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  Discussion on Poor freezer-- proven methods to improve results?
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Member:
ekaufman

Posted on Thursday, Apr 3, 2008 - 1:43 pm:

My 6 year-old stallion is having an exciting week at the CSU Repro. facility learning to be collected. I had hoped to find that his semen froze well, to have the option of gelding him in the future (he is a well-mannered stallion to handle and ride, but a miserable territorial fence pacer who is unsafe for turnout with other horses. Gelding him would likely improve his quality of life a lot).

His typical collection is 20B with 100% motility, 60% progressive. A test freeze with 2 different extenders showed progressive motility of 7% and 12% respectively, which is a terrible result.

This stallion is maintained on a free choice grass and alfalfa diet, with no current supplementation (except a trace mineral block, which he kicks around his paddock).

CSU is currently engaged in a study on an Omega supplement called Magnitude which claims to improve semen quality and cooling/freezing performance in sub-fertile stallions (my boy's fertility is unknown until my mares settle or not). Their impression mid-study is that the supplement will prove to help some stallions a lot, some a little, and some not at all. They feel there's no harm in trying. My vet agrees, and recommends a slow addition of flax seed to his diet as well. No one is optimistic that we can change his numbers enough to have confidence in his frozen semen (CSU's bottom line for frozen is 30% progressive).

My questions:
1) Is there any good research that shows a path to significant improvement in freezing performance?

2) Has anyone on the boards ever seen a significant improvement in freezing performance, and to what do you attribute the change?
Moderator:
DrO

Posted on Saturday, Apr 5, 2008 - 11:52 am:

Elizabeth can you tell us what procedures are being used? Without knowing what they are doing it is hard to make recommendations. We do have an interesting study recently published, Equine Reproduction » Horse Breeding & Artificial Insemination » New Technologies in Equine Reproduction » Research Summary: Improved freezing technique for semen.
DrO
Member:
ekaufman

Posted on Sunday, Apr 6, 2008 - 12:13 am:

Hi Dr. O,

I don't know what specific freezing techniques they are using, but will ask. I'll prepare myself in advance with the article. Thank you very much for the guidance.
Member:
mrose

Posted on Sunday, Apr 6, 2008 - 12:26 am:

Hi Elizabeth,
We just had semen frozen from our two stallions. both had excellent results. They each first had two "clean out" collections before the test collections, and the test collections were tested with several different extenders and different antibiotic combinations to see which gave the best results. The collections were done 2x/day the first two cleanout collections, then a day's wait before the other collecitons, which were spaced at 1x/day. I'm curious if the proceedure was the same at CSU. Of course, CSU is reknowned for the repro department, and the vet that oversees the repro end of the the facility we used is a CSU grad, so I'd think similar proceedures would be used.
Member:
ekaufman

Posted on Sunday, Apr 6, 2008 - 12:30 am:

Thanks Sara,

The schedule was similar, but my report only showed 2 test freeze samples (different extenders), which is part of what got me wondering whether we had explored all the options. Can you tell me what the variation was that you saw in progressive motility of your various frozen samples? Thanks.
Member:
mrose

Posted on Sunday, Apr 6, 2008 - 9:24 am:

I'm at a show so don't have any reports handy and can't tell you what extenders were used, but the motility rate was over 50% with each used for both stallions, which the vet said was very unusual. (He also said that Arabians and TB's prodduce more sperm per collection than other breads. It was news to me that one breed would produce more than another.)One of the stallion's sperm swam in little circles initially which would affect the motility reading, but they had a means of heating the sperm slightly, and when heated the sperm straightend out.

I haven't read Dr. O's article or done any research on the subject. I would wonder if it wouldn't be possible to combine the results of more than one collection to increase the density of the motile sperm. I haven't had my coffee yet this a.m., so am not sure that's a "smart thought" or not.

Of course, some stallions' just can't be frozen. I know of several top Arab stallions that even had a difficult time settling mares using cooled semen.

I'd call the head tech or vet that dealth with your stallion and have a talk with him; you pay enough for the testing that I wouldn't be shy about "bothering" them. I'd also be inclined to review the stallion's diet and put him on a good supplement making sure he gets plenty of "B" vitamins especially. How old is your guy? Have you used him for breeding?

If the freezing doesn't work out, all may not be lost if you want to use him for breeding in hand and on premise AI. The pacing can be managed; at least we were able to controll it with one of our boys that used to pace.

Sorry I don't know more details about the extenders used, etc. If I don't have it written down it just goes right out of the brain, esp. early in the a.m.
Member:
ekaufman

Posted on Tuesday, Apr 15, 2008 - 9:22 am:

Just an update on my guy, as food for thought for others who may find themselves trying to improve stallion freezing performance.

I have spoken to several repro. specialists and have received several shared impressions, and some solid but not especially optimistic advice.

Here is what I've gathered so far:

- A test freeze performed at CSU, even if it uses only 2 samples, is considered a fair test, provided one extender was egg-based and one milk-based (true). There are many possible variations, but they are refinements that would not significantly alter the result, though they would likely have tripled my bill. (Client observation-- CSU has always been very conscientious with my money).

- Some stallions seem to freeze better in late summer than in the spring. No one could point me to any research, but everyone felt it was true.

- Progressive motility is not fertility. Royal Vista stands a stallion with 5% progressive who settles every mare he covers. A second stallion at 65% failed to settle any mare and was voted off the island. If my stallion is very fertile, 12% on frozen would be better than good. If not, then frozen may not work anyhow.

- Breeding with frozen is more witchcraft than science, and many repro vets hate it. (My vet uses it in her own horses, and doesn't feel so strongly).

- The Magnitude supplement works very well in some stallions, and not at all in others.

So I have started my guy on the Magnitude supplement, hyped to the skies at https://www.magnitudedha.com/ Note that this supplement has palatability comparable to ranitidine-- it smells like the nasty fish oil that it is, even though they have thrown the kitchen sink (molasses, mint flavor, saccharin) in it to try to hide the taste. It is tricky to feed, especially if you respect their restrictions on limiting vegetable fats.

I will try my guy again in mid June, after about 10 weeks on the Magnitude, and see if we have better results. In the meantime, we'll see if he has settled the 2 mares we bred (if he does not, it's a red flag, since one is extremely reliably fertile).
Member:
mrose

Posted on Tuesday, Apr 15, 2008 - 2:08 pm:

Hi Elizabeth. Hmm very interesting. I've always believed that motility is related to fertility, though of course I know it only takes one "swimmer" to do the job. I'm also surprised at your being told that some stallions freeze better in summer. Were you told why? Does it depend on whether or not they've been doing in-hand breeding in the meantime?

Let us know about the mares you've already bred and keep us posted. I totally agree about freezing semen being "witchcraft." imo All breeding involves a large percentage of witchcraft inspite of all the scientific clinics I've taken!
Member:
ekaufman

Posted on Tuesday, Apr 15, 2008 - 3:19 pm:

Hi Sara,

Well, I think better motility *must* be a good thing, but it is not a direct measure/predictor of fertility. Since we can (at present) only measure fertility retrospectively (did he or didn't he settle mares), we substitute motility for trying to predict the future. The repro specialists I spoke to felt progressive motility was a relatively poor predictor for any given stallion, though they look at the numbers anyhow, since it's all we know how to do. The most vehement on this topic were the folks at Royal Vista, who make their living on repro. Note that they also were the most negative about the breeding value of frozen semen.

The "better breeder in summer" thing is an impression shared by several of the repro vets I talked to, but it is supported by their best guesses rather than any particular study. They feel that stallions (and mares) are highly seasonal in their breeding fitness, and that their fitness naturally improves, all things being equal, as they reach the "natural" peak of breeding season. So it had nothing to do with whether they were active in the Spring-- it is a hypothetical adjustment (possibly circadian) that they make with the season. In fact, they told me that they often send borderline stallions home to wait for summer, and they see improved results, for whatever reason.

Of course, my guy collects and cools extremely well, so he's hardly borderline in the traditional sense. I think there is also a part of this argument that is along the lines of "give this great guy another chance," rather than a seasonal analysis.

Going to be interesting to see how we go along. I have my own bias about what I think our results will be, but I'm going to follow the advice from CSU and hope we get lucky. (To be fair, no one has promised me an improvement, so they are advocating a long shot, not a cure.)
Member:
ekaufman

Posted on Wednesday, Apr 16, 2008 - 2:03 pm:

First mare checked in foal this morning. Yay!
Member:
mrose

Posted on Wednesday, Apr 16, 2008 - 8:58 pm:

Good news,Elizabeth!!
Member:
ekaufman

Posted on Monday, Jun 16, 2008 - 4:18 pm:

Here's an update. We performed several test freezes, all with poor post-thaw motility. His best number was around 15% progressive, with 40% motility. The general standard for high quality frozen semen at CSU is 30% progressive.

I put him on a 70 day trial of Magnitude HA, just completed, and we began another series of test freezes. Today's result came in at 24% post-thaw progressive, with an overall 60% motility. We'll repeat the test Wednesday, but it is an improvement that is inline with the best results seen in the research environments, and we were all surprised and pleased.

I will say that the Magnitude is very difficult to feed-- it is highly unpalatable, and there are restrictions on what you can hide it in. I wish they made it in a paste to make it easier to get into the horse.
Moderator:
DrO

Posted on Tuesday, Jun 17, 2008 - 7:10 am:

Good to hear the numbers are improving Elizabeth. What is in the Magnitude HA that they believe improves the freezing qualities of the semen?
DrO
Member:
ekaufman

Posted on Tuesday, Jun 17, 2008 - 9:57 am:

Hi DrO,

It's made out of snake oil, err, I mean fish oil.

The Magnitude is a fish-oil based Omega supplement. https://www.magnitudedha.com/ for all the vendor hooting and hollering.

For what it's worth, my vet felt the same nutritional objectives could be met with flax, but I didn't find any stallion research on flax, and figured I would try the supplement that had *some* research to support it. Why a fish would help a horse, who knows.


CSU studied a paste-based fish oil omega supplement and found that it could be shown to alter the composition of the membrane around the sperm cell. They also found that some stallions on that supplement saw improved cooling/freezing performance, some of which was dramatic (on the order of a 50% improvement in post-thaw motility). The improvement in motility seems correlated to the membrane changes. As far as I know, there isn't research yet that tries to describe the connection between the two.

The research seems to be ongoing, but I had the luck to have members of the research team collecting my stallion, and they felt it was worth a try, though all of us were skeptical that we would see any real change.
Member:
mrose

Posted on Tuesday, Jun 17, 2008 - 10:21 am:

VERY interesting. I knew you should feed all the B vitamins to horses you were breeding, but was unaware of how important Omega 3s seem to be. Guess it figures, since I know they are important to humans.
Moderator:
DrO

Posted on Wednesday, Jun 18, 2008 - 7:59 am:

Sara, it is not that simple. While essential fatty acid deficiency is a known problem in humans (and other species) deliberate attempts to produce deficiency in the horse have failed to do so and that was at levels 1/30th that of the normal diet of horses. It is quite clear that the dietary requirement for omega-3 or omega-6 is much lower in horses than other species for everyday health. However this research into deficiency states did not look at fecundity and so does not specifically conflict with Elizabeth's experience. On initial look it does not appear the simple supplementation of omega-3s from most sources will be on much benefit to these stallions but it is the specific supplementation of high levels of docosahexaenoic acid as found in fish oil.

The CSU's experience with the fish oil and it's correlation with improved membrane stability is very interesting Elizabeth and does provide us with a possible mechanism of action. It is true there is support already out there for this notion. Here is one of several research papers that supports its use in stallions of marginal fertility when "handled" semen and attributes the effect specifically to docosahexaenoic acid (DHA). This is an omega-3 that in humans does not require specific ingestion as it can be synthesized from other omega-3's but the process aparrently does not produce the same levels as a diet high in DHA. We do not know of the situation in horse metabolism. Currently fish oil is the richest source of DHA I know.
DrO
PS thank you for bringing this information to us, in my opinion one of the real strengths of Horseadvice is the way our thousands of members keep us in touch with the very edge of the practical application of the newest research.

Theriogenology. 2005 Mar 15;63(5):1519-27.
Effect of feeding a DHA-enriched nutriceutical on the quality of fresh, cooled and frozen stallion semen.

Brinsko SP, Varner DD, Love CC, Blanchard TL, Day BC, Wilson ME.
Department of Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4475, USA.

Eight stallions were used in 2 x 2 crossover study to determine if feeding a nutraceutical rich in docosahexaenoic acid (DHA) would improve semen quality. Stallions were randomly assigned to one of two treatment groups (n = 4 per group). Stallions were fed their normal diet (control) or their normal diet top-dressed with 250 g of a DHA-enriched nutraceutical. Feeding trials lasted for 14 week, after which a 14-week washout period was allowed and the treatment groups were reversed for another 14 week feeding trial. Feeding the nutraceutical resulted in a three-fold increase in semen DHA levels and 50% increase in the ratio of DHA to DPA in semen. Sperm motion characteristics in fresh semen were unaffected by treatment. After 24 h of cooled semen storage in an Equitainer, total and progressive motility did not differ between treatment groups, but sperm from stallions fed the nutraceutical exhibited higher velocity and straighter projectory (P < 0.05). After 48 h of cooled storage, increases in the percentages of sperm exhibiting total motility (P = 0.07), progressive motility (P = 0.06) and rapid motility (P = 0.04), were observed when stallions were being fed the nutraceutical. For a subset of four stallions, whose progressive sperm motility was <40% after 24 h of cooled storage when fed the control diet, feeding the nutraceutical resulted in improvements in mean progressive motility of sperm after 24 h (P = 0.10) and 48 h (P = 0.03) of storage. Feeding the nutraceutical resulted in similar improvements in motion characteristics being observed in frozen-thawed semen. While it appears that feeding the nutraceutical may improve the motion characteristics of cool-stored stallion semen, it may be most beneficial for stallions of marginal fertility whose sperm do not tolerate the rigors of cooling and storage. The nutraceutical also appeared to improve the freezability of semen. More dramatic improvements in semen quality may be observed if modifications in the main fat content of the diet are incorporated with the DHA supplement.
Member:
ekaufman

Posted on Wednesday, Jun 18, 2008 - 8:18 pm:

Thank you for the paper, DrO. I find the 14 day trial period interesting. CSU was emphatic that the Omega/DHA supplementation be continued for at least 60 days (because that is the rough term for spermatogenesis), and we didn't see changes in freezing motility until roughly 70 days after I had started supplementation.

(In fact, our test at 68 days showed the same poor results as pre-supplementation. I did not want to spend the money on further testing, so the successful test happened because a member of the research team got curious and did a test freeze at their own expense on our final collection. Just a little pitch for the dedicated folks at CSU.)
Moderator:
DrO

Posted on Thursday, Jun 19, 2008 - 8:04 am:

Umm, if it is the research piece you are referring to, that is 14 weeks not 14 days. Did I misunderstand your reference?
DrO
Member:
ekaufman

Posted on Thursday, Jun 19, 2008 - 10:28 am:

Sorry-- looks like my reading skills need some brushing up. Weeks makes more sense.

Our final test freeze also came back with comparable improvements.

For this stallion, this year, it certainly seems that the supplementation has been very helpful.

For future reference, I plan to investigate how these researchers accomplished the DHA supplementation in order to avoid the fishy food problems we had this summer. If I get any feedback, I'll post it up for others.

Thanks!
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