雞冷凍精液製備、繁殖力評估及冷凍前後精子蛋白質差異表現之研究

Abstract

精子冷凍保存在維持生物多樣性或保留性能優異種公雞種源扮演重要角色，然而，經冷凍解凍處理後精子的繁殖力會顯著降低。影響精子冷凍保存效果的因素包括稀釋液組成、冷凍降溫速率、抗凍劑種類、冷凍保存包裝型態等。本研究之目的為比較不同冷凍稀釋液製備雞冷凍精液之效果、初步建立雞精子蛋白質資料庫並分析公雞精子冷凍解凍後蛋白質表現量的改變，作為改善冷凍精液繁殖力的基礎。試驗一使用用36週齡L2品系台灣土雞及40週齡L2品系台灣土雞與選拔採食殘差品系之雜交公雞，取精子活力高於80%之精液混合後，以Lake、Schramm與Moce等三種稀釋液搭配dimethylacetamide為抗凍劑製備顆粒狀冷凍精液，記錄冷凍前後之精子活力與精子存活率，並將冷凍保存之精液解凍進行人工授精，評估其繁殖力。試驗結果顯示，L2品系台灣土雞與選拔採食殘差品系之雜交公雞精液經三種稀釋液製備冷凍精液之解凍後精子活力與存活率均顯著低於新鮮精液(P<0.05)，L2品系台灣土雞公雞精液則只有Moce稀釋液製備冷凍精液解凍後之精子活力與新鮮精液無顯著差異；冷凍精液解凍後進行人工授精的結果顯示，L2品系台灣土雞與選拔採食殘差品系之雜交公雞及L2品系台灣土雞精液以三種稀釋液所製備冷凍精液之受精率皆低於新鮮精液，L2品系台灣土雞與選拔採食殘差品系之雜交公雞冷凍精液的孵化率在處理間無顯著差異。試驗二使用36週齡L2品系台灣土雞新鮮精液建立雞精子蛋白質資料庫及比較L2品系台灣土雞精子冷凍解凍前後蛋白質之表現。取1 mg總蛋白質進行雙向電泳分析，電泳膠片以colloidal Coomassie blue染色可偵測到約500個蛋白質點，目前已成功鑑定310個蛋白質點之身份，分別屬於100個不同的蛋白質，經Gene Ontology分析後可知，此等蛋白質主要分佈於粒線體(27%)與細胞質液(14%)、參與細胞代謝過程(34%)與生物過程調節(12%)及具有蛋白質結合(19%)與氧化還原酶活性(12%)等分子功能。以蛋白質體學方法比較36週齡L2品系台灣土雞精子冷凍解凍前後蛋白質之表現差異之結果顯示，在所有定量的495個蛋白質點中有55個蛋白質點之表現量在冷凍前與解凍後精子間具顯著差異(P<0.05)，其中19蛋白質點在冷凍解凍後表現量降低，胜肽質量指紋分析成功鑑定其中45個蛋白質點之身份，分別屬於33個不同的蛋白質。經Gene Ontology分析後可知，此等蛋白質主要分佈於粒線體(31%)與微管(18%)、參與細胞代謝(28%)與細胞碳水化合物代謝(15%)等生物過程以及具有氧化還原酶活性(19%)與蛋白質結合(18%)等分子功能。本研究之結果確認冷凍解凍後雞精子活力、存活力與受精率均顯著下降，初步建立公雞精子蛋白質資料庫，而冷凍解凍後差異表現之精子蛋白質多與精子產能、鞭毛結構等有關，可能影響精子內部產能、活力與頭帽反應能力等，因此，這些差異表現蛋白質將可作為改善冷凍精液繁殖力之參考指標。

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.