The vaginal microbiota of healthy female cats.

The vaginal microbiota of the queen (i.e., female cat) has never been described using culture independent methods. The objectives of the present research were to describe the vaginal microbiota of healthy domestic shorthair queens using both 16S rRNA sequencing and culture, and to assess the effects of age, living environment, and reproductive season on its composition. Thirty queens undergoing elective ovariectomy were included in the study. The vaginal samples were collected just before surgery, from animals under general anaesthesia. Two consecutive mini-swabs were introduced in the queens' vaginal tract. A preliminary study with 10 healthy queens aimed to negate sampling order's effect. Two consecutive samples for sequencing (5 queens, 10 swabs) and culture (5 queens, 10 swabs) were collected, confirming a match (100 % in culture, Bray-Curtis P = 0.96 in sequencing). The experiment included 20 queens that were prospectively grouped based on age (prepubertal N = 10, adult N = 10), living environment (indoor N = 10, outdoor N = 10), and time of the year, whether during the reproductive season (N = 10) or during seasonal anoestrous (N = 10). Bacteria were identified through metataxonomic analysis, amplifying the V1-V2 regions of 16S rRNA gene, and through standard culture followed by MALDI-TOF MS. The feline vaginal microbiota is dominated by Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteria. Escherichia-Shigella, Streptococcus, and Pasteurella were the most abundant genera. Although culture underestimated bacterial richness and diversity compared to sequencing, Escherichia and Streptococcus were the most isolated bacteria. No bacterial growth was observed in 15 % of samples (N = 3/20), whereas growth of one or two bacterial species was observed in 64.7 % (N = 11/17) and 35.3 % (N = 6/17) of cases, respectively. No differences in terms of alpha (Kruskal-Wallis rank sum test P = 0.65) and beta diversity (Bray-Curtis, Unweighted and Weighted UniFrac analyses P > 0.5) were observed. Although a difference in alpha diversity based on phylogenetic tree (P = 0.02) was detected between indoor and outdoor queens. In conclusion, mixed and monoculture of Escherichia coli, Streptococcus canis, Staphylococcus felis, and Enterococcus spp. are normal findings within the cat vagina. Age and reproductive season do not influence the feline vaginal microbiota, whereas further research is needed to elucidate the role of the living environment.

The dogma of the sterile uterus revisited: does microbial seeding occur during fetal life in humans and animals?

In brief: Opposing conclusions have been drawn regarding the presence of viable bacteria in the healthy pregnant uterus. Current evidence in humans and animals suggests that fetomaternal tissues present only traces of bacteria whose viability is still to be proven. Abstract: The debate about the pioneer colonization of the fetus is still open, being the 'in utero colonization' hypothesis versus the 'sterile womb paradigm' the two opposing sides. The seed in this field of research sprouted in human medicine in the last decade and became a central topic in other mammals as well. We aimed to review the literature on bacterial colonization of the healthy placenta, amniotic fluid, and meconium as representatives of the fetal environment. What emerges is that confirming the colonization of fetomaternal tissues by viable bacteria is challenging in humans as well as in animals. Contamination represents the major risk in this type of research as it can be related to different parts of the study design. Sampling at natural parturition or postpartum introduces risk for colonization by the vaginal microbiome of the mother or from the environment. Culture does not reveal the presence of unculturable microorganisms, and sequencing does not allow confirming bacterial viability, while also introducing the variability associated with the data analysis. Therefore, on the basis of the present review, we provide some guidelines on the best practices when performing this type of studies. What emerges from the current literature in humans and animals is that fetomaternal tissues are characterized by a very low biomass, that the viability of bacteria eventually present is still to be confirmed, while massive colonization happens at birth, priming the individual, regardless of the species.

Birth weight, puppies' growth and health with limited-ingredient novel protein diet vs standard diet in late pregnancy.

Great attention has been given in the last years to the protein source of dog food, and commercial limited-ingredient diets with a single protein are available also for late pregnancy. This work compared the effect of a fish-based limited ingredient diet (LID), and of a standard mixed-protein diet (Mixed), fed to the bitches from the last three weeks of pregnancy and to the puppies at weaning, on birth weight, growth and health of the puppies. From a breeder's records, the weight of 22 Lagotto Romagnolo (LR) and 10 Appenzeller Cattle Dog (ACD) bitches on the day of mating, and of their 199 puppies, were extracted. The effect of diet on puppies' weight on day 0, 6, 30 and 60 was analyzed, considering litter size and sex. The analyses were repeated on puppies' weights normalized on the relative dam's non-pregnant bodyweight. Birth weight was available for 146 puppies, 82 LR and 64 ACD. Median birth weight of LR puppies was 287.5 g (170-400 g); sex ratio was 1.11 (males/females, N = 80). Median birth weight of ACD puppies was 390 g (240-525 g); sex ratio 1.15 (males/females, N = 58). Diet did not significantly affect birth weight in both breeds; however, it showed a significant effect on normalized birth weights (LR, P = 0.016; ACD, P = 0.034), with higher values for LID. At day 30, ACD puppies showed significantly higher weights with the Mixed diet (P = 0.002), and, at day 60, diet significantly affected the normalized weight in both breeds (LR, P = 0.019; ACD, P = 0.001), with higher values for the Mixed type. LID may help the dam to invest the energy in the growth of her litter, however, the same diet resulted in lower puppies' weights around weaning, compared to the Mixed diet. Although our results should be confirmed with larger numbers of animals and more breeds, they set some points worth to be further investigated. The choice of a limited-ingredient single-protein diet can affect litter weight and weight at weaning. Whether, administered to dams and puppies, it can prevent later pathologies, like chronic gastrointestinal diseases or food allergies, is a field of research deserving full attention.

Challenging the Hypothesis of in Utero Microbiota Acquisition in Healthy Canine and Feline Pregnancies at Term: Preliminary Data.

At present, there are no data on the presence of bacteria in healthy canine and feline pregnancies at term. Here, we investigated the uterine microbiome in bitches (n = 5) and queens (n = 3) undergoing elective cesarean section in two facilities. Samples included swabs from the endometrium, amniotic fluid, and meconium, and environmental swabs of the surgical tray as controls. Culture and 16S rRNA gene sequencing were used to investigate the presence of bacteria. Culture was positive for 34.3% of samples (uterus n = 3, amniotic fluid n = 2, meconium n = 4, controls n = 0), mostly with low growth of common contaminant bacteria. With sequencing techniques, the bacterial abundance was significantly lower than in environmental controls (p < 0.05). Sequencing results showed a species-specific pattern, and significant differences between canine and feline bacterial populations were found at order, family, and genus level. No differences were found in alpha and beta diversities between feto-maternal tissues and controls (p > 0.05). Dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria in different proportions based on tissue and species. Culture and sequencing results suggest that the bacterial biomass is very low in healthy canine and feline pregnancies at term, that bacteria likely originate from contamination from the dam's skin, and that the presence of viable bacteria could not be confirmed most of the time.

The evolution of dam-litter microbial flora from birth to 60 days of age.

BACKGROUND: Early bacterial colonization in puppies is still a poorly understood phenomenon. Although the topic is of considerable interest, a big gap in knowledge still exists on the understanding of timing and features of neonatal gut colonization. Thence, the purpose of this study was to evaluate the relationship between dam and litter microbial flora, in vaginally delivered puppies, from birth to two months of age. Bacteria were identified using MALDI-TOF, an accurate and sensitive method, and cluster analysis of data provided a new insight on the investigated topic. METHODS: Six dam-litter units of two medium size breeds were enrolled in the study. Vaginal and colostrum/milk samples were collected from dams after delivery and 48h post-partum, while rectal samples were taken from dams and puppies after delivery and at day 2, 30 and 60 (T2, T30 and T60, respectively) post-partum. Bacterial isolation and identification were performed following standard techniques, then the data were analyzed using a new approach based on bacterial genus population composition obtained using a wide MALDI-TOF screening and cluster analysis. RESULTS: Forty-eight bacteriological samples were collected from the dams and 145 from their 42 puppies. Colostrum/milk samples (n = 12) showed a bacterial growth mainly limited to few colonies. Staphylococci, Enterococci, E. coli, Proteus spp. were most frequently isolated. All vaginal swabs (n = 12) resulted in bacteria isolation (medium to high growth). Streptococci, Enterococci, E. coli were the most frequently detected. E. coli, Proteus mirabilis, Enterococcus spp., Streptococcus spp. were often obtained from dams' and puppies' rectal swabs. Clostridia, not isolated in any other sampling site, were rarely found (n = 3) in meconium while they were more frequently isolated at later times (T2: n = 30; T30: n = 17; T60: n = 27). Analysis of the bacterial genus pattern over time showed a statistically significant reduction (P < 0.01) in the heterogeneity of microbial composition in all time points if compared to birth for each dam-litter unit. These results were confirmed with cluster analysis and two-dimensional scaling. CONCLUSION: This novel data analysis suggests a fundamental role of the individual dam in seeding and shaping the microbiome of the litter. Thus, modulating the dam's microbiota may positively impact the puppy microbiota and benefit their health.

Trends in Small Animal Reproduction: A Bibliometric Analysis of the Literature.

Small animal reproduction (SAR) is a main research field in veterinary medicine and bibliometric analyses are useful to investigate trends in specific research areas. The objective of the present study was to conduct a bibliometric analysis of the literature of the last decade on SAR. A search equation was created, and documents were retrieved from the Web of Science database. Documents were manually revised, categorized and R software version 4.1.2 with Bibliometrix R package version 3.1 and MS Excel were used to perform the analyses. The included documents (n = 1470) were mainly research articles (78%). The top countries for the number of documents and citations were Brazil, United States, Italy, Poland, and Korea. These also account for the most prolific authors and institutions. Analyses by author keywords, categories, and recent reviews of the literature suggest that research on the canine species is more abundant than research on the feline one and that reproductive biotechnologies are a main research focus. Some clinical topics are still considered niche or neglected themes (e.g., semen collection in tomcats, neonatology). However, heterogeneity and ambiguity in keywords and categories are undeniable. This study offers interesting insights, providing definitions for main keywords in the field of SAR.

A retrospective study and survival analysis on bitches with mammary tumours spayed at the same time of mastectomy.

The aim of the present study was to retrospectively assess whether spaying at the same time of mastectomy increased disease-free survival (DFS) in bitches with mammary tumours and to investigate the utility of clinical data when designing a surgical plan that includes gonadectomy. Characteristics of 225 bitches carrying 489 tumours were retrieved. Of the116 bitches that underwent surgery, 52 bitches underwent mastectomy and ovariectomy, 46 bitches underwent mastectomy alone, whereas 18 bitches were already spayed. Analysis by Kaplan-Meier and in-between groups comparisons using Student's T, Chi-square, and one-way ANOVA tests were performed. DFS was longer for bitches that underwent ovariectomy and mastectomy compared to those that were left intact (p = .00064) or were already spayed (p = .0098). Spaying status affected tumour size (spayed: 2.75 cm ± 2.72; intact: 1.76 cm ± 2.04; p = .039), but not malignancy (p > .05). Differences in age were detected between animals with benign and malignant tumours (years: 9.1 ± 2.8 and 10 ± 2.3; p = .004), with multiple and single tumours (years: 10.18 ± 2.6 and 9.3 ± 2.8; p = .007), and between purebred and mixed breed bitches (years: 10.46 ±1.78 and 9.27 ±2.68; p = .005). Malignant tumours were larger than benign ones (2.17 cm±2.31 and 1.34 cm ±1.82; p = .005) and size increased according to the degree of malignancy. DFS was shorter for animals presenting tumours >2 cm in size (p < .006) and with tumours in the first pair of thoracic mammary glands (p = .00009). Gonadectomy should be suggested to owners of intact bitches carrying mammary tumours and age, size of the tumour, and location should be carefully considered when performing surgery.

Does Bacteria Colonization of Canine Newborns Start in the Uterus?

The assumption that requires the uterus to be a sterile environment to sustain a successful pregnancy has been recently challenged in humans, and is still under debate. The aim of this study was to assess whether bacteria can be isolated from the pregnant uterus and from amniotic fluid and meconium of healthy canine fetuses at term, delivered through cesarean section. Fifteen dams of different breed, age and parity, undergoing either elective (n = 10) or emergency (n = 5) cesarean section after a healthy pregnancy, were included in the study. Swabs for bacterial culture were collected from the uterus, and from amniotic fluid and meconium. Bacteria were isolated from all the sampled sites and materials, irrespective of cesarean type. In most cases, different bacteria were isolated from the different sites. Acinetobacter spp., coagulase-negative Staphylococci and Bacillus spp. were frequently found while Pseudomonas aeruginosa, Micrococcus spp., Moraxella spp., Macrococcus spp., Glutamicibacter spp., Stenotrophomonas spp. and Psychrobacter spp. were only occasionally identified. Our data show that uterus and fetuses may not be sterile in healthy term canine pregnancies.

Effect of sterilization on the canine vaginal microbiota: a pilot study.

BACKGROUND: Surgical sterilization is the most effective method of contraception for dogs. It also prevents pyometra and reduces the risk of mammary tumour development. However, this procedure also has negative effects, such as urinary incontinence. Steroid hormone deprivation following gonadectomy could also affect canine vaginal mucosa conditions and the microbial community colonizing the vaginal tract. This hypothesis was tested by comparing the vaginal cytology and microbial community of two groups of bitches, including 11 in anoestrus and 10 sterilized bitches (post-pubertal sterilization in the last 4 years). Bacteria were identified through metataxonomic analysis, amplifying the V3-V4 regions of 16S rRNA gene, and culturing methods. RESULTS: Vaginal mucosa cytology was suggestive of dystrophic conditions in sterilized bitches, whereas a typical anoestrus pattern with parabasal and intermediate cells was appreciable in anoestrous animals. Metataxonomic analysis revealed large inter-individual variability. Salmonella, Mycoplasma and Staphylococcus were present in moderate quantities in almost all the samples in both groups. Mollicutes (class level) and Tenericutes (phylum level) were commonly present in moderate quantities in anoestrus samples, whereas these microbes were present at high levels in a single sample from the sterilized group. Based on culturing, a higher number of different species were isolated from the anoestrous bitches, and Mycoplasma canis was exclusively identified in an anoestrous bitch. Staphylococcus spp. was the most frequently isolated genus in both groups, followed by Streptococcus spp., and, among gram-negative bacteria, Escherichia spp. and Haemophilus spp. A comparison of the numbers of the most frequently isolated genera of bacteria from vaginal cultures of bitches revealed that Pasteurella and Proteus were the most frequently identified in sterilized animals based on metataxonomic analysis (p-value = 0.0497 and 0.0382, respectively), whereas Streptococcus was significantly and most frequently isolated from anoestrous bitches using culture methods (p value = 0.0436). CONCLUSIONS: In this preliminary investigation, no global patterns of the vaginal bacteria community were noted that characterized the condition of the bitches; however, cytology suggested local modifications. Sterilization after puberty caused minimal alterations in the vaginal microbial community of bitches within 4 years after surgery.

Seroprevalence of Canine Herpesvirus-1 in Breeding Dogs with or Without Vaccination in Northwest Italy.

Canine herpesvirus-1 (CHV-1) can cause abortion and foetal and neonatal deaths in the bitch. The reactivation of latent infections with asymptomatic virus shedding represents a mechanism, whereby the virus can persist in a dog population. The aim of this study was to investigate the seroprevalence of CHV-1 in a population of breeding dogs in Piedmont, Northern Italy, and to investigate the distribution of herpesvirus vaccination. The study was carried out in 370 animals that were housed in 33 breeding kennels. Antibodies against CHV-1 in serum samples were measured by means of serum neutralization. Vaccination had been performed in 21.2% of the kennels and 8.4% of the dogs. The overall seroprevalence of CHV-1 was 50.3%. In ten kennels (30.3%), no seropositive dogs were identified. The percentage of seropositive dogs ranged from 7.1% to 100% in positive kennels. More than 40% of the seropositive dogs showed high titres. Sex had no significant effect on either seroprevalence or the category of the serum titre. The number of positive animals was significantly lower in the groups of prepuberal bitches and animals younger than 1.5 years. The majority of younger animals showed very high titres, suggesting recent contact with the virus. Our data show that CHV-1 is a common infection in breeding dogs in Piedmont. Vaccination is rarely performed but might be an option, because, although many animals of breeding age already show high antibody titres, seronegative pregnant bitches will be at high risk of contracting the infection due to viral circulation in kennels where the virus is enzootic.

Serological survey of canine parvovirus 2 antibody titres in breeding kennels in northern Italy.

BACKGROUND: Current guidelines recommend parvovirus revaccination of adult dogs no more frequently than every 3 years. The aim of this study was to determine the prevalence of dogs showing protective serum antibody titres against canine parvovirus 2 in breeding kennels in Northern Italy and to assess the effect of time from vaccination and the sex of the dog on antibody titres. The study was carried out on 370 animals of different breeds kept in 33 breeding kennels. Antibodies to canine parvovirus 2 in serum samples were measured with an indirect immunoenzymatic assay validated by the manufacturer in relation to the 'gold standard' haemagglutination inhibition test. The number of months that had elapsed since the last vaccination was calculated for each animal and categorized into the following classes: < 12 months; 13-24 months; 25-36 months; 37-48 months; and > 49 months. RESULTS: The prevalence of 'unprotected' dogs was 4.6%. A satisfactory solid herd immunity was present in the majority of breeding kennels, although some vaccination failures were detected. A significant negative correlation was found between antibody titre and months since last vaccination. Comparable antibody titres were found in the first 3 years after vaccination. Although the antibody titre over time was not affected by the sex of the dog, 'unprotected' females had been vaccinated more recently than males with analogous low titres. CONCLUSIONS: Parvovirus revaccination of adult dogs every 3 years, as currently recommended, is also the appropriate recommendation for breeding kennels. Serological tests could be a useful tool to assess the effectiveness of vaccination.

Evaluation of serum C-reactive protein concentration as a marker of impending parturition and correlation with progesterone profile in peri-partum bitches.

C-reactive protein (CRP) is one of the major acute phase proteins in dogs. It is produced by the liver and rapidly increases in response to an inflammatory stimulus. The aim of this study was to measure CRP concentrations around parturition and to verify whether this protein could be useful, together with progesterone (P), to detect the onset of parturition in bitches. The CRP and P concentrations were measured in 66 serum samples from 28 healthy pregnant bitches, collected between -5 and +2 days from parturition (day of parturition = day 0). The effect of 'days from parturition', parity, and litter size on P and CRP concentration was analyzed and the correlation between CRP and P values was calculated. The P and CRP values were affected by 'days from parturition'. While P decreased during the last days of pregnancy, CRP concentration was greater than the normal range (0-1.07 mg/dl) starting the parturition day with the increase starting on day -1. The CRP concentration profiles during the days around parturition have not been previously reported in dogs. The increase in CRP very near the time of parturition and the low magnitude of the increase do not allow for it to be useful in clinical practice to assess the onset of parturition in the bitch.

Occurrence and characterization of methicillin-resistant Staphylococcus pseudintermedius in successive parturitions of bitches and their puppies in two kennels in Italy.

BACKGROUND: Multi-drug methicillin-resistant Staphylococcus pseudintermedius (MRSP) detection is rapidly increasing in microbial specimens from pets across Europe. MRSP has also been isolated from bitches and newborns in dog breeding kennels. This study assessed whether MRSP lineage differs between breeding kennels and is maintained over time. Post-partum bitches (at day 3 vaginal and day 3, 9 and 35 milk samples) and their litters (at day 3, 9 and 35 oral and abdominal skin samples) from two Italian breeding kennels (A and B) were sampled and MRSP was subsequently characterized via whole-genome sequencing and antibiotic susceptibility testing. The study was carried out from October 2014 to March 2016 and included successive parturitions from the same animals. RESULTS: The analysis revealed different situations in both investigated kennels. In kennel A, circulating strains were from 7-locus sequence types ST688, ST258 and closely related isolates of ST71, which included most isolates. In kennel B, only a new isolate, ST772, was detected. In addition, most isolates from both kennels had multi-resistant antibiotic profiles. MRSP was only isolated from litters of MRSP-positive bitches, thus suggesting that bitch-litter transmission is likely. CONCLUSIONS: Our data show that MRSP circulation can differ in different settings, that several clonal lineages can circulate together, and that vertical transmission appears common. MRSP colonization did not affect the health conditions of the bitches or of their litters.

Comparison of different methods of semen cryopreservation in Melopsittacus undulatus.

Melopsittacus undulatus is largely used as a potential model for assisted reproduction in other endangered parrot species. Semen was collected from nineteen healthy adult males, by massage technique, during the breeding season. After preliminary evaluations through CASA analysis, eight birds were selected as semen donors and five ejaculates of each bird were utilized to evaluate the effect of 10% ethylene glycol (EG) addition, in two times, during 40min of equilibration time at 4°C, and to compare two programmable freezing curves (a rapid and a slow temperature descent rate) and freezing on nitrogen vapors. Diluted semen (modified TALP, pH 8.4) was divided into two aliquots, the first for freezing individual samples, the second to be mixed in a semen pool from the eight birds. Potential inseminating doses of 10µl, containing 1×106 spermatozoa, were frozen. The effect of EG addition on semen motility and kinetic parameters was analyzed and the three freezing methods were compared. EG addition caused a significant decline of semen motility in individual samples, not in semen pools. The three freezing curves resulted in significant differences in thawed-semen parameters, with nitrogen vapors showing the worst results, while the higher total and progressive motility values were obtained with the 'fast' protocol. Thawed-semen pools motility and kinetic parameters appeared lower than those of the individual ejaculates. The differences found between single ejaculates and semen pools emphasize the importance of performing artificial insemination tests in order to assess the in vivo performance of single ejaculates in the parrot species.

Comparison of celioscopy and histological examinations to assess male gonadal health and functionality in adults and immature wild raptors.

Celioscopy is routinely used in birds for sex determination and diagnostic purposes. Aim of this work was to validate celioscopy for the assessment of male gonads functionality in wild raptors, comparing the results of direct observation with morphometrical and histological characteristics. The work was done at the 'Centro Animali Non Convenzionali' of the University of Turin, Italy, on 31 endoscopically evaluated raptors that died or were euthanized. Through celioscopic observation, the birds were classified in adults or immatures and maturity categories were defined according to the adrenal-gonad size ratio and to the degree of blood filling of testicular vessels. The gonads were removed immediately after death/euthanasia and measured. Albuginea tunic thickness, diameter of seminiferous tubules, number of meiosis figures, tubular development degree, tubular degeneration degree and germinal cells production degree were evaluated. Testicular size tended to increase from immature to adult birds and from 'out of' to 'in' breeding season; albuginea tunic thickness tended to be higher out of the reproductive season while diameter of the seminiferous tubules, germinative epithelium thickness and number of meiosis figures were higher in the breeding season. In season adults generally showed higher values in tubular development and germinal cells production, and lower degrees of tubular cells degeneration and fibrosis. From the interpretation of all the morphometrical and histological aspects, a general reproductive degree of activity was given to the birds and compared with celioscopic results. A perfect concordance was found in 23 out of 31 cases and a good concordance in six ones; histology could describe obviously better sub-clinical conditions undetectable at direct observation. These preliminary results suggest that celioscopy could be a useful tool to assess male gonads functionality in wild raptors, with the future goal to select the better potential semen donors.

Aging and Veterinary Care of Cats, Dogs, and Horses through the Records of Three University Veterinary Hospitals.

The present article examines over 63,000 medical records belonging to the Veterinary Hospitals of the Universities of Bologna, Torino, and Padova, all in Northern Italy, and relative to dogs (approximately 50,000), cats (approximately 12,000), and companion horses (slightly less than 1,000). The animals of the three species were divided into age classes and categorized per sex into males, females, and neutered individuals. The mean age at visit and the effects of age classes and category (analyzed via ANOVA) are presented and discussed. The data indicate that many animals are presented to the hospitals either in the early phases of their life (presumably for vaccination and, in cats and dogs, gonadectomy) or in the advanced age (over 10 years in dogs, over 15 years in cats, and over 17 years in horses). The records of very old individuals of the three species are also reported. On the whole, the results suggest that a growing population of mature to old domestic carnivores or companion horses reaches ages that were considered exceptional only a few years ago. The data also testify an evolution in the animal-owner relationship and a renewed respect for the value of life in companion domestic mammals.

Semen Collection and Spermatozoa Characteristics in the Kea Parrot (Nestor notabilis).

We describe the seminal characteristics of the kea parrot (Nestor notabilis), an endangered species endemic to the South Island of New Zealand. Semen was collected in the full breeding season from 6 birds in the collection of an amateur aviculturist. The manual massage technique was used. A total of 25 ejaculates was collected and evaluated for volume, degree of contamination, and spermatozoa concentration; motility and kinetic parameters were assessed on diluted samples (modified Tyrode's Albumin Lactate Pyruvate, pH 8.2, temperature 37.5°C) with a computer-aided sperm analyzer. Four ejaculates were not analyzed because of an excessively high degree of contamination. Semen color ranged from transparent or turbid yellow to whitish. The geometric mean of spermatozoa number/ejaculate was 765.9 ± 2084.7 × 106. Total and progressive motility were 71.7% ± 20.0% and 59.8% ± 22.0%, respectively. Great variability was observed both among birds and among different ejaculates of the same subject. The seminal characteristics of kea are worth further investigation, with the aim of relating semen quality to fertility and defining a minimum inseminating dose for breeding purposes. A deeper knowledge of male reproductive biology also can increase the success of breeding programs and enable the use of the kea as a model species for other more threatened species, such as the kaka ( Nestor meridionalis ) and the kakapo (Strigops habroptila).

Semen evaluation in four autochthonous wild raptor species using computer-aided sperm analyzer.

At least 10 percent of the approximately 300 species of the order Falconiformes are listed as being globally threatened. The present work describes the seminal characteristics of three diurnal and one nocturnal raptor species. Semen was collected from clinically healthy Accipiter nisus (n = 1), Falco subbuteo (n = 6), and Falco tinnunculus (n = 5) adult males that were housed at the 'Centro Animali Non Convenzionali' of the Department of Veterinary Sciences of the University of Turin. The semen was collected after a period of recovery and before their release as well as from seven Bubo bubo males bred in captivity as part of a raptor conservation project. All the potential semen donors were trained in semen collection during the breeding season via a ritualized procedure. Ejaculation was achieved using a massaging technique. Each sample was evaluated for volume, degree of contamination, and spermatozoa concentration. The semen motility and kinetic parameters were assessed on diluted semen (modified tyroides albumin lactate pyruvate, pH 7.5, temperature 37.5 °C) using a computer-aided sperm analyzer. Semen collection was successful in all the diurnal species and in five B bubo individuals. The sperm motility and sperm kinetic parameters were very variable both among and within species. In contrast with previous studies that involved raptors bred in captivity and imprinted on humans, we worked with wild birds and attempted to overcome the problem of poor semen quality, which is strongly influenced by stress, by adopting a ritualized procedure that has never been reported for semen collection purposes.

Isolation of coagulase-positive staphylococci from bitches' colostrum and milk and genetic typing of methicillin-resistant Staphylococcus pseudintermedius strains.

BACKGROUND: Among the coagulase-positive, potentially pathogenic staphylococci, Staphylococcus pseudintermedius has been frequently isolated from bitches' milk. This organism colonizes the mammary gland or causes infection, while S. aureus has been only occasionally reported. The objective of this study was to investigate the occurrence and persistence of coagulase-positive staphylococci in the colostrum and milk of postpartum bitches, either treated or untreated with antimicrobials, and to assess the incidence, antibiotic resistance profile and genetic type of the methicillin-resistant strains. On postpartum D1, D7 and D15, drops of secretion were collected from the mammary glands of 27 postpartum bitches, nine of which were treated with antimicrobials. Coagulase-positive staphylococci were identified, antimicrobial susceptibility and the presence of mecA were tested and the genetic profile of methicillin-resistant strains was assessed. RESULTS: Staphylococcus pseudintermedius was the only coagulase-positive staphylococcus isolated, and its presence was detected in 21 out of 27 bitches and in 66 out of 145 swabs. In a single bitch, it caused puerperal mastitis. In untreated bitches, the frequency of isolation was lower in colostrum than in milk. All of the isolates except one were resistant to at least three antimicrobial classes, while 14 out of 66 S. pseudintermedius strains were methicillin-resistant mecA positive (MRSP) and were isolated from eight bitches housed in the same breeding kennel. A significant association was found between antimicrobial treatment and the presence of MRSP. Six of the 12 typed isolates belonged to spa-type t02 carrying SCCmec II/III, and another six were non-typeable with spa carrying SCCmec IV. The t02-SCCmec II/III isolates were sequence type (ST) 71; four NT-SCCmec IV isolates were ST258 and two were ST369. PFGE showed that isolates from the same dog had identical band patterns, while isolates from different dogs had unique band patterns. MRSP strains showed multidrug resistance profiles. CONCLUSIONS: Our results confirm that S. pseudintermedius is the most frequently isolated coagulase-positive staphylococcus from bitches' milk. The isolation of several different strains of MRSP with different genetic characteristics in the same kennel and the fact that two of the strains belonged to a sequence type (ST) described for the first time are noteworthy findings.

Sox9 duplications are a relevant cause of Sry-negative XX sex reversal dogs.

Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus.