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EVALUATION OF A GONADOTROPIN RELEASING HORMONE VACCINE
FOR THE HUMANE CONTROL OF FEMALE FERAL CATS
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF
FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
I would like to thank Dr. Julie Levy for the opportunity to study under her and for
her willingness to give second, even third, chances. She knew when I needed to be
pushed even when I did not know it. I have very much appreciated and been inspired by
Dr. Cynda Crawford's refusal to allow her indefatigable work habits to dull her sense of
humor. I am grateful for Dr. Lowell Miller's great patience while answering my
seemingly endless questions regarding the vaccine, and for welcoming me to his
laboratory in Colorado. I have benefited greatly from Dr. Karine Onclin's ability to
explain endocrinology to me and I thank her. Without the support and motivation of Dr.
Paul Chadik and Jim Crocker, I would not have returned to school and I can not thank
them enough. I would also like to thank Sylvia Tucker, Mike Reese, Kathy Kirkland,
Audria West, and Rob Schleich for their technical assistance. The girls, of course,
remind me every day why I have chosen this path.
This study was made possible by funding received from the Morris Animal
TABLE OF CONTENTS
A C K N O W L E D G M EN T S .............................................................. .....................ii
LIST OF TABLES ............................... ................. ....... .. ..............v
LIST OF FIGURES ............ .................................. .... ...... ................. vi
A B ST R A C T ............................................................................................................... vii
IM PA CT OF FERAL CA TS .................................. ..........................................
Human Health ........................................................................................................ 2
A nim al H health ..........................................................................................4
Predation on W wildlife ................................. ...................... ............... 5
METHODS TO MANAGE FERAL CATS ............. .............. ............... 8
L ethal M ethods ....................................................... 8
Non-Lethal M ethods ......... .... .......... ......... ...... ............... 10
A adoption ................................................ ............... 10
Relocation Programs .......... ...................... .. ............... 10
Sanctuaries ........................ .... ........ ............................... 10
TNR Programs .............................. ....... ... 11
N on-surgical Contraception...................................... .......... ......... 13
Feline reproductive endocrinology .......................................14
Steroids and G nRH agonists ............. ................................... 17
Cell destruction ............... .............................................18
IM M UNOCONTRACEPTION ....................................................... .... ........... 19
Im m unocontraception Targets ................................................... ........ 19
Riboflavin Carrier Protein............... ......... ............. .. .............. 19
Sperm Proteins ..... .. ........ .... ........................ ... .... ... ..... .... 20
Zona Pellucida .......................... .... ... .... ........... ........ ......20
Hypothalamic-Pituitary-Gonadal Axis ...........................................20
V accine D delivery ..................... .. ......................... .... .. ........... 2 1
GNRH VACCINE .................................... ..... .......... .............. .. 23
M ATERIALS AND M ETHODS......................................... .......................... 25
C ats ....................................................... 2 5
Vaccine Construction................................................ 25
Treatment .............. ......... ............. ......... 26
B lood C collection ............................................ .............. ....27
Detection of GnRH Antibodies.................. ........ ..............27
Determination of Serum Estradiol-173 and Progesterone
Concentrations ...................... ...... ........ ................ 29
B reeding T rial ............................................................2 9
E strus Induction ............................................. ........ .......... 29
Assignment of Cats to Study Groups ..................................30
F ertility and F ecundity .................................................... ............... 30
B ody Phenotype ...................................... .... ............. .. .............. .. ... 30
S statistical A n aly sis............ .... .......................................... ........ .............. 3 1
R E S U L T S ................................................................3 4
Reactions to Treatm ent ........................................................................... 34
B reedin g T rial ............................................................34
D election of E strus..................................... ............................ 34
F ertility and F ecundity .................................................... ............... 35
G nR H A ntibody Titers........................................................ ............... 35
H orm one C concentrations ........................................ .......................... 36
Progesterone ......... .. .. ......... .....................36
Estradiol-17.. ...... ....................................37
B o dy C o n d itio n ......... ....... .................................................... .. .... .... .. ....3 7
B ody W eight .................................... .... ...... .... .............. ..37
F alciform F at P ad ............................. .. ........ ...... .. .. .......... 38
DISCUSSION ......................................... ................ 43
R E F E R E N C E L IST ......................................................................... ....................48
BIOGRAPHICAL SKETCH .............................................................................55
LIST OF TABLES
1. Body weight and percent w eight gain........................................ ............... 42
2. Falciform fat pad depth and area ............................................... ............... 42
LIST OF FIGURES
1 Timeline ......... ................. ................... ...... 32
2 Lateral abdominal radiograph.......................... .......................... ....... ........ 33
3 B reedin g trial ...............................................................3 9
4 GnRH antibody titers for female cats ....................................... ............... 40
5 GnRH antibody titers for cats with high titers ............................................. 41
6 GnRH antibody titers for cats with variable titers .............................................41
7 GnRH antibody titers of nonresponders. ............................................................42
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
EVALUATION OF A GONADOTROPIN RELEASING HORMONE VACCINE
FOR THE HUMANE CONTROL OF FEMALE FERAL CATS
Chair: Julie Levy
Major Department: Veterinary Medicine
The unwanted cat population in the United States numbers in the tens of millions
and current control measures have only had limited success in reducing it.
Immunocontraception has the potential to humanely reduce this population. The purpose
of this study was to investigate the effectiveness of a GnRH-based vaccine for
immunocontraception of female cats. It was expected that the treated cats would produce
antibodies against GnRH and there would be a positive correlation between high titer and
contraception. Adult female cats were divided into a sham group (n = 5) and a treatment
group (n = 15) that was immunized once with 200 |tg of synthetic GnRH coupled to
keyhole limpet hemocyanin and combined with a mycobacterial adjuvant. GnRH
antibody titer and serum concentrations of progesterone and estradiol-173 were
determined monthly. For the duration of the study the daily photoperiod was
manipulated in an attempt to induce estrus. A male breeding cat was housed with the
females during the long-day periods, and continuous videography was used to monitor
for signs of estrus and breeding. GnRH antibodies were detected in all treated cats by
150 days after immunization, but when the titer in four cats fell below 16,000, they
became pregnant and were classified as nonresponders. The titers of the remaining 11
cats responderss) never decreased below 16,000. These cats displayed no signs of
behavioral estrus and did not become pregnant by the end of the study 24 months after
immunization. All five sham cats became pregnant within one month of the introduction
of the male cat. From 60 days after immunization until the end of the study, progesterone
concentrations in all responders remained at basal levels, and increased two months
before parturition in all cats that became pregnant. The responder cats gained more
weight than the nonresponders during the 14 months after immunization (P = 0.004),
which is the same response observed in surgically sterilized cats. A single dose of GnRH
vaccine resulted in contraception in 73% of the cats for at least 24 months.
IMPACT OF FERAL CATS
The domesticated cat is the most numerous companion animal in the United
States with 37.7 million households owning 90.5 million cats . It is estimated that
there are also an equal number of unowned, unwanted cats . Considerable debate
exists on what impact these cats have on human health through zoonotic diseases, animal
health by acting as reservoirs for diseases that affect pet cats and other species, and
wildlife through predation and competition. In addition, there is not agreement on the
quality of life of the cats. The debate extends to the actions that should be taken to
ameliorate the impact of these unowned cats.
Accurately assessing the impact and designing solutions is made more difficult
because cats are assigned to subpopulations according to different criteria. Cats are often
described according to their ownership status (owned or unowned), lifestyle (indoor,
indoor/outdoor, outdoor), or degree of socialization  (tame, feral). In addition, during
its lifetime an individual cat may move from one subpopulation to another. For instance,
a cat living indoors may be abandoned, forced to live outside, and over time become
untrusting of humans; this tame, indoor, owned cat has become an unowned, feral cat.
Many reports on the impacts of cats fail to clearly define which subpopulation of cats is
being studied. For the purposes of this thesis, a feral cat will be defined as any unowned,
free-roaming cat, regardless of its socialization status.
Concerns have been expressed that feral cats serve as a reservoir for diseases that
may be transmitted to humans, pet cats and wildlife [4,5]. In general, feral cats do not
appear to pose a greater risk to humans or other cats than pet cats [3,6]. Cats can transmit
disease directly through a bite or other physical contact; by shedding the pathogenic
organism into the environment; or by concentrating pathogens that can be transferred to
other hosts via vectors such as fleas, ticks and mosquitoes. The Center for Disease
Control and Prevention considers eradicating wildlife to reduce disease reservoirs
ineffective, but does support trap-vaccinate-release programs for wildlife .
The American Association of Feline Practitioners lists 40 potential feline zoonotic
agents, but transmission of the disease from cat to human has not been documented or
occurs only rarely with most of the organisms . Rabies is often cited as a risk from
feral cats, but none of the 57 human cases of rabies in the United States from 1980 to
2004 was attributable to cats [9,10]. The risk of rabies transmission from cat to human is
Bartonella henselae andBartonella clarridgeiae can both be passed from cats to
humans by biting or scratching. Infection by these bacteria is the most common direct
zoonosis associated with cats, and it results in 25,000 cases of cat scratch disease in the
United States each year . Cat scratch disease is generally a mild illness, but in rare
cases it can lead to serious disease. In Randolph County, North Carolina, 93 of 100
(93%) feral cats and 48 of 76 (63%) pet cats were seropositive for antibodies against B
henselae . In northern Florida 186 of 553 (33.6%) feral cats were seropositive for
antibodies against B henselae, which is within the range of values found in several
studies of pet cats .
Roundworms, hookworms and tapeworms can be shed by cats in their feces and
can potentially cause infection in humans. However, the larvae of roundworms and
hookworms must mature in the environment for at least 3 days and 3 weeks, respectively,
before they can infect a new host . Fleas containing tapeworm must be consumed for
infection to occur . The infection rate of roundworms in feral (21%) and pet (18%)
cats in Randolph County, North Carolina, was not statistically different . However, a
study of 80 feral and 70 pet cats in California found infection rates of roundworms and
tapeworms significantly higher in feral cats than pet cats. The roundworm infection rates
were 54% and 4% for feral and pet cats, respectively, and tapeworm infection rates were
26% and 4% for feral and pet cats, respectively .
The protozoa Toxoplasma gondii, Cryptosporidium parvum, and some species of
Giardia are shed in the feces of cats and can infect humans through the fecal-oral route.
Infection with Cryptosporidium spp. and Giardia spp. in humans is common, but they are
rarely directly linked to cats . In Randolph County, North Carolina, the feral and pet
cats did not have significantly different prevalences of infection with Cryptosporidium
(7% and 6%) or Giardia (6% and 5%) .
Domestic cats and other felids are the only definitive hosts to shed T gondii
oocytes in their feces, thus contaminating the environment with infectious organisms.
The seroprevalence of T gondii in feral cats (63%) in Randolph County, North Carolina,
was higher than for owned cats (34%) . Feral cats from Northern Florida were found
to have a seroprevalence of 12.1%, lower than seroprevalence rates found for pet cats in
the United States (30%) . Consumption of tissue containing T. gondii cysts allows
transmission between intermediate hosts, such as pigs and humans. The relative
importance of the two modes of infection in humans, ingestion of oocytes shed by cats
and consumption of infected tissue, is unknown.
The natural oropharyngeal flora of healthy cats includes Capnocytophaga and
Pasteurella, which are frequently found in cat bite infections. Untreated infections with
these organisms can cause death . However, most feral cat bites are provoked .
Advising the public to avoid direct contact with feral cats and implementation of trap-
vaccinate-return programs should reduce the zoonotic disease risk from feral cats.
The feline zoonotic diseases are transmissible between cats, but cats can also
transmit diseases that do not affect humans, including feline leukemia virus (FeLV),
feline immunodeficiency virus (FIV), and feline coronavirus (FCoV). Some feel that
kitten mortality rates, which may be as high as 75%, and the general health of feral
cats are compelling reasons to euthanize these cats. They believe that feral cats suffer
higher rates of injury and disease than owned cats. However, feral cats brought to
trap-neuter-return (TNR) clinics in northern Florida were lean, but not emaciated ,
had death rates due to complications of surgery comparable to pet cats, and had a
euthanasia rate for humane reasons of only 0.4%[14,15].
In addition, several studies have found that disease prevalence rates are very
similar between feral and owned cats[6,11,16,17]. In 2004, 5,259 feral cats and 12,779
pet cats were tested for feline immunodeficiency virus (FIV) and feline leukemia virus
(FeLV) in 145 animal shelters and 345 veterinary clinics in the United States, Puerto
Rico and Canada. The infection rates for the feral cats were 2.0% (FIV) and 1.6% (FeLV)
compared to the infection rates for the pet cats that were 2.7% (FIV) and 2.6% (FeLV)
. Between 1995 and 2000, a total of 1,876 feral cats from two trap-neuter-return
programs in Gainesville, Florida and Raleigh, North Carolina, had infection rates for FIV
and FeLV of 3.5% and 4.3%, respectively. In addition, 553 feral cats in northern
Florida had similar or lower prevalence rates of Mycoplasma haemofelis, Mycoplasma
Haemominutum and Bartonella henselae compared to previous reports in pet cats .
However, it is possible that the overall health of feral cats brought in to TNR
clinics is different than the health of feral cats in general.
Predation on Wildlife
It has been well-documented that cats can have adverse impacts on wildlife,
particularly on islands. Prey species on island ecosystems are especially vulnerable to the
introduction of non-native mammalian predators such as the cat . Cats have been
introduced to at least 65 island groups and are a major threat to many island bird species
. In 1949, cats were introduced to Marion Island, a 112-square mile sub-Antarctic
island in the Indian Ocean. By 1975, it was estimated that they were killing 450,000
burrowing petrels annually and probably had driven the common diving petrel to local
extinction . On an island in New Zealand, the last remaining Stephens Island Wren
(Xenicus [Traversia] lyalli) was killed by a cat .
There is evidence that cats contributed to the total extinction of the Little Barrier
snipe (Coenocorypha aucklandica barrierensis) and the local extinction of the North
Island Saddleback (Philesturnus carunculatus rufusater) on Little Barrier Island, New
Zealand . However, introduced rats also had a detrimental effect on the bird
populations, and the relative contributions from the cat and the rat could not be
determined . In addition, seven years after eradication of cats was completed on the
island, the bird numbers were similar to their numbers before the cat eradication program
began . It is believed that in some instances cats may actually protect island species
by keeping the rodent population in check [19,22].
Determining the impact of cat predation in mainland settings is even more
difficult than on islands because in most instances there are additional predators present.
Outside of Canberra, Australia, domestic cats were found to be opportunistic predators,
catching prey in proportion to the prey density. The conclusion reached was that cats had
little effect on the local ecosystem . Wildlife managers in Australia have been
working to remove rabbits, another exotic species. However, in many ecosystems rabbits
are the primary food for cats, and there is a fear that a sudden removal of rabbits will
cause the opportunistic cats to switch to native prey. Individual ecosystems must be
evaluated to determine the impact that cat predation has on wildlife and if removal is
appropriate or necessary.
Possible transmission of T. gondii from cats to California sea otters (Enhydra
lutris nereis) is of current concern. Between 1997 and 2001, the seroprevalence of T
gondii was 42% for live otters and 62% for dead otters . The source of the T gondii
is believed to be cat feces from sewage and surface runoff from coastal communities.
However, whether the feces came from litter boxes of indoor cats, from owned cats
allowed outdoors, or feral cats has not been investigated. There is also concern that
native marsupials in Australia, which has non-native cats, may be more susceptible to T.
gondii than ecosystems with native cats.
Although the true impact of feral cats on the health and welfare of the public,
animals, and the environment is difficult to quantify, it is appropriate public policy to
develop effective cat control programs. The best approach to cat control is another area
of intense controversy.
METHODS TO MANAGE FERAL CATS
The most common action taken regarding feral cats has been to do nothing .
Historically, lethal methods of control have been used in an attempt to eradicate
particular groups of cats. In the United Sates, euthanasia is commonly performed at
animal control shelters. Worldwide, however, trapping, poisoning, and hunting have
been the most common methods for eradication of feral cats. The use of biological
vectors has been utilized in at least three island eradications [20,27]. In some areas of the
world, including the United States, there has been strong public pressure to devise more
humane ways of dealing with feral cats, including trap and relocate, and TNR.
Since 1934, cats have been eradicated from 48 islands around the world. All but
five of these islands had cat populations of less than 100, and only 10 of the islands are
greater than 10 square kilometers . The largest eradication effort occurred on Marion
Island using trapping, poisoning, hunting with guns and dogs, and biological control, and
took 19 years to complete . It took three years to eradicate 151 cats from Little
Barrier Island using traps, poison, hunting with guns and dogs, and biological control
The poison used in most eradication campaigns was sodium monofluoracetate
(1080), which has been used since the 1950s in New Zealand for pest control .
Sodium monofluoracetate kills by disrupting the Krebs cycle, thus inhibiting energy
production by cells. Long-term exposure to sub-lethal doses can be harmful to people
handling the poison. Non-target animals are also at risk; dogs are especially susceptible to
1080 . For these reasons, the use of 1080 in the United States is restricted to the
protection of livestock from coyotes . Secondary poisoning by ingestion of rats
poisoned with the anticoagulant, brodifacoum, contributed to cat eradication on four
Feline panleukopenia virus (FPV) was the biological control agent used on Jarvis,
Marion, and Little Barrier Islands . FPV virus is spread through feces, urine, saliva,
and vomit. Felids are the primary hosts of FPV, but raccoons and a few other mammals
can also contract the disease. On Marion Island, 96 cats were trapped and inoculated
with 1000 TCLD5s of FPV and released by helicopter to 93 different locations on the
island. The total cat population declined at a rate of 26% per year for five years, but
stabilized as the population developed immunity . On Little Barrier Island, the use of
FPV was abandoned because the virulence among that population of cats was determined
to be too low to be effective . The use of biological controls has many inherent risks,
including accidental release as happened with the rabbit calicivirus in Australia in 1995
, and non-target susceptibility. As was demonstrated on Marion and Little Barrier
Islands, the target species may become resistant over time.
Eradication or reduction of cats on the mainland is more difficult than on islands
for several reasons. Continuous influx of cats into the area is likely, the risk of non-target
species death increases with poisoning and hunting with dogs, hunting with guns is
heavily restricted for human safety reasons, and biological control mechanisms would be
nearly impossible to contain.
Euthanasia at animal shelters is the leading cause of death of cats in the United
States, with an estimated 3 million cats per year [26,32]. Euthanasia has not been shown
to effectively reduce feral cat populations. In 1996 in Ohio 72.2% of cats admitted to
shelters were euthanized and in 2004, the euthanasia rate was 68.8% . It is
hypothesized that removing cats from a habitat simply allows other cats to fill the vacated
More humane solutions include trapping cats, sterilizing them, and adopting them
into homes, relocating them to a more acceptable outdoor location, placing them in
sanctuaries, or returning them to the location of their capture.
Seventy-five percent of the cats euthanized in animal shelters in the United States
are classified as adoptable , but there are not sufficient homes to accept them.
Increasing the pool of cats waiting to be adopted with less socialized cats is clearly not a
viable solution to the overall problem.
Relocation of cats to a non-enclosed site is difficult because it is time-consuming
to acclimate the cats to their new environment and the cats often have low survival rates
at the new sites .
Another alternative is to remove cats to permanent sanctuaries where they can live
out their lives in confinement. Although sanctuaries can be a small-scale solution, the
overall number of feral cats is too large to be accommodated in sanctuaries. Three
sanctuaries that have reached capacity and only accept cats on a very limited basis are
Best Friends Animal Sanctuary in Utah, the Chico Cat Coalition in California, and a
program instituted by the National Humane Education Society. In addition to their
sanctuaries, Best Friends Animal Sanctuary and National Humane Education Society
operate TNR programs .
The goal of TNR programs is to sterilize feral cats and return them to the location
where they were trapped. TNR programs are generally run as grass-roots operations that
depend on donations and volunteers. However, some municipalities, such as Orange
County, Florida, use TNR programs as a cost-effective alternative to trapping and
euthanizing cats and have incorporated TNR into their animal control efforts. The
average cost of sterilizing each cat was $56 compared to the estimated $139 per cat for
impounding, sheltering and processing . Other agencies that have incorporated TNR
into their animal control programs include Tomkins County Society for the Prevention of
Cruelty to Animals, New York; Maricopa County Animal Care and Control, AZ; New
York City Center for Animal Care and Control; San Francisco Society for the Prevention
of Cruelty to Animals; and the American Society for the Prevention of Cruelty to
Animals. At a minimum, TNR programs sterilize the cats and return them to their
colonies. Some programs provide food, shelter and veterinary care, vaccinate against
rabies and other diseases, and test for diseases such as FIV and FeLV. Removal of the tip
of an ear is recognized internationally as a sign that the cat has been sterilized. For
control to be effective it is essential that colonies are monitored for new arrivals and for
the birth of kittens. A TNR program was started and stopped on a Florida university
campus; once the program stopped, the feral cat population began increasing .
One long-term goal of many TNR programs is to reduce the colony size through
adoption and attrition. Complete elimination of large colonies is uncommon, but there
are many examples of colonies being greatly reduced in size. In 1991, a TNR program
was instituted on the University of Central Florida campus and by 2002, the original 11
colonies containing a total of 155 cats had decreased to eight colonies comprised of 23
cats . In addition to a decrease of 132 cats, other benefits of the program included
prevention of the birth of an estimated 300 to 700 kittens, and medical care or euthanasia
for sick and injured cats in the colonies. In a three-year period ending in 2002, 1,116
California Veterinary Medical Association veterinarians neutered 170,334 feral cats
through the Feral Cat Altering Program . Maddie's Fund awarded nearly $9.5
million to the California Veterinary Medical Association for the program . ATNR
program on a Texas university campus resulted in a 30% decrease in the feral cat
population in two years .
Some wildlife advocates believe TNR is inappropriate because the cats are not
removed from the wild, some critics believe euthanasia is more humane than returning
the cats to their colonies, and others believe TNR is ineffective. Indeed, critics of TNR
often cite two county parks in south Florida, A.D. Barnes Park and Crandon Marina, as
examples of TNR failure. The high visibility of the TNR programs in these parks
encouraged continual cat abandonment at the sites, resulting in a net increase in the
colony size despite a decrease in the original cat populations .
Drawbacks recognized by proponents of TNR include expenses for traps, surgical
equipment, medical supplies, and veterinary fees; intensive labor for trapping,
transportation, surgery, recovery and return to the colony; and requirement for the
technical expertise of veterinarians.
Mathematical models have been used to estimate the percent of feral cats that
would need to be neutered in order to cause an overall decline in a population. One
population model predicted that the annual percent sterilization necessary for stabilization
of population growth would be 14% of the 241,000 cats in San Diego County, CA and
19% for the 36,000 cats in Alachua County, FL . Another study  predicted that
there would have to be a 75% neutering rate annually in order to have a population
decline. This study predicted that a trap and euthanize program would only have to trap
50% of the population to have a population decline. A limitation to the mathematical
models is the difficulty of determining parameters that determine feral cat reproductive
capacity, such as the carrying capacity of a particular habitat .
While surgical TNR can be effective for reducing targeted cat populations, it is
extremely resource-intensive and difficult to implement on a regional or national scale.
Non-surgical methods of contraception or sterilization have the potential for more
efficient implementation, less risk to cats, lower cost, and lower technical requirement
when compared to surgery. Surgical sterilization achieves sterility by removal of the
gonads, thus preventing the production of gametes. Successful non-surgical
contraception or sterilization methods disrupt some aspect of the hypothalamic-pituitary-
gonadal axis or interfere with fertilization of the egg or maintenance of the pregnancy.
Feline reproductive endocrinology
Cats will only breed when they are in estrus, which is characterized by a rapid
increase in estradiol-173 concentrations. The estrogen is released by the follicles, which
are stimulated to grow by the gonadotrope, follicle-stimulating hormone (FSH). FSH is
synthesized and released at a basal level, but the rate of synthesis increases greatly with
the release of gonadotropin releasing hormone (GnRH). Female cats are photoperiod
sensitive and they begin estrous cycling with an increasing daylight-length, as occurs in
January and February in the northern hemisphere. Cats will stop estrus with a decreasing
daylight-length as occurs in September or October. Cats in the laboratory can be induced
to enter estrus by increasing the daylight-length to 14 hours or greater. It is possible that
the pineal gland responds to increasing daylight-length by decreasing the production of
melatonin, which inhibits the release of FSH . Conversely, decreasing the amount of
light appears to increase the production of FSH and the cats will enter anestrous, meaning
that estrous cycling stops.
During estrus, female cats are receptive to male cats and sufficient breeding
triggers a cascade that leads to ovulation. As an induced ovulating species, cats release
GnRH within minutes of genital somatosensory stimuli, as occurs during breeding .
GnRH causes the release of a second gonadotrope, luteinizing hormone (LH), within 5
minutes of the GnRH release . LH concentration increase does not necessarily lead
to ovulation, however. With a single copulation only about 50% of cats will ovulate, but
repeated copulations probably trigger successive GnRH releases which leads to
cumulative increments in LH levels .
GnRH is synthesized in hypothalamic neurons whose axons extend into the
pituitary stalk, which is not within the blood-brain barrier. Upon appropriate stimulation,
GnRH is released by exocytosis into the capillary plexus that emanates from the superior
hypophyseal artery. GnRH is carried down the pituitary stalk in portal veins which give
rise to a second capillary plexus that supplies the endocrine cells of the anterior pituitary.
GnRH binds to gonadotrophs which synthesize and release the two gonadotropins, LH
FSH. However, the duration and the amplitude of the gonadotropin release depends on
the pulsatile and periodic release of GnRH. For a proper functioning hypothalamic-
pituitary-gonadal axis (HPGA), GnRH must be released with the proper pulsatility and
periodicity; the characteristics of the GnRH release differ in male and female cats, with
the age of the cat, and the stage of estrus with the female cat.
LH binds to Leydig cells in the male cat and thecal cells in the female cat and
stimulates the synthesis and secretion of androgens. FSH binds to Sertoli cells in the
male cat and granulosa cells in the female cat and stimulates estrogen synthesis. FSH
also stimulates synthesis in the Sertoli and granulosa cells of various protein products,
including activin and inhibin, two proteins involved in feedback mechanisms within the
HPGA. In addition, FSH increases the number of LH receptors on the granulosa cells,
which amplifies the sensitivity of granulosa cells to LH.
If the female cat does not breed during estrus, or if there is insufficient breeding to
induce ovulation, then the estrogen levels decrease after three to sixteen days  and the
cat enters interestrous, or a phase between successive estrous periods. The cat will
continue to cycle between estrus and anestrous until ovulation occurs or the breeding
season comes to an end with decreasing daylight-length.
If the cat ovulates and the egg or eggs are successfully fertilized then the cat
begins a gestation period of approximately 65 days. If the cat ovulates, but the egg or
eggs are not fertilized, then the cat enters pseudopregnancy which lasts approximately 45
days. In both pregnant and pseudopregnant cats, the follicles that have matured have
released their eggs from the ovary. The portion of the follicles that remain behind are
termed corpus lutea and within 48 hours of ovulation they begin secreting progesterone.
Prior to the release of progesterone by the corpus lutea, the basal level of
progesterone is less than 2 ng/ml. Within 14 to 18 days after ovulation the progesterone
concentrations are greater than 20 ng/ml. The lifespan of the corpus lutea is
approximately 35 days, so the progesterone concentration in a pseudopregant cat returns
to baseline after 35 or 40 days. However, a pregnant cat maintains progesterone
concentrations above baseline because the placenta secretes the hormone after 30 days of
GnRH, the gonadotropes and the sex hormones form a complex feedback web.
GnRH induces the release of FSH and LH, which in turn induce the release of
testosterone and estrogen. However, testosterone and estrogen provide negative feedback
and inhibit the release of LH and GnRH. In addition, the gonads produce three other
hormones that act on the gonadotrophs. Activin stimulates the release ofFSH, inhibin
and follistatin inhibit release of FSH .
Additional hormones that are produced outside the gonads and affect fertility
include leptin, prolactin, growth hormone and insulin-like growth factor 1 (IGF-1).
Leptin is required for fertility , while high concentrations of prolactin inhibit the
secretion of FSH and LH in both sexes. Growth hormone stimulates synthesis of insulin-
like growth factor 1 which stimulates sex hormone synthesis .
The reproductive system in cats provides a number of mechanisms and potential
targets to inhibit fertility, including the use of steroid and peptide hormones to disrupt the
necessary hormone balance, destruction of reproduction-related cells, and
immunocontraception to bind a hormone or hormone receptor in the HPGA.
Steroid hormones and GnRH agonists
Steroids related to estrogen and progesterone have been used since the 1960's for
contraception of some species. Diethylstilbestrol (DES) is a synthetic estrogen that can
reduce fertility in animals, but it must be administered with precise timing, lasts a limited
amount of time, accumulates in body tissues, and has potential adverse health effects in
treated animals . These characteristics preclude DES and other estrogen-related
compounds from intense use in feral cat population control. Progestins, which are
synthetic progesterones, induce contraception, but the mechanism of contraception is not
well understood. The mechanism most likely involves one or more of the following:
negative feedback on GnRH release, disruption of oocyte transport and fertilization, and
altered receptivity of the endometrium . However, limited duration of contraception
and potential harm to target animals also preclude progestins from use in feral cats.
Administration of GnRH agonists disrupt the pulsatile release of GnRH necessary for
secretion of pituitary follicle-stimulating (FSH) and lutenizing hormones (LH) . This
method disrupts gonadal hormone production to produce contraception, but the short
duration of effectiveness makes this method unsuitable for feral cats.
Targeted destruction of specific cells or tissues can be used to disrupt the
reproductive process. A GnRH analogue conjugated to a cytotoxin, pokeweed antiviral
protein, resulted in lower serum testosterone concentrations in dogs . It is believed
that the GnRH analogue portion of the conjugate attaches to GnRH receptors on the
gonadotroph cells in the pituitary gland. The molecule is taken into the cells by
endocytosis and the cytotoxic portion of the conjugate destroys the cell's ability to
synthesize proteins, which leads to cell death. It was hoped that the effect would be
permanent, but a portion of the treated dogs demonstrated increasing FSH and LH serum
concentrations by week 36. Ovarian follicles in rats have been destroyed by
administration of 4-vinylcyclohexene diepoxide (VCD), a metabolite of an industrial
chemical . Female mammals are born with their entire lifetime supply of oocytes
contained within primordial ovarian follicles. VCD targets and destroys the primordial
follicles and the oocytes they contain leaving the animal devoid of oocyte stores and
unable to ovulate successfully. This method provides permanent sterility and cessation of
estrus cycling. However, VCD has only proven safe and effective in rodents and more
research needs to be done in other species. In addition, the current treatment requires
daily doses for 15 days, which is not practical for feral cats.
The goal of immunocontraception is to induce an immune response against
critical elements of the reproductive process that are not used for other functions. There
are several targets that meet this criterion and have been investigated for their
immunocontraceptive potential, including riboflavin carrier protein (RCP), sperm
proteins, zona pellucida (ZP), lutenizing hormone receptor (LH-R), and GnRH.
An acceptable immunocontraceptive agent for feral cats would be effective in
both sexes and only a single treatment should be used because retrapping of free-roaming
cats for repeated treatments would be impractical. In addition, the vaccine should block
the production of sex hormones so as to eliminate nuisance behaviors such as calling,
spraying, wandering, and fighting. In addition to these minimum standards, an ideal
agent would be permanent, have a quick onset of contraceptive effect, be effective in all
ages, be safe to the cat and the environment, be inexpensive, and be easy to administer.
Riboflavin Carrier Protein
RCP is the prime mediator of riboflavin supply to the developing zygote in
mammals and is necessary for maintenance of pregnancy. Three monthly treatments with
an RCP vaccine followed by repeated treatments every four months elicited high
antibody titers and interfered with pregnancy in Bonnet monkeys, but it was not
determined if conception or implantation was affected . The need for multiple
treatments and failure to block sex hormone production make this approach impractical
for feral cats.
Provoking an immune response against sperm proteins has the potential to be
effective in both sexes because antibodies to the proteins could interfere with sperm
development and viability in the male or after insemination in the female . Targeting
sperm would not interfere with sex hormone production, therefore,
immunocontraceptives against sperm proteins are not the best candidate for feral cats.
Zona pellucida (ZP) is a glycoprotein layer surrounding the mammalian egg and
is involved in sperm-egg interaction . The use of ZP antigens for
immunocontraception has been successful in preventing pregnancy in many species, but
not in cats [49,50]. When ZP from pigs, cows, ferrets, dogs and mink was used, the cats
developed high antibody titers to the xenogenic proteins, but these antibodies failed to
cross-react with the cats' native ZP. Immunization with antigens from feline zona
pellucida evoked a poor immune response in the cats, possibly because feline ZP was
recognized as a self protein.
Seven adult female cats immunized against LH-R and given four booster
treatments had absence of behavioral estrus for 500 days, at which point the cats showed
signs of recovering normal ovarian function . While immunization against LH-R has
the potential to block the production of sex hormones, multiple treatments were used and
the goal of a single treatment was not reached. However, it is possible that a variation on
this method that extended the duration of immunity could lead to an effective single-
In a previous study, six male cats immunized three to four times with GnRH
conjugated to tetanus toxoid produced GnRH antibody titers, but there was no resulting
decrease in testosterone concentrations below the contraceptive level . In another
study 10 female and four male cats were immunized against GnRH at eight weeks,
boosted four weeks and 100 weeks later, and housed with a proven male. GnRH
antibodies were detected in all 10 female cats. They had basal progesterone
concentrations, did not display estrous behavior and failed to become pregnant for the
entire observation period of 20 months. Three of the four males had castrate levels of
testosterone for the entire duration of the study. A rise in testosterone in the 4th male was
associated with a decline in GnRH antibody titer .
Vaccines can be delivered orally, by a biological vector, or by injection. Delivery
of the vaccine through bait would be easier than the other methods, but dose is much
more difficult to control, and consumption of the vaccine by non-target species would be
The advantages of a biological vector for immunocontraception include the low
expense for vectors that are self-disseminating; possibly low environmental impact
compared to poisons; and it is considered humane . Disadvantages include the risk to
non-target species and the inherent risk associated with releasing a potential pathogen
into the environment. As designed, the vector is not pathogenic, but there is always a
chance for reversion to virulence or unexpected effects on non-target species. There is
great concern that other species would be transfected by the vector, so this approach
could not be used where other potentially vulnerable species are present. This limitation
applies to almost any mainland area. Biological vectors may play a role in
immunocontraception of cats on islands where the risk of the vector reaching the
mainland is low.
Injection requires the targeting of individual animals, which increases the effort
required for control programs. Injectable vaccines are often delivered with a dart rifle for
large animals , but it requires a skilled marksman and may not be practical or safe for
cats due to their small size.
TNR programs typically use live traps to capture cats for transport to veterinary
clinics the cats to veterinarians for neutering. The same method of capture would allow
delivery of the vaccine by intramuscular or subcutaneous injection in the field without
need for transporting the cats to a central facility for surgical sterilization. An implant,
which would allow timed-release of a vaccine, could also be delivered in this way.
Blocking GnRH from binding to the gonadotrophs disrupts the hormone cascade,
leading to cessation of both fertility and sexual behavior. GnRH has the potential to
satisfy many of the requirements of an ideal antigen for an immunocontraceptive vaccine.
Since GnRH is a decapeptide, it is a hapten which is a very weak immunogen.
The National Wildlife Research Center (NWRC) developed a GnRH vaccine that uses
keyhole limpet hemocyanin (KLH) as a protein carrier and AdjuVacTM as an adjuvant.
The GnRH peptides are attached to the KLH in such a way as to mimic molecular
patterns found on many microorganisms; these patterns activate the innate immune
system . AdjuVacTM was developed as an alternative to Freund's complete adjuvant
(FCA). FCA is a very effective adjuvant, but it can result in harm to the immunized
animal including necrosis and development of granulomas. AdjuVacTM contains
inactivated Mycobacterium avium in oil.
In a previous pilot study in male cats, GnRH/KLH-AdjuVacTM resulted in
testosterone concentrations below the contraceptive level following a single treatment in
six of nine male cats for the duration of the 6-month observation period . A single
injection also prevented pregnancy in female feral pigs for 36 weeks  and was
effective in female deer .
The hypothesis was that a single-dose GnRH vaccine, (GnRH/KLH-AdjuVacTM),
would produce long-term immunocontraception in female cats. The specific objectives
were to determine the proportion of cats that respond, the duration of the response, and
the safety of the vaccine over a 24 month period.
MATERIALS AND METHODS
Twenty-four 8- to 14-month-old specific-pathogen-free female domestic shorthair
cats were acquired from a commercial vendor (Liberty Research, Waverly, NY, USA).
All 24 cats were group-housed in the Animal Care Services facilities at the University of
Florida College of Veterinary Medicine, which are accredited by the Association for
Assessment and Accreditation of Laboratory Animal Care. Cat housing consisted of one
large room with raised resting benches and was climate controlled to maintain ambient
temperatures between 21 and 23 C with controlled lighting. Food and water were
provided ad libitum. The experimental design was approved by the UF Institutional
Animal Care and Use Committee. All cats and their offspring underwent surgical
sterilization and were adopted to private homes at the conclusion of the study.
A GnRH vaccine was constructed using a synthetic GnRH peptide with the sequence
[pEHWSYGLRPGGGC-SH] produced by the Fmoc/tBU protection method (Global
Peptide Services, Fort Collins, CO, USA). Immunogenicity was enhanced by coupling
GnRH peptides to a protein carrier, keyhole limpet hemocyanin (KLH; Pierce Endogen,
Rockford, IL, USA), in a 1:3 GnRH:KLH mass ratio. The underlined amino acids
represent the native GnRH molecule and "pE" signifies pyro-glutamate. Two glycines
were added at the C terminus as a spacer and a cysteine was added to ensure consistent
alignment of the peptide to the maleimide-activated KLH. The aqueous-based GnRH-
KLH conjugate (200 [tg) was combined in a 1:1 ratio by volume with a novel adjuvant,
AdjuVac. AdjuVac was produced by diluting a USDA-licensed Johne's disease
vaccine containing inactivated Mycobacterium avium in mineral oil (Mycopar; Fort
Dodge Animal Health, Fort Dodge, IA, USA)
Upon arrival, the 24 cats were housed for 30 days in a photoperiod regime (8 hours
light: 16 hours dark) that is inhibitory of estrus. Fifty days prior to treatment (Day -50),
the photoperiod regime was reversed to 16 hours light:8 hours dark, which stimulates
estrous cycling within 15 days in 85% of cats . Serum hormones (progesterone and
estradiol-173) were measured on Day -60, then every other day from Day -50 to Day -30.
The magnitude, duration and rate of change of estradiol-173 concentrations in each cat
from Day -50 to Day -30 were used to confirm normal estrous cycling in cats selected for
Confirmation of normal hormonal responses to the lighting change, including
evidence of estrous cycling and docile temperament, were used to select 20 of the 24 cats
to continue in the study. The 20 cats were randomized into a sham group (n = 5) and a
treatment group (n = 15) based on maximum estradiol-173 concentrations. The sham
group received placebo vaccines containing all components except GnRH-KLH. The 15
treated cats received vaccines containing 200 [g GnRH-KLH. Brief anesthesia was
induced by administration of isoflurane (IsoFlo; Abbott Laboratories, North Chicago,
IL, USA) by face mask. The hair of the right cranial thigh was clipped, and the injection
site was cleaned with 70% isopropyl alcohol. The vaccine (0.5 mL) was injected into the
quadriceps muscle group. The right pinna was tattooed with a treatment code,
specifically, Cl through C5 for the sham cats and T through T15 for the treatment cats.
Potential adverse reactions to treatment were evaluated by daily physical
examination, including inspection of the injection site and measurement of body
temperature for one week following treatment.
Blood (4 mL) was collected by jugular venipuncture monthly into serum separator
tubes for determination of GnRH antibody titer and concentration of estradiol-173 and
progesterone. In addition, blood for estradiol-173 and progesterone concentration
determination was collected every other day for 20 days following each of four estrus-
inducing photoperiod changes. Serum was separated by centrifugation and stored at
-20 C until analysis.
Detection of GnRH Antibodies
Serum was tested for GnRH antibodies using an enzyme-linked immunoabsorbant
assay (ELISA). Bovine serum albumen (BSA) was coupled to GnRH in a 1:1
GnRH:BSA mass ratio and 200 ng of GnRH-BSA buffered with 50 [tl bicarbonate was
used to coat the wells in each 96-well microtiter plate and incubated overnight at 40 C.
GnRH-BSA was used so that only anti-GnRH antibodies would be detected, not
antibodies against the KLH component of the vaccine. The wells were washed twice
with 200 [tl of phosphate buffered saline (PBS) and 0.05% Tween 20 (Sigma Chemical
Co., St. Louis, MO, USA), blocked with salmon serum (SeaBlock, East Coast Bio, Inc.,
North Berwick, ME, USA) overnight at 40 C. The wells were aspirated and 100 [tl of cat
serum diluted 1:1,000 in PBS was added in the top row of the plate, with each column
containing a sample from a different cat. Two negative controls were run on each plate;
one negative control was buffer without cat serum and the other was pre-vaccination cat
serum. The last column was used as a positive control with serum from a cat with a
known high antibody titer.
All remaining wells were filled with 50 [tl PBS and serial dilutions were made from
1:1,000 to 1:128,000 by taking 50 [tl from each well in row 1 and mixing it into the
corresponding well in row two. This process was repeated for the remaining six rows.
The plates were then incubated for two hours at room temperature on a shaker. The wells
were washed twice with 200 [tl PBS/0.05% Tween 20.
Antibody to GnRH was detected by adding 50 [tl goat anti-cat IgG (Sigma Chemical
Co.) diluted 1:10,000 in PBS/1% Sea Block to each well. The plate was incubated at
room temperature on a shaker for 2 hours. The wells were washed twice with 200 [tl
PBS/0.05% Tween 20. Fifty microliters of rabbit anti-goat coupled to horseradish
peroxidase conjugate (Sigma Chemical Co.) diluted 1:3,000 in PBS/1% Sea Block was
added to each well and incubated for two hours at room temperature on a shaker. The
wells were washed three times with 200 [tl PBS/0.05% Tween 20. Fifty microliters of
Tetramethylbenzidine/phosphate-citrate buffer (Sigma Chemical Co.) was added to each
well and the plate was incubated for three to five minutes or until a blue color change
developed. Fifty microliters of 2M sulfuric acid was added to each well to stop the
reaction and the plates were read on a plate reader (Dynatech, Horsham, PA) at 450 nm.
The endpoint dilution was considered positive based on the positive control with the titer
equal to the reciprocal of the endpoint dilution.
Determination of Serum Estradiol-17p and Progesterone Concentrations
In cats, behavioral estrus is preceded by a surge in estradiol-173 released by
developing follicles. After ovulation progesterone concentrations rise significantly within
four days . Serum was tested monthly for estradiol-173 and progesterone
concentration. In addition, blood was collected every other day for 20 days following
each of four estrus-inducing photoperiod changes. Serum samples were analyzed for total
estradiol-17P and progesterone by radio-immunoassay (Coat-A-Count; Diagnostic
Products Corporation, Los Angeles, CA, USA) according to the manufacturer's
instructions. The manufacturer reports an estradiol-173 sensitivity of 8 pg/mL with
within-run coefficient of variation (CV) of 4-7% and between-run CV of 4-8%,
depending on the estradiol-17P concentration. The manufacturer reports a progesterone
sensitivity of 0.02 ng/mL with within-run CV of 2-9% and between-run CV of 4-10%,
depending on the progesterone concentration.
The effects of treatment on normal hormone responses and fertility were evaluated
beginning four months after treatment and continuing for the duration of the 26-month
study. The photoperiod length was alternated between the estrus-inhibiting regime (16
hours dark:8 hours light) and the estrus-inducing regime (8 hours dark: 16 hours light).
From Day 0 to Day 120, Day 330 to Day 360 and Day 510 to Day 540, the cats were
exposed to the estrus-inhibiting regime (26% of post-treatment time). For the remainder
of the study the cats were exposed to estrus-inducing light regime (74% of post-treatment
time) (Figure 1). The switch from the short-day photoperiod to the long-day photoperiod
was intended to induce estrus in any of the cats that were capable of an estrous response.
A breeding male was housed continuously with the females during all of the long-day
photoperiods after Day 120. Three breeding males were alternated to assure that inter-cat
incompatibility was not an issue. Time-lapse videography was used to monitor breeding
activity during periods of long-day photoperiod. The tapes were reviewed daily and the
number of attempted and successful breeding was recorded. Distinctive shaving of the
cats' fur was used to allow differentiation of similar-looking cats.
Assignment of Cats to Study Group
During the pre-treatment long-day photoperiod (Day -50 to Day -30), 19 of 24 cats
(79%) had a pattern of estradiol-173 concentrations consistent with estrus based on the
peak concentrations and the rate of change of the concentrations. These 19 cats and one
cat that did not display estrus were divided among the sham and treatment groups in such
a way that the two groups did not have significantly different maximum estradiol-17 3
concentrations (P = 0.4).
Fertility and Fecundity
Fertility was defined by the proportion of cats becoming pregnant. Fecundity was
defined as the number of live births. Pregnancy was scored as a treatment failure, and the
cats were removed from the study after parturition. The treated cats were divided into
responders, which failed to become pregnant during the 19 months following treatment,
and nonresponders, which became pregnant during the study.
It has been well-documented that cats gain weight after surgical neutering, but it is
unknown if immunocontraception has the same effect. To evaluate for this effect, body
weight was recorded for each cat on Day 0 and Day 420, and percent weight gain was
calculated. Radiographic measurement of the falciform fat pad was performed 14 months
(Day 420) after treatment for further evaluation of phenotypic changes. For fat pad
measurement, the cats were anesthetized with medetomidine 40 mcg/kg IM and
radiographed in right lateral recumbancy. Using computed radiography (Kodak,
Rochester, New York, USA), the depth (mm) of the falciform fat pad was measured by
dropping a perpendicular line from the center of the body of the 12th thoracic vertebra to
the ventral body wall and measuring the distance between the caudoventral angle of the
liver and the ventral body wall. The area (mm2) of the fat pad was defined as the area
outlined by the line used for the depth measurement, the ventral border of the liver, the
diaphragm, and the ventral body wall (Figure 2).
Descriptive statistics (mean, error, range) were calculated for each group for hormone
concentrations, GnRH antibody titers, fecundity and body weight. The fertility of the
sham and treatment groups was compared by the two-sided Wilcoxon rank sum test.
Fecundity of the sham and nonresponder groups was compared by one-way analysis of
variance. Differences in GnRH antibody titer and hormone concentrations between the
groups were tested by Kruskal-Wallis one-way analysis of variance on ranks (Kruskal-
Wallis ANOVA). Differences between the three groups (responder, nonresponder and
sham) with regard to body weight, percent change of body weight, falciform fat pad
depth, and falciform fat pad area were analyzed using Kruskal-Wallis ANOVA. The
Holm-Sidak method for pairwise comparison was used for all of the Kruskal-Wallis
ANOVA tests that indicated a difference between at least two of the groups. Differences
were considered significant when P < 0.05. All tests were performed using SigmaStat
statistics software, version 3.0.1 (SPSS Inc., Chicago, IL, USA).
* Blood collection
-60 60 180 300 420 540 660
Figure 1. Timeline for 26-month study showing alternating periods of long-day (16 hr
light:8 hr dark) and short-day (8 hr light: 16 hr dark); periods during which the
breeding male had access to the females (Breeding trial); blood collection times;
the treatment (Day 0); falciform fat pad radiograph; and body weight times.
figuree 2. Lateral abdominal radiograph ot a cat with talcitorm tat pad measurements
marked. The vertical line extends from the center of the body of T12 to the
ventral abdominal wall with the solid portion of the line measuring the fat pad
depth. The three solid lines demarcate the area for the falciform fat pad area
Reactions to Treatment
Body temperature in all of the cats remained normal after treatment, and there was
no inflammation or tenderness at the injection sites. One treatment cat, (T4), died
suddenly 45 days after treatment; necropsy revealed no gross or histological
abnormalities to explain the death. This cat was replaced with a two-year old cat of
proven fertility from the research colony which was tattooed with the code "T16" and
vaccinated with a vaccine containing GnRH. This replacement cat was exposed to the
same lighting regimen and blood collection schedule as the other 19 cats.
Twenty-four months after immunization a 3 cm x 4 cm mass was discovered by
palpation at the injection site. A biopsy was performed and the mass was found to be
neither cancerous or infected. It was concluded that the mass was a granuloma that had
formed in response to the adjuvant. One cat had multiple 1 cm masses near the injection
site and three other cats had a single 1 cm mass near the injection site.
Detection of Estrus
The breeding male cat was introduced on Day 120 at the beginning of the second
long-day regime. All sham cats displayed behavioral signs of estrus and were receptive to
breeding attempts by the male. Time-lapse videography revealed that the male bred each
of the sham cats at least 15 times between Day 122 and Day 155. One cat was observed
breeding in two intervals during gestation (days 7-11, 25-26 of gestation). Four treated
cats displayed estrous behavior and began breeding on Day 128 (T9), Day 418 (T8), Day
469 (T15), and Day 504 (T5). These four nonresponder cats were the treated cats with
the lowest GnRH antibody titers.
Fertility and Fecundity
Based on an average gestation period of 65 days, the date of parturition was used to
estimate the date of successful breeding. All five sham cats became pregnant between
five and 26 days after the male cat was introduced (Day 125 to Day 146) and four of
them within six days of each cat's first observed breeding. Nonresponder cat T9 was bred
in three different estrous intervals from Day 129 to Day 162 before becoming pregnant.
In contrast, nonresponder cats T8, T15, and T5 became pregnant in their first breeding
cycles (Day 418, Day 469, and Day 504, respectively) (Figure 3). The mean number of
live kittens per litter was 3.8 (range 3 to 5) in the sham group, which was significantly
higher (P = 0.03) than the mean of 2.3 (range 1 to 3) live kittens in the nonresponder
group. Based on maintenance of contraception in 11 of 15 treated cats over a 24-month
period, the vaccine had a 73% success rate.
GnRH Antibody Titer
The mean GnRH antibody titer for the responder group was significantly higher than
the titer of the nonresponder group by Day 150 and remained significantly higher for
most of the remainder of the study (Figure 4). All of the sham cats had negative titers
throughout the study.
Three patterns of antibody response were observed among the treated cats: high titers,
variable titers, and low titers. Five of the 11 responder cats (T1, T7, T10, T14, and T16)
had GnRH antibody titers of 128,000 by Day 30 and maintained a titer of 64,000 or
above for the remaining 20 months of the study (Figure 5). The antibody titers of five
other responders (T2, T6, T11, T12, and T13) peaked, then decreased and subsequently
increased again. None of these variable titers decreased below 16,000, and after a trough
level of three to six months, all the titers increased again to at least 64,000 by Day 540
(Figure 6). All four nonresponders became pregnant after their GnRH antibody titer
decreased to 8,000 or below (Figure 7).
For the last seven months of the study, 10 of 11 responders had a GnRH titer of
64,000 or above. The responder cat with the lowest GnRH antibody titer, T3, remained at
a titer of 16,000 for the last 13 months of the study. Two of the nonresponder cats had an
initial high titer that was not sustained and two had titers at 32,000 or below throughout
the study. These four cats became pregnant when titers decreased below 16,000, which
suggests that a titer of 16,000 is contraceptive in female cats. Poor antibody responses in
the nonresponder group delayed, but did not prevent pregnancy.
In cats, ovulation accompanied by fertilization results in pregnancy, whereas
ovulation without fertilization results in pseudopregnancy. In both cases, serum
progesterone increases from a baseline of < 0.5 ng/mL to a peak above 20 ng/mL
between 16 and 26 days after ovulation . In pregnancy, progesterone concentrations
are higher than in pseudopregnancy and remain above baseline for the entire gestation
period. In pseudopregnancy, progesterone concentrations return to baseline by 50 days.
The nine cats (five sham, four nonresponder) that became pregnant had substantial
increases in progesterone concentrations for two months prior to parturition. This finding
was expected given the average gestation period of 65 days. Prior to the introduction of
the male (Day 120), two of the sham cats, two of the responders, and two of the
nonresponders had serum progesterone concentrations higher than 2.0 ng/mL, indicating
ovulation. T9, the first nonresponder to become pregnant, had progesterone
concentrations indicative of pseudopregnancy on Day -30 and Day 60. A second
nonresponder, T5, had elevated progesterone concentration on Day 30. T1 and T2, both
responders, had progesterone concentrations of 7 and 20 ng/mL respectively, on Day 30.
It is likely that these cats experienced nonfertile ovulation and pseudopregnancy prior to
the development of contraceptive titers of GnRH antibody. After Day 30, none of the
responder cats had evidence of ovulation.
It was expected that the sham group's estradiol-173 concentrations would be
consistent with estrous cycling until they became pregnant. Four of the five control cats
became pregnant during the first sustained estradiol-173 elevation (the first estrous cycle)
following the introduction of the breeding male and one cat, C2, became pregnant during
the second estrous cycle.
The mean weights of the sham, nonresponder, and responder cats on Day 0 were not
significantly different from each other (P = 0.5). Similar to cats undergoing surgical
ovariohysterectomy, responding cats had a higher percent gain in body weight than the
nonresponder cats (P = 0.004) and sham cats (P = 0.02) (Table 1). The percent weight
gain was not significantly different between the sham and nonresponder groups (P =
0.08). The mean weight on Day 420 of the responder group was significantly greater than
the nonresponder group (P = 0.002), but not significantly greater than the sham group (P
= 0.1) (Table 1). The mean weights of the sham and nonresponder groups on Day 420
were not significantly different (P = 0.1).
Falciform Fat Pad
At 14 months post-treatment, the mean falciform fat pad depth for the responder
group was greater than the mean for the nonresponder group (P = 0.046) and sham group
(P = 0.03) (Table 2). The mean falciform fat pad areas of the three groups were not
significantly different from each other (P = 0.1) (Table 2).
Figure 3. Breeding trial. Change to a long day-length cycle (Day 120) resulted in the
rapid induction of behavioral estrus and breeding in all five sham cats, whereas
only one of 15 immunized cats was observed to be breeding. All six cats that
bred shortly after Day 120 became pregnant. During the same period three
additional immunized cats displayed behavioral estrus and became pregnant.
@ 120 T I X_
S- T I --Sham (n=5)
-o -Non-responder (n=4)
0 1 Responder (n=11)
30 130 230 330 430 530 630
Figure 4. GnRH antibody titer for female cats (mean + SE). The sham group had
baseline titer for the duration of the study. For nine of the 18 post-treatment
time points the responder cats (cats that did not become pregnant during the
study) had significantly higher GnRH antibody titers than the nonresponder
cats. (*P<0.05.) None of the responder cats had antibody titers that decreased
below 16,000 and all four nonresponder cats (cats that became pregnant during
the study) decreased below 16,000 and became pregnant within 100 days. The
contraceptive titer for these cats ( ) was 16,000.
0 1- -- -- 0--T14
30 130 230 330 430 530 630
Figure 5. GnRH antibody titers for cats with high titers from Day 30 through Day 630.
These five responders (cats that did not become pregnant during the study) (T1,
T7, T10, T14, T16) had GnRH antibody titers of 128,000 by Day 30 and none
decreased below a titer of 64,000 for the entire 24-month treatment period,
which was above the contraceptive titer of 16,000 ( ).
0 _-_ ., ,/__,_
30 130 230 330 430 530 630
Figure 6. GnRH antibody titers for cats with variable titers. These five responders (cats
that did not become pregnant during the study) (T2, T6, T11, T12, T13) had
titers that peaked, decreased, and then increased again. After the second
increase all five cats maintained this new titer through Day 630. The titers for
these cats did not decrease below the contraceptive titer ( ) of 16,000.
130 230 330 430 530 630
Figure 7. GnRH antibody titers of nonresponders (treated cats that became pregnant
during the study). Cats became pregnant on Day 569 (T5), Day 483 (T8), Day
229 (T9), and Day 534 (T15). The titer of these cats decreased below the
contraceptive titer ( ) of 16,000.
Table 1. Body weight and percent weight gain in cats over a 14-month period following
GnRH immunocontraception. The mean weights of the three groups were not
significantly different from each other on Day 0. The percent weight gain of the
responder group was significantly greater than the nonresponder group and
p Weight (kg), % Weight gain,
mean (range) mean (range)
Day 0 (Treatment Day) Day 420 Day 0 to Day 420
Sham 3.54 (3.30 4.18) 4.05 (3.48 -4.66) 15(1 -41)
Nonresponder 3.21 (2.77 3.50) 3.31 (2.62 4.00) 3.5 (-14 31)
Responder 3.34 (2.64 4.20) 4.63 (3.70 5.98) 40 (12-73)
Table 2. Falciform fat pad depth and area in cats 14 months after GnRH immunization.
Fat pad depth of the responder group was significantly greater than the
nonresponder group and sham group. The falciform fat pad areas were not
significantly different between the groups.
Fat Pad Depth (mm) Fat Pad Area (mm2)
Group mean (range) mean (range)
Sham 18.6 (13.2 23.8) 728 (367 1210)
Nonresponder 18.6 (15.2 22.4) 839 (543 1270)
Responder 27.8 (17.2 42.9) 1194 (489 2020)
In this study, 15 sexually intact female cats were immunized once against GnRH.
Twenty-four months after immunization it was discovered that five of the treated cats had
granulomas near their injection site. There was no lameness evident and the masses did
not appear to cause the cats any discomfort. It is possible that a lower dose of vaccine
would still be effective, but not cause this type of reaction at the injection site.
No systemic or local adverse reactions were noted during the 24-month observation
period. For the 24 months following treatment, 73% of the treated cats did not become
pregnant or display signs of behavioral estrus. The four nonresponders had GnRH
antibody titers at or below 8,000 when they became pregnant, while none of responders
had a titer below 16,000 during the study. This suggests that the contraceptive titer in
female cats is 16,000.
As expected, the responder cats reacted to the immunocontraceptive treatment in
ways similar to cats that have undergone surgical sterilization. Behavioral estrus was
suppressed and they gained more weight than the sham and nonresponder cats. However,
surgically sterilized female cats have estradiol-17 3 plasma concentrations consistent with
cats in anestrous, whereas the responder cats intermittently demonstrated estradiol-173
concentrations well above expected anestrous values and possibly indicative of estrus
cycling. However, the females were never receptive nor attractive to the male, so if the
cats were cycling, they were not cycling normally.
Many studies have demonstrated weight gain following surgical sterilization [61-63]
and the percent weight gain in these studies (28% to 39%) was consistent with the 40%
weight gain found in the responder cats in the present study.
The number of live kittens per litter born to the nonresponder cats was significantly less
than the number for sham cats. Immunization against GnRH may not prevent pregnancy
in all cats, but it might interfere with some aspect of pregnancy that results in lower
fecundity. One possibility is that decreased LH, FSH, and estradiol-173 led to fewer
follicles developing and fewer ovulated follicles. However, the sample size was very low
and the difference found may have been due to normal variation.
In a study of nine male cats immunized with the same vaccine used in the current
study (50 to 200 [tg), six cats had a GnRH antibody titer above 64,000, undetectable
testosterone concentrations and testicular atrophy. The three cats with an antibody titer
below 64,000 had measurable testosterone concentrations and normal semen quantity
In a study that targeted a hypothalamic-pituitary-gonadal axis component downstream
from GnRH, seven adult female cats were immunized with lutenizing hormone receptor
(LH-R) encapsulated in an implant and given four booster injections. LH-R antibodies
were detected, progesterone concentrations remained near basal levels, and no behavioral
estrus was observed for approximately 500 days, at which point antibody titers began to
decrease and the cats showed signs of recovering normal physiologic ovarian function.
Estradiol-17P concentrations were not significantly different between the sham cats and
treated cats, even though estrus was observed in the sham cats, but not in the treated cats
. Determining hormonal estrous cycling based on estradiol-173 concentrations
requires estrogen be measured very frequently over the time frame of interest. In the
current study, there were four clusters of 11 every other day blood collection over a 26-
month span. The four clusters of blood collections, each 20 days long, were likely to
reveal an estrous cycle if one occurred during one of those intervals. However, it is
possible that an estrous cycle occurred, but was missed, during the two- to seven-month
intervals between these clustered blood collections. In addition, the basal levels and
estrus profile of estradiol-173 concentrations are highly variable among cats and other
components such as plasma protein content and steroid hormone binding protein may
interfere with the measurement .
Increasing the frequency of estrogen measurement would aid in more precise
determination of estrous cycling regardless of basal and maximum concentrations.
However, continuous collection of blood samples would create welfare and health issues
for cats and monitoring of fecal estrogen would necessitate individual housing. To
increase the accuracy of the estradiol-173 assay, a portion of each sample can be used to
prepare an estradiol-173-free aliquot. The steroid is removed by passing the aliquot
through a charcoal column. The estradiol-173 concentration in each sample is
determined by subtracting the value in the aliquot (sample blank) from the estradiol-173
concentration value in the unfiltered portion 
Immunization against GnRH in other species has been used as an alternative to
gonadectomy to prevent undesirable behavior or characteristics caused by sex hormones
for more than 25 years . It has been primarily used in juvenile male food animals and
has resulted in decreased aggression, testosterone concentration, and testes size in bulls
[66-70], boars [58,71-73] and rams [74,75]. In female pigs [58,76], sheep  and horses
 estrus was suppressed; concentrations of lutenizing hormone, progesterone, and
inhibin A were decreased; and ovarian and uterine weights were reduced. Most of these
animals received multiple treatments, and the observation periods were short since most
of the animals were sent to slaughter.
GnRH immunization has been proposed as a humane solution to overpopulation of
wildlife species and has been tested in deer, bison, rats, swine, rabbits, squirrels, coyotes
and horses . In white-tailed deer, three or four treatments of GnRH vaccine resulted
in a fawning rate reduction up to 88% . GnRH vaccination was 100% effective with
three treatments in male and female Norway rats for up to 17 months . A single
treatment in male and female feral swine resulted in reduced testicular and ovarian sizes,
reduced concentrations of testosterone and progesterone, and a 90% reduction in
pregnancy for 36 weeks . A single treatment in bison led to a decrease in
progesterone concentrations and prevented pregnancies for one year .
There is a protein very similar to GnRH, named GnRH-II, whose function has yet to
be elucidated. However, there is concern that a vaccine against GnRH could also bind to
GnRH-II and disrupt a non-reproduction function . The safety of the GnRH vaccine
has been widely investigated, though, and no significant safety concerns have been
found. A study in male rats and rabbits found no differences in hematological or
biochemical findings between GnRH-immunized animals and surgically sterilized
animals . At necropsy the only abnormalities were detected in the reproductive
organs. Studies in many species including horses , deer , and male cats ,
found no health concerns associated with GnRH immunocontraception.
Contraceptive GnRH antibody titer and effective dose may be different for male and
female animals. The GnRH antibody titer needed to induce contraception in the current
study in female cats (16,000) was lower than the antibody titer needed for male cats
(64,000) treated with the same vaccine . A similar sex difference has been noted in
horses with a contraceptive titer in males of 1,000 and in females of 300 . The
effective dose of GnRH vaccine was found to be lower in male than female swine .
Sex differences may be related to the cyclic nature of hormone secretion in females that
is largely absent in males . Since males continuously produce GnRH, it is possible
that the store of anti-GnRH antibodies is depleted more quickly at the sites of interaction
or that GnRH must be suppressed to a greater degree to block reproductive functions in
males than in females.
GnRH has been shown previously to be an effective immunocontraceptive target in
many species, including cats, but in reports the contraceptive activity was not effective in
all of the animals, required multiple treatments, and faded over time. In contrast, a
practical contraceptive for feral cats must be effective in a large fraction of cats for a
substantial duration following a single treatment.
In this report, a single treatment of GnRH conjugated with KLH and adjuvanted with
M. avium and oil achieved long-term contraception in female cats, meeting the minimum
requirements for contraception in feral cats. Targeting of GnRH had the additional
benefit of curbing nuisance behavior associated with estrous cycling. Additional studies
are needed to investigate the full duration of immunity, rate of efficacy, and safety in
both sexes and all ages of cats.
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John Friary was born July 24, 1970, in Laconia, New Hampshire. He attended
secondary school in St. Petersburg, Florida, studied for one year at the Georgia Institute
of Technology in Atlanta and then moved to Gainesville, Florida. John graduated from
the University of Florida with a B.S. in environmental engineering in 2004. John became
interested in research to benefit feral cats after volunteering at Operation Catnip, a trap-
neuter-return clinic in Gainesville, Florida.
He entered a graduate program at the College of Veterinary Medical at the
University of Florida to investigate the potential for immunocontraception to reduce the
population of feral cats under the advisement of Dr. Julie Levy. He received his M.S. in
John will continue research related to the humane control of feral cats.