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Short Communications abstracts (in order of presentation) Ultrasonography- an important diagnostic tool in the work up of prostatic disease in the dog.
The Norwegian School of Veterinary Science Department of Companion Animal Clinical Sciences Radiology Section 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 clinicalpresentation 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) cysts3. Non-parenchymal (paraprostatic) cysts4. Bacterial prostatitis a. Acuteb. Chronic 5. Prostatic abscessation6. Neoplasia7. 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 MHzrespectively. The dogs were placed in dorsal recumbency and the caudoventral abdomen was clipped. Transabdominal scanning were performed in longitudinal and transverseplanes.
Discussion The ultrasongraphic appearance of the different prostatic diseases in clinical cases will be presented.
Short Communications abstracts (in order of presentation) The presence of a glucokinase-like activity in dog, but not in boar, sperm from fresh J.Ma. Fernandez-Novell', J. Ballester", A. Medrano", M. Rivera del Alamo", P.J. Otaequi", J.J.
Guinovart", J.E. Hodrlquez-Gif 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 ofVeterinary 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.
IntroductionVertebrate glucokinase (hexokinase type IV) is a member of the hexokinase protein family which acta as a sensitive and efficient control step for the maintenance 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 changes observed, at least in dog. Taking this into consideration, the main aim of this work isto 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 from their motion parameters to their life-span ejaculation. In these cells the the total hexokinase activity kinetics was determined, and then, with immunological like rat-liver glucokinase glucokinase regulatory protein were tested, in order to find some glucokinase-like regulation in sperm cells.
Results and conclussionsHexokinase kinetics from dog extracts showed the presence of a glucokinase-like (DSGLP), in the range of glucose concentrations of 4mM to 10 mM, whereas boar sperm didnot 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 distributed in the peri- and post-acrosomal zones of the head, and the midpiece and principalpiece of tail of dog spermatozoa.
Our results indicate that dog spermatozoa, cells, showed a clear glucokinase-like activity. This activity could be related to the dog- sperm's ability to specificially react in front of glucose or fructose. In this way, this activitycould be of the basis of sugar-related specific changes in dog-sperm function.
(1) Cardenas, M.L., Cornish-Bowden, A. And Ureta, T. (1998) Biochim. Biophys. Acta 1401, 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: 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 inejaculated 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 dogspermatozoa 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 wascentrifuged and the supernatant was discarded. The sperm pellet was diluted with an egg-yolk tris extender and chilled to 4°C for 2h (equilibration period). Prior to freezing, usingAndersen method (4) described as a two-step dilution protocol (5), the chilled semensamples 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 theincubation at 39°C. The percentage of total motile spermatozoa (TM%) was assessedsubjectively. Sperm plasma membrane integrity and acrosome integrity was evaluated usingCFDA / 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.
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 intactplasma membrane (44.8±10.3 and 45.5±8.1; with and without the addition of Equex STMpaste, respectively). At 2h after thawing, higher TM% (P=0.01) and greater number ofepididymal spermatozoa with intact acrosome (P=0.001) were observed in the samplescontaining Equex STM paste. The mean of TM%, intact plasma membrane and intactacrosome 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.
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 thepercentage of intact acrosome.
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 54: 859-875.
6.Rota et al. 1995. Theriogenology 44:885-900.
7.Axner et al. 2002. Proc. EVSSAR;yet :115. 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 theproblem, 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 lengthoutside 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 constrictionproduced 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% glucosatedserum 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 ofthe checking visit the lesion had improved its aspect, but the owner said he had observed acontinuous 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 ofthe 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 penilebase, 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 beexposed 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. Systemicantibiotherapy 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 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 ofthe penile protrusion was due to secondary edema caused by the fibrous constrictionappeared during the healing of the lesion. The (partial preputioraphy) preputioplasty wasperformed 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. Therecurrence of the problem when there is no lesion complicating the case can be avoided withorchiectomy, or progestagens compounds therapy as it reduces or eliminates the sexualexcitement. In this particular case, the owners wanted the dog for reproduction, and theseoptions were not a possible choice, after resolution.
References:
-Papazoglou L.G.; Idiopathic chronic penile protrusion in the dog: a report of six cases.
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 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 Ada Rota1, Lieta Marinelli2 1 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, althoughthe 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 andleading to normal fertility and pregnancy rates [6, 1, 5]. The aim of this work was to compareplasma prolactin concentrations in spontaneous and induced cycles, in particular duringpregnancy, 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 administrationof 5 og/kg cabergoline (Galastop, Centralvet-Vetem- Milan, Italy) until the progression ofprooestrous 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 bloodsampling 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 measuredusing 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 thepresumed LH peak) [4], were compared using a paired t-test.
All the bitches responded to treatment and prolactin concentration significantly decreased after cabergoline administration; unfortunately, one of the animals could not be followed untilthe 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 statisticallysignificant difference between basal and maximum expected value in the induced cycleswhile the difference was statistically significant (P<O.05) in the spontaneous cycles.
administration, oestrous onset [6] and also during the following dioestrous [2], but apparentlydata are lacking regarding pregnancy following an induced cycle. Our observations are only 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.
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) 2003, Reprod Dom An, 38, 440-443. 6) Verstegen J et al., 1999, Theriogenology, Short Communications abstracts (in order of presentation) THE ACCURACY OF FINE-NEEDLE ASPIRATION CYTOLOGY IN CANINE AND FELINE MAMMARY LESIONS: COMPARISON WITH HISTOPATHOLOGICAL Eva Hollon 1, Juana Martfn de las Mulas2 1 Clfnica Veterinaria Canymar. C/ Granja de San IIdefonso, n° 5. 11007 Cadiz.
2 Dpto. de A. y Anatomfa Facultad de Veterinaria. Universidad de Cordoba. Edificio de Sanidad Animal. Campus de Rabanales. 14014 Cordoba.
OBJECTIVETo 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 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 & Lumsden2000). The histopathologic study was performed on Haematoxylin and eosin-stained tissuesections 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 STARDinitiative).
Neoplasia / no neoplasia Malignant / benign efficacy SN: Sensitivity PPV: Positive predictive NPV: Negative predictive value NE: Not done.
Fine needle aspiration cytology is a reliable diagnostic tool for the diagnosis mammary lesions. In canine mammary lesions, on the contrary, FNA cytology should beused as an orientative diagnostic method exclusively because its diagnostic accuracy for the between malignant and benign neoplasia was low. Thus, benign tumours and dysplasias were classified as malignant tumours in half of the cases, approximately, whilethe 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.
REFERENCIASBaker & 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.
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 eitherin surgical cure, or in using hormonal molecules, like GnRH or hCG, in order toinduce luteinization (2), which bears the danger of promoting cystic endometrial hyperplasia or pyometra. The purpose of this clinical study was to test the efficiencyof 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 andshowing 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 offunctional follicular cysts: blood progesterone remaining between 2.59 and 4.36 during several days (2 cases), blood progesterone during oestrus decreasing from8.07 to 3.28 (1 case), abnormally prolonged heats (1 case), short interestrousintervals and prolonged anovulatory heats (1 case), persistent vaginal oestrussmears 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 indorsolateral recumbency. After shaving the site of puncture, the skin was cleanedand draped with aseptic precaution. Ultrasonographic approach was done using anATL 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 andtransverse planes to locate the ovary. Cyst aspiration was done, either with a fineneedle (22 or 23 gauge), or with a spinal needle, in the case of larger bitches. Theprogress of the needle was observed through the tissue on ultrasound until the tipwas visualised well within the cyst. The needle tip was monitored throughoutaspiration.
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 diagnosiswas performed between 21 to 25 days of pregnancy. Fertility and litter size were 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, bloodprogesterone remaining between 2.59 and 4.36 during several days, or bloodprogesterone during oestrus decreasing from 8.07 to 3.28.
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.
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.
(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. 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: 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 whelpedbefore 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, 50 mg once daily, were administered from day 1 till day 55 post breeding. Bitch n. 1 and n. 2were mated, while bitch n. 3 was inseminated with frozen semen. Aspirin was withdrawn 5days prior to whelping as it could cause an insufficient release of prostaglandins.
Blood tests were normal; IT3 - IT4 results were below average at the beginning of proestrusbut 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 fourlive puppies, one of which with spina bifida. Bitch n. 2 had three embryonic vesicles at day 25post breeding disappeared in three days. Bitch n. 3 was pregnant with four normal foetuses on ultrasoundtill day 55 post breeding, but only one puppy was born alive and normal; aspirin waswithdrawn in this bitch at day 25 because of a persistent bloody vulvar discharge. Aspirin cancause hemostatic defects especially in hypothyroid bitches.
The fact that reproductive failure in these 3 bitches (despite good reproductive management) wassolved 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 thyroidstatus had never been checked before in these 3 bitches, no definitive conclusions can be drawn on therole of aspirin, although it is interesting to note that bitch n. 3 lost 3/4 foetuses following aspirin 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 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 Apoptosis is a highly organized sequence of biochemical events that mediates cell death.
Together with cell proliferation, have been suggested to regulate ovarian and uterinefunction. At least two pathways have bee been identified in the control of apoptosis, oneinvolves a mitochondria-dependent pathway modulated by bcl-2 proteins, and the secondone 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 essentialnuclear 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, itsexpression in the reproductive tract of the bitch has not been investigated.The aim of thisstudy 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 anticaspase 3 (CPP32) Ab4 (Neomarkers). Samples were incubated overnight in a humidifiedchamber at 4°C. Primary antibody binding was detected using a biotynilated secondary goat-antirabbit IgG with aVidin-biotin-peroxidase complexing. The primary detection reagent was3-amino-9-ethylcarbazole AEC (Vector Labs). Sections were cover-slipped in aqueous-based mounted medium. Positive and negative controls were included in each experiment. Stainingintensity 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 bitchesthat 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 andSUb-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 theatretic 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 thenegative controls. Results from uterus showed more homogeneous staining pattern.
Caspase 3 was expressed in the glandular and superficial endometrial epithelia, myometriumand blood vessels. The tissues obtained from healthy bitches, and those obtained frombitches that exhibited CEH, showed similar staining scores. The presence of active caspase 3 in the genital tract of the bitch suggests that apoptosis could be involved in the regulation ofthe ovarian and uterine function in this species.
1. Roseto A, Brenner C (1999): Arch Argent Pediatr, 97(4): 253-75.
H, Simoens P(2001): Reproduction, Short Communications abstracts (in order of presentation) Tolerability of MILBEMAX® (milbemycin oxime and praziquantel) in breeding and
Rudolf Schenker, Robert Cody & GOntherStrehlau Novartis Animal Health Inc.
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, andclinical assessment, bodyweight, haematology and clinical chemistry in the resulting kittenswere 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 andlactation. 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 queenstreated 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 Short Communications abstracts (in order of presentation) Treatment of spontaneous pyometra in the bitch with a combination of cabergoline
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.
AbstractThis study evaluated the response of bitches with both open-cervix and closed-cervix pyometra to treatment with a combination of cabergoline and prostaglandin. Twenty-twobitches 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 pregnantand delivered normal litters, although the number of pups was smaller than the average forthe 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 treatedwith 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 wereexamined on alternate days and a number of assessments were made including clinical examination, haematology, biochemistry, plasma progesterone concentration and ultrasoundexamination of the reproductive tract.
ResultsNineteen of the bitches were successfully managed by a to-day treatment period. Twobitches 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 vulvaldischarge and a reduction in the uterine diameter. For the 21 bitches that were successfullytreated, haematological and biochemical profiles were normal within six and nine days oftreatment 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 subsequentdose 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. Twounmated bitches had a recurrence of the pyometra following the subsequent oestrus.
This study demonstrates the possibility of managing spontaneous pyometra using acombination of a prolactin antagonist with prostaglandin, either for patient stabilisation priorto surgery or for the potential maintenance of breeding capacity.
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 retrospectivestudy the success of treatment with depot preparations of GnRH analogues in 33 bitcheswith spaying induced incontinence was evaluated. Before and 8 to 11 weeks after treatmentthe plasma gonadotropin levels of these bitches were analyzed. The bitches, which werecontinent after treatment, were compared to the remaining dogs. The success of the therapywas 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 onspaying related incontinence. Bitches that had an inadequate or no response to GnRH wereadditionally treated with alpha-adrenergics. Their response to the combined treatment wascompared to what had been reported by the owners during the previous treatment of theseanimals with alpha-adrenergics alone. The purpose of this comparison was to determine ifdogs which responded neither to alpha-adrenergics nor to depot GnRH analogues becomecontinent 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-analoguetreatment 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 ofincontinence 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 ofspaying, duration of incontinence and changes in plasma gonadotropin levels, on thesuccess 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 hadalready been successfully pre-treated. After GnRH therapy 52% of the bitches werecontinent and a further 42% were continent with additional alpha-adrenergic medication. Thesuccess of treatment with depot GnRH analogues correlated neither with the data extractedfrom 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 respondinadequately to alpha-adrenergics. This observation indicates that there is a synergisticeffect of depot GnRH analogues and alpha-adrenergics. Possibly, the GnRH analogues restore the responsiveness of the alpha-adrenergic receptors.
Congress report (1975). Sequelae of bitch sterilisation: regional survey. Vet Rec 1975;96:371-372. [2] Holt PE, Thrusfield 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. MV, Holt PE, Muirhead RH. Acquired urinary incontinence in bitches: its incidence and relationship 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. M, ROsch P. Incontinentia bei der kastrierten HOndin: Haufigkeit Schweiz Arch Tierheilk 1989; 131 :259-263.
[10] Finco DR, Osborne CA, Lewis RE. Nonneurogenic causes of abnormal in the dog and cat. Vet Clin North Am [11] Osborne CA, Oliver JE, Polzin DE. Non-neurogenic In Kirk RW (ed.): Current Veterinary Therapy VII.
we Saunders, 1980;1128-1136.
[12] Reichler 1M, Pfeiffer E, Piche C, Jochle W, Roos M, Hubler M, Arnold S. Changes in plasma gonadotropin and urethral closure pressure in the bitch during the 12 months following ovariectomy.
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. Aims and scopeAt 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, asynthetic micro-opioid receptor agonist is a preferred anaesthetic agent for inductionand maintenance in human cardiac surgery. Continuous intravenous infusion offentanyl raises its plasma concentration in a linear fashion within 20 minutes. Thisstudy evaluated the quality of anaesthesia, cardio- respiratory and stress response induced by the combination of Fentanyl (Fentanyl 5ml Richter Gedeon Inc. ) andMidazolam (Dormicum 5mg EGIS Budapest Hungary).
Materials and MethodsCaesarean section due to dystochia has been performed in thirteen pregnant bitches from different breeds. Blood was collected prior to induction of narcosis and at theend of surgery from the cephalic vein in anticoagulant pre-treated (heparin)evacuated tubes for measurement of biochemical parameters and in tubes withoutanticoagulant 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 atO,04mg/kgof bw.and Midazolam at 0,2 mg/kg of bw. Maintenance of narcosis duringsurgery 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 aninfusion 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 CriticareSystems Inc. USA).
ResultsDuring 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, alkalinephosphatase activity, GGT did not vary significantly with anaesthesia (p<O,05)(Table).
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.
Short Communications abstracts (in order of presentation)Short Communication. Sunday 6th June. Paediatrics and exotics pets. MinorRoom 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.
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 thathave been stored for 8 days (Yu & Leibo 2002). All lynx that are killed at hunting or founddead 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. Insome animals, parts of the reproductive tract were accidentally removed in connection withpelting. The mean weight of the testes in each animal was calculated. Spermatozoa werecollected by mincing the cauda epididymidis in Dulbecco's PBS, Ham's F-10 or Kenney'sdilution 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 andwith 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 beevaluated 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 reachespuberty 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 thepresent study is necessary.
Axner E, Hermansson U, Linde-Forsberg C. Anim Reprod Sci 2004; in press.
Yu I, Leibo SP. Theriogenology 2002; 57:1179-1190.
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.
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. Insome animals, parts of the reproductive tract were accidentally removed in connection withpelting. The mean weight of the testes in each animal was calculated. Spermatozoa 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 andwith 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 toMarch (which is the hunting season). The present data indicates that the male lynx reachespuberty at between 13 and 20 kg of bw. To create gene banks from epididymal spermatozoa it is likely that a shorter interval between death and sperm collection than in thepresent study is necessary.
Axner E, Hermansson U, Linde-Forsberg C. Anim Reprod Sci 2004; in press.
Yu I, Leibo SP. Theriogenology 2002; 57:1179-1190.
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 of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Istanbul Campus, 34850, Avctier, Istanbul, Turkey. Telephone: +90212-473-70- 2Departmentof Nuclear Medicine, Cerrahpasa Medical Faculty, Istanbul University,lstanbul, 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 bydifferent 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 mineraldensity (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 consideredsuitable for longitudinal measurement of changes in bone mass over time(2, 6). The aim ofthis 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 andungonadectomized puppies across time.
Materials and MethodsAnimals: 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 litterwere 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 dogfood" individually twice daily. Surgeries were performed at 10 week. The procedures forovariohysterectomy and laparatomy were routine. BMD and BMC Measurements: In order todetermine the changes of the BMD and BMC, the calcaneus andthe accessory carpal boneof each pup was measured by the DXA using a bone densitometer" under sedation at onemonth intervals till 6 months of age after surgery. All scans were performed using AP SpineScan Selections Programme. And the scans were processed by Low Density Spine scanmode(V8.26a:5). Each pup was positioned in lateral recumbency and the calcaneus and theaccessory carpal bone were scanned in mediolateral direction. The estimated area for allmeasurements was 0,67 cm2• Statistical Methods: The Independent Samples t-test wasused 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 densitometerResultsIn 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 asignificantdifference 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 Our findings were in contrast with the study by Omi and Ezawa (10), but in accordance withthe 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 distalradius in ovariectomized ewes. But also, in the same study, BMD in the vertebrae (L4-L6/L5-L7) was found significantly lower in the ovariectomized group compared to sham. As weknow 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, wefound higher BMD and BMC values in gonadectomized puppies. In addition to this, as Milleret al.(8) indicated in their review, the skeleton may not be as responsive to the reduction ofovarian 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 themixed 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 demonstrated the effects of prepubertal gonadectomy on the BMD and BMC.
•• 0,8
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) Harris,S.S., Dawson-Hughes,B.: Weight, body composition, and bone mineral density in postmenopausal women. Calcif. Tissue Int. 59, 428-432, (1996) Koo,W.W.K., Hockman,E.M.: Physiologic predictors of lumbar spine bone mass in neonates. Pediat.Res. Kustritz,M.V.R.: Early spay and neuter. Managing Canine Reproduction, Veterinary Learning Systems. 29-31, (1998) 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) 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) Miller,S.C., Bowman,B.M.,Jee,W.S.S.: Available animal models of osteopenia-small and large. Bone. VoI.17(4), 117S- Ohtsuka,S., Kumasaka,T., Ishikawa,K., Watanabe,R., Mori,T., Inaba,N.: The effect of body weight on mineral density ofeach 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) AbstractsShort Communications abstracts (in order of presentation) in puppies of a new canine parvovirus vaccine using the variant CPV-2b : comparison with three conventional B. Martinet1, C. Morales Moliner, P. Mahl3, L. Gardey" - 1Ecole Nationale Veterinalred'Alfort, FR-94700 Maisons-Alfort Cedex; 2Universitat Autonoma de Barcelona, SP-08193 Bellaterra; 3Virbac SA, BP27, FR-06511 Carros Cedex, : Canine Parvovirus (CPV) infection is a major disorder in puppies especially in breeding kennels. Vaccination is the most effective means of preventionbut conventional vaccines show a lack of efficicacy during the critical period, due to the interference of maternally derived antibodies (MDA) with vaccinal antigens. Fieldstrains 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 wasdeveloped in order to allow an early and intense immune response and to bettercope the field serotypes. This study aimed to compare the immunogenicity of the newvaccine 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 (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 (HI) test. At DO, dogs were considered seropositive when MDA was equal or over 20. Seroconversion (SCo) at D14 and 28was defined as a four-fold increase or over in HI titers when positive prior tovaccination, and for titers over 20 when negative on DO. The efficacy was assessedby the SCo rate at D14 and 28, and the titers evolution. Fischer's exact test wasconducted 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 ingroup A as soon as D14 compared to others (P<0.05, Table 1). Highest titers wereobtained for each serotype in group A (P<0.05) whether the pups were seropositiveor not, whereas the presence of MDA led to low anti-CPV-2a and 2b titers in groupsB, C and D (Table 2).
Table 1 : SCo rates (%) regarding in the 4 vaccine groups on Table 2 : HI titers to CPV-2, CPV-2a and CPV-2b on 014 and 28 and influence of
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.
Short Communications abstracts (in order of presentation) LONGITUDINAL CLINICAL, SEROLOGICAL AND VIROLOGICAL
PATTERN OF CANINE
(CHV-1) IN BREEDING COLONIES.
VERSTEGEN J1.; RONSSE V1.; ONCLIN K1.; THIRY E2.; POULET H3.
1Department of Clinical Sciences, Section Small Animal Reproduction, College of Veterinary Medecine, Universite de Liege, Boulevard de Colonster 20, B44, 4000 Liege, Belgium; Tel.: Fax: 0032(0)43664231; 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 isassociated with fertility disorders and neonatal mortality. To advise dog breeders towards aneffective management of CHV-1 infected colonies, antibody and viral excretion patterns wereinvestigated 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 onserologic and viral excretion patterns was evaluated. The association between reproductivedisorders seroconverted and 46% of mated bitches experienced reproductive disorders. Encountereddisorders were embryonic no offspring after mating and mummification.
difference in antibody patterns was observed between mated and unmated bitches. In matedbitches, no difference in antibody patterns occured depending on reproductive disorders,although the majority of bitches with reproduction disorders had a decrease in antibody titerduring early or mid-di-oestrus. Significantly higher titers were observed at all cycle stages inkennels with more than 20 dogs. None of the vaginal and nasal samples and buffy coatswere positive on PCR analyis. The mixed image of clinical and sub-clinical carriage confirmsCHV-1 has a complex and difficult to predict clinical behavior. Preventive management withsystematic vaccination of reproducing bitches in kennels with reproductive disorders shouldbe advised. Also, CHV-1 related management should focus on prevention of clinical signs rather than on prevention from infection.
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 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 spermaticcord 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 hidentesticle fully drop, it would be necessary its scrotal removal. Ley y col.(2003) 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 anatural 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).
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 but only 4 of them (36%) needed a laparotomy for castration.
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.
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.
-Ley, W.B.; Holyoak, R.; Digrassie, A.; Partisano; D. (2003) In: Small Animal Theriogenology. Root Kustritz, M.V.Elsevier Science (USA). pp: 468 Poster abstracts (in order of presentation) Expression Of Androgen Receptor In The Canine Prostate: Differences Between Normal, Inflamed, Squamous Metaplasia, Hyperplastic And Neoplastic Glands F. Gallardo T. Mogas T. Bar62, R. Habanal", E. Taberner J. Morote", T. Rigau 1, J.
Heventos", and J. l.loreta".
1 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 inthe 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 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, and neoplastic prostate tissue of adult dogs (n=37) in paraffin sections. Polyclonal antibody against AR (NCL_Arp, 1:20, Novocastra) was used, along withconventional antigen retrieval methods and the Envision TM System. Tissue sections fromhuman prostate were used as positive controls. As a negative control, AR antibody wasreplaced by PBS.
Results: The results of the study confirmed that canine prostate is a target organ of sexualsteroids. AR was present in both the epithelial and stromal cells. lmmunostaining 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 the percentage of positive epithelial cells (::S;98%), and in the intensity of immunostaining.
negative controls, all nuclei were negative. Squamous metaplasic tissue showed a slightreduction 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 thesame sample. Presence of AR decreased in neoplasic cells, with a mean of 65%. Thepercentage 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 aprogressive decrease in hyperplasia, metaplasia, prostatitis and cancer.
Poster abstracts (in order of presentation) FERTILITY 24 HOURS AFTER BILATERAL VASECTOMY 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 humansand animals.':" Vasectomy prevents fertility by both obstructing ejaculation of spermatozoaand 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 thedogs 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 one day after surgery and by day 3 after occlusion azoospermic." It has been previously reported that azoospermia develop from 2 to 20 daysafter bilateral vasectomy in dogs.6,? To our knowledge there are no data on the specific timecourse of fertility waning off following bilateral vasectomy in this species. In this article a caseof 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 themoment of consultation.
Bilateral vasectomy was indicated and performed under general anesthesia according toHardie (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.
confirmed by ultrasonography 25 days after the mating and gestation was pharmaceutically The results here described agree with previous reports that showed that although spermcount one day after bilateral vasectomy is almost absent, azoospermia develops from day 2after surgery.5,6,? No data is available on fertility during these initial critical days. Knowledqeof 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 vasectomyin the domestic dog. Until further work on fertility of these cases is carried out strict physicalseparation of animals should be indicated for a minimum of 72 hours after surgery.
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 SEX1977; 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 ofbilateral 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 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 deEspinardo, University of Murcia, E-30071 Murcia, Spain. 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 moreexactly nonsperm and sperm particles when frozen-thawed spermatozoa are assessed. Theaim 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 protocoldescribed 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 UppsalaEquex-2 extenders (2). Straws were thawed in a water bath at 70°C for 8 sec and each strawdiluted in 1 ml of thawing medium. After thawing, 100 III of the semen samples (-6 x 10 6cells) were diluted with 400 III of PBS and stained with final concentration of 100 nM SYBR- 14, 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 twomeasurements per straw were used. To validate the method, 15 straws were measuredsimultaneously 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 BritishStandards Institution (BSI) repeatability coefficient was used (4).
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 ofagreements were d ± 2So- = (9.96; -6.32%) and (16.7; -8.7%) respectively, showing high The triple stain for the simultaneous assessment of membrane and acrosomal integrityshowed good repeatability and method-agreement; therefore, it can be a useful technique forthe evaluation of frozen-thawed canine spermatozoa.
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.
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 withtamoxiphen 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 whithan intermitent bloody urethral discharge problem. Rectal palpation showed that the prostategland was enlarged and irregular, but not painful. Ultrasound analisis showed the presenceof numerous intra-parenchimal cysts. The diagnosis was benign prostate hyperplasia with prostatic cysts. The dog was orchiectomized and medical treatment with tamoxiphen wasprescribed at 0.3 mglKg/24 hours/p.o. The ultrasound controls at 15 and 30 days post-surgery 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 sameproblem. Ultrasound showed the presence of cysts and an enlarged prostate, and diagnosisof benign prostate hyperplasia with prostatic cysts was made. Tamoxiphen was prescribed atthe 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 estrogencompounds.
The results of treatment with tamoxiphen in these two cases (presented) seem(s) to confirmthis. 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.
-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.
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 As in many species, canine semen has to be diluted before it can be analysed by computer-assisted systems because of the high concentration of freshly collected semen. The aim ofthis study was to evaluate the effects of four different extenders for dog semen on thedifferent parameters obtained by computer-assisted sperm analysis.
Materials and methods: A total of 28 ejaculates from 6 clinically healthy beagle dogs aged 2- 4 years were used. The sperm rich fraction was evaluated for estimated motility, pH andsperm 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 thewhole trajectory of the spermatozoon [4]. Progressive motility was defined by VCl and aminimal linearity (STR) of 90% [3]. Hypermotility was defined as VCl >118 urn/s, lateral headdisplacement (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 aregiven as percentages of spermatozoa with intact plasma membranes. Statistical analysis:Spearman's correlation was used for comparison of estimated motility with motilityparameters 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 and6.4, 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 andmotility 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 PBSand T, viability was 85.4 and 87.7%, respectively (P>0.05). After dilution with TM, viability ofsamples determined with SYBR-14/PI and CFDA staining was significantly correlated Conclusion: T can be recommended as dilution medium of canine semen for furtherlaboratory analysis because parameters are not changed. Dilution of semen with PSsignificantly influenced semen characteristics in comparison with freshly collected semen.
1. Aleporou-Mairinou V, Pappa H, Yalouris P, Patargias T (2001) Comp Biochem Phys B 128, 537-542.
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.
Poster abstracts (in order of presentation) Clinical test of pumpkin seed extract against benign hyperplasia of the prostate gland 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 Objectives: The aim of the study was to compare the effects of pumpkin seed extract, aphytopharmacon (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, revealed an enlarged prostate gland with regular structure but low grade increased density. In 80 % of dogs, erythrocytes and protein was detectable Treatment started after the first check with 1 pumpkin tablet SID, only one dog received 2tablets 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. Mainclinical signs: 5 with bloody preputial discharge, 2 with defecation difficulties. Initial treatment: injection of 3 mg/kg of Cyproteronacetate Dogs were clinically checked and blood samples were taken 1-2 weeks afterwards, then monthly until 6 monthsafter the first application. Testosterone 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 notaffected. 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 (heightx length) decreased not significantly in 5 of 8 dogs until the 6th visit, with varying values inbetween. Increased sexual activity increased the size despite therapy. Bloody preputialdischarge vanished in 1 dog after 6 months with 2 tablets SID, but not in any dogs given 1tablet SID. Side effects: 2 dogs had soft, green feces after 3 months of treatment (1 tabletSID).
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, anddefecation and micturition difficulties as well as preputial discharge vanished at the sametime. In no patient a relapse occurred within 6 months.
Conclusions: With 1 pumpkin tablet SID, libido was not affected, urine quality, defecation aswell as micturition difficulties improved within 4-6 months. Prostate gland size decreasednon-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.
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 IntroductionA transcervical intrauterine insemination (TCI) technique for dogs was developed in Norwayat the beginning of the 1970s (1). This nonsurgical has proven successful in canine AI (2,3). This study reports results with frozen semen TCI AI at our clinic from 1999 to2003.
Materials and methodsA total of 371 bitches of 90 breeds were inseminated with frozen-thawed 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 smearand serum concentration of progesterone, and inseminations performed two and three daysafter estimated time of ovulation.
Statistical analysisThe statistical in Epiinfo 6.03 and the results are given as (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.
ResultsA total of 281 bitches (76%) gave birth with a mean (±SEM) litter size of 6.0 ±0.19 pups. In3.5% 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 WRas 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) littersize of 6.1 (± 0.24) pups, and imported semen 74% WR and 5.7 (± 0.32) pups (n.s.).
Discussion and conclusionOur 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 of84% and litter size of 5.4 (± 0.3) reported by Linde Forsberg et al. (3). In the present studynone 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. Inaddition, the attempts to pass the catheter through the cervical canal might possibly inducechanges 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 Alswith large numbers of spermatozoa seemed to be beneficial (4).
on a new technique.
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 semen: a retrospective study. Theriogenology 1999:52;11-23. 4) Notling JO, Gerstenberg C and Volkmann DH. Success with intravaginal insemination of frozen-thawed retrospective study. J S Afr Vet Assoc 1995: 66; 49-55.
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 eliminateseveral drawbacks inherent to the conventional evaluation of sperm morphology and mayidentify subtle morphometric sperm characteristics which cannot be detected by visualevaluation. As internal image settings may influence the results considerably, the effect ofdifferent technical settings and semen handling procedures on morphometric parameters ofcanine semen, measured by the Metrix Oval Head Morphology software implemented in theHamilton Thorne Ceros (version 12.1; HTR 12.1 Metrix), was investigated. In the presentstudy, all semen samples were stained with Diff-Quick. Comparison of sperm morphometricmeasurements of 200 spermatozoa from pooled semen of 3 dogs at a magnification of x 40and x 60 demonstrated a more accurate identification of the sperm head boundaries at amagnification level x 60 (4 replicates). Dilution of the pooled semen samples to a spermconcentration of 50 x 106/mL allowed for a correct evaluation of the sperm cell dimensionswhereas 100 and 200 x 106/mL resulted in significantly larger sperm dimensions andsignificantly 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) wereobtained for all the evaluated sperm samples compared to the fresh semen samples (n=12ejaculates). 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 reliableand objective sperm morphometric measurements by the HTR 12.1 Metrix in canine. The large variations in morphometric dimensions between individual dogs suggest that greaternumbers of dogs might be necessary to obtain a reliable and accurate estimate of caninesperm dimensions. Possible explanations for the decreased morphometric dimensions aftercryopreservation are the progressive dehydratation of the sperm cell upon cooling and freezing, the high proportion of spermatozoa with a damaged acrosome with subsequent lossof 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 incanine.
This research was supported by the RUG Special Research Fund, Grant N° 011 B8698 andGrant N° 01109700. E-mail 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: Objective of the work. Ketamine alone or in mixture with xylazine or medetomidinehas been reported as anesthetic protocol for electroejaculation (EE), but their effectson sperm quality has never been studied. The aim of this study was to evaluate theeffect of ketamine and medetomidine on domestic cat sperm characteristics after EE.
Materials and Methods. In this study 3 experiments were performed. The effects ofketamine or medetomidine on sperm quality (Exp.1) and sperm flow (Exp.2) afterelectroejaculation, and sperm flow without electroejaculation (Exp.3) were evaluated.
Animals: european adult male random-source cats aged 2 to 7 years and weighing 3to 5 kg. EE protocol: ketamine (K group) 20 mg/kg and medetomidine (M group) 130- 140 IJg/kg IM were administrated and sperm was collected as previously described by Howard et aI., 1990. Semen volume, sperm concentration, motility andmorphology 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 bycystocentesis. 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 groupstreated 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 wereperformed using Student's t-test and ANOVA elaborated applying a softwarepackage (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: themean 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 K2group (18.2 ± 9.50; 1.08 ± 1.20 respectively). Significant differences (P<0.01) between thenumber of spermatozoa displaced in urethra from the storage sites (ducts deferentsand 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 thanC group (0.24 ± 0.27).
Conclusions. Medetomidine is an <X2-adrenergicagonist used routinaly in veterinarymedicine as a sedative and analgesic for dogs and cats. The data obtained show thatthe mean sperm concentration, mean total number of spermatozoa and spermatozoadisplaced in urethra from the storage sites are significatively higher usingmedetomidine instead of ketamine. Our study also shows that the mean number ofspermatozoa emitted in urethra without electroejaculation is significatively higherafter injection of medetomidine instead of ketamine. These results could be attributable to stimulation of <X2-adrenoreceptors of the vas deferentes andepididymes. Alpha-adrenoreceptors mediate the response to adrenergic agonists forcontraction 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 <X2-adrenoreceptor 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 aftermedetomidine or ketamine administration were found. In conclusion, the use ofmedetomidine, for sperm collection by EE, permits to obtain a good pharmacologicrestriction and a higher number of spermatozoa per ejaculate without increasing theretrograde flow in the urinary bladder.
Poster abstracts (in order of presentation) OF FRUCTOSE, BUT NOT GLUCOSE, ALLOWS FOR
A FEASIBLE MEDIUM-TERM STORAGE OF REFRIGERATED
DOG SEMEN
Ma. Montserrat Hlvera", Fanny Gallardo", Armando Oulntero-Moreno'', OlgaMadriqal", Joan E. Rodriguez-Gila and Teresa Rigaua 'Unit of Animal Reproduction Department of Animal Medicina and Surgery. School ofVeterinary Medicine. Autonomous University of Barcelona. E-08193 Bellaterra. SpainbUnit of Animal Reproduction. Faculty of Veterinary Science. University of Zulia. Box 15252, Maracaibo 4005-A. Venezuela IntroductionDog sperm shows aremarkable specificity in its modulatry mechanisms of energy metabolism. Thus, although these cells are able to utilize both glucose and fructoseas energy sources, the effects of both monosaccharides are separate and specific inconcrete sperm functions, such as motion parameters (1) or tyrosine-phosphorylationpattern (2). This direct link between a concrete sugar and a concrete function wouldaffect the storage ability of dog sperm subjected to restrictive energy condition, Le.
low concentrations of substrates. To test this possibility, we studied the survivalability of dog sperm stored at 4°C in a medium with low concentrations of an specificsugar as energy substrate.
Material and MethodsFresh 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 4days, and an aliquot was taken daily, in order to perfom the approppriate analysis.
Semen quality of samples was determined by analyzing the percentages of viabilityand 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 themain 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 failedin the attainment of this goal. A similar result was determined after observing the totalmotility. On the other hand, sperm maintained up to 4 days in a medium with fructoseshowed a less linear mean movement than those incubated with glucose. These results show that the specific energy utilization of fructose by dog sperm induces amore efficient survival estrategy in restricted conditions that that related to glucose.
References1. 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, PefiaA, 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.
Poster abstracts (in order of presentation) Inflammatory response in the female reproductive tract after natural mating in 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, Objective of the workThis study aimed to investigate the distribution of leukocytes in the mucosa, submucosa andblood 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 MethodsOestrus 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 wasconfirmed by the presence of corpora haemorrhagica or corpora lutea. A group of sixfemales was ovariohysterectomised at 30 min, 3 hr, 48 hr or 96 hr after mating. One side ofthe 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 reproductivesegment, 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 meannumbers 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) ofSAS (1996). The statistical models included times after mating (4 times), segments of theuterine 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 classof the factors and were compared by using Student's t-test. A P-value < 0.05 was consideredstatistically significant.
A marked number of lymphocytes and neutrophils were observed in the mucosa andsubmucosa 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 oflymphocytes and neutrophils were found in the cranial part of the uterus. A high number ofneutrophils 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 cranialand caudal uterus. The highest numbers of neutrophils were seen at 3 h after mating in thevascular layer of the cranial uterus and this was higher than at 30 min and 96 h (P<0.05). Afew eosinophils, plasma cells, monocytes and macrophages were found in the various reproductive segments but were not quantified.
ConclusionsAn 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 wasobserved at 48 hr after mating.
1 Over-all mean±S.E.
numbers of lymphocytes reproductive segments for all times after matingLeukocytes abc different superscript letters within a row indicate significant difference (P<0.05) 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 MethodsOestrus was induced in 24 female cats using 100 iu eCG. The cats were mated four timeswithin one hour to obtain a sufficient LH release for induction of ovulation. Ovulation wasconfirmed 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 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 reproductivesegment, five sections on coded slides were chosen for microscopic 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, plasma cells, monocytes and macrophages were done by the same person.
Numbers of leukocytes were compared using general linear mixed model (PROC MIXED) ofSAS (1996). The statistical models included times after mating (4 times), segments of theuterine 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 classof the factors and were compared by using Student's t-test. A P-value < 0.05 was consideredstatistically significant.
A marked number of lymphocytes 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 oflymphocytes and neutrophils were found in the cranial part of the uterus. A high number ofneutrophils 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 cranialand caudal uterus. The highest numbers of neutrophils were seen at 3 h after mating in thevascular layer of the cranial uterus and this was higher than at 30 min and 96 h (P<0.05). Afew eosinophils, plasma cells, monocytes were found in the various reproductive segments but were not quantified.
ConclusionsAn 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.
Table 1 Over-all mean±S.E. numbers of lymphocytes and neutrophils in the various
reproductive segments for all times after matingLeukocytes 1.1±O.3abc 1.4±O.3a abc different superscript letters within a row indicate significant difference (P<O.05) Poster abstracts (in order of presentation) USE OF LOGISTIC REGRESSION MODELS TO STUDY UL TRASONOGRAPHIC, MACROSCOPIC AND MICROSCOPIC FEATURES OF CANINE MAMMARY 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 MATERIAL AND METHODS: The tumours were diagnosed in 10 bitches of differentbreeds and ages between 7 and 13 that were presented to the Murcia's University Veterinary Teaching Hospital. The ultrasonographic 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 representedby the following variables: TNM Classification (W.H.O., 1980), Location (mammary gland affected) and Shape (spherical-oval or polymorphous).
appearence of the tumours is represented by the variable Diagnosis (mixed tumouror 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 thesemodels are estimated using the maximum likelihood method. In order to estimate theeffects of each explanatory variable on the probabilities of the ultrasonographic parameters hypothesis tests with probability a = 0.05 are performed, using theScaled Deviance, with Ji Square distribution.
In a first model the explanatory variables are the macrocospic characteristics of the and only the variable on the ultrasonographic parameters with value of the statistic 12,91 > 12,59 = X6;O,05 (with p-value = 0.044). Ina second model the explanatory probabilities of the ultrasonographic parameters with p-value = 0.043.
RESULTS: The tumours with spherical-oval shape have ultrasonographic with regular margin, diffuse distribution or heterogeneous.
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, diffusedistribution 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 showacoustic shadowing.
CONCLUSION: The ultrasonographic characteristics of canine mammary tumours donot have any relation with their location or TNM classification, significative association with the shape of the tumour and with the histopathological 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.
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- Flobbe K, Bosch AM, Kessels AG, Beets GL, Nelemans PJ, von Meyenfeldt MF,van Engelshoven JM. 2003. The additional diagnostic value of ultrasonography in thediagnosis 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.

Source: http://www.evssar.org/.pdf/Proceedings_4thEVSSARcongress_2004_part4.pdf