User:Nmills1/Box ol' sand

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. By testing a small representative sample of the sperm population, it possible to gain an indication of overall gamete quality in the sample, despite which parameter is measured. As a result of measuring gamete quality, the rate of successful fertilization could possibly be increased or accurately predicted, even if a relatively small population of sperm quality is maintained. It is difficult to determine which parameter should be used to test gamete quality because of the vast number of cellular processes that quality is dependent upon. However, by testing individual parameters and adjusting the seminal environment, the quality of the sperm can be maintained and successful fertilization can be achieved (Lahnsteiner et al., 1998). By obtaining an accurate indication of gamete quality, it is possible to select gamete samples that could result in successful fertilization, or give the ability to predict the likely outcome of fertilization or cryopreservation.

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 (Tiersch, 2000). 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 (Alvai et al., 2006). Gametes that are lacking a significant concentration of these components can result in gamete developmental mutations (Alvai et al., 2006). 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 (Tubbs and Thomas, 2008). 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 (Labbe and Maisse, 2001).

Cryopreservation Methods and Applications:

Cryopreservation is defined, according to Tiersch et al., 2006, 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 (Tiersch, 2000). 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 (Leibo, 2003). 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 (Tiersch et al., 2006). 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 (Leibo, 2003). 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 (Tiersch et al., 2006). 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 (Taylor, Adams, Boardman and Wallis, 1974). There several commercial applications of cryopreservation and it could be used to possibly in the creation of new industry. According to Tiersch et al., 2006, 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. Cryopreservation also has potential to become increasingly useful in regards to endangered species, protecting valuable genetic lines, and biomedical research (Tiersch et al., 2006). However, little research has been conducted to promote the usage of cryopreservation in these fields (Tiersch, 2001). 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 (Tiersch and Mazik, 2000). Possibly the largest barrier the future of cryopreservation faces is the lack of standardization involving practices and reporting (Tiersch et al., 2006).