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Effect of Time
of Exposure to Different Temperatures The Sperm Chromatin Structure Assay (SCSA) has been used to evaluate the integrity of the DNA of many species. In terms of clinical application, it has been used most extensively to explain the level of fertility in the stallion. Currently, samples are preserved immediately following ejaculation in either the raw or extended form, by placing aliquots immediately under freezing conditions (liquid nitrogen, or non-frost-free type freezers). These samples are then thawed and processed immediately using the protocol for the SCSA. In the United States and European Union countries the use of processed semen (cooled and frozen) is becoming more extensive as more breed registries allow their use. There are many factors that have the potential to negatively effect the quality of the sperm DNA when the spermatozoa is in contact with them for an extended period of time either in the cooled or frozen form. Some of these factors include length of exposure to a particular temperature, extender type, antibiotic type and amount of seminal plasma included in the extended semen. It is therefore, of interest to determine the effect of these potential stresses on the integrity of sperm DNA in the stallion, so that semen processing protocols can be developed to maximize sperm quality. The SCSA is also an important clinical test that is used to estimate the level of fertility expected from a stallion. In the United States, cooled stallion semen tends to be shipped longer distances than in Europe and there is concern about the quality of semen that is used in the shipping process because of the variability in the pregnancy rates. Therefore, it is of interest to determine if the SCSA can be used to evaluate changes in the DNA of cooled semen and whether this test could be of use when evaluating stored semen. There are several questions that must be answered: 1. What is the expected degree of change in the sperm DNA, if any, when stored at temperatures of 5°C, 20°C or 37°C? 2. What is the expected degree of change in the sperm DNA, if any, when stallion sperm is stored at different time intervals at 5°C, assuming the semen is handled in a proper and consistent manner? If there is no change over time under controlled conditions, then the results of clinical samples should either represent the initial intrinsic quality of the stallions DNA or the degree of alteration in the DNA over time due the stresses incurred by the semen sample since collection. 3. Does the sperm DNA from stallions of different fertility levels experience different rates of susceptibility to denaturation when exposed to different temperatures? If stallions of lesser fertility exhibit a higher rate of DNA denaturation than stallions of greater fertility, this would introduce another factor that should be considered when evaluating stallion fertility, especially those stallions considered for use with shipped semen. There have been no studies to evaluate stallion sperm DNA following incubation at different temperatures. The goal of this project was to describe the change in the susceptibility of stallion sperm DNA to denaturation over time when exposed to three different temperatures (5°C, 20°C, and 37°C) and determine whether the rate of change in the DNA is related to fertility status. Materials and Methods One ejaculate from each of 18 stallions was collected in a Missouri Model artificial vagina. The semen was extended and divided into one of three temperature treatment groups (i.e., 5°C, 20°C or 37°C). At certain time intervals (approximately 7, 20, 31, and 46 hours) samples were removed from the designated storage temperature conditions and frozen at -22°C until processing on the flow cytometer. Fertility data was acquired on 9 and 12 stallions for cycles per pregnancy and seasonal pregnancy rate respectively. For analysis, stallions were divided into two fertility groups, fertile and less fertile. Those categorized as less fertile were those presented clinically as stallions that were achieving pregnancy rates deemed unsatisfactory by the owners, whereas those stallions classified as fertile were actively breeding stallions with no history for fertility problems. Sperm samples for the SCSA were processed as previously described (Love and Kenney, 1998). Briefly, individual samples were removed from the freezer and thawed at approximately 35°C. A 5 ml aliquot was combined with 195 ml of a buffered solution (TNE) which was then combined with a low pH (~1.2) acid solution for 30 seconds. The acridine orange solution (1.2 ml at 4.0 micrograms/ml) was then added and the sample was processed on the flow cytometer. The term alpha-t is used to describe the relationship between the amounts of green and red fluorescence and essentially determines the ratio of red fluorescence to the total amount (red plus green fluorescence). Endpoints measured for the SCSA included the mean of alpha-t, standard deviation of alpha-t, and the cells outside the main population (COMP) of alpha-t. A mixed regression model with a two level error term was used. Stallion was treated as a random whole-plot factor to account for the repeated measures over time, of individual stallion-ejaculates and time was treated as a random sub-plot factor. The test statistic used for significant testing was the Likelihood Ratio Chi-square. Results There was a significant effect (p < 0.05) of incubation time, incubation time x temperature, and incubation time x incubation time x temperature interaction for all SCSA measures. These measures increased as incubation time increased, but the extent of rise was affected by the temperature, with those samples stored at 5 °C showing no change, while those at the two higher temperatures showing a rise in all SCSA values. The time x time x temperature interaction indicates that within temperature the slope of the rise tends to be quadratic in nature. A significant difference (p < 0.05) was detected between those stallions classified as fertile and less fertile for all SCSA measures at 5°C and 20°C. There was a significant time x time x fertility interaction for percent COMP at 5°C, but none of the other temperatures. The cycles per pregnancy rates were 1.37 and 5 for the fertile and subfertile stallions; and the seasonal pregnancy rate was 95% (range- 82-100) and 18% (range- 0-35) for the fertile and subfertile stallions, respectively. Discussion The study demonstrates that stallion sperm DNA denatures at different rates depending on the storage temperature. In this study for all stallions, sperm stored at 5°C showed no change in the rate of DNA denaturation up to 46 hours, whereas storage at 37°C showed the greatest rate of denaturation and storage at 20°C showed a more moderate level of denaturation. At 20°C and 37°C the Mean and COMP alpha-t values tended to rise within 7 hours after exposure to those temperatures, with those samples exposed to the higher temperature showing a more dramatic rise. This indicates that stallion sperm stored for 20 hours at 5°C should show no change in the degree of DNA denaturation from the initial collection time if the semen has been processed correctly, whereas storage at 20 and 37°C should be expected to show signs of denaturation. It further suggests that DNA may denature at temperatures less than 20°C as well as when exposed to these higher temperature for shorter time periods. The significant time x time x fertility interaction for the COMPa-t values at 5°C suggests that the curves for the fertile and less fertile groups are different and that these curves are quadratic in nature. When the curves for the fertile and less fertile groups are plotted there is no change in the COMPa-t of the fertile groups over the 46 hour time period, whereas, the less fertile group shows no change after 20 hours, at which time it rises at the 30 hours. Therefore, the sperm DNA of less fertile stallions may denature at a greater rate after 20-30 hours than sperm from more fertile stallions, suggesting an increased “sensitivity” of their sperm to environmental stresses. Therefore, sperm DNA from less fertile stallions, may initially be more susceptible to denaturation, it also has the potential denature further at a greater rate than sperm from more fertile stallions, when exposed to similar environmental conditions. Previously, the SCSA has been correlated with the stallion fertility when fresh frozen has been analyzed. This study shows that it may also be useful for the evaluation of semen that has been cooled to 5°C. It appears that the DNA of semen from all stallions, regardless of fertility status, can be maintained for at least 20 hours in its initial state (i.e., at the time of collection); however, certain stallions that appear to be less fertile may have a more dramatic rise in the rate of DNA denaturation following this time period. This 24-hour time period in critical because this is the approximate interval at which the cooled transported semen of many stallions in the United States is received and inseminated. If a sperm sample exhibits poor spermatozoal quality following cooling, this quality may reflect the quality of semen handling following semen collection, or it may reflect the quality of semen handling following semen collection. The results of the SCSA will reflect the quality of the sperm DNA at the time of insemination. It will therefore be up to the clinician to diagnose the direct cause of the poor quality DNA. References 1. Ballachey BE, Evenson DP, Saacke RG 1988. Sperm chromatin structure assay: relationship with alternate tests of semen quality and heterospermic performance in bulls. Journal of Andrology 9: 109-115. 2. Evenson DP, Jost LK, Marsham D, Zinaman MJ, Clegg E, Purvis K, deAngelis P, Claussen OP 1999. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Human Reproduction 14: 1039-1049. 3. Love CC, Kenney RM 1998. The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. Theriogenology 50: 955-972. Acknowledgments Financial support for this study was provided by the Link Equine Research Fund, Texas A & M University |
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