Of Mini-Pigs and Men?

It’s not just mice and rats that are used for metabolic research at CBMR. Research Assistant Ann Normann Hansen from the Barrès Group has been working with a different kind of animal to help her understand how diet can change the epigenetic marks that are passed onto offspring through sperm – the mini-pig!

First of all, what even is a mini-pig?

The miniature pig (generally referred to as a ‘mini-pig’) is an animal model that has been developed for biomedical research. The first attempt to develop miniature pigs goes back to the 1940s in the United States where scientists started to use pigs more frequently in biomedical research, and therefore the need for a smaller and more manageable version of the domesticated pig arose.

Compared to the 150-400kg body weight of an adult domestic pig, a mini-pig only weighs around 35-45kg. Besides the smaller size, which makes them more easy to handle, other advantages of small pigs are that they require less food and space, as well as the pharmacological products and anesthetics needed. And they are quite cute.

And what are you using them for?

We are using the mini-pigs as an animal model to investigate effects of chronic nutritional stress on the epigenetic landscape of spermatozoa. This is one project out of many within the GECKO international research consortium whose main research goal is to understand how a father’s diet before conception influences the metabolic health and predisposition to obesity in future offspring.

We think that specific paternal diets can modify the epigenetic information carried in the spermatozoa, which is then transferred to the oocyte at the point of fertilization. This contributes to an altered offspring phenotype through differential programming of the developing embryo.

The GECKO consortium is also particularly interested in finding out whether these epigenetic modifications represent a biological response that is evolutionary conserved across different species. In other words, do all animals respond in the same way at the epigenetic level to nutritional challenges, or does each species have a specific response? This is one reason why we have chosen to work with mini-pigs, as they will represent a species within our comparative analysis of vertebrate sperm epigenomes.

What exactly are you feeding them?

We are feeding the mini-pigs a “Western diet”, i.e. a diet rich in saturated fat, fructose and cholesterol over a period of three months to induce the nutritional overload/stress in the animals. We are collecting sperm samples at different time points throughout this dietary intervention, and will perform different epigenetic analyses – mainly DNA methylation, small RNA profiling and chromatin accessibility – to identify gene families/ or regions in the genome that are changing in response to the diet. 

Why are they useful for the kind of research you are doing? Why not mice?

Mini-pigs are pretty useful because they share a similar anatomy, physiology and metabolic profile with humans. Sadly, mini-pigs are – like humans – also very prone to obesity and upon extended high fat-sugar dietary intake, they develop a phenotype similar to the metabolic syndrome, including visceral adiposity, dyslipidemia, glucose intolerance, insulin resistance and high blood pressure. Therefore, they are a very good model for the human obese condition – although interestingly, pigs rarely become diabetic!

What do you hope to find out?

I hope this project will help us to better understand the molecular effects of a chronic high-caloric intake/nutritional overload on the epigenetic information carried in spermatozoa – of fathers to be – what genomic regions/genes are affected, and how this potentially translates to a differential programming and phenotype in a future developing offspring. 

How has COVID-19 affected the mini-pigs project?

We are conducting the study in collaboration with a private biomedical research facility, BIONEA LAB, which is located in the South of France. The animals are housed in their facility and they are responsible for the daily care and many of the experimental procedures, such as sperm collection. Due to the COVID-19 outbreak, I have only been able to visit the mini-pigs once, but I really hope to be able to go again in the near future and follow the last part of the study in person.

The BIONEA LAB in France.
The BIONEA LAB in France.

Despite the many challenges, I think we have been quite lucky that we were able to initiate and continue the study at BIONEA LAB during the lockdown in France. This was not the case for many other research projects across Europe and the rest of the world.  

You’ve told us a bit about sperm collection. I’m a little nervous to ask, but how exactly does this work?

A particularly important reason why mini-pigs are useful is that we can train them to deliver a natural ejaculate, thereby getting only the mature sperm cells, which mimics the human situation. A bonus here is also that the ejaculate volume in mini-pigs is very large – up to more than 100ml – so you can get plenty of sample material to work with!

Mini-pig on the ‘dummy sow’!
Mini-pig on the ‘dummy sow’!

But collecting semen from a mini-pig is not an easy task and requires a lot of training – just until the mini-pigs figure out what it’s all about! Normally, you train the mini-pigs to mount a dummy sow. The first step is to expose them to the dummy sow several times with daily intervals to make them acquainted with the dummy. After this, they should learn to deliver an ejaculate each time they are mounting the dummy. Sometimes, this doesn’t occur, typically because of distraction, so you need to establish an environment that facilitates the willingness and mating behavior of the mini-pigs.

For example, the room needs to be quiet – no talking! – with no food hoppers and waterers around. Moreover, it’s important that the semen collection from one mini-pig is made immediately after the collection from another trained mini-pig, as the pheromones and odor from the first can ‘trick’ the second to be excited.

At BIONEA, the technicians collect the semen in the same room as where all the mini-pigs are housed, precisely because the pheromones produced by the animals stimulate the others. When the mini-pigs are trained, the semen collection is easy and takes at most five minutes each.  Now the mini-pigs even run to the dummy themselves when they are let out of their box!

A virtual R course with Leo

Many of us find ourselves with the opportunity to learn a new skill while we are working from home. Bioinformatics is central to much of the research at CBMR, and is a specialist skill that takes many years of training and experience – so much so that many lab scientists don’t get the chance to train in bioinformatics (and vice versa). 

A photo of a laptop with the caption, Learning a new skill from the home office.
Learning a new skill from the home office

Leonidas Lundell and Lars Ingerslev, who are both bioinformaticians in the Barrès Group, decided to share their programming expertise and teach the basics of programming in R (a popular and useful programming platform) with three other members of the group. Unusually, they have a background as bench scientists, and so understand how challenging it can be when starting out.

They thought it would be a good idea to teach researchers to solve some common problems they face when processing their data – to take some of the workload off the bioinformaticians! For example, they taught the basics of plotting so that researchers can modify figures themselves and the basics of processing microarray data. Of course, the main goal was to pass on programming skills to researchers who might otherwise not have the chance to learn them. 

Ann Normann Hansen, who participated in the course, says: “We are learning things that are potentially very relevant for molecular biologists to use, such as visualizing data in different ways, sub-setting and converting data into different formats, and creating nice and consistent looking plots. Seen from a group-perspective it is also a win-win (hopefully!), as teaching us non-bioinformaticians some basic R programming skills could help relieve some of the very common tasks for the bioinformaticians. As we all know, they are very popular and busy guys!”

A screenshot of the program microsoft teams through which Leo is teaching the programming language R.

Learning to program in R on Microsoft Teams

During the course, Leo was surprised that the virtual format actually had a key strength over traditional classroom teaching. People can individually share their screens if they have a problem, and the other students get to follow the problem-solving process. This is in contrast with traditional classroom-settings,  where individual troubleshooting is rarely shared amongst the class.

Ann echoed this advantage: “A good thing about learning to code ‘virtually’ is that we are a small group of people, which makes it a lot easier to ask questions. I wouldn’t have necessarily had this opportunity if I was sitting in a big class with lots of people, and it also feels more comfortable. The small-format ‘meetings’ also allow us to easily share our screens and codes with each other, for example when Leo and Lars are going over new topics, when we discuss specific exercises, or if we are stuck on a question. Also, by learning to code this way, we have a good opportunity for seeing and discussing different ways of solving the same question.”

As well as this initiative from the Barrès group, there are lots of ways researchers can learn new things during the lockdown. There are a number of online seminar series, for example @EcoEvoSeminars, whichare open to researchers from all disciplines, giving people the chance to step out of their comfort zone and hear from experts. Leo also suggests ‘The Science of Well Being’ course, hosted by Yale University – if you fancy something a little different! Ann sums up how the lockdown has impacted her thinking: “Looking at all of this in a positive way: instead of being busy with many of the weekly tasks we would normally do if we were in the lab, we have more time and opportunity to learn new things – both individually and together, such as having our little R course”.