Abstracts

Abstracts
Short Communications abstracts (in order of presentation)
Ultrasonography- an important diagnostic tool in the work up of prostatic disease in the dog.
Nina Ottesen
The Norwegian School of Veterinary Science
Department of Companion Animal Clinical Sciences
Radiology Section
Introduction
In Scandinavia very few people have their male dog castrated routinely. Prostatic
disease is a therefore a common presentation not only in the older dog. The clinical
presentation will usually give an index of suspicion that the prostate may be involved.
There are at least seven diseases affecting the prostate:
1. Benign prostatic hyperplasia (BPH)
2. Parenchymal (retention) cysts
3. Non-parenchymal (paraprostatic) cysts
4. Bacterial prostatitis
a. Acute
b. Chronic
5. Prostatic abscessation
6. Neoplasia
7. Squamous metaplasia
A number of diagnostic procedures are used to make the diagnosis and transabdominal
ultrasonography is a powerful tool for imaging the prostate. Transrectal scanning is the
standard for evaluation of the human prostate gland and the use in the dog has been
reported. In the dog however the necessity of sedation or anesthesia is a disadvantage.
Materials and methods
Client owned intact male dogs were examined using Philps HOI 5000 ultrasound
machine and multifrequency curved linear -and linear probes, 8 - 5 MHz and 12 - 5 MHz
respectively. The dogs were placed in dorsal recumbency and the caudoventral abdomen
was clipped. Transabdominal scanning were performed in longitudinal and transverse
planes.
Discussion
The ultrasongraphic appearance of the different prostatic diseases in clinical cases
will be presented.
257
Abstracts
Short Communications abstracts (in order of presentation)
The presence of a glucokinase-like activity in dog, but not in boar, sperm from fresh
ejaculates
J.Ma. Fernandez-Novell',
J. Ballester", A. Medrano", M. Rivera del Alamo", P.J. Otaequi", J.J.
Guinovart", J.E. Hodrlquez-Gif and T. Rigau2*,
1Department of Biochemistry and Molecular Biology and IRBB, Barcelona Science Park.
University of Barcelona. E-08028 Barcelona. Spain.
2Unit of Animal Reproduction, Department of Animal Medicine and Surgery, School of
Veterinary Medicine, Autonomous University of Barcelona. E-08193 Bellaterra, Spain.
3Department of Biochemistry and Molecular Biology, School of Veterinary Medicine,
Autonomous University of Barcelona. E-08193 Bellaterra, Spain.
*Corresponding author.
E-mail: [email protected]
Introduction
Vertebrate glucokinase (hexokinase type IV) is a member of the hexokinase protein family
which acta as a sensitive and efficient control step for the maintenance of mammalian
glucose metabolism (1). The existence of a glucokinase-like activity in mammalian sperm
could be an efficient system to control the described above, i.e., hexose-specific functional
changes observed, at least in dog. Taking this into consideration, the main aim of this work is
to test the presence of a glucokinase or glucokinase-like activity in mammalian sperm.
Material and methods
For this work, sperm cells from dog and boar were used, as species which showed very
different functional characteristics, from their motion parameters to their life-span after
ejaculation. In these cells the the total hexokinase activity kinetics was determined, and then,
the presence of proteins with immunological properties like rat-liver glucokinase and
glucokinase regulatory protein were tested, in order to find some glucokinase-like activity and
regulation in sperm cells.
Results and conclussions
Hexokinase kinetics from dog extracts showed the presence of a glucokinase-like activity
(DSGLP), in the range of glucose concentrations of 4mM to 10 mM, whereas boar sperm did
not shown any DSGLP activity. Furthermore, dog-sperm cells, but not those of boar, showed
the presence of a protein which specifically reacted against a rat liver anti-glucokinase
antibody. This protein also had a molecular weight equal to that observed in rat-liver extracts,
suggesting a close similarity between both proteins. This glucokinase-like
protein was
distributed in the peri- and post-acrosomal zones of the head, and the midpiece and principal
piece of tail of dog spermatozoa. Our results indicate that dog spermatozoa, but not boar
cells, showed a clear glucokinase-like activity. This activity could be related to the dogsperm's ability to specificially react in front of glucose or fructose. In this way, this activity
could be of the basis of sugar-related specific changes in dog-sperm function.
REFERENCES
(1) Cardenas, M.L., Cornish-Bowden,
242-264
A. And Ureta, T. (1998) Biochim. Biophys. Acta 1401,
259
Abstracts
Short Communications abstracts (in order of presentation)
Effects of Equex STM paste on cryopreserved epididymal dog spermatozoa.
Ponglowhapan S., Chatdarong K., Suwimonteerabutr J. and Lohachit C.
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Sciences,
Chulalongkorn University, Bangkok, Thailand, E-mail: [email protected]
Introduction
There are few reports regarding cryoprotocols of epididymal dog spermatozoa, and the
results demonstrated that post-thaw semen quality was lower in epididymal sperm than in
ejaculated sperm (1, 2). Addition of Equex STM paste to a freezing extender has been
shown to improve viability, motility, longevity and acrosome integrity of ejaculated dog
spermatozoa after freezing and thawing (3). The aim of this study was to investigate whether
Equex STM paste improve post-thaw epididymal dog sperm parameters.
Materials and Methods
Within 15 min after routine castration, sperm were recovered from the cauda epididymides of
13 dogs. A blunted 25 ga needle connected to a 3-mL syringe was inserted into the vas
deferens and sperm were flushed with 1.5-mL flushing medium, tris-fructose citrate solution,
in a retrograde direction from the epididymides. Sperm suspension from each dog was
centrifuged and the supernatant was discarded. The sperm pellet was diluted with an eggyolk tris extender and chilled to 4°C for 2h (equilibration period). Prior to freezing, using
Andersen method (4) described as a two-step dilution protocol (5), the chilled semen
samples were divided into 2 equal portions that were frozen either with or without addition of
Equex STM paste (3). Thawing was done at 39°C for 30 sec. Sperm parameters were
evaluated after collection, before freezing and after thawing at Oh and 2h during the
incubation at 39°C. The percentage of total motile spermatozoa (TM%) was assessed
subjectively. Sperm plasma membrane integrity and acrosome integrity was evaluated using
CFDA / PI staining (6) and FITC-PNA / PI (7) staining, respectively.
Two-hundred spermatozoa were assessed for evaluation of plasma membrane integrity and
acrosome integrity. Data were statistically analyzed using analysis of variance (PROC
MIXED) (SAS institute Inc., Cary, NC, USA) and presented as means ± standard deviations.
Results
Immediately after thawing, Equex STM paste had no effect on TM% (34.2±12.3 and
33.8±14.8; with and without the addition of Equex STM paste, respectively) and intact
plasma membrane (44.8±10.3 and 45.5±8.1; with and without the addition of Equex STM
paste, respectively). At 2h after thawing, higher TM% (P=0.01) and greater number of
epididymal spermatozoa with intact acrosome (P=0.001) were observed in the samples
containing Equex STM paste. The mean of TM%, intact plasma membrane and intact
acrosome of epididymal sperm were 77.7 (±7.2), 80.0 (±5.5) and 93.5 (±7.7) (n=13). TM%
was significantly decreased to 66.9 (±9.9) after equilibration. In overall, freezing and thawing
resulted in a reduced TM% (34.0±13.4) and the percentage of intact plasma membrane
(45.1±9.0) (P<0.05) while acrosome integrity was not affected significantly.
Conclusion
The results from the present study showed that addition of Equex STM paste to a freezing
extender increased longevity of the thawed epididymal dog spermatozoa as well as the
percentage of intact acrosome.
261
References
1.Hewitt et al. 2001. Anim. Reprod. Sci. 67: 101-111.
2.Hori et al. 2004. J. Vet. Med. Sci. 66 (1): 37-41.
3.Rota et al. 1997. Theriogenology47:1093-1101.
4.Andersen K. 1975. Zuchthyg 10: 1-4.
5.Peiia A. and Linde-Forsberg C.2000. Theriogenology
6.Rota et al. 1995. Theriogenology 44:885-900.
7.Axner et al. 2002. Proc. EVSSAR;yet :115.
54: 859-875.
262
Abstracts
Short Communications abstracts (in order of presentation)
Paraphimosis in a toy Yorkshire Terrier. A clinical Case.
lIIas, J; Sa16, F; Arus, J; Vinaixa, F
Veterinaris SL, Terrassa, Spain
Clinical case
A 6 months old Toy Yorkshire Terrier intact male arrived to our clinic presenting a
problem of penile protrusion. The owners brought the dog as soon as they detected the
problem, but they said that it could happen between 3 and 6 hours before bringing the dog to
the clinic. The physical exam was normal, but the dog presented 2/3 of the penile length
outside the prepuce. The penis wasn't erected and the aspect was edematous and swollen
(inflamed). At the exposure we found a bride of hair constricting the penis. This constriction
produced a severe but not bleeding lesion in the penile mucose. The resection of this hair
was made and the penis was reinserted into the prepuce sheath, using cold 50% glucosated
serum to minimize the edema. The dog was sent home with topical antibiotic treatment (2%
c1indamicine pomade {Oalacin Vaginal~), (with) systemic antibiotherapy (cephalexin, 20
mg/kg/8 hours/p.o. [Rilexine~) and wearing an isabellian collar (which was maintained until
the complete resolution of the case); and it was called to revision 2 days later. At the time of
the checking visit the lesion had improved its aspect, but the owner said he had observed a
continuous protrusion of the penis outside the prepuce sheath with difficulties to hide again.
The combined, systemic and topical, medical treatment was working fine, but the severity of
the constricting lesion was the reason of the difficulties of the penis to hide inside the
prepuce sheath when it protruded, as it was creating a chronic edema in the distal portion of
the penis. By the moment we continued with the same treatment and called for next revision
in 2 days. At the third visit, the medical management had achieved the mucosal healing but
failed in avoiding the penile protrusion, because of the fibrous cicatritial ring in the penile
base, consequence of the constriction lesion. The chronic edema didn't allow the penis to
hide when it protruded. At that point we considered to make a partial preputial occlusion,
leaving the penis retained inside the prepuce sheath for 3 weeks; the penis only could be
exposed when the owner forced it. During this time, the owner exposed the penis twice a day
to spread the topical antibiotic pomade and to clean the mucosal surface. Systemic
antibiotherapy was maintained during 2 weeks, but after that we discontinued the treatment
because there had been a total healing of the mucosal wound. After this period of 3 weeks
the mucose was completely healed and no edema was present so we made a preputiotomy
in order to obtain a normal preputial aperture again. After 10 days we retired the sutures and
the dog was considered absolutely recovered. No recurrence of the problem was detected 6
months post-operation.
Discussion:
Paraphimosis is a relatively frequent problem in young intact males, due to
masturbation, sexual activity, trauma or constriction of preputial hair around the penis,
neurological problem, fracture of the os penis, or balanoposthitis. The partial amputation of
the penis was an option because of the gravity of the site lesion, but it wasn't allowed
because the owners expected the dog to be a breeding male .In our case, the recurrence of
the penile protrusion was due to secondary edema caused by the fibrous constriction
appeared during the healing of the lesion. The (partial preputioraphy) preputioplasty was
performed in order to support the medical treatment until the absence of the edema. When
the appearance of the penis was normal, the reconstructional preputiotomy was practiced
giving the preputial opening a normal size. Now the dog has a normal sexual activity. The
recurrence of the problem when there is no lesion complicating the case can be avoided with
orchiectomy, or progestagens compounds therapy as it reduces or eliminates the sexual
excitement. In this particular case, the owners wanted the dog for reproduction, and these
options were not a possible choice, after resolution.
263
References:
-Papazoglou L.G.; Idiopathic chronic penile protrusion in the dog: a report of six cases.
Jou.SmaIlAn.Pra, 2001 ;42:510-513
-Somerville
M.E.;
Phallopexy for treatment of paraphimosis
in the dog.
Jou.Am.An.Hosp.Ass., 2001;37:397-400
-V.Root Kustritz M.; Disorders of the canine penis. Vet.Clin.NorthAm.:SmaIlAn.Prac.,
2001;V31num.2:247-257
264
Abstracts
Short Communications abstracts (in order of presentation)
Short Communication. Saturday 5th June. Female. Minor Room B.
Comparison between plasma prolactin concentration in spontaneous and cabergoline
induced oestrous cycles in the bitch
1
Ada Rota1, Lieta Marinelli2
Department of Veterinary Clinical Sciences, 2 Department of Experimental Veterinary
Sciences - Faculty of Veterinary Medicine, University of Padua, Italy
1. Objective of the work
Oestrus induction in the bitch using cabergoline has entered the general practice, although
the exact mechanism of action of dopamine agonists is not completely clarified and is not
limited to suppression of prolactin secretion [3]. Induced cycles are apparently normal and
leading to normal fertility and pregnancy rates [6, 1, 5]. The aim of this work was to compare
plasma prolactin concentrations in spontaneous and induced cycles, in particular during
pregnancy, around parturition and the beginning of lactation, or dioestrous.
2. Materials and Methods
Four late anoestrous adult German Shepherd bitches were treated with a daily administration
of 5 og/kg cabergoline (Galastop, Centralvet-Vetem- Milan, Italy) until the progression of
prooestrous into oestrous. Blood was collected at least at ten days interval, from the
beginning of treatment until about two weeks after parturition or after the end of dioestrous,
by venipuncture of the cephalic vein, between 8 hand 10 a.m .. The frequency of blood
sampling was higher during oestrous and the bitches were mated once, when suggested by
plasma progesterone concentration.
As control, six spontaneous oestrous cycles, including four pregnancies and two dioestrous
phases, were monitored in the same way. Plasma prolactin concentrations were measured
using a canine immuno-enzymatic assay (MKVCP1, Milenia Biotec, Germany). In both
induced and spontaneous cycles, differences in prolactin concentration between anoestrous
(basal) and the interval when the maximum value is expected (66 - 86 days from the
presumed LH peak) [4], were compared using a paired t-test.
3. Results
All the bitches responded to treatment and prolactin concentration significantly decreased
after cabergoline administration; unfortunately, one of the animals could not be followed until
the end of pregnancy and another one failed to conceive. The number of born puppies was 8
± 0 in the induced cycles and 7.2 ± 1.7 in the spontaneous ones. Pairwise comparisons
between the prolactin concentrations using the paired t-test, showed no statistically
significant difference between basal and maximum expected value in the induced cycles
while the difference was statistically significant (P<O.05) in the spontaneous cycles.
4. Conclusions
Previous works investigated plasma prolactin concentrations during cabergoline
administration, oestrous onset [6] and also during the following dioestrous [2], but apparently
data are lacking regarding pregnancy following an induced cycle. Our observations are only
265
preliminary and the data available at the moment are too few for exhaustive analysis;
however, if confirmed, they suggest a lasting effect of cabergoline that affects prolactin
concentration during dioestrous, parturition and lactation.
5. Bibliography
1) Gobello C et al, 2002, J Am Vet Med Assoc 220, 1653-1654. 2) Jeukenne P & Verstegen
J, 1997, J Reprod Fertil Suppl51, 59-66. 3) Okkens AC et al, 1997, J Reprod Fertil Suppl51,
55-58.4) Onclin K & Verstegen JP, 1997, J Reprod Fertil Suppl 51,203-208.5)
Rota A et aL,
2003, Reprod Dom An, 38, 440-443. 6) Verstegen J et al., 1999, Theriogenology, 51, 597611.
266
Abstracts
Short Communications abstracts (in order of presentation)
THE ACCURACY
MAMMARY
OF FINE-NEEDLE
ASPIRATION
LESIONS: COMPARISON
Eva Hollon
1,
CYTOLOGY
IN CANINE AND FELINE
WITH HISTOPATHOLOGICAL
DIAGNOSIS
Juana Martfn de las Mulas2
Clfnica Veterinaria Canymar. C/ Granja de San IIdefonso, n° 5. 11007 Cadiz.
Dpto. de A. y Anatomfa Patoloqlca Comparadas. Facultad de Veterinaria. Universidad de
Cordoba. Edificio de Sanidad Animal. Campus de Rabanales. 14014 Cordoba.
1
2
OBJECTIVE
To analyse the diagnostic accuracy of fine needle aspiration (FNA) cytology in canine and
feline mammary lesions comparing the FNAC and histopathologic diagnoses of the same
lesion.
MATERIALS AND METHODS
FNA biopsies were taken from 120 mammary gland lesions (101 canine, 19 feline) (Cowell &
Tyler 1999), stained with Diff-Quik and evaluated (Cowell & Tyler 1999; Baker & Lumsden
2000). The histopathologic study was performed on Haematoxylin and eosin-stained tissue
sections of routinely processed biopsy samples from the same lesions (WHO classification of
Misdorp et ai, 1999). Diagnostic accuracy of FNAC was evaluated using the method of
Gerstman & Cappucci (1986) according to the recommendations of Bossuyt and co-workers
(2003) for the complete and accurate reporting of studies of diagnostic accuracy (the STARD
initiative).
RESULTS
Neoplasia / no neoplasia
canine
feline
Cell origin
canine
feline
canine
feline
78
100
77
92
DE (%)
88
SN (%)
90
100
79
100
SP (%)
70
60
73
NO
PPV (%)
96
80
92
100
NPV (%)
47
100
48
NO
DE: Diagnostic
efficacy
NPV: Negative predictive
.82
Malignant / benign
SN: Sensitivity
SP: Specificity
PPV: Positive
predictive
value
value NE: Not done.
CONCLUSIONS
Fine needle aspiration cytology is a reliable diagnostic tool for the diagnosis of feline
mammary lesions. In canine mammary lesions, on the contrary, FNA cytology should be
used as an orientative diagnostic method exclusively because its diagnostic accuracy for the
267
differentiation between malignant and benign neoplasia was low. Thus, benign tumours and
dysplasias were classified as malignant tumours in half of the cases, approximately, while
the opposite occurred in a similar percentage of cases. In addition, FNA cytology did not
allow the identification of the cellular origin of the neoplasias, which can be used as a
therapeutic decision criterion as well as a prognostic indicator.
REFERENCIAS
Baker & Lumsden (2000), Color Atlas of Cytology of the Dog and Cat, Mosby Inc.
Bossuyt et ai, (2003) British Medical Journal 326:41-44.
Cowell & Tyler (1999), Diagnostic Cytology and Hematology of the Dog and Cat, Mosby Inc.
Gerstman & Cappucci (1986) JAVMA 188:248-251.
Klaasen (2002) The Veterinary Clinics of North America 32:1237-1266.
Misdorp et ai, (1999) WHO, Washington DC, Armed Forces Institute of Pathology.
268
Abstracts
Short Communications abstracts (in order of presentation)
Ultrasound-guided
aspiration in management of ovarian follicular
Cysts in the bitch.
G. Bassu', O.Raule, J.Besso2, N.Marseloo1 and A.Fontbonne1
1: Animal Reproduction, 2: Diagnostic Imaging
National Veterinary College, Alfort (Paris).France
Up to now, reported methods to treat follicular cysts in breeding bitches consist either
in surgical cure, or in using hormonal molecules, like GnRH or hCG, in order to
induce luteinization (2), which bears the danger of promoting cystic endometrial
hyperplasia or pyometra. The purpose of this clinical study was to test the efficiency
of fine-needle aspiration as an alternative treatment of ovarian cystic lesions.
Materials and methods.
6 bitches of different breeds, aged 2-6 years, suffering from fertility disorders and
showing anechoic ovarian lesions sized 1.2 to 3.8 em in diameter, were included in
this clinical trial. All of them had ovulation problems, suggesting a potential role of
functional follicular cysts: blood progesterone remaining between 2.59 and 4.36
during several days (2 cases), blood progesterone during oestrus decreasing from
8.07 to 3.28 (1 case), abnormally prolonged heats (1 case), short interestrous
intervals and prolonged anovulatory heats (1 case), persistent vaginal oestrus
smears with > 90% superficial cells several days after progesterone increasing above
10ng/ml (1 case).
The animals were sedated intravenously using propofol or xylazine, and placed in
dorsolateral recumbency. After shaving the site of puncture, the skin was cleaned
and draped with aseptic precaution. Ultrasonographic approach was done using an
ATL HOI 3500 ultrasound machine and a high frequency transducer (7.5 to 10 MHz).
The caudal pole of the kidney and the adjacent area were scanned in sagittal and
transverse planes to locate the ovary. Cyst aspiration was done, either with a fine
needle (22 or 23 gauge), or with a spinal needle, in the case of larger bitches. The
progress of the needle was observed through the tissue on ultrasound until the tip
was visualised well within the cyst. The needle tip was monitored throughout
aspiration.
Further ultrasonographic survey of the ovaries was performed to detect ovulation of
the remaining follicles (4 cases) or any recurrence of the cystic lesions. All ovulating
bitches were mated or artificially inseminated and a ultrasound pregnancy diagnosis
was performed between 21 to 25 days of pregnancy. Fertility and litter size were
recorded.
Re.sults.
Aspiration and emptying the cystic ovarian lesions were successful in all bitches.
There were no procedure-related complications and no recurrence or persistence of
lesions after several days.
Ovulation occurred in the 4 bitches showing either abnormally prolonged heats, blood
progesterone remaining between 2.59 and 4.36 during several days, or blood
progesterone during oestrus decreasing from 8.07 to 3.28.
269
3 of these 4 bitches conceived, litter size was respectively 7,8, 1 and 0 pups. The 2
other bitches were not pregnant at early pregnancy diagnosis.
Conclusion.
As in women (1), fine needle aspiration was shown to be an easy, safe and effective
technique to aspirate ovarian cystic lesions in bitches, provided that a good
ultrasound machine with an improved image quality and a high frequency probe are
available.
Further investigations are required to evaluate the clinical efficiency of these
technique in restoring ovulation and fertility, compared to hormonal or surgical
treatments of follicular cysts.
References:
(1): S.Mittal et al. Ultrasound-guided aspiration in mamagement of cystic lesions of the ovary: IntJ.Gynecol.and Obstetrics.
1998, 62, 261-267.
(2): S.D. Johnston, MRoot-Kustritz and P.N.S.Olson. Canine and Feline Theriogenology. Philadelphia,2001, we Saanders.
270
Abstracts
Short Communications abstracts (in order of presentation)
Infertility in Miniature Bull Terriers bitches
Ludovica Salamon, DVM
Clone Italy, Via Cadorna 2 - S. Alessio 27016 (PV) Italy - E-mail: [email protected]
Objective of the work
Efficacy of a combined treatment with tiroxina and low dosage aspirin during proestrus,
estrus and pregnancy in three infertile hypothyroid Miniature Bull Terriers (MBT).
Materials and Methods
Three MBT bitches were monitored from proestrus till day 25 post breeding. Bitch n. 1 and n.
2 were 7-year old full sisters, previously mated four times at different estrous, never whelped
before in spite of a correct serum progesterone (P4) rising. Bitch n. 3, not related to n. 1 and
n. 2, two years old, mated once in the past, never whelped before. Blood work-up: CBC,
serum chemistry panel, IT3 and IT 4 repeated two weeks after onset of oral treatment with
tiroxina (Eutirox™, Bracco) 50 Jlg, bitch n. 1 and n. 2 one tablet am and one pm, bitch n. 3
half tablet am and half tablet pm from proestrus for 120 days. P4 was assayed (MINIVIDAS,
ELFA) around ovulation time and during pregnancy. Aspirin tablets (Cardloaspirinw, Bayer),
50 mg once daily, were administered from day 1 till day 55 post breeding. Bitch n. 1 and n. 2
were mated, while bitch n. 3 was inseminated with frozen semen. Aspirin was withdrawn 5
days prior to whelping as it could cause an insufficient release of prostaglandins.
Results
Blood tests were normal; IT3 - IT4 results were below average at the beginning of proestrus
but later raised to within the normal range for the rest of pregnancy. P4 raised normally
around ovulation and remained above the minimum for pregnancy. Bitch n. 1 whelped four
live puppies, one of which with spina bifida. Bitch n. 2 had three embryonic vesicles at day 25
post breeding on ultrasound, then showed a bloody vulvar discharge and embryos
disappeared in three days. Bitch n. 3 was pregnant with four normal foetuses on ultrasound
till day 55 post breeding, but only one puppy was born alive and normal; aspirin was
withdrawn in this bitch at day 25 because of a persistent bloody vulvar discharge. Aspirin can
cause hemostatic defects especially in hypothyroid bitches.
Discussion
The fact that reproductive failure in these 3 bitches (despite good reproductive management) was
solved by administering a thyroid replacement therapy confirms the role of hypothyroidism in canine
infertility. However, in the author's experience hypothyroid bitches will often fail to carry a pregnancy to
term regardless of proper thyroid replacement therapy. The rationale for using aspirin in human
pregnancy is to keep under control the level of antiphospholipid and antithyroid antibodies. As thyroid
status had never been checked before in these 3 bitches, no definitive conclusions can be drawn on the
role of aspirin, although it is interesting to note that bitch n. 3 lost 3/4 foetuses following aspirin
withdrawal.
271
References
Giacomucci E, et al. 1994. J. Steroid. Biochem. Mol. BioI. Jun; 49(2-3): 107-21 - Kutteh WHo 2002. Minerva Ginecol. Jun; 54(3):
217-24 - Sher G, et al. 1998. Am. J. Reprod. Immunol. Apr; 39(4): 223-5 - Zeigler ZR, et al. 1986. Am. J. Hematol. Dec; 23(4):
391-9
272
Abstracts
Short Communications abstracts (in order of presentation)
Expression of active caspase 3 in the reproductive tract of the bitch
Iglesias M.1,Oliva J.1,2,Garcla-Botey C.1, Perez J.F.1
1Department of Medicine and Surgery, School of Veterinary Medicine, Complutense
University of Madrid, 28040 Madrid, Spain. 2INIFAP, Mexico
Introduction
Apoptosis is a highly organized sequence of biochemical events that mediates cell death.
Together with cell proliferation, have been suggested to regulate ovarian and uterine
function. At least two pathways have bee been identified in the control of apoptosis, one
involves a mitochondria-dependent pathway modulated by bcl-2 proteins, and the second
one is a pathway triggered by the activation of tumor necrosis factor (TNF) group receptors.
Both pathways induce caspase 3 activation, a cysteine protease that cleaves essential
nuclear proteins and structural nuclear lamins inducing apoptosis (1). Caspase 3 has been
detected in the reproductive tract of the hen, quail, mice and humans; however, its
expression in the reproductive tract of the bitch has not been investigated.The aim of this
study was to determine the active caspase 3 localization in the reproductive tract of the bitch
Materials and methods: Reproductive tracts from 15 bitches, 10 from healthty bitches and 5
from bitches that exhibited cystic endometrial hyperplasia (CEH); were collected at surgery,
fixed in 10% formalin for 24 hours at 4°C, and processed routinely with paraffin embedding.
Sections were cut in 51lm sections mounted on poly-L-Iysine coated slides, dried,
deparaffinized in xylenes and rehydrated in graded ethanol solutions. Sections were stained
with hematoxylin/eosin. Immunocytochemistry was performed using a 1:20 dilution of anti
caspase 3 (CPP32) Ab4 (Neomarkers). Samples were incubated overnight in a humidified
chamber at 4°C. Primary antibody binding was detected using a biotynilated secondary goatantirabbit IgG with aVidin-biotin-peroxidase complexing. The primary detection reagent was
3-amino-9-ethylcarbazole AEC (Vector Labs). Sections were cover-slipped in aqueous-based
mounted medium. Positive and negative controls were included in each experiment. Staining
intensity and proportion of cells stained in each group of cells were evaluated according to a
subjective scale (2). Statistical analysis: Differences between healthy bitches and bitches
that presented CEH, were analysed by a Wilconxon test using SAS software.
Results and discussion: Active caspase 3 was localized in the ovary and in the uterus of the
bitch. Caspase 3 was expressed in all ovarian structures. Interstitial cells, blood vessels and
SUb-superficial cells showed the highest staining intensity scores. Thecal cells from healthy
follicles showed higher intensity than the granulosa cells and the ovary, whereas in the
atretic follicles granulosa cells and ovary showed higher intensity than the theca cells. No
immunoreactivity for caspase 3 was detected in the luteinized theca cells, stroma nor in the
negative controls. Results from uterus showed more homogeneous staining pattern.
Caspase 3 was expressed in the glandular and superficial endometrial epithelia, myometrium
and blood vessels. The tissues obtained from healthy bitches, and those obtained from
bitches that exhibited CEH, showed similar staining scores. The presence of active caspase
273
3 in the genital tract of the bitch suggests that apoptosis could be involved in the regulation of
the ovarian and uterine function in this species.
1. Roseto A, Brenner C (1999): Arch Argent Pediatr, 97(4): 253-75.
2.Vermeirsch H, Simoens P(2001): Reproduction, 122: 73-83.
274
Abstracts
Short Communications abstracts (in order of presentation)
Tolerability of MILBEMAX® (milbemycin oxime and praziquantel)
lactating queens
in breeding and
Rudolf Schenker, Robert Cody & GOntherStrehlau
Novartis Animal Health Inc.
CH-4002 Basel
Switzerland
The tolerability of MILBEMAX (milbemycin oxime and praziquantel) given weekly at the
highest recommended dose was evaluated in queens during anoestrus, proestrus,
pregnancy and lactation. The treatment schedule ensured that the test article was
administered on each day of pregnancy. The effect of this treatment on the resulting kittens
until weaning was assessed.
Two groups of 15 queens each were treated either with placebo or MILBEMAX. During each
phase of reproduction the queens were clinically assessed and their bodyweight,
haematology and clinical chemistry evaluated. Reproductive performance of the queens, and
clinical assessment, bodyweight, haematology and clinical chemistry in the resulting kittens
were also measured.
In queens no clinically relevant differences between the two treatment groups for all
parameters measured were found in the pre-study evaluation, and during pregnancy and
lactation. Reproductive performance in terms of length of pregnancy, number of kittens,
number of kittens alive or dead and congenital abnormalities was not different between the
two groups. Dystocia did not occur.
In kittens up to 56 days of age there were also no clinically relevant differences between the
two groups for the parameters measured.
This study demonstrated that there are no treatment related effects on reproducing queens
treated with MILBEMAX and the resulting kittens. MILBEMAX is very well tolerated by
reproducing queens and has no effect on reproductive performance and the resulting kittens
275
Abstracts
Short Communications abstracts (in order of presentation)
Treatment of spontaneous
pyometra in the bitch with a combination of cabergoline
and prostaglandin
G.C.W. England* and M. Russo. *Guide Dogs for the Blind Association, Tollgate House,
Banbury Road, Bishops Tachbrook, Warwickshire, and Department of Clinical Sciences,
Section of Clinical Obstetrics, Faculty of Veterinary Medicine, University of Naples, Italy.
Abstract
This study evaluated the response of bitches with both open-cervix and closed-cervix
pyometra to treatment with a combination of cabergoline and prostaglandin. Twenty-two
bitches were recruited to the study, of which 21 successfully responded to the medical
treatment regime. Of these, 11 bitches were subsequently mated and 7 became pregnant
and delivered normal litters, although the number of pups was smaller than the average for
the breed. Two unmated bitches had a recurrence of pyometra after the subsequent oestrus.
Materials and methods
Twenty-two bitches with ultrasonographically diagnosed spontaneous pyometra were treated
with a combination of cabergoline (5 mcg/kg/day), c1oprostenol (5 mcg/kg every third day)
combined with the twice-daily administration of potentiated sulphonamide. Bitches were
examined on alternate days and a number of assessments were made including clinical
examination, haematology, biochemistry, plasma progesterone concentration and ultrasound
examination of the reproductive tract.
Results
Nineteen of the bitches were successfully managed by a to-day treatment period. Two
bitches required a further three days of treatment and in one bitch with a partial uterine
torsion the treatment was not successful and an ovariohysterectomy was performed.
Bitches with both open and closed-cervix pyometra showed a rapid clinical improvement
associated with a reduction in plasma progesterone concentration, increased vulval
discharge and a reduction in the uterine diameter. For the 21 bitches that were successfully
treated, haematological and biochemical profiles were normal within six and nine days of
treatment respectively.
Adverse effects of treatment were transient, limited to the 45 minutes immediately after
prostaglandin administration, and included retching, vomiting, mild abdominal straining,
diarrhoea and panting. The incidence of adverse effects was reduced with each subsequent
dose of prostaglandin.
Eleven of the 21 successfully treated bitches were mated at the subsequent oestrus and
seven became pregnant. The litter size was smaller than published breed averages. Two
unmated bitches had a recurrence of the pyometra following the subsequent oestrus.
277
Conclusion
This study demonstrates the possibility of managing spontaneous pyometra using a
combination of a prolactin antagonist with prostaglandin, either for patient stabilisation prior
to surgery or for the potential maintenance of breeding capacity.
278
Abstracts
Short Communications abstracts (in order of presentation)
Treatment of urinary incontinence after spaying with GnRH analogues
1M Reichler, Veterinary Faculty, Zurich, Switzerland
A novel therapeutic approach for the treatment of urinary incontinence: In a retrospective
study the success of treatment with depot preparations of GnRH analogues in 33 bitches
with spaying induced incontinence was evaluated. Before and 8 to 11 weeks after treatment
the plasma gonadotropin levels of these bitches were analyzed. The bitches, which were
continent after treatment, were compared to the remaining dogs. The success of the therapy
was evaluated with reference to possible influencing factors, such as weight, age, duration of
incontinence, changes in the plasma gonadotropin levels or response to the pre-treatment.
The objective of this analysis was to determine the mode of action of depot GnRH on
spaying related incontinence. Bitches that had an inadequate or no response to GnRH were
additionally treated with alpha-adrenergics. Their response to the combined treatment was
compared to what had been reported by the owners during the previous treatment of these
animals with alpha-adrenergics alone. The purpose of this comparison was to determine if
dogs which responded neither to alpha-adrenergics nor to depot GnRH analogues become
continent with a combined therapy.
FSH and LH concentrations were determined with a canine specific LH radioimmunoassay
and a canine specific FSH immunoradiometric assay. The effect of the GnRH-analogue
treatment on the plasma gonadotropin levels was analysed with a paired sign-test. All data,
including duration of continence after GnRH treatment alone, body weight, duration of
incontinence before treatment and age at the time of spaying were recorded as mean values
with a 95% confidence interval. The influence of various factors, such as age at the time of
spaying, duration of incontinence and changes in plasma gonadotropin levels, on the
success of GnRH treatment were analysed by the Mann-Whitney test. Additionally, the
number of bitches, which were continent after GnRH application alone or in combination with
alpha-adrenergic drugs, was compared descriptively with the number of bitches, which had
already been successfully pre-treated. After GnRH therapy 52% of the bitches were
continent and a further 42% were continent with additional alpha-adrenergic medication. The
success of treatment with depot GnRH analogues correlated neither with the data extracted
from their histories nor the changes in plasma gonadotropins.
Depot preparations of GnRH analogues alone or in combination with phenylpropanolamine
are suitable alternative medical treatments for urinary incontinence in bitches, which respond
inadequately to alpha-adrenergics. This observation indicates that there is a synergistic
effect of depot GnRH analogues and alpha-adrenergics. Possibly, the GnRH analogues
restore the responsiveness of the alpha-adrenergic receptors.
References:
[1]B.SAVA
Congress report (1975). Sequelae of bitch sterilisation: regional survey. Vet Rec 1975;96:371-372.
[2] Holt PE, Thrusfield
MV. Association
in bitches between breed, size, neutering and docking, and acquired urinary
incontinence due to incompetence of the urethral sphincter mechanism. Vet Rec 1993;133:177-180.
[3] Krawiec DR. Diagnosis and treatment of acquired canine urinary incontinence. Camp Anim Pract 1989;19:12-20.
[4] Ruckstuhl B. Die Incontinentia urinae bel der HOndin als Spatfolge der Kastration. Schweiz Arch Tiertleilk 1978;120:143-148.
[5] Thrusfield MV. Association between urinary incontinence and spaying in bitches. Vet Rec 1985;116:695.
[6] Thrusfield MV, Holt PE, Muirhead RH. Acquired urinary incontinence in bitches: its incidence and relationship to neutering
practices. J Small Anim Pract 1998;39:559-566.
[7] Arbeiter K. Harnblaseninkontinenz
nach der Ovariohysterektomie
bei der Hundin. Kleintierpraxis 1986;31 :215-222.
[8] Blendinger C, Blendinger K, Bostedt H. Die Harninkontinenz nach Kastration bei der HOndin. Tierarzt Prax 1995;23:291-299.
279
(9) Arnold S, Arnold P, Hubler M, Casal M, ROsch P. Incontinentia
urinae bei der kastrierten HOndin: Haufigkeit und
Rassedisposition.
Schweiz Arch Tierheilk 1989; 131 :259-263.
[10] Finco DR, Osborne CA, Lewis RE. Nonneurogenic causes of abnormal micturition in the dog and cat. Vet Clin North Am
1974;4:501-516.
.
[11] Osborne CA, Oliver JE, Polzin DE. Non-neurogenic urinary incontinence. In Kirk RW (ed.): Current Veterinary Therapy VII.
Philadelphia: we Saunders, 1980;1128-1136.
[12] Reichler 1M, Pfeiffer E, Piche C, Jochle W, Roos M, Hubler M, Arnold S. Changes in plasma gonadotropin concentrations
and urethral closure pressure in the bitch during the 12 months following ovariectomy. Theriogenology in press.
280
Abstracts
Short Communications abstracts (in order of presentation)
Experiences with total intravenous anaesthesia (TIA) of Fentanyl - Midazolam
combination induced narcosis for caesarean section in dogs
Aniko Tibold, Krisztina Biro, Lajos Balogh, Julianna Thuroczy
SzlU, Faculty of Veterinary Science, Department of Obstetrics and Reproduction
H-1400 Budapest, P.O.Box: 2. [email protected]
Aims and scope
At the time of caesarean section, the mother may suffer respiratory depression,
hypotension, increased gastric acidity, increased predisposition to regurgitation while
the newborn may suffer anaesthetic-induced respiratory depression. Fentanyl, a
synthetic micro-opioid receptor agonist is a preferred anaesthetic agent for induction
and maintenance in human cardiac surgery. Continuous intravenous infusion of
fentanyl raises its plasma concentration in a linear fashion within 20 minutes. This
study evaluated the quality of anaesthesia, cardio- respiratory and stress response
induced by the combination of Fentanyl (Fentanyl 5ml Richter Gedeon Inc. ) and
Midazolam (Dormicum 5mg EGIS Budapest Hungary).
Materials and Methods
Caesarean section due to dystochia has been performed in thirteen pregnant bitches
from different breeds. Blood was collected prior to induction of narcosis and at the
end of surgery from the cephalic vein in anticoagulant pre-treated (heparin)
evacuated tubes for measurement of biochemical parameters and in tubes without
anticoagulant for cortisol assay (BD Vacutainer, Belliver Industrial Estate, Plymouth,
UK). Blood was stored at 4 ec, centrifuged within 1 hour for hormone determination.
Serum was stored at -18 "C till assayed by ELISA (Active Cortisol DSL Inc. USA
Texas). Induction of narcosis was performed in bolus of combination of Fentanyl at
O,04mg/kgof bw.and Midazolam at 0,2 mg/kg of bw. Maintenance of narcosis during
surgery was completed with continuous intravenous infusion of Fentanyl at 0,001
mg/kg of bw. combined with Midazolam at 0,02 mg/kg of bw. per minutes by an
infusion pump (ASCOR spolka zo.o Poland). The intravenous narcosis was
complemented till end of surgery with O2 inhalation. Hearth frequency and 02
saturation was monitored by a pulse-oxymeter (504 DX Digital Oximeter Criticare
Systems Inc. USA).
Results
During fentanyl-midazolam infusion complete surgical anaesthesia was obtained.
Mean± SD duration of the infusion was 29,83 ± 5,77 minutes for dogs undergoing
caesarean section. Significant difference was present only between lowest and
highest O2 saturation 79,5 ±11,16, 98,83±0,88. Values for cortisol, ALT, alkaline
phosphatase activity, GGT did not vary significantly with anaesthesia (p<O,05)
(Table).
281
Mean
±SD
Hearth
frequency
Ind.
Ter
m
70,8
81
3
13,5
15,3
3
Saturation
Cortisol
Ind.
Ind.
90,1
6
9,11
Ter
m
85,6
6
12,7
7
ALT
279,
6
Ter
m
333,
6
83,4
7
74,5
7
Ind.
Alk.ph.
Ind.
GGT
53,5
8
Ter
m
51,0
8
251,
4
Ter
m
219,
6
16,1
8
19,2
9
139,
0
128,
6
Ind.
6,18
Ter
m
5,56
4,41
4,27
Discussion
Results suggest that induction of anaesthesia with Fentanyl-Midazolam bolus and
maintenance with continuous infusion of the same components can be considered
as a safe anaesthetic procedure in bitches and puppies undergoing caesarean
section. The combination was suitable for obtaining surgical anaesthesia during
hysterotomy.
282
Abstracts
Short Communications abstracts (in order of presentation)
Short Communication. Sunday 6th June. Paediatrics and exotics pets. Minor
Room B.
Reproductive physiology in the male lynx (Lynx lynx)aaB
Axner E1, Linde-Forsberg C1, Morner T2, Ulhorn H2,Agren E2.'Department of Obstetrics and
Gynaecology, Swedish University of Agricultural Sciences, P.O Box 7039, SE-750 07
Uppsala. 2Departmentof Wildlife, National Veterinary Institute, SE-751 89 Uppsala, Sweden.
Introduction
This abstract presents preliminary data from a study on the reproductive function in the male
Eurasian lynx and evaluates the possibility to retrieve live spermatozoa from dead lynx to
create gene banks. In dogs motile spermatozoa can be recovered from epididymides that
have been stored for 8 days (Yu & Leibo 2002). All lynx that are killed at hunting or found
dead in Sweden are sent to the Swedish Veterinary Institute (SVA).
Material and methods: Reproductive organs from 36 lynx were collected from November
2002 to May 2003. Time between death and evaluation ranged between 2 and 21 days. In
some animals, parts of the reproductive tract were accidentally removed in connection with
pelting. The mean weight of the testes in each animal was calculated. Spermatozoa were
collected by mincing the cauda epididymidis in Dulbecco's PBS, Ham's F-10 or Kenney's
dilution media at 37°. Acrosome integrity was evaluated with FITC-PNA and the sperm
membranes with EthD-1 and Sybr-14 (Axner et al. 2004). Penile spines were present in
19/20 animals in which the penis could be extruded from the prepuce.
Results and discussion: The mean weight of the testes was significantly correlated with body
weight (bw) in animals with a bw<20 kg (rs=0.88, P<0.001, n=19) but not in animals with a bw
~20 kg (r=0.017, P>0.2, n=17). The lowest bw of an animal from which spermatozoa could
be recovered from the cauda epididymidis was 13 kg and the lowest mean weight of the
testes was 1.5 g. Spermatozoa were found in all males with a mean testis weight >2 g and
with a bw ~O kg. The mean percentage of membrane intact spermatozoa was 15.9±21.5%
(range from 0 to 68.5%, n=22). The mean percentage of acrosome intact spermatozoa was
17.2±14.6% with a range from 0 to 45.5% (n=21). Sperm motility was 5% in one male, 1% in
two males and 0% in 19 males. The effect of season on testicular activity could not be
evaluated because of the skewed distribution with 30/36 males that died during January to
March (which is the hunting season). The present data indicates that the male lynx reaches
puberty at between 13 and 20 kg of bw. To create gene banks from epididymal lynx
spermatozoa it is likely that a shorter interval between death and sperm collection than in the
present study is necessary.
Weight group
(range)
Number of
animals
Mean testes
weight, range
Number of
animals with
s ermatozoa
7
0.16-0.94g
0
6 (1)
12
0.38-3.20g
8
1 (2)
17
1,59-3.10g
17
0(6)
283
Axner E, Hermansson U, Linde-Forsberg C. Anim Reprod Sci 2004; in press.
Yu I, Leibo SP. Theriogenology 2002; 57:1179-1190.
284
Abstracts
Short Communications abstracts (in order of presentation)
Reproductive physiology in the male lynx (Lynx lynx)aaB
Axner E1, Linde-Forsberg C1, Marner T2, Ulhorn H2, Agren E2. 1Department of Obstetrics and
Gynaecology, Swedish University of Agricultural Sciences, P.O Box 7039, SE-750 07
Uppsala. 2Department of Wildlife, National Veterinary Institute, SE-751 89 Uppsala, Sweden.
Introduction
This abstract presents preliminary data from a study on the reproductive function in the male
Eurasian lynx and evaluates the possibility to retrieve live spermatozoa from dead lynx to
create gene banks. In dogs motile spermatozoa can be recovered from epididymides that
have been stored for 8 days (Yu & Leibo 2002). All lynx that are killed at hunting or found
dead in Sweden are sent to the Swedish Veterinary Institute (SVA).
Material and methods: Reproductive organs from 36 lynx were collected from November
2002 to May 2003. Time between death and evaluation ranged between 2 and 21 days. In
some animals, parts of the reproductive tract were accidentally removed in connection with
pelting. The mean weight of the testes in each animal was calculated. Spermatozoa were
collected by mincing the cauda epididymidis in Dulbecco's PBS, Ham's F-10 or Kenney's
dilution media at 37°. Acrosome integrity was evaluated with FITC-PNA and the. sperm
membranes with EthD-1 and Sybr-14 (Axner et al. 2004). Penile spines were present in
19/20 animals in which the penis could be extruded from the prepuce.
Results and discussion: The mean weight of the testes was significantly correlated with body
weight (bw) in animals with a bw<20 kg (rs=0.88, P<0.001, n=19) but not in animals with a bw
~20 kg (r=0.017, P>0.2, n=17). The lowest bw of an animal from which spermatozoa could
be recovered from the cauda epididymidis was 13 kg and the lowest mean weight of the
testes was 1.5 g. Spermatozoa were found in all males with a mean testis weight >2 g and
with a bw ~20 kg. The mean percentage of membrane intact spermatozoa was 15.9±21.5%
(range from 0 to 68.5%, n=22). The mean percentage of acrosome intact spermatozoa was
17.2±14.6% with a range from 0 to 45.5% (n=21). Sperm motility was 5% in one male, 1% in
two males and 0% in 19 males. The effect of season on testicular activity could not be
evaluated because of the skewed distribution with 30/36 males that died during January to
March (which is the hunting season). The present data indicates that the male lynx reaches
puberty at between 13 and 20 kg of bw. To create gene banks from epididymal lynx
spermatozoa it is likely that a shorter interval between death and sperm collection than in the
present study is necessary.
Weight group
(range)
Number of
animals
Mean testes
weight, range
Number of
animals with
s ermatozoa
7
0.16-0.94g
0
6 (1)
12
0.38-3.20g
8
1 (2)
17
1,59-3.10g
17
0(6)
Axner E, Hermansson U, Linde-Forsberg C. Anim Reprod Sci 2004; in press.
Yu I, Leibo SP. Theriogenology 2002; 57:1179-1190.
285
Abstracts
Short Communications abstracts (in order of presentation)
The Effect of Prepubertal Gonadectomy on Bone Mineral Density and
Bone Mineral Content of Calcaneus and Accessory Carpal Bone in Puppies
Ekici H\ Sontas B.H.1,Toydemir T.S.F.1, Senmevsim 6.2, Kabasakal L.2,Imre y.2
1Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Istanbul
University,Avcllar Campus, 34850, Avctier, Istanbul, Turkey. Telephone: +90212-473-7070/[email protected]
2Department of Nuclear Medicine, Cerrahpasa Medical Faculty, Istanbul University,lstanbul,
Turkey
Introduction
Early age neuter or spay or prepubertal gonadectomy refers to the surgical sterilization of
sexually immature animals (5). In dogs, various effects of early age neutering on growth,
obesity, secondary sex characters, urinary tract and behaviour have been demonstrated by
different researches (1, 11). But the effect of prepubertal gonadectomy on bone mineral
density hasn't been investigated. Recently, it has been possible to measure the bone mineral
density (BMD) of small animals, accurately using dual-energy x-ray absorptiometry (DXA),
which is an accurate and precise method for clinical measurement of BMD and is considered
suitable for longitudinal measurement of changes in bone mass over time(2, 6). The aim of
this study was to compare the bone mineral density (BMD) and the bone mineral content
(BMC) values of the calcaneus and the accessory carpal bone of gonadectomized and
ungonadectomized puppies across time.
Materials and Methods
Animals: Fifteen 6-8 week old mixed breed female pups that weighed 1.160-3.600 kg (mean
weight 2.504 kg) were selected from animal shelters. Study Design: The pups from each litter
were randomly assigned to 2 groups; group I, (n=8) was gonadectomized and group II, (n=7)
had only laparatomy. All animals were vaccinated against infectious diseases and given an
anthelmentic.They were housed with their Iittermates in an indoor facility and fed with dry dog
food" individually twice daily. Surgeries were performed at 10 week. The procedures for
ovariohysterectomy and laparatomy were routine. BMD and BMC Measurements: In order to
determine the changes of the BMD and BMC, the calcaneus andthe accessory carpal bone
of each pup was measured by the DXA using a bone densitometer" under sedation at one
month intervals till 6 months of age after surgery. All scans were performed using AP Spine
Scan Selections Programme. And the scans were processed by Low Density Spine scan
mode(V8.26a:5). Each pup was positioned in lateral recumbency and the calcaneus and the
accessory carpal bone were scanned in mediolateral direction. The estimated area for all
measurements was 0,67 cm2• Statistical Methods: The Independent Samples t-test was
used to analyze the differences between group I and group II. P<0,05 was considered to be
statistically significant. a ANF Advantage Puppy, ANF Specialities, Inc., Nacogdoches, TX,
USA. b Hologic QDR-4500W Elit X-ray bone densitometer
Results
In gonadectomized puppies(group I), compared with the control group(group II), higher
values for the BMD and BMC of the calcaneus and accessory carpal bone, were found
(Fig.1, 2, 3, 4). But the differences between the groups were statistically unimportant. Only
the 4 th measurements of the accessory carpal bone for BMD and BMC displayed a
significantdifference between the groups. Altough the control group displayed a significant
increase for the BMD and BMC from the 3rd month to the 4 th, there was no difference
between the groups from 4 th month through the 6th•
Discussion
287
Our findings were in contrast with the study by Omi and Ezawa (10), but in accordance with
the study by Turner et al.(12) Omi and Ezawa(10) found significantly lower BMD values of
the lumbar spine and tibial proximal metaphysis in ovariectomized aged and growing rats.
However, Turner et al.(12) found no significant change in the BMD of the calcaneus or distal
radius in ovariectomized ewes. But also, in the same study, BMD in the vertebrae (L4-L6/L5L7) was found significantly lower in the ovariectomized group compared to sham. As we
know from the numerous studies on humans (neonates, pre-peri- and post-menopausal
women)(3, 4, 7, 10) and dogs(6), body weight (BW) is one of the principal determinants of
BMD. In our study, BW values of gonadectomized puppies were higher than the control
group in all measurements. Because of the positive correlation between BW and BMD, we
found higher BMD and BMC values in gonadectomized puppies. In addition to this, as Miller
et al.(8) indicated in their review, the skeleton may not be as responsive to the reduction of
ovarian hormones after ovariectomy in animals with longer anestrus periods. In conclusion,
prepubertal gonadectomy or early-age neutering does not effect the bone mineral density
(BMD) and the bone mineral content (BMC) of calcaneus and accessory carpal bone of the
mixed breed puppies until six months of age. Additional studies with longer experimental
time, different breeds and sufficient animal material are needed.As far as we know, the
present study is the first which used dual energy x-ray absorptiometry on puppies,
demonstrated the effects of prepubertal gonadectomy on the BMD and BMC.
Fig.1
Fig.2
BMD - Calcaneus
1
0,8
~ 0,6
E 0,4
III 0,2
1:
BMC - Calcaneus
IIGroup I
_Group
o
3
4
5
II
1
•• 0,8
E 0,6
!! 0,4
III 02
'0
6
3
Months
4
5
6
Months
Fig.3
Fig.4
BMD - Accessory
Carpal Bone
BMC - Accessory
Carpal Bone
1
0,8
..!:! 0,6
•• 0,4
~ 0,2
1:
o
3
4
5
Months
6
Months
References
1.
2.
Bloomberg,M.S.: Surgical neutering and nonsurgical alternatives. JAVMA Vol 208, (4),517-519, (1996)
Emmerson,T.D., Lawes,T.J., Goodship,A.E., Rueux-Mason,C., Muir,P.: Dual energy X-ray absorptiometry measurement of
bone-mineral density in the distal aspect of the limbs in racing Greyhounds. AJVRVol61,
(10) 1214-1219, (2000)
3. Harris,S.S., Dawson-Hughes,B.: Weight, body composition, and bone mineral density in postmenopausal women. Calcif.
Tissue Int. 59, 428-432, (1996)
4. Koo,W.W.K., Hockman,E.M.: Physiologic predictors of lumbar spine bone mass in neonates. Pediat.Res. 48(4) 485489,(2000)
5. Kustritz,M.V.R.: Early spay and neuter. Managing Canine Reproduction, Veterinary Learning Systems. 29-31, (1998)
6. Martin,R.K., Albright,J.P., Jee,W.S., Taylor,G.N., Clarke,W.R.: Bone loss in the beagle tibia: influence of age, weight and
sex. Calcif. Tissue Int. 33(3), 233-238, (1981)
7. Mazess,R.B., Barden,H.S.: Bone density in premenopausal women:effects of age, dietary intake, physical activity, smoking
and birth-control pills. AJCN. 53,132-142,(1991)
8. Miller,S.C., Bowman,B.M.,Jee,W.S.S.: Available animal models of osteopenia-small and large. Bone. VoI.17(4), 117S123S, (1995)
9. Ohtsuka,S., Kumasaka,T., Ishikawa,K., Watanabe,R., Mori,T., Inaba,N.: The effect of body weight on mineral density of
each vertebral bone with in perimenopausal women. Maturitas. VoI.27,SuppI.1,129,(1997)
10. Omi,N., Ezawa,l.: The effect of ovariectomy on bone metabolism in rats. Bone. Vol.17 (4), 163S-168S, (1995)
11. Salmeri,K.R., Bloomberg,M.S., Scruggs,S.L., Shille,V.M.: Gonadectomy in immature dogs: Effects on skeletal, physical
and behavioral development. JA VMA Vol 198, (7), 1193-1203, (1991)
12. Turner,A.S., Alvis,M., Myers,W., Stevens,M.L., Lundy,MW.: Changes in bone mineral density and bone-spesific alkaline
phosphatase in ovariectomized ewes. Bone. Vol.17 (4), S395-S402, (1995)
288
Abstracts
Short Communications abstracts (in order of presentation)
Immunogenicity
in puppies of a new canine parvovirus vaccine using the
variant CPV-2b : comparison with three conventional
monovalent vaccines
B. Martinet1, C. Morales Moliner, P. Mahl3, L. Gardey" - 1Ecole Nationale Veterinalre
d'Alfort, FR-94700 Maisons-Alfort Cedex; 2Universitat Autonoma de Barcelona, SP08193 Bellaterra; 3Virbac SA, BP27, FR-06511 Carros Cedex, [email protected]
Introduction
: Canine Parvovirus (CPV) infection is a major disorder in puppies
especially in breeding kennels. Vaccination is the most effective means of prevention
but conventional vaccines show a lack of efficicacy during the critical period, due to
the interference of maternally derived antibodies (MDA) with vaccinal antigens. Field
strains have been evolving and the original CPV-2 serotype doesn't prevail at
present. Therefore, a modified live vaccine (MLV) containing the CPV-2b variant was
developed in order to allow an early and intense immune response and to better
cope the field serotypes. This study aimed to compare the immunogenicity of the new
vaccine with three CPV-2 MLV marketed in Europe.
Materials and methods: 5-week old puppies were recruited near Barcelona (Spain).
All puppies of a same litter were randomly assigned to a vaccine group: A (Canigen
Puppy2b®, Virbac, including the CPV-2b variant), B (Primodog®, Merial), C
(Vanguard CPV®, Pfizer), D (Nobivac Parvo®, Intervet). Pups were vaccinated at 5,
7 and 9 weeks of age: CPV on DO and D14, and CHPPi/L on D28 respectively.
Antibody titers to CPV-2, CPV-2a and CPV-2b were determined at DO, 14 and 28
using the hemagglutination
inhibition (HI) test. At DO, dogs were considered
seropositive when MDA was equal or over 20. Seroconversion (SCo) at D14 and 28
was defined as a four-fold increase or over in HI titers when positive prior to
vaccination, and for titers over 20 when negative on DO. The efficacy was assessed
by the SCo rate at D14 and 28, and the titers evolution. Fischer's exact test was
conducted on SCo rates, titers were compared by ANOV A. P values less than 0.05
were considered significant.
Results : In total, 181 puppies were included. Highest SCo rates were achieved in
group A as soon as D14 compared to others (P<0.05, Table 1). Highest titers were
obtained for each serotype in group A (P<0.05) whether the pups were seropositive
or not, whereas the presence of MDA led to low anti-CPV-2a and 2b titers in groups
B, C and D (Table 2).
Table 1 : SCo rates (%) regarding
014 and 28
CPV-2a
CPV-2
ABC
D14
D28
96
92
68
73
55
75
the 3 serotypes
0
ABC
49
68
96
96
65
63
53
65
in the 4 vaccine
groups
on
CPV-2b
0
ABC
49
68
96
98
D
65
63
55
60
49
68
289
Table 2 : HI titers to CPV-2, CPV-2a and CPV-2b on 014 and 28 and influence of
MOA
MDA
D14
negativ
e
D28
D14
MDA
positive D28
CPV-2
A
B
D
C
127
370 322
7
8
667
2
116
118 353
9
898 3
3
542
185 57
94
6
229
0
315 71 184
CPV-2a
B
A
C
D
472
575 65 130
5
284
2
125 113 509
755
0
63
67
39
224
5
40
39 37
CPV-2b
A
B
C
D
724
2
487 104 126
230
9
113 166 474
570
4
73
60
40
294
7
43
36
51
Conclusion : This report demonstrates that Canigen Puppy2b® stimulates an early
and very strong immune response to all CPV serotypes. These findings suggest that
an early vaccination with Canigen Puppy2b® starting as soon as 5 weeks of age may
be the most effective approach for reducing the critical period.
290
Abstracts
Short Communications abstracts (in order of presentation)
LONGITUDINAL
CLINICAL, SEROLOGICAL AND VIROLOGICAL PATTERN OF CANINE
HERPESVIRUS-1 (CHV-1) IN BREEDING COLONIES.
VERSTEGEN J1.; RONSSE V1.; ONCLIN K1.; THIRY E2.; POULET H3.
1 Department of Clinical Sciences, Section Small Animal Reproduction, College of Veterinary
Medecine, Universite de Liege, Boulevard de Colonster 20, B44, 4000 Liege, Belgium; Tel.:
0032(0)43664233; Fax: 0032(0)43664231; E-mail: [email protected]
2Department of Infectious and Parasitic Diseases, Section of Virology, Epidemiology and
Pathology of Viral Diseases, College of Veterinary Medicine, Universite de Liege
3Merial, Laboratoire de Lyon Gerland France
Canine herpesvirus-1 ·(CHV-1) is presumed to be enzootic in the dog population and is
associated with fertility disorders and neonatal mortality. To advise dog breeders towards an
effective management of CHV-1 infected colonies, antibody and viral excretion patterns were
investigated in field conditions. Twenty seven breeding bitches were sampled at fixed
moments during 1 reproductive cycle. The effect of cycle stage, kennel size and mating on
serologic and viral excretion patterns was evaluated. The association between reproductive
disorders and CHV-1 antibodies and viral excretion was also analysed. All dogs
seroconverted and 46% of mated bitches experienced reproductive disorders. Encountered
disorders were embryonic resorption, no offspring after mating and mummification. No
difference in antibody patterns was observed between mated and unmated bitches. In mated
bitches, no difference in antibody patterns occured depending on reproductive disorders,
although the majority of bitches with reproduction disorders had a decrease in antibody titer
during early or mid-di-oestrus. Significantly higher titers were observed at all cycle stages in
kennels with more than 20 dogs. None of the vaginal and nasal samples and buffy coats
were positive on PCR analyis. The mixed image of clinical and sub-clinical carriage confirms
CHV-1 has a complex and difficult to predict clinical behavior. Preventive management with
systematic vaccination of reproducing bitches in kennels with reproductive disorders should
be advised. Also, CHV-1 related management should focus on prevention of clinical signs
rather than on prevention from infection.
.
291
Abstracts
Poster abstracts (in order of presentation)
The Scientific Committee of the Congress will make an award to the short
communication with the best scientific quality. The short communication
awarded will be communicated to the delegates during the Congress
Posters Sesion Friday 4th June. MALE.
TREATMENT OF CRYPTORCHIDISM ON DOGS WITH HCG BEFORE CASTRATION
Falceto, M.V.; Martinez, S.; Ciudad, M.J.; Cruz, J.1.
Facultad de Veterinaria de Zaragoza. Spain.
Bilateral orchydectomy or orchydectomy and vasectomy are the possible elective methods
for the treatment of chryptorquidism in the dog. The last mentioned method is often well
accepted by the owners and prevents the possibility of tumours development and spermatic
cord torsion. It does not limit the normal growing of the animal.
Exploratory laparotomy is necessary for intraabdominal testicle removal but only a superficial
pre-scrotal skin incision is required if the testicle is subcutaneously retained. Treatment with
hCG before 1 year of age is cheap and helps the testicle to migrate from intraabdominal to
subcutaneous position, making its surgical removal easier.
The owner is well informed about the limitations of medical treatments and even if the hiden
testicle fully drop, it would be necessary its scrotal removal. Ley y col.(2003)
found no
succes with hCG in dogs but we found a testicular descent in dogs older than 16 weeks.
MATERIAL AND METHODS
A total of 30 clinical cases diagnosed as chryptorquids are reviewed in this study. 15 of them
underwent through a hCG treatment at time of diagnosis and the rest were left to evolve in a
natural way. Castration was recommended to all patients before one year of age. All dogs
were treated with 35 UI of hCG /kg bw sc for 6 days with a 48 hrs interval between doses
(Ganlvet, 1992).
RESULTS
15 untreated dogs remained chryptorquids at one year old.
Evolution of the 15 treated animals was as follows: 3 with full scrotal descent (20%), 8 partial
descent (53%) and 4 with no descent (27%)
11 cases out of the 15 treated cases (73.33%) were diagnosed as intraabdominal
of them (36%) needed a laparotomy for castration.
but only 4
4 cases (26.67%) out of the 15 treated cases were diagnosed as deep inguinal. 1 of them
(25%) became scrotal and 3 (75%) became superficial inguinal.
293
12 (80%) of the 15 treated animals were above 16 weeks old. In 2 of them (16.7%) a full
testicular descent was obtained, in 7 of them (58.3%) partial descent was obtained and in 3
of them (25%) no descent was obtained.
-Ganivet, A. (1992) Ectopie testiculaire chez Ie chien. In: Les indispensables de la'animal de compagnie: Reproduction du chien
et du chat. Dumon, C. y Fontbonne, A. P.M.CAC. ed. pp:203
-Ley, W.B.; Holyoak, R.; Digrassie, A.; Partisano; D. (2003)
Science (USA). pp: 468
In: Small Animal Theriogenology. Root Kustritz, M.V.Elsevier
294
Abstracts
Poster abstracts (in order of presentation)
Expression Of Androgen Receptor In The Canine Prostate: Differences Between Normal,
Inflamed, Squamous Metaplasia, Hyperplastic And Neoplastic Glands
1
F. Gallardo \ T. Mogas \ T. Bar62, R. Habanal", E. Taberner \ J. Morote", T. Rigau 1, J.
Heventos", and J. l.loreta".
Facultat de Veterinaria, Universitat Autonorna de Barcelona; 2 Hospital del Mar, Universitat
Pompeu Fabra and 3 Hospital Vall Hebr6n, Barcelona, Spain.
Objective of the work: . The presence of androgen receptors (AR) and their importance in
the normal development of the canine prostate is well established. The knowledge of the
localization and expression of androgen receptors in differents prostatic diseases may lead
to a better understanding of their hormonal regulation. The aim of this study was to
investigate the specific localization
of androgen
receptor in canine prostate by
immunohistochemistry and to compare the percentage of AR expressing cells in different cell
compartments from samples of normal or pathological canine prostate.
Materials and Methods: AR expression was assessed in samples of normal, inflamed,
metaplastic, hyperplastic, and neoplastic prostate tissue of adult dogs (n=37) in paraffin
sections. Polyclonal antibody against AR (NCL_Arp, 1:20, Novocastra) was used, along with
conventional antigen retrieval methods and the Envision TM System. Tissue sections from
human prostate were used as positive controls. As a negative control, AR antibody was
replaced by PBS.
Results: The results of the study confirmed that canine prostate is a target organ of sexual
steroids. AR was present in both the epithelial and stromal cells. lmmunostaining was
localized in the nuclei of normal, inflamed, metaplastic, hyperplastic and neoplasic epithelial
and stromal cells and minimal cytoplasmic staining was also observed in all samples. In
normal and hyperplastic canine prostatic glands, AR expression was homogeneous both in
the percentage of positive epithelial cells (::S;98%), and in the intensity of immunostaining. In
negative controls, all nuclei were negative. Squamous metaplasic tissue showed a slight
reduction in the percentage and intensity of positive epithelial cells (73,3%). Similarly, tissues
with prostatitis showed a 74,1 % of positivity in epithelial cells. Most of the tumours had a
heterogeneous staining intensity, varying from an intense positivity to no staining within the
same sample. Presence of AR decreased in neoplasic cells, with a mean of 65%. The
percentage of positive stromal cells was always under 25% in all pathologies studied.
Conclusions: Expression of AR in canine prostatic tissue is highest in normal glands, with a
progressive decrease in hyperplasia, metaplasia, prostatitis and cancer.
295
Abstracts
Poster abstracts (in order of presentation)
FERTILITY 24 HOURS AFTER BILATERAL VASECTOMY
A CASE REPORT
IN A DOG:
Giordano A; Baschar H; Arias D; Tortora M; Gobello C
Faculty of Veterinary Medicine, La Plata, Argentina.
Vasectomy has been used as a permanent male sterilization technique either en humans
and animals.':" Vasectomy prevents fertility by both obstructing ejaculation of spermatozoa
and by causing secondary testicular atrophy." Studies in humans have shown that the time
to achievement of azoospermia is variable and may take 4 weeks to 6 months." Because the
dogs lack seminal vesicles, there is not likely to be any reservoir to store sperm and
therefore, time to azoopermia might be shorter.
In a recent study in which 6 dogs were vasectomized, semen was almost absent (mean 1
spermatozoa/ml) one day after surgery and by day 3 after occlusion all dogs were
azoospermic." It has been previously reported that azoospermia develop from 2 to 20 days
after bilateral vasectomy in dogs.6,? To our knowledge there are no data on the specific time
course of fertility waning off following bilateral vasectomy in this species. In this article a case
of fertility 24 hours after vasectomy was reported in a dog.
A previously fertile, 3 years old, male Giant Schnauzer was diagnosed local demodectic
mange. The male lived with a female of the same breed which was in proestrus at the
moment of consultation.
Bilateral vasectomy was indicated and performed under general anesthesia according to
Hardie (1984) removing a segment of the ductus deferens and ligating the severed ends.'
Although, physical separation of the animals was indicated, the male escaped and mated
the already estrous bitch once within the first 24 hours after surgery.
Pregnancy was
confirmed by ultrasonography 25 days after the mating and gestation was pharmaceutically
interrupted.
The results here described agree with previous reports that showed that although sperm
count one day after bilateral vasectomy is almost absent, azoospermia develops from day 2
after surgery.5,6,? No data is available on fertility during these initial critical days. Knowledqe
of the dynamics of "the waning off period of fertility" in can ids would be of interest to prevent
unwanted fertile mating either in domestic and wild dogs kept in captivity.
It is concluded from this case that fertility is possible within 24 hour after bilateral vasectomy
in the domestic dog. Until further work on fertility of these cases is carried out strict physical
separation of animals should be indicated for a minimum of 72 hours after surgery.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
HARDIE E UROGENITAL SURGERY. VET CUN NORTH AM SMALL ANIM PRACT 1984; 14: 105 -131.
EMPERAIRE JC, AUDERBERT A, MATTEI A. MALE CONTRACEPTION AND VASECTOMY CONTRACEPT FERTIL SEX
1977; 5 (4): 335·342
Vare AM, Bansal, PC Changes in the canine testes after bilateral vasectomy- an experimental study. Fertil Steril 1973; 24:
793-797
Freund M, David J Disappearance rate of spermatozoa from the ejaculate following vasectomy Fertil Steril 1978; 20: 163-165
Schiff J D, Li PS, Schlegel PN, et al. Rapid disappearance of spermatozoa after vasal occlusion in the dog. J Androl 2003; 24
(3): 361-363
Brueschke EE, Wingfield JR, Burns N, et al. 1974 Development of a reversible vas deferens occlusive device II Effects of
bilateral and unilateral vasectomy on semen characteristic in the dog. Fert Steril 2001; 25: 673-686
Pineda MH, Reimers TJ, Faulkner LC Disappearance of spermatozoa from the ejaculates of vasectomized dogs. J Am Vet Med
Ass 1976; 168: 502·503
297
Abstracts
Poster abstracts (in order of presentation)
Simultaneous assessment of membrane and acrosomal integrity of frozen-thawed
canine spermatozoa by flow cytometry.
M Hernandez, X Lucas, J.M. Vazquez, E.A. Martinez and J.Roca.
Department of Medicine and Animal Surgery. Faculty of Veterinary Medicine, Campus de
Espinardo, University of Murcia, E-30071 Murcia, Spain. E-mail:[email protected]
Objetive
Flow cytometry appears as suitable tool to evaluate different characteristics of sperm quality,
but it is necessary to accurately identify the sperm population of the amount of particles
suspended in the medium. DNA dyes combinations are preferred to discriminate more
exactly nonsperm and sperm particles when frozen-thawed spermatozoa are assessed. The
aim of this study was to validate the effectiveness of a new triple stain combination including
DNA stains (SYBR-14 and PI) and PE-PNA for the simultaneous evaluation of membrane
and acrosome integrity of frozen-thawed canine spermatozoa based on the protocol
described by Nagy et ~1.(2003) for bovine spermatozoa (1).
Materials and Methods
Sperm samples were frozen using the Uppsala two-step dilution method and the Uppsala
Equex-2 extenders (2). Straws were thawed in a water bath at 70°C for 8 sec and each straw
diluted in 1 ml of thawing medium. After thawing, 100 III of the semen samples (-6 x 10 6
cells) were diluted with 400 III of PBS and stained with final concentration of 100 nM SYBR14, 1.25 Ilglml of PE-PNA and 7.4 11Mof PI. Samples were incubated 10 min at 37°C. To
investigate the repeatability of the method, 15 straws from different dogs with two
measurements per straw were used. To validate the method, 15 straws were measured
simultaneously by the triple stain, CFDAIPI and FITC-PNAIPI (3) at 0 and 3h after thawing.
Fluorescent data of 10000 events per sample with similar scatter parameters to spermatozoa
were collected by flow cytometer (Coulter Epics XL™) and percentages of different
population were analysed. To assess the repeatability and the method-agreement the British
Standards Institution (BSI) repeatability coefficient was used (4).
Results
The BSI repeatability coefficient (2SD) for the triple stain was 1.70%, indicating a high
repeatability. The mean of the differences ± SD between the tr.iple stain vs CFDAIPI and
triple stain vs FITC-PNAIPI were 1.82% ± 4.075% and 4.10% ± 6.30%, and 95% limits of
agreements were d ± 2So- = (9.96; -6.32%) and (16.7; -8.7%) respectively, showing high
agreement.
Conclusions
The triple stain for the simultaneous assessment of membrane and acrosomal integrity
showed good repeatability and method-agreement; therefore, it can be a useful technique for
the evaluation of frozen-thawed canine spermatozoa.
References
1. Nagy S, Jansen J, Topper EK, Gadella BM. Bioi Reprod 2003; 68: 1828-1835.
2. Peter AA, Linde-Forsberg C. Theriogenology 2003; 59: 1525-1532.
3. Peiia A, Johannisson A, Linde-Forsberg C. Theriogenology 1999; 52: 965-980.
4. Bland JM, Altman DG. Lancet, 1986; 1: 307-310.
299
Abstracts
Poster abstracts (in order of presentation)
Title: "Use of tamoxiphen for treatment of two cases of benign prostate hyperplasia
with prostatic cysts"
Authors: Sal6 F., lIIas J., Arus J., Vinaixa F.
Institution: Arvivet Veterinaris SL, Terrassa, Spain
Objective of the work
Prostatic cysts are a common pathology in veterinary medicine that have no medical
treatment described. We present two cases of dogs with prostatic cysts, one treated with
tamoxiphen and the other one combining tamoxiphen treatment and orchiectomy.
Tamoxiphen is a non-steroidal antiestrogen used in human medicine for the treatment of
breast cancer. Its action mechanism is not completely understood, but it is thought to act at
the level of the captation of endogenous estrogens, inhibiting it.
The first case was an 8 years old intact Spanish Mastin male that arrived to our clinic whith
an intermitent bloody urethral discharge problem. Rectal palpation showed that the prostate
gland was enlarged and irregular, but not painful. Ultrasound analisis showed the presence
of numerous intra-parenchimal cysts. The diagnosis was benign prostate hyperplasia with
prostatic cysts. The dog was orchiectomized and medical treatment with tamoxiphen was
prescribed at 0.3 mglKg/24 hours/p.o. The ultrasound controls at 15 and 30 days postsurgery showed a size diminution of both the prostate gland and the cysts.
The second case was a 10 years old intact crossbred male, wich was visited for the same
problem. Ultrasound showed the presence of cysts and an enlarged prostate, and diagnosis
of benign prostate hyperplasia with prostatic cysts was made. Tamoxiphen was prescribed at
the same dosage than in the first case, and ultrasound controls were realized the same days
post-initiation of treatment. The prostate and the cysts diminished its size.
Discussion and conclusion:
Tamoxiphen is used as an anti-estrogen in human breast cancer. This property could be
beneficial in the treatment of prostatic cysts in dogs, since the origin of them, though unclear,
seems to be secondary to squamous cell metaplasia induced by endogenous estrogen
compounds.
The results of treatment with tamoxiphen in these two cases (presented) seem(s) to confirm
this. The size of both the prostate and the cysts disminish with castration and tamoxiphen,
but with medical treatment alone the results are similar.
More studies have to be done to confirm these results, in order to evaluate if tamoxiphen is
able to obtain the same results as orchiectomy, and how long does it take to achieve it.
References:
-Shirley D.Johnston, Margaret V. Root Kustritz, Patricia N.S. Olson "Canine and Feline
Theriogenology" W.D. Saunders 2001
-Various authors "Current Veterinary Therapy XI" 1992 Saunders Co.
301
Abstracts
Poster abstracts (in order of presentation)
Effect of four different semen extenders on motility and viability of dog spermatozoa
assessed by computer-assisted sperm analysis
Sabine Schafer-Sorni, Christine Aurich
Centre for Artificial Insemination and Embryo Transfer, Department of Animal Breeding and
Reproduction, University of Veterinary Medicine, Vienna, Austria
Abstract
As in many species, canine semen has to be diluted before it can be analysed by computerassisted systems because of the high concentration of freshly collected semen. The aim of
this study was to evaluate the effects of four different extenders for dog semen on the
different parameters obtained by computer-assisted sperm analysis.
Materials and methods: A total of 28 ejaculates from 6 clinically healthy beagle dogs aged 24 years were used. The sperm rich fraction was evaluated for estimated motility, pH and
sperm concentration. Four aliquots of the sperm rich fraction were diluted with either saline
(S), PBS, prostate secretion (PS) or a Tris extender (T; 3.025 g TRIS + 1.7 g citric acid +
1.25 g fructose + 0.1 g streptomycin ad 100 ml distilled water, + 0.06 g benzyl penicillin in 0.3
ml distilled water; pH 6.76, 324 mOsm; [2]). Semen was diluted to a final concentration of
100 x 106 spermatozoa/ml. Total motility, progressive motility, hypermotility and viability were
evaluated with a computer-assisted semen analysis system (Sperm Vision, MinitOb,
Germany). Total motility was defined as percentage of spermatozoa with curvilinear velocity
(VCl) >15 urn/s, VCl being the instantaneously recorded sequential progression along the
whole trajectory of the spermatozoon [4]. Progressive motility was defined by VCl and a
minimal linearity (STR) of 90% [3]. Hypermotility was defined as VCl >118 urn/s, lateral head
displacement (AlH; mean width of sperm head oscillation; [1]) > 6.5 11m,and linearity (LIN) <
50%. Viability was investigated with SYBR-14/PI and CFDA fluorescence stains. Results are
given as percentages of spermatozoa with intact plasma membranes. Statistical analysis:
Spearman's correlation was used for comparison of estimated motility with motility
parameters of diluted semen, and Wilcoxon test for analysis of differences between aliquots.
A P-value < 0.05 was considered significant.
Results: Osmolarity and pH of semen diluted in PBS, PS, T and S was 292 and 7.0, 307 and
6.4, 309 and 6.3 and 277 and 6.5, respectively. Dilution with PS increased total motility,
progressive motility and hyperactivity significantly in comparison to estimated motility and
motility after dilution with PBS, S, and T. Only in samples diluted with T, motility was
significantly correlated with estimated motility (P<0.01). After dilution of samples with PBS
and T, viability was 85.4 and 87.7%, respectively (P>0.05). After dilution with TM, viability of
samples determined with SYBR-14/PI and CFDA staining was significantly correlated
(P<0.01).
Conclusion: T can be recommended as dilution medium of canine semen for further
laboratory analysis because parameters are not changed. Dilution of semen with PS
significantly influenced semen characteristics in comparison with freshly collected semen.
References
1. Aleporou-Mairinou V, Pappa H, Yalouris P, Patargias T (2001) Comp Biochem Phys B
128, 537-542.
303
2. Linde-Forsberg C (2001). International Veterinary Information Services (www.ivis.org).
Ithaca, New York, USA. Document No. A 1209.0501. (15 sid)
3. Pena AI, Lopez-Lugilde L, Barrio M, Becerra JJ, Quintela LA, Herradon PG (2003) Reprod
Dom Anim 38, 27-35.
4. Rigau T, Farre M, Ballester J, Mogas T, Pena A, Rodriguez-Gil JE (2001) Theriogenology
56, 801-815.
304
Abstracts
Poster abstracts (in order of presentation)
Clinical test of pumpkin seed extract against benign hyperplasia of the prostate gland
in dogs
S. Schater-Somi', K. Zitterl-Eglsee~, M. Juql-Ohizzola", Ch. Franz"
'Ollnlc for Obstetrics, Gynecology and Andrology, 21nstitute for Applied Botany, University for
Veterinary Medicine Vienna, Veterlnarptatz 1, A-121 0 Vienna, Austria
Abstract
Objectives: The aim of the study was to compare the effects of pumpkin seed extract, a
phytopharmacon (Cucurbitae peponis semen, Fa. Apomedica) with the effect of Androcur®
(Fa. Schering) in dogs with benign prostate gland hyperplasia.
Material and Methods: 15 male dogs were assigned to two groups, according to clinical
symptoms and use. Experimental group: 8 dogs of different breeds, aged 6.4 ± 2.5 years,
body weight 31.4 ± 18.7 kg, all breeding dogs. Main clinical signs: 6 with blood in the third
fraction of the ejaculate, 1 with defecation and 1 with micturition difficulties. In all cases,
ultrasonography revealed an enlarged prostate gland with regular structure but low grade
increased density. In 80 % of dogs, erythrocytes and protein was detectable in urine.
Treatment started after the first check with 1 pumpkin tablet SID, only one dog received 2
tablets SID. Dogs were clinically checked and blood samples were taken on average 19.6,
43.6, 81.4, 103.8 and 136.8 days after the first visit.
Control group: 7 male dogs, 7.1 ± 3.3 years, body weight 27.4 ± 11.3 kg, all pets. Main
clinical signs: 5 with bloody preputial discharge, 2 with defecation difficulties. Initial treatment:
1 subcutaneous injection of 3 mg/kg of Cyproteronacetate (Androcur"), Dogs were clinically
checked and blood samples were taken 1-2 weeks afterwards, then monthly until 6 months
after the first application. Testosterone concentration in serum samples from both groups
was measured by Enzyme Immunoassay (SR1 Analyzer; Serono Diagnostics S.A.).
Results: in the experimental group, all dogs showed normal libido. Serum testosterone
concentration as well as sonographical density and structure of the prostate gland were not
affected. In 1 dog, preputial discharge, defecation and micturition difficulties, and decreased
libido improved within 6 weeks. After 3 weeks of treatment, only in 1 urine sample low
amounts of haemoglobin and protein were detectable. The size of the prostate gland (height
x length) decreased not significantly in 5 of 8 dogs until the 6th visit, with varying values in
between. Increased sexual activity increased the size despite therapy. Bloody preputial
discharge vanished in 1 dog after 6 months with 2 tablets SID, but not in any dogs given 1
tablet SID. Side effects: 2 dogs had soft, green feces after 3 months of treatment (1 tablet
SID).
In the control group, 2 dogs received a 2nd injection after 2 weeks. In all other cases, the size
decreased significantly within 1-2 weeks (p < 0.05). Structure and density normalized, and
defecation and micturition difficulties as well as preputial discharge vanished at the same
time. In no patient a relapse occurred within 6 months.
Conclusions: With 1 pumpkin tablet SID, libido was not affected, urine quality, defecation as
well as micturition difficulties improved within 4-6 months. Prostate gland size decreased
non-significantly, depending on sexual activity. Blood in seminal plasma vanished in 1 dog
after 6 months, but he received 2 tablets SID.
It is therefore assumed, that dosage adjustment during sexually active phases, prolonged
treatment up to 6 months and regular controls are necessary for this alternative therapy for
benign prostatic hyperplasia in breeding dogs.
305
Abstracts
Poster abstracts (in order of presentation)
Nonsurgical artificial insemination with frozen semen in dogs:
Results from 1999-2003.A retrospective study.
Thomassen R. Andersen Berg K, Krogenres A, Fougner JA and Farstad W.
Department of Production Animal Clinical Sciences, Norwegian School of Veterinary
Science, PO box 8146 Dep 0033 Oslo Norway
Introduction
A transcervical intrauterine insemination (TCI) technique for dogs was developed in Norway
at the beginning of the 1970s (1). This nonsurgical technique has proven successful in
canine AI (2,3). This study reports results with frozen semen TCI AI at our clinic from 1999 to
2003.
Materials and methods
A total of 371 bitches of 90 breeds were inseminated with frozen-thawed semen from 250
dogs. The animals were privately owned breeding dogs and not selected for this study.
Imported semen was used to inseminate 146 bitches and 225 bitches were inseminated with
domestic semen. Timing of AI was based on clinical evaluation, including a vaginal smear
and serum concentration of progesterone, and inseminations performed two and three days
after estimated time of ovulation.
Statistical analysis
The statistical analysis was performed in Epiinfo 6.03 and the results are given as
percentage (whelping rate) and mean ±SEM (litter size). Chi-square tests were used to
compare whelping rates, and litter sizes were compared using Student's t-test.
Results
A total of 281 bitches (76%) gave birth with a mean (±SEM) litter size of 6.0 ±0.19 pups. In
3.5% of cases TCI failed and the AI was done in the cranial vagina (IV). Welping rates (WR)
were 79% and 0%, respectively (p<0.05). Bitches inseminated only once had the same WR
as bitches inseminated twice (79%), but had a smaller mean (±SEM) litter size, 5.4 (± 0.3) vs.
6.4 (± 0.25) pups (p=0.01). Domestic semen gave a WR of 78% and a mean (±SEM) litter
size of 6.1 (± 0.24) pups, and imported semen 74% WR and 5.7 (± 0.32) pups (n.s.).
Discussion and conclusion
Our results using TCI have improved from the previously reported (2) 70% to 76% WR
(p<0.07) and from 5.3 (± 0.20) to 6.0 (± 0.19) pups (p=0.02), and are similar to the WR of
84% and litter size of 5.4 (± 0.3) reported by Linde Forsberg et al. (3). In the present study
none of the bitches inseminated IV gave birth. This is in contrast to other reports were IV AI
yielded reasonably good results (3,4). This could be due to that the bitches in which we were
unable to perform TC IU AI were late in oestrus and that the cervical canal was closed. In
addition, the attempts to pass the catheter through the cervical canal might possibly induce
changes in the vaginal environment detrimental to the semen placed in the vagina. In one of
the reports in which vaginal AI was used successfully the Als were planned and multiple Als
with large numbers of spermatozoa seemed to be beneficial (4).
References
1) Andersen K.
Insemination with frozen dog semen based on a new technique.
Zuchthygiene 1975: 10;1-4. 2) Thomassen R, Farstad W, Krogenres A, Fougner JA and
Andersen Berg K. Artificial insemination with frozen semen in dogs: a retrospective study. J
Reprod Fertil 2001: Suppl 57; 341-346. 3) Linde-Forsberg C, Strom-Holst, Band Govette G.
Comparison of fertility data from vaginal vs intrauterine insemination of frozen-thawed dog
semen: a retrospective study. Theriogenology 1999:52;11-23. 4) Notling JO, Gerstenberg C
and Volkmann DH. Success with intravaginal insemination of frozen-thawed dog semen: a
retrospective study. J S Afr Vet Assoc 1995: 66; 49-55.
307
Abstracts
Poster abstracts (in order of presentation)
Automated sperm morphometry and morphology analysis of canine semen by the
Hamilton-Thorne analyser
T Rijsselaere, A Van Soom, G. Hoflack, D Maes, A de Kruif
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine,
Ghent University, Merelbeke, Belgium
computer-assisted sperm morphometry analysis systems have the potential to eliminate
several drawbacks inherent to the conventional evaluation of sperm morphology and may
identify subtle morphometric sperm characteristics which cannot be detected by visual
evaluation. As internal image settings may influence the results considerably, the effect of
different technical settings and semen handling procedures on morphometric parameters of
canine semen, measured by the Metrix Oval Head Morphology software implemented in the
Hamilton Thorne Ceros (version 12.1; HTR 12.1 Metrix), was investigated. In the present
study, all semen samples were stained with Diff-Quick. Comparison of sperm morphometric
measurements of 200 spermatozoa from pooled semen of 3 dogs at a magnification of x 40
and x 60 demonstrated a more accurate identification of the sperm head boundaries at a
magnification level x 60 (4 replicates). Dilution of the pooled semen samples to a sperm
concentration of 50 x 106/mL allowed for a correct evaluation of the sperm cell dimensions
whereas 100 and 200 x 106/mL resulted in significantly larger sperm dimensions and
significantly higher percentages of rejected spermatozoa due to overlapping (4 replicates;
P<0.05). No significant differences were found in all evaluated morphometric sperm
dimensions when 100 or 200 spermatozoa were evaluated for each of 15 dogs. The mean
morphometric parameters of fresh canine spermatozoa, based on the ejaculates of 23 dogs,
were: major axis 6.65 ± 0.20 urn: minor axis 3.88 ± 0.14 urn: area 20.66 ± 1.04 /-lm2;
elongation 58.64 ± 2.58%; perimeter 17.57 ± 0.43 urn and tail length 48.93 ± 10.16 urn.
Large variations in morphometric dimensions were detected between and within individual
dogs. After cryopreservation, significantly lower morphometric dimensions (P<0.05) were
obtained for all the evaluated sperm samples compared to the fresh semen samples (n=12
ejaculates). Finally, a correlation of 0.82 (P<0.05) was established for the percentage of
normal spermatozoa assessed by subjective evaluation and by the HTR 12.1 Metrix (n = 39
semen samples). In conclusion, this study highlights that several technical parameters can
influence the results obtained by the HTR 12.1. Metrix system. Therefore, dilution of the
semen samples to approximately 50 x 106 spermatozoa/mL and a magnification of x 60,
analysing at least 100 spermatozoa, are the technical settings proposed to obtain reliable
and objective sperm morphometric measurements by the HTR 12.1 Metrix in canine. The
large variations in morphometric dimensions between individual dogs suggest that greater
numbers of dogs might be necessary to obtain a reliable and accurate estimate of canine
sperm dimensions. Possible explanations for the decreased morphometric dimensions after
cryopreservation are the progressive dehydratation of the sperm cell upon cooling and
freezing, the high proportion of spermatozoa with a damaged acrosome with subsequent loss
of acrosomal contents and overcondensation of the sperm chromatine which was previously
related to a decreased sperm head surface in human. Additionally, our present data suggest
that this system might also be useful to determine the percentage of normal spermatozoa in
canine.
This research was supported by the RUG Special Research Fund, Grant N° 011 B8698 and
Grant N° 01109700. E-mail correspondingauthor:[email protected]
309
Abstracts
Poster abstracts (in order of presentation)
The effect of ketamine and medetomidine on quality of domestic cat sperm
after electroejaculation
Zambelli D., Baietti B., Prati F., Belluzzi S.
Veterinary Clinical Department, Obstetrical and Gynaecological Section - University
of Bologna, Via Tolara di Sopra 50,40064 Ozzano Emilia, Italy. e-mail:
[email protected]
Objective of the work. Ketamine alone or in mixture with xylazine or medetomidine
has been reported as anesthetic protocol for electroejaculation (EE), but their effects
on sperm quality has never been studied. The aim of this study was to evaluate the
effect of ketamine and medetomidine on domestic cat sperm characteristics after EE.
Materials and Methods. In this study 3 experiments were performed. The effects of
ketamine or medetomidine on sperm quality (Exp.1) and sperm flow (Exp.2) after
electroejaculation, and sperm flow without electroejaculation (Exp.3) were evaluated.
Animals: european adult male random-source cats aged 2 to 7 years and weighing 3
to 5 kg. EE protocol: ketamine (K group) 20 mg/kg and medetomidine (M group) 130140 IJg/kg IM were administrated and sperm was collected as previously described
by Howard et aI., 1990. Semen volume, sperm concentration, motility and
morphology were evaluated in the sperm and spermatozoa count and urinary volume
were evaluated in the urine. Exp.1: eight cats were electroejaculated after ketamine
(K1) administration and 2 days later after medetomidine (M1) administration. Exp.2:
ten cats divided into two groups were anesthetized with ketamine (K2. n=5) and
medetomidine (M2.n=5). Before EE 0.5 to 1.5 ml of pre-EE urine were collected by
cystocentesis. After EE the urinary bladder was emptied by cystocentesis to collect
the post-EE urine. Exp.3: seventeen cats were assigned to one of three groups
treated with ketamine (K3, n=6), medetomidine (M3, n=6) and control group (C, n=5).
A volume of 0.5 to 1.5 ml of urine were collected by cystocentesis and than the
urinary bladder was emptied with a 4F urinary cat catheter. Statistical analysis were
performed using Student's t-test and ANOVA elaborated applying a software
package (Statistica for Windows- Stat Soft Inc, Tusla, USA).
Results. Exp.1: the mean sperm concentration in M1(196,25 ± 20,49 x 106 Iml) was
significatively higher (P<0.01) than in K1 group (111.75 ± 13.48 x 106 Iml). Exp.2: the
mean sperm concentration and the mean total number of spermatozoa in M2 (72.8 ±
17.72; 4.46 ± 1.80 respectively) were significatively (P<0.01) higher than in K2 group
(18.2 ± 9.50; 1.08 ± 1.20 respectively). Significant differences (P<0.01) between the
number of spermatozoa displaced in urethra from the storage sites (ducts deferents
and epydidimes) in K2 group (3.72 ± 3.03) and M2 group (17.90 ± 13.26 ) were
observed. Exp.3: the number of spermatozoa displaced in urethra in M3 group (19.95
± 17.46) was significatively higher (P<0.05) than in Ksgroup (0.03 ± 0.06) and than
C group (0.24 ± 0.27).
Conclusions. Medetomidine is an <X2-adrenergicagonist used routinaly in veterinary
medicine as a sedative and analgesic for dogs and cats. The data obtained show that
the mean sperm concentration, mean total number of spermatozoa and spermatozoa
displaced in urethra from the storage sites are significatively higher using
medetomidine instead of ketamine. Our study also shows that the mean number of
spermatozoa emitted in urethra without electroejaculation is significatively higher
after injection of medetomidine instead of ketamine. These results could be
311
attributable to stimulation of <X2-adrenoreceptors of the vas deferentes and
epididymes. Alpha-adrenoreceptors mediate the response to adrenergic agonists for
contraction of the ducts deferentes and are believed to partecipate in contraction of
the trigone and sphincter of urinary bladder during ejaculation. The results of the
present work are in accord with previous studies that describe the use of other <X2adrenoreceptor agonists in dog, cat and horse. As previously reported a retrograde
flow of spermatozoa into urinary bladder is a normal component of the ejaculatory
process in cats. No significant differences in the percentage of retrograde flow after
medetomidine or ketamine administration were found. In conclusion, the use of
medetomidine, for sperm collection by EE, permits to obtain a good pharmacologic
restriction and a higher number of spermatozoa per ejaculate without increasing the
retrograde flow in the urinary bladder.
312
Abstracts
Poster abstracts (in order of presentation)
LOW CONCENTRATIONS OF FRUCTOSE, BUT NOT GLUCOSE, ALLOWS FOR
A FEASIBLE MEDIUM-TERM STORAGE OF REFRIGERATED DOG SEMEN
Ma. Montserrat Hlvera", Fanny Gallardo", Armando Oulntero-Moreno'', Olga
Madriqal", Joan E. Rodriguez-Gila and Teresa Rigaua
'Unit of Animal Reproduction Department of Animal Medicina and Surgery. School of
Veterinary Medicine. Autonomous University of Barcelona. E-08193 Bellaterra. Spain
bUnit of Animal Reproduction. Faculty of Veterinary Science. University of Zulia. Box
15252, Maracaibo 4005-A. Venezuela
Introduction
Dog sperm shows aremarkable specificity in its modulatry mechanisms of energy
metabolism. Thus, although these cells are able to utilize both glucose and fructose
as energy sources, the effects of both monosaccharides are separate and specific in
concrete sperm functions, such as motion parameters (1) or tyrosine-phosphorylation
pattern (2). This direct link between a concrete sugar and a concrete function would
affect the storage ability of dog sperm subjected to restrictive energy condition, Le.
low concentrations of substrates. To test this possibility, we studied the survival
ability of dog sperm stored at 4°C in a medium with low concentrations of an specific
sugar as energy substrate.
Material and Methods
Fresh sperm-rich fraccions of ejaculates from healthy, adult Beagle dogs were
obtained by manual stimulation and immediately diluted in a basal Tris-Citrate
medium added with 20% (vlv) egg yolk, and containing either glucose of fructose to a
final concentration from 10 mM to 20 mM. Samples were then stored at 4°C during 4
days, and an aliquot was taken daily, in order to perfom the approppriate analysis.
Semen quality of samples was determined by analyzing the percentages of viability
and altered acrosomes after an Eosin-Nigrosin stain, HOS test, as in (3) and motion
parameters after a Computer-Assisted System Analysis (CASA), as in (1).
Results and Discussion
The addition of fructose to a final concentration of 10mM-20mM to a simple,
isoosmotic and pH-neutral medium without any other energy source maintained the
main parameters of dog seminal quality (percentages of viability, altered acrosomes,
total motility and HOS Test) at levels nearly to that determined in fresh samples for,
at least, 3-4 days of storage at 4°C. However, similar concentrations of glucose failed
in the attainment of this goal. A similar result was determined after observing the total
motility. On the other hand, sperm maintained up to 4 days in a medium with fructose
showed a less linear mean movement than those incubated with glucose. These
results show that the specific energy utilization of fructose by dog sperm induces a
more efficient survival estrategy in restricted conditions that that related to glucose.
References
1. Rigau T, Farra M, Ballester J, Mogas T, Pefia A, Rodriguez-Gil JE (2001)
Theriogenology 56: 801-815.
2. Rigau T, Rivera M, Palomo MJ, Fernandez-Novell JM, Mogas T, Ballester J, Pefia
A, Otaegui PJ, Guinovart JJ, Rodriguez-Gil JE (2002) Reproduction 123: 579-591.
3. Rodriguez-Gil JE, Montserrat A, Rigau T (1994) Theriogeneology 42: 815-829.
313
Abstracts
Poster abstracts (in order of presentation)
Inflammatory response in the female reproductive tract after natural mating in
domestic cats
Chatdarong, K.1, Rungsipipat, A.\ Tummaruk, P.1 And Linde-Forsberg, C.2
Institution: 1Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand;
2Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala,
Sweden
Objective of the work
This study aimed to investigate the distribution of leukocytes in the mucosa, submucosa and
blood vessels in the muscular layer of different segments of the uterine tubes and uterus at
various times after natural mating in domestic cats.
Materials and Methods
Oestrus was induced in 24 female cats using 100 iu eCG. The cats were mated four times
within one hour to obtain a sufficient LH release for induction of ovulation. Ovulation was
confirmed by the presence of corpora haemorrhagica or corpora lutea. A group of six
females was ovariohysterectomised at 30 min, 3 hr, 48 hr or 96 hr after mating. One side of
the reproductive tract was excised into seven segments: infundibulum, ampulla, isthmus,
uterotubal junction (UTJ), cranial and caudal uterine horn, and uterine body and then fixed in
3% neutral buffered formalin and processed for routine histology. From each reproductive
segment, five sections on coded slides were chosen for microscopic examination using
magnification of x400. In each section, four areas of 0.0625 rnm" of the various tissue layers
(mucosa, submucosa and blood vessels of muscular layer) were examined and the mean
numbers of leukocytes were used in the analyses. Counting of lymphocytes, neutrophils,
eosinophils, plasma cells, monocytes and macrophages were done by the same person.
Numbers of leukocytes were compared using general linear mixed model (PROC MIXED) of
SAS (1996). The statistical models included times after mating (4 times), segments of the
uterine tube and uterus (7 segments), tissue layers (3 layers), interaction between times and
segment and between times and layers. Least-square means were obtained from each class
of the factors and were compared by using Student's t-test. A P-value < 0.05 was considered
statistically significant.
Results
A marked number of lymphocytes and neutrophils were observed in the mucosa and
submucosa of the reproductive tract after mating. The mean number of leukocytes in various
reproductive segments at all times after mating is shown in Table 1. The highest numbers of
lymphocytes and neutrophils were found in the cranial part of the uterus. A high number of
neutrophils was also observed in the uterine tubes. On average, more lymphocytes were
found in the mucosa and submucosa than in the vascular layer (P<0.05), whereas more
neutrophils were found in the vascular layer, than in the mucosa and submucosa (P<0.05).
The numbers of lymphocytes were highest at 48 hr after mating in the mucosa of the cranial
and caudal uterus. The highest numbers of neutrophils were seen at 3 h after mating in the
vascular layer of the cranial uterus and this was higher than at 30 min and 96 h (P<0.05). A
few eosinophils, plasma cells, monocytes and macrophages were found in the various
reproductive segments but were not quantified.
Conclusions
An inflammatory response in the female reproductive tract was found at 3 h after mating in
the form of neutrophils, whereas the immune response in the form of lymphocytes was
observed at 48 hr after mating.
315
Table 1 Over-all mean±S.E. numbers of lymphocytes and
reproductive segments for all times after mating
Leukocytes
Infundibulum
Ampulla
Isthmus
UTJ
neutrophils
in the various
Cranial
Caudal
uterine
uterine
horn
horn
Lymphocyte
1.4±O.3abc
1.1±0.3abc
1.4±O.3a
1.4±O.3ab
2.4±0.3c
2.1 ±O.3b
Neutrophil
1.9±O.2a
1.5±0.2b
1.7±0.2a
1.3±O.2b
1.9±0.2a
1.4±0.2b
abc different superscript letters within a row indicate significant difference (P<0.05)
Body of
uterus
1.8±0.3b
0.4±O.2c
Objective of the work
This study aimed to investigate the distribution of leukocytes in the mucosa, submucosa and
blood vessels in the muscular layer of different segments of the uterine tubes and uterus at
various times after natural mating in domestic cats.
Materials and Methods
Oestrus was induced in 24 female cats using 100 iu eCG. The cats were mated four times
within one hour to obtain a sufficient LH release for induction of ovulation. Ovulation was
confirmed by the presence of corpora haemorrhagica or corpora lutea. A group of six
females was ovariohysterectomised
at 30 min, 3 hr, 48 hr or 96 hr after mating. One side of
the reproductive tract was excised into seven segments: infundibulum, ampulla, isthmus,
uterotubal junction (UT J), cranial and caudal uterine horn, and uterine body and then fixed in
3% neutral buffered formalin and processed for routine histology. From each reproductive
segment, five sections on coded slides were chosen for microscopic examination using
magnification of x400. In each section, four areas of 0.0625 rnrn" of the various tissue layers
(mucosa, submucosa and blood vessels of muscular layer) were examined and the mean
numbers of leukocytes were used in the analyses. Counting of lymphocytes, neutrophils,
eosinophils, plasma cells, monocytes and macrophages were done by the same person.
Numbers of leukocytes were compared using general linear mixed model (PROC MIXED) of
SAS (1996). The statistical models included times after mating (4 times), segments of the
uterine tube and uterus (7 segments), tissue layers (3 layers), interaction between times and
segment and between times and layers. Least-square means were obtained from each class
of the factors and were compared by using Student's t-test. A P-value < 0.05 was considered
statistically significant.
Results
A marked number of lymphocytes and neutrophils were observed in the mucosa and
submucosa of the reproductive tract after mating. The mean number of leukocytes in various
reproductive segments at all times after mating is shown in Table 1. The highest numbers of
lymphocytes and neutrophils were found in the cranial part of the uterus. A high number of
neutrophils was also observed in the uterine tubes. On average, more lymphocytes were
found in the mucosa and submucosa than in the vascular layer (P<0.05), whereas more
neutrophils were found in the vascular layer, than in the mucosa and submucosa (P<0.05).
The numbers of lymphocytes were highest at 48 hr after mating in the mucosa of the cranial
and caudal uterus. The highest numbers of neutrophils were seen at 3 h after mating in the
vascular layer of the cranial uterus and this was higher than at 30 min and 96 h (P<0.05). A
few eosinophils, plasma cells, monocytes and macrophages were found in the various
reproductive segments but were not quantified.
Conclusions
An inflammatory response in the female reproductive tract was found at 3 h after mating in
the form of neutrophils, whereas the immune response in the form of lymphocytes was
observed at 48 hr after mating.
316
Table 1 Over-all mean±S.E. numbers of lymphocytes and neutrophils in the various
reproductive segments for all times after mating
UTJ
Leukocytes
Infundibulum Ampulla
Isthmus
Cranial
Caudal
Body of
uterine
uterine
uterus
horn
horn
Lymphocyte 1.4±O.3abc
1.1±O.3abc 1.4±O.3a
1.4±O.3ab 2.4±O.3c
2.1±O.3b
1.8±O.3b
Neutrophil
1.9±O.2a
1.5±O.2b
1.7±O.2a
1.3±O.2b
1.9±O.2a
1.4±O.2b 0.4±O.2c
abc different superscript letters within a row indicate significant difference (P<O.05)
317
Abstracts
Poster abstracts (in order of presentation)
USE OF LOGISTIC REGRESSION MODELS TO STUDY UL TRASONOGRAPHIC,
MACROSCOPIC AND MICROSCOPIC FEATURES OF CANINE MAMMARY
TUMOURS
Authors: Francisco de Membiela, Xiomara Lucas, Amalia Agut.
Institution: Veterinary Teaching Hospital. University of Murcia. 30100. Espinardo
(Murcia). Spain.
The objetive of this study is to evaluate the association between the ultrasonographic
patterns of 16 canine mammary tumours and their macrocospic and microscopic
features.
MATERIAL AND METHODS: The tumours were diagnosed in 10 bitches of different
breeds and ages between 7 and 13 that were presented to the Murcia's University
Veterinary Teaching Hospital. The ultrasonographic characteristics of the tumours
were examinated before surgery with a 11 MHz transducer and they are given by the
multinomial variable .Ultrasonography, that includes the parameters Margin (regular
or irregular), Distribution of the tumours within the mammary gland (focal or diffuse),
Echogenicity (hypoechoic, hyperechoic or heterogeneous) and Acoustic shadowing
(presence or absence). The macrocospic appearence of the tumours is represented
by the following variables: TNM Classification (W.H.O., 1980), Location (mammary
gland affected) and Shape (spherical-oval or polymorphous). The microscopic
appearence of the tumours is represented by the variable Diagnosis (mixed tumour
or not mixed tumour).
In order to evaluate the association between Ultrasonography and the rest of the
variables of this study logistic regression models are used. The coefficients of these
models are estimated using the maximum likelihood method. In order to estimate the
effects of each explanatory variable on the probabilities of the ultrasonographic
parameters hypothesis tests with probability a = 0.05 are performed, using the
Scaled Deviance, with Ji Square distribution.
In a first model the explanatory variables are the macrocospic characteristics of the
tumours and only the variable Shape has influence on the ultrasonographic
parameters with value of the statistic 12,91 > 12,59 = X6;O,05 (with p-value = 0.044). In
a second model the explanatory variable is Diagnosis, which determines the
probabilities of the ultrasonographic parameters with p-value = 0.043.
RESULTS: The tumours with spherical-oval shape have ultrasonographic images
with regular margin, diffuse distribution and hyperechoic or heterogeneous. The
polymorphous tumours have regular or irregular margin, diffuse distribution.. are
heterogeneous or hypoechoic and show acoustic shadowing in 33,3% of the cases.
The mammary mixed tumours do not have echopatterns of irregular margin, diffuse
distribution and heterogeneous
echogenicity
or with regular margin, diffuse
distribution and hyperechoic, but they can show the rest of possible combinations of
results of the ultrasonographic examination. The tumours that are not histologically
classified as mammary mixed tumours have diffuse distribution and do not show
acoustic shadowing.
CONCLUSION: The ultrasonographic characteristics of canine mammary tumours do
not have any relation with their location or TNM classification, but they show a
significative association with the shape of the tumour and with the histopathological
319
diagnosis. However, the association between these variables and the
ultrasonographic parameters of canine mammary tumours requires further study.
- Gonzalez de Bulnes A, Garcia Fernandez P, Mayenco Aguirre AM, Sanchez de la
Muela M. 1998. Ultrasonographic imaging of canine mammary tumours. Vet Rec.
143(25):687-9.
- Flobbe K, Bosch AM, Kessels AG, Beets GL, Nelemans PJ, von Meyenfeldt MF,
van Engelshoven JM. 2003. The additional diagnostic value of ultrasonography in the
diagnosis of breast cancer. Arch Intern Med. 163(10):1194-9.
- Mehta TS. 2003. Current uses of ultrasound in the evaluation of the breast. Radiol
Clin North Am. 41(4):841-56.
320
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