A composite pain scale for assessing and quantifying pain in rabbits (CANCRS) has been designed merging the Rabbit Grimace Scale (RbtGS) and a scale including clinical parameters (CPS). Construct validity and inter-rater reliability were assessed for CANCRS, for RbtGS and for CPS, in order to test their potential to detect pain in a clinical setting. Rabbits (n = 116) were either hybrids or purebreds and they were independently evaluated by two raters, who could be veterinarians (V) or veterinary medicine students (S). Score intervals determined four pain classes (No pain, Discomfort, Moderate pain and Severe pain) that matched presumptive pain classes associated with some pathological conditions. A chi-square test was used to assess the construct validity of the scales by checking how frequently scale results and presumptive pain classes matched. Sixty-nine patients were evaluated by one V and one S, whereas forty-seven rabbits were assessed by two V, in order to test inter-rater reliability. An intra-class correlation coefficient (ICC) was used to test reliability of the scales, whereas Cohen’s kappa tested inter-rater agreement for each parameter of the CANCRS. Construct validity results show that CANCRS and RbtGS efficiently reveal pain (P ≤ 0.05), while CPS does not (p > 0.05). Inter-rater reliability was very good for both CANCRS and CPS (ICC 0.88 V-V, 0.94 between V-S; ICC 0.97 V-V, 0.91 V-S) and good for RbtGS (ICC 0.77 V-V, 0.88 V-S); therefore, CPS reproducibility was better between veterinarians and students than between veterinarians. Inter-rater agreement between veterinarians and veterinary medicine students was moderate to very good for all the parameters included in the CANCRS (Cohen’s kappa >0,60). In conclusion, it is possible to state that the CANCRS has construct validity and it is a reliable tool for use in clinical practice, when coping with many rabbits with morphological differences. It is easy and fast to use and enriches the RbtGS with some clinical parameters that should be monitored during any clinical examination, allowing for capture of the multidimensional aspect of pain.

: 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. 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.

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.

Anti-Müllerian hormone (AMH) has been suggested to be involved in spermatogenesis. The aim of this study was to investigate the relationship between blood serum AMH concentration and semen quality in dogs. Moreover, this study sought to find the optimal cut-off point value of serum AMH with the greatest sensitivity and specificity to predict semen quality. Forty-five clinically healthy dogs were included in the study and their age as well as the following semen parameters were determined and correlated to serum AMH concentration: total sperm output, normal morphology, plasma membrane integrity, total motility, progressive motility, and velocity parameters. Statistical analysis for correlations were performed using Spearman’s correlation coefficients. Moderate negative associations were found between serum AMH and semen total motility (r = −0.38, p = 0.01), progressive motility (r = −0.36, p = 0.01), and normal morphology (r = −0.36, p= 0.02). Based on these associations, an AMH concentration of 5.54 µg/L was found to be the optimal cut-off point value to obtain the greatest summation of sensitivity (86%) and specificity (63%) to predict semen quality. The serum AMH assay may therefore be a potential hormonal marker to predict which dogs would require further semen analysis. Future research is however needed to confirm these preliminary results.

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.

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.