發(fā)布時間：2018-07-30 08:50

【摘要】：用液態(tài)稀釋液延長豬精液的保存時間對豬人工授精的推廣及豬優(yōu)良種畜的繁育非常關鍵,但是用稀釋液保存精子的過程中會遇到一些問題:稀釋液中添加成分及添加量的多樣性;精子在保稀釋液中存活時間較短;受精率低等問題。解決精液液態(tài)保存過程中保存效果不佳可從兩個方面出發(fā),一是精子在代謝過程中的能量供應,二是及時清除掉精子在代謝過程中生成的不利物質(zhì)。然而,在豬精液的液態(tài)保存過程中關于射精精子在體外長時間培養(yǎng)期間的糖代謝研究尚少,不同哺乳動物精子PPP途徑是否存在,三羧酸循環(huán)是否必需,并沒有明確的結(jié)論。因此,對豬精液液態(tài)保存過程中精子果糖代謝途徑的研究無論對于豐富精子能量代謝基礎理論,還是對于提高精液液態(tài)保存時間、提高精子活力、擴大豬人工授精的推廣,提供了有利的理論支撐和重要的實踐意義。本試驗以不含任何能量物質(zhì)的磷酸緩沖鹽溶液(PBS)為保存稀釋液,通過添加果糖和糖代謝中間產(chǎn)物以及糖酵解、PPP途徑和電子傳遞的抑制劑,在15℃下液態(tài)保存豬精液不同時間檢測精子活力、活率、頂體完整率、DNA損傷率、抗氧化能力與線粒體膜電位高低的變化等指標,研究豬精子在稀釋液中液態(tài)保存過程中對果糖的利用情況和代謝途徑,并以此為基礎,研制改進精子能量代謝的保存體系,延長豬精液液態(tài)保存時間。結(jié)果發(fā)現(xiàn):(1)在15℃下,在PBS保存稀釋液中添加3mM的果糖對精子的保存最有利,精子活力保持30%以上的保存時間達到5天。(2)在15℃下,在PBS保存稀釋液中添加5mM的丙酮酸對精子的保存最有利,精子活力保持30%以上的保存時間達到5天。(3)在15℃下,在PBS保存稀釋液中添加3mM果糖作為對照組,對比組在此基礎上添加碘醋酸鈉(糖酵解途徑抑制劑),發(fā)現(xiàn)對比組精子活力顯著降低、精子保存時間顯著縮短、精子線粒體功能完整性顯著下降。(4)在15℃下,在PBS保存稀釋液中添加3mM果糖作為對照組,對比組在此基礎上添加6-氨基煙酰胺(磷酸戊糖途徑抑制劑),發(fā)現(xiàn)對比組精子活力與保存時間無顯著變化,但精子的抗氧化能力卻顯著下降。(5)在PBS保存稀釋液中加入3mM果糖作為對照組,對比組在此基礎上添加魚藤酮(電子傳遞鏈的抑制劑),精子活力、保存時間顯著下降通過以上實驗結(jié)果本研究得出以下結(jié)論:1)在15℃下,PBS保存稀釋液中添加3mM的果糖有利于豬精子的存活,添加過量的果糖會減少豬精子的體外保存時間2)豬精子不但可以利用果糖進行代謝,還能利用糖代謝過程中的中間產(chǎn)物用丙酮酸。3)糖酵解途徑是維持豬精子活力、保持線粒體功能不受影響的重要保證。4)磷酸戊糖途徑不影響精子活力,但可增加精子的抗氧化能力。5)有氧氧化途徑有助于豬精子活力的維持。
[Abstract]:Prolonging the preservation time of porcine semen with liquid diluent is very important for the popularization of artificial insemination and breeding of fine breeds of pigs. However, there are some problems in the process of sperm preservation with diluent, such as the diversity of the added components and amount in the diluent, the short survival time of sperm in the diluent, the low fertilization rate, and so on. There are two ways to solve the problem, one is the energy supply in the process of sperm metabolism, the other is to remove the unfavorable substances produced by the sperm in the course of metabolism in time. However, there are few studies on glucose metabolism of ejaculated sperm during the liquid preservation of porcine semen. There is no clear conclusion as to whether PPP pathway exists in different mammalian sperm and whether tricarboxylic acid cycle is necessary. Therefore, the study on the pathway of fructose metabolism in the process of porcine semen liquid preservation is not only for enriching the basic theory of sperm energy metabolism, but also for increasing the time of liquid sperm preservation, improving sperm motility and expanding the popularization of artificial insemination in pigs. It provides favorable theoretical support and important practical significance. In this experiment, the phosphate buffer solution (PBS), which contains no energy substance, was used as the diluent, and the inhibitors of fructose and carbohydrate metabolites, glycolysis and electron transport were added. Sperm motility, acrosome integrity, DNA damage rate, antioxidant capacity and mitochondrial membrane potential were measured at 15 鈩，

本文編號：2154468