Studies on canine semen preservation

• Main Authors: Huang, Kuo-Yu, 黃國祐
• Other Authors: Fung Hang-Pong
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/63906094954726307436

碩士 === 國立中興大學 === 獸醫學系 === 86 === Semen preservation combined with artificial insemination in dogs have been recently applied as clinical techniques in veterinary medicine. However, as compared to fresh or chilled semen the survival rate of frozen-thawed spermatozoa has been reported to be unsatisfactory due to freezing injury. Individual male has been also found as an important factor on the semen freezing outcomes. The unsatisfactory freezing outcomes largely contribute to the poor fertility of frozen-thawed semen. Freezing outcomes might be improved by involving different components in designed extenders, particularly for certain important males. Therefore, apart from freezing procedures and storage methods recent studies have beenfocused on using different component extenders to achieve satisfactory sperm quality after preservation. Egg yolk that can prevent spermatozoa from cold shock, is the most commonly accepted component for many species. Lately, fetal bovine serum has been found to be beneficial for spermatozoa for its a number of nutrients, growth factors. Use of fetal bovine serum has resulted in satisfactory mice spermatozoal viability after thawing. The aim of this study was to compare the effect of fetal bovine serum and egg yolk as the major component in dog semen extenders during chilled and cryo preservation. Six physiologically healthy dogs with sperm motility more than 70% were used for this study. Sperm rich fractions were collected and divided into 2 aliquots. One aliquot was used to study fetal bovine serum extender and another was used for egg yolk. Spermatozoa in two different extenders were preserved at 4℃ for 7 days and taken daily for sperm quality evaluation. Spermatozoa in two different extenders were chilled and frozen by a programmed freezer and stored in liquid nitrogen for 7 days until sperm quality evaluation. Motility was assessed under a microscope equipped with a heating stage held at 37℃. Viability was evaluated with the combined fluorescent probes SYBR 14 and propidium iodide, while acrosomal integrity was evaluated by fluorescent FITC labeled peanut agglutinin (PNA). Morphology was examined using eosin/nigrosin staining. In addition, spermatozoa obtained from different time points of the freezing process were ultrastructurally examined using scanning and transmission electron microscopy. The results showed that except for sperm morphology, there was a moderate tendency of decrease in sperm motility, viability and acrosomal integrity during 7 days preservation at 4℃. But, no significant difference (P>0.05) was detected between spermatozoa preserved in two extenders for all mentioned parameters. In the occasion of frozen-thawed spermatozoa, a significant decrease (P<0.05) in sperm motility, viability, acrosomal integrity as well as morphology were observed in both extenders. However, no significant difference (P>0.05) was detected between these two extenders. Ultrastructurally, the plasma membrane of spermatozoa subjected to 4℃treatment appeared to be intact whereas after thawing, the spermatozoa presented various degrees of damages such as partial to total deprivation of plasma membrane and the acrosome. In summary, fetal bovine serum provides the similar protection effect as egg yolk does during chilling and cryopreservation of dog spermatozoa. Therefore, fetal bovine serum might be an alternative component for dog semen extenders when egg yolk is not beneficial for certain dogs. Combined use of SYBR 14 and propidium iodide staining can distinguish the live and dead dog spermatozoon at the presence of fetal bovine serum or egg yolk. And, use of FITC-PNA staining can monitor the changes of acrosomal integrity of dog spermatozoa during preservation. Future study should be toward investigation on the fertility of fetal bovine serum preserved dog semen.