Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Endocannabinoids as endometrial inflammatory markers in lactating Holstein cows

The objective of this study was to consider endocannabinoid system as inflammatory markers in bovine endometrium to better understand the role of this system in regulating many of the functions that are related to inflammatory condition. At day 26 post‐partum, fourteen cows were divided into two groups depending on the inflammatory condition: 1‐ subclinical endometritis (n = 7, with purulent or mucopurulent uterine discharge detectable in the vagina) and 2‐ healthy (n = 7, No (muco)) purulent discharge. Blood samples were collected at 26 and 30 days relative to calving to determine plasma tumour necrosis factor (TNF) and lipopolysaccharide‐binding protein (LBP) concentrations; moreover, uterine biopsy was carried out on day 26 post‐partum to measure mRNA abundance of TNF, interleukin‐1B (IL1B), interleukin‐6 (IL‐6), C‐X‐C motif chemokine ligand 8 (CXCL8), endocannabinoid receptor (CNR2), N‐acyl phosphatidylethanolamine phospholipase D (NAPEPLD), fatty acid amide hydrolase (FAAH), N‐acylethanolamine acid amidase (NAAA) and monoglyceride lipase (MGLL) by real‐time PCR. Results showed mean plasma concentrations of TNF and LBP were lower in healthy cows compared to subclinical endometritis cows (p < .05). Relative mRNA expression for NAAA and FAAH was decreased (p < .05), and relative mRNA expression for CNR2 and NAPEPLD increased in cows with subclinical endometritis compared to healthy cows. In conclusion, relative mRNA expression of TNF, IL1B and CXCL8 and plasma concentration of LBP increased during inflammatory condition along with decreased endocannabinoids hydrolyzing enzyme (NAAA and FAAH), increased enzymes that synthesize endocannabinoids (NAPEPLD) and relative gene expression of the endocannabinoid receptor; together, these contribute to increased endocannabinoids levels during inflammation. Overall, we provide evidence that endocannabinoid system is altered in endometrium tissue during inflammation through increased mRNA expression of CNR2 and synthesis enzyme and decreased mRNA expression of hydrolyzing enzymes interfere with pro‐cytokine production and signalling, which may interfere with the onset and progression of inflammation.     

Accessory nictitating membrane in a Lipizzaner colt

A 2‐year‐old Lipizzaner colt presented for removal of a mass over the left eye. The colt had no blepharospasm or significant ocular discharge, but the cornea underlying the mass was mildly oedematous. The mass protruded from the conjunctiva at the dorsotemporal aspect of the globe and was covered in normal conjunctiva. It had a gross appearance similar to the cartilaginous flap of a nictitating membrane, but in an aberrant location. It was moveable to cover the dorsal aspect of the cornea when the globe was retropulsed. Excision of the mass under general anaesthesia was elected. Histopathology supported a diagnosis of an accessory nictitating membrane, consisting of a cartilaginous band surrounded by glandular epithelium. The colt recovered from surgery without complication and no problems were reported by the owner at 8.5 months post‐operatively. To the author's knowledge, there have not been previous reports of accessory or ectopic nictitating membranes in equine or other species.     

Genomics in equine veterinary medicine

Veterinary medicine is marching forward with genomics grasped firmly in one fist. Genomics has been part of our veterinary tool bag for some time in various guises, but the breadth and depth of its ramifications can be daunting. This article is designed to provide toe‐holds for veterinarians to enjoy a better understanding of genomics – a truly fascinating area of science.     

Saquayamycin B1 Suppresses Proliferation,Invasion, and Migration by Inhibiting PI3K/AKT Signaling Pathway in Human Colorectal Cancer Cells

Moromycin B (Mor B), saquayamycin B₁ (Saq B₁), saquayamycin B (Saq B), and landomycin N (Lan N), four angucyclines produced by the marine-derived actinomycete Streptomyces sp., are a class of polyketone compounds containing benzanthracene. Here, the structure–activity relationship of these four compounds was analyzed in human colorectal cancer (CRC) cells. Saq B₁, which showed the strongest cytotoxicity with an IC₅₀ of 0.18–0.84 µM for CRC cells in MTT assays, was employed to test underlying mechanisms of action in SW480 and SW620 cells (two invasive CRC cell lines). Our results showed that Saq B₁ inhibited CRC cell proliferation in a dose- and time-dependent manner. Notably, lower cytotoxicity was measured in normal human hepatocyte cells (QSG-7701). Furthermore, we observed proapoptosis, antimigration, and anti-invasion activities of Saq B₁ in CRC cells. At the same time, the protein and mRNA expression of important markers related to the epithelial–mesenchymal transition (EMT) and apoptosis changed, including N-cadherin, E-cadherin, and Bcl-2, in Saq B₁-treated CRC cells. Surprisingly, the PI3K/AKT signaling pathway was shown to be involved in Saq B₁-induced apoptosis, and in inhibiting invasion and migration. Computer docking models also suggested that Saq B₁ might bind to PI3Kα. Collectively, these results indicate that Saq B₁ effectively inhibited growth and decreased the motor ability of CRC cells by regulating the PI3K/AKT signaling pathway, which provides more possibilities for the development of drugs in the treatment of CRC.     

Stability of Saxitoxin in 50% Methanol Fecal Extracts and Raw Feces from Bowhead Whales (Balaena mysticetus)

In recent decades, harmful algal blooms (HABs) producing paralytic shellfish toxins (including saxitoxin, STX) have become increasingly frequent in the marine waters of Alaska, USA, subjecting Pacific Arctic and subarctic communities and wildlife to increased toxin exposure risks. Research on the risks of HAB toxin exposures to marine mammal health commonly relies on the sampling of marine mammal gastrointestinal (GI) contents to quantify HAB toxins, yet no studies have been published testing the stability of STX in marine mammal GI matrices. An understanding of STX stability in test matrices under storage and handling conditions is imperative to the integrity of toxin quantifications and conclusions drawn thereby. Here, STX stability is characterized in field-collected bowhead whale feces (stored raw in several treatments) and in fecal extracts (50% methanol, MeOH) over multiple time points. Toxin stability, as the percent of initial concentration (T0), was reported for each storage treatment and time point. STX was stable (mean 99% T0) in 50% MeOH extracts over the 8-week study period, and there was no significant difference in STX concentrations quantified in split fecal samples extracted in 80% ethanol (EtOH) and 50% MeOH. STX was also relatively stable in raw fecal material stored in the freezer (mean 94% T0) and the refrigerator (mean 93% T0) up to 8 weeks. STX degraded over time in the room-temperature dark, room-temperature light, and warm treatments to means of 48 ± 1.9, 38 ± 2.8, and 20 ± 0.7% T0, respectively, after 8 weeks (mean ± standard error; SE). Additional opportunistically analyzed samples frozen for ≤4.5 years also showed STX to be relatively stable (mean 97% T0). Mean percent of T0 was measured slightly above 100% in some extracts following some treatments, and (most notably) at some long-term frozen time points, likely due to evaporation from samples causing STX to concentrate, or variability between ELISA plates. Overall, these results suggest that long-term frozen storage of raw fecal samples and the analysis of extracts within 8 weeks of extraction in 50% MeOH is sufficient for obtaining accurate STX quantifications in marine mammal fecal material without concerns about significant degradation.