I work in a company where the most important are fertility results of pigs. As we all know, this part of pig production has a decisive influence on the whole profitability of the business. In our times, when production costs like feeding, medicines and transport grow so fast, we have to be absolutely sure that semen what we use for our sows is excellent. This is why we decided to buy two units of CASA from Microptic and I want to share my knowledge with you to help you with a difficult but very fascinating part of the pig business – semen production.

My adventure with CASA started when I was boar stud manager for a few thousand sows. Then my boss signed a contract with an external auditor from Holland, who was great AI specialist, and he visited me a few times with his CASA, to check semen and confirm that we produce insemination doses with the highest standard and he also helped us in all cases. Some day he said that CASA is necessary in that size of production, if we want to move forward with our results of conception rates and born alive. We chose Microptic unit because of a few reasons: recognized brand, good mobility and attractive price. After a few months we bought the second one, and we put one in the biggest boar stud permanently.

What about using CASA? First I have to say that the basic thing is training. Microptic program called SCA is not complicated, but it is very important to learn using this in the right way from the beginning, to avoid mistakes in the future. I started to learn with my AI manager and our external auditor a few weeks before I started to be responsible my for semen which can be used or not on our farms. The first tries failed, especially with semen concentration tests, because of my weak skills with automatic pipette or forgetting about the steps like mixing the semen. All these things disappeared after a few days, and now – after several years with CASA – I can’t imagine working without this unit.

In our company we have a few boar studs, mostly with terminal boars, but also with breeding ones. We use post-cervical insemination almost on 100% our sows (gilts have classical manner of insemination). We split one 90ml dose for three inseminations, so our concentration of cells in a dose is high – it’s 3.3 billion in 90ml dose. This amount gives us a guarantee that every sow gets above one billion cells straightly to the uterus, and the chance for successful conception is the highest. The extender which we use is long terminated, and we keep the doses in 16 degrees Celsius and use them maximum 4 days after collection. In the case of terminal boars, we produce mixes from 2-3 semens and the information about boars used in insemination is saved in our global computer program. With breeding boars we prepare and use only single ejaculates to avoid inbred and to allow the breeding work.

How we use the CASA.

During production of semen, we collect plenty boars in a very short time, and every ejaculate is tested by SCA on Leja slide. We test already full diluted semen from a single boar to check movement, morphology and also concentration, to score every ejaculate in the scale from 1 to 5.

We test 600-800 cells for movement on alive semen and 100 cells for morphology on dead semen (semen is killed inside the chamber of Leja by short contact with a high temperature). For terminal boars, when we have 3-4 ejaculates already tested, we prepare mixes of 2-3 ejaculates, according to the following rule: score 1 (the best) or 2 can be mixed with every semen, but score 3 can be mixed only with score 1. This protects the final mix against mistakes in quality. The components have to be very similar in temperature- the maximum difference is 1 degree.

Sometimes we use also CASA to check concentration in pure semen, to compare the result with the spectrophotometer- then we prepare one probe of pure semen diluted 1:10 in probe, and start the test.

The most important test is the final checking- we check the prepared mix from 2-3 single ejaculates for quality of movement, and first of all for concentration, because we have to have 3,3 billion cells in every single dose. If concentration is lower because of any reason, we add additionally one more concentrated semen to thicken the mix; in the case of too high concentration, we weigh the mix, count the right volume and add extender to have 3,3 billion in dose.

I need Microptic CASA also for the next thing- testing the semen after 24, 48 and 72 hours. We leave one dose from every ejaculate sent to the farm to watch cells mortality; if the quality of semen goes down, we call the farm and ask them not to use that semen. For this test I use only movement test, because concentration and morphology are on the same level, but the percentage static cells grow every day.

Testing movement: for movement we check 600-800 cells; to have a good concentration test we do the first slide from the back side of the Leja chamber, but sometimes movement can look worse there than in the other area; we take the next 3 slides moving with the objective from the back to the front of the chamber in order to make the last photo at the beginning of the chamber- in many cases we can see that the movement looks the best in this place. Anyway the average result is decisive: there has to be minimum 90% of motile cells and 60% of progressive cells in single semen to have score 3, but minimum 95% of motile and 65% of progressive in the final mix. Movement also depends on the temperature of the heating table, but it’s very important to remember that some ejaculates have different optimal temperature points – I mean one semen can have excellent movement in 35 degrees (if we keep Leja chamber in an empty-heating place on a microscope table where the objective is, the temperature can be lower), but the second semen needs minimum 38 degrees. It is very important to check semen again with short heating on the table, if the first result of movement test was not good. Motility properties that we use are the following: velocity curvilinear- 50 micrometer/sec, straightness of path- 45%.

Testing morphology: 100 cells rated for basic defects such as protoplasm droplets (distal and proximal), head defects (acrosome damaged, wrong shape of head), tail defects (broken tail, coiled tail) and incomplete sperms (without head or tale).

It is important to watch cells when they are static, because it’s very difficult to see some defects when sperm is motile. To do it we use the lighter fire- a very short contact of the bottom of the chamber with the middle part of the flame not to smoke the slide, or a special tool with a high temperature tip to touch the chamber for 1-2 seconds from the top side of chamber. A longer contact with high temperature can make more coiled tails. The contact with temperature should be on whole chamber length, because if we touch only the middle part of chamber, alive cells from front and back part can move to this area later. It is also possible to kill the sperm with formalin, and then fill the chamber with dead semen, but it results in additional chamber being wasted.

SCA program has very comfortable manner of scoring cells, where every cell is marked with a square: the green one is a good cell, the red one is a bad cell. The red square can be also signed with number of defect.

Testing concentration: this is the part of evaluating semen, where I had the biggest doubts at the beginning of my work with CASA. The giant problem for me was the repeatability of tests and sampling the representative probes from semen. The best available method of checking the number of cells in ejaculate is flow cytometer, but because of a very high price of that kind of equipment, everyone uses spectrophotometers in the lab. After thousands of ejaculates tested, I can confirm that the CASA system can test concentration really accurately, better than any spectrophotometer. Here are the main rules of concentration testing to have reliable results:

  • fast delivery of the semen from the dose to the slide
  • test has to be done as fast as possible: moving cells go to the edges of the chamber, and stay there, so with every second the number of cells in the middle of the chamber is lower
  • minimum 3-4 slides have to be checked; slides from the whole length of the chamber
  • semen has to be stirred accurately just before a test
  • for the pipette the best choice is 1-10 microliter tip, but the pipette has to be set to take a little bigger probe than the chamber needs

Reporting: it is very important to have clear data from semen production, especially in big companies, where thousands of sows are inseminated every month. SCA has a very nice solution of reports, and we use two types of them. The first is a summary report where you can see basic information like the number of boar and date and important parameters like motility and progressive movement, morphology defects, concentration and number of doses. The second report which we use in our company is boar report: there you can find all the details about the boar which you put in SCA program and all the values from a single test, including single photos with cells and paths of movement. This kind of report is useful in situation, when we received a boar from our supplier, and his semen is still bad- then we save all reports and after a few weeks we can make a complaint and ask for a refund. These reports are proofs for bad quality of semen.

My observations:

  • to have reliable concentration result very important is practice, but also kind of pipette, kind of tip for pipette, time between taking the sample and injecting to chamber (the faster the better), stirring the semen every time before taking the sample, manner of pressing the pipette (it should be one, stable and full movement, without breaks to the end of Leja chamber)
  • very important is the quality and clearness of chambers; slides should be closed all the time, opened only if it’s necessary; our supplier of slides takes care to supply us only with freshly produced slides
  • cells with proximal cytoplasm droplets can have excellent results in movement test; in my experience I had situations when movement test in single semen was very good and I didn’t see any morphology defects on moving semen, but after making the semen static, I found 50-60% of proximal droplets so according to current knowledge that semen was almost infertile- it means that to be absolutely sure about the quality of the semen, it’s necessary to look at the morphology all the time

at the beginning I had problems with flowing semen; after application semen to the chamber, all sperms were moving vertically and straight together with plasma- test in this moment shows like the 90% progressive even if semen was dead; to avoid this problem we only need to take off the drop of semen, that is left after application at the beginning of the chamber (in the place of entry to the chamber): semen has to be only inside the chamber, not outside

it is very important to watch what program classifies as a cell: some contamination in shape of cells can be classified as a cell, especially if the semen is more contaminated; in that rare case, when semen is contaminated but looks good and sperms are alive, with no morphology defects in high percentage, it’s better to consider only motile cells in counting the concentration- in this moment we can be sure that we have the required concentration of cells

movement test on cold semen can be failed with progressive parameter because cells are moving fast but they are moving in circles; depending on what type of diluent is used, semen has to be warmed to 36 degrees slowly- in the case of the extender which we use, warming of the dose takes more than one hour

Lukasz Marchel
Production Manager of Boar Stud
Agri Plus Poland
Murphy Brown Group