A. Pecile

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.

Effectiveness of electroacupuncture analgesia compared with opioid administration in a dog model: a pilot study

BACKGROUND Although opioid analgesics are the usual drugs to treat post-surgical pain, acupuncture has also been demonstrated to relieve various pain syndromes. The present pilot study aims to investigate the efficacy of electroacupuncture compared with a conventional opioid compound, butorphanol, for postoperative pain treatment in dogs undergoing elective ovariohysterectomy. METHODS Twelve dogs were randomly allocated into two groups. Dogs received either electroacupuncture stimulation (16 and 43 Hz) at Shen Shu, Chang Shu, He Gu, Tai Yuan, Zu San Li, Yang Ling Quan, and Bai Hui acupoints, while control dogs were treated with butorphanol. Cardiovascular and respiratory parameters were recorded for both groups during operation. Plasma β-endorphin concentrations were evaluated before surgery (baseline) and up to 24 h later. For each dog, pain was measured according to a dedicated subjective pain scoring system. RESULTS Plasma β-endorphin levels in dogs receiving electroacupuncture increased significantly against baseline values after 1 and 3 h after surgery. Moreover, the end-tidal isoflurane concentration needed for second ovary traction was significantly lower in acupuncture-treated dogs than control animals. All animals having electroacupuncture experienced prolonged analgesia, over 24 h at least, while four out of six dogs treated with butorphanol needed post-surgical ketorolac and tramadol supplementation to their pain relief. CONCLUSIONS The results obtained from the present investigation showed some evidence for electroacupuncture as an alternative technique to provide postoperative analgesia in dogs.

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.

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.

Prognostic potential of amniotic fluid analysis at birth on canine neonatal outcomes.

Glucose, lactate and cortisol concentrations in amniotic fluid were measured at birth in 95 pups and related to neonatal viability based on Apgar scoring and to neonatal mortality. Neither amniotic parameters nor neonatal mortality were associated with the Apgar score. Stillborn pups showed high lactate (P < 0.001) and cortisol (P < 0.05) but low glucose amniotic concentrations (P < 0.001). No amniotic fluid differences were observed between normal and malformed pups. Amniotic glucose (P < 0.001), lactate (P < 0.05) and cortisol (P < 0.05) concentrations were higher in pups delivered by vaginal parturition than by Caesarean section. Birth weight was higher in live pups than in pups dying within 48 h (P < 0.05). Although these are preliminary results, the analysis of amniotic fluid collected at birth could be a valuable predictor of neonatal outcomes in dogs.

The role of birth weight on litter size and mortality within 24h of life in purebred dogs: What aspects are involved?

In humans, scientific evidence emphasizes the role of birth weight on neonatal welfare, morbidity and mortality. In canine species, defining normal ranges of birth weight is a harder issue due to a great morphological variability in size, body weight and breed. The aim of this study was to correlate birth weight with litter size and mortality within 24h of life in 789 pups from 140 litters of purebred dogs and to investigate the aspects that might affect these factors. Birth weight was influenced by maternal size, weight and age (P<0.001). The lightest pups were from toy sized or weighing up to 10 kg bitches. Conversely, bitches aged 2-8 years whelped heavier pups than younger and older mothers. Birth weight was also related both to litter size, with heavier pups in smaller rather than in larger litters from medium sized bitches, and breed (P<0.05). Unexpectedly, birth weight did not differ between live born and stillborn pups. However, birth weight was lower in pups dying within 24h of life (P<0.05). High mortality of pups was related both to short pregnancies (P<0.05), also showing lighter litters (P<0.001), and to dystocic parturitions (P<0.001). Litter size was associated with parity, type and number of mating, and length of pregnancy (P<0.001). Low birth weight appears to predispose to early neonatal mortality suggesting a predominant role of the breed rather than size and weight in determining birth weight in pups.

Clinical pharmacokinetics of tramadol and main metabolites in horses undergoing orchiectomy

Background: Tramadol is a synthetic codeine analogue used as an analgesic in human and veterinary medicine. It is not approved for use in horses, but could represent a valid tool for pain treatment in this species. Objectives: The serum pharmacokinetic profile and urinary excretion of tramadol and its metabolites (O-desmethyltramadol [M1], N-desmethyltramadol [M2] and N,O-desmethyltramadol [M5]) was investigated in a multidrug anaesthetic and analgesic approach for orchiectomy in horses. The evaluation of the degree of cardiovascular stability, the intraoperative effect and postoperative analgesia obtained by the visual analogue scale are also reported. Animal and methods: Tramadol (4 mg/kg BW) was administered intravenously to eight male yearlings as a bolus over 60 seconds, 5 min after intubation and 15 min prior to surgery. Drug quantification was performed in serum and urine for tramadol, M1, M2 and M5 by high-performance liquid chromatography with fluorimetric detection. Results: Mean tramadol concentration was 14.87 ± 11.14 μg/mL at 0.08 h, and 0.05 ± 0.06 μg/mL at 10 h. Serum concentrations of M1 and M2 metabolites were quite limited. For M1 and M2, median maximum concentration (Cmax) and time to achieve maximum concentration (Tmax) were 0.05 μg/mL and 0.75 h, and 0.08 μg/mL and 2 h, respectively; M5 was never detected. In urine, tramadol was the most recovered compound, followed by M1, M2 and M5. Conclusions and clinical relevance: Showing no adverse events and based on the kinetic behaviour, pre-operative tramadol IV at a dose of 4 mg/kg BW might be useful and safe as analgesic in horses undergoing surgery.

Genome-wide analysis of DNA methylation in hypothalamus and ovary of Capra hircus

BackgroundDNA methylation is a frequently studied epigenetic modification due to its role in regulating gene expression and hence in biological processes and in determining phenotypic plasticity in organisms. Rudimentary DNA methylation patterns for some livestock species are publically available: among these, goat methylome deserves to be further explored.ResultsGenome-wide DNA methylation maps of the hypothalamus and ovary from Saanen goats were generated using Methyl-CpG binding domain protein sequencing (MBD-seq). Analysis of DNA methylation patterns indicate that the majority of methylation peaks found within genes are located gene body regions, for both organs. Analysis of the distribution of methylated sites per chromosome showed that chromosome X had the lowest number of methylation peaks. The X chromosome has one of the highest percentages of methylated CpG islands in both organs, and approximately 50% of the CpG islands in the goat epigenome are methylated in hypothalamus and ovary. Organ-specific Differentially Methylated Genes (DMGs) were correlated with the expression levels.ConclusionsThe comparison between transcriptome and methylome in hypothalamus and ovary showed that a higher level of methylation is not accompanied by a higher gene suppression. The genome-wide DNA methylation map for two goat organs produced here is a valuable starting point for studying the involvement of epigenetic modifications in regulating goat reproduction performance.

A National Census of Birth Weight in Purebred Dogs in Italy

Simple Summary Birth weight is a key factor for neonatal mortality and morbidity in most mammalian species. The great morphological variability in size, body weight and breed, as well as in skeletal and cranial conformation makes it challenging to define birth weight standards in dogs. A total of 3293 purebred pups were surveyed to study which maternal aspects can determine birth weight considering head and body shape, size, body weight and breed in bitches, as well as litter size and sex in pups. In our sample, multivariate analysis outcomes suggested that birth weight and litter size were directly proportional to maternal size. The maternal body shape influenced both birth weight and litter size, whereas the maternal head shape had impact only on birth weight. Sex differences in birth weight were found. Birth weight and litter size also varied among breeds. The results of the present study could have practical implications allowing one to identify pups in need of admission to intensive nursing care, as occurs in humans. A deeper knowledge of the factors that significantly influence birth weight could positively affect the canine breeding management helping to prevent and reduce neonatal mortality. Abstract Despite increasing professionalism in dog breeding, the physiological range of birth weight in this species remains unclear. Low birth weight can predispose to neonatal mortality and growth deficiencies in humans. To date, the influence of the morphotype on birth weight has never been studied in dogs. For this purpose, an Italian census of birth weight was collected from 3293 purebred pups based on maternal morphotype, size, body weight and breed, as well as on litter size and sex of pups. Multivariate analysis outcomes showed that birth weight (p < 0.001) and litter size (p < 0.05) increased with maternal size and body weight. Birth weight was also influenced by the maternal head and body shape, with brachycephalic and brachymorph dogs showing the heaviest and the lightest pups, respectively (p < 0.001). Birth weight decreased with litter size (p < 0.001), and male pups were heavier than females (p < 0.001). These results suggest that canine morphotype, not only maternal size and body weight, can affect birth weight and litter size with possible practical implications in neonatal assistance.