This summer I have been given the opportunity to spend 8 weeks working in the Griffin Lab at the University of Kent working on Pig IVF and embryo sexing thanks to the Biochemical Society summer studentship (woohoooo science!). After already spending last summer here as a volunteer I have the advantage of knowing everyone (I'm part of the furniture) which got rid of some of the nerves. However, since last time I was here I was working on a different project, I have absolutely no idea what I am doing! The first job of all was to get reading! I was given a pile of relevant research papers and I spent a good amount of time highlighting and asking loads of ridiculous questions. For the first week I have been focused on getting to grips with the pig IVP protocol which involves a vast amount of pipetting, a huge amount of waiting and then (the fun bit) using the micromanipulator!
After a couple of days delay the pig ovaries finally arrived, albeit slightly too warm for comfort at a toasty 39˚C (they should be a few degrees less after their little journey- probably a bit hot going in!). Opening the flask released the stench of pig juice that soon filled the room (I could even smell it through my cold!). After a bit of getting used to the smell, the ovaries were taken out and an aspirator was used to extract the oocytes from the follicles. Only the larger, juicy follicles were pierced and the dark, nasty looking ones were left where they were. Some of the warm handling medium needed to be aspirated regularly to maintain the warm environment for the removed oocytes. The handling medium and oocytes were then poured onto a pre-warmed petri dish and, using a really cool EZ grip pipette with a bendy needle on the end, about 200 good oocytes (with 2/3 layers of compact cumulous cells) were moved to a nunc plate. Porcine oocytes differ from human oocytes in that they are much darker and thicker in appearance. These cells were then transferred to a maturation plate containing some hormones, along with some other bits and bobs, and placed in a CO2 controlled incubator until the next day.
Evi (Masters) being a scientist and some cute piggies!
The next few steps involved some moving of media and oocytes into different incubators but, seeing as this was quick to be done, I got to spend the rest of the afternoon on the micromanipulator! Just for a bit of practise, I spent some time figuring out the different contraptions such as the fine and coarse controls as well as the micro tool holders and then played around sucking dead pig oocytes into the holding pipette, moving things around with the biopsy pipette and making holes in the zona pellucida(s/ae?) with the laser (which had a fantastic huge red “FIRE” button on the remote). The coarse controls let you move the micro tools further across the field of view than the fine controls, which are used for the fiddly bits. I tried my hand at then sucking out the cytoplasm of some of the cells (pretending that I was extracting a blastocyst) by pushing the biopsy pipette through the hole created by the laser and then using the suction control to pull out the contents of the cell. This was really tricky because of the rubbery texture of the cells and it took a few stabs with the pipette and a couple of extra laser blasts to break through. My supervisor, Kate, is going for a lecturer interview tomorrow so we all spent a bit of time in the afternoon quizzing her and making her practise her presentation!
Fertilisation day arrived! Yet to see how this has panned out but the sperm motility was pretty poor (they were mostly dead or just having a little wiggle on the spot). Lots of testing of the media had to be done to ensure good pH and osmolarity levels. After a small mishap with a slight pH overshoot after adding too much NaOH all was forgiven and both levels were on track for success! The only other issue was a slight spill of half of the sperm wash stash (it was definitely one of those days) but we luckily still had enough for what we needed to do! One of my favourite aspects of today’s protocol was that you had to give the sperm their morning fix of caffeine so that they would get a bit of a move on, you can do it boys! And also…Kate got her job, YEYYYY!
Kate being happy about her new job!
Next week: PCR, FISH and more Piggiessssss!