Proliferation and apoptosis dynamics of the normal canine mammary gland during the oestrous cycle evaluated by Ki-67 and Caspase-3 indexes.

Understanding the normal physiology of the canine mammary gland (CMG) is crucial, as it provides a foundational reference for understanding canine mammary neoplasms. The relation between the Proliferation Index (PI) indicated by Ki-67 expression, along with the Apoptotic Index (AI) determined through Caspase-3 expression during the oestrous cycle, is inadequately documented in existing literature. This study seeks to offer insights into the interplay between PI and AI in the CMG across oestrous cycle phases. An extensive investigation was conducted on a diverse case series of bitches (n = 18). Oestrous cycle stages were determined through vaginal cytology, histological examination of the reproductive tract and serum progesterone and oestradiol concentrations. The entire mammary chain was histologically examined, and proliferation and apoptosis were assessed via double immunohistochemistry employing anti-Ki-67 and Caspase-3 antibodies. PI and AI were evaluated through a systematic random sampling approach, counting a minimum of 200 cells for each cell type. There was a significantly higher PI during early dioestrus in all mammary gland components, with a greater proportion of positive cells observed in epithelial cells compared to stromal cells. The highest PI was detected in epithelial cells within the end buds. Significant differences were found in Ki-67 labelling across the cranial mammary glands. A positive and strong correlation was noted between progesterone concentration and PI in epithelial cells. The AI remained consistently low throughout the oestrous cycle, with few differences observed across histological components. Caspase-3 labelling displayed the highest positivity in caudal mammary pairs. A negative and moderate correlation was identified between progesterone concentration and AI in interlobular mesenchymal cells. This study highlights the influence of endocrine regulation on cell proliferation indices in mammary tissue, emphasizing the need to consider these hormonal variations in toxicopathological studies involving canine mammary gland.

Macro and microscopic characteristics of the placenta and its relationship with the weight and the Apgar score of canine neonates.

The placenta is the main organ of pregnancy and is directly related to the proper development of the fetus. The correlation among some placental measurements and their respective neonates is widely studied in the human species. However, the studies regarding bitches are still limited. Therefore, the aim of this work was to evaluate if there is a relationship between placental weight and volume and the weight of neonates at birth in the canine species, as well as its influence on their viability. In this work, 7 bitches, 18 neonates and their placentas were evaluated. The weight of the placentas was measured using an analytical balance and the volume was calculated by measuring the volume of water displaced after placing it in a container of water. The neonates were weighed and classified according to the Apgar score after birth. Samples from each placenta were fixed in formalin and embedded in paraffin, then placed on slides and stained in hematoxylin and eosin. From these samples, the microvascular density (MVD) was calculated, as well as the presence or absence of necrosis, calcification and haemorrhage, classified in scores from 0 to 2. Data were analyzed using Kendall's test. The mean weight of the placentas was 29.11 ± 11.06 g and the volume was 21.33 ± 10.65 cm³. The mean weight of the neonates was 282.94 ± 123.28 g and the Apgar score was 8.83 ± 2.06. The mean MVD of the placentas was 0.04 ± 0.01. A positive correlation was observed between birth weight and placental weight and volume. Placental weight also positively correlated with placental volume. Also, no significant correlation was found between MVD and alterations with placental weight and volume and with the weight and Apgar score of neonates. Among the microscopic changes, only necrosis showed a moderate correlation with placental weight and volume. It can be concluded that the placenta has an influence on the weight of neonates, which is essential for its development in intra and extrauterine life. However, more studies are required in the described species, to better elucidate these questions.

CA 15-3, CRP, and LDH correlates with prognostic parameters in canine mammary neoplasms.

The identification of putative prognostic factors in canine mammary neoplasms (CMNs) has been focused on tissue-specific biomarkers, but the serum biomarkers, including cancer antigen 15-3 (CA 15-3), c-reactive protein (CRP), and lactate dehydrogenase (LDH) have been demonstrated to display clinical application in cases of CMNs. The aim of the study was to evaluate the levels of these serum biomarkers and their association with well-established prognostic factors in CMNs. Samples from 15 female canines with CMNs and 15 clinically healthy ones were collected. The results were evaluated using the Tukey's, Pearson, or Spearman tests. The cut-off point, sensitivity, specificity, and area under curve (AUC) were evaluated using the receiver operating characteristic (ROC) curve analysis in a logistic regression model (P<0.05). The levels of CA 15-3, CRP and LDH were significantly higher in the serum of female dogs with CMNs compared to the healthy ones. Moreover, these factors were positively correlated with ulceration, tumor size, histopathological grade, metastatic lymph node, and clinical staging. Female dogs with CMNs were found to exhibit highest serum levels of CA 15-3, CRP, and LDH. Therefore, they can be applied to improve the efficacy of the diagnosis and prognostic evaluation in casas of CMNs.

Proteomic characterization of canine seminal plasma.

The present study was conducted to identify the major proteome of the sperm-rich fraction and prostatic fraction of canine seminal plasma. Three semen samples from four healthy dogs were obtained by digital manipulation. The pre-sperm fraction, sperm-rich fraction and prostatic fraction were separated from each ejaculate. Immediately after sperm analysis, a protease inhibitor was added to the sperm-rich fraction and prostatic fraction, and the fractions were separately centrifuged and frozen at -80 °C. The samples were thawed, re-centrifuged, and the total protein concentration was determined. Samples were subjected to 1D SDS-PAGE and Coomassie-blue stained gels, were analyzed by Quantity One 1D Analysis Software. Bands detected in the gels were excised and proteins subjected to digestion with trypsin. Proteins were identified by nano-HPLC-MS and tools of bioinformatics. Tandem mass spectrometry allowed the detection of 268 proteins in the gels of sperm-rich fraction and prostatic fraction of canine ejaculate. A total of 251 proteins were common to the sperm-rich and prostatic fractions, while 17 proteins were present in the sperm-rich fraction and absent in the prostatic fraction. The intensity of the bands detected in range 1 and 2 represented 46.5% of all of the band intensities detected in the 1D gels for proteins of the sperm-rich fraction and 53.0% of all bands in the prostatic fraction. Arginine esterase and lactotransferrin precursor were the protein with the highest intensity observed in the both fractions. Among the proteins present only in the sperm-rich fraction, the proteins UPF0764 protein C16orf89 homolog and epididymal-specific lipocalin-9 were the most abundant. In conclusion, canine sperm-rich fraction and prostatic fraction express a very diverse set of proteins, with unique biochemical properties and functions. Moreover, although most proteins are common to both sperm-rich fraction and prostatic fraction, there are some exclusive proteins in sperm-rich fraction.