Wenbin Yue

Effect of elemental nano-selenium on semen quality, glutathione peroxidase activity, and testis ultrastructure in male Boer goats.

The objective of this experiment is to study the effects of novel elemental nano-selenium in the diet on testicular ultrastructure, semen quality and GSH-Px activity in male goats. Forty-two 2-month-old bucks were offered a total mixed ration which had been supplemented with nano-Se (0.3mg/kg Se) or unsupplemented (the control group only received 0.06mg/kg Se-background), for a period of 12 weeks (from weaning to sexual maturity). Results showed that the testicular Se level, semen glutathione peroxidase and ATPase activity increased significantly in the nano-Se supplementation group compared with control (P<0.05). The semen quality (volume, density, motility and pH) was not affected by added Se in diets, however, the sperm abnormality rate of control bucks was significantly higher than Se supplemented bucks (P<0.05). The testes of 5 goats in each group were examined by transmission electron microscopy (TEM), and showed that in Se-deficient bucks the membrane was damaged, and showed the occurrence of abnormalities in the mitochondria of the midpiece of spermatozoa. In conclusion, selenium deficiency resulted in abnormal spermatozoal mitochondria, and supplementation with nano-Se enhanced the testis Se content, testicular and semen GSH-Px activity, protected the membrane system integrity and the tight arrayment of the midpiece of the mitochondria. Further studies are required to research the novel elemental nano-Se with characterization of bioavailability and toxicity in small ruminants.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of ram seminal plasma proteins and their correlation with semen characteristics

The present study was conducted to investigate fertility-associated proteins in ram seminal plasma and the correlation between specific protein and semen characteristics in sheep. Thirty-eight German merino sheep clinically proven healthy were chosen and divided into three groups according to fertility. Ejaculates were collected by an artificial vagina and semen characteristics (volume, pH value, motility, viability and concentration) were recorded. Seminal plasma was harvested by centrifugation and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis in parallel with molecular weight standards. Fifteen protein bands with different molecular weights, ranging from 15.13 to 116.20kDa, were identified on the gel. The results showed that the relative content of eight protein bands was significantly different between the high-fertility group (H-group) and the low-fertility group (L-group). Although the remaining seven protein bands showed no fertility-associated changes in their relative content, some of them were negatively or positively correlated with some semen quality parameters (motility, viability, concentration or pH value). Thus, this study indicates that ram seminal plasma contains specific proteins that are associated with fertility and semen characteristics. Also, these proteins could be utilised in developing a reliable and simple method to determine the ram fertility or semen quality.

Effects of maternal and dietary selenium (Se-enriched yeast) on oxidative status in testis and apoptosis of germ cells during spermatogenesis of their offspring in goats.

To investigate the effect of maternal and dietary selenium on antioxidant status in testis and apoptosis of germ cells during spermatogenesis of their offspring, selected Taihang Black Goats (n=119) were randomly allotted to four treatment groups. They were fed the experimental diet with different Se levels (from Se-enriched yeast) for 174 d from 60 d prior to lactation to weaning of kids. The treatments were: (1) Group 1 (control), basal diet without Se supplementation, (2) Group 2, the same basal diet supplemented 0.5mg Se/kg DM, (3) Group 3, the same basal diet supplemented 2mg Se/kg DM and (4) Group 4, the same basal diet supplemented 4 mg Se/kg DM. Thirty days after weaning, testis samples of the young male goats were collected for mRNA expression and analyzing the antioxidant status and Se concentration, as well as the population of apoptotic germ cells by TUNEL assay. The results show that mRNA expression of apoptosis genes (Bcl-2, Bax and Caspase 8) were significantly higher in Groups 1 and 4 than that in Groups 2 and 3. The same trend was observed in the population of apoptotic cells analyzed by TUNEL assay. GSH-Px activity and Se concentration in testis of offspring was progressively increased with the increasing Se level in diet of dams. However, there was no significant difference in GSH-Px activity between Groups 3 and 4. The lowest MDA content was obtained in Group 2 and a significant decrease was observed in Groups 1, 3 and 4. These data suggest that doe maternal and dietary Se could influence antioxidant status in the testis of their offspring and the oxidative stress related to Se from the dam could modulate mRNA expression of apoptosis genes and apoptosis of germ cells during spermatogenesis. It is possible that Se supplementation of the dams diet during gestation and lactation could be a way to supply the Se necessary for normal development of reproductive function of their offspring.

Effects of different levels of dietary selenium on the proliferation of spermatogonial stem cells and antioxidant status in testis of roosters

The objective of this study was to investigate the different levels of dietary Se (from sodium selenite) on the proliferation of SSCs (spermatogonial stem cells) in testis of roosters. Also, the antioxidant status and Se content in blood plasma and testis were evaluated. A total of eighty 12-week-old Hy-Line Variety white roosters at an averaged body weight of 1.38 ± 0.2 kg were selected and randomly divided into four experimental groups. They were fed with the basal diet (0.044 mgSe/kg DM) supplemented with 0 (control), 0.5, 1.0 or 2.0 mgSe/kg DM (from sodium selenite). After the feeding experiment, blood and testis samples were collected for analysis of the antioxidant status and Se concentration. The testis samples were also used to examine the Thy-1 and β1-integrin mRNA expression by RT-PCR and detect the population of SSCs by immunofluorescence analysis. The results show that Se concentration in blood and testis of the animals was progressively increased with the increasing Se level in diet. The highest GSH-Px (glutathione peroxidase) activity and lowest MDA content in blood and testis was obtained in the treatment of 0.5mg/kg. RT-PCR analysis showed that mRNA expression of SSCs markers were significantly lower in the control and 1.0mg/kg groups when compared with that in the treatment of 0.5mg/kg. A similar trend was observed in the population of SSCs analyzed by immunofluorescence assay. These data suggest that dietary Se can influence the population of SSCs of roosters during spermatogenesis and that oxidative stress can modulate SSCs behavior through regulating some key factors during spermatogenesis.

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation.

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2-3h after collection. Cumulus-oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4-6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4-6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.

Effects of maternal and dietary selenium (Se-enriched yeast) on the expression of p34cdc2 and CyclinB1 of germ cells of their offspring in goats

The aim of the study was to evaluate the effect of selenium on the expression of p34(cdc2) and CyclinB1 (two components of MPF regulating cell cycle) of germ cells of their offspring in goats. A herd of 119 Taihang Black Goats, which was randomly divided into 4 treatments, received experimental diet with different Se levels (from Se-enriched yeast) for 174d. The four treatments, fed with a basal diet, were supplemented with 0 (control), 0.5, 2 and 4 mgkg⁻¹ DM Se. Testis samples were collected from the young male goats of each treatment group at the end of the study (30d after weaning) for mRNA expression using real-time PCR and for protein expression by immunohistochemistry assay. Results show that a significant decrease was observed in mRNA expression of p34(cdc2) and CyclinB1 in the testis of Se-deficient (Group 1) and Se-excess (Group 4) animals compared with that in Groups 2 and 3. However, no significant changes were found in mRNA expression of p34(cdc2) between Se-deficient (Group 1) and Se-excess (Group 4). Also the immunohistochemistry assay detected similar results of protein expression of these two genes. These results suggest, that maternal and dietary Se-induced oxidative stress can modulate the mRNA and protein expression of the cell cycle related genes (p34(cdc2) and CyclinB1) in the testis of their offspring. In addition, Se deficiency and Se excess could prevent the completion of the cell cycle.

Phylogenetic Analysis of S1 Gene of Infectious Bronchitis Virus Isolates from China

SUMMARY. Between 2006 and 2009, seven strains of infectious bronchitis (IB) virus (IBV) were isolated from vaccinated chicken flocks on different chicken farms in China. The pathogenic characters of seven IBV strains were assessed. Each of the seven strains was infective to the test chickens and could induce an immune response. The results from chicken embryo cross-neutralization assays showed that these strains were antigenically distinct from classic IBV strains of H120, M41, Conn, and Gray. Compared to H120 vaccine strain, point mutation, short insertion, and deletion occurred at many positions in the S1 protein of the seven strains. Five of the seven strains had the motif (HRRRR), which was identical to that of the epidemic IBV strains in China. Two new motifs (HRLRR and RRIRR) emerged in the isolated strains. The homology of the nucleotide and amino acid sequences of the S1 gene among the seven isolates was 81.7%–99.7% and 79.0%–99.4%, respectively. These seven strains were also genetically different from the vaccine strains and non-China IBV strains but closely related to large numbers of Chinese strains. The seven isolates and 36 reference IBV strains were clustered into six distinct groups (I–VI). The seven strains were categorized into groups I, II, and III, forming a big phylogenetic branch, which is closely related to Chinese IBVs, whereas the vaccine strains belonging to group VI are genetically distant from groups I, II, and III. The results from this study indicate that different IBV strains cocirculate in the chicken population in China.

Effect of oocyte selection, estradiol and antioxidant treatment on in vitro maturation of oocytes collected from prepubertal Boer goats.

Development of improved procedures for in vitro maturation of oocytes collected from prepubertal goats has applications for in vitro embryo production and accompanying strategies for genetic improvement. The objective of described studies was to determine the effects of oocyte grade, in vitro maturation time, antioxidant supplementation and concentrations of estradiol in the maturation medium on in vitro maturation of oocytes harvested from 1–6 mm follicles present on the ovaries (obtained from an abattoir) of 1–6-month old prepubertal Boer goats. Rates of progression to metaphase II were greater for Grade 1 oocytes (>3 compact layers of cumulus cells and evenly granulated cytoplasm) than Grade 2 oocytes (<3 layers cumulus cells and evenly granulated cytoplasm) and were lowest for Grade 3 oocytes (devoid of cumulus cells with abnormal cytoplasm). No significant effects of maturation for 24 vs 27 vs 30 h on progression of oocytes to metaphase II were observed. The addition of 5 µM β-mercaptoethanol to maturation medium increased the proportion of oocytes progressing to metaphase II and glutathione content in such oocytes, whereas supplementation with 10 mM hypotaurine was without effect. A significant inhibitory effect of in vitro maturation in the presence of high concentrations of estradiol (10 and 100 µg/mL) on progression to metaphase II was observed, and no effect was observed in response to 1 µg/mL estradiol treatment as compared with control. Results suggest that oocyte selection and β-mercaptoethanol supplementation can positively influence progression to metaphase II of oocytes harvested from ovaries of prepubertal goats, whereas high concentrations of estradiol are inhibitory to in vitro maturation.

Enhanced fat consumption potentiates acrylamide-induced oxidative stress in epididymis and epididymal sperm and effect spermatogenesis in mice.

Acrylamide (ACR) and high contents of fat could be found co-existent in many foods processed by high temperature, such as deep-frying and roasting. This study investigated the effect of enhanced fat consumption on deficits of spermatogenesis induced by ACR, and explored potential mechanisms of oxidative damage involved in this pathology in mice. Results show that enhanced feeding of corn oil and pork fat on mice potentiated the decreases of spermatogonia along with mature sperms after treatment of ACR, and that spermatozoa quality is significantly reduced as a result of enhanced feeding of corn oil and pork fat on mice treated with ACR. Moreover, enhanced consumption of corn oil and pork fat potentiated the up-regulation of malondialdehyde (MDA) level in epididymal sperm and cauda epididymides, also up-regulated level of Protein carbonyls (PCOs) in cauda epididymides, of mice after treatment of ACR. Last, enhanced consumption of corn oil and pork fat potentiated the reduced activity of superoxide dismutases (SOD) in epididymal sperm, corpus, and cauda epididymides, also reduced activity of glutathione peroxidase (GPx) in cauda epididymides, of mice treated with ACR. These data suggest that enhanced feeding of corn oil and pork fat on mice potentiates ACR-induced oxidative stress in the epididymis and epididymal sperm and a subsequent effect on spermatogenesis.

Effects of AY9944 A-7 on gonadotropin-induced meiotic resumption of oocytes and development of parthenogenetic embryos in sheep

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus-oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7-induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes.