D. Groppetti

Endometrial cytology and computerized morphometric analysis of epithelial nuclei: A useful tool for reproductive diagnosis in the bitch

New diagnostic approaches are required to recognize early canine hypofertility or infertility. We suggest that the identification of different cytologic types, cellular aspects, and nuclear features of the endometrial epithelial cells may be suitable for this purpose. This study was performed on the bitch (Canis familiaris) during the physiologic reproductive cycle and in uterine diseases. We also applied computerized cytomorphometry to evaluate nuclear area, perimeter, diameter, density, aspect, and roundness of endometrial epithelial cells in healthy dogs (N=35) at different stages of the reproductive cycle (before puberty, during proestrus, estrus, diestrus, and anestrus) and in bitches affected by uterine disorders (N=10). The stage of the estrous cycle was determined by vaginal cytology and progesterone evaluation and also confirmed by clinical and histologic observations. Samples for endometrial cytology were collected in vivo by uterine flushing with transcervical uterine cannulation. After uterine sampling, each dog underwent OHE or uterine stump revision. Cytologic analyses were compared with histologic examinations to verify the uterine condition. The uterine cellular population was represented by endometrial epithelial cells, erythrocytes, neutrophils, lymphocytes, eosinophils, macrophages, plasma cells, and cervical or incidental vaginal cells. Bacteria and amorphous material were observed. The proportion of different cells and nuclear features in the cytologic samples varied throughout the stages of the reproductive cycle and between normal and pathologic uterine conditions. The computer-assisted nuclear morphometry, performed in cytologic specimens by means of the six nuclear parameters chosen to evaluate the endometrial epithelial cell population, proved to be useful for determining the stage of the reproductive cycle. Furthermore, this system was demonstrated to be a valid support to diagnose and distinguish uterine disorders.

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.

A collaboration of aquaporins handles water transport in relation to the estrous cycle in the bitch uterus

Fluid movement through uterine cell membranes is crucial, as it can modulate the tissue imbibition pattern in the different phases of the estrous cycle. To gain insight into the mechanisms underlying steroid-controlled water handling, the presence and distribution of aquaporins (AQPs), integral membrane channel proteins permitting rapid passive water movement, was explored in bitch uterine tissues. Immunohistochemistry and Western immunoblot analysis were used to study the presence of AQP1, AQP2, and AQP5 in the layers of the bitch uterine wall during the different estrous phases. Presence of endothelial nitric oxide-generating enzyme NO synthase (NOS3) was also investigated, as it is known that the vasodilator NOS3 might be involved in the development of uterine edema. The results demonstrated the following: (1) AQP1, AQP2, and AQP5 were present in the uterus of cycling bitches. (2) AQP1 was localized within uterine mesometrial, myometrial, and endometrial blood vessels and in the circular and longitudinal layers of myometrium. AQP1 localization and expression were unaffected by the estrous cycle. (3) The estrogenic milieu was probably at the basis of AQP2 expression in the glandular and luminal epithelium of the endometrium. (4) AQP5 water channels were present in the apical plasma membrane of uterine epithelial cells in coincidence with plasma progesterone increase. (5) NOS3 was localized in the myometrial and epithelial tissues as well as in blood vessels indicating a contribution of this vasoactive peptide to the uterine imbibition processes. Thus, we can hypothesize that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different functional uterine phases.

Mutations in the RSPO1 coding region are not the main cause of canine SRY-negative XX sex reversal in several breeds.

This report details a case of SRY-negative XX sex reversal in a mixed breed dog and surveys affected dogs of several breeds for mutations in RSPO1 coding regions. Genomic DNA from the mixed breed case was evaluated for mutations in candidate genes. Sequencing identified a homozygous G to A transition in RSPO1 exon 4 that changes a highly conserved amino acid codon in the thrombospondin domain. The possibility that this was a single nucleotide polymorphism (SNP) could not be excluded by genotyping family members. Therefore, the coding region of RSPO1 was sequenced in a survey of affected dogs, which identified a T to C transition (exon 3) in some, the above G to A transition (exon 4) in others, and no change in the remaining affected dogs. Genotypes at these base pair positions were not uniquely associated with the affected phenotype in any breed, indicating the identified transitions are most likely SNPs, not causative mutations for this canine disorder. However, the possibility that polymorphisms play a modifier role, such as changing threshold or severity of phenotypic expression in a mixed breed dog, cannot be excluded. This study emphasizes the importance of canine pedigree, breed, and population studies in evaluating candidate mutations.

Vaginal bacterial flora and cytology in proestrous bitches: Role on fertility

The study of canine vaginal cytology underwent limited evolution over the years. Presence and significance of inflammatory cells in vaginal smears are little considered aspects in the bitch. Moreover, occurrence of vaginal bacteria in breeding bitches during follicular phase of the reproductive cycle, in absence of clinical signs of infection, involves the difficult question of antibiotics administration. The aim of this study was to relate findings in vaginal cytology (presence of neutrophils, lymphocytes, eosinophils, erytrocytes and bacteria) and microbial environment during proestrus with fertility outcomes (development of pregnancy, uterine infection, resorption, abortion and neonatal mortality). Bacteria sensitivity to antibiotics normally used in small animal practice was also evaluated. Bacteria isolated from vagina, in order of frequency, were Enterococcus faecalis, Streptococcus β-haemolyticus, Pasteurella multocida, E. coli, Klebsiella pneumoniae, Proteus mirabilis, E. coli haemolyticus, Arcanobacterium pyogenes, Streptococcus spp., Staphylococcus spp. and Acinetobacter spp. No mycoplasmas were observed. The present study showed that proestrous cytological aspects do not affect fertility. Eosinophils were never detected, while erythrocytes were always detected. During diestrus, E. coli was found in all pregnant bitches that developed clinical symptoms of uterine disorders (n = 3), resulting in uterine infection, resorption or abortion, but without statistical significance. Vaginal presence of Streptococcus spp. in proestrus was instead negatively associated with development of uterine infections (P = 0.005). Therefore, Streptococcus spp. could have a protective competitive role against more dangerous pathogens affecting fertility of the bitch. Among the 12 antibiotics tested, Gram-negative bacteria showed a significant sensitivity towards the amoxicillin and clavulanic acid association (P = 0.038). However, antibiotic treatment before mating, on the basis of positive culture, yet in the absence of clinical signs, seems to be unnecessary besides harmful leading to imbalance in vaginal commensal flora with adverse effects on fertility. In conclusion, vaginal bacteria, neutrophils, lymphocytes and erytrocytes should be considered as physiological aspect in the bitch during proestrus that does not require antibiotic therapy when asymptomatic.

Breed-specific fetal biometry and factors affecting the prediction of whelping date in the German shepherd dog

To date many studies have been published about predicting parturition by ultrasonographic fetal measurements in the bitch. Given that accuracy in such prediction is a key point for clinicians and breeders, formulas to calculate the whelping date were mainly obtained from small and medium sized dogs, which means poor accuracy when applied to large or giant breeds. Based on the evidence that ethnicity significantly affects fetal biometry in humans, this study aimed at developing a breed-specific linear regression model for estimating parturition date in the German shepherd dog. For this purpose, serial ultrasonographic measurements of the inner chorionic cavity diameter (ICC) and the fetal biparietal diameter (BP) were collected in 40 pregnant German shepherd bitches. The quality of the regression models for estimating parturition date was further verified in 22 other pregnant German shepherd bitches. Accuracy related to the prediction of parturition date was higher than previously reported: 94.5% and 91.7% within ±2 days interval based on ICC and BP measurements, respectively. Additional investigation was performed on the effects of maternal weight, age and litter size in relation to fetal biometry and to accuracy of parturition estimation. Moreover, the study included a comparison between hormonal and fetal ultrasound (ICC and BP) measurements connected to the estimation of whelping date. We suggest that specific equations from a single breed are likely to offer excellent accuracy, comparable to that of periovulatory progesteronemia, in parturition prediction and to avoid morphological variables present in dogs of different breeds even with the same size/weight.

An insight into testis and gubernaculum dynamics of INSL3–RXFP2 signalling during testicular descent in the dog

Insulin-like 3 (INSL3) plays a prominent role in male development and is supposed to induce the growth of the gubernaculum testis (g.t.), thus being directly involved in testicular descent in humans and rodents. This happens through activation of the RXFP2 receptor (GREAT or LGR8). The INSL3-RXFP2 complex is reputed to play an additional paracrine role in the testis, possibly acting as part of an autocrine feedback loop. The present work provides evidence of the immunolocalisation of INSL3 in the Leydig cells of canine fetuses and of the expression of RXFP2 receptor in different tissues of the g.t. of the same specimens. RXFP2 was localised at the cell membrane of g.t. muscle and connective cells, as well as in the epithelial cells of the developing excurrent ducts. Notably, RXFP2 immunoreactivity of the g.t. was limited to fetuses at ~35-45 days of gestation, which is also the fetal period when the endocrine compartment of the dog testis is active endocrinologically, as confirmed by the anti-P450c17 and anti-INSL3 immunoreactivities of the fetal Leydig cells, and by anti-Müllerian hormone immunoreactivity of the Sertoli cells. The same immunoreactivities were also evaluated in the testes of cryptorchid dogs of different ages. RXFP2 immunoreactivity was absent from genital tracts of cryptorchid testes and g.t. remnants.

Morpho- and Histometric Evaluations on the Testis and Epididymis in Buffalo Bulls During the Different Reproductive Seasons

Some morphometric parameters of the buffalo gonad and epididymis were compared in the mating (late autumn-winter) versus non-mating (late spring to the beginning of autumn) seasons. Organs from mature bulls of proven fertility were collected at slaughtering in February and September and used for this study. Testicular and epididymal diameters and weights were measured. The diameters of the testicular seminiferous tubules, as well as the diameters of the ductal lumen in sections taken from caput, corpus and cauda, were compared on haematoxylin and eosin-stained sections. The height of epididymal principal cells was also measured, from the basement to the apical membrane. The data obtained in the two seasons were analyzed by ANOVA, co-variating the histometric data for testicular and epididymal weight, respectively. Season-related differences were observed. Testicular and epididymal weights were higher during the mating period, as well as the organ lengths. Spermatogenesis was conserved in the resting period, but the testis showed smaller tubular diameters. In the epididymal caput and corpus the tubular diameters and epithelial cell height were significantly diminished in summer. On the contrary, tubular diameter and epithelial cell height of cauda epididymis increased during the summer, due to accumulation of non-ejaculated spermatozoa. Seasonal variations of genitalia recorded in buffalo bulls could represent an interesting aspect affecting fertility, which might influence the quality of semen and, in turn, the chance of cryopreservation. Morphometric studies of reproductive organs could contribute to better develop the reproductive potential in this species.

Periovulatory time in the bitch: What's new to know?: Comparison between ovarian histology and clinical features

The ability to recognize specific events happening in the ovaries during periovulatory time allows optimal management of canine reproduction. The objective of this study was to assess the efficacy of vaginal cytology and blood progesterone (P4) assay to identify accurately the changes occurring at the ovarian structures, mainly during the fertile period. Tertiary follicles, corpora hemorrhagica (CHs) and corpora lutea (CLs) from forty healthy bitches undergoing ovariohysterectomy were evaluated by histo-morphometry based on their aspect, number and size. The tertiary follicles distribution (small, medium and large) was statistically different (P<0.002) among all the stages of the reproductive cycle, except for small follicles (<2mm), which were always observed from proestrus to anestrus. Very large follicles (>4mm) were predominant (P=0.008) around ovulation when P4 mean level was 6.1±1.7ng/mL. The early postovulatory estrous period was characterized by CHs (P<0.002) and P4 level of 16.7±5.9ng/mL. The end of the fertile period - start of diestrus - coincided with the development of CLs (P=0.001) associated with a P4 mean level of 73.9±9.9ng/mL. The small (P<0.001) and medium (P<0.05) follicle diameters were positively correlated with the bitch size. The number of follicles larger than 4mm was significantly lower in bitches younger than 4 years (P<0.02). This study provides insight into some critical steps in the canine reproductive processes in the periovulatory phase and the end of the fertile period, essential to plan breeding programs.

Identification of C-kit-positive interstitial cells in the dog lower urinary tract and relationship with smooth muscle and nerves. Hypotheses for a likely pacemaker role.

The aim of this work was to give an evidence of the likely presence of interstitial cells in the canine lower urinary tract and to study their possible interactions with the musculature and the intramural innervation. Cryosections of normal canine bladder and urethra were immunofluorescently labelled with c-kit, a transmembrane, tyrosine kinase growth factor receptor, known to be expressed on the interstitial cells of Cajal (ICCs) of the gut. The relationship with antiactin positive smooth muscle cells and PGP9.5-positive intramural innervation was also investigated by confocal microscopy. Anti-c-kit labelling demonstrated a network of elongated and branched c-kit positive cells, which were located in interstitial spaces, oriented in parallel to the smooth muscle bundles that form the bladder muscular layer, irrespective of dog sex. Cells with a similar localization were also PAS- and NADPH-diaphorase-positive. A contact between c-kit immunofluorescent cells and intramural innervation was demonstrated, too. The roles of interstitial cells might include regulation of smooth muscle activity of the bladder detrusor, integrating neuronal signals during urine storage and voiding.