AAEP Convention 2004 Wrap-Up: Reproduction

Pregnancies from Euthanized Mare Ovaries

"You think breeding mares on foal heat is tough, try dead mares!" said Elaine Carnevale, DVM, PhD, professor in equine reproduction in the department of biomedical sciences at Colorado State University (CSU), with a laugh as she began her talk following a presentation on foal heat breeding success. Her presentation covered CSU's work with deceased mares' ovaries and oocytes, and their success rates.

"Death or euthanasia of a valuable mare results in loss of her genetic potential," she said. "However, when a mare dies, her ovaries still contain potentially viable oocytes that can be harvested to produce additional offspring. Some mares seem to have a lot of viable oocytes, while others have very few."

Factors that can affect this are:

  • Donor variability;
  • Age and fertility;
  • Time of year (seasonal variations in ovarian activity);
  • Quality of oocytes;
  • Medical history and treatment;
  • Interval from death to harvest and use; and
  • Transportation interval and conditions.

"This technique also has clinical use," Carnevale added. "In 2001, a Quarter Horse mare was euthanized, and her ovaries were transported to CSU. We transferred five oocytes, had five embryonic vesicles in a recipient's uterus, and got a healthy foal."

She further reported that in this retrospective study, this technique had been used with ovaries from 27 mares that were four to 30 years old. The ovaries are very sensitive, so they need to be kept isolated and padded during transport, she said. Two mares that were more than 20 years old yielded zero viable oocytes or one of poor quality, so no oocytes were transferred from them. From the remaining 25 mares, a total of 226 oocytes were harvested. Oocyte transfer was done with 191 (72%) of the oocytes; one to three recipients per donor (a total of 46 recipients) had one to 14 donated oocytes placed in their oviducts.

"Thirty percent (14/46) of recipients were diagnosed as pregnant with one or more embryonic vesicles by Day 16," she reported. "Six of the 14 pregnancies (43%) were lost by Day 60. To date, no pregnancy losses have occurred after Day 60. Currently, seven foals have been born."

She noted that their work had shown an embryo loss rate of 43% for these mares. "Many (donor) mares were older, sick, and/or ovaries had been exposed to very low temperatures," she said. Also, ovarian activity at the time of death varied greatly. But the researchers had foals or ongoing pregnancies for 24% of donors.

"All of the foals have been normal and healthy," she said. "Most problems end up in embryo loss; once they get on the ground, they're pretty normal."

She described in detail procedures for removing and transporting the ovaries, as well as harvesting the eggs and working with the recipients. She noted that with a transport interval of less than one hour (between ovary harvesting and oocyte recovery work beginning at CSU), they found a pregnancy rate of 36% per oocyte recovered. However, if the transport time lengthened to eight to 26 hours, the pregnancy rate per oocyte dropped to 10%. She described the optimal temperature for long transport intervals as 12-22ºC (53.6-71.6ºF).

She summarized that roughly one of four donors had yielded pregnancies, and commented, "For younger mares, such as those dying from traumatic injury, for which transportation time was short, I think (pregnancy) rates will be quite good."

Future considerations for donor selection might include age, ovarian activity, and medical treatment, Carnevale said. Future considerations for offering the service might include increased locations to minimize transport distance and time.

One attendee asked if euthanasia solution might be problematic for the work. "Possibly, we just don't really know yet," she answered. "We don't yet know if that's actually decreasing our pregnancy rate.

"When faced with the death of a valuable mare, owners can be given the option to try to obtain additional pregnancies," Carnevale concluded. "If possible, euthanasia should be planned to minimize the transportation interval and the time that the ovaries remain in the mare after death. A semen shipment must be arranged, and hair follicles should be collected if needed for DNA analysis for the respective breed registry."

More Options

She also mentioned cryopreservation of oocytes for getting foals from subfertile mares. While this has resulted in two normal, healthy foals, Carnevale noted that the problem with this technique is, "the efficiency of the procedure is so low it probably won't help us with these already compromised oocytes."

Intracytoplasmic sperm injection (ICSI) is another option in which sperm is injected into the egg. "It reduces the amount of semen required, reduces potential cost, and might increase efficiency. It's potentially a very good way to go," she commented.

More information: www.TheHorse.com/ViewArticle.aspx?id=5400.

Stallion Fertility

While a large part of microscopic semen evaluation centers around motility (directional sperm movement) and morphology (sperm structure), some reproductive specialists think there's more to the story. In an attempt to better understand stallion fertility, Steven P. Brinsko, DVM, MS, PhD, Dipl. ACT, associate professor of theriogenology at Texas A&M University, presented the results of research investigating the relationships between sperm membrane integrity, motility, and morphology.

"Motility is important and many still look at it heavily, including me, but in a majority of studies there has been a poor correlation between motility and fertility," he said. "Similarly, morphology doesn't have as good a correlation with fertility as we might think.

"Integrity of the plasma membrane is essential for the proper sperm functions that lead to capacitation (a change in sperm that gives it the ability to acrosome react), the acrosome reaction (fusion of the outer acrosome membrane with the plasma membrane, which facilitates penetration of the egg), and fertilization," he went on. "Assessment of sperm membrane integrity has been reported to be a more accurate predictor of fertility than sperm motility. Although some sperm may seem morphologically normal and motile, membrane damage could render them incapable of fertilization."

The first hypothesis of the study was that motility, morphology, and membrane integrity are related, Brinsko said. The second was that relationships differ between the first and second ejaculates (i.e., sexually rested and sexually active samples). One hundred sperm per ejaculate were evaluated at random.

Semen from 10 stallions (presented for routine breeding soundness examination, with 10-80% per cycle pregnancy rates) was collected twice an hour apart following at least a week of sexual rest, then again the next day. "Many (sperm) looked quite morphologically normal, but stained as having disrupted membranes," Brinsko reported. Conversely, he added, "I have often been amazed at how many sperm with poor morphology stained as membrane-intact."

The results? Between normal morphology and sperm membrane integrity, there was no association whatsoever. "There was a high percentage of morphologically normal sperm with membrane damage and vice versa (morphologically abnormal, but no membrane damage)," reported Brinsko.

As for the relationship between total sperm motility and membrane integrity, again no linear relationship was found. "Some stallions had very high progressive motility, but much lower membrane integrity," he noted. "Progressive motility and normal morphology had a much better correlation (positive linear relationship).

"Overall, the percentage of normal sperm and membrane-intact sperm was not different between the first and third ejaculates," he went on. "In the first ejaculates, morphologically normal sperm were just as likely to be membrane-damaged as they were to be membrane-intact. However, the percentage of normal sperm that were membrane-damaged was much lower in the third ejaculate, where morphologically normal sperm were almost twice as likely to be membrane-intact compared to the first ejaculate."

Brinsko stated the following observations based on the study:

  • Membrane-damaged sperm can be motile.
  • Morphologically normal is not equal to membrane-intact.
  • Stored epididymal sperm can deteriorate without concomitant alteration in morphology. Therefore, good sperm morphology should not be used alone to assume that semen quality is acceptable in ejaculates of sexually rested stallions.

He also offered the following recommendations for practitioners evaluating stallion fertility:

  • Examine multiple ejaculates.
  • Examine multiple sperm attributes to give the best idea of semen quality.

In conclusion, Brinsko said, "Although fertility was not assessed in the present study, the data presented here may help to explain results of earlier studies where correlations between fertility and motility, or fertility and morphology, were poor or non-existent. These findings also raise concerns over using only the number of progressively motile sperm or number of morphologically normal sperm in an ejaculate as an assessment of semen quality.

"This would be especially worrisome if these methods were employed on the first ejaculate obtained after sexual rest to determine its suitability for cooling or freezing," he went on. "Stresses associated with cooling or freezing could magnify membrane damage that existed before processing. When this occurs, fertility could be much lower than would be expected based on the morphologic and motion characteristics of the sample."

More information: www.TheHorse.com/ViewArticle.aspx?id=5397.

Embryo Recovery, Transfer

"With the continued changes in the equine industry, particularly the reproductive industry, we need to have a reasonable embryo recovery rate," said Jason J. Hudson, DVM, veterinary resident at Colorado State University (CSU). "With multiple foals able to be registered per year, along with superovulation in the mare and advances in cryopreservation of equine embryos, we need to have the ability to have enhanced embryo recovery rates."

Hudson found that embryo recovery can be enhanced by slight modifications of the standard embryo flush technique, and by verifying that the embryo was not retained in the tip of the sheath with which the veterinarian performs the embryo transfer.

In the commercial embryo transfer program at CSU, veterinarians attempted to recover 334 embryos in 2002-2004. A standard flush of three infusions using 4 liters of media yielded a 35% success rate in recovering embryos. After instituting an extra flush, 46 more embryos were recovered (14%). The overall recovery rate was 49% (163/334).

In a recent study at CSU where Hudson modified the flush technique (see the following description), 73 flushes were performed after 255 ovulations (these mares were superovulating due to equine follicle stimulating hormone provided by Bioniche). Hudson said 148 embryos were recovered (107 embryos were recovered after the standard flush and 41 were recovered after the "extra" flush), which worked out to a 58% embryo recovery rate. He found:

  • There was an enhanced embryonic recovery rate if after the veterinarian added additional flushing media to the initial amount in the uterus, it was allowed to stay in the mare for three minutes, and the mare was given oxytocin (which helps contract the uterus and recover the fluid);
  • Age, fertility of donor mare, quality of the sire's semen, day of recovery, the number of ovulations, and the clinician's experience influence the embryo recovery rates;
  • The tip of the embryo transfer sheath should be rinsed with media post-transfer to ensure that the embryo hasn't been retained; and
  • Flushes of embryos on Day 6.5 (following ovulation) are much more difficult to retrieve than on Day 7 or early on Day 8.

The modified embryo recovery procedures were economically significant. The cost of additional media, oxytocin, and the clinician's time added up, but is minimal compared to the potential value of a recovered embryo. Hudson said it would be useful to find out if the additional flush, the three-minute waiting period, the oxytocin, or the combination of all three was the catalyst in improving recovery procedures.

More information: www.TheHorse.com/ViewArticle.aspx?id=5416.

Ovulation and hCG

Managing a mare's estrous cycle is an integral part of breeding management, and human chorionic gonadotropin (hCG) is one hormone option for doing just that. Patrick McCue, DVM, PhD, Dipl. ACT, associate professor of equine science at Colorado State University, discussed the use of hCG to manage ovulation.

"Most of us have used hCG throughout our practice lifetimes," he began. "It's used to induce a timed ovulation in in-heat mares. We expect that 75-85% of mares will ovulate within 48 hours after hCG administration, and the average interval to ovulation is about 36 hours."

He also briefly discussed the history of another ovulation induction agent--Ovuplant (deslorelin acetate, an analog of gonadotropin-releasing hormone or GnRH). The difference between the two is that hCG mimics the activity of luteinizing hormone (LH), which hastens the maturation and induces ovulation of the dominant follicle in a mare.

In comparison, deslorelin stimulates the pituitary gland to secrete endogenous LH, which will in turn cause follicle maturation and ovulation. Ovuplant was an implant that was designed to be inserted under the skin. When it was first commercially available, it was noted that some mares induced to ovulate with Ovuplant had a delay in their return to estrus. Studies showed that when the implant was removed after ovulation was detected, the incidence of failure to return to estrus was reduced. However, he said that by the end of 2003 the implant was no longer available in the United States. Compounded deslorelin has been used in the equine breeding industry in the past few years, but future availability of the compounded product is uncertain.

Consequently, hCG might once again be the only approved drug available for induction of ovulation in mares. McCue indicated that "It is controversial as to whether or not repeated use of hCG within a breeding season is associated with a loss of efficacy at inducing a timed ovulation. Repeated administration of hCG has been documented to result in formation of anti-hCG antibodies. Adverse effects, if any, are presumed to be due to the presence of these antibodies." Some studies have shown a reduced efficacy with multiple use, while others have failed to show an adverse effect.

The goal of the CSU study he presented was to evaluate data from a large number of mares given hCG to determine the effects of repeated administration during a given breeding season on efficacy, and to determine if a relationship exists between mare age and hCG effectiveness in inducing a timed ovulation. Their study found that 78% of all mares ovulated within 48 hours of hCG administration on the first cycle of the year, and about 4.4% failed to ovulate in response to hCG.

When hCG was given multiple times within a single breeding season, the percentage of mares ovulating within 24 hours after administering hCG increased and the percentage of mares ovulating between 24 and 48 hours decreased, McCue reported. In addition, the percentage of mares ovulating three to four days after hCG administration increased in later cycles during the breeding season. It was noted that a previous study had reported a seasonal effect in which an increase in the frequency of ovulation less than 24 hours after hCG administration occurred later in the spring.

The CSU study also reported a decrease in the efficacy of hCG in inducing a timed ovulation with increased mare age. He noted that a similar decrease in efficacy with age was reported for Ovuplant.

McCue noted that it was common practice to use hCG for the first cycle or two during a breeding season, then switch to an alternative ovulation-inducing agent, such as deslorelin (when available), for subsequent cycles. But, "It is unknown whether any form of deslorelin will be available during the 2005 breeding season," he concluded. "If hCG is the only agent to be used to induce ovulation in mares bred during multiple estrous cycles within a single breeding season, such as donor mares in an embryo transfer program, the ability to accurately predict the time of ovulation may be reduced later in the year."

More information: www.TheHorse.com/ViewArticle.aspx?id=5398.

Reducing Drug Costs, Side Effects

A major use of prostaglandin is to manipulate mares' estrous cycles. A veterinarian's most commonly used prostaglandin treatments are usually Lutalyse (dinoprost tromethamine or PGF2) and the prostaglandin analogue (a similar compound) Estrumate (cloprostenol or CLO). Some side effects observed when mares are treated with labeled doses of these medications include sweating and colic, and as a result owner distaste for using the drugs is growing.

Gary J. Nie, DVM, PhD, consultant for World Wide Veterinary Consultants in Rochester, Minn., conducted a study while at Auburn University where he compared "micro" doses of both products with standard doses for their ability to affect luteolysis and short-cycle mares. He also evaluated the ability of microdoses to reduce side effects observed with standard doses.

Nie found that a microdose of CLO is equally as effective at short-cycling mares as standard doses of PGF2 or CLO, yet it virtually eliminates unwanted side effects and reduces drug costs by 1,000-2,000%. He does not recommend a single microdose of PGF2, however, because of a delayed progesterone drop observed in the treated mares.

Prostaglandin's function in this case is to lyse the progesterone-producing corpus luteum left after ovulation, and return the mare to estrus. Thus, a progesterone drop is indicative of prostaglandin efficacy.

In the study, Nie treated 17 mares (in 57 cycles) intramuscularly with either the standard doses or microdoses of CLO (Nie administered only 5-10% of the standard dose of CLO as the microdose--25 micrograms rather than 250 to 500 micrograms), of PGF2 (0.5 mg instead of 10 mg), or the control (sterile water) on Day 6 after ovulation. Their progesterone levels and ovarian activity were monitored daily.

Nie said, "Their progesterone concentrations were quite interesting. We sampled out to 96 hours after treatment at 24-hour intervals. Our control mares had no change after the sterile water injections. All the prostaglandin-treated mares had significantly lower progesterone levels than the controls.

"All (of the mares' progesterone levels) dropped quickly," he said. "But the microdose of PGF2 didn't drop the progesterone concentrations like the other treatments did." He added that the progesterone levels were statistically higher than the other prostaglandin treatments. "It's like we injured the corpus luteum, but we didn't completely lyse it (cause its regression)."

When measuring systemic response to the prostaglandin treatments, he found that the microdoses caused significantly fewer and less severe side effects than either standard dose, but were very similar to the response seen in the control mares. In contrast, "Both standard doses of prostaglandin did cause significant elevations in heart rate," he said. Also, rectal temperature of the mares treated with standard doses dropped rather quickly until 90 minutes post-administration, and had not recovered to the level of control mares by 240 minutes. Nie said the drop in rectal temperature observed probably reflected heat loss through evaporation of sweat.

Nie said he has used the same microdose of CLO in a 900-pound Arabian mare as he has used in a 1,600-pound Warmblood mare, with much success in both.

More information: www.TheHorse.com/ViewArticle.aspx?id=5411.


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