Association of Sphingosine‐1‐phosphate (S1P)/S1P Receptor‐1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma

Background

Sphingosine‐1‐phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs.

Hypothesis/Objectives

S1P/S1P₁ signaling will contribute to the progression of hemangiosarcoma (HSA).

Animals

Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used.

Methods

This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT‐PCR, immunohistochemistry, and immunoblotting were performed to examine S1P₁ expression. S1P concentrations were measured by high‐performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca²⁺ mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining.

Results

Canine HSA cells expressed higher levels of S1P₁ mRNA than nonmalignant endothelial cells. S1P₁ protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P₁, decreased S1P₁ protein expression and induced apoptosis of HSA cells.

Conclusions and clinical importance

S1P/S1P₁ signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P₁ or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.     

Achievements and future perspectives of embryo transfer technology in pigs

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non‐surgical ET and short‐ and long‐term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non‐surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short‐term use in pig production.     

Concentration and prevalence of Escherichia coli O157 and Salmonella serotypes in sheep during slaughter at two Australian abattoirs

Objective This study aimed to determine the presence and concentration of Escherichia coli O157 and Salmonella spp. on fleece, faeces and carcases of sheep during slaughter. Procedure Faeces, fleece and pre-chill carcase samples were collected from 164 sheep slaughtered at two Australian abattoirs. The presence of E. coli O157 and Salmonella spp. were determined by use of automated immunomagnetic separation (AIMS) with enumeration by use of the ‘most probable number’ (MPN) method. Results Escherichia coli O157 was isolated from 5% of faeces, 3% of fleeces and 0.6% of pre-chill carcases. The mean log₁₀ count of E. coli O157 positive faecal samples was 2.32 MPN/g, but counts on fleeces and carcases were below the countable limit (−1 log₁₀ MPN/cm²). Salmonella spp. were isolated from 20% of faeces, 13% of fleeces and 1.3% of pre-chill carcases. The mean log₁₀ count of Salmonella spp. in faeces was 1.43 MPN/g and on fleece was −0.24 MPN/cm², but counts on carcases were below the countable limit (−1 log₁₀ MPN/cm²). Conclusion The prevalence and concentration of pathogens were low in the sheep tested in this study, indicating a low risk of human infection from products derived from these animals.     

Feline parathyroid hormone: validation of hormonal assays and dynamics of secretion

Validated assays for quantification of intact parathyroid hormone (I-PTH) are no longer available. Moreover, the third-generation PTH assay that only detects the whole PTH molecule (W-PTH) has never been tested in cats. The work presented here is aimed to validate a commercially available assay for measurement of I-PTH and W-PTH in cats and to study the dynamics of PTH secretion in healthy cats. Our results show that both assays are reliable for the measurement of feline PTH. In healthy adult cats W-PTH concentration (15.1 ± 1.6 pg/mL) was greater (P < 0.001) than I-PTH concentration (9.1 ± 0.7 pg/mL). The dynamics of PTH secretion in response to changes in extracellular calcium (Ca(2+)) were investigated in 13 cats by studying PTH-Ca(2+) curves. PTH-Ca(2+) curves were obtained by intravenous infusion of disodium ethylenediaminetetraacetic acid and CaCl(2). PTH was measured using both I-PTH and W-PTH assays. During hypocalcemia a sigmoidal curve that was similar when measured with I-PTH or W-PTH was obtained. The maximal PTH concentration in response to hypocalcemia was greater with W-PTH (179.6 ± 41.9 pg/mL) than with I-PTH (67.6 ± 10.5 pg/mL; P = 0.01). However, hypercalcemia resulted in an equivalent PTH inhibition, with both assays yielding PTH concentrations as follows: W-PTH = 4.0 ± 0.4 pg/mL and I-PTH = 4.9 ± 0.3 pg/mL (NS). Parameters of the feline PTH-Ca(2+) curve are similar to what has been previously reported in dogs.     

Consequences of Intrauterine Enrofloxacin Infusion on Mare Endometrium

Endometritis is an important cause of infertility in mares. Enrofloxacin is a broad-spectrum antibiotic to which most equine endometritis pathogens are not resistant. The objective of this study was to determine whether enrofloxacin is safe to use as a conventional intrauterine infusion treatment. Nine healthy mares received intrauterine infusions of enrofloxacin (Baytril 100, 100 mg/mL, Bayer Health Care LLC, Animal Health Division) at 2.5 mg/kg daily for 3 days. Ultrasonographic examination and vaginal examinations were performed during the study. Endometrial biopsies were performed before treatment (S0) and 24 hours after the last treatment (S1) to evaluate acute effects. For evaluating chronic effects, biopsies were performed at 14 days (S2) and 60 days posttreatment (S3). Biopsies were graded histologically by the Kenny and Doig category scale. Difference in histological biopsy grade before and after treatment was compared between biopsies by using a repeated-measures one-way analysis of variance. and significant changes in grades were used to assess treatment effects. The vaginal and ultrasonographic examination after intrauterine infusion of enrofloxacin showed that all mares had severe purulent vaginitis and uterine fluid accumulation of ≥2 cm, with ≥1.5-cm thickening of the endometrial wall which persisted in most mares until the end of the study. Histologically, there was acute endometrial ulceration, necrosis, and hemorrhage in biopsy S1 in all mares, categorized as grade III. In biopsy S2, most mares developed fibrosis and inflammation graded as IIb (four of nine mares) or III (four of nine mares). In biopsy S3, fibrosis was extensive and had variable inflammation, graded as IIb (two of nine mares) or III (five of nine mares), with some mares healing to grade IIa (two of nine mares). There was an overall worsening of endometrial biopsy grade from I to III at S3 compared with S0 (P < .001). These results confirm that enrofloxacin is not suitable for conventional intrauterine infusion treatment in mares.     

Early luteal development in Santa Inês ewes superovulated with reduced doses of porcine follicle‐stimulating hormone

The aim was to compare the early luteal development in ewes superovulated with different doses of pFSH. Twenty‐nine Santa Inês ewes received a progesterone device (CIDR®) for 8 days. Gonadotrophic treatment started on Day 6: G200 (control, n = 9, 200 mg); G133 (n = 10, 133 mg); and G100 (n = 10, 100 mg of pFSH). On Day 6, all females received eCG (300 IU). B‐mode and spectral Doppler ultrasonography were performed daily during the early luteal phase (Days 11–15) to monitor the development of corpora lutea (CLs; dimensions) and ovarian arteries indices. CLs were also classified as normal or prematurely regressed (PRCL) on Day 15 by videolaparoscopy. Ewes from G100 and G133 showed gradual increase in luteal diameter during the early luteal phase (p < 0.001), whereas G200 animals presented increase from Day 11 to Day 13, and then decrease on Days 14 and 15 (p < 0.001). The G200 females showed greater percentage of PRCL (45.20%) than those of the other groups (p < 0.001). The normal CLs number was greater in G100 than in G133 (p = 0.04), while the PRCL number was greater in G200 than in the other groups (p = 0.03). Resistive index (RI) was greater in G200 than in G100 (p = 0.02). RI was lower in Day 12 than Day 15 (p = 0.02). Pulsatility index (PI) was greater on Days 14 and 15 (p < 0.01). In conclusion, the lowest dose of pFSH (100 mg) can be considered sufficient for an efficient superovulatory response in sheep, producing better CLs development dynamic in early luteal phase and ovarian blood perfusion and smaller number of PRCL than the traditional (200 mg) pFSH dose.     

Serologic and Urinary PCR Survey of Leptospirosis in Healthy Cats and in Cats with Kidney Disease

Background

Although there is serologic evidence of exposure of cats to Leptospira spp., clinical disease is rarely reported in cats.

Objective

To compare the seropositivity and urinary polymerase chain reaction (PCR) status for Leptospira spp. between healthy (H) cats and cats with kidney disease (KD), to investigate the serovars potentially involved, and to evaluate potential risk factors.

Animals

Two hundred and forty client‐owned cats.

Methods

Cats were prospectively recruited and classified based on physical examination, complete blood count, serum biochemistry profile, and urinalysis (125 H and 115 KD cats). Leptospira spp. serology (titers ≥1 : 100 considered positive) and urinary PCR were performed in all cats. Data assessing risk factors, obtained from a questionnaire, were evaluated using logistic regression models.

Results

Seropositivity for Leptospira spp. was statistically different between groups: 7.2% (9/125) and 14.9% (17/114) in the H and KD, respectively (P = .05). The proportion of PCR‐positive cats was not. The most common serovars detected serologically were Pomona (n = 16) and Bratislava (n = 8). Risk factors for seropositivity included outdoor and hunting lifestyles (P = .03 and P < .001, respectively), the presence of another cat in the household (P < .01), and the sampling period, with the greatest number of cases identified between June and August (P =.02).

Conclusions

Seropositivity was significantly greater in KD cats, suggesting that the role of Leptospira spp. in KD in cats should be further investigated. The detection of urinary shedding of leptospires in several cats identifies a potential role in the transmission of the organism.     

Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle

Background: A major,unresolved issue is how the uterine microenvironment determines pregnancy success in cattle.Before implantation,conceptus development depends on the uterine secretome(i.e.,histotroph).Despite its pivotal role,little is known about the dynamics of histotroph synthesis and changes in composition throughout the early diestrus and the relevance to pregnancy establishment.We hypothesize that disturbances on histotroph composition affect the establishment of pregnancy.Aim was to disturb histotroph composition at early diestrus and verify the effects on:(Exp.1) timing to restore its composition; and(Exp.2) pregnancy rate after multiple-embryo transfer.Estrous cycle of multiparous Nelore cows were synchronized and estrus was considered d 0(D0) of the experiments.Disturbance was through flushing each uterine horn with 30 m L of DMPBS and collecting the resulting uterine luminal flushing(ULF) on D1; D4; D7; D1 + D4 + D7.Control group remained not-collected.In Exp.1,ULF was collected on D7.5 from all animals and used for quantification of total protein concentration and abundance of albumin.In Exp.2,three in vitro-produced embryos were transferred to the uterine horn ipsilateral to the ovary containing the CL on D7.5 and pregnancy was checked on D25 by ultrasound.Results: In Exp.1,ULF collection on D4 or D7 increased(1.5-to 2.2-folds) the total protein concentration and albumin abundance.ULF collection on D1 did not alter(P 0.10) these endpoints.In Exp.2,ULF collected on D4 or D7 decreased pregnancy rates to approximately half of that measured in the remaining groups.Conclusions: Subtle perturbations imposed to the native intrauterine milieu,such as those caused by a single,low-volume collection of ULF,profoundly disturbs intrauterine composition and pregnancy success.At least 4 d were necessary for the uterus to recover its composition and the functional capacity to carry post-implantation gestation.