A method for freezing bovine lymphocytes

A simple two-stage technique for preserving bovine lymphocytes is described. Lymphocytes from animals chosen at random were used. The experiments indicate that the optimum temperature for freezing and the optimum concentration of dime-thylsulphoxide (DMSO) as cryoprotectant were in the range -29 °C to -31 °C and 17.5 % to 20 % respectively. These concentrations of DMSO are much greater than those reported in most other studies.     

A method for freezing bovine lymphocytes

A simple two-stage technique for preserving bovine lymphocytes is described. Lymphocytes from animals chosen at random were used. The experiments indicate that the optimum temperature for freezing and the optimum concentration of dimethylsulphoxide (DMSO) as cryoprotectant were in the range -29 degrees C to -31 degrees C and 17.5% to 20% respectively. These concentrations of DMSO are much greater than those reported in most other studies.     

Optimisation of freezing conditions for bovine spermatozoa

The use of a programmable forced vapour biological freezer to optimise freezing conditions for bovine semen is described. The results showed that for most bulls, optimisation is possible, including bulls whose semen shows poor survival with conventional freezing methods. The importance of inducing ice crystallisation before any significant degree of supercooling occurs is also demonstrated, as the majority of sperm are lost in this region of the freezing curve. Comparison with static vapour freezing shows enhanced post thaw sperm survival, particularly with bulls whose semen shows poor survival using conventional freezing methods.     

Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports

The aim of this study was to improve the freezing protocol of bull sperm, by investigating the influence on sperm viability after freeze/thawing of different freezing medium components, as well as the effect of cooling rates in the different stages of the cooling protocol, in single factor experiments. The experimental variables were: (1) salt-based versus a sugar-based medium (Tris versus sucrose); (2) glycerol concentration; (3) detergent (Equex) concentration; (4) presence of bicarbonate; (5) rate of cooling from 22 degrees C to holding temperature (CR1); (6) holding temperature (HT); (7) rate of cooling from holding temperature to -6 degrees C (CR2); (8) rate of cooling from -10 to -100 degrees C (CR3). All experiments were performed using five bulls per experiment (three ejaculates per bull). Sperm motility after freezing and thawing was assessed by CASA system, and sperm membrane integrity was assessed by flow cytometry. Sucrose-based medium did not offer a clear significant benefit compared to Tris medium. The concentration of Equex that gave the best results in Tris-based media group and sucrose-based media group was in a range between 2-7 and 4-7 g/l, respectively. In both media groups, a glycerol concentration of 800 mM was the best in any post-thaw viability parameters. In the Tris media group, the presence of bicarbonate had a negative effect on sperm viability. CR1 and CR2 had no significant effect on any of the post-thaw sperm viability parameters, but a CR1=0.2 degrees C/min and CR2=4 degrees C/min appeared to give better results in both media. The holding temperature (HT) that gave the best results was found to be in the range of 5-9 degrees C. There was a significant disadvantage of using a low CR3 of 10 degrees C/min, while 150 degrees C/min appeared to be the best cooling rate for either medium.     

High-presssure shift freezing. Part 2. Modeling of freezing times for a finite cylindrical model

A comprehensive vision of the heat transfer process involved in high-pressure shift freezing (HPSF) is shown in comparison to the process at atmospheric pressure. In addition, a mathematical model to predict the freezing times is presented. This model takes into consideration the dependence of the thermophysical properties relating to temperature and pressure and the supercooling reached by liquid water at atmospheric pressure after adiabatic expansion in the HPSF process. Experimental and theoretical data appear to agree.     

桂花抗冻种质的筛选及抗冻机理初探

采用田间调查及人工模拟冰冻鉴定的方法,比较测定了4个桂花品种的抗冻性,其抗冻性强弱顺序为金桂（Osmanthusfragransvar．thunbergii）＞银桂（O．fragransvar.latifolius）＞丹桂（O．fragransvar．aurantiacus）＞四季桂（O．fragransvar.semperflorens）。筛选出5株抗冻性最强的单株,它们的致死温度都在－26℃左右。桂花叶片膜脂脂肪酸组成随季节呈周期性变化,随着气温下降亚麻酸（18∶3）含量增加而亚油酸（18∶2）含量降低。不同品种均表现出一致的变化趋势,抗性强的品种这种变化则更为显著。在自然脱锻炼过程中,叶面喷施脱落酸（200ppm）能使亚麻酸含量保持稳定,延缓桂花的脱锻炼。     

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.     

A method for one-step freezing of mouse embryos

A simple one-step method of freezing mouse embryos directly in liquid nitrogen is described. The main objective of this study was to assess post-thaw survival following predehydration in various mixtures of glycerol and sucrose. Also investigated was pretreatment with glycerol prior to dehydration and effects of embryo stage. When sucrose was held constant (0.25 M) and glycerol concentration varied (1.0-4.0 M), post-thaw survival was best (67%) in 2.0 M glycerol. Pretreatment in glycerol provided no advantage over no pretreatment. When glycerol was held constant (2.0 M) and sucrose concentration varied (0-1.0 M), optimum post-thaw survival (81%) was found in 0.5 M sucrose. Morulae survived better than blastocysts (86% vs 72%, respectively). Transfer of thawed embryos frozen by the optimum treatment (2.0 M glycerol + 0.5 M sucrose) resulted in a birthrate of 41%, compared to 54% for fresh controls. This technique could find application in freezing and thawing of livestock embryos on the farm.     

Evolution of freezing susceptibility and freezing tolerance in terrestrial arthropods

Arthropods have evolved various adaptations to survive adverse seasons and it has long been discussed why some arthropods are freezing-susceptible and some are freezing-tolerant. However, which mode of frost resistance came first during the course of evolution? A commonly held opinion is that no choice of strategy has been offered in evolution, because each species of arthropod may have its own evolutionary and natural history, leading to cold-hardiness. Freezing tolerance is more frequent in holometabolous insect orders and partially used by certain vertebrates, like some terrestrially hibernating amphibians and reptiles. Supported by phylogenetic, ontogenetic and ecological arguments, we suggest here that freezing tolerance is more recent than freezing susceptibility in the course of arthropods evolution. In addition, we observe that three basic modes of freezing resistance in insect species exist in the field: (i) permanent or year-round freezing-susceptible species, (ii) alternative or seasonal freezing-susceptible/freezing-tolerant species, (iii) permanent or year-round freezing tolerant species.