PROTOCOLS FOR MARE MANAGEMENT WHEN USING FROZEN SEMEN
(The same protocols can be used for cooled transported semen, with some timing relaxations)
By Jos Mottershead
USING NO PHARMACOLOGICAL MANIPULATION
Obviously one major key ingredient in any breeding program from a mare owner's point of view, is the awareness of when the mare
is in estrus. This is especially so in a breeding program that uses artificial insemination of cooled transported, or frozen semen. This can be achieved by careful monitoring with palpations and/or
ultrasound, the "teasing" of mares, or - most effectively - a combination of both. Geldings can often be used to tease mares, but if mares are commonly housed with the geldings, or in an adjacent
pasture, it may be necessary to remove one or other from the area for a major portion of the day, and monitor the response when the animal is re-introduced.
One common error that occurs with mare owners who monitor their mares without the use of a stallion to tease, is the belief that when the mare first shows estrus, she is just "entering" the
estrus phase of the cycle. In many instances, the mare is in fact in an advanced state of estrus when she finally "shows" - her response is a desperate plea for the attention of a stallion NOW!
Often this response is not seen until the mare is very close to ovulation. Misinterpretation of this being the start of estrus will result in missing the ovulation.
If you have a mare who you are sure is just entering estrus, it is safe to wait until about day three of the cycle before a veterinarian is needed to evaluate her state. This is assuming that
the mare is a mare that shows a "normal" estrus duration of 5 to 7 days. Most mares will ovulate 24 to 48 hours prior to the end of estrus as evidenced by "standing heat". This means that a mare
that is examined at about day 3 of estrus will be "mid-estrus", and will probably have a follicle of around 3-4 centimeters size. If the mare has not had a uterine swab sample taken on a previous
cycle, this may be taken at this time, and a cytology smear - looking for the presence of neutrophils - can be used to evaluate the possible presence or absence of an infection. If neutrophils are
present, then a culture of the swab must be prepared and read, with a sensitivity test of the resulting growth to various antibiotics performed (as this takes time, there is the possibility of
missing breeding on this cycle, so the evaluations are better performed on the cycle prior to intended breeding). An examination of the mare at this stage in her cycle should be performed with
ultrasound. A review of follicular size and activity, presence of any fluid in the uterus noted and treated, and the observation and recordation for future reference of the position of any cysts
should be carried out.
If uterine fluid is seen at this stage, providing neutrophils have not been detected in the cytology smear, (which would indicate inflammatory response, and possible infection), the use of
oxytocin may be adequate to clear the fluid. This may be coupled with estrogen to encourage relaxation of the cervix, aiding in the expulsion of the fluid. Poor uterine tone may be aided by a
similar treatment, or with the use of oxytocin alone. If the fluid depth exceeds 2.5 cm, uterine lavage with either sterile saline or Lactated Ringer's solution coupled with oxytocin treatment is
indicated prior to commencing breeding.
If all appears to be in good shape, and a follicle in the 3 to 4 cm range is found, the mare can be "followed" using rectal palpation. Ultrasound can also be used to "follow the follicle", but
repeated ultrasounds can increase breeding costs significantly, and some veterinarians do not routinely transport their ultrasounds with them on farm calls, so a combination of palpations and
ultrasound examinations may be both cost-effective and efficacious. The number and frequency of checks will vary depending upon the results of the initial ultrasound examination, and often the
value of the semen! If the follicle size is tending towards smaller at the initial evaluation, it will probably be safe to wait up to 2 days before further evaluation. If the follicle is larger, or
noted to be suggestive of being closer to ovulation, then the next check should be carried out much sooner.
When imminent ovulation is anticipated as a result of ultrasound evaluation showing a change in shape, thickening of the follicular wall, and reduction in uterine edema; or the "softening" of a
large follicle per rectal palpation, checks should be carried out as often as every 6 hours. This will fit nicely into the time frame demanded for the artificial insemination of frozen semen - no
more than 12 hours prior to, or 6 hours after ovulation. The ideal time for insemination is at the time of ovulation, or within the 6 hours preceding. There is a greater incidence of early
embryonic loss in mares inseminated greater than 6 hours after ovulation.
If ultrasound is available, its use the day after insemination (assuming palpation has determined a timely ovulation) is valuable to evaluate fluid build up, if present. Some mares will show a
greater inflammatory response and associated fluid build up than others. It is particularly noted in older mares, and is believed to be more of an indication of the mare's inability to clear the
fluid, rather than an excessive response itself. The actual causative agent of the response has been shown to not be the egg yolk or glycerol in the extender as was previously thought, but rather
the sperm themselves.
If fluid is present post breeding, the use of oxytocin can be beneficial at that time also. Multiple treatments of 10-20 IU, 6 hours apart can be carried out until as
late as 3½ days post-ovulation. Some like to routinely use 10-20 IU of oxytocin 4 hours after breeding with frozen or cooled transported semen, as the natural release of oxytocin present
with live cover is reduced or does not occur with AI. The use of prostaglandin F2α or one of its synthetic analogues such as cloprostenol
("Estrumate™" - Schering-Plough Corporation Kenilworth, NJ USA) - which has a longer half-life and can therefore be used every 12 hours, rather than every six hours, such as
oxytocin requires - to aid post-breeding clearance has been shown to interfere with the successful formation of a fully functional corpus luteum, and its use is therefore contra-indicated after 24 hours
post-ovulation, although prior to that it may be of value.
USING PHARMACOLOGICAL MANIPULATION:
PROSTAGLANDIN F2 alpha (PGF2α)
The use of prostaglandin (PGF2α) - in North America found in the common trade names of
"Lutalyse" or "Prostin F2 Alpha" (Pharmacia & Upjohn Company, Kalamazoo, MI), or "ProstaMate" (Phoenix Pharmaceutical, Inc St. Joseph, MO) - is probably the commonest form of
hormonal manipulation of the mare used to induce estrus (commonly referred to as short cycling). The problem with the use of this hormone is that it will only be reliable if given at a certain
point in the mare's cycle. There must be an active Corpus Luteum (CL) for the PGF2α to act upon, so use within about 4-5 days after an ovulation, or in an anestrus
mare, will not result in estrus. One can simply rely on luck and external observation to ensure that this drug is given a minimum of 6 days after the mare has ovulated, but even assuming that you
are fortunate enough to be able to achieve this, there is still the possibility that the mare will produce a "mid cycle follicle". If the PGF2α is given with a large
active follicle present, there is the possibility that the mare will enter estrus and ovulate within 3 days after dosage, with lowered fertility1.
In order to ensure there is an active CL present, it is possible to carry out a blood-progesterone assay. If progesterone is present in levels of 4 ng/ml or greater, this is a guarantee that
there is an active CL present. Lesser amounts may indicate that the mare is either entering or leaving estrus, or in estrus or a state of anestrus, and the testing should be repeated in 2-3 days.
If the progesterone levels have increased then, it is indicative of leaving estrus, and the mare may well already have, or will shortly have a CL receptive to prostaglandin. If the progesterone
level has dropped on the subsequent test, she is entering estrus, and normal monitoring procedures may be followed. Onset of estrus is normally anticipated 3 to 5 days following the administering
of PGF2α at the right stage in an estrous cycle, with no active mid-cycle follicle present.
The successful use of prostaglandin to synchronize mares is limited, although a reasonable degree of synchronicity is seen if mares are given two full doses of prostaglandin 14 days apart. In
that case, by the time of the second dose being administered, both mares should have a responsive CL present. Although not researched or demonstrated, use of the two micro-doses of 2α on days 0 and 1 and again on days 13 and 14 should produce the same effect without the unpleasant side-effects (see below).
A common cause for concern connected with the use of prostaglandin to short cycle mares are the potentially dramatic side effects. Transient sweating and colic-like symptoms are frequently seen
commencing 5 to 10 minutes after dosage. These symptoms have usually disappeared an hour later, but often not before causing significant alarm for the mare owner! The cause of the side effects is
constriction of the smooth muscle of the body, which includes not only muscle under the skin (hence the - at times - profuse sweating), but also the uterus (hence colic-like symptoms as a result of
cramping). It is extremely rare that these side-effects cause significant problems, although occasionally they do, most commonly as the affected horse reduces water consumption resulting in a
full-blown colic. The use of cloprostenol ("Estrumate" - Schering-Plough Corporation, Kenilworth, NJ), rather than prostaglandin may avoid these side-effect, although not in all animals. It
should be noted that Estrumate is not labelled for use in the equine, but is commonly used off-label for this purpose. It has been shown that the use of one-tenth of the normal dose of
prostaglandin given on two consecutive days at least 6 days after the previous ovulation will result in a return to estrus in the same 3-5 day interval as the full dose, but without the side
effects2. It is believed that the lack of side effects is as a result of this compounding of a low dose more closely mimicking the natural
release of prostaglandin.
PROGESTERONE OR ALTRENOGEST:
Progesterone is available in the USA as a compounded product from a variety of compounding pharmacies, or as the synthetic progestin analogue altrenogest.
Altrenogest is commercially produced as RegumateTM (Intervet - Millsboro, DE, USA; Whitby, ON, Canada) or in a compounded biorelease 12 or 30 day formulation
(BET Pharm, Lexington, KY USA). One of the most important points for consideration in the use of progestins is that, although they will suppress estrus, they will not necessarily suppress
ovulation. This means that their use is not a guaranteed method of inducing timely estrus, as if the mare is producing what in effect becomes a mid-cycle ovulation, it will throw her natural
cyclical activity into an erratic state. Consequently, the mare may not come into estrus at the anticipated "normal" 3 days post cessation of the treatment.
The administering of a combination of progesterone and estradiol - specifically estradiol 17β - produces a high
degree of control of the mares estrus cycle3,4,5. The estradiol (estrogen) portion of the mixture suppresses the follicular development and ovulation of any follicle present, and allows the
progesterone to achieve the effect that is desired - but often doesn't occur - by its use alone. There is currently no "brand name" product combining these 2 hormones, but it can be obtained from
some veterinary compounding pharmacies. It is most commonly used in an injectable form that requires injections once a day for 10 days. Prostaglandin should be administered in conjunction on the
10th day, or if using the reduced dosage, we have found that it may be given on the 9th and 10th days, resulting in no unpleasant side-effects,
and no negative impact on the P&E protocol. Ovulation will occur, in over 80% of mares, when combined with an ovulation stimulator, 9 days after the last administration of the P&E. The use
of this hormonal combination is discussed at greater length in an article elsewhere on this web site.
The drawback of 10 daily injections may be overcome by the use of a biorelease formulation of P&E (BET Pharm, Lexington, KY, USA), although the timing of the resulting ovulation may not
be as accurate as that of the daily dosing system. Oral dosing has been attempted, but results have been unpredictable. The use of a vaginal sponge, or dosing directly into the vagina has been seen
to produce reliable results6, although a transient vaginitis may be present that resolves spontaneously before the onset of estrus. Early
research suggests that the biorelease formulation of altrenogest (BET Pharm, Lexington, KY, USA) may produce similarly accurate timing of ovulation as P&E7.
Promoting ovulation may be beneficial, offering a more accurate prediction of the timing of the ovulation and a reduction in necessary monitoring. Commonly used
ovulation stimulatory agents include hCG; a synthetic form of GnRH, deslorelin; and recombinant LH.
Human Chorionic Gonadotropin (hCG)
Human chorionic gonadotropin (hCG) is commercially available as Chorulon in North America (Intervet - Millsboro, DE, USA; Whitby, ON, Canada). In
order for hCG to produce reliable timing of ovulation, it must be given when there is a 35 mm follicle present. The usual dosage for a 1,000-1,500 lb. horse is considered to be anywhere between
1,500 and 3,300 IU, although 1,500 IU has been shown to be an adequate dose to achieve timely ovulation8. Overdosing - at 5,000 IU or greater
- has been seen to suppress ovulation rather than stimulate it. It is worthy of note that hCG is commonly packaged in 5,000 and 10,000 IU vials, representing multiple doses, and rather than wasting
the unused portion, freezing individual doses in suitable-sized syringes for use within the same breeding season makes the product particularly cost-effective. Repeated use of hCG in the same mare
within the breeding season has been suggested as resulting in a reduction in reliability of ovulation induction, especially in older mares9,
although this has been debated8, and clinical evidence strongly suggests an individual response.
GnRH and its analogues
The synthetic GnRH deslorelin was presented in a commercially prepared time-release implant in the product "Ovuplant™" (Wyeth
Animal Health, Guelph, ON, Canada; Peptech Animal Health, Sydney, NSW, Australia) but shortly after introduction to the US market it became apparent that in the event of failure of pregnancy
establishment, return to subsequent estrus - especially if PGF2α was used in an attempt to induce estrus once pregnancy establishment failure was identified - could
be seriously delayed10. Despite developments of techniques wherein the delay was eliminated, involving removal of the implant 48 hours after
implantation11, the product was removed from the US market and remains unavailable as of 2007.
A compounded version of deslorelin in a time-release formulation is available (BET Pharm, Lexington, KY, USA) and has been shown to be efficacious in induction of ovulation within 48 hours
if given to an estrus mare in the presence of a 30 mm follicle, with possibly improved conception rates when compared to other ovulation induction agents12.
New to the market for 2007, recombinant eLH (Aspen BioPharma, Castle Rock, CO, USA) was demonstrated as having properties
similar to hCG without development of the antibodies that may or may not result in reduction of efficacy of hCG if used repeatedly in the same breeding season13. 750 µg of reLH seems to be an optimum dose for inducing ovulation within 48 hours of treatment of a mare with a 35 mm follicle present.
MONITORING OF THE MARE UNDERGOING PHARMACOLOGICAL MANIPULATION:
If there is every reason to believe that the mare has entered estrus at the anticipated point, then the
same protocol as is followed for the non-manipulated mare may be followed for the mare that has undergone treatment with hormones. Where the problems arise are in those mares that do not respond to
hormone use as anticipated, most commonly as a result of the hormones being administered at the wrong point in the cycle (especially PGF2α), or the presence of a
large mid-cycle follicle. The use of "P&E" is strongly recommended to avoid these misadventures.
With the use of "P&E", a high percentage of mares (in the region of 80%) will enter estrus, and ovulate almost universally 9 days after the last dose of the hormones if an ovulation
stimulating agent is given on the morning of day 8, if the follicle then present is between 35 and 40 mm in size. Larger follicles may result in an earlier ovulation. Administering hCG or reLH when
follicles are smaller than 35 mm, or deslorelin when follicles are smaller than 30 mm, will have an unreliable or no effect on induction of ovulation.
The mare should receive an ultrasound examination 6 or 7 days after the administering of the last dose of P&E. If not previously taken, a uterine swab sample may be obtained at this point,
for the purposes described above. There may be a follicle of approximately 30 to 40 mm present at this stage, and if so the ovulation stimulatory agent may be administered. Palpations or
ultrasounds should be increased to as often as every 6 hours in the period commencing 24 hours after this.
It is worth noting that unless one is fortunate enough to observe such items as the "pear shaping" of a follicle, the thickening of the follicular wall, and/or the peaking and reduction of
uterine edema, it may not be easy to predict the impending ovulation using ultrasound alone. Rectal palpation to assess the "softness" of the follicle is resuming popularity as a reliable method of
assessing the likelihood of impending ovulation, although the ultrasound can be very useful to confirm ovulation did occur if a definitive ovulatory depression is not palpated.
Pharmacological manipulation - at the very least use of an ovulatory stimulating agent - also opens the door to the use of timed insemination protocols14, thereby reducing the number of ultrasounds and/or palpations required. This can reduce breeding costs significantly, although the need for multiple
insemination doses may become a factor if buying semen "by the dose".
POST OVULATORY MONITORING:
It is important to assess the mare to ensure that ovulation did indeed occur at the anticipated time. Mares are notorious for proving even
the experienced palpator wrong. Palpation or ultrasound should be used to detect ovulation, although palpation alone should be followed with ultrasound to evaluate the uterine environment as
Breeding the mare with frozen or cooled transported semen without the use of pharmacological manipulation can be successfully achieved, but may prove more difficult, costly
and less successful, especially in the absence of a "teaser" stallion or gelding. The level of success will depend greatly on the degree of monitoring, and the experience level of those monitoring
the mare. Pharmacological management if correctly performed can offer a more predictable timing for onset of estrus and the resulting ovulation in many, if not most, instances of use. This can make
the level of success greater, and often the breeding process cheaper in the long run.
NOTE: The hormones used in manipulation of the mare are the same hormones that are present in humans, and they can be absorbed through the skin if spilled. All asthmatics regardless of
gender, and pregnant women, must be particularly careful when handling prostaglandin. Progesterone, altrenogest, or Regumate must be handled extremely carefully by all women to avoid potentially
significant disruption of menstrual cycles.
1:Lindenburg H, Koskinen E, Huhtinen M, Reilas T, Perttula H, Katila T. 2002; Influence of PG
administration and follicle status on the number of conceptuses Theriogenology, 58 2-4; 571-574. 2:Wüstenhagen A, Handler J, Kindahl H and Aurich C. 2002; Luteal function and estrous cycle characteristics in mares treated with
subtherapeutic doses of prostaglandin F2α Theriogenology, 58: 2-4; 537-539. 3:Loy, R.G., Pemstein, R., O'Canna, D., and Douglas, R.H. 1981; Control of ovulation in cycling mares with ovarian steroids and prostaglandin. Theriogenology
15:191-200. 4:Varner, D.D., Blanchard, T.L., and Brinsko, S.P. 1988; Estrogens, oxytocin and ergot alkaloids - Uses in reproductive management of mares. Proc. Am.
Assoc. Equine. Pract., 219-241. 5:Lofstedt, R.M. 1988; Control of the estrous cycle of the mare. The Veterinary Clinics of North America - Equine Practice, 189-190. 6:Lofstedt, R.M. 2002; personal communication. 7:Burns P.J. 2007; personal communication. 8:Gastal E.L., Silva L.A., Gastal M.O., Evans M.J. 2006; Effect of different doses of hCG on diameter of the preovulatory follicle and interval to ovulation
in mares; Animal Repro. Sci. 94 (2006) 186-190. 9:McCue P., Hudson J.J., Bruemmer J.E., Squires E.L. 2004; Efficacy of hCG at Inducing Ovulation: A New Look at an Old Issue; Proc. AAEP 1492:1204. 10:Johnson, C.A., Thompson, Jr. D.L., Kulinski K.M., and Guitreau A.M. 2000; Prolonged interovulatory interval and hormonal changes in mares following the
use of Ovuplant™ to hasten ovulation. J. Equine Vet. Sci. 20:331-336. 11:Farquhar V.J., McCue P.M., Carnevale E.M., Nett T.M, Squires E.L. 2002; Deslorelin acetate (Ovuplant) therapy in cycling mares: effect of implant
removal on FSH secretion and ovarian function; Equine Veterinary Journal 34:4;417-420 12:Kölling M, Allen W.R. 2005; Ovulation induction for embryo transfer: hCG versus GnRH analogue; Proc. International Equine Gametes Group Workshop
II 13:Niswender K., Roser J.F., Boime I., Colgin M. 2006; Induction of Ovulation in the Mare With Recombinant Equine Luteinizing Hormone; Proc. AAEP
387:388 14:Barbacini S. 2000; Management of mares for frozen semen. Proc. 14th Int. Congr. Anim. Reprod. AI; 308.