A study on the preparation of frozen semen, fertility evaluation and the differential protein expression of sperm before and after freezing in chickens

• Main Authors: Pin-Rong Chen, 陳品蓉
• Other Authors: 黃三元
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/79964511088378311548

碩士 === 國立中興大學 === 動物科學系所 === 100 === Cryopreservation of avian gamete is important for maintaining genomic diversity or preserving genetic resources of elite roosters. Nevertheless, the fertility of spermatozoa decreased significantly after freezing and thawing. The factors affecting the effect of cryopreservation include composition of extenders, rate of cooling to freezing, cryopresentants, and type of frozen semen. The purposes of this study were to compare the effect of different extenders on the semen quality and fertility of frozen semen in chickens, to establish a protein database of chicken sperm, and to analyze the change of sperm proteins before and after freezing-thawing treatment. In experiment I, 40-wk-old F1 crossbred roosters between a chicken line selected for low residual feed consumption and L2 strain Taiwan country chicken and 36-wk old roosters of L2 strain Taiwan country chicken were used. The semen with motility higher than 80% was mixed for frozen semen preparation. The semen was diluted with extenders of Lake, Schramm, and Moce and subjected to frozen semen preparation using dimethylacetamide as cryoprotectant. The sperm motility and viability before freezing and after thawing were recorded. The frozen semen was thawed for artificial insemination and evaluating fertilization rate and hatchability. The result indicated that sperm motility and viability of freezing-thawing semen prepared from the three extenders were significantly lower than those in fresh semen (P<0.05). The fertilization rate of frozen semen prepared by the three extender were significantly lower than that of fresh semen in both F1 crossbred roosters between a chicken line selected for low residual feed consumption and L2 strain Taiwan country chicken and roosters of L2 strain Taiwan country chickens. The hatchability of F1 crossbred roosters between a chicken line selected for low residual feed consumption and L2 strain Taiwan country chicken was not significantly different among treatments (P>0.05). In experiment II, A total of 1 mg sperm protein from 36-wk old L2 strain Taiwan country chicken were used to establish protein profile and protein database of chicken sperm. There were around 500 protein spots detected using Coomassie blue staining. There were 310 protein spots identified by peptide mass fingerprinting and belong to 100 different proteins. Gene ontology annotation revealed that most of the chicken sperm proteins located in mitochondria (27%) and cytosol (14%), participating in cellular metabolic process (34%) and regulation of biological process (11%), and with molecular function of protein binding (19%) and oxidoreductase activity (12%). Results of proteomic analysis of sperm proteins revealed that there were 55 out of 495 quantified protein spots differed significantly before and after freezing-thawing treatment (P<0.05). Among the 55 protein spots, the expression IV of 19 proteins decreased after treatment. The identities of 45 differentially expressed protein spots were identified and belong to 33 unique proteins. Gene ontology analysis revealed that most of the differentially expressed proteins located in mitochondria (31%) and microtubule (18%), involved in cellular metabolism process (28%) and cellular carbohydrate metabolism process (15%), and with molecular function of oxidoreductase activity (19%) and protein binding (18%). The results of this study confirmed that freezing-thawing treatment significantly decreased the motility, viability and fertility of chicken sperm. A protein database of chicken sperm containing about 500 protein spots was established. The differentially expressed proteins before and after freezing-thawing treatment were mostly associated with sperm energy production and structure of flagellum. These proteins may affect intracellular energy production, sperm motility, and acrosome reaction of sperm. Therefore, the differentially expressed proteins may be candidates for improving the fertility of frozen semen of roosters.