Programmed Death-Ligand (PD-L1), Epidermal Growth Factor (EGF), Relaxin, and Matrix Metalloproteinase-3 (MMP3): Potential Biomarkers of Malignancy in Canine Mammary Neoplasia.

Gene expression has been suggested as a putative tool for prognosis and diagnosis in canine mammary neoplasia (CMNs). In the present study, 58 formalin-fixed paraffin-embedded (FFPE) paraffined canine mammary neoplasias from 27 different bitches were included. Thirty-seven tumours were classified as benign, whereas thirty-one were classified as different types of canine carcinoma. In addition, mammary samples from three healthy bitches were also included. The gene expression for vascular endothelial growth factor-α (VEGFα), CD20, progesterone receptor (PGR), hyaluronidase-1 (HYAL-1), programmed death-ligand 1 (PD-L1), epidermal growth factor (EGF), relaxin (RLN2), and matrix metalloproteinase-3 (MMP3) was assessed through RT-qPCR. All the assessed genes yielded a higher expression in neoplastic mammary tissue than in healthy tissue. All the evaluated genes were overexpressed in neoplastic mammary tissue, suggesting a role in the process of tumorigenesis. Moreover, PD-L1, EGF, relaxin, and MMP3 were significantly overexpressed in malignant CMNs compared to benign CMNs, suggesting they may be useful as malignancy biomarkers.

Importance of Water Transport in Mammalian Female Reproductive Tract.

Aquaporins (AQPs) are involved in water homeostasis in tissues and are ubiquitous in the reproductive tract. AQPs are classified into classical aquaporins (AQP0, 1, 2, 4, 5, 6 and 8), aquaglycerolporins (AQP3, 7, 9, and 10) and superaquaporins (AQP11 and 12). Nine AQPs were described in the mammalian female reproductive tract. Some of their functions are influenced by sexual steroid hormones. The continuous physiological changes that occur throughout the sexual cycle, pregnancy and parturition, modify the expression of AQPs, thus creating at every moment the required water homeostasis. AQPs in the ovary regulate follicular development and ovulation. In the vagina and the cervix, AQPs are involved mainly in lubrication. In the uterus, AQPs are mostly mediated by estradiol and progesterone to prepare the endometrium for possible embryo implantation and fetal development. In the placenta, AQPs are responsible for the fluid support to the fetus to maintain fetal homeostasis that ensures correct fetal development as pregnancy goes on. This review is focused on understanding the role of AQPs in the mammalian female reproductive tract during the sexual cycle of pregnancy and parturition.

Evaluation of the Probiotic In Vitro Potential of Lactic Acid-Producing Bacteria from Canine Vagina: Possible Role in Vaginal Health.

Lactic acid-producing bacteria (LAB) are being widely studied due to their probiotic potential. The aim of the present study was to determine and identify the presence of LAB from canine vaginal samples, as well as to evaluate their probiotic in vitro potential. Ninety-four bitches were included in the study. Vaginal samples were obtained by means of a sterile swab and streaked on Man Rogosa Sharpe agar plates. A total of 100 LAB strains were obtained and submitted to Gram stains and basic biochemical tests, which included catalase, oxidase and haemolysis tests. Thirteen strains belonging to the genera Lactobacillus (n = 10), Lactococcus (n = 2) and Pediococcus (n = 1) were selected as potential probiotics and further subjected to evaluation of resistance to gastrointestinal conditions (pH, lysozyme, bile salts and hydrogen peroxide) and safety and efficacy in vitro (resistance to antibiotics and antimicrobial capacity). Only three strains, one Lactobacillus lactis and two Lactobacillus plantarum, accomplished the requirements for being considered as potential in vitro probiotics.

Inflammatory Markers in Uterine Lavage Fluids of Pregnant, Non-Pregnant, and Intrauterine Device Implanted Mares on Days 10 and 15 Post Ovulation.

Intrauterine devices (IUDs) are used in mares to suppress oestrous behaviour, but the underlying mechanism is yet to be elucidated. The presence of an embryo or an IUD prevents cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin (PG) release and luteolysis. However, inflammation may also be involved. Endometrial inflammatory markers in uterine lavage fluid were measured on Day 10 (EXP 1, n = 25) and Day 15 (EXP 2, n = 27) after ovulation in inseminated mares, non-pregnant or pregnant, and in mares in which a small plastic sphere had been inserted into the uterus 4 (EXP 1) or 3 days (EXP 2) after ovulation. Uterine lavage fluid samples were analysed for nitric oxide (NO), prostaglandin E2 (PGE2) (only EXP 1), prostaglandin F2α (PGF2α), inhibin A and cytokines, and blood samples for progesterone and oestradiol. On Day 10, the concentration of PGF2α was lower (p < 0.05) in the IUD group than in pregnant mares. The concentration of the modulatory cytokine IL-10 was significantly higher in the IUD group in comparison to non-pregnant mares, and inhibin A was significantly higher in IUD mares than in the pregnant counterparts on Day 15. The results suggest that the presence of IUD causes endometrial inflammation which is at a resolution stage on Day 15.

Microbial Prevalence and Antimicrobial Sensitivity in Equine Endometritis in Field Conditions.

Endometritis is one of the main causes of infertility in mares. In the present study, 363 mares with a history of repetitive infertility, and positive endometrial cytology and/or vaginal discharge were included. An endometrial swab for microbiological purposes plus sensitivity test was obtained from each mare. A positive culture was obtained in 89% of mares. The main isolated genera were Staphylococcus (25.1%), Streptococcus (18.2%), Escherichia (17.3%) and Pseudomonas (12.1%). With regard to species, the most isolated microorganism was Escherichia coli (17.3%), Staphylococcus spp. (15.6%) and Streptococcus spp. (13.5%). Sensitivity tests showed that the most efficient antimicrobial was amikacin (57.3% of cultures), followed by cefoxitin (48.6%) and gentamicin (48.3%). When sensitivity test was analyzed in terms of Gram+ and Gram- bacteria, Gram+ were highly resistant to cephaloridine (77.3% of cultures), apramycin (70.8%) and penicillin (62.3%), whereas Gram- were highly resistant to penicillin (85.8%), followed by cephaloridine (78.9%). In conclusion, the present study shows the most prevalent microorganisms isolated from equine endometritis, which were found to be resistant to ß-lactam antimicrobials. Likewise, these results highlight the significance of performing microbiological analyses as well as sensitivity tests prior to applying an antimicrobial therapy.

Endometrial Status in Queens Evaluated by Histopathology Findings and Two Cytological Techniques: Low-Volume Uterine Lavage and Uterine Swabbing.

Endometritis is associated with fertility problems in many species, with endometrial biopsy being the main diagnostic tool. In feline queens, the reduced size of the uterus may make it difficult to obtain representative diagnostic samples. Endometrial cytology may represent a valuable diagnostic tool for evaluating the health status of the endometrium in queens. Fifty domestic shorthair queens were included and divided into two cytological diagnostic technique groups, the uterine lavage (UL; n = 28) and uterine swabbing (US; n = 22) groups. Cytological results were compared with histopathological and bacteriological information. Changes in the histopathological patterns were also evaluated and compared with progesterone levels to confirm previous published data. Furthermore, the results from both cytological sampling methods were compared to evaluate the utility of each method. Endometritis was ruled out in all queens by means of histology and microbiology. Leukocyte counts and red blood cell/endometrial cell ratios were significantly higher in US than UL samples. Additionally, UL sampling is less affected by blood contamination and cells are better preserved. The combination of endometrial cytology and uterine culture might be useful for evaluating the endometrial characteristics in queens. The UL evaluation method is more representative of the actual endometrial status than the US technique.

Changes in Acute Phase Proteins in Bitches after Laparoscopic, Midline, and Flank Ovariectomy Using the Same Method for Hemostasis.

Acute phase proteins (APP) are biomarkers of systemic inflammation, which allow monitoring the evolution of diseases, the response to treatments, and post-operative complications. Ovariectomy (OVE) is frequently performed in veterinary medicine and can be a useful model to evaluate surgical trauma and inflammation in the bitch. The objective was to investigate and compare the acute phase response (APR) after applying three different OVE techniques by measuring serum levels of C-reactive protein (CRP), haptoglobin (Hp), albumin (Alb), and paraoxonase-1 (PON-1). Forty-five intact bitches were included in the study, being randomly distributed into three groups: laparoscopic OVE (L), midline OVE (M), and flank OVE (F). Serum CRP, Hp, Alb, and PON-1 were measured before surgery, 1, 24, 72, and 168 h post-intervention. CRP levels increased significantly 24 h post-surgery in the M and F groups, but no significant variation was observed in the L group at any time of the study period. Hp was significantly higher in group L than in group F 72 h post-surgery. Alb and PON-1 showed no statistical difference among groups or among sampling periods. CRP response suggests that the use of laparoscopic procedures produce lower inflammation compared to open conventional approaches when performing OVE in the bitch.

Red LED Light Acts on the Mitochondrial Electron Chain of Mammalian Sperm via Light-Time Exposure-Dependent Mechanisms.

This work analyzes the effects of red LED light on mammalian sperm mitochondrial function, using the pig as an animal model. Liquid-stored pig semen was stimulated with red-light for 1, 5 and 10 min in the presence or absence of oligomycin A, a specific inhibitor of mitochondrial ATP synthase, or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific disruptor of mitochondrial electron chain. Whereas exposure for 1 and 5 min significantly (p < 0.05) decreased total motility and intracellular ATP levels, irradiation for 10 min induced the opposite effect. Oligomycin A abolished the light-effects on intracellular ATP levels, O2 consumption and mitochondrial membrane potential, whereas compared to non-irradiated samples, FCCP significantly (p < 0.05) increased O2 consumption when sperm were irradiated for 1 min. Both oligomycin A and FCCP significantly (p < 0.05) decreased total motility. Red-light increased cytochrome c oxidase activity with a maximal effect after 5 min of irradiation, which was abolished by both oligomycin A and FCCP. In conclusion, red-light modulates sperm mitochondrial function via electron chain activity in an exposition, time-dependent manner.

Medium-term effects of the diluted pig semen irradiation with red LED light on the integrity of nucleoprotein structure and resilience to withstand thermal stress.

This study sought to evaluate the effects of irradiating pig seminal doses with red LED light irradiation on their quality and longevity over liquid-storage at 17 °C. For this purpose, boar ejaculates were diluted in a commercial extender at a final concentration of 3 × 107 sperm/mL and stored at 17 °C for 96 h. Upon arrival to our laboratory (5-6 h within collection), 1.5 mL-aliquots were subjected to irradiation with a temperature-controlled red light-emitting diode (LED) for 1 min, 5 min or 10 min. Controls consisted of non-irradiated spermatozoa. Aliquots were then stored at 17 °C for 96 h, and plasma membrane and acrosome integrity, motility and free cysteine radicals of sperm head proteins were evaluated every 24 h. In addition, the sperm resilience to withstand thermal stress following irradiation was evaluated at 24 h, 48 h, 72 h and 96 h by incubating stored seminal doses at 37 °C for 120 min. In our experimental conditions, light-stimulation for 5 min and 10 min counteracted the decrease in thermal stress observed in non-irradiated samples during the first 48 h of storage. Moreover, all irradiation protocols counteracted the decrease in percentages of spermatozoa with altered acrosomes observed in non-irradiated samples after 72 h of storage. The effects of light-stimulation upon sperm motility parameters were less consistent. While liquid-storage also led to an increase in the free cysteine levels of sperm head proteins, this increment was partially mitigated through light-stimulation for 5 min and 10 min. Our results suggest that effects linked with red LED light irradiation would be consistently maintained in our experimental conditions for the first 48 h. Finally, the maintenance of light effect appears to depend upon the specific experimental design, the analyzed sperm parameters and the utilized irradiation patterns.

Urine glucose concentration: A useful parameter as a surrogate for glycaemia on the first day of life in canine neonates.

INTRODUCTION: Hypoglycaemia is a well-known risk factor in neonatal puppies and kittens; glycaemia control is crucial during the first days of life. Kidneys immaturity provokes the presence of physiological glycosuria during the first 2-3 weeks of life in small animals. OBJECTIVES: The aim of this study was to evaluate the potential of glycosuria as a predictor of glycaemia in neonatal puppies during the first two weeks of life. METHODS: Prospective study. Thirty-three client-owned healthy neonatal puppies admitted to the Veterinary Teaching Hospital, Autonomous University of Barcelona, were included in the study and divided into four different groups according to the day of sampling (1, 4, 7, and 11 days post-delivery). Glucose levels in blood and urine samples were evaluated and compared between groups. Correlation between glucose levels in blood and urine was also determined. RESULTS: Hypoglycaemia was diagnosed in 17.14% of the puppies and only on day 1 after delivery. A positive and significant correlation between blood and urine glucose concentration on day 1 after delivery was observed. No significant correlation between blood and urine glucose was observed on days 4, 7 and 11 after delivery. CONCLUSIONS: Urine concentration of glucose is a useful parameter to establish glycaemic status on the first day of life in canine puppies.

In vitro assessment of egg yolk-, soya bean lecithin- and liposome-based extenders for cryopreservation of dairy bull semen.

The study was conducted to compare the effect of four commercially available extenders (Triladyl®- egg yolk-based; Andromed® and Bioxcell®-plant based and Optixcell®-liposome-based) on post-thaw sperm quality and functionality variables evaluated using computer-assisted sperm analysis and flow cytometry. A total of 30 ejaculates from five bulls were analysed. With use of Triladyl®, sperm had a greater post-thaw total motility than with use of Bioxell® and Optixcell® but there was no difference as compared with use of Andromed® with the greatest (P < 0.05) percentage of progressively motile cells. With use of Optixcell®, there was a greater (P < 0.05) percentage of sperm with an intact membrane than with use of Triladyl® and Bioxcell®, but values were similar with use of Andromed®. Acrosome damage in semen preserved with use of Optixcell® was less than with use of Bioxcell® and Andromed®. With use of Optixcell®, there was a greater percentage of viable spermatozoa with a lesser lipid disruption (P < 0.05) when compared with the other extenders. Production of peroxides was greater for sperm cryopreserved with use of Triladyl® and Optixcell® while less superoxide was produced in the samples cryopreserved with the egg yolk-based extender. Optixcell® appears to be a promising alternative to replace traditional egg yolk extenders. With use of Optixcell®, however, there were greater peroxide concentrations after thawing. With use of Andromed®, there were similar results as with use of Optixcell®, therefore, it could be an effective substitute for egg-yolk based media due to the greater proportion of highly and progressively motile spermatozoa at thawing.

Tyrosine phosphorylation is not a relevant mechanism to modulate aquaporin 2 activity in gestational queen endometrium and placenta.

Aquaporins have been shown to be regulated by phosphorylation of serine residues, but the possible role of tyrosine residues phosphorylation has not been evaluated. Changes in the localization of aquaporin 2 (AQP2) in the queen endometrium have been related to serum progesterone levels. The aim of this study was to determine whether these AQP2-localization changes are mediated by variations in its tyrosine phosphorylation levels. Twelve queens were included in the study and divided into (a) non-macroscopically pregnant with low levels of progesterone; (b) non-macroscopically pregnant with high levels of progesterone; (c) 30 days of pregnancy; and (d) 60 days of pregnancy. Samples from endometrium and placental transference zone were obtained, immunoprecipitated and analysed by immunoblotting to determine the abundance of AQP2 and its relative levels of tyrosine phosphorylation. No significant differences in the tyrosine phosphorylation levels of immunoprecipated-AQP2 were observed between groups. We can thus conclude that changes in the localization of AQP2 in the queen endometrium are not modulated by tyrosine phosphorylation.

Cyclooxygenase-2 is inhibited in prolonged luteal maintenance induced by intrauterine devices in mares.

Treatment with intrauterine devices (IUD) prolongs luteal phases in mares, but the mechanism for this has not been fully elucidated. The aims of the present study were to examine how IUDs affect the uterus to induce longer luteal phases, particularly the role of cyclooxygenase-2 (COX-2) in the maintenance of the corpus luteum (CL). Twenty-seven reproductively normal mares were included: 12 were inseminated (AI), and 15 were fitted with IUDs. Blood samples for progesterone were obtained on Days 0, 3, 5, 7, 9, 11, 13, 14, and 15 (relative to day of ovulation). The groups were further divided into non-pregnant (AI-N, n = 4), pregnant (AI-P, n = 8), normal (IUD-N, n = 8) and prolonged luteal phase (IUD-P, n = 7) based on ultrasonic examinations and serum progesterone concentrations on Days 14 and 15. Blood sampling to quantify the PGF2α metabolite (PGFM) was performed through a catheter hourly from 15:00 to 20:00 h on Day 14, and from 6:00 until 13:00 h on Day 15. On Day 15, a low-volume uterine lavage followed by an endometrial biopsy was performed. Estradiol concentration in the Day 15 serum and lavage fluid was determined, while the abundance of COX-2 was evaluated in the biopsy specimens using western blotting (WB) and immunohistochemistry (IHC). All pregnant mares were negative for COX-2 in IHC samples and 5 of 8 were negative in WB samples while all mares of the IUD-N group were positive for COX-2. Of the seven mares in the IUD-P group, five and four were negative for COX-2 with the IHC and WB samples, respectively. The results from this study indicate that IUDs, when effective, suppress COX-2, leading to the inhibition of PGF2α release and maintenance of CL.

Placental and uterine expression of GLUT3, but not GLUT1, is related with serum progesterone levels during the first stages of pregnancy in queens.

The present study investigated the expression of GLUT1 and GLUT3 in the uterus and placental transference zone of non-pregnant and pregnant queens throughout different pregnancy ages, using immunohistochemistry and immunoblotting techniques. Both GLUT1 and GLUT3 were expressed in both uterine glandular and luminal epithelia and myometrium in pregnant and non-pregnant queens. While endometrial endothelia showed expression of GLUT1 in both pregnant and non-pregnant queens, GLUT3 was only expressed in the pregnant counterparts. Regarding placental structures, GLUT3 was present in cytotrophoblasts, syncytiotrophoblasts and chorionic endothelia and GLUT1 showed a similar location but was absent in cytotrophoblasts. The presence of GLUT1 (55 kDa) and GLUT3 (60 kDa) was confirmed in both uterine and placental tissues through immunoblotting. When the expression of both GLUT1 and GLUT3 were analysed as a whole in the total of the pregnancy period, no significant differences in the relative content of both GLUTs were observed between pregnant and non-pregnant queens. However, when GLUTs expression was analysed in a time-period basis and related with progesterone levels, results were different. Thus, whereas the relative content of GLUT1 showed no correlation with serum progesterone levels, a significant (P < 0.05) and negative correlation was found between the relative GLUT3-content in the uterus on days 30 and 40 of pregnancy as well as in the placental transference zone on day 30 and serum progesterone levels. In summary, our results indicate that whereas GLUT1 could be considered as a basal, constant sugar intake system for the whole of pregnancy in queens, GLUT3 is specially required for optimizing glucose uptake during the first half of pregnancy in this species through a progesterone-related mechanism.

Pro-inflammatory cytokines: Useful markers for the diagnosis of canine mammary tumours?

The aim of the present study was to analyse the expression of 60 pro-inflammatory cytokines as possible markers of malignancy in canine mammary tumours using a human cytokine antibody array. The cytokines were grouped into two different categories: (1) cytokines in which expression indicated the presence of a mammary tumour and (2) cytokines in which expression differentiated between simple mammary adenoma, tubulopapillary carcinoma or complex carcinoma. These data suggest that specific pro-inflammatory cytokines could be useful as tools for the diagnosis of canine mammary tumours.

Effects on the equine endometrium of cervical occlusion after insemination.

Cervical patency is considered to be important for uterine drainage after mating or artificial insemination (AI), and failure to relax or premature tightening of the cervix can lead to persistent endometritis. This study investigated the hypothesis that cervical occlusion after AI increases accumulation of fluid, polymorphonuclear leukocytes (PMNs), and cytokines in the uterine lumen. Endometrial swabs were obtained from 29 normal cyclic mares during the first, third, and fifth estrus and biopsies during the first and fifth estrus. All mares were inseminated during the second and fourth estrus. In either the second or fourth estrus, a clamped catheter was inserted into the uterus immediately after AI. Accumulation of intrauterine fluid was evaluated by transrectal ultrasonography at 0, 6, 25, and 48 hours. Fluid was drained from the catheter at either 25 hours (TxA) or 6 and 25 hours after AI (TxB). In the control estrus (TxC, no catheters), fluid was obtained by a tampon at 25 hours after AI. The uteri were then lavaged with Ringer's solution, after which the catheters were withdrawn. Sequences of treatments in the second and fourth estrus were A followed by C, C followed by A, B followed by C, and C followed by B in groups AC, CA, BC, and CB, respectively. Five mares lost their catheters and were excluded from the study. Scores for total inflammation, gland dilation, and lymphatic lacunae in the uterine biopsies did not differ significantly between groups or estrous periods. In contrast, periglandular fibrosis scores increased in all groups during the experiment. At 25 hours after AI in the second estrus, the mares with the catheters had larger accumulations of fluid (P < 0.05) and higher concentrations and total numbers of PMNs in uterine fluid (P < 0.05) than the mares without catheters. In the fourth estrus, the total number of PMNs was lower in TxB than in TxA at 25 hours (P < 0.05). Concentrations of PMNs in TxC were 10 times higher in the fourth estrus than the second. Within mare groups AC and BC, total numbers of PMNs in treatment C (fourth estrus) were as high as in TxA and B (second estrus). Expression of IL-1ß, IL-6, IL-10 and TNF-α, analyzed by Western blotting, did not differ significantly between the treatments or estrous periods. It is concluded that a closed cervix after insemination results in pronounced inflammation of the mare's endometrium. Furthermore, this kind of severe insult may lead to permanent pathologic changes in the endometrium, including fibrosis.

Coagulation parameters do not change during luteal phase and pregnancy in cats.

Changes in coagulation parameters depending on reproductive status and pregnancy have been previously reported in both human and other veterinary species. The objective of this study was to determine if different reproductive status affects coagulation parameters in queens. Blood samples from 66 queens submitted to spay surgery were obtained. A hemostatic panel including platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen concentration, and D-dimer and also progesterone concentrations were measured before surgery. According to progesterone results and embryo vesicles diameter, four groups were established: (1) nonpregnant queens with low (≤1 ng/mL) progesterone concentration (LP) (n = 33); (2) nonpregnant queens with high (≥2 ng/mL) progesterone concentration (n = 8) (HP); (3) first half of pregnancy (n = 12); and (4) second half of pregnancy (n = 13). None of the evaluated parameters showed statistically significant differences among the different groups. There was no significant linear correlation between progesterone values and coagulation parameters. In conclusion, neither the presence of the embryo nor the higher values of progesterone concentration induced statistically significant changes in the coagulation profile studied.