A Case of Pseudopregnancy in a Bitch

A case of pseudopregnacy was presented to Veterinary Teaching Hospital, Michael Okpara University of Agriculture Umudike, Nigeria. Eighteen (18) months old bitch weighing 15kg was presentedclinical signs suspected to be pseudopregnacy, which was observed four weeks after the bitch came on heat (estrus). The clinical signs observed were restlessness, anorexia, aggression, nesting behaviour and licking of mammary gland. On clinical examination it was observed that bitch had enlarged mammary glands and milk secretion. The physiological parameters were found to be within the normal range. Abdominal palpation, ultrasonography and vagina cytology were carried out to confirm pregnancy. Due to absence of foetuses on abdominal palpation and ultrasonography and presence of scanty epithelial cells in vaginal cytology, it was confirmed and concluded to be a case of pseudopregnancy. Causes, treatment and other associated factors with pseudopregnancy were further discussed.

Comparative Study of Serum Profile in Bitches Treated for Transmissible Venereal Tumor by Autohaemotherapy

A total of 18 bitches in the period in the period of six month diagnosed for transmissible venereal tumor in Teaching Veterinary Clinical Complex, Nagpur by per vaginal examination and vaginal cytology. These bitches were divided randomly into three groups, viz., Group I (Inj. Vincristine @ 0.025mg/kg slow IV in DNS and Injection Botropase 1ml IM for 1st week), Group II (Inj. Vincristine @ 0.025mg/kg slow IV in DNS and Tab. Amoxycillin and Potassium Clavinate for first 7 days orally) and Group III (treated with autohaemotherapy@10 ml whole blood IM) for once in week and continue for 5 week. At the time of treatment, liver and kidney function tests were performed. During the course of treatment significant variation was noted in Group I and II in BUN values while creatinine values changes significantly in all treatment groups. In case of SGPT only significant changes observed in Group I. Rest of the changes in serum prolife found to be non significant. Also from recovery point of view we noted that, it was faster in Group I and II compare than Group III. Side effects observed only in Group I and II. From the study we concluded that autohaemotherapy will be an alternate option for treatment of TVT in bitches who shows the excessive side effects of Vincristine Sulphate.

Assessing urinary odours across the oestrous cycle in a mouse model using portable and benchtop gas chromatography-mass spectrometry

For female mammals, communicating the timing of ovulation is essential for reproduction. Olfactory communication via volatile organic compounds (VOCs) can play a key role. We investigated urinary VOCs across the oestrous cycle using laboratory mice. We assessed the oestrous stage through daily vaginal cytology and analysed urinary VOCs using headspace gas chromatography-mass spectrometry (GC-MS), testing a portable GC-MS against a benchtop system. We detected 65 VOCs from 40 samples stored in VOC traps and analysed on a benchtop GC-MS, and 15 VOCs from 90 samples extracted by solid-phase microextraction (SPME) and analysed on a portable GC-MS. Only three compounds were found in common between the two techniques. Urine collected from the fertile stages of the oestrous cycle had increased quantities of a few notable VOCs compared with urine from non-fertile stages. These VOCs may be indicators of fertility. However, we did not find significant differences in chemical composition among oestrous stages. It is possible that changes in VOC abundance were too small to be detected by our analytical methods. Overall, the use of VOC traps combined with benchtop GC-MS was the more successful of the two methods, yet portable GC-MS systems may still have utility for some in situ applications.

Histomorphometry and uterine proteomics during the normal reproductive cycle in bitches

We aimed to evaluate the histomorphometry and proteomic profile of the canine uterus during all stages of the reproductive cycle. Eighteen healthy female dogs had their estrous cycle identified by clinical evaluation, vaginal cytology, and serum progesterone levels, which were allocated to the proestrus (n=5), estrus (n=5), diestrus (n=5), and anestrus (n=3) groups. All were submitted to elective ovariosalpingohysterectomy, and the uteri were collected for histomorphometric measurement (Image J software). For proteomic analysis, fragments of the uterine horns were subjected to protein measurement (Bradford method) and extraction by 2D electrophoresis (PDquest software). The results showed that the diestrus promoted greater values of thickness in the uterine structures (μm): uterine wall (2,223.8±229.8), endometrium (819.7±109.1), and myometrium (1,392.6±294.2). Uterus showed a protein profile with good reproducibility per phase (pI: 3.5–9.0; PM: 24–150 KDa), with 11 spots in all phases. Despite the greatest histomorphometric changes in the diestrus, we observed a greater number of spots in the estrus (253±45), followed by the proestrus (185±21), diestrus (113±39), and anestrus (80±21). This finding showed probable participation of these proteins in the uterine preparation for receiving gametes for fertilization. Our results showed greater uterine thickness in the diestrus, and greater protein secretion in the estrus, contributing to the prospection of identification of proteins responsible for the biological reproduction processes.

Loss of ASPM Disrupts Female Folliculogenesis in Mice

Background: The abnormal spindle-like, microcephaly-associated (ASPM) gene is a causative gene of autosomal recessive primary microcephaly (MCPH) 5 in humans, which is characterized by a reduction in brain volume. It was previously reported that truncated ASPM proteins in transgenic mice caused major defects in the germline, a severe reduction in ovary weight and the number of follicles accompanied by reduced fertility. However; it remains unknown whether a loss of ASPM induces abnormal ovarian function, resulting in female infertility. Methods: In order to assess the ovary function, we examined vaginal smear cytology from the age of 7 weeks to 100 weeks in CAG-mediated Cre-loxP conditional Aspm-/- knockout mice and control female mice. In addition, we evaluated the ovarian size, fibrosis ratio and the number of follicles (primordial, primary, secondary, antral and atretic follicles) in mice from 15 weeks to 100 weeks old by image analyses. Mann-Whitney U-test was used for statistical analysis. Results: The size of the ovary was significantly reduced in Aspm knockout mice at 15-20 weeks, 40-50 weeks and 70-80 weeks old. compared with the control mice. Furthermore, at all stages, we found a severe decrease in the number of developing follicles at 10-15 weeks, 40-50 weeks and 70-80 weeks old, accompanied by disrupted cyclic changes of vaginal cytology. Conclusion: The results showing that folliculogenesis was significantly decreased and associated with abnormal vaginal cytology in Aspm knockout female ovaries suggested that ASPM might play an important role in the folliculogenesis and estrous cyclicity of the postnatal ovary.

A reappraisal of mesenchymal-to-epithelial transition within the endometrium

A controversial topic, mesenchymal-epithelial transition (MET) is thought to be a mechanism involved in regeneration of the uterine epithelial layer following pregnancy and menstruation. Little is known about this process though, requiring further exploration. Previously, MET was thought to only occur as a damage-repair mechanism following parturition and menses-like events in mouse models. However, in the current study we hypothesized that MET would also occur in other endometrial epithelialization events as a mechanism of homeostatic epithelial turnover. To identify mesenchymal-derived cells within the adult uterine epithelium, an Amhr2-Cre; Rosa26-EYFP reporter mouse line was used. Mice were staged by vaginal cytology prior to the isolation of uterine epithelial and stromal cells, which were then stained with EpCAM to identify epithelial cells and analyzed by flow cytometry. EpCAM+YFP+ cells were identified in all stages of the estrous cycle except diestrus, indicating a proportion of epithelial cells were derived from the stroma (i.e., mesenchyme). Up to 80 percent of the uterine epithelia was EpCAM+YFP+ during estrogen-dominant stages (proestrus and estrus) of the cycle, with negligible amounts found during progesterone-dominant stages (metestrus and diestrus), suggesting this population may be responsive to estrogen. Uteri were also evaluated direct fluorescence and immunofluorescence in tissue sections. Immunofluorescence for EpCAM, forkhead box protein A2 (FOXA2), Ki67, estrogen receptor alpha (ESR1), and progesterone receptor (PGR) was performed to assess epithelial characteristics and potential functionality. To further investigate the role of MET in epithelialization and assess the temporal origin of mesenchymal-derived epithelial cells, we evaluated key postnatal (P) developmental time points using Amhr2-Cre; Rosa26-tTA; H2B-GFP reporter mice. Flow cytometry data indicated that MET may initially occur immediately after birth at P 0.5, with results varying from negligible amounts (0.21 percent) to approximately 82 percent. Similar results were found at P 3, but with decreasing variation; the highest EpCAM+GFP+ population representing approximately 50 percent of the epithelium. Between P 3 and early adenogenesis at P 8, this population decreased to average less than 2 percent. By the completion of adenogenesis initiation at P 14, approximately 10 percent of epithelial cells were EpCAM+GFP+, suggesting MET may occur during adenogenesis initiation and is maintained through P 21. Together, these results suggest that MET may be a more ubiquitous mechanism of epithelialization than originally thought and is likely hormone regulated. This research will help elucidate the role of MET in uterine epithelialization with potential for insights into dysregulation of MET in diseases such as endometrial adenocarcinoma and endometriosis.

Comparative Durability of Common Stains Used for Exfoliative Vaginal ‎Cytology

In a study to compare the durability of commonly used stains (Giemsa, Leishman, Wright, Eosin, Nigrosin and Gentian violet) for exfoliative vaginal cytology, vaginal smear was obtained from eleven apparently healthy West African Dwarf (WAD) female Goats and processed according to standard technique. Scores (0-3) were given on four parameters namely background of smears, overall staining pattern, cytoplasmic staining and nuclear staining. Quality index one (QI-I) was calculated from the ratio of score achieved to the maximum score possible (12), immediately post staining while quality index–II (QI-II) was obtained 35 days after. Calculation for durability index (DI) was self-derived and equalled to ratio of QI-II to QI-I. The data were presented as mean ± SEM. Multinomial logistic regression model was generated for the QI-I and QI-II using durability index as reference category. Giemsa, Leishman and Wright stains were more durable than others with their mean DI values significantly (P < 0.05) higher than Gentian violet, Nigrosin and Eosin.The model showed 89.2% overall model accuracy for the multinomial logistic regression model and 81.5% for the multinomial Bayes Naïve regression model. In conclusion, Giemsa, Leishman and Wright stains were more reliable and durable for exfoliative vaginal cytology compared to the other stains.

Safety Evaluation of KP-10 (Metastin 45–54) Following once Daily Intravenous Administration for 14 Days in Dog

Kisspeptin-10 (previously referred as metastin 45–54), an active fragment of the endogenous full-length kisspeptin-145, is a potential therapeutic agent for reproductive disorders such as infertility, amenorrhea, and pubertal delay. A safety evaluation of KP-10 was conducted in dogs at the doses of 30, 100, and 1,000 μg/kg, given once daily intravenously for 14 days with a 14-day recovery period. There were no overt signs of drug-related toxicity observed in clinical signs, body weights, food consumption, clinical pathology, histopathology, urinalysis, electrocardiogram, or respiratory rate. Due to very rapid clearance of the peptide, luteinizing hormone (LH) levels were measured as a surrogate marker to demonstrate KP-10 exposure. The LH response reached a maximum concentration at 5 minutes post-dose and remained relatively unchanged for at least 30 minutes after dosing with no gender effect. LH concentrations on Day 1 were generally greater than on day 14. Vaginal cytology results indicated all dogs were in anestrous throughout the dosing period. There were also no KP-10–related findings observed in recovery animals on Day 29. In conclusion, KP-10 demonstrated favorable safety profile in dog where 1,000 μg/kg dose was considered as a no-observed-adverse-effect level dose when administered IV once daily for 14 days.

Laboratory Findings, Vaginal Cytology and Histopathology in Bitches with Cystic Endometrial Hyperplasia – Pyometra Complex

Cystic Endometrial Hyperplasia -Pyometra complex, a common pathological condition of uterus in bitches, it is attributed hormonal and bacteriological causes. It is studied in 10 bitches of cross and pure bred and aged from 1-13 years. In this study, the average age of animals was 8.33±1 year. Hematological and biochemical lab findings were evaluated in bitches with or without vaginal discharge. Lab findings were leukocytosis, neutrophilia and normocytic, normocromic anemia. Biochemical results shown increased of ALT and ALKP and a moderate of BUN. An increased number of parabasal and intermediate cells were detected in vaginal cytology. Histopathologic diagnosis confirmed thickness of uteri wall, a lot of cystic glands of it and the pus presented in lumen of uterus and its horns. The purposes of this study were that through different techniques to diagnose earlier Cystic Endometrial Hyperplasia – Pyometra complex in bitches, in order to welfare of animals.