective of this study was to examine the post-thaw effects of three cryoprotective extenders (Tris-fructose-citric acid extender, Tris-glucose-citric acid extender, and lactose extender), three linear freezing rates (-1, -6, and -20 degrees C/min), and three thawing rates (37 degrees C water bath for 120s, 60 degrees C water bath for 30s, and 70 degrees C water bath for 8s) on coyote spermatozoa. After thawing, the findings supported that cryopreservation of coyote (Canis latrans) spermatozoa frozen at a moderate freezing rate (-6 degrees C/min), in either a Tris-fructose or Tris-glucose extender, and thawed at a slow rate (37 degrees C water bath for 120s) or moderate rate (60 degrees C water bath for 30s), resulted in a more vigorous post-thaw motility (range, 57.5-44.0%) and viability (range, 64-49.6%) with the least amount of morphological and acrosomal abnormalities.     

Incubation at 37 degrees C prior to cryopreservation decreases viability of liver slices after cryopreservation by rapid freezing

Precision-cut liver slices are to some extent resistant to ice formation induced by rapid freezing. Susceptibility to rapid freezing damage has been shown to be (partly) dependent on intrinsic properties of cells. In the present study an attempt was made to decrease the susceptibility of rat liver slices for rapid freezing damage: the slices were pre-incubated at 37 degrees C under oxygen, prior to cryopreservation to recover from low ATP levels, impaired ion regulation and cell swelling induced by their preparation. It was shown that, unexpectedly, recovery of cellular homeostasis prior to the cryopreservation procedure by the 37 degrees C pre-incubation markedly decreased viability of rapidly frozen slices (in which ice was formed), but not of vitrified slices (in which no ice was formed), in a time- and temperature-dependent manner. UW was found to protect slices from this 'warm pre-incubation phenomenon.' Apparently, pre-incubation prior to freezing causes certain cellular alterations that render slices more susceptible to rapid freezing damage.