* Collected AI * used kenny extender on one collection * EZ mix on the second * checked in Microscope, all was fine
* shipped out first shipment, on arrival at breeding farm, sperm was checked, sperm was swimming backwards, and much was dead * second shipment, next day, good motility, good count, active sperm. shipped to same breeding farm, found to have a high percentage of sperm swimming back-wards
* checked semen bag residue which was discarded in garbage 7 hours later, less than 10 % moving backwards, 30% dead, balance OK
conditions: last 3 days 30-31C and very humid.
Am stumped, what caused this sperm to move backwards.
Never had a problem before
* used 2 different AV liners * new Syringes * 2 different lubes * 2 different extenders * 1st express container, second equi container
Many things can cause this to happen. Usually the first thing to do is go back and carefully examine all of your equipment, cleaning procedures, etc. Then examine your semen handling and packaging protocols. If you do this long enough it´s bound to happen to everyone at one time or another. It usually turns out to be something very subtle with the protocols.
I had a similar thing happen a few years ago and it turned out the horse suddenly became susceptible to the preservative in Priority Care sterile Lube. But it could be any number of things. On one occasion someone had changed a few of the fluorescent light bulbs in the lab and replaced them with those that produce the same spectrum of light as the sun.
Just some areas that need to be closely examined. make sure that none of your equipment has been exposed to iodine or alcohol in any form. Make sure that all of your non disposables are clean and free of any residue. Make sure that the soap used to clean your equipment is non residual. Such as Alconox, Nolvasan solution, plain Ivory dish washing liquid, etc. Then make sure it is rinsed at least three times and air dried. I have found a simple inexpensive and safe way to sterilize all your non disposable breeding equipment. After washing and when it is completely dry, simply place it in the microwave for 30 seconds on the high setting. This will effectively kill any bacteria.
Another avenue to examine is extender time studies. Gather a couple of samples of every extender and combination of antibiotics you can find. Collect the horse and divide the ejaculate into multiple small doses (does not need to be an insemination dose). Record progressive motility, then place a representative sample of gel free semen into each extender at a 4:1 to 5:1 ratio. Then place all the samples into and Equitainer (preferably all the samples in one Equitainer). Every 24 hours examine each sample for progressive motility and note the figures for each extender daily. Ideally this should take three days. After which you should have one or two extenders that clearly are superior for this horse. Contrary to common belief there is no such thing as a universally appropriate extender. Many stallions will work just fine in these so called universal extenders, however most will benefit from an extender or combination of extender/antibiotics, that is more specific to them.
These are just a few of the things that could be causing your problem. There are many more. Best of luck with the mystery.
P.S. One thing we all tend to forget over time is, the sperm cells are dying the moment they leave the stallion. The rate at which they die depends largely on how we handle it.
It´s good to remind our selves of this from time to time.
Posted on Monday, June 18, 2001 - 02:43 pm:
Anonymous- I spoke with my Reproductive Specialists, and he told me that the tail of the sperm is bent around towards the head which makes it appear to "swim backwards". The most common cause assoiciated with this is shock. Most probable cause would be from cooling too fast, take the stages down slower. Any process that involves cooling or warming could shock the sperm to react in this manner.
It is worth a try, Good Luck.
Posted on Monday, June 18, 2001 - 08:01 pm:
Sorry to hear that you had some problems. Obviously my suggestions have to be taken a little in the abstract, as without actually seeing the situation it is a little difficult to be conclusive, but hopefully I may be able to be of some help.
[I also received a private e-mail on this matter, which mentioned that the ejaculate volume was lower than normal, so this was also addressed in my private answer which I am reprinting here for everyone's information]
Ejaculate volume is actually a variable parameter which may or may not be significant in it's changes. If it is extremely low it might suggest a secondary sex gland problem, but often it is more closely linked to the amount of teasing that the stallion received prior to ejaculation - the more teasing, the more seminal plasma. The important figure that should not change dramatically, unless there is a change in the frequency of collection, would be the total number of sperm in the ejaculate, consequently, the higher the volume, the lower the concentration should be.
There is however one important possibility relative to lowered volume, which is that it may be associated with a partial ejaculate, which can cause a change in semen pH (acidity/alkalinity) towards the alkaline, and which in turn may produce changes in sperm activity. A partial ejaculation may result in the presentation of the full numbers of sperm, but a significantly reduced amount of seminal plasma, which is what can alter the pH dramatically (not all the secretions are present from the accessory sex glands). pH should be between 6.8 and 7.4, but if there is a change to the alkaline, and lowered volume, one should suspect partial ejaculation. Such a change in pH could result in a change in either longevity of the sperm, or possibly a change in motility - although it would not be specifically linked to retrograde motion.
You do not indicate if the low volume was seen with both ejaculates or not, but if it was, you may wish to evaluate your collection procedures for changes - specifically those related to changes in AV temperature or tension - or alternatively evaluate the stallion for possible soreness which could lead to a partial ejaculatory process.
[This reference is to apparent poor motility at the breeding (receiving) farm]
When lecturing, I always joke (although sadly it's not really a joke!) that one of the leading causes of poor motility in transported cooled semen is the failure of the evaluator at the receiving end to warm the sample to body temperature for a sufficient duration (usually about 5 - 10 minutes) prior to the evaluation occurring. It would be worth making sure that this was indeed done in this case - even the most reputable establishments can sometimes make mistakes......
It does, I admit, seem extremely strange.
Am I right in saying that the sample that you looked at at 3:00 was an extended sample that had been cooled in the same manner as the shipped dose? Another thought that springs to mind is to wonder what temperature you have your extender at when adding it to the semen? There is great emphasis placed on having the extender in the incubator (or water bath) and having it come into contact with the semen at body temperature only - this however is actually erroneous unless the semen itself is also at body temperature. If the semen has cooled to room temperature (which it may do quite rapidly without damage) and then has extender added that is at body temperature, heat shocking of the sperm may occur and this of course may possibly result in subsequent irregular motility. If subjected to a cooled period, such damage may become more demonstrable.
Another area that could cause problems would be a rapid change in osmolarity. Such a change can occur as a result of adding greater than an equal portion (by volume) of extender to semen without a pause for equilibration. In other words, if you are using 10 ml of semen, and 40 ml of extender, add 10 ml of extender to the semen sample, pause for about a minute mixing gently, then add 20 ml more of extender and pause for another minute before adding the final 10 ml. Never add semen to extender - always the other way around.
[The sender here referenced heat/cold shock in their e-mail]
I think you are working on the right premise here, but of course it is difficult to establish exactly where/when the shocking has happened. The fact that the raw sample from the liner that you evaluated appeared normal is obviously suggestive of it's happening at the time of, or after addition of the extender. The fact that you have changed extenders rules out the possibility of a problem with the extender itself, but still leaves a possibility of an adding process problem.
The transport situation is of course another area where heat shock could occur, but you have changed containers and especially it seems that, as the shipment was picked up, damage at this stage is unlikely.
Evaluation at the receiving end is the next question.... Ensuring sufficient pre-heating prior to evaluation I have already mentioned, but what about contaminants on the slides and/or cover slips that are being used? I had one particularly unpleasant moment myself some years ago when I did an evaluation on some slides that I reused, but had cleaned using alcohol - allowing the slides to air-dry after cleaning. The result was very similar to that which you are indicating - high percentages of dead sperm, and erratic mobility. Upon (rapidly and panic-stricken!!) changing to a box of new slides, all returned to normal..... This therefore has to be a consideration at the evaluation level.
It will be interesting to see if the mare gets pregnant regardless, which would suggest that perhaps there was a problem in this respect.
In addition to a complete overall evaluation of your procedures, I would:
Evaluate the ejaculate for changes in pH, and if one is seen check for changes to AV temp/tension and possible soreness or damage to the stallion which could cause a partial ejaculation;
Monitor temperatures of semen/extender and ensure they are closely matched at time of addition;
Ensure there are no dramatic changes in osmolarity associated with such addition;
Package samples using the same containers and evaluate those at the same stage at the sample being inseminated is being evaluated;
Package samples in the same containers as were used for these problem shipments and see if there is a problem associated with those specific containers;
(Attempt to!) ensure that the evaluation at the receiving end is conducted with pre-warmed equipment and after suitable warming of the sample, and that there are no possible toxins present on the slides and/or cover slips or any other evaluation equipment (e.g. rubber-stoppered syringes being used to draw a sample).
I'm not sure if this is of help or if you have already checked all these parameters, but I hope perhaps it may be of a little use.
Very Informative.. I dont' have this problem, but I really enjoyed reading ALL your well wrote answers
Please note that opinions, product information, advice or suggestions posted on this bulletin board are not necessarily those of the management at Equine-Reproduction.com nor does the maintenance of the post position indicate an implicit or any endorsement of that information, opinion or product.
Further, although we have the greatest respect for the posters offering assistance here, you are advised to seek a consultation with your veterinarian prior to using information obtained from this board if it is of a veterinary nature.Proud to be sponsored and supported by: