Harvesting Sperm and Artificial Insemination of Mice


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This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

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


Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination.

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female’s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.


Artificial Insemination

Artificial insemination is used instead of natural matings to increase the chances that all the females have will become pregnant. 2 male was used for every 1 superovulated females or a female known to be in estrous. The caudae epididymides from mature male mice were placed in a 9% milk suspension and sliced open with 18 gauge needles. The sperm was allowed to swim freely for a few minutes and then the epididymides were taken from the mixture. The suspension was evaluated with a microscope for confluence and motile sperm. A blunt 22 gauge 1½ inch long needle was used to administer the sperm. A 120° bend was placed about 3/4th the way down the needle. The needle was inserted into the vagina up to the bend and 0.025 - 0.05mls injected. If an assistant is used to hold the female in the appropriate position, the use of anesthetics is not needed.


  1. Use 4 males for 6 females
  2. Euthanize males at the vivarium
  3. Prepare about 5 ml of 9% milk from powdered milk
  4. Put 1.5 ml in small Petri dish
  5. Cut out the epididymus and vas deferens
  6. Put in 1.5ml of 9% milk
  7. Using two 18 gauge needles, and using long cutting motion with needles, slice open the epididymus and vas deferens to release the sperm
  8. Take out left over tissue and check for sperm under microscope


  1. Use blunt needle at 120° angle
  2. Hold mouse in hand and inject mixture. If uncomfortable with one person holding and injecting, two people can be used. No anesthetic should be used, as on one knows how this might influence the procedure.
  3. Use 0.05ml of sperm/milk mix per female

Recipient females

  1. Do vaginal smear on recipient females (by this time the recipient females should be in proestrus/estrus)
  2. Females that have not reached estrus by Day 5 should probably not used

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I would like to thank Mike Dewey from the Peromyscus Genetic Stock Center for his valuable insight, patience, and knowledge on rodent reproduction.


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




  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.

    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

    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)

    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  

    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.

    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

    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

    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    

    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  

    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.

    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.

    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

    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?

    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

    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.

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

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