AN ABSTRACT OF THE THESIS OF
Jennifer Ann Bartell for the degree of Master of Science in Animal Science presented
on December 5, 2011
Title: Porcine Zona Pellucida Immunocontraceptive Vaccine for Horses
Abstract approved: ______________________________________________________
Alfred R. Menino, Jr.
The Bureau of Land Management (BLM) maintains a growing number of feral
horses on public rangelands. With population growth rates as high as 22% annually,
herds are exceeding their carrying capacity and millions of dollars are spent maintaining
captured horses in holding facilities awaiting adoption. To manage the feral horse
population, the BLM is seeking a contraceptive that is safe, can be remotely delivered,
requires only a single administration and is effective for several years. Contraceptive
strategies have been developed for feral horses that include hormone implants, chemical
intrauterine devices, and immunocontraception. Porcine zona pellucida (pZP)
immunocontraceptive vaccines have shown great potential for providing safe, long-term
contraception in feral horses. ImmunoVaccine Technologies (Halifax, Nova Scotia,
Canada) has developed a liposome encapsulated pZP formulation known as SpayVac™
(SpayVac), which after a single-dose provides multi-year contraceptive efficacy. In a
continued effort to optimize the acceptability and efficacy of SpayVac, ImmunoVaccine
Technologies developed alternative adjuvant preparations using either killed
Mycobacterium butyricum (Modified Freund’s Adjuvant; MFA) or a proprietary nonMycobacterium based adjuvant (IVT) that are proposed to have less of the undesirable
side-effects associated with Mycobacterium tuberculosis. Therefore, the objective of
this research was to evaluate SpayVac in different adjuvant formulations for efficacy of
contraception as measured by pZP titers and estrous cyclicity in treated mares.
Domestic mares (n=28) were randomly assigned to four treatments (7 mares per
treatment): adjuvant alone or saline (Control) or SpayVac vaccines in one of three
adjuvant preparations: IVT or MFA in either an aqueous (MFA aq) or non-aqueous
(MFA non-aq) suspension. Pre-immune blood samples were collected from each mare
and mares were injected in the neck with a single injection of the Control or SpayVac.
Subsequent blood samples were collected at weekly intervals for 26 weeks. Sera were
analyzed for pZP titers and progesterone using ELISA. At the conclusion of the study,
ovaries were recovered by ovariectomy (16 mares) or at necropsy (12 mares) for
histologic analysis and collection of morphometric data and oocytes. Titers for pZP
were greater (P<0.05) in IVT and MFA mares compared to Control mares and for MFA
compared to IVT mares. Mares vaccinated with MFA aq had greater (P<0.05) pZP titers
at 2 weeks post-injection compared to mares injected with IVT or MFA non-aq and at 3
weeks post-injection compared to mares injected with IVT. MFA non-aq mares had
greater (P<0.05) pZP titers at 6 weeks post-injection compared to IVT mares and,
although not significantly different, titers in MFA non-aq mares remained greater
during weeks 8, 10, 14, 18 and 22 compared to IVT and MFA aq mares. Mean serum
progesterone concentrations were greater (P<0.05) in Control compared to MFA non-aq
mares. Mean ovarian weights, oocyte diameters, zona pellucida thicknesses and the
number of horse sperm bound to oocytes recovered from vaccinated mares were greater
(P<0.05) in Control mares compared to IVT and MFA mares. As judged by pZP titers
and serum progesterone, these results suggest that SpayVac suspended in the MFA nonaqueous formulation exerted the greatest contraceptive effects in treated mares. This
preparation of SpayVac may meet the criteria cited by the BLM for their most desirable
immunocontraceptive.
©Copyright by Jennifer Ann Bartell
December 5, 2011
All Rights Reserved
PORCINE ZONA PELLUCIDA IMMUNOCONTRACEPTIVE VACCINE FOR
HORSES
By
Jennifer Ann Bartell
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented December 5, 2011
Commencement June 2012
Master of Science thesis of Jennifer Ann Bartell presented on December 5, 2011.
APPROVED:
Major Professor, representing Animal Science
Head of the Department of Animal Sciences
Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Jennifer Ann Bartell, Author
ACKNOWLEDGEMENTS
This is my chance to take a moment to thank those who aided in the completion
of this project. There are many people that aided in the accomplishment of finishing my
thesis; family, friends, co-workers, advisers and others. To be able to thank everyone
involved is impossible, but all should know I appreciated their efforts and thank them. I
first and foremost want to thank Oregon State University for the opportunity to
experience this amazing opportunity. If it was not for Dr. Menino offering me the
opportunity to work with him and allowing me to follow my love of horses by working
on this equine project, I would not have met my goal in such an interesting and
educational way.
My primary investigator of the study, Dr. Bechert allowed me this unique
opportunity to work with her on this study and indulge my curiosity by exploring new
parameters of the project, as well as providing me with great assistants on the ranch to
do some difficult horse wrangling every Monday, rain or shine. Scott Oeffner,
Makensie Anderson and Amanda Kyser were troupers while we tamed some very wild
equids to get all the samples needed. Dr. Kutzler, our consulting veterinarian for this
study, was a great asset to the team, providing incredible insight and information to aid
us at the ranch and throughout the study.
In the lab, to get through all the crazy, and often times smelly tasks that were
required, I owe TEAM REPRO a huge thank you for dealing with all my “ovary
smoothie” smells, and helping when and wherever needed. Everyone in the lab provided
me with more help than I can thank them for.
I also could not have completed this phase in my life if it was not for some great
support from my family and friends. They were there to give me physical, emotional
and even financial support when needed. It was their encouragement that helped me
pursue this incredible opportunity, taking the leap to move across the state with little
notice or foreknowledge of what lay ahead while working on my degree. It was a scary
thing for me to leave everyone in my small rural community, to move to the “big city”
of Corvallis, but it was an incredible opportunity that I would not trade for the world. I
learned so much, got to experience things that I never dreamed of doing, and came out
of it with many new friends and memories.
I again want to whole heartedly thank Dr. Menino for providing me with the
chance of a lifetime by offering me the opportunity to work in his laboratory as his
graduate student. He was very patient with me and my project, and taught me
invaluable lessons about research, labs, and teaching that I will benefit from for years to
come. I don’t know how to put in words how incredible it was that he gave me the
chance to work on this horse project.
TABLE OF CONTENTS
Page
INTRODUCTION……………………………………………………………………… 1
REVIEW OF THE LITERATURE………………………………………………..…… 5
Female contraception ………………………………………………………….. 5
Immunocontraception ………………………………………………………….. 6
Adjuvants ……………………………………………………………… 7
Gonadotropin-releasing hormone immunocontraceptive vaccine ……..10
The zona pellucida as a contraceptive target …………………………. 11
SpayVac as a pZP immunocontraceptive vaccine ……………………. 18
MATERIALS AND METHODS …………………………………………………..… 19
Animals …………………………………………………………………….… 19
Measurement of pZP antibody titers …………………………………………. 20
Progesterone assay ……………………………………………………………. 21
Ovarian weights and fluid collection …………………………………………. 21
Oocyte dimensions …...…………………………………………………….… 22
Sperm binding assay …………………..…………………………………….... 22
Bovine …………………………………………………………….…... 22
Equine …………………………………………………………….…... 24
Equine anti-pZP binding assay…………………………………………………25
Statistical analysis …………………………………………………………..... 25
RESULTS …………………………………………………………………………….. 27
PZP antibody titers .…………………………………………………………... 27
Progesterone ……….…………………………………………………………. 29
Ovarian weights ……………………..………………………………………... 33
PZP antibody titers from collected fluids …………………………………..… 33
Oocyte dimensions …...…………………………………………………….… 33
TABLE OF CONTENTS (Continued)
Page
Sperm binding assays…….…………………………………………………… 36
Bovine ………………………………………………………………… 36
Equine ………………………………………………………………… 36
Equine anti-pZP binding assay ..……………………………………………… 38
DISCUSSION……………………………………………………………………….… 40
pZP antibody titers …………………………………………………………… 40
Progesterone ………………………………………………………………….. 41
Ovarian weights and fluid collection ………………………………………… 41
Oocyte dimensions …………………………………………………………… 42
Sperm binding assays ………………………………………………………… 43
Bovine ………………………………………………………………... 43
Equine ……………………………………………………………….... 43
Equine anti-pZP binding assay ……………………………………………..… 44
BIBLIOGRAPHY ………………………………………………………………….… 46
LIST OF FIGURES
Figure
Page
1. Mean anti-pZP titers in mares injected with vehicle (Control) or SpayVac in
three different adjuvant preparations (IVT, MFA aq, and MFA non-aq)…….. 28
2. Anti-pZP titers in mares injected with vehicle (Control) or SpayVac in three
different adjuvant preparations (IVT, MFA aq, MFA non-aq) .……………… 28
3. Serum progesterone concentrations in mares injected with vehicle (Control) or
SpayVac in three different adjuvant preparations (IVT, MFA aq, MFA nonaq).…………………………………………………………………………..… 30
4. Serum progesterone expressed as area under the curve for mares injected with
vehicle (Control) or SpayVac in three different adjuvant preparations (IVT,
MFA aq, MFA non-aq) ………………………...…………………………...… 32
5. Weights of ovaries collected from ovariectomized and necropsied mares injected
with vehicle (Control) or SpayVac in three different adjuvant preparations (IVT,
MFA aq, MFA non-aq) ……...………………………………..……………..... 34
6. Anti-pZP titers in follicular, oviductal and uterine fluids collected from mares
injected with vehicle (Control) or SpayVac in three different adjuvant
preparations (IVT, MFA aq, MFA non-aq) …………………...…………….... 34
7. Diameters and zona pellucida thicknesses of oocytes recovered from mares
injected with vehicle (Control) or SpayVac in three different adjuvant
preparations (IVT, MFA aq, MFA non-aq) .…...……………………….…….. 35
8. Numbers of bovine sperm bound to bovine oocytes incubated in DPBS (Saline)
or DPBS with 0.5 or 50% pooled sera recovered from pre-immune, control or
high pZP titer mares …………………….………………….………………… 37
9. Numbers of equine sperm bound to oocytes recovered from mares injected with
vehicle (Control) or SpayVac in three different adjuvant preparations (IVT,
MFA aq, MFA non-aq) …………………………………..…………………….37
LIST OF FIGURES (Continued)
Figure
Page
10. Phase contrast (inset) and fluorescent images of bovine oocytes incubated in
DPBS or pooled sera recovered from pre-immune, control or high pZP titer
mares and exposed to rabbit anti-horse IgG-FITC conjugate. Magnification of
images in photomicrograph and inset are 100X and 200X, respectively ……...39
11. Mean relative fluorescence of bovine oocytes incubated in DPBS (Saline) or
pooled sera recovered from pre-immune, control or high pZP titer mares and
exposed to rabbit anti-horse IgG-FITC conjugate ……………………………..39
LIST OF TABLES
Table
Page
1. Differences in serum progesterone concentrations in mares injected with vehicle
(Control) or SpayVac in three different adjuvant preparations [IVT, MFA aq
(AQ), MFA non-aq (NAQ) (P<0.05)]. ND indicates no difference among means
(P>0.10). ………………………………...………..………………………….. 31
1
INTRODUCTION
Wild horses have long been a symbol of the independence of the American West
(Government Accountability Office, 2008). Historically, in North America, wild horse
populations were controlled by local citizens who captured wild horses for use as beasts
of burden, pet food, and rodeo stock (Kirkpatrick et al., 1993; Warren et al., 1993).
Public sentiment and interest arose after the great population decreases in the early
1900s due to reduced habitat and round-ups to make room for livestock and farming
operations (Warren et al., 1993). By 1971, only about 9,500 wild horses were thought to
live on public rangelands (Government Accountability Office, 2008). The passage of
the Wild Horse and Burro Act in 1971 by the U.S. Congress provided almost complete
protection to wild and feral equids on public lands (Kirkpatrick et al., 1993). The act
placed the Bureau of Land Management (BLM) responsible for protecting and
managing wild free-roaming horses and burros, maintaining a thriving natural
ecological balance on the public lands (Government Accountability Office, 2008). The
act was meant to protect dwindling populations of feral horses, however, it neither
provided a means for managing the populations once their numbers had been reestablished nor did it allow for any type of management that involved death of the
animals (Willis et al., 1994). By 1985, the numbers had increased to an estimated
42,756 horses and 7,665 burros. In 1986, it was estimated the appropriate management
levels resided between 25,000 and 30,000 animals (Boyles, 1986). The large population
increase indicated wild horses and burros were contributing to overgrazing of
rangelands.
2
In 1985, as an attempt to provide some form of population control, the BLM
initiated the “Adopt-a-Horse” program. Horses were gathered and adopted by people
interested in acquiring a wild horse (Kirkpatrick et al., 1993). However, many of the
collected animals were not desirable for adoption due to age, disposition and sex. Since
the BLM issued a moratorium in 1982 to end the destruction of excess unadoptable
animals, unadoptable horses could not be sold or destroyed. Those not re-homed
through the “Adopt-A-Horse” program remained in holding pens indefinitely (Garrot,
1991; Willis et al., 1994; Government Accountability Office, 2008). The high cost of
the Adopt-a-Horse program and its inability to remove sufficiently large numbers of
horses and the increased reproductive success of the remaining horses led to steady
population increases (Kirkpatrick et al., 1993). The agencies were charged with the
dilemma to control the feral horse populations to reduce overgrazing on native
vegetative communities and to comply with the Wild Horse and Burro Act in a manner
logistically and fiscally feasible (Warren et al., 1993). Current management strategies of
removal and adoption are expensive, logistically challenging, and minimally effective in
reducing and maintaining wild horse populations at a desired level (Killian et al., 2008).
Garrot (1991, 1992) concluded implementing contraceptive strategies would reduce the
number of horses required for population removal from federal lands to reach the
targeted 30,000 animals.
Fertility control to manage the growth of the wild horse population started with
attempts to develop contraceptives for sterilization of harem stallions (Turner and
Kirkpatrick, 1996), including steroid hormone treatment, castration and vasectomy
3
(Deals and Hughes, 1995; Kirkpatrick et al., 1993). Sterilization of the dominant
stallion (harem sire) has been shown to be ineffective as a contraceptive because
bachelor stallions breed up to one third of the mares of a harem despite efforts of the
dominant stallion to guard his harem (Kirkpatrick et al., 1993).
A committee of scientists representing the National Research Council concluded
female-oriented contraceptive techniques would have a higher probability of success
when applied on a management scale (Garrot, 1992). Selection of the most appropriate
technique for limiting feral horse populations depends on which aspects of the
management program are considered the most important (Garrott, 1992), whether it be
through reproductive inhibition or fertility control (Garrott, 1991) via contraception. To
be a practical and ideal contraceptive method, it must be applied in a single treatment,
be effective for years without significant loss of future fertility, not pose any other
serious problems to the health of the animal and be easy to administer (Deals and
Hughes, 1995; Killian et al., 2008). While a reversible contraceptive is the ideal form
of fertility control, some suggest chemosterilization is a plausible form of population
control if it can be provided in a safe, humane and cost-effective manner (Garrot, 1991).
Garrott (1991) concluded with simulations that contraceptive programs could
substantially reduce population growth rates. The study provided some insight to the
process of designing mare contraceptive programs for feral horse populations but gave
no direction to a successful contraceptive method. Although chemically or
immunologically induced inhibition of fertility in equids has been studied since 1988
4
(Plotka et al., 1988), overall, such approaches have been researched little (Kirkpatrick et
al., 1993). It has not been until recently that interest had increased to a point of
providing productive methods. This increased interest was largely due to the
uncontrolled populations of free-roaming wild horses and feral burros (Kirkpatrick et
al., 1993).
5
REVIEW OF THE LITERATURE
Female Contraception
Plotka et al. (1988) implanted 30 captive wild mares in Nevada with
subcutaneous implants containing various doses of estradiol and progesterone. Despite a
high incidence of rejected and lost implants, remaining implants were reported to
decrease estrus incidence, however over 80% of the mares bred and conceived when
placed with a stallion. In 1992, Plotka and coworkers used implants containing
ethinylestradiol, estradiol 17-β, progesterone, or progesterone with high
ethinylestradiol. Implants containing ethinylestradiol resulted in 3 years of
contraception with an estimated efficacy period of up to 5 years. Contraception
occurred regardless of route of implant delivery (subcutaneous, intramuscular, or
intraperitoneal).
Results achieved with estradiol, progesterone, and ethinylestradiol implants
brought to focus advantages and disadvantages of natural versus synthetic steroids for
contraception in equids (Kirkpatrick et al., 1993). Hormonal treatments have had
limited success due to dosage requirements and ineffective delivery systems (Deals and
Hughes, 1995). Steroids native to the mare, such as estradiol and progesterone, are
recognized by the mare’s metabolic enzymes and degraded so rapidly that contraceptive
doses are so large they are difficult or impossible to administer. Synthetic steroids, such
as ethinylestradiol, may delay metabolic degradation and permit sustained contraceptive
effects thereby proving useful fertility inhibition in certain instances. Any risk, however
6
small, of the passage of these synthetic steroids to humans or other wildlife may make
registration by regulatory agencies such as the Food and Drug Administration (FDA),
the U.S. Department of Agriculture (USDA), or the Environmental Protection Agency
(EPA) unlikely or difficult (Kirkpatrick et al., 1993).
Deals and Hughes (1995) studied the effectiveness of an intrauterine device
(IUD) for fertility control in mares. The mechanism of contraceptive action of the IUD
is a chronic low-grade uterine inflammation. Deals and Hughes (1995) manually placed
a silastic O-ring shaped IUD into the uterus of 6 mares with no tools. One mare lost an
IUD shortly after placement, but the remaining mares did not conceive when exposed to
a stallion throughout the yearlong study and returned to normal fertility following
removal. Although this study supported the efficacy of IUD as a viable contraceptive in
mares, there are limitations to this method for feral horses because each mare would
need to be rounded up, chemically and physically restrained, and monitored for loss of
the device.
Immunocontraception
Immunocontraceptives have numerous advantages over contraceptive steroids
because they can be delivered remotely, making them more feasible for field application
than methods requiring capture and immobilization. Also, a protein-based vaccine
would likely be deactivated if ingested orally by non-target organisms, in contrast to
persistent tissue residues often characterizing synthetic steroids (Warren et al., 1993).
Immunocontraception attempts to stimulate the target animal’s immune system to
7
interfere with some critical reproductive event (Kirkpatrick and Turner, 1991). Upon
injection of an antigen into the target animal, antibodies are produced against molecules
requisite for successful reproduction (Kirkpatrick and Turner, 1991).
Immunocontraception can be aimed at specific molecules in the reproductive process,
including hypothalamic releasing hormones, pituitary gonadotropins, and sperm or egg
receptor molecules necessary for fertilization (Kirkpatrick and Turner, 1991).
Adjuvants
A key ingredient in immunocontraceptive vaccine development is the adjuvant.
Adjuvants are compounds used to evoke or increase an immune response to an antigen
(Schijns, 2000). Adjuvants are responsible for altering the immune system to recognize
infection and continually stimulating the immune system to create antibodies to attack
and destroy any foreign bodies. These antibodies can be detected through the
development of high antibody titers (Killian et al., 2004; Perry et al., 2006). The
mechanism of action of adjuvant emulsions includes the formation of a depot at the
injection site, enabling the slow release of antigen and the stimulation of antibody
producing plasma cells (Petrovsky and Aguilar, 2004). Adjuvants can cause injection
site reactions that become metastatic however it is the general inflammatory response
causing the lesion and not the adjuvant per se (Lyda et al., 2005). The issue of injection
site reactions must be reviewed carefully for adverse reactions, but also placed within
the framework of benefits versus hazards because adjuvants are critical components in
vaccines for the development of high titers (Lyda et al., 2005; Miller et al., 2009).
8
The original adjuvant, Freund’s Complete Adjuvant (FCA) was developed in
1945, and is an emulsion of water, mineral oil and dried fractionated cell walls of
Mycobacterium tuberculosis (Petrovsky and Aguilar, 2004; Miller et al., 2004). FCA
acts as a powerful nonspecific immune stimulant, but its use has been discontinued due
to the associated side effects and lack of approval by the U.S. FDA (Miller et al., 2009).
A prominent negative side effect of FCA is the high incidence of injection site
reactions, including open abscesses. The second drawback to FCA is the false-positive
tuberculosis (TB) test resulting in treated animals because of the immune response
generated against the M. tuberculosis cell walls. The potential of a false-positive TB test
associated with FCA-treated animals created an issue for captive exotic species in zoos
and a potential issue in other domestic animals (Lyda et al., 2005).
Freund’s Complete Adjuvant is often followed by booster vaccines using
Freund’s Incomplete Adjuvant (FIA) (Lyda et al., 2005). FIA is the oil and water
emulsion without the added mycobacterium making it immunoreactive (Petrovsky and
Aguilar, 2004). FIA acts similarly to FCA through formation of a depot at the injection
site, but has a weaker immune response due to the lack of bacterium (Petrovsky and
Aguilar, 2004; Stills, 2005).
To eliminate the problem of the false-positive TB test results caused by M.
tuberculosis in FCA, Freund’s Modified Adjuvant (MFA) was developed. MFA
includes the freeze-dried fractionated cell walls of Mycobacterium butyricum, a
bacterium commonly found in rancid butter with no known associated pathologies
9
(Lyda et al, 2005). MFA is a common adjuvant used in rats when studying rheumatoid
arthritis and suppression methods. It is advantageous because of its ability to evoke an
immune response with a single low concentration dose without inflammation when
administered intraperitoneally (Conforti et al., 1997).
The addition of Mycobacterium in Freund’s adjuvants provides a vital
immunoreactive response by the immune system which is the key to Freund’s success
(Miller et al., 2004) as well as providing a waxy coat protecting the cell walls from
rapid macrophage destruction. Because M. tubuculosis in FCA can cause false positive
TB tests, the USDA National Wildlife Research Center (NWRC) in Fort Collins,
Colorado worked to create a new adjuvant (Perry et al., 2008). AdjuVacTM is a water
and oil emulsion containing a small quantity (<200µg) of killed Mycobacterium avium
(Perry et al., 2008; Miller et al., 2009). M. avium are common in the environment and
its likely most domestic and wildlife species have been exposed to the bacteria, thereby
strengthening the initial immune response (Miller et al., 2004). Miller and co-workers
(2004) demonstrated it took approximately 2 to 3 weeks for specific antibodies to be
developed against an antigen when inoculating with AdjuVacTM. With the active
bacterium being so common in the environment, it is assumed to have a natural booster
effect by utilizing the animal’s continual exposure to the bacteria and naturally
extending the high titer responses beyond the benefit of the waxy cell walls providing
multi-year responses (Perry et al., 2008).
10
Gonadotropin-releasing hormone (GnRH) immunocontraceptive vaccines
The use of GnRH vaccines for suppression of fertility as well as aggressive and
sexual behaviors has shown promising results (Botha et al., 2008). GnRH is the most
important hypothalamic neuropeptide governing reproduction because it acts on the pars
distalis to control the release of the two gonadotrophins, follicle stimulating hormone
(FSH) and luteinizing hormone (LH) (Senger, 2003).
One immunologically based contraceptive strategy involves blocking release of
GnRH from the hypothalamus, thereby preventing pituitary secretion of FSH and LH
and their subsequent trophic actions on the gonads (Kirkpatrick et al., 1993). The GnRH
peptide is identical in all mammals and is not immunogenic because it is considered
“self” by the immune system and its small size (Miller et al., 2004). When conjugated to
a larger protein and combined with an adjuvant, GnRH can be used to inhibit ovulation
in females (Warren et al., 1993). Anti-GnRH antibodies bind to endogenous GnRH and
inhibit natural binding of this molecule to its receptors in the pituitary, thereby
suppressing secretion of both FSH and LH (Botha et al., 2008). Vaccination against
GnRH is potentially safe, effective, and, eventually, reversible. However, the duration
of suppression of reproductive activity may not be as predictable or manageable as with
other treatments (McCue, 2003).
In 2008, Killian and co-workers (2008) reported on a four year study using
GonaConTM, a GnRH contraceptive vaccine developed at the NWRC. GonaConTM
includes a GnRH-keyhole limpet hemocyanin (KLH) conjugate emulsified in
11
AdjuVacTM and is an effective single-injection contraceptive in a variety of species
(Mauldin and Miller, 2007). Fifteen mares were given a single-injection GnRH vaccine
of either 1800 µg or 2800 µg GonaConTM and exposed to a fertile stallion during the
breeding seasons. During their four year study, Killian et al. (2008) reported annual
pregnancy rates measured by rectal ultrasonography of 93%, 64%, 57%, and 43%,
respectively, with a significant decline in mean antibody titers over the last three years.
This study reported the greatest and longest lasting contraception rates in mares after
being vaccinated with the GnRH vaccine, GonaConTM.
Because GnRH must be linked to a large protein to make it immunogenic,
developing a long-acting vaccine has presented some downfalls. The new formulation
of GonaConTM has been shown to be an effective single-injection immunocontraceptive
in females with effects lasting 1-4 years depending on dose (Miller et al., 2004). With
the goal of the ideal contraceptive vaccine being a single injection effective for several
years, GnRH vaccines are approaching this point (Killian et al., 2008). However,
according to Miller and coworkers (2004), current GnRH vaccines are still not capable
of providing feral horses with lifelong contraception from a single injection.
The zona pellucida as a contraceptive target
The zona pellucida (ZP) is a complex glycoprotein matrix that surrounds the
mammalian oocyte and early embryo (Dean, 1992; Miller et al., 1992). This
extracellular matrix plays a pivotal role in fertilization and protects the oocyte and early
embryo during its transit through the reproductive tract (Barber et al., 2000). The ZP is
12
formed during early folliculogenesis and is laid down between the granulosa cells and
the oocyte. The oocyte and granulosa cells have both been implicated as contributing to
ZP synthesis, but it is now evident that the oocyte is responsible for its synthesis in most
species (Skinner et al., 1996; Senger, 2003). Granulosa cells possess FSH receptors and
are important for oocyte maturation and estrogen, inhibin and follicular fluid production
(Senger, 2003).
The ZP’s role in fertilization includes such processes as sperm attachment,
binding and penetration, induction of the acrosome reaction and polyspermy prevention
(Sacco et al., 1989). It is comprised of three major glycoproteins, ZP1, ZP2, and ZP3,
that are evolutionarily conserved among mammals and encoded by single-copy genes
located on different chromosomes (Wassarman, 2008). The genes are named as ZPA,
ZPB, and ZPC and each encodes a ZP protein arranged by the size of the cDNA. The
porcine (p) pZP is limited on the amount of ZP2 and instead has ZP3α and ZP3β (Sacco
et al., 1989). The ZP3 or pZP3α serves as a receptor for sperm and is the acrosome
reaction-inducer (Sacco et al., 1989; Wassarman et al., 1999). As the oocyte grows, ZP
thickness increases as well until, for horses, it reaches a thickness of 7-10 µm which is
comparable to the pig and cow ZP (Miller et al., 1992; Wassarman, 2008)
As the target of sperm binding, the ZP plays a pivotal role in fertilization. It has
therefore become a desirable target for immunocontraception (Barber et al., 2000). The
ZP matrix has been shown in numerous studies to be specific to the ovary, decreasing
the possibility of adverse side effects to immunization with ZP (Skinner et al., 1996).
13
Kirkpatrick and coworkers (1996) have shown that pZP does not cross-react with
somatic tissues or protein hormones in equids. By injecting raw ZP protein or ZP3, the
female can raise antibodies against the ZP and be an effective contraceptive or
sterilization agent in several mammalian species (Kirkpatrick and Turner, 1991; Skinner
et al., 1996; Barber et al., 2000).
Although this vaccine induces an immunocontraceptive effect in many species,
the actual mechanism is not completely understood. The current theory is injected pZP
causes an immune reaction in which anti-pZP antibodies are generated and recognize a
determinant or determinants on the ZP surface of the treated animal’s oocyte thereby
blocking sperm binding. However, long term treatment results in the alteration of
ovarian function in pZP-treated mares leading to a permanent period of anestrus
(Kirkpatrick et al., 1992). When antibodies remain elevated for long periods of time,
they indirectly prevent fertilization by depleting the oocyte pool causing sterilization
(Barber, 2000). Skinner and coworkers (1984) were the first to report a reduction in
ovarian function and primordial follicle counts in rabbits after 20 weeks of titer levels.
VandeVoort and coworkers (1995) proposed a mechanism for sterilization
where antibodies bind to ZP antigens as soon as they are expressed and secreted into the
space between the oocyte and granulosa cells. These antibodies interfere with the
junctional complexes between the oocyte and differentiating granulosa cells causing the
oocyte to degenerate. Without the developing oocyte, follicle cells cannot differentiate
and, ultimately, no antral or mature Graafian follicles develop. Without positive
14
feedback signals from Graffian follicles to inhibit follicular maturation, most, if not all
primordial follicles are recruited overtime. In the presence of antibodies, however, the
recruitment results in death of the oocytes. Killian and coworkers (2008) proposed that
IgG levels may decrease when the mare naturally enters seasonal anestrus. When the
breeding season resumes, new ZP proteins are produced with the initiation of follicular
development, stimulating a renewed immune response. Over a period of time,
therefore, oocytes are depleted and steroid-producing cells do not differentiate
(VandeVoort et al., 1995; Skinner et al., 1996). Factors playing a significant role in
long-term alterations of ovarian function are the duration of contraceptive efficiency,
which is directly related to the amount of pZP antigen administered, the resulting
antibody titers, and the degree of ovarian dysfunction, which may likewise be related to
pZP dose (Henderson et al., 1988; Millar et al., 1989).
The first documented trial vaccinating mares with pZP was performed by Liu
and coworkers (1989) who used 10 captured feral mares between three to ten years old
with palpable normal reproductive tracts. Mares were given a series of four
intramuscular injections, containing either 2000 or 5000 heat-solubilized pZP mixed
with either aluminum hydroxide gel or Freund’s as adjuvants. The adjuvant change was
due to a low immune response with the first adjuvant. Two months after the last
injection, all the mares injected with pZP had high antibody levels, but by ten months,
two mares had titers reduced to undetectable levels. The remaining eight feral mares
were exposed to fertile stallions for eight months but no palpable pregnancies were
detected. An in vitro sperm binding experiment was performed using horse anti-serum
15
with pig oocytes and boar semen. Pre-incubation of oocytes in antisera reduced sperm
binding relative to the antibody levels with the strongest antiserum preventing sperm
binding in 99% of exposed oocytes. While only a preliminary study, Liu and coworkers
(1989) showed that the use of pZP in mares may be an attractive form of contraception
for controlling the wild horse populations.
Kirkpatrick and coworkers (1990) began their long immunocontraceptive
interest with feral horses on the Assateague Islands in Maryland. Kirkpatrick et al.
(1990) vaccinated 26 mares with 5,000 heat-solubilized pZP (64.3 µg protein)
emulsified with FCA for the first injection and FIA for the following two booster
injections. Three of the mares developed injection site reactions but a 50% decrease in
foaling rate was reported one year after vaccination. A very important part of the study
was no deleterious effects were observed on existing pregnancies because 14 mares
vaccinated with pZP delivered healthy foals shortly after injection.
Over the next two years, Kirkpatrick and coworkers (1991; 1992) monitored the
herds and administered annual booster vaccinations two years after the initial injection.
The first annual booster yielded a significant decrease in foaling rates; 7% for treated
mares and 50 and 44% for control and untreated mares, respectively (Kirkpatrick et al.,
1991). The third year of the study resulted in 100% contraception for treated mares. The
majority of treated mares (5/7) also did not express any estrous behavior or ovarian
function as determined from collected urine samples. Kirkpatrick and coworkers (1992)
16
concluded that three consecutive years of treatment was the primary factor in
determining ovarian dysfunction.
To aid in the reduction of handling costs associated with rounding up feral
mares to inject contraceptive vaccines, Willis and coworkers (1994) developed a new
pZP formulation that could be remotely delivered in a bio-bullet. Mares received an
initial vaccination of either 200 or 400 µg pZP per dose and 4.5 weeks later all were
given a booster vaccination of 200 µg pZP. The vaccine was prepared in synthetic
trehalose dicorynomycolate glycolipid and squalene oil adjuvant frozen into a biobullet.
Vaccines were administered at distances of 20 feet from a 0.25 caliber air gun. No
abscesses were reported and serum antibody titers persisted for 40 weeks after the initial
vaccination. Of the six mares vaccinated with the two vaccine preparations, none
became pregnant for up to two years after the first injection.
The use of pZP as a contraceptive method in mares has been studied since 1988.
The most popular and successful preparation of the vaccine was adding whole pZP to
FCA with booster injections of FIA. After FCA use raised concerns because of side
effects associated with the vaccination, injection site reactions and false positive TB
tests, Lyda and coworkers (2005) decided to test the efficiency of the vaccine prepared
with MFA. MFA had been used in many zoo species as a substitute adjuvant to FCA for
vaccinations without the effects of the misleading TB tests (Lyda et al., 2005). Even
though there currently is not a reliable TB test for horses, USDA has pushed to move
away from the use of FCA. MFA had not been examined for efficacy as a substitute
17
adjuvant in pZP vaccines so Lyda and coworkers (2005) vaccinated seven mares with
the previous preparation of pZP plus FCA followed by a booster injection of pZP with
FIA. They also vaccinated eight mares with pZP in MFA, which contained 0.85 mg/ml
of bacterial cell suspension in mannide monooleate oil (Lyda et al., 2005). Less than
one month later, a booster vaccination of pZP plus FIA was given to aid in antibody
production. Over the 11 month study, MFA-treated mares were reported to have higher
titers than FCA treated mares, although the difference was not significant. This study
also demonstrated pZP could be administered to pregnant females without harming the
fetus, regardless of the adjuvants tried so far.
The use of pZP as a female contraceptive has shown promising results, but the
need for multiple inoculations by hand injection makes the use impractical for feral
horse population control (Liu et al., 2005). To work towards finding an ideal method of
contraception for wild horses that is safe, potentially reversible, and effective for several
years following a single practical administration with minimal behavioral effects (Deals
and Hughes, 1995), Killian and coworkers (2008) selected a new pZP preparation,
SpayVac™ (SpayVac). The SpayVac preparation contained isolated ZP antigens
encapsulated in liposomes containing phospholipon 90G and cholesterol in saline
(Brown et al., 1997). SpayVac was administered with the new adjuvant AdjuVac and
injected as a 1-ml dose. Killian and coworkers (2008) monitored the mares after the
single injection for four years, analyzing contraception rates from rectal palpations and
biannual titers. Contraception rates of 100%, 83%, 83%, and 83% during years 1-4
respectively, were observed over the study. Titers of contracepted mares remained
18
above pregnant treated mares throughout the 4 year study. Killian et al. (2008) reported
a possible self-boosting immune response seen as a fluctuation in titers from year to
year and provided evidence a long-term, single injection contraceptive vaccine was
possible through the use of SpayVac.
SpayVac as a pZP immunocontraceptive vaccine
SpayVac is currently the only long-lasting, single-dose pZP contraceptive
vaccine with proven multi-year efficacy. The use of the liposome encapsulated
technology provides a unique self-boosting effect post-immunization that is essential to
the single-dose multi-year efficiency (Maudlin et al., 2007). SpayVac has traditionally
been formulated with the adjuvant AdjuVac™ because the adjuvant has a great effect
on immune response efficiency (Petrocsky and Aguilar, 2004). However, efficacy of
SpayVac in different adjuvants needs to be analyzed. Therefore, the objective of this
study was to evaluate SpayVac in three different adjuvant formulations, MFA in either
an aqueous (MFA aq) or non-aqueous (MFA non-aq) suspension or a proprietary nonMycobacterium based adjuvant (IVT) for pZP titers, estrous cyclicity and ovarian
activity. We hypothesized SpayVac administered in an MFA solution would provide
greater contraception by inducing higher titers and decreasing estrous cyclicity, ovarian
weights and the number of ovarian oocytes. It was also of interest in this study to
confirm binding of equine anti-pZP antibodies to the ZP of both bovine and equine
oocytes because the proposed mechanism of contraception with anti-pZP is through
inhibition of sperm binding.
19
MATERIALS AND METHODS
Animals
Twenty-eight reproductively sound mares of varied breed, size and weight with
ages ranging from 3-15 years were used in this study. Mares were either purchased or
donated and housed at an off-campus location. An initial health check and baseline
blood collection occurred soon after arrival at the facility. Mares were maintained on
pasture and had access to shelter and water ad libitum, fed daily rations of either grass
or alfalfa hay, and had a salt/mineral block provided free choice. Mares were randomly
sorted into groups and maintained throughout the study in these groups unless a
problem arose. Mares in this study were neither monitored for behavioral estrus and
receptivity nor mated.
Mares were haltered, chemically restrained with 0.3-1.5 ml Dormosedan
(detomidine hydrochloride; 10 mg/ml, Pfizer Animal Health, Exton, PA) iv and placed
into stocks for vaccinations, rectal palpation, ultrasonography per rectum and jugular
blood sampling. Mares were randomly assigned to four treatments (7 mares per
treatment): adjuvant alone or saline (Control) or SpayVac vaccines in one of three
adjuvant preparations, IVT, MFA aq or MFA non-aq. Doses of 1 ml were administered
intramuscularly in the right lateral neck of each mare. Weekly jugular blood samples (7
ml) were collected for quantifying serum progesterone and antibody titers. Blood was
refrigerated overnight to allow clotting and centrifuged at 2000 RPM for 15 min. Serum
was recovered and frozen at -20°C until assayed. All procedures involving animals were
20
approved by the Institutional Animal Care and Use Committee of Oregon State
University.
Measurement of pZP antibody titers
Endpoint antibody titers were determined by ELISA. Briefly, lyophilized pZP
(kindly provided by ImmunoVaccine Technologies, Halifax, NS, Canada) was
dissolved in Dulbecco’s Phosphate Buffered Saline (DPBS) to a final concentration of
1.0 mg/ml, gently shaken and heated for 30 min at 37°C. PZP was diluted to 1 µg/ml
with coating buffer (1.59% NaHCO3, 2.93% Na2CO3 and 0.2% NaN3). One hundred
microliters of pZP in coating buffer were aliquoted into each well of a flat-bottom
Costar® 96 well EIA/RIA plate (Corning Incorporated, Corning, NY, USA), covered,
and incubated overnight at 4°C. The plate was washed 5 times with Tris-buffered
saline-Tween 20 (TBS-T; 0.001M Tris, 0.004M Tween-20 and 0.15M NaCl) and
blocked with 100 µl of 5% skim milk in TBS-T for 1 h at 37°C. Following 2 washes
with TBS-T, 100 µl of serial serum dilutions ranging from 1:10 to 1:2,048,000 were
aliquoted into the wells and the plate was incubated overnight at 4°C. The plate was
washed 5 times with TBS-T, 100 µl of alkaline phosphatase (AP)-conjugated Protein G
(EMD Chemicals, Inc., Gibbstown, NJ, USA) in AP buffer (0.01M Tris, 0.1M NaCl
and 0.01M MgCl2) were added to each well and the plate was incubated for 1 h at 37°C.
The plate was washed 5 times with TBS-T and twice with AP buffer, and 100 µl of 1
mg/ml p-nitrophenyl phosphate, disodium salt (EMD Chemicals, Inc.) in AP buffer
were added to each well. The plate was incubated in the dark for 1 h at 37°C and read at
21
an absorbance of 405 nm using a Benchmark microplate reader (Bio-Rad Laboratories
Inc., Hercules, CA). Endpoint titers were defined as the reciprocal of the highest
dilution above a cutoff value calculated from the measured absorbances. Cutoff values
were calculated using the 95% confidence interval as described by Frey et al. (1998).
Using one or two pre-immune columns (n=8 or 16) from the plate, the cutoff value was
calculated using the following equation: c
̅
(
√
( )).
Progesterone assay
Serum progesterone concentrations were determined using the Alpha Diagnostic
International (San Antonio, TX) progesterone ELISA kit. Cross-reactivity of the
antiserum was 100% to progesterone with the next highest cross-reactivities of: 1.5% to
11-deoxycorticosterone, 0.7% to 17-α-hydroxyprogesterone, 0.15% to pregnenolone,
and less than 0.1% for all others. Intra- and inter-assay coefficients of variation were 9.0
and 14.2% and assay sensitivity was 0.2 ng/ml.
Ovarian weights and fluid collection
Ovaries from Control and SpayVac-treated mares were removed either by
ovariectomy (n=16 mares) or necropsy (n=12 mares). Ovaries were cleaned of excess
tissue and weighed. Follicular fluid from any accessible antral follicle was aspirated
with an 18-g needle and syringe. A section of the ovulation fossa was removed for
another project and ovaries were individually frozen at -20°C until oocyte collection.
One horn and oviduct from each necropsy tract was randomly selected to be flushed for
22
anti-pZP detection. For oviducts, the infundibular end was cannulated with intramedic
tubing and 5 ml of DPBS were irrigated into the isthmic end of the oviduct just cranial
of the utero-tubular junction using a 5-ml syringe and 23-g needle. For uterine horns,
the caudal end was cannulated with Tygon tubing and 20 ml of DPBS were irrigated
into the tip of the uterine horn just caudal of the utero-tubular junction using a 30-ml
syringe and a blunt 16-g needle. Collected fluids were centrifuged at 2000 RPM for 15
min, aliquoted into sample tubes and frozen at -20°C until analyzed for pZP titers.
Oocyte dimensions
Oocytes recovered from Control and SpayVac treated mares fixed in 2%
paraformaldehyde microdrops were measured using an ocular micrometer mounted on
the eyepiece of a Leitz inverted stage-phase contrast microscope. Oocyte diameters and
ZP thicknesses were measured at 100 and 200X, respectively. Oocyte diameters were
measured from the outside edges of the ZP and ZP thicknesses were measured from the
exterior to interior edge of the ZP.
Sperm binding assays
Bovine
Bovine ovaries were collected at slaughter from an abattoir and stored at -20°C
until use. Frozen ovaries were thawed at room temperature prior to oocyte collection.
To isolate ZP-bound oocytes, ovaries were macerated in DPBS using a tissue
homogenizer. The macerated extract was strained through cheese cloth to remove large
23
debris and poured through a Teflon filter screen with a grid size of 72 µm. The Teflon
filter screen was inverted over a glass collection dish and the screen sprayed with 60 ml
DPBS to wash oocytes into the dish. Intact ovarian tissue was returned to the
homogenizer and the procedure was repeated until all ovarian tissue was thoroughly
macerated. Collection dishes were searched using a dissecting microscope at 10X
magnification and oocytes were removed by aspiration with a small glass micropipette
and placed in DPBS with 0.1% BSA (DPBS-BSA) until use. As many as fifteen oocytes
without any cumulus cells were washed through three 50-µl microdrops of DPBS-BSA
under paraffin oil. Oocytes were incubated at 39°C for 2 h in 50 µl of one of the
following treatments: DPBS or 0.5 or 50% pooled sera from pre-immune, control, or
high pZP-titer mares. Oocytes were washed through three drops of DPBS-BSA
followed by three drops of pre-incubated, sperm TALP (Parrish et al., 1988). Oocytes
were added to 80-µl microdrops containing 50,000 sperm in sperm TALP and incubated
for 4 h at 39°C in a humidified atmosphere of 5% CO2 in air. Oocytes and sperm were
placed into a microcentrifuge tube with 100 µl DPBS-BSA and vortexed at a fixed low
speed for 15 seconds. After washing through three drops of DPBS-BSA, oocytes were
placed in 50-µl microdrops of 2% paraformaldehyde for at least one h. To count the
number of sperm bound to the oocytes, oocytes were washed through three drops of
DPBS-BSA and incubated for 10 min at room temperature in the dark with 1% Hoechst
33342 in 25% ethanol in sodium citrate dihydrate solution (Pursel et al., 1985).
Following three washes in DPBS-BSA, sperm bound to oocytes were counted using
24
epifluorescence on a Leitz inverted stage-phase contrast microscope at 200X
magnification.
Equine
Frozen equine ovaries were thawed at room temperature and oocytes from
individual mares were collected as described for bovine oocytes. Equine oocytes were
washed through three 50-µl microdrops of DPBS-BSA under paraffin oil followed by 3
wash drops of pre-incubated, sperm TALP (Parrish et al., 1988). Oocytes were added to
80-µl microdrops containing 25,000 sperm in sperm TALP and incubated for 4 h at
39°C in a humidified atmosphere of 5% CO2 in air. A lower sperm concentration was
used for the equine oocytes because of the fewer number of oocytes in each microdrop.
Oocytes and sperm were placed into a microcentrifuge tube with 100 µl DPBS-BSA
and vortexed at a fixed low speed for 15 seconds. After washing through three drops of
DPBS-BSA, oocytes were placed in 50-µl microdrops of 2% paraformaldehyde for at
least one h. To count the number of sperm bound to the oocytes, oocytes were washed
through three drops of DPBS-BSA and incubated for 10 min at room temperature in the
dark with 1% Hoechst 33342 in 25% ethanol in sodium citrate dihydrate solution
(Pursel et al., 1985). Following three washes in DPBS-BSA, sperm bound to the
oocytes were counted using epifluorescence on a Leitz inverted stage-phase contrast
microscope at 200X magnification.
25
Horse anti-pZP binding assay
Bovine oocytes were collected as described above, washed three times in DPBSBSA and placed in 2% paraformaldehyde for at least 1 h. Oocytes were washed three
times with DPBS-BSA, blocked for 1 h with 5% heat treated cow serum, and washed
through three 50-µl microdrops of DPBS-BSA under mineral oil at room temperature.
Oocytes were incubated at room temperature for 1 h in 50 µl of one of the following
treatments: DPBS or 0.5% pooled pre-immune, control or high pZP titer sera. Oocytes
were washed through three drops of DPBS-BSA and incubated with rabbit anti-horse
IgG-flourescein isothiocyanate (FITC) (Sigma-Aldrich, St. Louis, MO) at a
concentration of 1:64 for 1 h at room temperature. Oocytes were washed through three
drops of DPBA-BSA and evaluated using epifluorescence on a Leitz inverted stagephase contrast microscope with a FITC filter. Fluorescent intensity of each oocyte was
quantified using a Leitz photometer.
Statistical analysis
Differences due to treatment in pZP antibody titers and serum progesterone were
analyzed by repeated measures ANOVA where treatment (Control, IVT, MFA aq and
MFA non-aq), time (weeks) and the treatment X time interaction were the main effects.
Because of unequal variance, pZP antibody titers were analyzed using the log (titers +
1) transformation. Total serum progesterone was also analyzed by computing the areas
under the curve for plots of serum progesterone concentrations by weeks of blood
withdrawal. Areas under the curve for progesterone, ovarian weights, oocyte diameters,
26
zona pellucida thicknesses, pZP titers in follicular, uterine and oviductal fluids, numbers
of equine sperm bound to equine oocytes and fluorescent intensities of bovine oocytes
were analyzed for differences due to treatment using one-way ANOVA. Numbers of
bovine sperm bound to bovine oocytes were analyzed using two-way ANOVA where
treatment (DPBS and pooled sera from control, pre-immune or high pZP titer mares),
percent sera (0.5 and 50%) and the treatment X percent sera interaction were the main
effects. If significant effects were observed in the ANOVA, differences between means
were determined using Fisher’s least significant difference procedures. All analyses
were performed using the Number Cruncher Statistical System software program
(NCSS; 2000; Hintze, JL; Kaysville, UT).
27
RESULTS
pZP antibody titers
Anti-pZP titers were greater (P<0.05) in IVT and MFA compared to Control
mares and in MFA compared to IVT mares (Figure 1). Significant titers were not
observed in Control mares. Overall, anti-pZP titers were higher (P<0.05) on weeks 6, 8,
10, 14, 18 and 22 compared to weeks 0, 2 and 3 (Figure 2). Mares vaccinated with MFA
aq had greater (P<0.05) anti-pZP titers at 2 weeks post-injection compared to mares
injected with IVT or MFA non-aq (Figure 2). At 3 weeks post-injection, mares
vaccinated with MFA aq or MFA non-aq had greater (P<0.05) anti-pZP titers compared
to mares injected with IVT. Anti-pZP titers in MFA non-aq mares were greater
(P<0.05) at 6 weeks post-injection compared to IVT mares and, although not
significantly different, remained greater during weeks 8, 10, 14, 18 and 22 compared to
IVT and MFA aq mares.
28
180
c
Titers (X1000)
150
120
c
90
60
b
30
a
0
Control
IVT
MFA aq MFA non-aq
Treatment
Figure 1. Mean anti-pZP titers in mares injected with vehicle (Control) or SpayVac in
three different adjuvant preparations (IVT, MFA aq and MFA non-aq).
a,b,c
Means without common superscripts differ (P<0.05).
6
a
Log (titers+1)
5
a
a
4
a,b
a
Treatment
IVT
MFA aq
MFA non-aq
b
b
3
2
b
1
b
0
0
2
3
4
6 8 10 14 18 22
Weeks
Figure 2. Anti-pZP titers in mares injected with SpayVac in three different adjuvant
preparations (IVT, MFA aq and MFA non-aq).
a,b
Means without common superscripts differ (P<0.05).
29
Progesterone
Mean serum progesterone was greater (P<0.05) in Control compared to MFA
non-aq mares (5.9 ± 0.5 and 1.6 ± 0.3 ng/ml, respectively) and neither differed (P>0.10)
with IVT and MFA aq mares (3.4 ± 0.4 and 3.7 ± 0.5 ng/ml, respectively). Overall,
serum progesterone was greatest (P<0.05) on weeks 8, 9, 10 and 11 compared to weeks
0, 2, 3, 4, 16, 17, 20, 22, 23, 25 and 26 post-vaccination (Figure 3). Reductions
(P<0.05) in serum progesterone in MFA non-aq injected mares compared to Control
mares were observed as early as week 12 post-vaccination (Figure 3; Table 1). In
contrast, reductions (P<0.05) in serum progesterone in IVT and MFA aq injected
mares compared to Control mares were not observed until week 15 post-vaccination.
Progesterone area under the curve mirrored mean serum progesterone concentrations
where areas were greater (P<0.05) for Control mares compared to MFA non-aq mares
(Figure 4).
30
IVT
Figure 3. Serum progesterone concentrations in mares injected with vehicle (Control) or
SpayVac in three different adjuvant preparations (IVT, MFA aq and MFA non-aq).
31
Week
0
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Control
ND
ND
ND
ND
ND
ND
ND
AQ
ND
ND
AQ
NAQ
ND
ND
AQ/NAQ/IVT
ND
ND
AQ/NAQ/IVT
AQ/NAQ
ND
AQ/NAQ/IVT
ND
NAQ
AQ/NAQ
ND
ND
IVT
ND
ND
ND
ND
ND
ND
ND
AQ
NAQ
ND
AQ
ND
ND
ND
Control
ND
ND
Control
AQ/NAQ
ND
Control
ND
ND
NAQ
ND
ND
Treatment
MFA aq (AQ)
ND
ND
ND
ND
ND
ND
NAQ
Control/IVT/NAQ
NAQ
ND
Control/IVT/NAQ
NAQ
ND
NAQ
Control
ND
ND
Control
Control/IVT
ND
Control
ND
ND
Control
ND
ND
MFA non-aq (NAQ)
ND
ND
ND
ND
ND
ND
AQ
AQ
Control/IVT/AQ
ND
AQ
Control/AQ
ND
AQ
Control
ND
ND
Control
Control/IVT
ND
control
ND
Control
Control/IVT
ND
ND
Table 1. Differences in serum progesterone concentrations in mares injected with
vehicle (Control) or SpayVac in three different adjuvant preparations [IVT, MFA aq
(AQ), MFA non-aq (NAQ) (P<0.05)]. ND indicates no difference among means
(P>0.10).
32
200
a
Progesterone (area)
175
150
125
a,b
a,b
100
75
b
50
25
0
Control
IVT
MFA aq MFA non-aq
Treatment
Figure 4. Serum progesterone expressed as area under the curve for mares injected with
vehicle (Control) or SpayVac in three different adjuvant preparations (IVT, MFA aq,
MFA non-aq).
a,b
Means without common superscripts differ (P<0.05).
33
Ovarian weights
Ovaries collected from Control mares weighed more (P<0.05) than both MFA
groups but were similar (P>0.10) to ovaries from IVT mares (Figure 5). Ovaries from
IVT mares weighed more (P<0.05) than ovaries from MFA non-aqueous mares.
PZP antibody titers from collected fluids
Anti-pZP titers in follicular fluid were lower (P>0.05) in Control mares
compared to IVT and MFA aq mares (Figure 6). Likewise, anti-pZP titers in uterine
fluid were lower (P<0.5) in Control mares compared to IVT, MFA aq and MFA non-aq
mares. Although anti-pZP titers were detected in oviductal fluid from mares injected
with SpayVac preparations, no differences (P>0.10) were detected compared to Control
mares.
Oocyte dimensions
Oocyte diameters and zona pellucida thicknesses were greater (P<0.05) in
oocytes recovered from Control compared to SpayVac vaccinated mares (Figure 7). No
differences (P>0.10) in oocyte diameters and zona pellucida thicknesses were observed
among IVT, MFA aq and MFA non-aq mares.
34
a
60
Ovarian weight (g)
50
40
a,b
b,c
30
c
20
10
0
Control
IVT
MFA aq MFA non-aq
Treatment
Figure 5. Weights of ovaries collected from ovariectomized and necropsied mares
injected with vehicle (Control) or SpayVac in three different adjuvant preparations
(IVT, MFA aq, MFA non-aq).
a,b,c
Means without similar superscripts differ (P<0.05).
1200
Treatment
Control
IVT
MFA aq
MFA non-aq
b
1000
Titers
800
600
b
b
400
b
200
0
a
a
b
Follicular(X10)
Oviductal
Uterine(X10)
Fluid
Figure 6. Anti-pZP titers in follicular, oviductal and uterine fluids collected from mares
injected with vehicle (Control) or SpayVac in three different adjuvant preparations
(IVT, MFA aq, MFA non-aq).
a,b
Means without common superscripts differ (P<0.05).
35
160
a
Treatment
Control
IVT
MFA aq
MFA non-aq
Diameter/thickness (microns)
140
120
b b
100
b
80
60
40
a
20
b b b
0
Oocyte Zona pellucida
Oocyte/zona pellucida
Figure 7. Diameters and zona pellucida thicknesses of oocytes recovered from mares
injected with vehicle (Control) or SpayVac in three different adjuvant preparations
(IVT, MFA aq, MFA non-aq).
a,b
Means without common superscripts differ (P<0.05).
36
Sperm binding assays
Bovine
No differences (P>0.10) in the numbers of sperm bound to oocytes were
observed due to serum concentration and the serum x treatment interaction was not
significant. More (P<0.05) sperm bound to oocytes incubated in DPBS and pre-immune
sera compared to oocytes incubated in sera from high titer mares (Figure 8). Although
the number of sperm bound to oocytes incubated with control sera was two-fold the
number of sperm bound to oocytes incubated with high titer sera, no differences
(P>0.10) were observed.
Equine
Regardless of adjuvant composition, oocytes collected from SpayVac-treated
mares had fewer (P<0.05) sperm bound to oocytes compared to oocytes from Control
mares (Figure 9). Oocytes collected from Control mares had at least 5-10 fold more
sperm bound compared to oocytes collected from SpayVac-treated mares.
37
Figure 8. Numbers of bovine sperm bound to bovine oocytes incubated in DPBS
(Saline) or DPBS with 0.5 or 50% pooled sera recovered from pre-immune, control or
high pZP titer mares.
a,b
Means without common superscripts differ (P<0.05).
Number of equine sperm bound
30
a
25
20
15
10
b
5
a
b
a
0
Control
IVT
MFA aq
Treatment
b
a
MFA non-aq
Figure 9. Numbers of equine sperm bound to oocytes recovered from mares injected
with vehicle (Control) or SpayVac in three different adjuvant preparations (IVT, MFA
aq, MFA non-aq).
a,b
Means without similar subscripts differ (P<0.05).
38
Horse Anti-pZP binding assay
Fluorescent intensity was greater (P<0.05) in oocytes incubated in high pZP titer
sera compared to oocytes incubated in pre-immune sera or DPBS (Figures 10 and 11).
Oocytes treated with Control sera exhibited greater (P<0.05) fluorescent intensity than
oocytes incubated in DPBS. Although fluorescent intensity was greater in oocytes
incubated with high pZP titer sera compared to oocytes incubated with control sera, no
significant differences was observed.
39
Figure 10. Phase contrast (inset) and fluorescent images of bovine oocytes incubated in
DPBS or 0.5% pooled sera recovered from pre-immune, control or high pZP titer mares
and exposed to rabbit anti-horse IgG-FITC conjugate. Magnification of images in
photomicrograph and inset are 200X and 100X, respectively.
Figure 11. Mean relative fluorescence of bovine oocytes incubated in DPBS (Saline) or
0.5% pooled sera recovered from pre-immune, control or high pZP titer mares and
exposed to rabbit anti-horse IgG-FITC conjugate.
a,b,c
Means without similar superscripts differ (P<0.05).
40
DISCUSSION
pZP antibody titers
SpayVac vaccinated mares developed substantial pZP antibodies during the
course of the study. Antibodies were detected as early as two weeks and achieved
plateaus by six to eight weeks post-vaccination. This differs from other reports where
Willis and coworkers (1994) reported maximum titers reached by two to three weeks
following booster vaccination and Turner and coworkers (2008) reported peak titers by
week three. Development of a more gradual immune response may be occurring with
SpayVac suspended in the adjuvants used in this study compared to the reports of Willis
et al., (1994) and Turner et al., (2008). Interestingly, mares vaccinated with MFA aq
initially developed antibodies at a much faster rate than either IVT or MFA non-aq but
titers reached an earlier acme and were eventually superseded by MAF non-aq. Mares
injected with IVT developed titers more slowly and mean titers were less compared to
mares injected with either MFA suspension. Killian and coworkers (2004) expressed a
range of titers among SpayVac vaccinated mares and reported that all antibody titers
were high enough to provide contraception. The present study only tracked titers for 2226 weeks post-vaccinations but Killian and coworkers (2004) recorded titers for at least
four years following vaccination with SpayVac. These authors reported an apparent
natural re-boostering effect between years 3 and 4 post-vaccination.
41
Progesterone
As evidence by the cycling concentrations of serum progesterone, Control mares
encountered recurring estrous cycles throughout the study. During the 26-week postvaccination period, a reduction in progesterone and estrous cyclicity was observed in
SpayVac vaccinated mares as early as 12 weeks post-vaccination with a steep reduction
in total progesterone in MFA non-aq vaccinated mares compared to the Controls.
While frequent serum progesterone sampling in SpayVac vaccinated mares has not been
reported in the literature, Killian and coworkers (2004) reported a decrease in serum
concentrations from four consecutive fall blood collections in vaccinated mares
compared to controls. These data suggest SpayVac vaccination interrupts
folliculogenesis thereby inhibiting ovulation and subsequent corpus luteum
development.
Ovarian weights and fluid collection
Kirkpatrick and coworkers (1992) have the only published report on the effects
of pZP vaccination in mares on ovarian function. Using urinary endocrine and
behavioral data, Kirkpatrick et al., (1992) observed altered ovarian function due to the
absence of a luteal phase and reduced estrous behavior after continual annual booster
vaccinations of pZP. Effects of SpayVac vaccination on ovarian weights have not been
published. This study demonstrated a reduction in ovarian weights in ovaries collected
from MFA-vaccinated compared to Control mares. SpayVac combined with MFA in
42
either the aqueous or non-aqueous formulation appears to exert a greater effect on
ovarian function than SpayVac in the IVT formulation.
Likewise, the presence of pZP antibodies in reproductive tract fluids has not
been reported in the literature from pZP vaccinated mares. This study demonstrated the
presence of antibodies in follicular fluid collected from IVT and MFA aq-treated mares.
Follicular fluid could not be recovered from MFA non-aq ovaries so an analysis for
these mares could not be conducted. Anti-pZP antibodies were detected in both the
oviduct and uterine horns of SpayVac treated mares. Although no significant
differences in anti-pZP titers were observed due to treatment in oviductal fluids,
antibodies were present in all SpayVac treated mares. Fluids collected from SpayVac
treated mares all had anti-pZP antibodies higher than fluids collected from Control
mares. The presence of pZP antibodies in follicles, oviducts and uteri is an important
discovery regarding the localization of potential mechanistic effects of vaccinating
mares with SpayVac. Considering the extent of antibody permeation, it is apparent
antibodies can bind to the oocytes at any site throughout the reproductive tract.
Oocyte dimensions
Effects on oocyte morphology in pZP vaccinated mares have also not been
previously reported in the literature. This study demonstrated reduced oocyte size and
ZP thicknesses in all mares vaccinated with SpayVac. VandeVoort and coworkers
(1995) and Killian and coworkers (2008) both proposed a mechanism in which females
vaccinated with pZP are sterilized due to antibodies binding to the ZP and interfering
43
with gap junction formation required for oocyte and granulosa cell maturation and
development. Alterations in oocyte diameters and ZP thicknesses support the hypothesis
that pZP antibodies are interfering with oocyte development.
Sperm binding assays
Bovine
Liu and coworkers (1989) treated porcine oocytes with sera collected from pZP
vaccinated mares and incubated them with boar sperm. These authors reported a 99%
prevention of sperm binding in oocytes incubated with high titer serum. The use of
bovine oocytes in a horse anti-pZP sperm binding assay has not been previously
reported in the literature. A decrease in number of sperm bound to oocytes incubated
with high titer sera was observed when compared to pre-immune sera. Serum
concentrations of 0.5 and 50% were evaluated in the current study, but reductions in the
number of sperm bound did not reach the 99% inhibition reported by Liu et al., (1989).
Equine
Liu and coworkers (1989) also reported on attempts to conduct in vitro sperm
binding assays with equine oocytes incubated in high titer serum, but were unable to
complete the experiments due to poor recovery of oocytes from mare ovaries (4 oocytes
per ovary). Barber and coworkers (2001) also reported difficulty in collecting oocytes
from mare ovaries thereby preventing experiments with equine oocytes. Successful
attempts to conduct in-vitro sperm binding assays on oocytes collected from pZP treated
44
mares has not yet been reported in the literature. With a fairly large number of ovaries
to collect oocytes from in this study, sufficient numbers of oocytes were recovered to
demonstrate regardless of adjuvant, significantly fewer sperm bound to SpayVac
oocytes compared to Control oocytes. These results, taken together with the reductions
in ZP thicknesses, clearly support aberrant ZP formation at the ovarian level resulting
from SpayVac treatment.
Equine anti-pZP binding assay
As evidenced by fluorescence microscopy, bovine oocytes bound pZP
antibodies generated in mares vaccinated with SpayVac. The extent of equine anti-pZP
binding to bovine oocytes was greater compared to pre-immune sera but did not differ
from control sera. Why control sera exhibited an intermediate range of fluorescent
intensity between oocytes exposed to high titer and pre-immune sera is not known.
However, oocyte size is a factor in measuring fluorescent intensities and contributed to
some degree of variation among the measurements. Also, within the same replicate,
fluorescent intensity among oocytes in a microdrop varied suggesting the ZP of some
oocytes may have had reduced antibody infinity compared to others. In a similar study,
Liu et al., (1989) incubated porcine oocytes with serum collected from pZP vaccinated
mares followed by treatment with rabbit anti-horse IgG FITC conjugate and confirmed
binding of equine anti-pZP antibodies to porcine ZP.
Results from the present study suggest that SpayVac suspended in the MFA
non-aq adjuvant provided greater contraceptive efficacy compared to other adjuvants.
45
Titer development was greater, loss of estrous cyclicity occurred earlier and ovarian
weights were lower in MFA non-aq vaccinated mares compared to the other adjuvants.
Differences in titer development were observed between MFA aq and MFA non-aq
mares suggesting the mineral oil in MFA non-aq may initially retard titer development
yet eventually enhance the antibody response. Mares injected with IVT did not appear
to have as drastic effects on estrous cyclicity and ovarian weights as mares injected with
MFA despite significant anti-pZP titers and obvious effects on oocyte quality. In this
regard, the IVT adjuvant may be a good candidate for a vaccine in which some degree
of reversibility is desired. The question of longevity and reversibility of the adjuvant
preparations used in the present study was not addressed but is an essential component
of pZP immunocontraception warranting further research.
46
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