收获的小鼠精子和人工授精

Published 4/28/2007
15 Comments
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Biology

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Summary

此协议表明,从男性的睾丸中提取精子,然后inseminating雌性小鼠的方法。此过程需要精确的时间是在发育的研究,以及转基因工作时非常有用。

Cite this Article

Copy Citation

Duselis, A. R., Vrana, P. B. Harvesting Sperm and Artificial Insemination of Mice. J. Vis. Exp. (3), e184, doi:10.3791/184 (2007).

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Abstract

鼠类属Peromyscus(鹿鼠)是最普遍的原生北美的哺乳动物。 Peromyscus物种的研究范围广泛,包括毒物学,流行病学,生态,行为,和遗传研究。在这里,他们提供了一个有用的模型示范人工授精。

此处显示的方法类似先前已被用于一些鹿小鼠研究,但没有详细的协议已经出版。在这里,我们展示了人工受精的基本方法。这种方法需要从啮齿类动物中提取的睾丸,然后隔离精子从附睾和输精管的。在排卵期,精子的成熟,现在在一个牛奶混合后,放置在女性生殖道。施肥是算作0天的胚胎发育的时间。胚胎可以在需要的时间点检索和操纵。

人工授精可用于在各种啮齿类动物是至关重要的,或难以取得确切的胚胎时机。这种技术是重要的物种或品系(包括最Peromyscus)可能无法立即交配和/或交配是很难评估。此外,人工受精胚胎发育的确切时间,无论是在映射发展进步和/或转基因工作。因为施肥是保证,可用于动物数量减少。这种方法已经进一步Peromyscus系统至关重要,以及将有望使他人受益。

Protocol

人工授精

人工授精代替自然交配,以增加成功的机会,所有的女性会怀孕。用于每1超排的女性或在发情已知一名女2男。 caudae附睾成熟的雄性小鼠被放置在了9%的牛奶悬挂和切片18表针开放。精子被允许自由游泳几分钟,然后是附睾从混合物。悬架被评估为合流和活动精子的显微镜。钝的22号1 ½英寸长针是用来管理精子。 120 °弯曲放置约3 / 4 ,上下针方式。弯曲和0.025 - 0.05mls注射针插入阴道。如果一个助理是用来装的女性,在适当的位置,不需要使用麻醉药。

制备

  1. 使用女6男4例
  2. 安乐死在男性的动植物公园
  3. 准备从9%的牛奶约5毫升奶粉
  4. 在小培养皿中放入1.5毫升
  5. 剪出epididymus和输精管
  6. 9%的牛奶在1.5毫升
  7. 使用两个18表针,并使用针长切割运动,切片打开epididymus和输精管释放精子
  8. 留过组织在显微镜下检查精子

注塑

  1. 使用120 °角的钝针
  2. 拿在手里的鼠标和注入的混合物。如果一个人持有和注射感到不舒服,两个人都可以使用。没有麻醉剂的使用,应在一个知道如何这可能会影响程序。
  3. 使用每个女性的精子/牛奶混合0.05毫升

收件人女性

  1. 对受援国的女性阴道涂片(此时收件人女性应在发情前期/发情)
  2. 都没有达到第5天发情女性可能不应该使用

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Acknowledgements

我想感谢迈克杜威从他宝贵的见解Peromyscus遗传股票中心,耐心,和啮齿类动物繁殖知识。

Materials

Name Type Company Catalog Number Comments
Petri Dish Tool Fisher Scientific 08-772-30
18 gauge needle Tool BD Biosciences 305195
Powdered milk Reagent Any Supplier
1 ml syringe Tool National Scientific Company S7510-1
Forceps Tool Roboz Surgical Instruments Co. RS-5060
Scissors Tool Roboz Surgical Instruments Co. RS-5882
Dulbecco’s Phosphate Buffered Saline Reagent GIBCO, by Life Technologies 14200-075
22 gauge blunt needle Tool Give to me

DOWNLOAD MATERIALS LIST

Comments

15 Comments

  1. can you tell me the resolution power of the microscope at which you have shown the video and the make of the mcroscope used.

    Reply
    Posted by: Anonymous
    September 17, 2007 - 12:56 AM
  2. The total magnification to do the AI is between 10-²0x depending on the user for basic dissection. The sperm can be seen at 400x

    Reply
    Posted by: Anonymous
    November 1, 2007 - 2:09 PM
  3. Hello Amanda!  Thank for this article. I d like if you can give me more information about the powered milk, and if there is any posibility of create "make in home" powered milk. Too , if you can please give me the aprox. milimeters that need introduce the nedlle into the vagina. You understand my english? Thank you. Carolina (from Buenos Aires, Argentina)

    Reply
    Posted by: Anonymous
    August 29, 2008 - 9:09 AM
  4. Hi Carolina, The milk is a commercial powdered milk bought from a regular grocery store. I am not sure what it contains if you wanted to make it at home. A little less than 1 cm of the needle was inserted into the vagina for sperm delivery but if you want to test it...I would use a dead mouse to check the insertion length when making a new needle. Good luck Amanda  

    Reply
    Posted by: Anonymous
    September 15, 2008 - 12:19 PM
  5. Would there be any difference in the dimention of the insemination needle for rats. Is there a non-invasive method of extracting sperm in rats. How can the vaginal smear indicate prŒstrus/estrus.

    Reply
    Posted by: Anonymous
    September 26, 2008 - 2:28 AM
  6. I know it is been a while since you posted the video. But what is the success rate of this protocol? thanks Ming

    Reply
    Posted by: Anonymous
    December 11, 2008 - 11:45 AM
  7. Amanda, Our lab is just beginning to look into artificial insemination.  After watching your video and looking at a few other artificial insemination protocols, I have a couple of questions: (1) How to you assess or induce ovulation? (²) How do you induce pseudopregnancy? Thank you, Rob

    Reply
    Posted by: Anonymous
    January 2, 2009 - 6:02 PM
  8. Hola, respuesta para Robert Stiles, mira si tu problema es matar a los machos, lo que puedes hacer castrar a los animales, de esa manera no es necesario sacrificarlos. Por lo tanto puedes obtener los espermas necesarios sin sacrificar al animal.   Saludos desde México Roberto Lazzarini, Biologo experimental    

    Reply
    Posted by: Roberto L.
    January 29, 2009 - 8:45 PM
  9. Hy Amanda, I would like to know  some things: 1 - what is your success rate? ² - Do you use the vaginal tampon like described by LECKIE et al, 1973 (Biology of Reproduction, Vol 9, 4²0-4²5)? 3 - I tested the techinique using dye solution and, afterwards, I sacrified the female, but I didn't see any solution inside the uterus. Do you introduce the needle in the cervix (like in human intra-uterine insemination) or you just put the needle very closely to it. Because don't have how to have sure about introducing into the cervix, unless using a special speculus. Thank you Daniela  

    Reply
    Posted by: Daniela F.
    March 4, 2009 - 9:44 AM
  10. Please send me the full video of collection of sperm from mice. Because i could not able to see the full video content in my institution so send full video to my mail. thanking you.

    Reply
    Posted by: Anonymous
    June 18, 2009 - 1:18 AM
  11. Hi, please contact us at support@jove.com and we'll see what we can do.

    Reply
    Posted by: Anonymous
    June 18, 2009 - 3:19 PM
  12. hi Amanda ,please Iwant a copy of the video and any related subjects if you can support them to me I will very gratful .
    with my regards

    Reply
    Posted by: Anonymous
    August 9, 2009 - 9:16 AM
  13. do you induce pseudopregnancy in the recipient female or do
    you literally just inject the donor sperm??
    If you induce how do you do this?
    What are the pregnancy rates like from using this AI?

    Reply
    Posted by: Anonymous
    January 29, 2010 - 4:50 AM
  14. Hi dear Amanda, I want to collect semen from rat, but when I removed the seminal vesicles and I cut them, semen was coagulated immediately. . In fact I need a protocole for rat semen collection. Thanks for help.
    Ameneh immunology MS student

    Reply
    Posted by: Anonymous
    December 12, 2011 - 8:52 AM
  15. Good day maam! Can i ask the procedure how to extract the sperm form the cauda epididymis? thank you.

    Reply
    Posted by: jamee d.
    November 23, 2012 - 6:55 AM

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