Beneficial Influence of Soybean Lecithin Nanoparticles on Rooster Frozen-Thawed Semen Quality and Fertility.

The present study aimed to investigate the impact of different concentrations (0%, 0.5%, 1.0%, 1.5%, and 2.0%) of nano-soybean lecithin (SL) in the extender on sperm quality, sperm motion characteristics, and fertility outcomes of post-thawed rooster semen. Adult Ross broiler breeder roosters (n = 20) were subjected to semen collections twice a week for three weeks. At each collection, semen samples were pooled and allocated into five treatments corresponding to different nano-SL concentrations (control, SL0.5, SL1.0, SL1.5, and SL2.0). Sperm parameters, including motility (collected using a computer-assisted sperm analysis system), plasma membrane and acrosome integrities, and mitochondrial activity were assessed. Sperm malondialdehyde (MDA) and antioxidant activities (total antioxidant capacity (TAC); superoxide dismutase (SOD); glutathione peroxidase (GPx)) were evaluated. The fertility and hatchability obtained with frozen-thawed rooster semen supplemented with the optimum nano-SL concentration were assessed after artificial insemination. The results showed that the addition of 1% nano-SL into the extender led to a higher semen motility in roosters, improved plasma membrane and acrosome integrities, and higher mitochondrial activity of post-thawed rooster semen in comparison to controls (p < 0.05). The MDA levels in the SL0.5 and SL1.0 groups were lower than the other groups (p < 0.05). TAC activities in SL0.5, SL1.0, and SL1.5 groups were significantly higher than those in the other groups (p < 0.05). It was observed that the concentration of SOD was higher in the SL1.0 group than in the other groups (p < 0.05). The activity of GPx was not influenced in any of the cases (p > 0.05). Moreover, the percentages of fertility and hatchability in the SL1.0 group were higher (56.36% and 58.06%) than those in the control group (42.72% and 40.43%). In summary, the addition of nano-SL to the extenders enhanced the post-thawed semen quality and fertility of roosters by reducing the level of oxidative stress. The optimum nano-SL concentration was 1.0%. These results may be beneficial for improving the efficacy of semen cryopreservation procedures in poultry breeding.

Effect of Rhodiola sachalinensis Aqueous Extract on In Vitro Maturation of Porcine Oocytes and Subsequent In Vitro Embryonic Development.

Oxidative stress can impede maturation of the nucleus and cytoplasm of oocytes during in vitro maturation (IVM). Rhodiola sachalinensis, an herb commonly used in traditional Chinese medicine, conveys antioxidative effects to cryopreserved bovine sperm. Therefore, the aims of this study were to evaluate the effects of different concentrations of R. sachalinensis aqueous extract (RSAE) on IVM and subsequent in vitro embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT). The results showed that RSAE supplementation (6 and 60 mg/L) significantly increased intracellular glutathione levels, but had no effect on maturation rates or reactive oxygen species. After in vitro culture, greater blastocyst formation was observed in PA embryos (6 mg/L RSAE), as well as in IVF and SCNT embryos (60 mg/L) matured in RSAE-supplemented IVM media. In conclusion, although there was no significant improvement in the maturation rate, RSAE supplementation conveyed an antioxidative effect during IVM, and improved subsequent embryonic development in vitro. Further studies are needed to explore gene expression pattern in oocytes and embryos treated with RSAE.

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation.

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3-5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.

Anti-inflammatory effects of tanshinone IIA on atherosclerostic vessels of ovariectomized ApoE mice are mediated by estrogen receptor activation and through the ERK signaling pathway.

AIMS: Estrogen plays a protective role in atherosclerosis. Our preliminary work demonstrated that the active conformation of Tanshinone IIA(TanIIA) is similar to the 17ß-estradiol and it can bind to the estrogen receptor. Here, we hypothesized that Tanshinone IIA might have anti-inflammatory and anti-oxidative effects in atherosclerosis, mediated through estrogen receptor activation. METHODS: Subjects for this study were 120 apoE(-/-) female mice and 20 C57/BL female mice. The apoE(-/-) mice were ovariectomized (OVX) and the C57/BL mice were sham ovariectomized. The sham OVX mice were maintained on a normal diet (NOR) group. The OVX apoE(-/-) mice were fed a high fat diet and randomly divided into 6 groups: Model (MOD) group which was fed a high fat diet only, E2 group were given estrogen (E2) 0.13 mg/kg/d; E2+ICI group were given E2:0.13 mg/kg/d and ICI182780:65 mg/kg/m; TLD group (TanIIA low dose) were given TanIIA: 30 mg/kg/d; THD group (TanIIA high dose) were given TanIIA:60 mg/kg/d; and TLD+ICI group were given TanIIA 30 mg/kg/d and ICI182780 65 mg/kg/m. After three months of treatment, the aorta and the blood of the mice from each group was collected. The aorta were used for testing the lipid deposition by using hematoxylin and eosin(HE) and oil red O staining and for testing the expression of p-ERK1/2 by Western blot. The blood was used for testing the serum cholesterol, superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA), nuclear factor kappa (NF-κB), soluble intercellular cell adhesion molecule-1 (sICAM-1), activating protein-1 (AP-1), E-selectin and 17ß-estradiol in serum. RESULTS: Tanshinone IIA significantly reduced the lipid deposition in aorta, decreased the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), very low density lipoprotein (VLDL), MDA, NF-κB, sICAM-1, AP-1, and E-selectin in serum but increased the levels of high density lipoprotein (HDL) and SOD in serum. Tanshinone IIA also suppressed the expression of p-ERK1/2. Tanshinone IIA had no effect of level of serum 17ß-estradiol levels. All of the effects of Tanshinone IIA were similar to estrogen and were inhibited by the estrogen receptor antagonist ICI182780. CONCLUSION: Tanshinone IIA may play an anti-inflammatory and anti-oxidative stress role in OVX atherosclerotic apoE(-/-) mice by activating the estrogen receptor through the ERK signaling pathway. Therefore, Tanshinone IIA, as a phytoestrogen, could be used for estrogen replacement therapy for cardiovascular disease of postmenopausal women.

Effect of Chinese drugs for activating blood circulation and detoxifying on indices of thrombosis, inflammatory reaction, and tissue damage in a rabbit model of toxin-heat and blood stasis syndrome.

OBJECTIVE: To observe the regulatory effect of Chinese drugs for activating blood circulation (ABC) and for activating blood circulation and detoxifying (ABCD) on indices of thrombosis, inflammatory reaction, and tissue damage in a rabbit model of toxin-heat and blood stasis syndrome. METHODS: Fifty-four rabbits were randomized into the normal control group, model group, simvastatin group (simvastatin, 0.93 mg/kg per day), ABC group [Xiongshao Capsule, 0.07 g/kg per day], and ABCD group [Xiongshao Capsule, 0.07 g/kg per day, and Huanglian Capsule, 0.14 g/kg per day]. All except the normal control group received a single injection of bovine serum albumin and were fed with high-fat diets for 6 weeks. At the end of week 4 of giving high-fat diets, a dose of endoxitin was given by ear vein injection, and a randomized 2-week treatment was initiated. At the end of treatment, blood lipids, circulating endothelial cells, and the pathological changes of the aortic arch were assessed. The serum levels of matrix metalloproteinases (MMP-9), tissue inhibitors to metalloproteinase (TIMP-1), granule membrane protein-140 (GMP-140), plasminogen activator inhibitor-1 (PAI-1), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and tumor necrosis factor-α(TNF-α) were determined. RESULTS: Compared with the model group, ABCD group showed decreased serum triglyceride (TG) level, improvement in the pathological change in the aortic arch, and reduction in the number of circulating endothelial cells (4.00 ± 1.41 per 0.9 µL for ABCD group vs 7.83 ± 1.72 per 0.9 µL for the model group). In addition, the levels of serum GMP-140, PAI-1, and IL-6 in ABCD group were also significantly reduced [0.79 ± 0.20 ng/mL, 5.23 ± 1.39 ng/mL, 40.64 ± 10.11 pg/mL for ABCD group vs 1.08 ± 0.31 ng/mL, 7.28 ± 2.01 ng/mL, 54.44 ± 13.56 pg/mL for the model group, respectively, P < 0.05]. A trend showing improvement in the indices of thrombosis, inflammatory reaction, and tissue damage was observed in the ABC group when compared to the model group, but the changes were not statistically significant (P > 0.05). CONCLUSIONS: Chinese drugs for activating blood circulation and detoxifying have beneficial effects on regulating indices of thrombosis (GMP-140 and PAI-1) and inflammatory reaction (IL-6) in rabbit model with toxic-heat and blood stasis. The effect of the activating blood circulation and detoxifying drugs in regulating the levels of serum GMP-140, PAI-1, and IL-6 was superior to that of the activating blood circulation drugs.

Correlation between Fc γ R III a and aortic atherosclerotic plaque destabilization in ApoE knockout mice and intervention effects of effective components of chuanxiong rhizome and red peony root.

OBJECTIVE: To explore the correlation between Fc γ RIII A (CD16A) and aortic atherosclerotic plaque destabilization in apoE knockout (apoE KO) mice and the intervention effects of effective components of chuanxiong rhizome and red peony root. METHODS: Eight 8-week-old male C57BL/6J mice were selected as the control group. Forty 8-week-old male apoE KO mice were randomly divided into the model group, apoE KO + intraperitoneal injection immunoglobulin group (IVIG), apoE KO + simvastatin group (Sm), apoE KO + high dosage of xiongshao capsule (XSC) group (XSCH), and apoE KO + low dosage of XSC group (XSCL), 8 mice in each group. Mice in the control group were put on a normal diet, and others were fed with a high-fat diet. After 10-week different interventions, monocyte CD16 expression was detected by flow cytometry, aortic matrix metalloproteinase-9 (MMP-9) mRNA expression was detected using reverse transcription polymerase chain reaction, and serum tumor necrosis factor (TNF)-α level was detected using enzyme-linked immunosorbent assay. RESULTS: Compared with the control group, monocyte CD16 expression, aortic MMP-9 mRNA expression, and serum TNF-α level in the model group increased obviously (P<0.01). Injections of apoE KO mice with intraperitoneal immunoglobulin during a 5-day period significantly reduced the monocyte CD16 expression, aortic MMP-9 mRNA expression, and serum TNF-α level (P<0.01 or 0.05) over a 10-week period of high-fat diet. Indices above in the Sm group, XSCH group, and XSCL group decreased in a different degree. Of them, the aortic MMP-9 mRNA expression in XSCH group was lower than that in Sm group (P<0.05) and the monocyte CD16 expression and serum TNF-α level showed no significant difference between XSCH group and Sm group (P>0.05). Correlation analyses suggested positive correlation between monocyte CD16 expression and aortic MMP-9 mRNA expression or serum TNF-α level in IVIG group, XSCH group, and XSCL group. CONCLUSIONS: FcγR III A mediates systemic inflammation in the progression of coronary heart disease with blood stasis syndrome. XSC could stabilize atherosclerotic plaque by suppressing inflammation and its target was relative with FcγRIII A.

[Effects of Chinese herbal drug-containing serum on oxidative damage and apoptosis of umbilical vein endothelial cells induced by oxidized low-density lipoprotein].

OBJECTIVE: To investigate the effects of Chinese herbal drug-containing serum, prepared by administration of Chinese herbal medicine for activating blood (Xiongshao Capsule, XS) or for activating blood and detoxifying (Xiongshao Capsule plus Huanglian Capsule, XSHL) in rats, on cell viability, oxidative damage and apoptosis of human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL). METHODS: Thirty-two rats were randomly divided into 4 groups: control group, positive control group (simvastatin 1.8 mg/kg), activating blood (XS, 0.135 g/kg) group, and activating blood and detoxifying (XS Capsule 0.135 g/kg and Huanglian Capsule 0.135 g/kg, XSHL) group. Corresponding drugs were continuously administered to the rats for 7 days and then drug-containing serum was harvested 1 hour after the last administration. HUVECs isolated from newborn children by collagenase digestion were stimulated by ox-LDL (100 µg/mL) [corrected] and incubated with corresponding drug-containing serum for 24 hours. Untreated HUVECs were also used as a normal control. The morphology and structure of HUVECs were observed by an inverted microscope. Cell viability was measured by methyl thiazolyl tetrazolium method, and cell membrane damage was determined by lactate dehydrogenase (LDH) leakage. Activity of superoxide dismutase (SOD) was examined by spectrophotometry, and content of malondialdehyde (MDA) in the cell lysate was examined by thiobarbituric acid assay. HUVECs were stained with Annexin V-fluorescein isothiocyanate and propidium iodide and analyzed on a flow cytometry to determine apoptosis. RESULTS: Compared with the normal HUVECs, the cell viability and the activity of SOD were significantly decreased while the content of MDA and apoptosis rate were significantly increased after 24-hour ox-LDL stimulation (P<0.01, P<0.05). Simvastatin-, XS-, and XSHL-containing serum significantly promoted the ox-LDL-stimulated HUVEC viability and inhibited early apoptosis (P<0.01, P<0.05), while had no significant effect on LDH leakage. Simvastatin-containing serum and XS-containing serum also showed significant decrease in MDA content and increase in SOD activity, while XSHL-containing serum showed no significant effects. There was no significant difference between the XS-containing serum group and the XSHL-containing serum group. CONCLUSION: Both sera containing XSHL and XS show protective action against the oxidative damage and apoptosis of HUVECs induced by ox-LDL.

Synergistic protection of Danhong injection (丹红注射液) and ischemic postconditioning on myocardial reperfusion injury in minipigs.

OBJECTIVE: To explore the synergistic protection of Danhong Injection (丹红注射液, DHI) and ischemic postconditioning on myocardial reperfusion injury in minipigs. METHODS: Acute myocardial infarction model was made by balloon occlusion in left anterior descending coronary artery (LAD) of minipigs, and then postconditioning was simulated through inflation/deflation of the angioplasty balloon. Minipigs were divided into four groups: the sham operation group (SH group), the ischemia/reperfusion group (I/R group), the ischemic postconditioning group (POC group) and DHI combined with ischemic postconditioning group (PAD group, DHI 20 mL through ear vein), six in each group. After 24-h continuous observation, myocardial infarction size was assessed by triphenyltetrazolium staining (TTC). Morphological changes of ischemic myocardium were observed by light microscopy, and cardiomyocyte ultrastructure was studied with electron microscopy. The superoxide dismutase (SOD) and malondialdehyde (MDA) activity in heart homogenates were measured by a biochemical method. RESULTS: The myocardial infarction size was smaller in the POC group than in the I/R group (0.26 ± 0.02 vs. 0.37 ± 0.09, P<0.05), and the PAD group (0.14 ± 0.08) displayed a significantly reduced infarction size relative to the I/R group (P<0.01) and POC group (P<0.05). The damage of myocardial tissue was severe in the I/R group shown by light and electron microscopy: myocardial fibers disorder, sarcoplasmic dissolution, myofilament fracture, mitochondria swelling and even vacuolization formation and a large number of inflammatory cell infiltrations. Compared with the I/R group, reduction of reperfusion injury in the PAD group included more orderly arranged myocardial fibers, less infiltration of inflammatory cells and maintenance of mitochondrial integrity. Compared with the I/R group, the damage of myocardial tissue in the POC group was improved, but not as significant as that in the PAD group. SOD levels in the POC group and the PAD group were significantly higher than those in the I/R group (96.96 ± 13.43, 112.25 ± 22.75 vs. 76.32 ± 10.63, P<0.05), and MDA was significantly lower in the POC group and the PAD group compared to the I/R group (1.27 ± 0.19, 1.09 ± 0.21 vs. 1.47 ± 0.16, P<0.05). CONCLUSION: DHI and ischemic postconditioning show a synergistic cardioprotection on myocardial reperfusion injury in minipigs.

Effects of active components of Red Paeonia and Rhizoma chuanxiong on angiogenesis in atherosclerosis plaque in rabbits.

OBJECTIVE: To investigate the effects and mechanism of the active components of Red Paeonia and Rhizoma chuanxiong (Xiongshao Capsule, XSC) on angiogenesis in atherosclerosis plaque in rabbits. METHODS: Fifty New Zealand rabbits were randomly divided into the normal group, the model group, and the three medicated groups treated respectively with Simvastatin (2.5 mg/kg per day), low-dose (0.24 g/kg per day) and high-dose (0.48 g/kg per day) XSC, 10 in each group. Rabbits in the normal group were fed with regular diet. To those in the other four groups, high fat diet was given, and a balloon angioplasty was performed two weeks later to establish abdominal aortic atherosclerosis model. Then, the model rabbits were fed continuously with high fat diet, and to those in the medicated groups, the testing drugs were added in the forage correspondingly for 6 successive weeks. Levels of blood lipids were measured at the end of the experiment. Meantime, serum levels of high sensitivity C-reactive protein (hsCRP) and tumor necrosis factor alpha (TNF-alpha) were detected with enzyme-linked immunoassay; the plaque area (PA), cross-sectional vascular area (CVA) and correcting plaque area (PA/CVA) were determined quantitatively using imaging software; and the protein expression of vascular endothelial growth factor (VEGF) and factor VIII related antigen (FVIIIRAg) in plaque was detected using immunohistochemical method. RESULTS: As compared with the model group, the content of total cholesterol (TC) in the three medicated groups, and contents of triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the Simvastatin group were lower to various extents (P<0.05, P<0.01). The serum level of hsCRP in all modeled rabbits was higher than that in the normal group, but in the three treated groups it was significantly lower than that in the model group (P<0.05, P<0.01). Expressions of VEGF and FVIIIRAg, as well as PA/CVA in the three medicated groups were significantly lower than those in the model control group (P<0.05, P<0.01). CONCLUSION: The active components of Red Paeonia and Rhizoma chuanxiong have definite effects in delaying the genesis and development of atherosclerosis, its mechanism might be related with the inhibition on angiogenesis in plaque, and also with its actions of lipo-metabolism regulation and anti-inflammation.