Supplementation with long-acting injectable progesterone 3 days after TAI impaired luteal function in buffaloes.

Three experiments were conducted to evaluate the effects of long-acting injectable progesterone (iP4) in buffalo cows. In Experiment 1, ovariectomized buffaloes received 300 mg (iP300) or 600 mg (iP600) of iP4, and serum P4 concentrations were evaluated. In experiment 2, three groups were compared: control or administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after timed artificial insemination (TAI). On day 16, reproductive tract was recovered for conceptus, endometrium, and corpus luteum (CL) analysis. In experiment 3, pregnancy per AI (P/TAI) and proportion of pregnancy losses were evaluated after administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after TAI in lactating buffaloes. In experiment 1, serum P4 concentrations remained over 1 ng/mL for ~ 3 days in both groups. The 300 mg dose was used in subsequent experiments. In experiment 2, CL weight and endometrial glands density were decreased, and conceptus length was increased in iP4-D3 compared to control and to iP4-D6 (P < 0.05). Transcript abundance of Prostaglandin F Receptor (FP) and ISG15 in CL and of ISG15 and MX1 in endometrium was greater in iP4-D3 when compared to control and to iP4-D6 (P < 0.05). In experiment 3, there was no difference among experimental groups for P/TAI at D30 and pregnancy losses (P > 0.1); however, iP4-D3 presented a lower P/TAI at day 60 (41.7%) when compared to control (56.8%) and iP4-D6 (57.7%; P = 0.07). In conclusion, administration iP4 at 3 days after TAI affects CL development and consequently decreases final pregnancy outcome in buffaloes.

Description of an Ultrasound-Guided Erector Spinae Plane Block and Comparison to a Blind Proximal Paravertebral Nerve Block in Cows: A Cadaveric Study.

The proximal paravertebral nerve block is commonly used to provide anaesthesia to the flank during standing surgical procedures in adult cattle. It has been reported that additional anaesthetic infiltration may be necessary to provide complete anaesthesia. In humans as well as animal species, another technique-the ultrasound (US)-guided erector spinae plane block (ESPB)-has been described. The goal of the present study was to develop and investigate an US-guided ESPB in comparison to a blind proximal paravertebral nerve block (PPNB) in cow cadavers. In 10 cadaver specimens, injections of methylene blue-lidocaine (1:1) were performed at the level of T13, L1 and L2 vertebras, on one side doing an ESPB block and, on the other side, a PPNB. Five cadavers were injected with high (40 mL per injection for PPNB and 20 mL for ESPB) and five with low (20 and 15 mL, respectively) volumes of injectate. For the ESPB, the ultrasound probe was oriented craniocaudally, and the ventral-cranial aspect of the articular processes (T13, L1 and L2) was targeted for injection. The dye spreading was evaluated by dissection. The landmarks for US-guided injection were easily visualized; however, injections were accidentally performed at T12, T13 and L1. Nevertheless, L2 was stained in 60% of ESPBs. Epidural spreading was observed with both techniques and all volumes. Viscera puncture was reported in two PPNBs. The ESPB resulted in similar nerve staining compared to the PPNB while using a lower volume of injectate. Even better staining is expected with a T13-L2 instead of a T12-L1 ESPB approach. Further studies are warranted to evaluate the clinical efficacy.

Insulin induces steroidogenesis in canine luteal cells via PI3K-MEK-MAPK.

Glucose uptake increases in canine luteal cells under insulin treatment. We hypothesize that insulin also increases luteal cell steroidogenesis. Dogs underwent elective ovariohysterectomy from days 10-60 post ovulation and their corpora lutea (CL) and blood samples were collected. Deep RNA sequencing determined differentially expressed genes in CL; those related to insulin signaling and steroidogenesis were validated in vivo by qPCR and their respective proteins by Western blotting and immunofluorescence. Next, luteal cell cultures were stimulated with insulin with or without inhibition of MAPK14, MAP2K1 and PI3K. Studied proteins except P450 aromatase showed the same expression pattern of coding genes in vivo. The expression of HSD3B and CYP19A1 was higher in insulin-treated cells (P < 0.005). Following respective pathway blockades, the culture medium had decreased concentrations of progesterone (P4) and 17b-estradiol (E2) (P < 0.01). Our results indicate that insulin increases HSD3B and CYP19A1 expression via MAPK and PI3K, and contributes to the regulation of P4 and E2 production in canine luteal cells.

Global transcriptome analysis implicates cholesterol availability in the regulation of canine cyclic luteal function.

Considering the key role of the corpus luteum in the regulation of the canine diestrus, the present study aimed to investigate changes in the luteal transcriptome of pseudopregnant dogs (n = 18) from days (D) 10, 20, 30, 40, 50 and 60 post-ovulation. After RNAsequencing was performed, data was analyzed by resorting to several informatic tools. A total of 3300 genes were differently expressed among all samples (FDR < 0.01). By comparing different time points, enriched biological processes as response to estradiol and lipids (D20 vs D10) and intracellular cholesterol transport (D40 vs D60) were observed. Moreover, LXR/RXR (liver X receptor- retinoid X receptor) signaling appeared as an overrepresented pathway in all comparisons. Thus, the expression of 19 genes involved in intracellular cholesterol availability was further evaluated; most were affected by time (P < 0.05). Adding to the deep transcriptomic analysis, presented data implies the importance of cholesterol regulation in luteal physiology of pseudopregnant dogs.

In vitro decidualisation of canine uterine stromal cells.

BACKGROUND: The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology. METHODS: Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n = 6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated. RESULTS: The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ERα were observed in decidualised cells; PRL and ERß remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable. CONCLUSION: An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

GLUT4 protein is differently modulated during development of obesity in monosodium glutamate-treated mice.

The aim of the present study was to investigate the GLUT4 protein expression during the development of obesity in monosodium glutamate- (MSG) treated mice. Control (C) and neonatally MSG-treated 2-month-old (2-mo), 4-month-old (4-mo) and 7-month-old (7-mo) mice were analyzed. Anthropometric data, basal glycemia and insulinemia were measured; and the GLUT4 protein was assessed by Western blotting in white adipose tissue (WAT), skeletal muscle gastrocnemius (SM) and heart (H). Compared to age-matched C mice, the 2-mo and 4-mo MSG mice were already obese, but metabolically they showed increased or preserved whole-body insulin sensitivity, respectively. At these ages they showed unchanged total GLUT4 content in SM and H. However, in plasma membrane fraction from WAT, the MSG showed increased GLUT4 content at both 2- (by 60%) and 4-month (by 45%) of age. When the GLUT4 protein was expressed by unit of adipocyte surface area the protein amount was increased by 36 and 220% in 2-mo and 4-mo MSG mice, respectively. At 7 months of age, obesity was fully established in MSG mice, showing a strongly insulin resistant condition. Additionally, in the 7-mo MSG-mice the GLUT4 protein was reduced in SM (by 40%), H (by 28%), PM and M fractions of WAT (by approximately 70%), and PM expressed by unit of adipocyte surface area (by 92%). The data demonstrate that early, during the accelerated development of obesity in MSG-treated mice, the GLUT4 content was increased in WAT, and that may play a key role in the development of obesity. Later on, when obesity is fully established, the GLUT4 protein was reduced in SM, heart and WAT, and that may be involved in the insulin resistance present in this condition.