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.

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.

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.

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.

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.

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.

Biological characteristics and metabolic profile of canine mesenchymal stem cells isolated from adipose tissue and umbilical cord matrix.

Despite the increasing demand of cellular therapies for dogs, little is known on the differences between adult and fetal adnexa canine mesenchymal stem cells (MSCs), and data on their metabolic features are lacking. The present study aimed at comparing the characteristics of canine adipose tissue (AT) and umbilical cord matrix (UC) MSCs. Moreover, for the first time in the dog, the cellular bioenergetics were investigated by evaluating the two main metabolic pathways (oxidative phosphorylation and glycolysis) of ATP production. Frozen-thawed samples were used for this study. No differences in mean cell proliferation were found (P>0.05). However, while AT-MSCs showed a progressive increase in doubling time over passages, UC-MSCs showed an initial post freezing-thawing latency. No differences in migration, spheroid formation ability, and differentiation potential were found (P>0.05). RT-PCR analysis confirmed the expression of CD90 and CD44, the lack of CD14 and weak expression of CD34, mostly by AT-MSCs. DLA-DRA1 and DLA-DQA1 were weakly expressed only at passage 0 by UC-MSCs, while they were expressed at different passages for AT-MSCs. There was no difference (P>0.05) in total ATP production between cell cultures, but the ratio between the "mitochondrial ATP Production Rate" and the "glycolytic ATP Production Rate" was higher (P<0.05) in AT- than in UC-MSCs. However, in both MSCs types the mitochondrial respiration was the main pathway of ATP production. Mitochondrial respiration and ATP turnover in UC-MSCs were higher (P<0.05) than in AT-MSCs, but both had a 100% coupling efficiency. These features and the possibility of increasing the oxygen consumption by a spare respiratory capacity of four (AT-MSCSs) and two (UC-MSCs) order of magnitude greater than basal respiration, can be taken as indicative of the cell propensity to differentiate. The findings may efficiently contribute to select the most appropriate MSCs, culture and experimental conditions for transplantation experiments in mesenchymal stem cell therapy for companion animals.

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.

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).