CryoStore diary - Paulina Pyrek

Apr 21

Secondment at INIAV, Portugal February - April 2025

Mid-February, I began my first secondment in the CryoStore project at INIAV - the National Institute for Agrarian and Veterinary Research. Leaving Norway at that time of year, after many months of winter, should be a happy moment for most. However, as I had completely fallen in love with cross-country skiing, saying goodbye was not easy. Nevertheless, my longing did not last long, as upon arrival at Lisbon airport, I was welcomed by blue skies, green trees, exotic plants, and a temperature of 15°C. The change in climate and landscape was huge and stunning! Equally surprising was my confusion about Portuguese flora, especially the orange and lemon trees. In theory, everyone knows how they grow but seeing them with your own eyes as a very common sight is a completely different story.

One might think this introduction sounds like the beginning of a nice holiday, but don't be mistaken – I arrived here to work and learn! My goals were to gain more insights into the cryopreservation of bovine oocytes and in vitro embryo production in cattle. From the very first days, I was warmly welcomed and taken care of by Prof. Rosa M.L.N. Pereira, the head of the embryology lab, and her entire team, including Linde, Filipa, Vanessa, and Pedro.

(From left) Rosa, Paulina and Filipa in the lab during a break in oocytes selection

In between all the in vitro embryo production steps, we were able to freeze bovine oocytes using a very fast method called vitrification. This technique requires excellent cell manipulation skills. You need to be quick! I suppose a bit of special powers like bilocation or time travel would make this task a bit easier, especially for beginners.

A very important part of my secondment was learning new methods for evaluating oocytes in terms of DNA maturation and integrity, as well as mitochondrial activity. During my PhD, I will focus extensively on cryodamage, and the ability to evaluate it using different staining protocols is essential. I am looking forward to implementing both acetazolamide and JC-1 dyes in my future experiments.

I am very grateful to all the employees at INIAV for their openness to scientific discussions and for sharing their knowledge with me. It is always a refreshing experience to be shown around and introduced to other laboratories’ routines. This exposure makes you think about alternatives and improvements for your own facilities.

A huge thank you also goes to my CryoStore colleague, Linde Rombaut. Your support, both at work and after hours, meant a lot to me! Some people say that "PhD friendships are built on trauma bonding and the shared delusion that we'll finish". I would perhaps also add broken and missed oocytes, tons of frustrations, and the correct number of characters accepted in a conference abstract!

Long days with Linde in the lab without light – oocytes do not like it! (from left: Paulina, Linde)

Our working schedule depended on the oocyte rhythm of the day (or maybe better said - week). It is extremely important to monitor the timing when culturing embryos. To produce a calf, properly grown and prepared cells need to match. In the case of cattle, their female gametes – oocytes – must mature in a special medium for 21-24 hours before being inseminated with spermatozoa (male gametes). Then, an 18-24 hour incubation is required for the sperm and oocyte to connect, followed by another 48 hours for the inseminated cells to divide into zygotes (2-cell embryos). Developed embryos that could be transferred to a recipient cow can be observed on day 7 or 8 after fertilization.