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Theriogenology 66 (2006) 122125

Artificial insemination in domestic cats (Felis catus)

Toshihiko Tsutsui *
Department of Reproduction, Nippon Veterinary and Animal Science University 7-1,
Kyonan-cho, 1 chome, Musashino-shi, Tokyo 180-8602, Japan

Artificial insemination (AI) in cats represents an important technique for increasing the contribution of genetically valuable
individuals in specific populations, whether they be highly pedigreed purebred cats, medically important laboratory cats or
endangered non-domestic cats. Semen is collected using electrical stimulation, with an artificial vagina or from intact or excised
cauda epididymis. Sperm samples can be used for AI immediately after collection, after temporary storage above 0 8C or after
cryopreservation. There have been three and five reports on intravaginal and intrauterine insemination, respectively, and one report
on tubal insemination with fresh semen. In studies using fresh semen, it was reported that conception rates of 50% or higher were
obtained by intravaginal insemination with 1050  106 spermatozoa, while, in another report, the conception rate was 78% after
AI with 80  106 spermatozoa. After intrauterine insemination, conception rates following deposition of 6.2  106 and 8  106
spermatozoa were reported to be 50 and 80%, respectively. With tubal insemination, the conception rate was 43% when 4  106
spermatozoa were used, showing that the number of spermatozoa required to obtain a satisfactory conception rate was similar to that
of cats inseminated directly into the uterus. When frozen semen was used for intravaginal insemination the conception rate was
rather low, but intrauterine insemination with 50  106 frozen/thawed spermatozoa resulted in a conception rate of 57%.
Furthermore, in one report, conception was obtained by intrauterine insemination of frozen epididymal spermatozoa. Overall,
there have been few reports on artificial insemination in cats. The results obtained to date show considerable variation, both within
and among laboratories depending upon the type and number of spermatozoa used and the site of sperm deposition. Undoubtedly,
future studies will identify the major factors required to consistently obtain reliable conception rates, so that AI can become a
practical technique for enhancing the production of desirable genotypes, both for laboratory and conservation purposes.
# 2006 Elsevier Inc. All rights reserved.
Keywords: Artificial insemination; Cat; Deposition site; Semen

1. Introduction
1.1. Background
In cats, artificial insemination (AI) may be necessary
when natural mating is not successful, or when the male
and female are housed at separate locations. The
technique is also potentially applicable to aiding in the

* Tel.: +81 422 39 7340; fax: +81 422 39 7340.

E-mail address:
0093-691X/$ see front matter # 2006 Elsevier Inc. All rights reserved.

conservation of rare felids on the verge of extinction [1].

During normal mating, cat semen is deposited
intravaginally; however, knowledge on semen storage
sites after natural mating is limited. Using AI, three
sperm deposition sites intravaginal, intrauterine and
intratubal are possible. The number of spermatozoa
necessary for conception may vary according to the
insemination site. Both spermatozoa can be inseminated
either immediately after recovery, after cryopreservation or after temporary storage at above 0 8C, although
there are no reports yet available using the latter
method. Furthermore, results may depend on whether or

T. Tsutsui / Theriogenology 66 (2006) 122125

not the female experienced a natural estrus at the time of

AI or whether estrus was induced by treatment with
gonadotropic hormones.
In the first report on AI in cats by Sojka et al. [2],
freshly collected spermatozoa were deposited intravaginally. Since then, AI of fresh as well as frozen
spermatozoa, into the vagina, uterus and uterine tube
has been reported. Cat semen has been collected both by
electroejaculation and voluntary ejaculation using an
artificial vagina. Spermatozoa can be recovered from
the epididymides either by percutaneous epididymal
sperm aspiration (PESA) or by transmigration from the
excised cauda epididymides into surrounding medium.
These collection methods may affect fertility in
different ways. The purpose of the present paper is to
review the background and current status of artificial
insemination in cats, including factors influencing the
success rate, with a particular focus on site of sperm
deposition in the reproductive tract.


only other study in which fresh spermatozoa were used

[3] was not published until 30 years after the first report
[2]. In the recent study, a single AI was performed at 15,
20 or 30 h after hCG administration. The conception
rate following AI was 1/16 (6%) with 20  106
spermatozoa, 6/18 (34%) with 40  106 spermatozoa
and 7/9 (78%) with 80  106 spermatozoa. There was
no correlation between the fertilization rate and the time
from hCG administration to AI. Three or four kittens
were born per litter regardless of the number of
spermatozoa inseminated. Even though Sojka et al. [2]
and Tanaka et al. [3] used similar methods for AI, the
results of the latter study indicated that higher sperm
numbers were required to achieve a satisfactory
pregnancy rate as compared to that of the original
report, 80  106 spermatozoa versus 550  106 spermatozoa, respectively. Also, as mentioned earlier,
another difference between the two studies was that
anesthesia was not used in the original report [2], while
the cats in the second study were anesthetized for AI [3]

2. Intravaginal artificial insemination

2.2. Frozen spermatozoa
In cats, intravaginal AI is performed deep into the
vagina using a fine needle, either without [2] or with
anesthesia [3,4], at various intervals after administration
of hCG for induction of ovulation. Including the initial
report in 1970 [2], of a total of three reports on intravaginal
AI in cats, fresh spermatozoa were used in two [2,3] and
frozen spermatozoa were used in the third [4].
2.1. Fresh spermatozoa
In both studies using fresh semen, ovulation was
induced by administering hCG to females in natural
estrus. In the first study on AI in cats that resulted in
conception [2], fertilization was achieved in one of three
females after deposition of 1.25  106 spermatozoa, but
not with 0.5  106 spermatozoa. A conception rate of
54% (14/26) was obtained after insemination with 5
50  106 spermatozoa and two to four offspring were
born per litter. In the same report [2], a preliminary trial
was done to determine if a second AI would result in a
higher pregnancy rate. Eight females were inseminated
with 5  106 spermatozoa at the time of the first hCG
treatment (50 IU) and again 24 h later, at which time a
lower level of hCG (10 IU) was given. Six pregnancies
(75%) were produced in this follow-up trial, which
should have been interpreted as encouraging evidence
for further studies on intravaginal AI in cats. However,
although more than three decades have elapsed since the
initial report on intravaginal AI, there have only been
two additional reports in domestic cats. The second and

In the first report of AI in cats with frozen semen [4],

the samples were frozen by pelleting on blocks of dry
ice before storage in liquid nitrogen. Upon thawing, 50
100  106 motile spermatozoa were intravaginally
inseminated into anesthetized females on the second
and third days of estrus using a 16 g lavage needle (9 cm
long) attached to a 1 ml syringe. Some females were
inseminated during a natural estrus after induction of
ovulation with hCG and other females were given FSH
and hCG to induce estrus and ovulation, respectively,
before AI. Pregnancy was established after AI in 6 of 56
(11%) cats, 4 of which were from the natural estrus
group and 2 were from the induced estrus group. A total
of 12 kittens were born, with litter sizes ranging from 1
to 4 kittens.
3. Intrauterine artificial insemination
Intrauterine insemination in cats has been performed
both by laparoscopy [5] and mid-line laparotomy [68].
There have been four studies on intrauterine AI in cats,
three using fresh semen (Howard et al. [5], Tsutsui et al.
[6,7]) and one using frozen spermatozoa (Tsutsui et al.
3.1. Fresh spermatozoa
In the study by Howard et al. [5], estrus was induced
by administration of 100 IU eCG, followed by induction


T. Tsutsui / Theriogenology 66 (2006) 122125

of ovulation with hCG 80 h later. Bilateral insemination

into the uterine horns was performed with 2.4
19.2  106 motile spermatozoa (mean = 6.2  0.9 
106) under laparoscopic observation. The conception
rate was 2 of 14 (14%) females when the AI was done
before ovulation occurred (2533 h after hCG) and 9 of
18 (50%) females inseminated after the ensuing
ovulation (3141 h after hCG). Howard et al. [5]
suggested that anesthesia with ketamine suppressed
ovulation and recommended that AI be conducted after
ovulation had occurred.
Tsutsui et al. [6] reported conception rates of 13% (2/
16), 31% (5/16) and 80% (8/10) following intrauterine
unilateral AI with 2  106, 4  106 and 8  106
spermatozoa, respectively. A single AI was performed
after accessing the uterus by laparotomy at 15, 20 or
30 h following hCG administration. The conception rate
when AI was done before ovulation was 10/18 (56%),
which was significantly higher than that obtained with
AI after ovulation (5/24, 21%), results that were in
direct contrast to those previously reported by Howard
et al. [5] in which ovulation was induced with
exogenous gonadotropins. The results of Tsutsui
et al. [6] suggest that anesthesia with ketamine and
halothane during the period of ovarian follicle maturation in a natural estrus did not suppress ovulation. Since
the number of spermatozoa used in the study of Howard
et al. [5] ranged widely from 2.4  106 to 19.2  106,
the number of spermatozoa required for conception by
intrauterine AI in their study could not be directly
compared with the results of Tsutsui et al. [6]. The
results of the latter study indicated that conception by
intrauterine AI in cats was possible with 1/10th of the
number of spermatozoa required for conception by
intravaginal AI, a similar ratio to that reported in dogs
[9]. The number of offspring obtained in the studies of
Howard et al. [5] and Tsutsui et al. [6] was not compared
because ovulation was induced by eCG/hCG in the
former study and AI was done at various intervals after
hCG treatment during a natural estrus in the latter study.
3.1.1. Unilateral versus bilateral insemination
Although previous results had demonstrated that
satisfactory conception rates could be achieved after
unilateral insemination of adequate numbers of motile
spermatozoa, it was not clear whether fertilization rates
of oocytes ovulated from the ovary contralateral to the
inseminate site were equivalent to that of oocytes from
the ipsilateral ovary. In an effort to answer this question,
Tsutsui et al. [8] examined whether ova could be
fertilized by unilateral deposition of either 2, 4 or 8  106
spermatozoa into the contralateral uterine horn after

either aspiration of all mature follicles or removal of the

ovary from the side of insemination. When 8  106
spermatozoa were used, the conception rate was 71% (5/
7), as compared to 14% (3/22) of the cats inseminated
with 2 or 4  106 spermatozoa. Embryo survival rate was
100% in three of the five pregnant females (11 kittens
from 11 ovulations) and 25% in the other two pregnant
females (2 kittens from 8 ovulations), for an over all
survival rate of 68% (13 kittens from 19 ovulations) in
cats inseminated with 8  106 spermatozoa.
3.2. Frozen spermatozoa
Tsutsui et al. [10] reported a conception rate of 57%
(8/14 females) after unilateral uterine AI with 50  106
frozen/thawed ejaculated spermatozoa. Recently, Tsutsui et al. [11] reported that of 11 females, 3 (27%)
established pregnancies after unilateral intrauterine
insemination with 5  107 cryopreserved epdidiymal
spermatozoa. Oocytes from the ovary contralateral to
the site of insemination were fertilized in at least one of
the pregnant females. The demonstration that pregnancies can be produced after insemination with cryopreserved epdidymal spermatozoa in domestic cats could
be technically important for the preservation of male
gametes from endangered non-domestic cats.
4. Intratubal artificial insemination
In the only study on intratubal AI in cats [12],
spermatozoa were deposited bilaterally into the ampulla
of the uterine tube. Fertilization was not observed after
AI with 5  103 or 5  105 spermatozoa, but two of
eight females (25%) inseminated with 2  106 spermatozoa and three of seven (43%) females inseminated
with 4  106 spermatozoa did conceive. The number of
spermatozoa required for conception by intratubal AI
was almost the same as that of intrauterine AI [6].
Hence, when performing intratubal AI it is necessary to
pay attention to the number of spermatozoa as well as
the normal capacitating conditions in the cat oviduct.
5. Conclusions
Although AI in cats cannot be sufficiently evaluated
because of the limited number of studies, it has been
confirmed that insemination is possible with both fresh
and frozen semen. Studies have also shown that
spermatozoa inseminated into one horn are able to
migrate to the other side and fertilize ova from the
contralateral ovary. However, large number of spermatozoa are required for surgical intrauterine AI even with

T. Tsutsui / Theriogenology 66 (2006) 122125

fresh semen, and the conception rate by intrauterine AI

with frozen semen is low. Surprisingly, the results of a
single study suggest that high numbers of spermatozoa
may be needed for intratubal AI despite the migratory
ability of spermatozoa, which suggest that oviductal
capacitating conditions may affect fertilization. Therefore, to consistently achieve satisfactory conception
rates by AI and to increase applicability, it is important
to develop non-surgical (i.e. transvaginal) AI techniques
for deposition of spermatozoa into the uterus. The
recent development of transvaginal techniques by
Chatdarong et al. [13] and Zambelli et al. [14] using
a small malleable catheter similar to that originally
designed by Swanson and Godke [15], are potentially
promising approaches.
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