My name is Amanda Dli from the laboratory of Dr.Paul Verana at the University of California Irvine. Today I'm gonna be demonstrating the procedure of artificial insemination. This entails extracting the sperm from the male and then taking that sperm and and putting it into an ovulating female.
The reason you might wanna do this is because the male and female may not mate at the time of which you need or they may not mate at all. Again, this can also be used with frozen sperm if a mouse line is very important. This procedure is very important to see demonstrated because a lot of manuals and journals do not show the proper procedure for handling the mouse.
The mouse can be handled and injected with one person without using any anesthetic on the female. This entails that the female is not injured in any way and ensures that the fertilization can take the best possible route. The dissecting tools that we'll use are two sets of sharp forceps and one pair of sharp scissors.
These will be used to isolate the best deference all the way through the testes and the fat fed. Once that's done, we'll be using the forceps to isolate the epididymus. 9%milk is used to shuttle the sperm into the female.
It is made up of one XPBS in powdered milk found from your local grocery store. This is I put into a small Petri dish in an isolate fashion so that the sperm is in a localized area. 18 gauge needles are used to slice the epididymus open so that the sperm can swim out in the 9%milk solution of the Petri dish.
One that is achieved and co fluencies found the sperm in 9%milk solution are taken up by a 22 gauge needle, which is blunt ended, and at 120 degree angle. This is attached to a one milliliter syringe and then 0.025 milliliters is injected into the female peanut mouse is, or rodent is polyus. Para cus are actually the most abundant endogenous North American mammal Polyus is found in the Florida southeastern region of the United States.
As you can see, it's a little bit larger in the eyes than mu musculus. In addition, it has a lighter coat. This is also helps to blend in with the sand dunes found in the Florida region.
What I'm gonna be doing is isolating the entire testis region of this mouse. It's 1-year-old here cus have been known to breed and captivity up to four years old. And the lifespan is actually unknown in captivity because they've been alive for up to seven years.
So the first thing that I'm gonna be doing is dousing the lower region of this male with 70%ethanol. It ensures that the hair doesn't get in the way. As you can see, the bulges here are the testes.
I'm gonna be making an incision through the outer layer of the skin, make a small incision on the inner wall and make very large horizontal cuts. Actually, I find it easier to simply push out the testes at this point because the fat pad is very large and easy to see. Once the testes are out, you want to make two cuts, one up by the fat pad to isolate that, and then below the epididymus, which connects it to the wall on the other side, wanna cut low enough so that you don't harm the epidemics.
The testes is made up of this big fall here. In addition, the epididymus is found through here, follows the testes up to this point up here. The epididymus also looks like a file of spaghetti at the bottom, and then the fat pad is found at the top.
The first thing I do is cut the epidermis away from the testis. Once that's done, a slight polling occurs to separate fat pad in the epidermis. Once this is done, I rotate the testes over and try to isolate as much of the epididymus away from the fat pad as possible.
Once this is done, it's very easy to slightly tear the epididymus away from the testes. And so doing isolate the entire epididymus in those testes. The epididymus has actually broken along the side of the testes.
So there's two different sections to isolate here. One on the bottom, which is still connected, and then the top portion, which I'm gonna again flip over so it's isolated in my left hand so that I can cut with my right and cut the fat right away. So you have the two different portions of the EPIs.
I'm gonna pick up the EPIs and then just place it in the 9%milk where I'll harvest this sperm. So at the moment I'm just organizing by isolated, so I can do these all individually. I basically, you just want to, most of this is fat, and here's the point.
You wanna make long strokes so that the sperm can swim out into the milk mixture. That's mostly fat. This one is a nice long, pretty much destroy the tissue.
I try and make enough cuts through here to access all the different, the different pockets where the sperm will be stored. The other thing you don't wanna do is get too small in pieces because when you try and pick up the sperm later on, I think it's clogged in the 22 gauge needle. I'm gonna let em sit for one minute to ensure that all the sperm has left the epidemo.
The squiggly lines that you see that are moving, causing the liquid to move are the actual sperm. The blobs are the milk deposit granules, which is why the image is a little bit darker because it is in a milk suspension. As you can see, the sperm is still active with the tails moving.
So the next thing that we're gonna do is suck up the 9%sperm solution into the syringe. You wanna tilt it down its side because there's not much liquid in here to begin with. You simply suck it up.
If you do get a clot, it's very easy to remove. Just use a chem light to wipe off the end of your tip. Once you've sucked up some of the solution, depending on how many mice that you have, you wanna get as much of the air out of the syringe as possible.
This is simply done by flicking the syringe to get the air bubble the top. These females are not anesthetized in any way, and so you can see that it's able to be injected without using any serpent anesthetic. You wanna inject the tip of the point in a vertical fashion.
The vagina's actually in a J shape, so if you inject and move the syringe back, then you want to inject at most 0.05, which I did. As you can see, it's plenty because it's going back out. I've just shown you how to artificially inseminate a female.
What we've done is isolated sperm from the para mucus polyon as male, and injected this into an ovulating female. Once this is done, fertilization should take place in the OV female at or soon after the artificial insemination. 12 hours After this a T cell embryo should be developed.
