ILAR 41 (4)

Inroduction. ILAR 41 (4): 183.
[Reviewer's Note - Any parts of this introductory article which recapped specific articles published in this ILAR issue were not reviewed as other LABSG members will be summarizing these articles for the LABSG list.] The increasing numbers of new laboratory animal genotypes brings with it an increase in new phenotypes. The scientific community is creating numerous new animal models but is lagging behind in comprehensively characterizing these models, in part due to the resource-intensive nature of comprehensive phenotyping. The first genome resource banks (GRBs) were established for domestic animal genetic management programs following the development of methods to cryopreserve domestic bull spermatozoa. Breeding programs for dairy and beef cattle by artificial insemination (AI) exploit this technology. During the early 1970s, Peter Mazur established mammalian embryo cryopreservation at Oak Ridge National Laboratory for mouse embryos. Live births from many species using frozen-thawed embryos have since been reported. GRBs also store additional cells and tissues such as ovarian tissue, blood cells, and skin biopsies, which can be used for rederivation, infectious pathogen screening, and somatic cell nuclear transfer, respectively. Currently, the USDA has established a National Animal Germplasm program to identify and preserve important genetic resources from all of the major domestic animal agriculture species. Conservation biologists have developed similar strategies to preserve genetic resources from rare and endangered populations. Two categories of approaches in this context have been taken to protect and preserve the Earth's biodiversity: (1) in situ programs, which involve preservation or restoration of large areas of protected habitat; and (2) ex situ programs, which commonly involve zoos, botanical gardens, aquaria, or private farms for the preservation of populations outside of their natural habitat. The development of GRBs for ex situ preservation of biodiversity has also been evolving under the umbrella of the Conservation Breeding Specialist Group of the International Union for the Conservation of Nature and Natural Resources' Species Survival Commission. Table 1 (p.184) summarizes the factors to be considered in developing a GRB for laboratory animals. Such factors include: summary; justification; current knowledge of life history and reproduction; current knowledge of assisted reproduction; status of the model; accessibility of existing animals for banking; type and amount of germplasm to be preserved; technical germplasm collection, processing and storage; use and distribution; ownership; resources and funding. Another area in which cryopreservation of reproductive cells and tissues are commonly used is human reproductive medicine. Human assisted reproductive technology (ART) laboratories routinely process and store human spermatozoa and embryos as part of the treatment of infertility. With respect to animals, infrastructural resource limitations necessitate selection of some populations and the de facto exclusion of others. In the context of laboratory animal research GRBs, processes for selecting specific stocks, strains, and lines of animals to be maintained and preserved must be predicted on meeting the research needs of the biomedical research community generally as well as individual PIs specifically. Development of these selection processes requires evaluation of many factors, including usefulness of the model, availability of both animals and effective methods for ART, and cryopreservation of reproductive cells and tissues.
1. Which of the following groups are frequently used as donor females for zygote production:
a. FVB, BALB/c, C57BL/6
b. C57BL/6, FVB, CD-1
c. C57BL/6, FVB, 129/Sv
d. FVB, C57BL/6, ICR
e. All of the above
2. _________ provides long-term storage and preservation of a transgenic line in case of disease outbreaks, accidents, and the cessation or alteration of genetic expression. Preimplantation embryos (2 to 8 cell stage) are usually frozen.
3. Describe the following nomenclature for this animal: C57BL/6-TgN(APOA1-2)1Rub
1. C.
2. Cryopreservation.
3. C57BL/6 (background), Tg (transgenic animal), N (mode of insertion, nonhomologous), (APOA1-2) = transgene, 1 ( # assigned by lab), Rub (lab code assigned by ILAR)

 Mechanisms of Cryoinjury in Living Cells. ILAR 41 (4): 187.
Cryobiology is the study of the physical and biological behaviors of cells and tissues (including their interactions with environment) at low temperatures. The major steps in cryopreservation can be summarized as follows:
1. Add cryoprotective agents (CPA) to cells/tissues before cooling.
2. Cool the cells/tissues toward a low temperature (eg., at -196 celsius, the liquid nitrogen temperature at pressure of 1 atm) at which the cells/tissues are stored.
3. Warm the cells/tissues.
4. Remove the CPAs from the cells/tissues after thawing.
Contrary to popular belief, the challenge to cells during crypreservation is not their ability to endure storage at low temperature; rather, it is the lethality of an intermediate zone of temperature (-15 to -60 degrees celsius) that cells must traverse TWICE - once during cooling and once during warming. The mechanisms underlying cellular damage differ between rates of freezing. In a two-factor hypothesis, Mazur et al. offer that at slow cooling rates, the cryoinjury occurs due to solution effects (i.e., the solute/electrolyte concentration, severe cell dehydration, and reduction of unfrozen fraction in the extracellular space). At high cooling rates, cryoinjury occurs due to lethal intracellular ice formation. Whether a given cooling rate is too high or low for a given cell type depends on the ability of water to move across the cell membrane. The cellular response to warming is highly dependent on the freezing conditions and cell type. Slow warming has been reported to be optimal for samples frozen slowly; however, the contrary is not true probably because the cells were killed by intracellular ice formation during fast cooling.
Although the avoidance of intracellular ice formation is necessary for cell survival, most cells also require the presence of CPAs. Glycerol remains one of the more effective and commonly used CPAs. Others include DMSO, ethylene glycol, methanol, propylene glycol, and dimethylacetamide. These harbor the ability to permeate the cell and decrease the concentration of electrolytes during freezing and decrease the extent of osmotic shrinkage at a given low temperature. A second class of CPAs is composed of nonpermeating solutes such as sugars and higher molecular weight compounds such as polyvinylpyrrolidone, hydroxyethyl starch, polyethylene glycols, and dextrans. These agents augment the effectiveness of a permeating CPA or permit the use of lower concentrations of permeating CPAs. Survival of the cell may require not only the presence of a CPA but also an optimal concentration that is non-toxic.
Progress in understanding mechanisms of injury to cells during cryopreservation has been furthered by physical modeling and mathematical formulations simulating a cell's response to environmental change during cryopreservation. These will aid in predicting optimal cryopreservation conditions. Other areas of study in cryobiology include mechanisms and modeling of cryoinjury and cryoprotection to multicellular systems (tissues/organs) and mechanisms of new potential CPAs.
1. Cellular damage during cryopreservation most often occurs during
a. cell storage
b. cell cooling
c. cell warming
d. b and c
e. all of the above
2. At _____ cooling rates, cryoinjury is due to solute effects, while at
_____ cooling rates, cryoinjury occurs due to lethal intracellular ice formation.
a. medium/fast
b. fast/slow
c. slow/fast
d. slow/medium
3. All CPAs are always beneficial during the cryopreservation process (T/F)?
3.False; high concentrations of CPAs can be toxic and some cryoprotectants are toxic to certain cell types (bull/boar/human sperm are damaged by glycerol and oocytes are damaged by DMSO and 1,2-propanediol)

 Cryopreservation of murine spermatozoa. ILAR 41 (4): 197.
Successful freezing of any cells requires that the critical cooling rate be slow enough to prevent intracellular ice formation and fast enough to prevent being damaged by the solution. The cooling rate can be calculated mathematically if one knows the permeability properties of the cells, the surface:volume ratio, and their tolerance to osmotic solutions. The cryobiological factors of spermatozoa appear to vary greatly among species and among mouse strains. Mouse and boar sperm appear to have "exquisitely narrow" osmotic limits, while human sperm is more tolerant. Osmotic limits can be widened by the use of cryoprotective solutions based on glycerol, DMSO, ethylene glycol, egg yolk or skim milk. In fact, the authors claim that the vast majority of research over the last 50 years has been on these extenders rather than on the basic cryobiology of cell types, and that knowledge of spermatozoal cryobiology lags far behind that of other cell types. Relatively successful freezing of mouse spermatozoa can be achieved without using cryoprotectants at all.
Differences between murine sperm and that of other species may be based in part upon the lipid content of the cell membrane. One reason mouse sperm may be so sensitive to freezing damage is that the cytoskeleton anchors the plasma membrane to the internal structure of the cell. Mouse spermatozoa also appear to be very easily damaged by routine mechanical factors such as mixing, centrifugation and pipetting.
The success of cryopreservation can be measured in many ways (such as sperm viability, sperm motility, sperm fertility in vitro, and live births), and there is not a linear relationship among these measurements. For example, although the sperm plasma membrane may remain intact in various osmotic concentrations, motility seems to be much more sensitive to osmotic changes. Strain differences are evident: for example, in vitro fertilization of B6C3F1 oocytes was successful in 61% if B6C3F1 sperm were used, 17% if 129/J sperm were used, and only 3% if C57BL/6J sperm were used. In general, spermatozoa from hybrids can more successfully be frozen than spermatozoa of inbred mice.
There has been even less research on rat spermatozoa, but unpublished work from these authors suggests that there are similarities between mouse and rat freezing success.
Two special techniques were mentioned that are not yet in common use. The first is a method of cell vitrification in which the freezing rate is so rapid that intracellular "glass" is formed rather than ice, and warming is rapid enough so that the cytoplasm remains vitreous. This has been used in mouse and Drosophila embryos, but not sperm. The second is the use of intracytoplasmic sperm injection, a special technique that can employ even freeze-dried or non-viable sperm. This has successfully produced live mice in one laboratory at the University of Hawaii.
1. Common methods used to cryopreserve rat spermatozoa include:
a. cell vitrification by rapid supercooling
b. addition of cryoprotectant such as glycerol followed by cooling to -196C
c. freeze-drying followed by intracytoplasmic injection
d. none-- they have not been successfully cryopreserved
2. Which of the following cryobiological factors must be known in order to mathematically predict the success of cryopreservation of a cell?
a. membrane permeability for water, surface:volume ratio, nuclear:cytoplasmic ratio
b. activation energy, nucleation temperature, percent lipid content
c. membrane permeability for water, activation energy, surface:volume ratio
d. activation energy, nucleation temperature, nuclear:cytoplasmic ratio
1. d
2. c (this type of question is a common format)

 Cryopreservation of murine oocyte and ovarian tissue. ILAR 41 (4): 207.
This review discusses the developing technology of cryopreservation of oocytes and ovarian tissue. This technology has been developing since the 1980's. Methods developed for oocyte and ovarian tissue cryo preservation must protect structural and functional viability. Strategies have been employed to preserve an immature oocyte( isolated from the ovary) or a mature oocyte
(after ovulation).
The mammalian ovary is divided into two regions, the outer cortex and inner medulla.
For cryopreservation the cortex is more important because it contains the primordial follicles. The oocyte is the largest
( 80-120 mm diameter) single mammalian cell. Immature oocytes are present in the antral follicle of ovaries and arrested in the prophase of the first meiotic division . These cells have a distinct large prophase nucleus called the germinal vesicle (GV1). After gonadotropin stimulation, the GV breaks down, a metaphase plate forms, and the oocyte is arrested at the metaphase of the second meiotic division (MII1) characterized by the presence of the first polar body Fundamental changes in the structure and function of oocytes occur as they develop from the GV stage (in which the chromosomes are well protected in their condensed form) to the MII stage (when the chromosomes and connected spindle fibers exist freely in the cytoplasm). The other important components are the cytoskeletal elements (microtubules and microfilaments) and cortical granule vesicles in the cytoplasm.
The steps in cryopreservation are as follows, initial exposure of cells or tissue to cryoprotective agents( CPAs), cooling to subzero temperatures, storage, thawing, and removal of CPA with return to normal environment allowing further development.
Two types of damage can occur to biological materials during the freezing process. 1. intracellular ice formation which occurs when tissue is cooled " too quickly", and water is trapped inside the cells. 2. damage due to high-solute concentrations when water precipitates as ice. This happens when the tissues are cooled " too slowly"
The three types of cooling which have produced viable ova are quasiequilibrium ( rapid cooling ,`~200degrees C/min),
non-equilibrium freezing( ultrarapid cooling, at ~2500 degrees C/ min and equilibrium freezing. Object is to produce vitrification
of the tissue, which is when an aqueous solution transitions from liquid to solid, but bypasses the crystalline solid state.
Ovarian tissue cryopreservation was first attempted in the 1950's. Glycerol was the only cryoprotectant used. In the 1990s other
CPAs such as Dimethyl sulfoxide( DMSO), ethylene glycol( EG) and propylene glycol (PG) were introduced. At the present time DMSO is the most commonly used cryoprotectant. In general across all mammalian species it appears that the slow cooling procedures appear to be the optimal method to cryopreserve ovarian tissue.
In humans there is concern that if a cancer patient saves ovarian tissue, and the patient is treated successfully for cancer that the subsequent autografting of ovarian tissue could result in reintroduction of the tumor.
Most of the cryopreservation work has been done in rodents, particularly mice. It has been shown that both adult and fetal cryopreserved ovaries can be used to restore fertility in mice. Reports show that cryopreservation of ovaries/ova is a very useful procedure for banking mouse genomes, particularly mouse strains with a low fertility rate.
The ability to bank primordial follicles has two advantages, there are thousands of potentially usable oocytes in prepubertal animals most of which will not be used, and cryopreserved primordial follicles may be easier than cryopreserving oocytes, The follicles are smaller in size , are arrested in prophase meiosis, and do not have clear zona pellucida, cortical granules, and spindle fibers all which are reportedly to be sensitive to cryopreservation.
To determine whether cryopreserved ova and ovarian tissue are viable it can be transplanted into a host animal, usually an immunodeficient mice. The two types of mice used most often are the athymic-nude mice( nu/nu) and the SCID mouse.
The paper reports the results of many studies to determine what the best CPA is, as well as comparing
different concentrations of CPAs, and comparing types of cooling methods.
Whole ovaries from mice and rats can be frozen due to their small size, this is much more difficult in larger species.
In 1977 Whittingham published the first results of live-birth from frozen-thawed mouse oocytes. Few attempts have been made to cryopreserve rat oocytes.
A.Name the two regions of the rodent ovary.
1. ooplasm and zona pellucida
2. outer cortex and inner medulla
3. corpus lutea, primordial follicles
4. oolema, and oocytes
B.What is the range of approximate diameter of a mammalian oocyte?
1. 20-80 mm
2. 90-110 mm
3. 80-120 mm
4. 60-120 mm
C. During freezing of tissue if intracellular ice formation occurs it is due to...
1. cooling the sample " too slowly"
2. cooling the sample" too quickly"
3. using the wrong concentration of CPA
4. using glycerol instead of DMSO
D. What is the most commonly used cryoprotectant, according to this paper?
1. PEG
2. glycerol
3. propylene glycol
E. What mice have been used as recipients for thawed ova/ovarian tissue?
1. Balb/C
2 nu/+
4. C57/B6
A.)2, B.)3 C.)2 D.)4 E.)3.

 Factors affecting the efficiency of embryo cryopreservation and rederivation of rat and mouse models. ILAR 41 (4): 221.
With the explosion in the number of rat and mouse strains which serve as models of human disease, the ability to cryopreserve embryos becomes critically important by allowing the preservation of valuable strains without the expense of maintaining continuous breeding colonies. However, not all genotypes are created equal and it is important to know how many embryos must be preserved (banked) to guarantee rederivation and how many embryo donors are required to obtain that number of embryos.
For a model strain to be considered 'banked'. four conditions must be met:
1. 500 embryos carrying the gene of interest are cryopreserved
2. Pups are rederived from these banked embryos
3. Rederived pups exhibit the desired genotype
4. Rederived pups breed and produce pups
The determination of the need for 500 embryos was based on previous reports of an overall rederivation efficiency of not less than 10%. This would yield approximately 50 pups. Utilizing 5 to 10 pups per rederivation, a particular genotype could be reestablished 5 to 10 times.
The authors looked at 111 rat models and 57 mouse models to see how genotype affected the number of embryo donors required to get 500 embryos. Standard embryo collection techniques were utilized. Embryos judged morphologically normal were selected for crypreservation.
Rats: varied from 49 to 612 embryo donors to obtain 500 embryos. Median = 83 (6/rat)
Mice: varied from 23 to 256 embryo donors to obtain 500 embryos. Median = 70 (7/mouse)
In both rats and mice, the genotype of donor females was found to affect the efficiency of embryo collection in 2 ways:
1. Variability in the proportion of females yielding embryos
Rats: 16 - 100%
Mice: 24 - 95%
2. Variability in the number of embryos recovered from each female
Rats: 4 - 10.6
Mice 5.3 - 32.2
This variability could reflect differences in the efficiency of estrus cycle synchronization (rats) or efficiency of superovulation (mice), male and female infertility, behavioral incompatibilities, or anatomical abnormalities.
The second step was to observe how genotype affected the efficiency of rederivation. To do this, embryos were thawed, transferred to recipient females, and the number of normal pups born counted. In rats, efficiency of rederivation ranged from 10 to 58%, with an average of 30%. In mice, efficiency of rederivation ranged form 11 to 53%, with an average of 33%. These results, showing the lowest efficiency to be 10 -11% for rat and mouse rederivation, agree with previously published values and support an embryo bank size of 500 embryos.
1. How was the genotype of the donor female found to affect efficiency of embryo collection?
2. How is the efficiency of rederivation best measured?
1. Variability in the proportion of females producing embryos
Variability in the number of embryos produced per female
2. Total number of normal pups born divided by the number of thawed embryos = efficiency of rederivation

 Genome resource banking for wildlife research, management, and conservation. ILAR 41 (4): 228.
Cryobiology can offer an opportunity to assist in the management and study of wildlife and endagnered species. In addition to preserving heterozygosity, genome resource banks(GRBs) can assist in genentic management of rare species held in captivity, frozen repositories to help preserve wild population against natural and man made catastrophes.
3 reasons for conserving biodiversity are:
1. biodiversity is the earth's life support system.
2. biodiversity provides economic resources such as food, chemicals,fiber, clothings, structural materials, energy, recreation and medicine.
3. man has a moral responsibility to protect/preserve wild populations
Advantages of GRBs
1. preserves genetic diversity within a species
2. valuable genetic resource composed of many biologicals such as germplasm, embryos, blood products, tissue and DNA
3. may probive economic opportunity for improving quality of life
4. Allows easy movement of genetic material
5. Increased efficiency in captive breeding programs
6. Reduces genetic problems by helping to prevent inbreeding
7. Fewer space problems by preserving species through cyropreservation
Priorities for Achieving Effective GRB's for Wildlife
1, Advocacy and support for developing and maintaining GRBs
2. Emphasis on Basic Research in wildlife species
3. Cooperation and sharing between countires
4. Genesis of individual banks
1. What organization has taken the lead in working towards the development of GRBs for wildlife?
a. Cites
b. World Conservation organization for species survival
c. Conservation Breeding specialist group
d. U.S. Fish and Wildlife Service
2. A GRB should preserve which of the following
a. germplasm
b. blood products
c. DNA
d. all of the above
e. none of the above
1. c 2. d