Multidrug-resistant Escherichia coli causing canine pyometra and urinary tract infections are genetically related but distinct from those causing prostatic abscesses.

Despite extensive characterisation of uropathogenic Escherichia coli (UPEC) causing urinary tract infections (UTIs), the genetic background of non-urinary extraintestinal pathogenic E. coli (ExPEC) in companion animals remains inadequately understood. In this study, we characterised virulence traits of 104 E. coli isolated from canine pyometra (n = 61) and prostatic abscesses (PAs) (n = 38), and bloodstream infections (BSIs) in dogs (n = 2), and cats (n = 3). A stronger association with UPEC of pyometra strains in comparison to PA strains was revealed. Notably, 44 isolates exhibited resistance to third-generation cephalosporins and/or fluoroquinolones, 15 were extended-spectrum ß-lactamase-producers. Twelve multidrug-resistant (MDR) strains, isolated from pyometra (n = 4), PAs (n = 5), and BSIs (n = 3), along with 7 previously characterised UPEC strains from dogs and cats, were sequenced. Genomic characteristics revealed that MDR E. coli associated with UTIs, pyometra, and BSIs belonged to international high-risk E. coli clones, including sequence type (ST) 38, ST131, ST617, ST648, and ST1193. However, PA strains belonged to distinct lineages, including ST12, ST44, ST457, ST744, and ST13037. The coreSNPs, cgMLST, and pan-genome illustrated intra-clonal variations within the same ST from different sources. The high-risk ST131 and ST1193 (phylogroup B2) contained high numbers of ExPEC virulence genes on pathogenicity islands, predominating in pyometra and UTI. Hybrid MDR/virulence IncF multi-replicon plasmids, containing aerobactin genes, were commonly found in non-B2 phylogroups from all sources. These findings offer genomic insights into non-urinary ExPEC, highlighting its potential for invasive infections in pets beyond UTIs, particularly with regards to high-risk global clones.

Advancing canine mammary tumor diagnostics: Unraveling the diagnostic potential of Cytokeratin 19 through droplet digital PCR analysis.

Cytokeratin 19 (CK19) is a complex intracytoplasmic cytoskeletal protein primarily localized in the ducts of the mammary gland and skin epithelial cells. In humans, the expression of CK19 gene within circulating tumor cells (CTCs) extracted from blood samples of breast cancer patients reflects tumor cell activity, offering valuable insights for predicting early metastatic relapse or monitoring treatment effectiveness. However, knowledge of serum tumor markers is limited in veterinary oncology. Recently, droplet digital PCR (ddPCR), has been employed to explore rare target genes due to its heightened sensitivity and accuracy as a novel molecular diagnostic tool. The objectives of this study were to investigate the expression of the CK19 mRNA in CTCs, non-neoplastic mammary tissues, and both benign and malignant canine mammary tumors (CMTs) through ddPCR analysis. In Study I, we optimized the discard volume for blood samples to reduce CK19 contamination from skin epithelial cells post-venipuncture. The results revealed that discarding the initial 3 mL of blood was adequate and effective in eliminating CK19 mRNA contamination. In Study II, after the removal of the initial 3 mL of blood, we investigated CK19 mRNA-positive CTCs in the peripheral blood of normal healthy dogs, including those with benign and malignant CMTs. Intriguingly, CK19 mRNA was undetectable in all blood samples. The expression of CK19 mRNA in mammary tissues was investigated in Study III. The copy number (CN) ratios of the CK19 gene in non-neoplastic mammary tissues (14.77 ± 14.65) were significantly higher (P < 0.05) than those in benign (4.23 ± 3.35) and malignant groups (6.56 ± 5.64). Notably, no difference was observed between the benign and malignant groups. In conclusion, CK19 mRNA appeared unlikely to be a suitable candidate as a biomarker in the peripheral blood of CMTs, while the CN ratio in mammary tissues could serve as a potential discriminator between non-neoplastic and CMT groups, complementing the gold standard of histopathological examination.

Serum Anti-Müllerian Hormone Levels and Estrous Monitoring of GnRH Agonist Deslorelin-Induced Estrus in Bitches: A Pilot Study.

This study was performed to monitor estrous patterns and, more importantly, changes in anti-Müllerian hormone (AMH) concentrations during the peri-ovulatory period in deslorelin-induced estrous bitches. Healthy anestrous bitches (n = 4) were used. Estrus and ovulation were monitored after deslorelin implantation. Blood samples were collected for analysis of progesterone, estradiol-17ß and AMH concentrations before implantation (day 0) and on days 6, 8, 10, 12, 14, 16, 18, 20 and 22 after implantation. Six days following treatment, all bitches showed estrus signs. Ovulation took place between days 12 and 15. Circulating AMH concentrations varied among bitches from 0.12 to 3.08 ng/mL. However, no significant differences in AMH levels (mean ± SD) were observed between day 0 and days following post-implantation (p > 0.05). There were no significant correlations between AMH and estradiol or AMH and progesterone (p > 0.05). Ultrasonographically, the number of clearly identifiable ovarian follicles was higher before ovulation and the area of ovaries increased after ovulation (p < 0.05). Except for AMH, changes in vaginal cytology, estradiol-17ß and progesterone levels observed in our study were similar to naturally occurring estrus. Large intra- and inter-individual variation in AMH were observed suggesting that AMH is currently not suitable as a canine fertility marker to monitor ovarian response to deslorelin treatment for estrus induction.

Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland.

In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (Ta) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues.

The evaluation of non-anesthetic computed tomography for detection of pulmonary parenchyma in feline mammary gland carcinoma: a preliminary study.

BACKGROUND: Thoracic radiography in awake cats is a common procedure for the evaluation of pulmonary metastasis in feline mammary gland carcinoma (MGC). However, due to poor sensitivity, computed tomography (CT) is progressively taking its place. To perform CT in animals, general anesthesia is normally preferred but can cause lung atelectasis, affecting lung interpretation. Besides, MGC is often found in senile cats that are concurrently affected with other diseases, increasing anesthetic risk. Therefore, this study was aimed at comparing the effect of anesthesia on lung atelectasis observed through CT in clinically healthy cats and comparing the feasibility of non-anesthetic CT with non-anesthetic radiography in the detection of lung lesions in feline MGC. Thoracic CTs from anesthetized, clinically healthy cats and non-anesthetized either clinically healthy cats or MGC-affected cats were reviewed. In clinically healthy cats, motion artifacts and characteristics of lung atelectasis were observed and compared. In MGC-affected cats, motion artifacts were observed and compared to clinically healthy cats, and the number of MGC-affected cats, the number and characteristics of lung lesions were compared between non-anesthetic thoracic CT and radiography. RESULTS: Anesthesia significantly increased lung CT attenuation (P = 0.0047) and was significantly correlated with lung atelectasis (OR = 15; CI 2.02-111.18; P = 0.0081), particularly of the cranial lung lobe. Nonetheless, significantly higher motion artifacts in the caudal thoracic area were found in non-anesthetized healthy cats (P = 0.0146), but comparable low motion artifacts were observed in anesthetized healthy and MGC-affected cats. Non-anesthetic CT revealed higher numbers of MGC-affected cats and pulmonary nodules with a significantly lower nodular diameter (P = 0.0041) than those observed on radiographs. The smallest nodular diameters detected on radiographs and CT were 2.5 and 1.0 mm, respectively. Furthermore, CT showed additional information such as intra-thoracic lymphadenopathy, that could not be seen on radiographs. CONCLUSIONS: Despite the motion artifacts, CT without anesthesia is a sensitive technique as it provides better lung inflation. Furthermore, compared to non-anesthetic radiography, non-anesthetic CT provided more information such as higher number of pulmonary nodules of a smaller size, including more distinct intra-thoracic lesions, such as lymphadenopathy, in MGC-affected cats.

Support vector regression algorithm modeling to predict the parturition date of small - to medium-sized dogs using maternal weight and fetal biparietal diameter.

BACKGROUND AND AIM: Fetal biparietal diameter (BPD) is a feasible parameter to predict canine parturition date due to its inverted correlation with days before parturition (DBP). Although such a relationship is generally described using a simple linear regression (SLR) model, the imprecision of this model in predicting the parturition date in small- to medium-sized dogs is a common problem among veterinarian practitioners. Support vector regression (SVR) is a useful machine learning model for prediction. This study aimed to compare the accuracy of SVR with that of SLR in predicting DBP. MATERIALS AND METHODS: After measuring 101 BPDs in 35 small- to medium-sized pregnant bitches, we fitted the data to the routine SLR model and the SVR model using three different kernel functions, radial basis function SVR, linear SVR, and polynomial SVR. The predicted DBP acquired from each model was further utilized for calculating the coefficient of determination (R2), mean absolute error, and mean squared error scores for determining the prediction accuracy. RESULTS: All SVR models were more accurate than the SLR model at predicting DBP. The linear and polynomial SVRs were identified as the two most accurate models (p<0.01). CONCLUSION: With available machine learning software, linear and polynomial SVRs can be applied to predicting DBP in small- to medium-sized pregnant bitches.

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment.

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine® , 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.

Impact of gonadal status on ultrasonographic renal parenchymal dimensions in healthy cats.

OBJECTIVES: The aim of this study was to evaluate the effect of gonadal status on ultrasonographic renal parenchymal dimensions in healthy cats. METHODS: Forty healthy cats (10 intact males, 10 intact females, 10 castrated males and 10 spayed females) presented to the Division of Obstetrics, Gynecology and Reproduction, and the Diagnostic Imaging Unit at The Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University. They were ultrasonographically examined to assess renal length, aortic luminal diameter, cortical thickness and medullary thickness. RESULTS: Regardless of gonadal status, the renal length, aortic luminal diameter, cortical thickness and medulla thickness of males were greater than those of females (P <0.05). In general, neutered cats had thicker medullae (0.36 ± 0.08 cm) and higher mean renal length:aortic luminal diameter ratio (12.15 ± 1.48) than intact cats (0.32 ± 0.08 cm and 11.22 ± 1.37 cm, respectively) (P <0.05), but no differences were observed in renal length, cortical thickness or aortic luminal diameter. Interestingly, when comparing between sexes with relatively equal body weight, only sex had an impact on renal length. CONCLUSIONS AND RELEVANCE: Gonadal status has an effect on medullary thickness and mean renal length:aortic luminal diameter ratio.

Prenatal diagnosis of foetal hydrocephalus and suspected X-linked recessive inheritance of cleft lip in a Chihuahua.

A 3.5-year-old, 2.9 kg, multiparous Chihuahua presented with abdominal distension; pregnancy was diagnosed. On Day 7 before parturition, prenatal sonograms showed anechoic bilateral dilated cerebral lateral ventricles, suggesting fluid-filled regions (ventriculomegaly) in one foetus. A Caesarean section was performed and the male newborn had an abnormally enlarged dome-shaped head and a cleft lip, and died 6 days after birth. According to the family pedigree, the X-linked recessive inheritance of an orofacial cleft from the unaffected mother was suggested. This report clearly demonstrates that canine foetal ventriculomegaly (hydrocephalus) can be diagnosed in utero. For dog breeds predisposed to congenital ventriculomegaly, early detection is important for the prediction of perinatal survival and adequate supportive care can be applied at delivery.

Gonadotropin-releasing hormone-modified chitosan as a safe and efficient gene delivery vector for spermatogonia cells.

The use of male gonadal tissue as a site for the local delivery of DNA is an interesting concept. Previously, we reported synthesis, physiochemical and biological properties of gonadotropin-releasing hormone (GnRH)-conjugated chitosan as a carrier for DNA delivery to GnRH receptor-overexpressing cells. In this study, the application of modified chitosan as a potential vector for gene delivery to testicular cells was carried out. Transfection efficiency was investigated in mouse-derived spermatogonia cells (GC-1 cells) using green fluorescent protein as a reporter gene. GnRH-conjugated chitosan exhibited higher transfection activity and specificity compared to the unmodified chitosan. Furthermore, the GnRH-modified chitosan showed less cytotoxicity. In conclusion, we have developed and successfully tested the GnRH-modified chitosan for delivery of a transgene of interest to spermatogonia cells in vitro. Such vector could be useful in particular for testis-mediated gene transfer.

Age-related changes in interstitial fibrosis and germ cell degeneration of the canine testis.

Fifty-five healthy medium-sized dogs were divided into four groups; young (1-3 years old, n = 14), adult (>3 to 6 years old, n = 12), old (>6 to 9 years old, n = 14) and senile (>9 years old, n = 15). After routine orchiectomy, testes were collected, and the degree of white streak areas on cut surfaces was subjectively assessed. Later, testicular tissue sections were stained with haematoxylin and eosin and Masson's trichrome for evaluation of germ cell degeneration and the proportion of interstitial connective tissue, respectively. Semiquantitative severity scoring of germ cell degeneration and quantitative analysis of spermatogenic cells for spermatic index (SI) and Sertoli cell index (SEI) was performed. The score of white streak on cut surface area of the testes increased with age, being higher (p < 0.05) in senile dogs than other age groups; no difference was found between adult and old dogs. The proportion of testicular interstitial fibrosis was highest (p < 0.05) in senile dogs. Positive correlations between age and white streak area (rho = 0.77, p < 0.01) as well as age and interstitial fibrosis (rho = 0.63, p < 0.01) were observed. The severity of germ cell degeneration gradually increased with age and differed among age groups (p < 0.05). Age positively correlated with atrophy of seminiferous tubules (rho = 0.93, p < 0.01). The SI was lower (p < 0.05) in senile dogs compared to other age groups, and SI was not different among young, adult and old dogs. Conversely, SEI was significantly higher in senile dogs compared to young, adult and old dogs. A negative correlation between age and SI (rho = -0.69) and a positive correlation between age and SEI (rho = 0.68) were significant (p < 0.01). In conclusion, influence of age on testicular interstitial fibrosis and germ cell degeneration/depletion were pronounced in dogs over 9 years old.

Formulation, physical, in vitro and ex vivo evaluation of nanomedicine-based chemosterilant for non-surgical castration of male animals.

The overpopulation of free-roaming companion animals has become the global crisis. The development and application of a suitable, effective, non-surgical approach for animal sterilization would have an enormous advantage over the current surgical method. The main purpose of this study was to develop and evaluate a novel nanomedicine-based chemosterilant for non-surgical castration of male companion animals. In this study, we first sought to investigate the testicular toxicity of different apoptosis-inducing agents. We next synthesized and characterized nano-sized particles which encapsulated the most potent testicular toxicants and evaluated in vitro sterilant properties. Our result showed that doxorubicin exhibited the highest cytotoxic activity against mouse spermatogenic cells. We therefore synthesized and characterized doxorubicin-encapsulated nanoemulsion. The negatively charged particle of doxorubicin-encapsulated nanoemulsion exhibited the anti-proliferative activity towards spermatogetic cells. Apoptosis studies revealed activation of Caspases 3 and 7 as well as annexin V expression. In addition, doxorubicin-encapsulated nanoemulsion exhibited anti-inflammatory activity in lipopolysaccharide-stimulated macrophages. Cell death was observed following treatment of isolated and cultured rat seminiferous tubules with doxorubicin-encapsulated nanoemulsion. In conclusion, nanoemulsion can be a potential carrier for prolonged release and to enhance activity of doxorubicin that may have utility in non-surgical castration of male animals.

Nanocarrier-mediated delivery of α-mangostin for non-surgical castration of male animals.

The overpopulation of abandoned and stray companion animals has become a global crisis. The main purpose of this study was to develop a novel nanomedicine-based antifertility compound for non-surgical castration of male animals. Mangosteen (Garcinia mangostana L) pericarp extract has been shown to exhibit anti-fertility property. α-mangostin (AM)-loaded nanostructured lipid carrier (AM-NLC) was developed to improve male germ cell apoptosis. This study was conducted to investigate physicochemical properties of AM-NLC and determine the biological effects of AM-NLC on spermatogonia cells and testicular explants obtained from castrated testes. AM-NLC was produced through a hot homogenization technique. The negatively charged particle of AM-NLC was nano-sized with a narrow dispersity. AM-NLC exhibited antiproliferative activity towards spermatogonium cells. It induced apoptosis in the cells. In addition, AM-NLC exhibited anti-inflammatory activities in lipopolysaccharide-activated macrophages. Abnormal anatomy of seminiferous tubule was noted following treatment of testicular explant with AM-NLC. This nanomedicine-based sterilant would be a promising platform that may have utility in non-surgical castration of male animals by intra-testicular injection.

Chitosan-based DNA delivery vector targeted to gonadotropin-releasing hormone (GnRH) receptor.

The main purpose of this study was to investigate the application of modified chitosan as a potential vector for gene delivery to gonadotropin-releasing hormone receptor (GnRHR)-expressing cells. Such design of gene carrier could be useful in particular for gene therapy for cancers related to the reproductive system, gene disorders of sexual development, and contraception and fertility control. In this study, a decapeptide GnRH was successfully conjugated to chitosan (CS) as confirmed by proton nuclear magnetic resonance spectroscopy (1H NMR) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The synthesized GnRH-conjugated chitosan (GnRH-CS) was able to condense DNA to form positively charged nanoparticles and specifically deliver plasmid DNA to targeted cells in both two-dimensional (2D) and three-dimensional (3D) cell cultures systems. Importantly, GnRH-CS exhibited higher transfection activity compared to unmodified CS. In conclusion, GnRH-conjugated chitosan can be a promising carrier for targeted DNA delivery to GnRHR-expressing cells.

Collagen and glycosaminoglycan profiles in the canine cervix during different stages of the estrous cycle and in open- and closed-cervix pyometra.

The extracellular matrix of the cervix that comprises collagen, elastin, proteoglycans and glycosaminoglycans (GAGs) is thought to have an essential role in cervical relaxation. This study investigated the proportion of collagen and smooth muscle as well as the GAGs in cervices obtained from healthy bitches at different stages of the estrous cycle and bitches with open- and closed-cervix pyometra. Cervices were collected after ovariohysterectomy. The proportion of collagen to smooth muscle was determined using Masson's trichrome staining. Alcian blue staining was used to evaluate the relative distribution of cervical GAGs. The proportion of cervical collagen relative to smooth muscle was higher at estrus compared to anestrus (P≤0.05). It was also higher (P≤0.05) in bitches with open- compared to those with closed-cervix pyometra. Overall, hyaluronan (HA) was the predominant GAG in the canine cervix. In the luminal epithelium, the staining intensity for HA was stronger in estrus than in anestrus (P≤0.05), but not in diestrus (P>0.05). On the contrary, the intensity for the combined keratan sulfate (KS) and heparan sulfate (HS) was stronger in anestrus than in estrus and diestrus (P≤0.05). In bitches with pyometra, the staining intensity of the stroma for KS and HS was weaker in open- compared to closed-cervix pyometra (P≤0.05). Collectively, the different profiles of collagen and GAG suggest that the metabolism of both collagen and GAGs in the canine cervix is associated with hormonal statuses during the estrous cycle and cervical patency of bitches with pathological uterine conditions, such as pyometra.

The effects of the prostaglandin E analogue Misoprostol and follicle-stimulating hormone on cervical penetrability in ewes during the peri-ovulatory period.

Two experiments in parous Welsh Mountain ewes determined the pattern of natural cervical relaxation over the peri-ovulatory period and investigated FSH and Misoprostol as cervical relaxants to facilitate transcervical passage of an insemination pipette into the uterine cavity. Following synchronisation of oestrus using progestagen sponges and PMSG (500 IU) the depth of cervical penetration was determined using a modified cattle insemination pipette as a measuring device. Penetration of the cervix was least at the time of sponge removal and increased to a maximum at 72 h after sponge removal and then declined. Intra-cervical administrations of either ovine FSH (Ovagen; 2mg) or Misoprostol (1mg; a Prostaglandin E(1) analogue) facilitated cervical penetration. Ovagen given 24h after sponge removal allowed transcervical intrauterine penetration in 100% of ewes at 54 and 60 h after sponge removal while Misoprostol given 48 h after sponge removal allowed trans-cervical penetration in 100% of ewes at 54 h. A combination of Ovagen and Misoprostol was as effective but not more so than Ovagen or Misoprostol alone. These results show that there is natural relaxation of the cervix at oestrus and that maximum relaxation occurs 72 h after sponge removal, which is too late for the correct timing of insemination. The intra-cervical administration of FSH or Misoprostol enhanced relaxation of the cervix and both were able to relax the cervix to allow intrauterine penetration 54 h after sponge removal, the optimum time for insemination. The results also show that FSH is biologically active after intracervical, topical application.

The effect of ACTH stimulation on cortisol and progesterone concentrations in intact and ovariohysterectomized domestic cats.

The objective was to evaluate the adrenocortical capacity for cortisol and progesterone production in female cats, both while intact and after ovariohysterectomy. Five privately owned female cats, 1-3 years old, were used in two trials while intact at an inactive stage of the cycle, and again in two trials, 2 weeks after ovariohysterectomy. The four trials were: intact saline injection control trial; intact ACTH injection (0.125 mg); ovariohysterectomized saline injection control trial; and ovariohysterectomized ACTH injection. Blood samples were obtained by an indwelling cephalic vein catheter at -30 and 0 min (immediately before injections) and at 60, 90, 120 and 180 min after injection. The mean basal pre-treatment concentrations of cortisol in the intact and ovariohysterectomized cats were 33 +/- 19 and 32 +/- 19 nmol/L, respectively; the corresponding values for progesterone were 1.1 +/- 0.6 and 0.7 +/- 0.6 nmol/L, respectively. Saline did not alter the serum cortisol or progesterone concentrations. In contrast, both cortisol and progesterone were elevated after ACTH, with peak values at 90 min and returned to basal levels at approximately 180 min. There was a positive correlation between cortisol and progesterone concentrations (r = 0.8, P < 0.05). In some instances, the procedure used to restrain the cats during blood collection induced increases in cortisol and progesterone of the same magnitude as when the ACTH was administered; these effects of restraint could alter the results of assisted reproduction efforts.

The use of propofol for electroejaculation in domestic cats.

The objective was to evaluate the use of propofol as an anesthetic drug for electroejaculation in the domestic cat. Cortisol concentrations, heart rates and respiratory rates of 20 male domestic cats were examined. The animals were randomly allocated into three groups. Group A (n = 8), were anesthetized with propofol (10 mg/kg) and underwent electroejaculation. Group B (n = 6), were pre-medicated with buprenorphine (0.01 mg/kg), anesthetized with propofol (5 mg/kg) and underwent electroejaculation. Group C (n = 6), the cats were anesthetized with propofol (10 mg/kg) without electroejaculation (control group). Blood samples were collected at four time points (30 min before propofol administration, immediately after the surgical plane of anesthesia was induced, immediately post-electroejaculation, and at the onset of anesthetic recovery). In the control group, the sampling time coincident with the end of electroejaculation was assigned as 21 min after the induction of anesthesia. The mean (+/- S.E.M.) duration time for electroejaculation was 18 +/- 3 min. The duration of anesthesia did not differ (P > 0.05) among the three groups of cats (26 +/- 2 min). Most of the cats (17/20) recovered smoothly. Pre-anesthetic medication with buprenorphine did not reduce the requirement of propofol for anesthesia. The cortisol concentrations, heart rates and respiratory rates of the three groups of cats did not differ (P > 0.05). A marked decline in cortisol levels was observed immediately post-electroejaculation. Propofol was a useful anesthetic for electroejaculation in felids with rapid onset, optimal duration of anesthesia for performing electroejaculation, and smooth recovery.

A short sperm-oocyte incubation time ZBA in the dog.

The fertilising capacity of a semen sample can be predicted by evaluation of spermatozoa with in vitro tests. The zona pellucida binding assay (ZBA) accounts for several parameters and interprets the interaction between the spermatozoa and the oocyte. The present study was made in two parts. The aim of the first experiment was to evaluate whether the sperm binding capacity of oocytes varies between different oocyte pools. Each zona binding was made with oocytes from different bitches, using pooled frozen-thawed semen from the same two dogs. The sperm-oocyte complexes were incubated for 1h. There was a significant difference between the six replicates in the number of sperm bound to the zona pellucida (ZP), which indicates that the sperm binding capacity of the ZP differs between oocyte pools. The aims of the second experiment were to evaluate the effects of five different treatments of the spermatozoa on the ZBA, and to evaluate two different incubation times of the sperm-oocyte complexes. ZBAs were made with: fresh semen; semen kept chilled for 1 or 2 days prior to the ZBA; and with semen that had been frozen with or without Equex. The oocytes and spermatozoa were incubated for 1 or 4h. For fresh semen and for semen frozen without Equex, incubation for 1h resulted in a higher number of bound spermatozoa per oocyte than incubation for 4h (P<0.0001). When the effect of the different sperm treatments on the number of spermatozoa bound to the ZP was evaluated, it was found that this number was higher for fresh spermatozoa than for chilled or frozen-thawed spermatozoa both after 1 and 4h of co-incubation (P<0.0001). After 1-h incubation of the sperm-oocyte complexes, spermatozoa chilled for 1 day showed better zona binding capacity than spermatozoa chilled for 2 days, and spermatozoa frozen without Equex had a better zona binding capacity than spermatozoa frozen with Equex. Sperm motility and sperm plasma membrane integrity were higher in fresh than in chilled and frozen-thawed semen. The acrosome integrity was high in all groups of treated semen. In conclusion, 1-h incubation of the sperm-oocyte complexes seems to be sufficient for fresh and chilled semen. Further studies are required to establish the optimal incubation time for sperm-oocyte complexes when frozen-thawed semen is evaluated, as a comparison between semen frozen with Equex and semen frozen without Equex gave different results depending on whether the incubation time was 1 or 4h (in the present study), or 6h [Ström Holst B, Larsson B, Linde-Forsberg C, Rodriguez-Martinez H. Evaluating chilled and frozen-thawed dog spermatozoa using a zona pellucida binding assay.

Freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days.

An experiment was conducted to investigate the freezing ability of canine epididymal spermatozoa after cool storage at 5 degrees C for 2 or 4 days. Spermatozoa were collected from the caudae epididymidis from 16 dogs. Total motility, plasma membrane integrity and acrosome integrity were evaluated immediately on harvesting, and after 2 and 4 days of storage at 5 degrees C, and at 0 and 2 h post-thaw at 37 degrees C. Sperm motility decreased significantly during cold storage, compared to freshly harvested spermatozoa (P < 0.001). Although there was no significant effect of pre-freeze storage time on post-thaw motility, there was a tendency towards decreased motility in spermatozoa that had been stored for 4 days, compared to spermatozoa that were frozen immediately after collection (P = 0.09). The number of post-thaw spermatozoa with an intact plasma membrane was decreased in spermatozoa cold-stored for 4 days (P < 0.001). There was no significant effect of pre-freeze storage time on the acrosomal status of post-thaw spermatozoa. In conclusion, canine epididymal spermatozoa were stored at 5 degrees C for up to 4 days without a clear detrimental effect on post-thaw motility and acrosome integrity, but storage may have decreased post-thaw motility. Results were, however, generally low.