As a stallion owner, I have a couple of clients whose veterinarians use both syringes on the same day. One uses one right after the other, the other vet waits a few hours.
I would like your opinion on this method. The argument they give is that the semen is better off in the mare, than waiting "cooled" in the container for inseminating the next day. Do you agree? My stallions semen keeps fine for a next day breeding. In fact, we have reason to believe that his semen has lasted for several days before ovulation, and gotten a pregnancy.
This week, June 20, one of my clients used both syringes on a 40 follicle. It was also discovered that the mare is building a second follicle, so they are planning on ordering more semen to catch that follicle, on June 23. The justification was that when this occurs, the "second" follicle is always the stronger. It was also stated that they were afraid they had inseminated too early to catch the first (40) follicle.
My argument was that they were flirting with producing twins. I'm not comfortable with this idea, or with using both syringes in a single day. I would really like for you to clear this up for me. Maybe my thinking is wrong.
This has been an "eternal debate" for some time in the world of AI, and I have little doubt that those who favour one method over another will be likely to continue with their beliefs regardless. We however like to look at peer-reviewed published research that is relative to the matter, and make our decisions based on hard evidence rather than anything else. I unfortunately do not have the specific titles and references available to me, but all of these points have been published in peer-reviewed publications such as Theriogenology and Animal Science journals:
Inseminating greater than 500 million progressively motile, morphologically normal sperm (PMMNS) does not increase pregnancy rates;
All [healthy] mares develop a post-breeding inflammatory response that is significantly under way by 6 hours post-breeding, peaks at about 16 hours, and has dissipated significantly by 24 hours;
A good number of sperm inseminated into a mare 16 hours after an initial breeding with only seminal plasma present (no extender) will make their way successfully to the oviducts;
Most sperm inseminated into a mare 16 hours after an initial breeding without seminal plasma will be destroyed by the inflammatory cells present and not get to the oviducts;
Addition of semen extender dilutes the protective qualities of the seminal plasma, and semen extended - even thought there is seminal plasma present - will fall into the second category where the sperm are destroyed in significant numbers before reaching the oviducts.
From all those points, the protocol that we have developed - and we are not unique in this, as many people follow the same protocol - is we will inseminate a single dose upon receipt. If at least 24 hours later the mare has not yet ovulated, we will evaluate the second dose of semen, and if there are >100 million PMMNS present, we will inseminate the second dose. In a perfect world of course, the mare will already have ovulated!
WRT breeding on a second follicle:
Remember that once the first follicle has ovulated, a CL is forming, and once that CL actively increases progesterone levels (by about 3 days after ovulation) the cervix will be closing/closed. This will prevent passage of post-breeding fluids back out of through the cervix after breeding, and typically result in breeding-induced endometritis. Breeding on a second follicle that ovulates >3 days after the first is therefore likely to be undesirable.
As far as concerns of a second pregnancy (twinning), that is a non-issue as long as there is someone capable of reducing the second pregnancy by pinching. Indeed, as sperm may live as long as 6 days in the oviducts, not rebreeding really isn't going to make a huge difference in some cases - you will still get twins. In fact, double ovulations are desirable (subject to the observation above re rebreeding):
The single ovulation pregnancy rate is approximately 60% on average;
The double ovulation pregnancy rates is approximately 86% on average;
The recorded success rate of twin reduction is 97% if an experienced person is performing it;
That means that even is all the double ovulations resulted in twins, with proper management there would still be about an 84% singleton pregnancy rate!
Thank you so much Jos. This answers a lot of questions.
As I finished reading this, the mare owner phoned. She told me that the first follicle was "gone" and the mare's second follicle is still, today (Sun), at a 30 (as it was Friday), but her uterus is much softer. I suppose we will go ahead and ship semen for a Tues. & Wed. breeding. Unfortunately, I will be away (important) and can't ship again until Thursday, when it would probably be too late.
Can you explain "gone"? It confuses me that Friday she had a healthy 40 follicle, + a 30 follicle, was bred that day (double dosed) and Sunday the 40 is missing and the 30 is a 30. Do you think the ultrasound missed something. Wouldn't be the first time.
Another question. I centrifuge all my semen, simply because I would prefer sending very large doses in regard to sperm count, than less. It isn't unusual for one mare to receive 4 or more billion sperm cells. Once it was 6 billion per syringe. In reading this, you make it clear that there is no advantage to this. Is there a disadvantage?
What do you think of routinely centrifuging, especially in a case where it isn't necessary to get a good dose? I use an awful lot of expensive Inra. I don't mind, as I know how expensive this venture is for my clients. On the otherhand, if there is no advantage, or worse yet - a disadvantage, then not doing it is probably best for my clients.
I learn from you more every season. It's why I like to take your course every other year. Just wish you'd move Arkansas back to Febuary (or January) as March gets too busy for me to leave the farm.
New email from the client asks.... "I wonder, IF the first follicle which has already ovulated was fertile and IF the first breeding on Friday was successful, what happens when I give M---} a second shot of Chorulon tomorrow night? Would that have any impact on an impregnated follicle that was in the process of implanting? There is just too much I don't know!"
I'm almost inclined to advise this mare owner to not breed her again, and short cycle her for next heat. What say ye? She's in Canada.
"Gone" has to mean either regressed or ovulated - and as your subsequent post seems to indicate, "ovulated" is the answer.
WRT centrifugation of semen: Sperm are the irritant that cause the post-breeding uterine inflammatory response, while seminal plasma is the mitigant that reduces the response. Centrifugation removes the seminal plasma (mitigant) and concentrates the irritant (sperm) - that's one of the reasons why we sometimes see more of an inflammatory response when using frozen semen. Centrifugation of semen also runs the risk of damaging the sperm, so that factor needs to be considered as well. On top of that, once centrifuged, one needs to add extender to bring the sperm back to a concentration of between 25 and 50 million/ml, and as you note that can use a lot of (expensive) extender. In fact, in order to obtain that desired diluted concentration, if shipping an insemination dose of 4 billion sperm, the final diluted volume should be between 80 and 160 ml (which is of course WAY too much!)!!! Granted removal of the seminal plasma may allow for a slightly greater final sperm concentration, but I'm sure you get my point. There are therefore several reasons to not centrifuge semen unless needed. As a general rule, the two most common reasons for centrifugation are low initial concentration, or a significant toxicity of seminal plasma. Centrifugation for centrifugation's sake is not needed. Having said that, "the proof of the pudding is in the eating" - if you are getting excellent pregnancy rates, then maybe another adage comes into play: "If it ain't broke, don't fix it"!!
A second dose of hCG - presuming that the first dose was a "standard" dose of between 2,500 and 3,300 i.u. - is neither needed, nor desirable. In fact it has been shown that giving >5,000 i.u. of hCG in a cycle can actually suppress ovulation in some instances. Additionally, we have to go back to what I observed earlier - that breeding >3 days after an ovulation has the potential to be detrimental because the cervix will be closing/closed in response to the rising levels of progesterone secreted by the primary CL. To breed >3 days after ovulation may well cause or exacerbate BIE and reduce the chances of an established or maintained pregnancy.
I'm not clear why you suggest short cycling the mare...? From what it appears, this mare was bred with perfect timing relative to the ovulation and has every chance of establishing a pregnancy. To not wait the 14 or 15 days and pregnancy check would be very counter-productive in my opinion unless there is something else I am not privy to or I am missing.
If this were my mare I would sit tight, not rebreed, and check in 14 days... Of course, if the mare then came up "open" everyone will want to blame someone for bad advice...
"If this were my mare I would sit tight, not rebreed, and check in 14 days... Of course, if the mare then came up "open" everyone will want to blame someone for bad advice..."
Yep, and you are just right behind me (stallion) on that one. HA! Great breeding stallion, top expert for advice, but these horses have a way of playing with our heads... AND pocketbooks!
This mare in question is a 10 year old mare, and has only had one foal (2007), although to my knowledge, they have tried throughout her life.
Last shipment, the vet reported a large number of motile sperm, at a 60 percent motility rate, next day after collection. She was monitored, ovulated right on the money...... but no pregnancy. Those cases are hard to figure out.
As for centrifuging, I will take what you said to heart, and just use it when I need to ship to a number of mares in a day.
BTW, I use and love you calculator! Thank you Jos for spending so much time for us here. I'll keep you posted on the progress.
"The veterinarian said the HCG concern is a non-issue because her standard dose is <2500 so even after the second shot we will still be less than 5,000 i.u. She is also convinced the first follicle was not viable and there is no primary CL present. She is quite confident that the state of Mona's cervix and uterus indicates progesterone levels are not rising. She feels all signs seem to indicate Mona is much more likely to conceive with this follicle."
The semen has been ordered. Let's hope she (veterinarian) is right.
Jos, I wanted to pass on the results of taking your advice about using the centrifuge, when not needed. (okay.... and brag a bit!) We were getting good results then, but as you know using that method takes a lot more time and money (Inra96). I decided to send some shipments without using the centrifuge, putting about 10+ cc's of semen to 50 cc's of Inra96. Last collection..ct:339, vol.60: motility..I won't claim that it is more than 85%, so put that on my paperwork, but honestly it often looks like as much as 95%.
I checked on the 3 inseminations, and all had the same report. By phone (shipped same day (Fri)/same collection), they said that 48 hours after collection, motility was still at 75%. Can't complain about that. Here is the email from the other. I had collected at 9 am on Monday. The mare was bred Tues & Wed...
"I just received word about the semen. Doc L--- bred Rose, with the second dose, just after 6am on Wednesday, but he wasn't able to scope it until after 8:15am. He said it was about as much motility as he has ever seen, and looked like it had just been collected!"
Given that his sample obviously laid around a couple of hours before looking at it, I guess there's little reason to use this new toy much anymore!
Standing a stallion can be frustrating when your boy produces this well, but some mares just won't co-operate no matter what you do.
Jos, your previous post brought another question to mind.
Apparently, as ultrasound would indicate, sperm can live somewhere in the mare for several days. Is it that the sperm, who has made it into the ovaduct, is protected there, and can live and wait there for ovulation but basically the guards at the door (inflammatory cells) don't permit anyone newcomers (sperm) to get that far, during the first 24 hours?
Well, sort of... but it's not quite as cut-and-dried as you outline.
Some stallions may have sperm that survive for a prolonged period in some mares.
There have been confirmed pregnancy establishments achieved with an ovulation that occurred as much as 6 days after breeding.
"Sperm reservoirs" have been identified in the region of the oviductal junction that seem to have a protective quality for sperm, but they do not appear to be present in all mares, or at least identified in all mares, so the person saying "my stallion has really good sperm. We've established pregnancies as much as 6 days after breeding" should in fact be saying "my mare has a really good sperm protective ability. We've established pregnancies as much as 6 days after breeding".
In other words, maybe it's not the stallion that's the fertility god...
Having said that, there is little doubt that there are some stallions whose semen does not survive for prolonged periods under any conditions, so the stallion certainly has some say in the matter, so perhaps it would be better to day that he alone is not the fertility god.
As far as the inflammatory cell issue... the uterine inflammatory response should be peaking at about 16 hours after breeding, and dissipated by 24-30 hours in a reproductively healthy mare. On top of that, the seminal plasma does offer a protective quality for the sperm, so sperm introduced during the inflammatory period with only seminal plasma present (no extender) will tend to be able to make it through to the oviducts and potentially achieve fertilization. In the absence of seminal plasma, or with diluted seminal plasma however, the inflammatory cells will attach to the sperm and destroy a significant proportion of them prior to their gaining the protective oviductal region.
No. Although extender dilutes the mitigating properties of the seminal plasma, it also contains antibiotics that will (hopefully) destroy bacteria that are introduced into the semen/uterus during the breeding procedure.
So using an antibiotic extender, a calculated dose of semen (not the whole ejaculate), and breeding once every 48 hours will give you a better result in pretty much all cases. This is proven by the fact that well-performed on-farm AI will result in higher pregnancy rates than live cover.
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