Inhibitory Effect of Bovine Adipose-Derived Mesenchymal Stem Cells on Lipopolysaccharide Induced Inflammation of Endometrial Epithelial Cells in Dairy Cows.

Endometritis is a disease that affects reproductive health in dairy cows and causes serious economic damage to the dairy industry world-wide. Although in recent years, the application of mesenchymal stem cell (MSC) therapy for the treatment of inflammatory diseases has attracted much attention, there are few reports of the use of MSCs in dairy cows. In the present study, our objective was to explore the inhibitory effects of bovine adipose-derived mesenchymal stem cells (bAD-MSCs) on lipopolysaccharide (LPS) induced inflammation in bovine endometrial epithelial cells (bEECs) along with the potential underlying molecular mechanisms. We characterized isolated bAD-MSCs using cell surface marker staining and adipogenic/osteogenic differentiation, and analyzed them using immunofluorescence, flow cytometry (surface marker staining), and adipogenic and osteogenic differentiation. Furthermore, to understand the anti-inflammatory effects of bAD-MSCs on LPS induced bEEC inflammation, we used a bAD-MSC/bEEC co-culture system. The results showed that bAD-MSC treatments could significantly decrease LPS induced bEEC apoptosis and pro-inflammatory cytokine expression levels, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Furthermore, our results showed that bAD-MSC treatments could also significantly downregulate LPS induced p38, IkB-a, and JAK1 phosphorylation and Bax protein expression levels, which are closely related to inflammatory progress and cellular apoptosis in bEECs. Our findings demonstrate that bAD-MSCs play an inhibitory role in LPS induced bEEC inflammation and provide new insights for the clinical therapy of endometritis in dairy cows.

Blood urea nitrogen may serve as a predictive indicator of retained placenta in dairy cows.

Retained placentae (RP) results in significant economic losses to dairy farmers. In Experiment 1, to screen biochemical indicators of RP, 21 cows with RP and 21 cows with no retained placenta (NRP) were selected as a control group, and blood was collected at -7 d, 0 h (parturition) and 12 h. Serum biochemical indicators were ascertained. Results indicate serum concentrations of phosphorus (P) and blood urea nitrogen (BUN) in cows of the RP group were markedly greater than in cows of the NRP group at -7 d (P < 0.01). In Experiment 2, to evaluate predictive indicators for RP, 34 cows with RP and 34 cows with NRP were selected, and there was blood sampling at -15 d, -10 d, -7 d, -4 d, and -1 d. Serum P, BUN, and total protein (TP) were evaluated. Associations of values among the three indicators and occurrence of RP were analyzed using binary logistic regression. Results indicate there was a negative correlation between only the values for BUN and RP (P = 0.016). In Experiment 3, to test hypothesis that relatively greater concentrations of BUN effects immune function in placental tissues, four cows were selected, placentae were collected at 0 and 12 h, and hematoxylin-eosin (HE) staining was performed. Results indicated that the extent of inflammatory cell infiltration and vascular proliferation were less at the 12 than 0-hour timepoint. Taken together, BUN at -7 d may serve as a predictive indicator of RP in cows.

Different heavy metals have various effects on Picea wilsonii pollen germination and tube growth.

Heavy metal pollution has became one of the realistic matters of globality. Previous reports indicated that heavy metals could significantly inhibit pollen germination and tube growth. In the present study, comparative studies on the effects of different heavy metals (As, Hg, Cd, Cr and Cu) on in-vitro picea wilsonii pollen germination and tube growth were carried out. Microscopic evaluation revealed that different heavy metals had various degree of toxicity on P. wilsonii pollen tube development. As showed the most toxic effects on pollen germination, which was followed by Hg and Cd, while Cr and Cu showed relatively lower toxicity. Besides, pollen tubes showed varying shapes in response to different heavy metal stress. Pollen tubes treated with Cd, Hg and As were usually characterized by irregularly increasing diameters and swelling tips with distinct cytoplasimic vacuolation. On the other hand, except for the slightly increased diameters, no obvious abnormal shape were observed in tubes treated with Cr or Cu. Lyso-Tracker Green staining indicated that only Cd-treated pollen tubes showed numerous vacuole-like acidic organelles, though cytoplasmic vacuolization were also observed in pollen tubes treated with Hg and A. In brief, our data indicated that different heavy metals have various effects on Picea wilsonii pollen germination and tube growth, and that in-vitro pollen culture might be used as a competent system for biomonitoring of air pollution.

Spatiotemporal expression of Wnt signaling pathway components during bovine placental development.

It is well established that trophoblasts play a crucial role in pregnancy establishment and maintenance through production of various biological substances. In this regard, Wnt signaling is an important regulator of embryo implantation and placentation in various species. However, the role of the Wnt signaling pathway during bovine placental development has remained largely unknown. Employing multiple approaches, we herein found that Wnt2 mRNA was more abundant in cotyledon tissues compared with caruncle tissues, whereas Wnt5b mRNA was more abundant in caruncle tissues compared with cotyledon tissues. Moreover, the Wnt receptor Fzd4 was detected in caruncle epithelial cells and binucleate trophoblasts, but not in uninucleate trophoblasts. In addition, ß-catenin, an integral cell-cell adhesion adaptor protein as well as transcriptional co-regulator of Wnt canonical pathway, was spatiotemporally expressed in bovine trophoblasts, with high levels of cellular accumulation and nuclear translocation, particularly in binucleate trophoblasts. Lymphoid enhancer factor-1 mRNA was more abundant in caruncle tissues compared with cotyledon tissues, which was well correlated with the expression profile of Dickkopf-1, a secreted antagonist of the canonical Wnt signaling pathway. These results provided new evidence that precisely regulated canonical Wnt activation may have a very important physiological role during fetal-maternal recognition and pregnancy maintenance in cattle.

Intermittent nutritional stimulus by short-term treatment of high-energy diet promotes ovarian performance together with increases in blood levels of glucose and insulin in cycling goats.

The aim of this study was to determine if short-term intermittent treatments of high-energy diet have any stimulatory effects on ovarian function and metabolic status in goat. Cycling Shiba goats were divided into treatment (TG; n=6) and control (CG; n=6) groups. After the detection of ovulation (1st ovulation, Day 0) by ultrasonography, a high-energy diet (250% of maintenance) was fed to the TG from Day 12 to Day 15 (4 days) and from Day 18 to Day 21 (4 days). The high-energy diet comprised 1000 g hay-cubes and 300 g of concentrated feed/head/day (approximately 15 MJ of digestible energy/day). The CG was offered maintenance diet throughout the experiment. Transrectal ultrasound examinations were conducted every other day during the luteal phase and were conducted daily during the follicular phase. Blood samples were collected daily from Day -2 to 7 days after ovulation (2nd ovulation) following the nutritional treatment for analysis of follicle stimulating hormone (FSH), progesterone, oestradiol, glucose and insulin in plasma. Two wave-like rises in the concentrations of glucose and insulin appeared in response to the intermittent nutritional stimulus. Mean plasma concentrations of glucose and insulin were significantly (P<0.05) higher in the TG than in the CG on almost all days during each treatment period. For ovarian performance, the total number of follicles that developed on the day prior to the 2nd ovulation in the TG (7.5 ± 2.7) was significantly higher than that of the CG (4.5 ± 2.6). Ovulation rate of the 2nd ovulation (4.5 ± 1.0) in the TG was significantly higher than that of the 1st ovulation (3.0 ± 0.6) of the TG and than both 1st (2.5 ± 0.5) and 2nd (2.7 ± 0.5) ovulations of the CG. No significant difference was detected in the concentrations of FSH, progesterone and oestradiol throughout the experiment between TG and CG. The present experiment demonstrated that short-term intermittent nutritional stimulus from the luteal phase increased the total number of ovulatory follicles and the ovulation rate in association with increasing plasma concentrations of glucose and insulin in goats.

Responses of gonadotropin secretion to short-term dietary supplementation in ovariectomized goats with different body weights.

The responses of luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion to acute dietary supplementation were studied in goats with different body weights. Ovariectomized Shiba goats (n=11) were used and were maintained with a feed of 100% of their energy requirement. They were implanted subcutaneously with an oestradiol capsule and were divided into light (LBW; 24kg, n=5, mean+/-S.D., 32.0+/-6.3kg) groups on the basis of their body weights at 8 days before the start of treatment. At the start of treatment (Day 1), the level of a feed changed to 250% of their energy requirement and this level was maintained for 7 days in both groups. Blood samples were collected daily from Day -7 to Day 7 for the analysis of FSH, glucose, and insulin profiles in plasma. Frequent blood samples were also collected at 10min intervals for 6h on Day 0, Day 3, and Day 7 for analysis of LH pulses. LH pulse frequency increased significantly on Day 3 as compared with that on Day 0 in both the HBW (7.4+/-0.5pulses/6h vs. 6.2+/-0.8pulses/6h, p<0.05) and LBW (6.5+/-0.8pulses/6h vs. 5.5+/-0.5pulses/6h, p<0.05) groups, whereas it decreased on Day 7 (HBW, 6.4+/-0.9pulses/6h; LBW, 6.3+/-1.6pulses/6h, p>0.05 vs. Day 0). Plasma glucose and insulin concentrations increased temporarily from Day 2 to Day 4 and then decreased to the level before the start of dietary supplementation in both groups. There was no significant difference in the LH pulse frequency or daily concentrations of FSH, glucose, or insulin between the HBW and LBW groups throughout the experimental period. The present study indicated that acute dietary supplementation stimulates pulsatile LH secretion in parallel with a rise of blood glucose and insulin levels. However, the influence of body weight on these responses between light and heavy animals was not observed.

Ultrasonographic uterine changes and vaginal discharges following intrauterine infusion of liquid paraffin in cows.

This study was conducted to examine uterine changes and uterocervical discharges following intrauterine infusion with liquid paraffin (LP) during the luteal phase by ultrasonic and vaginoscopic examinations in cows. Multiparous dairy cows (n=10) were infused with 50 ml physiological saline (PS group; n=5) or liquid paraffin (LP group; n=5) on day 10 or 11 after ovulation (day 0: ovulation). Vaginoscopic, rectal and ultrasonogaphic examinations were carried out at 0.25, 1, 2, 3, 4, 6, 8, 12 and 24 h after the LP and PS infusion and then at daily intervals until subsequent ovulation after the infusions. The mean volumes of recovered discharges from the vagina within 6 h after infusion were significantly greater (P<0.05) in the LP group than in the PS group (33.0 +/- 9.9 vs.14.0 +/- 13.9 ml). Yellowish-white discharge was first observed at 3.2 +/- 0.5 and 3.6 +/- 0.6 h after infusion and lasted for 12.2 +/- 2.9 and 2.1 +/- 1.5 days for the LP and PS groups, respectively, showing a significant difference (P<0.05) in duration. Subsequently, transparent discharge appeared again 2-3 days before the subsequent ovulation after the treatments in both groups and disappeared on the day prior to or the day of ovulation. During the immediate examination after the infusion, the cavity of the uterine horn appeared anechoic and dilated in the images of both groups. The anechoic images changed to echoic images at 2.2 +/- 0.8 and 2.6 +/- 0.9 h after the infusion in the LP and PS groups, respectively, and the echoic images lasted for 12.2 +/- 2.9 and 2.1 +/- 1.5 days in the LP and PS groups, respectively. These results suggest that the appearance and disappearance of intrauterine anechoic and echoic images reflect the appearance and disappearance of the characteristics of the recovered LP/PS-like liquid and yellowish-white and transparent discharges from the vagina.

The effects of short-term nutritional stimulus before and after the luteolysis on metabolic status, reproductive hormones and ovarian activity in goats.

The effect of short-term nutritional supplementation on hormonal and ovarian dynamics was studied in goats. Cycling Shiba goats were divided randomly into maintenance (group M, n=4) and high-energy (group H, n=4) groups. After the detection of the ovulation (Day 0, 1(st) ovulation), group H received a high-energy diet providing 2.5 times of the maintenance energy requirement for 7 days from Day 7 to Day 13 and were administered 2 mg of prostaglandin F(2alpha) (PGF(2alpha)) on Day 10 to induce luteal regression followed by the follicular phase. Follicular and luteal dynamics were monitored using ultrasonography daily or every other day, and blood samples were collected daily from Day 0 to the third ovulation (3(rd) ovulation) following the second ovulation (2(nd) ovulation) induced by PGF(2alpha) administration. Blood samples were also collected at 10-min intervals for 6 h on Day 9 and Day 11 for analysis of pulsatile LH secretion. The mean concentrations of glucose and insulin were significantly (P<0.05) higher in group H than in group M on Days 8, 9, 12, 13 and Days 8, 9 and 10, respectively. For both the 2(nd) and 3(rd) ovulations, no significant difference was detected in ovulation rate between groups M and H. On the other hand, the interpeak interval for wave-like patterns of FSH in group H was significantly (P<0.05) shorter than in group M during the period between the 1(st) and 2(nd) ovulations (4.3 +/- 0.3 vs. 6.5 +/- 1.5 days). The mean LH pulse frequency in group H was significantly (P<0.05) greater than in group M on Day 11 (4.5 +/- 0.6 vs. 3.3 +/- 0.5 pulses/6 h). The present study clearly demonstrated that short-term (7 days) nutritional supplementation promoted pulsatile LH and wave-like FSH secretions in cycling goats. However, no significant increase in ovarian performance was found under such endocrine and metabolic conditions.