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Using cryopreservation as a means of conservation of endangered species.

Intro:
Artificial fertilization and sperm cryopreservation are commonly used techniques for propagation of aquatic species in North America and worldwide for a number of purposes. These include breeding of endangered and recreational species to replenish or augment natural populations, production of commercial species for aquaculture, and breeding of laboratory species and genetic lines for biomedical research. It is possible that future work in the cryopreservation field could lead to the restoration of endangered species and anthropogenically damaged habitats.

Cryopreservation is defined as the process of cooling and storing various cells, tissues, or organs at low or freezing temperature in order to use them in the future. The methodology of cryopreservation various depending on the type of material being stored. It takes a large amount of experimentation to determine every parameter of cryopreservation for a certain species. Each type of material requires different temperatures, chemicals, and storage solution to ensure for a successful storage of the material.

Gamete Assessment:
The ability of a scientist to control reproduction is an essential factor to successful artificial fertilization in aquaculture species. The greatest limitation of this control is the ability to determine the quality of the sperm and egg. There are several ways for the quality of gametes to be determined, but the results will vary depending on the species, the tests used, and the scientist that conducts the assessment. These variations require the development of an overall standard to be implemented in all aquatic species. Though there are several ways to assess gamete quality, there are several pros and cons to each assessment.

Examination of the molecular and cellular characteristics of the gametes is considered to be an acceptable method to determine gamete quality. When determining egg quality, the amount of vitamins, proteins, maternal mRNA, and hormones can be used as indicators of the quality of the gamete. Gametes that are lacking a significant concentration of these components can result in gamete developmental mutations. Because of the lack of research conducted on these molecular characteristics, it is not an entirely accurate method of determining gamete quality.

The composition of the sperm plasma membrane can be examined to provide an indication of the quality of the gamete. The plasma membrane detects ionic changes in the solution, allowing for sperm activation in water. This activation is seen by the beating of the flagella and the acquisition of motility. Understanding the molecular components that comprise the plasma membrane is useful in determining the viability of the gamete. Based on the environment that the species is naturally found in, the plasma membrane will vary in composition of cholesterol, lipids, and proteins. Changes in the relative proportion of these components may affect sperm quality and reduce fertilization ability.

Cryopreservation Methods and Applications:
Cryopreservation is defined, according to Tiersch et al., 2007, as a process that uses ultra- low temperatures to preserve biological materials, such as sperm cells, that remain viable after warming to temperatures over the 0 degrees C threshold. Liquid nitrogen is used to lower the sample temperature to -196 degrees C and is used a storage medium. The process of sperm cryopreservation is typically conducted as a series of steps that involve gamete extraction and suspension, gamete quality assessment, addition of cyroprotectants, cryopreservation equilibration, freezing, thawing and fertilization. Each one the steps involved in the cryopreservation procedures has the potential to destroy or damage cellular components. Because cryopreservation involves a series of steps, it is essential that each step is completes with utmost care. Failure to complete a step correctly or the admittance of a step may result in the loss of all viable cells. Fertilization and assessment of early developmental stages of the embryo can be used as a measurement of gamete quality and as a measurement of cryopreservation success. It is also necessary to complete a range of assays of cellular function in order to determine the survival and normality of the cryopreserved sperm after the removal of cryprotectant agents (CPA) from the sample.

Typical cryopreservation procedure includes the use of cryprotectants, which are defined as chemicals that protect the integrity of the cells during the freezing and thawing process. It believed that the use of cryoprotectants lower the freezing point of solution in the cell, prevent the formation of intercellular crystals, and reduce the osmotic shock by replacing the intercellular water. It is important to control the cyroprotectant set dilution and sperm concentration ratio during the cryopreservation process. In the event of too much cyroprotectant, the cells will undergo osmotic swelling, leading to cell rupture during the thawing and dilution, and the overall toxicity effects to the cell. On the opposite end of spectrum, the addition of too little cyroprotectants leads to the formation of intracellular ice.

Cryopreservation Uses in Conservation
Cryopreservation also has potential to become increasingly useful in regards to endangered species, protecting valuable genetic lines, and biomedical research. However, little research has been conducted to promote the usage of cryopreservation in these fields.

Audubon Center for Research of Endangered Species:

The Audubon Center for Research of Endangered Species (ACRES) is currently using cryopreservation procedures to help the critically endangered African black-footed cat. The decline of this species is primarily being caused by the decrease in habitat by conversion to grazing pastures and agriculture fields. There has also been a decrease in their primary food sources, small rodents and ground nesting birds. This species is also facing persecution by local famers, who see the cats as menaces to their crops. There is a current estimate of approximately 10,000 cats in the wild and there are no more than 1,000 individuals found in a population. Currently, there are 40 individuals that are in captivity and are being used in an attempt to increase the population through fertilization processes.

ACRES first started using cryopreservation as a means of conservation of these animals in 1999. A previous frozen embryo was inserted into a surrogate domestic cat. This procedure resulted in the both of a the birth of a single kitten named Jazz. Recently the procedure has slightly changed by using actual black-footed female cats as the surrogate. During 2003 a male African black-footed cat's sperm was collected and cryogenically preserved. This frozen sperm was then combined with eggs that were collected and frozen in 2005. These gametes were then thawed and inserted into a surrogate African black-footed female the falling December. This in vitro procedure produced two male kittens that were able to survive into adulthood and are know able to be used in future procedures to increase the genetic diversity in the species. The ACRES Institute is looking to continue in this project by expanding it to include cloning the African black-footed cats and inserting the embryos into domestic cats as surrogates.

Aquaculture:

There several commercial applications of cryopreservation and it could be used to possibly in the creation of new industry. According to Tiersch et al., 2007, cryopreservation could be used to provide on simpler way to have an on-demand supply of sperm in commercial hatcheries, decrease the number of males kept on the premise allowing for more resources to be used on female and larvae. This method also had the potential to cut back on the amount of money that is spent raising the animals, as well decrease the amount of waste that is produced during the process; an example of this is seen in the channel catfish, Ictalurus punctatus. According to the Department of Wildlife and Fisheries, fifty percent of the cost of raising catfish is consumed by feeding. By implementing the cryopreservation technique, it is possible to decrease the amount of fish needed to be kept on site and still maintain the amount reproduction and fingerlings produced. This also had the potential to decrease that amount of waste that is produced by farm raising these animals.

New Industry:

Currently there is research going on that is using cryopreservation as a means to produce genetically modified animals. The eastern oyster, Crassostrea virginica, naturally is a diploid animal. Through cryopreservation, researchers have been able to produce tetraploid animals, which can be breed with the diploid animals to produce triploid animals. These triploid animals have an extended glycogen stores. Glycogen stores are what gives the oysters its sweet, salty taste that is considered desirable by consumers. This is a method that can be used to extend the harvesting season for the farmers, which is typically decreased in the summer months when the oysters lose their glycogen stores. This methodology is only done in the aquaculture facilities, which has the potential of decreasing the amount of fossil fuels that are consumed during the normal harvesting process because the oysters are kept in tanks nearby.

Conclusion:
Examining the results of the research conducted involving cryopreservation shows that there is a large amount of vacations between the current procedures and excepted results. There are many reasons that could be attributed to the source of the variations. Many of problems, such as technical problems, small sperm volumes, decreased access to the necessary technology, create barriers to the possible expansion in application of cryopreservation. Possibly the largest barrier the future of cryopreservation faces is the lack of standardization involving practices and reporting.

Possible Images: An anatomical structure of a sperm cell- to give an idea of what we are actually talking about deal with A simplified version of the cryopreservation process represented through a flow chart. This could help remove some of the complexity from the procedure. A picture of one of the species being conserved using the cryopreservation process. This might give a "face" to the methods.