Open Journal of Obstetrics and Gynecology, 2013, 3, 722-726 OJOG
http://dx.doi.org/10.4236/ojog.2013.310133 Published Online December 2013 (http://www.scirp.org/journal/ojog/)
Effect of administration of phenylephrine immediately
after low dose insemination on pregnancy rates in mares*
Theresa Beachler1, Renan Sper1, C. Scott Bailey1#, James Shewmaker2, C. Nick Buchanan1,
Anne Josson-Schramme1,3, Michael Whitacre1
1College of Veterinary Medicine, North Carolina State University, Raleigh, USA
2School of Veterinary Medicine, Ross University, Basseterre, Saint Kitts and Nevis
3Current Address: VetAgro Sup, Université de Lyon, Lyon, France
Received 17 October 2013; revised 15 November 2013; accepted 23 November 2013
Copyright © 2013 Theresa Beachler et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
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ABSTRACT
There is considerable pressure on equine veterinari-
ans to achieve good pregnancy rates with very small
doses of semen. Phenylephrine administration in the
rabbit increased numbers of oviductal, uterine, and
cervical sperm, myometrial contractions, and fertil-
ized ova after low dose insemination. The use of
phenylephrine to enhance uterine contractility and
fertility has not been investigated in the mare. Thus,
the objective of this study was to determine if pheny-
lephrine administration would result in clinically ac-
ceptable pregnancy rates in mares bred by low dose
insemination. The hypothesis (Ha) was that preg-
nancy rates would be significantly higher in mares
receiving phenylephrine compared to saline controls.
Six pony mares and eight horse mares were enrolled
in this study. Mares were inseminated within 24
hours of ovulation with 30 million progressively mo-
tile spermatozoa from a single fertile stallion. Imme-
diately following insemination, mares were adminis-
tered either phenylephrine (0.06 mg/kg) or 1mL of
saline via IV injection. Pregnancy status was deter-
mined 14 days following ovulation via transrectal
ultrasonography. Pregnancy rates in phenylephrine
treated mares were 44% (4/9) while 22% (2/9) in sa-
line-treated mares (P > 0.05).
Keywords: Artificial Insemination; Low Dose
Inseminati on; Eq ui ne; Phenylephrine
1. INTRODUCTION
Low dose insemination provides the opportunity to in-
crease the number of offspring a valuable stallion can
produce in a season, and extends the availability of fro-
zen semen from deceased or infertile stallions. In addi-
tion, the development of new advanced reproductive
techniques, such as gender selection of equine sperm and
the use of density gradients for centrifugation, places
pressure on the equine industry to achieve acceptable
pregnancy rates with very low numbers of sperm. It is
known from work in horses as well as other species that
only a fraction of the original inseminate arrives in the
oviducts following natural service or standard artificial
insemination [1-3]. Therefore, techniques that maximize
the percentage of sperm that reach the oviduct may im-
prove pregnancy rates when fewer sperm are used. Tech-
niques that have been previously described in the mare to
enhance sperm delivery to the oviduct include rectally
guided deep horn insemination and hysteroscopic in-
semination [4-7]. As both of these are technically de-
manding and not widely available, there is a need for
alternate methods of reducing the total number of sperm
in a breeding dose while maintaining acceptable per cy-
cle pregnancy rates. One such method would be the phar-
macologic stimulation of sperm transport through en-
hancement of uterine contractility at the time of breed-
ing.
In the mare, the exogenous administration of both
prostaglandin E2 (PGE2) and oxytocin has been investi-
gated to facilitate gamete transport with disap pointing or
inconsistent results [8,9]. In contrast, administration of
alpha adrenergic agonists has resulted in increased fertil-
ity after low dose insemination in the rabbit and ewe
[10-12].
*CONFLICT OF INTEREST: None of the authors has any financial or
p
ersonal relationships that could inappropriately influence or bias the
content of the paper.
#Corresponding author.

T. Beachl e et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 722-726 723
Phenylephrine, an alpha adrenergic agonist, is rela-
tively selective for α1 receptors over α2 receptors. It is
commonly used in animals as a decongestant, mydriatic
agent, and to diagnose Horner’s Syndrome. The primary
effect of activation of α1 adrenoceptors is to increase
smooth muscle tone, corresponding to an increase in in-
tracellular calcium. In rabbit does, phenylephrine has
been shown to increase frequency and amplitude of pri-
mary uterine contractions and the frequency of secondary
contractions [10]. The administration of phenylephrine at
the time of breed ing resulted in th e recovery of increased
numbers of spermatozoa from the cervices, uterus and
oviducts of sacrificed does [10]. The same authors dem-
onstrated that phenylephrine administration increased the
frequency of uterine contractions in ewes, but did not
find improved sperm recovery from the oviducts of sac-
rificed animals [11,12]. Fertility trials have not been
performed in the ewe, but administration of phenyleph-
rine at time of low-dose insemination has been shown to
increase recovery of fertilized ova from rabbit does [10].
No previous work has been conducted investigating this
aspect of phenylephrine administration in the mare.
Therefore, the objectiv e of this study was to determine
whether administration of phenylephrine immediately
after insemination would result in clinically acceptable
pregnancy rates in mares that had been inseminated with
low numbers of sperm. We hypothesized that pregnancy
rates would be significantly higher in mares that received
phenylephrine compared to those that received a saline
control.
2. MATERIALS AND METHODS
Six pony mares and eight Quarter Horse or Thor-
oughbred mares, as well as one Quarter Horse stallion,
were used in this study during the months of June
through August. Mar es were three to 15 years of age and
were housed on pasture at two univer sity-own ed farms in
central North Carolina. Seven mares were housed at the
Teaching Animal Unit in Raleigh NC, and seven mares
were housed at the Equine Health Center at Southern
Pines. The stallion was maintained in an individual pas-
ture on the grounds of the Equine Health Center at
Southern Pines. All procedures were in accordance with
North Carolina State University’s Institutional Animal
Care and Use Committee’s guidelines for the humane
treatment of research animals (IACUC ID#10-048-O).
All animals had a complete breeding soundness exam
prior to enrollment in the study, including ultrasound
exam of the uterus, speculum and digital exam of the
vagina and cervix and a uterine biopsy. Mares were cy-
cling regularly and had no evidence of reproductive
complications on examination. At the onset of the study,
mares were randomly divided into two separate groups,
one receiving phenylephrine and one receiving saline. A
cross-over study design was used in which treatment
groups were switched for subsequent estrous cycles, re-
sulting in a total of 28 cycles in the study. Transrectal
palpation and ultrasonographic examinations of the re-
productive tract were performed every other day for
breeding management. Mares identified in early to mid
diestrus were given Prostaglandin F2α (Lutalyse, Pfizer, 5
mg IM) to induce estrus. Late diestral mares were al-
lowed to return to estrus without treatment. When mares
were detected in estrus and exhibited a dominant follicle
greater than 30 - 35 mm on transrectal ultrasound, 1500 -
2000 international units (IU) of human chorionic gonad-
otropin (hCG, Chorulon, Intervet Schering Plough Ani-
mal Health Corp), depending on body weight, were ad-
ministered intravenously to induce ovulation. Twenty-
four hours later, mares were inseminated with 30 million
progressively motile spermatozoa extended in commer-
cial semen extender (INRA 96, IMV Technologies) in
the uterine body.
Semen was collected from a single fertile Quarter
Horse stallion using a Colorado-model artificial vagina.
Immediately following collection, laboratory analysis of
raw and extended semen was performed, including pho-
tometric measurement of sperm concentration (Densime-
ter; Animal Reproduction Systems) and measurement of
motility and morphology via ligh t microscopy (Figure 1).
The fresh semen was extended with INRA 96 (IMV
Technologies) up to a maximum volume of 10 mL, with
the goal of obtaining a final concentration of 25 million
sperm/mL. Each insemination dose contained 30 million
progressively motile spermatozoa and was protected
from light until insemination. Seven mares housed onsite
were inseminated with fresh extended semen, whereas
semen was cooled for up to 8 hours in a commercial
shipper (Equine Express II Cooled Semen Shipper;
Exodus Breeders Corporation) prior to insemination of
seven mares located in Raleigh.
Figure 1. Equine semen sample stained with Eosin-Nigrosin
Stain for evaluation of morphologic charac teristics.

T. Beachl e et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 722-726 724
Mares were restrained in stocks with their tails wrapp-
ed and tied to the side away from the vulva and perineum
for artificial insemination. After removing feces from the
rectum, the perineal area was washed with beta di ne scrub,
rinsed with water, and dried with a paper towel to re-
move fecal contamination. Insemination of all mares was
performed by a single individual. Wearing a sterile lubri-
cated glove, an insemination pipette was passed through
the vulvar lips into the vaginal vault. The insemination
pipette was then guided through the external cervical os
and cervix and advanced 1 to 2 cm into the uterine body
before depositing the semen. Immediately following in-
semination, mares were given either phenylephrine (0.06
mg/kg) or 1 mL of saline via intravenous injection. On
the following day, mares were examined via transrectal
ultrasonography to detect ovulation. If ovulation had not
occurred, mares were inseminated and treated a second
time as described above. Mares that did not ovulate
within 72 hours of the hCG administration or which had
evidence of endometritis were excluded from this study.
Pregnancy status was determined 14 days following
ovulation via transrectal ultraso nographic examination to
identify the presence of an embryonic vesicle (Figure 2).
At this time, pregnant animals received Prostaglandin F2α
(Lutalyse, Pfizer, 5 mg IM) to terminate the pregnancy.
A priori analysis using the program GPower 3.1 (G*
Powe r, Dü ss eld or f, G e rman y) w as pe rf or med p r ior to the
onset of the study. Based on previous work with the stal-
lion enrolled in this study and the results of Hawk and
coworkers, power analysis indicated the need for 14 ani-
mals, examined over 2 cycles. At the conclusion of the
study, data were statistically examined for differences
between groups, differ ences between the first and second
cycle in the study, differences between farms, and dif-
ferences between mares inseminated once or twice. The
statistical software program Statistix 8.1 (Statistix®,
Analytical Software Inc, Tallahassee FL) was utilized for
all analyses and data were tested using Wilkoxon Rank
Sum tests. Differences were considered significant at P <
0.05.
3. RESULTS
Out of a total of 28 possible cycles from 14 mares during
the period of the study, 18 cycles qualified for inclusion
in the analysis. Three cycles were excluded because the
mares failed to ovulate within 72 hours of hCG admini-
stration, 4 cycles were excluded because mares ovulated
unexpectedly before insemination, and 3 cycles were
excluded due to ultrasonographic or cytologic evidence
of endometritis. Of the 18 included cycles, the pregnancy
rate was 44% (4/9) for phenylephrine treated cycles and
22% (2/9) for the saline-treated cycles (P = 0.62; Table
1). No differences were detected in pregnancy rates by
location (3 pregnant mares in each location), cycle (4
Figure 2. Ultrasonographic image of an
equine embryonic vesicle 14 days post-ovu-
lation.
Table 1. Pregnancy rates in mares after insemination with 30
million spermatozoa and treatment with phenylephrine or sa-
line.
Pregnant Open
Total
Estrous
Cycles Pregnancy Rates
TXa 4 5 9 44%
CTb 2 7 9 22%
aPhenylephrine HCl (0.06mg/kg) injected intravenously immediately
after insemination; b1 ml of Saline injected intravenously immediately
after insemination.
pregnancies were detected during the first cycle, 2 during
the second), insemination number (3 pregnant mares
were insemi nated once, 3 wer e inseminated twi ce).
4. DISCUSSION
Altering contractility may be a viable means of improv-
ing pregnancy rates in mares when suboptimal doses of
sperm or compromised sperm are used for insemination.
This is supported by studies in rabbits and sheep, which
demonstrate that phenylephrine and other uterotonic
agents enhance uterine contractility, sperm numbers at
the site of fertilization and fertilization rates [10-12].
These studies also demonstrated, however, that any posi-
tive effect on pregnancy rates is likely dose-specific. In
mares, convincing evidence also exists to suggest that
uterine contractility is an important component of sperm
transport [13-17]. Artificial insemination alone has been
shown to cause an initial increase in the myoelectrical
activity of the uterus, resulting in transport of sperm
through the female tract [18]. A second increase in
myoelectrical activity, beginning 4 hours post breeding
and lasting up to 12 hours, results from an inflammatory
reaction to the semen and is associated with semen
clearance from the reproductive tract [18]. Current re-
search in mares is limited by the difficulty of objectively
quantifying uterine contractility in response to insemina-
tion or drug administration. Most techniques described

T. Beachl e et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 722-726 725
are invasive or not applicable to fertile breedings
[14,15,18]. While less invasive, ultrasonographic evalua-
tion of uterine contractility is subjective and may not be
sufficiently sensitive to differentiate propulsive contrac-
tions from those that serve to evacuate the uterus. Con-
sequently, the outco me selected for this and prior studies
has been pregnancy rates in treated and untreated mares.
While no statistical differences were detected in the cur-
rent study, a doubling in pregnancy rates from 22% to
44% in phenylephrine-treated mares may be clinically
important. Varner and coworkers demonstrated similar
numerical improvements in pregnancy rates with PGE
infused into the tip of the u terus [9 ], wher eas intr avenou s
and intramuscular oxytocin administration resulted in a
numerical decrease in pregnancy rates compared to sa-
line controls [8]. While higher pregnancy rates would be
desired, a 44% pregnancy rate approaches that of other
methods described for low dose insemination [4,6,9] and
may be clinically useful, particularly given the ease of
administration.
There were several limitation s to the current stud y: the
exclusion of 10 cycles from analysis reduced the statisti-
cal power of the study; further, the relatively high preg-
nancy rate found in the control group was unexpected
based on previous work with this stallion. A larger sam-
ple population of mares and a smaller dose of motile
sperm (5 million or fewer) may have resulted in statisti-
cal differences.
Altering the phenylephrine dose may enhance propul-
sive contractions and improve pregnancy rates in future
studies. The current dose was selected based on its prior
use in horses to induce contraction of the spleen in the
treatment of nephrosplenic entrapment, however the
uterine effects of this dose have not been evaluated. In
rabbits, Hawk and coworkers demonstrated that 5mg of
phenylephrine, but not a higher or lower dose, was effec-
tive at enhancing sperm delivery to the oviduct [10].
Further, the route of administration selected in this study
may have affected the outcome. While phenylephrine is
generally administered to horses intravenously to treat
nephrosplenic entrapments, the drug was administered
intramuscularly in the rabbit and ewe. Changing the
route of administration may result in an alteration of the
intensity or timing of uterine contractions to promote
sperm-transport in the desired direction.
5. CONCLUSION
In conclusion , although this stud y failed to demonstrate a
statistical improvement in pregnancy rates, the numerical
increase may be clinically relevant. The results suggest
that pregnancy rates after low dose insemination of
mares might be improved by the use of uterotonic agents
including phenylephrine. A sensitive and non-invasive
technique is needed to measure uterine contractile pat-
terns in response pharmacologic stimuli, to best deter-
mine the clinically app ropriate dose.
6. ACKNOWLEDGEMENTS
The authors thank Jori Vasgaard for her invaluable technical and edito-
rial assistance.
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LIST OF ABBREVIATIONS
mg: milligram
IM: intramuscular
IU: intrauterine
hCG: hum a n chori o ni c go nadotropin
mL: milliliter