Effect of administration of phenylephrine immediately after low dose insemination on pregnancy rates in mares

doi:10.4236/ojog.2013.310133

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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

Email: #[email protected]

Received 17 October 2013; revised 15 November 2013; accepted 23 November 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-

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

ersonal relationships that could inappropriately influence or bias the

content of the paper.

#Corresponding author.

OPEN ACCESS

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.

REFERENCES

[1] First, N.L., Short, R.E., Peters, J.B. and Stratman, F.W.

(1968) Transport and loss of boar spermatozoa in repro-

ductive tract of sow. Journal of Animal Science, 27,

1037-1040.

[2] Overstreet, J.W. and Cooper, G.W. (1978) Sperm trans-

port in reproductive-tract of female rabbit .1. Rapid tran-

sit phase of transport. Biology of Reproduction, 19, 101-

114. http://dx.doi.org/10.1095/biolreprod19.1.101

[3] Bader. H. (1981) Sperm population in the genital-tract of

the mare. Zuchthygiene-Reproduction in Domestic Ani-

mals, 16, 72-73.

[4] Lindsey, A.C., Bruemmer, J.E. and Squires, E.L. (2001)

Low dose insemination of mares using non-sorted and

sex-sorted sperm. Animal Reproduction Science, 68, 279-

289. http://dx.doi.org/10.1016/S0378-4320(01)00165-8

[5] Mari, G., Santi, A., Merlo, B., Iacono, E., Mislei, B. and

Roberts, W. (2006) Pregnancy rates obtained with low

numbers of frozen spermatozoa inseminated into the tip

or the body of the uterus in mares. Animal Reproduction

Science, 94, 378-380.

http://dx.doi.org/10.1016/j.anireprosci.2006.04.029

[6] Brinsko, S.P., Rigby, S.L., Lindsey, A.C. Blanchard, T.L.,

Love, C.C. and Varner, D.D. (2003) Pregnancy rates in

mares following hysteroscopic or transrectally-guided

insemination with low sperm numbers at the utero-tubal

papilla. Theriogenology, 59, 1001-1009.

http://dx.doi.org/10.1016/S0093-691X(02)01123-8

[7] Morris, L.H.A., Hunter, R.H.F. and Allen, W.R. (2000)

Hysteroscopic insemination of small numbers of sper-

matozoa at the uterotubal junction of preovulatory mares.

Journal of Reproduction and Fertility, 118, 95-100.

[8] Rigby, S., Hill, J., Miller, C., Thompson, J., Varner, D.

and Blanchard, T. (1999) Administration of oxytocin

immediately after insemination does not improve preg-

nancy rates in mares bred by fertile or subfertile stallions.

Theriogenology, 51, 1143-1150.

http://dx.doi.org/10.1016/S0093-691X(99)80017-X

[9] Woods, J., Rigby, S., Brinsko, S., Stephens, R., Varner, D.

and Blanchard, T. (2000) Effect of intrauterine treatment

with prostaglandin E-2 prior to insemination of mares in

the uterine horn or body. Theriogenology, 53, 1827-1836.

http://dx.doi.org/10.1016/S0093-691X(00)00318-6

[10] Hawk, H.W., Cooper, B.S. and Conley, H.H. (1982) In-

creased numbers of sperm in the oviducts and improved

fertilization rates in rabbits after administration of

phenylephrine or ergonovine near the time of insemina-

tion. Journal of Animal Science, 55, 878-890.

T. Beachl e et al. / Open Journal of Obstetrics and Gynecology 3 (2013) 722-726

726

OPEN ACCESS

[11] Hawk, H.W. and Conley, H.H. (1985) Effect of pros-

taglandin-F2-alpha, phenylephrine and ergonovine on

uterine contractions in the ewe. Journal of Animal Sci-

ence, 60, 537-543.

[12] Hawk, H.W. and Cooper, B.S. (1984) Improvement by

ergonovine of sperm transport, fertilization and preg-

nancy rates in ewes in natural or prostaglandin-induced

estrus. Journal of Animal Science, 59, 754-763.

[13] Katila, T. (1999) Uterine contractility in nonpregnant

mares. Pferdeheilkunde, 15, 574-578.

[14] Katila, T. (2001) Sperm-uterine interactions: A review.

Animal Reproduction Science, 68, 267-272.

http://dx.doi.org/10.1016/S0378-4320(01)00162-2

[15] Troedsson, M.H.T., Liu, I.K.M. and Crabo, B.G. (1998)

Sperm transport and survival in the mare: A review (vol

49, pg 905, 1998). Theriogenology, 50, 807-818.

[16] Campbell, M.L.H. and England, G.C.W. (2002) M-mode

ultrasound imaging of the contractions of the equine

uterus. Veterinary Record, 150, 575-577.

http://dx.doi.org/10.1136/vr.150.18.575

[17] Campbell, M.L.H. and England, G.C.W. (2004) Effect of

teasing, mechanical stimulation and the intrauterine infu-

sion of saline on uterine contractions in mares. Veterinary

Record, 155, 103. http://dx.doi.org/10.1136/vr.155.4.103

[18] Troedsson, M.H.T., Steiger, B.N., Ibrahim, N.M., King,

V.L., Foster, D.N. and Crabo, B.G. (1995) Mechanism of

sperm-induced endometritis in the mare. Biology of Re-

production, 52, 133-133.

LIST OF ABBREVIATIONS

mg: milligram

IM: intramuscular

IU: intrauterine

hCG: hum a n chori o ni c go nadotropin

mL: milliliter