Clinical approach to prostatic diseases in the dog.

In small animal practice, prostatic diseases are increasingly encountered. All dogs may experience prostatic disease, but particular care should be addressed to breeding dogs, in which prostatic affection may lead to decrease in semen quality and fertility. The most common prostatic disease is the benign prostatic hyperplasia (BPH) followed by prostatitis, prostatic neoplasia and prostate squamous metaplasia. These diseases do not have pathognomonic symptoms, therefore, making a correct diagnosis may not be easy. An accurate clinical examination and a correct diagnostic protocol are essential in order to begin the most appropriate treatment, and also to do a good prophylaxis where it is possible. BPH therapy is usually recommended when mild-severe signs are present or if symptoms disturb the patient. New therapeutic approaches, both medical and surgical, allow to maintain fertility in most animals with prostatic disorders. Prostate cancer is relatively infrequent. Elective therapy is the surgical one, but it is considered palliative and can result in important post-operative complications. The aim of this paper is to lay down the most appropriate diagnostic process describing the aetiologies of prostatic disease, their symptoms, the right investigative tools and therapy.

Evaluation of methadone concentrations in bitches and in umbilical cords after epidural or systemic administration for caesarean section: A randomized trial.

OBJECTIVE: To measure plasma methadone concentrations in bitches and the umbilical cords of their puppies after systemic or epidural administration. STUDY DESIGN: Prospective, randomized, clinical study. ANIMALS: A total of 27 healthy pregnant female dogs undergoing caesarean section, 4.3 ± 2.3 years of age and weighing 19.9 ± 13.2 kg. METHODS: The dogs were randomly divided into three groups: 1) intramuscular methadone (0.3 mg kg-1) (group MET; n = 9); 2) epidural methadone (0.1 mg kg-1) (group METEPI; n = 9); and 3) epidural lidocaine (4.4 mg kg-1) [group CON (control group); n = 9]. Ten minutes before induction, methadone was administered intramuscularly to the group MET dogs. Anaesthesia was induced with propofol and maintained with isoflurane. Cardiovascular and respiratory parameters were monitored throughout the anaesthesia. After induction, epidural anaesthesia was administered to dogs in groups METEPI and CON. Before any treatment (T0) and, as soon as the last foetus was removed from the uterus (T1), venous blood samples were collected from each dog into heparinized tubes; the umbilical cords were collected and stored at -80 °C until pharmacological analysis was carried out. The samples were analysed using ultra performance liquid chromatography. RESULTS: The cardiorespiratory parameters of the bitches and of the puppies at birth, and the Apgar scores did not differ significantly between groups. At T1 both the median maternal methadone plasma concentration and the median methadone umbilical cord concentration were higher in group MET compared to group METEPI (p = 0.0018 and p = 0.004, respectively). The maternal plasma concentration was higher than the concentration in the umbilical cords (p = 0.05) in group METEPI but not in group MET (p = 0.25). CONCLUSIONS AND CLINICAL RELEVANCE: Epidural methadone (0.1 mg kg-1) administered to bitches undergoing caesarean section is associated with lower umbilical cord methadone concentrations as compared with intramuscularly administered methadone at higher dosages (0.3 mg kg-1).

Pharmacokinetics of buprenorphine following constant rate infusion for postoperative analgesia in dogs undergoing ovariectomy.

OBJECTIVE: To investigate the pharmacokinetics of buprenorphine and its main active metabolite, norbuprenorphine, after administration of an intravenous loading dose followed by constant rate infusion (CRI) in dogs. STUDY DESIGN: Prospective, clinical study. ANIMALS: A total of seven healthy dogs undergoing elective ovariectomy. METHODS: Buprenorphine was administered as a loading dose (intravenous bolus of 15 µg kg-1) followed by CRI (2.5 µg kg-1 hour-1 for 6 hours). Moreover, intraoperative analgesia was supplemented by an intramuscular carprofen (4 mg kg-1) injection, administered prior to surgery, and by lidocaine, administrated through subcutaneous infiltration and through a splash on the ovarian vascular pedicle during surgery. Pain and sedation were scored for all animals throughout the 24-hour study period and rescue analgesia was administered when a visual analogue scale score was > 40 mm. Blood samples were collected from a jugular catheter at regular intervals, and plasma concentrations of buprenorphine and norbuprenorphine were determined by a validated liquid chromatography-tandem mass spectrometry method. RESULTS: Buprenorphine showed a two-compartment kinetic profile. Maximum concentration was 23.92 ± 8.64 ng mL-1 at 1 minute (maximum time); elimination half-life was 41.87 ± 17.35 minutes; area under the curve was 486.68 ± 125.66 minutes ng-1 mL-1; clearance was 33.61 ± 13.01 mL minute-1 kg-1, and volume of distribution at steady state was 1.77 ± 0.50 L kg-1. In no case was rescue analgesia required. Norbuprenorphine resulted below the lower limit of quantification in almost all samples. CONCLUSIONS AND CLINICAL RELEVANCE: The results suggest that a buprenorphine CRI can be a useful tool for providing analgesia in postoperative patients, considering its minor side effects and the advantages of a CRI compared to frequent boluses. The negligible contribution of norbuprenorphine to the therapeutic effect was confirmed.

Semen Collection and Evaluation in Two Tigers (Panthera tigris) and Two Leopards (Panthera pardus).

Assisted reproduction techniques play a significant role in veterinary medicine, and although they are widely used in domestic animals, they are also becoming increasingly relevant in clinical practice for wild felids, especially in the conservation efforts for endangered species. In this study, the result of two semen collection techniques, urethral catheterization after pharmacological induction (Ur.Ca.P.I.) and electroejaculation, are described, aiming to provide new practical information about sperm collection using the Ur.Ca.P.I. technique and electroejaculation in tigers and leopards, describing the authors' experience and presenting new data and observations. The following descriptive study included two subjects of Panthera tigris species and two of Panthera pardus. These subjects, after general anesthesia, underwent sperm collection initially with Ur.Ca.P.I. and, subsequently, with electroejaculation. Sampling was made possible in both species thanks to the use of electroejaculation. Sperm volumes in leopards ranged from 0.3 to 0.5 mL and in tigers from 0.5 to 2.177 mL. Sperm concentration in leopards ranged from 136 × 106 to 280 × 106 sperm/mL, and in tigers, from 21.5 × 106 to 354 × 106 sperm/mL. Urethral catheterization gave positive results in leopards, with sperm volumes ranging from 25 up to 150 µL and a concentration ranging from 110 × 106 up to 1082 × 106 sperm/mL. In tigers, unlike in leopards, the use of the Ur.Ca.P.I. technique encountered difficulties that did not allow satisfactory results to be obtained. Therefore, it would be useful to test the feasibility of urethral catheterization on a larger group of individuals in order to have more meaningful feedback. Finally, because electroejaculation always allowed semen collection in tigers, with a higher sperm quality than samples collected by Ur.Ca.P.I., we currently consider it the technique of choice for the collection of semen material in this species.

Cushioned and high-speed centrifugation improve sperm recovery rate but affect the quality of fresh and cryopreserved feline spermatozoa.

The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints. The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.

Multicentric aggressive unclassified hematopoietic neoplasm involving the placenta in a pregnant bitch.

Hematopoietic neoplasms are common in dogs; however, their association with pregnancy has not been previously reported in veterinary medicine. This rare occurrence presents a variety of diagnostic, therapeutic, prognostic, and ethical challenges. We report a case of a 3-year-old pregnant Bernese Mountain Dog diagnosed with multicentric aggressive unclassified hematopoietic cancer associated with paraneoplastic hypercalcemia during pregnancy. The dog died 7 days after diagnosis, and at Day 36 of pregnancy before any treatment decision could be made. Post-mortem evaluation, including histology, immunohistochemistry, and clonality analysis, led to the diagnosis of an unclassified hematopoietic cancer affecting the uterus and placenta, with no evidence of fetal involvement. The placenta likely acted as a barrier, preventing neoplastic involvement of the fetuses. Alternatively, the pregnancy might have been too early for the hematopoietic neoplasm to affect the labyrinth zone of the placenta and the fetuses. The dramatic disease progression could be explained by compromised cell-mediated immunity during pregnancy. This immunodeficient state is induced by embryonic, maternal, and hormonal factors, which suppress the response to mitogens to prevent rejection of the placenta and the conceptuses. Thus, pregnant dogs might exhibit increased vulnerability to cancer and infectious diseases that rely on cell-mediated immunity for host defense.

Increasing the heme-dependent respiratory efficiency of Lactococcus lactis by inhibition of lactate dehydrogenase.

The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism.

Innovative models for teaching reproduction in small animals: The experience at DIMEVET of Bologna University.

For a long time, the main way to acquire the skills necessary for good veterinary practice has been the traditional apprenticeship model (observe, assist, and perform under supervision). However, in the last years, more creative and innovative teaching models have been adopted by academic institutions and, parallelly, the opportunities to gain hands-on experience for clinical and surgical procedures are becoming more limited. For that reason, the introduction of the models can provide a potential solution to the ethical and legal implications related to the use of live animals for educational purposes and the biosafety risks deriving from the manipulation of human/animal cadavers. The activity on this topic at DIMEVET and, in particular, the experience about teaching reproduction includes in-house production and use of models for learning skills both for degree (fifth and third years of course) and post-graduate courses. Our models are designed on the basis of two fundamental aspect: the student level and the teacher target. The aim of this study was to evaluate, for the first time in literature, how much the use of simulators impacts on practical teaching in degrees and post graduate courses at DIMEVET, considering different learners with different levels of knowledge and skills. Namely, effectiveness, adequacy and quality of simulators have been checked assessing students' learning experience and teacher's opinion by specific satisfaction questionnaires and considering the attendance at the training sessions and the results of a final examination (PHY group) and an in vivo test (POST group). The rate of satisfaction was high among the three groups considered and the participants that had used the models had a higher success, both in the final examination and in vivo test. Data collected have been useful in order to show an improvement in teaching at DIMEVET in the field of reproduction, despite the relatively short years of experience using simulation. With this study we demonstrate that the adequacy of the models is not correlated to how simulators is built, whether with basic materials or not faithfully in terms of anatomical appearance, but it is important that it is realistic in terms of psychomotor procedure. In conclusion, the use of models permits the student to gain the hand-eye coordination and dexterity necessary to perform certain skills and the models proposed are meeting the desired educational goals.

Clinical and Histological Findings of Male Uterus (Uterus Masculinus) in Three Dogs (2014-2018).

Data from three cases of uterus masculinus were retrieved from 2014 to 2018. Two out of three cases presented clinical signs compatible with systemic infection, as observed in bitches with pyometra. Ultrasound examination revealed a tubular fluid-filled structure with a thin irregular wall located cranially to the prostate and in continuity with the cranial part of the gland. In two cases, two other tubular fluid-filled structures were visualized in the caudal part of the abdominal cavity, ventrally to the prostate gland and urinary bladder. After surgical removal of these, histological examination revealed the presence of a uterine structure morphologically similar to the female counterpart. Various types of epithelial cell lining were found, including simple columnar, simple stratified and squamous epithelium associated with glands in the underlying stroma. Immunohistochemistry to anti-Müllerian hormone (AMH) produced a positive result on glands, while multifocal expression was found in the lining epithelium. AMH seems involved in the pathogenesis of uterus masculinus, but its role is not fully understood. Thorough clinical and ultrasonographical examinations, followed by a histological confirmation, are necessary to properly diagnose uterus masculinus in dogs.

Artificial neural networks for the definition of kinetic subpopulations in electroejaculated and epididymal spermatozoa in the domestic cat.

This study was designed for the identification of different sperm kinetic subpopulations in feline semen using artificial neural networks (ANNs) and for the evaluation of the effect of ejaculation on motility patterns of these subpopulations. Seven tomcats presented for routine orchiectomy were electroejaculated, and after 5 days, orchiectomized and epididymal tail sperms were collected. Sperm motility characteristics were evaluated using a computer-assisted sperm analyzer that provided individual kinetic characteristics of each spermatozoon. A total of 23 400 spermatozoa for electroejaculated and 9200 for epididymal tail samples were evaluated using a multivariate approach, comprising principal component analysis and ANN classification. The multivariate approach allowed the identification and characterization of three different and well-defined sperm subpopulations. There were significant differences before (epididymal tail spermatozoa) and after (electroejaculated sperm) ejaculation in sperm kinetic subpopulation characteristics. In both epididymal and ejaculated samples, the majority of subpopulation was characterized by high velocity and progressiveness; however, the electroejaculated samples showed significantly higher values, suggesting that the microenvironment of the epididymal tail could affect the sperm motility or, alternatively, seminal plasma could increase the kinetic characteristics of the spermatozoa, indicating that only after ejaculation, the spermatozoa express their motility potential. Nevertheless, further studies are required to clarify the functional significance of each kinetic subpopulation.

Artificial Insemination in Queens in the Clinical Practice Setting: Protocols and challenges.

PRACTICAL RELEVANCE: Despite substantial advances in assisted reproductive techniques having been recently reported in cats, the use of these is limited and routine application is still far from being a reality in veterinary clinics. Nevertheless, there is an increasing demand from domestic cat breeders for artificial insemination (AI) techniques that are already commonly used in dogs. Where natural breeding is not possible in tom cats and queens of high breeding value, AI could offer a solution. Clinical challenges: AI in cats is more difficult than in other species - both in terms of semen collection/handling and oestrous cycle management given that ovulation must be induced. AIM: For practitioners wishing to perform AI in queens, there are challenges to overcome, and a good understanding of the techniques and procedures involved is pivotal. This review aims to contribute to improved knowledge by providing an overview of AI protocols, encompassing choice of breeding animals, procedures for semen collection, oestrus and ovulation induction, AI techniques and equipment. EQUIPMENT AND TECHNICAL SKILLS: Depending on the animals involved and the specific AI technique chosen, essential equipment may include an artificial vagina, electroejaculator, endoscope (sialendoscope, which can be fairly expensive) and special catheters for transcervical insemination. Other instrumentation and materials needed are typically readily available in a veterinary clinic. In general, no particular skills are needed to perform the procedures described in this review, with the exception of endoscopic transcervical catheterisation, where the ability to use an endoscope is required. EVIDENCE BASE: The information and advice/recommendations provided are based on specific feline research and reviews published in scientific peer-reviewed journals, animal reproduction textbooks, and presentations at national and international congresses. The authors also drew on their own clinical experience with regard to the choice of protocols and procedures presented in this review.

Partial Vaginectomy, Complete Vaginectomy, Partial Vestibule-Vaginectomy, Vulvo-Vestibule-Vaginectomy and Vulvo-Vestibulectomy: Different Surgical Procedure in Order to Better Approach Vaginal Diseases.

Total or partial vulvo-vaginectomy or vaginectomy are not routinely performed due to the complexity of the techniques and because they are considered radical treatments. Little information can be found in the literature, as the same technique is often named in a different way by different authors, confusing the reader. Therefore, the aim of this essay is to describe five different surgical techniques: partial vaginectomy, complete vaginectomy, partial vestibule-vaginectomy, vulvo-vestibule-vaginectomy and vulvo-vestibulectomy. All techniques are described on the basis of the correct identification of the anatomical nomenclature related to structures involved in surgery, in order to give a more precise and unambiguous description and execution of surgical techniques. Moreover, possible intraoperative and perioperative complications and the authors' clinical experience in 33 dogs are described. All techniques are well tolerated and could be curative in case of benign or malignant tumours that have not yet metastasized and palliative in other cases. Moreover, they are also useful for therapeutic purposes for chronic vaginitis, severe vaginal cysts or congenital abnormalities. It is our opinion that having five different available techniques to approach vaginal disease is useful to perform the best surgery according to the clinical findings, patient's characteristics, technique invasiveness and whether it is palliative or not.

Total Perineal Prostatectomy: A Retrospective Study in Six Dogs.

Perineal hernia refers to the herniation of pelvic and abdominal viscera into the subcutaneous perineal region through a pelvic diaphragm weakness: a concomitant prostatic disease is observed in 25-59% of cases. Prostatectomy involves the removal of the prostate, either partially (partial prostatectomy) or completely (total prostatectomy). In case of complicated perineal hernia, staged procedures are recommended: celiotomy in order to perform colopexy, vasopexy, cystopexy, and/or to treat the prostatic disease, and perineal access in order to repair the perineal hernia. Very few reports relate prostatectomy using a perineal approach and, to the extent of the author's knowledge, this technique has not been thoroughly investigated in the literature. The aim of this article is to retrospectively describe the total perineal prostatectomy in dogs presenting perineal hernia with concomitant prostatic diseases which required the removal of the gland. The experience in six dogs (three dogs with the prostate within hernial contents and three dogs with intrapelvic prostate) is reported as well as advantages, disadvantages, and limitations of the surgical procedure. In the authors' clinical practice, total perineal prostatectomy has been a useful surgical approach to canine prostatic diseases, proven to be safe, well tolerated, and effective.

Benign prostatic hyperplasia in the dog.

In small animal practice, prostatic diseases are increasingly encountered. All dogs may experience prostatic disease, but there should be particular attention to breeding dogs, as prostatic disease may decrease semen quality and fertility. The most common prostatic disease is benign prostatic hyperplasia (BPH), a non-cancerous enlargement of the gland affecting intact adult dogs, part of an aging process, including both an increase in cell numbers (hyperplasia) and in cell size (hypertrophy). Acute and chronic prostatitis, prostatic abscess, prostatic neoplasia and prostate squamous metaplasia also occur in dogs, in order of frequency. These diseases often lack pathognomonic clinical signs; therefore, a thorough clinical examination and a correct diagnostic protocol are essential to determine the most appropriate treatment or prophylaxis. Frequently dogs with BPH are asymptomatic, but when clinical signs are present, the most common is a serous to sanguineous urethral discharge. BPH therapy includes various options and is usually recommended when mild-severe signs are present or if clinical signs disturb the dog. In most dogs with this disorder, it is possible to maintain fertility by avoiding castration and choosing alternative therapeutic approaches.

Canine Placenta Histological Findings and Microvascular Density: The Histological Basis of a Negative Neonatal Outcome?

Placenta is essential for the development of the fetus, and its impaired function can lead to a negative outcome (i.e., neonatal mortality). In dogs, investigations on placenta histology and neonatal outcome in healthy bitches are lacking, and a contribution is provided in this study to emphasize the use of placenta histology in practice. Fifty-one placentas from 11 litters were collected during cesarean section, classified according to the litter size (large (L) or small (S)) and the outcome, this latter as healthy (Group 1) or dead within 7 days (Group 2). The placenta/puppy weight ratio (PPR) was calculated, and specimens were formalin-fixed and paraffin-wax embedded, and on the resulting histological slides, capillary density (CD) was quantified. Among necrosis, calcification, and intravascular leucocytes, only the presence of multifocal-confluent necrosis (significantly more frequent in Group 2) was associated with a higher risk of death within 7 days (odds ratio = 30.7). Mixed logistic regression ruled out the effect on death both of a bitch and cesarean type (programmed vs. emergency). PPR and CD values were associated with litter size; large litters had lower PPR (p < 0.01) and higher CD (p < 0.05) than small litters. The relationship between PPR and CD was negative and significant (p < 0.01). Necrosis was a frequent finding in canine placentas, but only when multifocal-confluent was it associated with a poor outcome. The litter size influenced PPR (lower in L) and CD (higher in L), and this is likely due to the plasticity of placenta adaptation.

Plasma Concentration Rise after the Intramuscular Administration of High Dose Medetomidine (0.13 mg/kg) for Semen Collection in Cats.

High dose medetomidine 0.13 mg/kg can be used for semen collection in cats with variable results in terms of quantity and quality. Therefore, a variation in terms of distribution and elimination among patients has been hypothesised. The aim of the study was to characterise the pharmacokinetics of medetomidine (0.13 mg/kg) administered intramuscularly (IM) in healthy male cats. Eighteen male cats undergoing castration were included, and medetomidine (0.13 mg/kg) was administered IM. Venous blood samples were collected at 20, 30, 40, 50, 60, 75 and 90 minutes after medetomidine administration. Before orchiectomy, at T20, sperm collection was attempted. Plasma medetomidine concentrations were determined by liquid chromatography/mass spectrometry analysis. Semen collection was successful in 15/18 cats. The medetomidine plasma concentration following the IM administration of a bolus was best described using a non-compartment model. Time of maximum concentration was observed at 40 minutes (range 20-90); maximum concentration was 32.8 ng/mL (range 26.8-51.2). The median apparent clearance was 11.9 mL/kg/minute (range 0.7-43.8). In conclusion, medetomidine administered IM at 0.13 mg/kg reached its peak plasma concentration slowly and with variability among patients. In addition, it was characterised by low total body clearance probably due to the cardiovascular alterations associated with medetomidine administration.

Is the protective effect of egg yolk against osmotic and cryogenic damage on dog spermatozoa dose-dependent?

Egg yolk (EY) is conventionally used to reduce sperm cryodamage, however, there has not be evaluation of whether there is a dose-dependent effect with inclusion of EY in semen extender. To enhance the knowledge about the protective effect of EY during cryopreservation of dog semen, a specific study was designed to evaluate the dose-dependent protection of the EY against osmotic and cryogenic damage of dog sperm. In the first experiment, sperm stored in an extender that contained graded EY concentrations (0 %, 5 %, 10 %, and 20 %) were diluted with hypo- or hyper-osmotic solutions (final osmolality of 75, 150, 300, 500, 1000 mOsm/kg). Results from sperm kinetic, membrane integrity (MI), mitochondrial activity, and normal morphology evaluations indicated osmotic stress has especially marked effects on the kinetic capacity of spermatozoa, however, there were no direct effects on mitochondrial activity. In both hypo- and hyper-osmotic conditions, EY had a protective effect regardless of concentration. In the second experiment, semen samples were diluted in extenders at increasing EY concentrations (0 %, 5 %, 10 %, and 20 %) and cryopreserved. Effects on sperm kinetics, membrane and acrosome integrity and mitochondrial membrane potential indicated there was improved sperm viability after thawing when the EY concentration was 5 % and 10 %, and lesser viability when it was 20 %. These results indicate, for the first time, that EY reduces osmotic and cryogenic damage when used at 5 % or 10 % concentrations, and that these concentrations can be used to protect dog spermatozoa more effectively than the conventionally used concentration (20 %).

Quantitative analysis of telomerase in feline mammary tissues.

The purpose of this study was to validate immunohistochemistry (IHC) as an alternative to telomerase repeat amplification protocol (TRAP) analysis to detect telomerase activity. TRAP-enzyme-linked immunosorbent assay (ELISA) reactivity was compared with telomerase reverse transcription (TERT) IHC staining in 22 feline mammary tissues (6 normal mammary glands, 2 dysplastic mammary glands, 1 fibroadenoma, and 13 malignant neoplasms [6 solid mammary carcinomas, 2 squamous-cell carcinomas, 4 tubulopapillary mammary carcinomas, and 1 mammary carcinosarcoma]). TERT IHC staining revealed enzymatic expression in nuclear, nucleolar, cytoplasmic, and combined nuclear and nucleolar staining patterns that were separately quantified by image analysis and expressed as the absolute number (average) of positive cells or percentage of positive cells with respect to overall cellularity. With TERT IHC staining, the absolute number and percentage of cells with positive nuclei and nucleoli within the same cell were the variables with the greatest discrimination between benign and malignant mammary lesions (analysis of variance [ANOVA], average P < 0.0001; percentage P < 0.001). For TRAP-ELISA-positive versus TRAP-ELISA-negative tissues, a positive test result provided greater differentiation between malignant versus benign mammary lesions (ANOVA, average P = 0.00038; percentage P = 0.0022). The same IHC pattern of expression showed a proportional and significant (average P = 0.004; percentage P = 0.002) but low (average R = 0.60; percentage R = 0.63) correlation with TRAP-ELISA by the Pearson test. The correlation coefficients obtained show that IHC and TRAP cannot be considered interchangeable because the 2 methods are more complementary than exclusive.

Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes' zona pellucida.

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 µM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity.

In vitro production of cat blastocysts of predetermined sex using flow cytometrically sorted semen.

Sex preselection in cats can have applications for both breeding purposes and as an experimental model for endangered felids. The present study examined the ability to produce cat embryos from in vitro fertilization (IVF) of in vitro matured (IVM) cat oocytes with flow cytometrically sorted spermatozoa and to verify the sex of the embryos obtained from sexed spermatozoa by PCR. In the first experiment, a total of 224 oocytes were fertilized with spermatozoa from six ejaculates sorted without sex separation. The sorting process did not influence the cleavage rate (sorted 44.0% versus unsorted 46.1%), day 6 morula-blastocyst rate (sorted 26.6% versus unsorted 29.6%) and day 7 blastocyst rate (sorted 16.5% versus unsorted 16.5%). In the second experiment, a total of 84 IVM oocytes were fertilized with sorted X- and Y-chromosome bearing spermatozoa from four ejaculates in order to obtain embryos of preselected sex. Embryonic sex determination by PCR revealed that 21 out of 24 embryos reaching morula/blastocyst stage (87.5%) were of the desired sex. In particular 12 out of 14 embryos (85.7%) derived from X-bearing spermatozoa were female and 9 embryos out of 10 (90%) derived from Y-bearing spermatozoa were male. Our results show, for the first time, that X- and Y-chromosome bearing spermatozoa sorted by high-speed flow cytometry can be successfully used in an IVM-IVF system to obtain cat embryos of a predetermined sex.