17β-Estradiol Regulates SKP2 Expression in Cultured Immature Boar Sertoli Cells Mainly via Estrogen Receptor β, cAMP-PKA and ERK1/2

Estrogen plays an important role in regulating testicular Sertoli cell number. Furthermore, S-phase kinase-associated protein 2 （SKP2） plays a central role in mammalian cell cycle progression. The objective of this study was to determine whether 17β-estradiol can regulate the expression of SKP2, and the Sertoli cell cycle, via estrogen receptor β （ERβ）, the cyclic adenosine monophosphate （cAMP）-protein kinase A （PKA） and extracellular signal-regulated kinase （ERK1/2） pathway. When cultured immature boar Sertoli cells were treated with 17β-estradiol, a time-dependent increase in SKP2 mRNA and protein level was observed by real-time PCR and Western blot, and 17β-estradiol activity peaked at 30 min. Treatment with ICI182780 and ERβ antagonist reduced 17β-estradiol-induced expression of SKP2 and proliferating cell nuclear antigen （PCNA）, while increasing the protein concentration of p27kip1. However, the effect of ERa antagonist on these parameters was lower than that of ICI 182780 and ERβ. Forskolin had a similar effect as 17β-estradiol on the expression of SKP2, PCNA and p27kip1, Rp-cAMP, H-89 and U0126 treatment reduced 17β-estradiol-induced changes, while H-89 also inhibited ERK1/2 activation. Therefore, 17β-estradiol mainly regulates SKP2 mRNA and protein expression via ERβ-cAMP-PKA and ERK1/2 activation. SKP2 and PCNA expression were positively correlated, while increased SKP2 expression likely resulted in p27kip1 degradation.     

Effects of Estrogen on ER, NGF, and ChAT Expression in Cerebellum of Aging Female Sprague-Dawley Rat

This article discusses the effects of estrogen on the expression of estrogen receptor （ER）, nerve growth factor （NGF）, and choline acetyltransferase （CHAT） in the cerebellum of rats. The model of aging female rat was established to study the expression and distribution of ER, NGF, and ChAT in the cerebellum following 17β-estradiol treatment using the technique of immunohistochemical ultrasensitive SP in sprague-dawley rat. The immunoreactive productions were distributed in stratum Purkinje cell, nucleus dentatus, nucleus interpositus, and nucleus fastigii of cerebellum, and the ER positive production was mainly located in the plasma, cytoplasmic membrane, and neurite, and also existed in nucleus. The general tendency of the expression of ER, NGF, and ChAT positive production in the cerebellum cortex and nuclei of aging rat significantly decreases, while the intensity and quantity of the immunoreactive production ascends predominantly after 17β-estradiol treatment. Simultaneously, the positive neurite of Purkinje cell shows a similar tendency. The above- mentioned results suggest that the estrogen upregulates the expression of NGF and CHAT, and plays a vital role in sustaining and protecting the structure and function of cerebellum neurons. Furthermore, the similarity of their changing tendency implies that they were correlated and cooperated during the course in effect of estrogen on cerebellum. It also showed that the action of estrogen in cerebellum could be via genomic and nongenomic mechanism.     

Effect of Metallothionein on Cell Cycle,Apoptosis Rate and Subsets Distribution of Lymphocytes in Peripheral Blood of Dairy Cattle under Heat Stress

[Objective] This study aimed to research the effect of metallothionein on cell cycle, apoptosis rate and subsets distribution of lymphocytes in peripheral blood of dairy cows under heat stress, so as to perfect the regulative mechanism re- searches of MT to anti-heat stress. [Method] Twenty lactating Chinese Holstein cows were randomly divided into four groups （A, B, C and D）, and injected with 0, 4.0, 8.0 and 12.0 mg Zn-metallothionein, respectively by intravenous route. Blood sam- ples were collected at 1＂, 16~, 31~, 46~ and 61~ day, and the dynamic changes of cell cycle, apoptosis rate and subsets distribution of lymphocytes were determined. [Result] The apoptosis rate of cells in group B and C was lower than those in group A by 26.63% （P〉0.05） and 24.84% （P〉0.05） respectively. The number of cells in the Gc/G1 phage in trial groups was increased and the number of cells in the S and GJM phages tended to decrease, but there were no significant differences （P〉 0.05）. The number of CD3~ T cell in three trial groups was greater than those in group A by 7.02% （P〉0.05）, 5.45% （P〉0.05） and 3.85% （P〉0.05） respectively, while the number of CD4~ T cell in trial groups was higher than those in control group by 31.04% （P〈0.05）, 35.68% （P〈0.05） and 39.34% （P〈0.05） respectively. The number of CD8＇ T cell and the levels of CD4＊/CD8~ in trial groups were increased observ- ably, but significant difference （P〈0.05） was observed in the levels of CD4~/CD8~ between groups A and C only. It demonstrated that exogenous Zn-metallothionein can decrease apoptosis rate, improve cell cycle and regulate subsets distribution of lymphocytes in dairy cattle in a dose-dependent manner. [Conclusion] This study will provide scientific basis for safe utilization of MT in dairy industry.     

Relationship Between the mRNA Expression Level of TGF-β Receptor Genes in Tissues and Ovulation Rate in Hu Sheep

Eight ewes of Hu sheep which bred multi-lamb were used as the high-fecundity group and the other eight ewes of Hu sheep which bred single lamb were used as the control group to investigate the relationship between the mRNA expression level of TGF-β receptor genes in tissues and ovulation rate in Hu sheep. Cloprostenol sodium was injected to make the synchronization of estrus treatment, then all ewes were slaughtered within 24-36 h after empathema and the ovaries were collected. Furthermore, the number of ovulation points was counted to determine ovulation rate for each sheep. Tissue expression analysis was conducted by RT-PCR for one ewe form the high-fecundity group and the relationship between the mRNA expression of genes encoding TGF-β receptors and ovulation rate was detected by real-time fluorescent quantitative PCR. The results showed that the relative expression level of TGF-flR I gene in the reproductive organ was significantly higher than in the lung and muscle （P 〈 0.01）, while relative expression level of TGF-βR H in reproductive organ was significantly higher than that of other tissues （P 〈 0.01）, indicating that these are highly expressed genes in the ovary. In addition, mRNA expression level of TGF-βR I and TGF-flRH in the ovaries of the high-fecundity group were significantly higher （P〈 0.01） and obviously higher （P= 0.011） than the control group, respectively. The mRNA expression level of TGF-βR I and TGF-βR H had a positive correlation with ovulation rate and the correlation coefficients were 0.562 （P〉 0.05） and 0.711 （P〈 0.05）, respectively. It is suggested that TGF-β receptors have close relationship with highfecundity in Hu sheep.     

Cell Production and Expansion in the Primary Root of Maize in Response to Low-Nitrogen Stress

Maize plants respond to low-nitrogen stress by enhancing root elongation. The underlying physiological mechanism remains unknown. Seedlings of maize （Zea mays L., cv. Zhengdan 958） were grown in hydroponics with the control （4 mmol L-1） or low-nitrogen （40 μmol L-1） for 12 d, supplied as nitrate. Low nitrogen enhanced root elongation rate by 4.1-fold, accompanied by increases in cell production rate by 2.2-fold, maximal elemental elongation rate （by 2.5-fold）, the length of elongation zone （by 1.5-fold）, and ifnal cell length by 1.8-fold. On low nitrogen, the higher cell production rate resulted from a higher cell division rate and in fact the number of dividing cells was reduced. Consequently, the residence time of a cell in the division zone tended to be shorter under low nitrogen. In addition, low nitrogen increased root diameter, an increase that occurred speciifcally in the cortex and was accompanied by an increase in cell number. It is concluded that roots elongates in response to low-nitrogen stress by accelerating cell production and expansion.     

Factors Affecting the Efficiency of Embryonic Cell Nuclear Transfer in Rabbits

Factors affecting the efficiency of nuclear transfer （NT） in rabbits were examined in the present study. When 100 V mm of pulse strength and 15 us of pulse duration were employed, 3 and 4 electronic pulses resulted in significantly more cytoplasts fused with donor cells compared with 2 electronic pulses （P〈 0.05）, but no significant difference was found in the cleavage rate of reconstructed embryos among the three groups （P〉0.05）. When the duration and number of electronic pulse were fixed at 15 ps and 3 times, increase of pulse intensity from 100 V mm 1 to 150 V mm^-1 and 200 V mm^-1 resulted in a significantly decrease in the cleavage rate of reconstructed embryos （P〈 0.05）, although the fusion rate did not significantly differ among the three groups （P〉 0.05）. Significantly more reconstructed embryos cleaved and developed to blastocysts when they were derived from donor embryos at the 8-16-cell stage, in comparison with the reconstructed embryos derived from donor embryos at the compact morula stage （P 〈 0.05）, although the fusion rate was similar （P 〉 0.05）. Activation of cytoplasts prior to fusion increased the cleavage rate （P〈 0.05） and blastocyst development （P〈 0.05） of reconstructed embryos, but decreased the fusion rate （P 〈 0.05） compared with cytoplasts activated post fusion. More reconstructed embryos developed to blastocysts when they were cultured in TCM ＋ 3% OCS at the first 48 h and then cultured in TCM199＋ 10% FCS, in comparison with the reconstructed embryos cultured in either TCM199＋ 10% FCS or TCM199＋3% OCS （P 〈 0.05）. When 22 NT embryos were transferred into the oviducts of one recipient rabbit, one recipient rabbit delivered a female rabbit at 34 days of gestation. In conclusion, either electrofusion parameter or developmental stage of donor embryos have a significant effect on the efficiency of NT, NT embryos require different concentration of serum at their different development stages.     

ALK Family Inhibitor A83-01 Promotes the Proliferation of Mouse Male Germline Stem Cells （mGSCs） Under Serum- and Feeder-Free Conditions

A83-01 is a selective inhibitor of the TGF-β type I receptor ALK,which inhibits the TGF-β-induced epithelial-to-mesenchymal transition(EMT) via the inhibition of Smad2 phosphorylation.Previous studies have showed that A83-01 promoted somatic cellular reprogramming significantly.Male germline stem cells(mGSCs),as an alternative resource of pluripotent stem cells derived adult testis,have promising valuable in clinic medicine and regeneration,however,the derivation of mGSCs was complex and difficult.What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown.In this study,combined with A83-01 and knockout serum replacement(KSR) medium,we obtained a relatively feeder-and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25 μmol L-1.After continuous culturing,the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well.Results showed that,A83-01 dramatically increased the number of mGSCs and AP positive colonies,and the mitosis index according to the BrdU assay.A83-01 could also increase the expression of pluripotent markers including Oct4,Klf4,Nanog and c-Myc,analyzed byreal-time quantative PCR.mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies(EBs),which consisted of three embryonic layers detected by immunofluorescence and RT-PCR.Remarkably,the results demonstrated 0.25 μmol L-1A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder-and serum-free systems.     

Myristic Acid （MA） Promotes Adipogenic Gene Expression and the Differentiation of Porcine Intramuscular Adipocyte Precursor Cells

Intramuscular fat （IMF） content is considered to be a key factor that affects the marbling, tenderness, juiciness and lfavor of pork. To investigate the effects of myristic acid （MA） on the differentiation of porcine intramuscular adipocytes, cells were isolated from longissimus dorsi muscle （LDM） and treated with 0, 10, 50 or 100μmol L-1 MA. The results showed that MA signiifcantly promotes the differentiation of intramuscular adipocytes in a dose-dependent manner. MA also led to a parallel increase in the expression of peroxisome proliferator activated receptor-γ（PPARγ） and adipose-related genes, such as glucose transporter 1 （GLUT1）, lipoprotein lipase （LPL）, adipocyte fatty acid binding protein 4 （FABP4/aP2）, fatty acid translocase （FAT）, acetyl-CoA carboxylaseα（ACCα）, adipose triglyceride lipase （ATGL） and fatty acid synthase （FASN）. However, no signiifcant effects of MA were observed on the expression of CAAT enhancer binding protein-α（C/EBPα） or hormone sensitive lipase （HSL）. The expression of pyruvate dehydrogenase kinase 4 （PDK4） was increased by MA during the early stages of differentiation （day 1-3）. In addition, MA also increased the absolute content of C14 （P〈0.001） and saturated fatty acids （SFA） （P〈0.05） to varying degrees, but no effects were observed on other fatty acids. These results suggest that MA might be able to enhance the IMF content of pork and increase the accumulation of myristic and myristoleic acid in muscle, which might have beneifcial implications for human health.     

The Differentiation-and Proliferation-Inhibitory Effects of Sporamin from Sweet Potato in 3T3-L1 Preadipocytes

The aim of this study was to investigate the effect of different concentrations of sporamin on the differentiation and proliferation of 3T3-LI preadipocytes, providing the theoretical basis for the development of food to treat obesity and diabetes, The isolation and purification of sporamin from sweet potato species 55-2 were performed by ammonium sulphate precipitation in combination with ion-exchange and gel filtration chromatography. With berberine as a positive control, different concentrations ofsporamin （0.000, 0.125, 0.025, 0.250, 0.500, and 1.000 mg·mL^-1 were used to treat 3T3-L1 preadipocytes. Intracellular fat accumulation and the degree of adipogenesis were quantified using Oil Red O staining and colorimetry, Preadipocytes differentiation was measured by 3（4,5-dimethylthiazolyl-2-yl）-2,5-diphenyltetrazolium bromide （MTT） spectrophotometric assay. Two sporamin proteins, which were separated into sporamin A （31 kD） and sporamin B （22 kD）, could be purified by ion-exchange and gel filtration chromatography. After being treated by different concentrations of sporamin, the differentiation of 3T3-L1 preadipocytes was significantly inhibited, compared with the positive control. When the sporamin solution concentration was 0.500 mg mL-1, the accumulation of lipid droplets within the cells was significantly decreased and the optical density （OD） value of the solution from destained Oil Red O reached to 0.35, which was the lowest value （P〈 0.05）. The proliferation of 3T3-L1 preadipocytes was significantly inhibited by treating at higher sporamin concentrations. In addition, the inhibitory effect was more obvious with the prolonged treatment time （P〈 0.05）. The differentiation and proliferation of 3T3-L1 preadipocytes could be inhibited significantly by the addition of higher concentration sporamin. It was, therefore, suggested that the sporamin was potentially effective for weight loss.     

Effect of immunopotentiator of the Chinese herbal medicine on the quantity of T lymphocyte in chicken

In order to study the mechanism of immunopotentiator, the quantity of T lymphocyte was observed. Total 240 1-day chikens were divided into 3 groups randomly: one contrast group and two groups being drunk immunopotentiator according to the concentration 10 mL·L-1 and 5 mL·L-1 lasting for 48 d. The number of T lymphocyte in blood was measured by E-rosette when the chikens were at 12-, 24-, 36-, and 48-day. The results showed that the percent of T lymphocyte in the trial group was obviously higher than that of the contrast, and the 10 mL·L-1 group was higher than that of the 5 mL·L-1 group; the quantity distribution of T lymphocyte in intestinal mucosa lymphoid tissue of 14-day, 21-day chiken was surveyed in the contrast group and the 10 mL·L-1 group with the method of routine histology-slices and ANAE, the results showed that the quantity of T lymphocyte in the 10 mL·L-1 group was significantly higher than that of the contrast, which indicated that immunopotentiator increased markedly the quantity of T lymphocyte, and the 10 mL·L-1 group was higher than that of the 5 mL·L-1 group.     

Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody （designated 3B2 and 3F3） of IgG1 and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1：105 detected by enzyme-linked immunoadsorbent assay （ELISA）. The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains＇ affinity constants were 4.63 × 10^9 and 3.75 × 10^9 （mol L^-1）-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.     

miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene

MicroRNAs(miRNAs) are implicated in swine spermatogenesis via their regulations of cell proliferation, apoptosis, and differentiation. Recent studies indicated that miR-34 c is indispensable in the late steps of spermatogenesis. However, whether miR-34 c plays similar important roles in immature porcine Sertoli cells remain unknown. In the present study, we conducted two experiments using a completely randomised design to study the function roles of miR-34 c. The results from experiment I demonstrated that the relative expression level of miR-34 c in swine testicular tissues increased(P=0.0017) quadratically with increasing age, while the relative expression level of SMAD family member 7(SMAD7) decreased(P=0.0009) with curve. Furthermore, miR-34 c expression levels showed a significant negative correlation(P=0.013) with SMAD7 gene expression levels. The results from experiment II indicated that miR-34 c directly targets the SMAD7 gene using a luciferase reporter assay, and suppresses(P0.05) SMAD7 mRNA and protein expressions in immature porcine Sertoli cells. Overexpression of miR-34 c inhibited(P0.05) proliferation and enhanced(P0.05) apoptosis in the immature porcine Sertoli cells, which was supported by the results from the Cell Counting Kit-8(CCK-8) assay, the 5-Ethynyl-2′-deoxyuridine(EdU) assay, and the Annexin V-FITC/PI staining assay. Furthermore, knockdown of SMAD7 via small interfering RNA(siR NA) gave a similar result. It is concluded that miR-34 c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene.     

Comparsion of the Effects of Succinate and NADH on Postmortem Metmyoglobin Redcutase Activity and Beef Colour Stability

In two experiments, the effects of succinate and NADH（reduced nicotinamide adenine dinucleotide） on metmyoglobin reductase activity and electron transport chain-linked metmyoglobin reduction were investigated and compared. In experiment 1, metmyoglobin（MetMb）, substrate and inhibitors were incubated with mitochondria. Comparsion of the effects of succinate and NADH on MetMb reduction was investigated. The MetMb percentage in sample treated with 8 mol L-1 succinate decreased by about 69% after 3 h incubation, and the effect was inhibited by the addition of 10 mol L-1 electron transfer chain complex II inhibitor malonic acid; the MetMb percentage in samples treated with 2 mol L-1 NADH decreased by 56% and the effect was inhibited by the addition of 0.02 mol L-1 electron transport chain complex I inhibitor rotenone. These results indicated that electron transport chain played an important role in MetMb reduction. Both complex II and complex I take part in the MetMb reduction in mitochondria through different pathways. NADH-MetMb reduction system was less stable than succinateMetMb system. In experiment 2, the beef longissimus dorsi muscle was blended with different concentrations of succinate or NADH. Enhancing patties with higher concentration of succinate or NADH improved colour stability in vacuum packaged samples（P〈0.05）. These results verified that mitochondria electron transport chain is related to the MetMb reduction in meat system.     

Interactive Effect of GA_3 ,N and P Ameliorate Growth, Seed and Fibre Yield by Enhancing Photosynthetic Capacity and Carbonic Anhydrase Activity of Linseed： A Dual Purpose Crop

Linseed (Linum usitatissimum L.) is an important dual-purpose, industrial crop. Its seeds are used for the extraction of oil and stem for fibres. However, the production of linseed is not going parallel with the increasing demand of its products. The present work was carried out with an aim to find out whether exogenous application of gibberellic acid (GA3) with or without graded levels of nitrogen (N) and phosphorus (P) could improve the performance of three linseed genotypes Parvati, Shekhar and Shubhra together with minimizing the costly fertilizer input and losses. Four combinations of N and P, viz., 0 mg N+0 mg P kg-1 soil (N0P0), N13.4 P4.46 , N26.8P8.94 and N40.2P13.4 were constituted. Half dose of each combination was applied basally at the time of sowing and remaining half dose was given at 40 d after sowing (DAS) as foliar spray along with 10-6 mol L-1 GA3 . Prior to sowing, the seeds of each linseed genotype were grouped in to two, one group of seeds was soaked in 0 mol L-1 GA3 (control) and the other group was soaked in 10-6 M GA3 solution, each for 8 hours. Treatments were comprised of (i) 0 mol L-1 GA3+N0P0 (T0 , control), (ii) 10-6 mol L-1 GA3 + N13.4P4.46 (T1), (iii) 10-6 mol L-1 GA 3 +N 26.8 P 8.94 (T2) and (iv) 10-6 mol L-1 GA3+N40.2P13.4 (T3). The crop performance was assessed in terms of growth, physiological and biochemical parameters at 60 and 75 DAS and yield attributes at harvest (175 DAS). The results showed a parallel increase in most of the parameters with increasing levels of N and P. However, application of 10-6 mol L-1 GA3 in association with N26.8P8.94 proved best, it enhanced seed yield, oil yield and fibre yield by 83.3, 97.3 and 78.7%, respectively accompanied with increase in net photosynthetic rate, carbonic anhydrase activity and dry matter accumulation. Among the genotypes tested, Shubhra performed best, while Parvati the least for most of the parameters studied. Thus, combined application of 10-6 mol L-1 GA3 plus N26.8P8.94 proved best and can be recommended to exploit the linseed as a dual-purpose crop for good yield of seed and fibre.     

Effect of Crossbreed on the Muscle Quality （Chemical Composition） in Yun- Ling Black Goats

Twenty castrated male goats, each of Yun-Ling Black goats （YLB goat）, N × YLB hybrid goats （Nubian ♂ ×Yun-Ling Black goats 9） and B × YLB hybrid goats （Boer ♂× Yun-Ling Black goats ♀）, were used to evaluate the effect of crossbreeding on the meat chemical composition in the YLB goats of China. After weaning of 90 days, all the experimental goats were reared on natural pasture when they were slaughtered at an age of 730 days. The longissimus dorsi （LD） and biceps femoris （BF） muscles were sampled from each carcass to determine chemical compositions. Both hybrid goats had higher protein content （P 〈 0.01） and lower fat content （P 〈 0.05） than the YLB goats in the two types of muscle. The inosinic acid contents of LD muscle for the YLB goats and the B × YLB hybrid goats were significantly higher （P 〈 0.05） than that for the N × YLB hybrid goats. The LD muscle from the YLB goats contained higher essential amino acid （P 〈 0.01）, total amino acid （P 〈 0.01）, and some individual amino acid （P 〈 0.05） than those from the hybrid goats. The concentration of unsaturated fatty acid （USFA） of LD muscle from the N × YLB hybrid goats was significantly higher than the other two goat breeds （P〈0.05） but did not differ between the other two goat breeds （P〉0.05）. The YLB goats had significantly higher （P〈0.05） concentrations of oleic acid （C18：1） and linolenic acid （C18：3） of LD muscle than the hybrid goats, had significantly higher （P 〈 0.05） proportion of mono-unsaturated （Sum for C 16：1 and C 18：1） than the B ×YLB hybrid goats, and tended to be higher than the N × YLB hybrid goats （P 〉 0.05）. In contrast, the proportion of poly-unsaturated in the YLB goats was significantly lower （P 〈 0.05） than that in the hybrid goats.     

Effect of GPAG Supplement on Porcine Oocyte Maturation and Its Following Embryonic Development by Parthenogenetic Activated

The aim of this study was to compare the effect of GPAG and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after the fertilization. COCs were aspirated from follicles and cultured for 16, 24, 32, 40 and 48 h in TCM-199 medium either with GPAG or FCS. After 24 h with GPAG, 89.4% of oocytes reached M Ⅰ stage while in the medium supplemented with FCS, only 27.7% of oocytes reached the same stage （P〈0.05）. Prolonged incubation for up to 32 h clearly demonstrated that some of oocytes cultured in GPAG medium were at M Ⅱ stage （35.7%）, few of oocytes from FCS medium were at M Ⅱ stage （7.5%） （P〈0.05）. Both groups of oocytes reached the same stage of maturation within 48 h. After 48 h of culture, the oocytes with extruded polar bodies were inseminated. Fertilized oocytes were cultured in PZM3 medium supplemented with 3 mg.mL of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPAG significantly increased their subsequent developmental ability when compared with FCS supplementation （29.2% ： 18.9% of blastocysts, P〈0.05）. However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number （39 ： 38） and the ICM/total cell ratio （0.26 ： 0.28）     

Preparation of Mouse Embryonic Stem Cells and Cardiomyocyte Differentiation Induced with Retinoic Acid and Ascorbic Acid

The experiment was designed to study effects of retinoic acid and ascorbic acid on differentiation of mouse embryonic stem cells to cardiomyocytes. Embryonic bodies(EB) were developed from m ESC in suspension culture, different levels of concentration of retinoic acid and ascorbic acid were used to determine the optimal conditions for EB formation. The results showed that the optimal concentrations were 10-9 mol · L-1 and 0.1 mg · m L-1 for retinoic acid and ascorbic acids, respectively. 50% of EB which was significantly(p0.05) different from the control group developed to cardiomyocytes. In conclusion, retinoic acid and ascorbic acid had strong ability to promote cardiomyocyte differentiation of mouse embryonic stem cells. 10-9 mol · L-1 retinoic acid and 0.10 mg · m L-1 ascorbic acids were recommended to induce differentiation of mouse ES cells toward cardiomyocytes.     

Binding Capacity for Aflatoxin B1 by Different Adsorbents

In this article, in vitro adsorption of aflatoxin B1 （AFB1） onto different adsorbents was characterized and the result was verified by comparing the growth performance and serum protein levels of broilers exposed to aflatoxin-contamination feed. Main components of adsorbents selected were yeast cell extracts （Product A）, HSCAS （Product B）, and a mixture of yeast product and HSCAS （Product C）, respectively. A total of 240 broilers were assigned to eight treatments, and the effects of three types of adsorbents on growth performance and serum protein levels were evaluated. Results indicated that Product B had the highest in vitro affinity for AFB1, followed by Product C and Product A. Product B bound 97.69% of the AFB1 in solution in 10 min, and it remained over 96.03% in 60 min at pH 8.0. The B-AFB1 complex was much stronger than the other two complexes in vitro condition （P 〈 0.05）. Feed intake （FI） and average daily gain （ADG） decreased （P 〈 0.05） and feed gain ratio increased （P 〈0.05） in the treatment fed aflatoxin-contaminated feed versus treatment on the basal feed. Serum total protein （TP）, albumin （ALB）, and globulin （GLOB） levels were significantly decreased （P〈0.05）. Product B （0.15%） increased growth performance and improved serum protein levels, Product A and Product C were not as effective as Product B. Three adsorbents tested here had sufficient potential to AFBt in some extents and Product B could bind AFB1 more effectively than Product A and Product C. These results indicated that Product B could alleviate some of the AFBt toxic effect in broilers.     

Follicle-stimulating hormone is expressed in ovarian follicles of chickens and promotes ovarian granulosa cell proliferation

Follicle-stimulating hormone(FSH), an important hypothalamic-pituitary-gonadal axis(HPG) hormone, is secreted by the pituitary gland. This study confirms that FSH is expressed in chicken follicles at different stages, and positive FSHβ mRNA signals were stronger(P0.05) in granulosa cells than in oocytes. The 369 bp coding sequence of FSHβ in ovaries is 100% identical to that in the pituitary gland. The experiment in vitro revealed that the ovary possessed FSH secretory capacity. Further, FSHβ mRNA was significantly upregulated(P0.05) in follicles and significantly higher(P0.05) than that in the pituitary gland by approximately 2–23 times with the development. The number of granulosa cells decreased significantly(P0.05) in the cells with siRNA treatment, confirming that the ovarian FSH could promote granulosa cell proliferation. This view was supported by cell cycle analysis and CCND2 and CCNE2 expression. Further research indicated that no difference(P0.05) was observed between the number of granulosa cells treated with FSHβ siRNA and in exogenous FSH. However, the number of granulosa cells without FSHβ siRNA transfection was significantly higher(P0.05) for exogenous FSH. This finding suggests that the proliferative effect of exogenous FSH on ovarian granulosa cells depend on endogenous FSH. This study demonstrated that the FSH gene was expressed in chicken follicles and promoted ovarian granulosa cell proliferation, which enriched the theory on HPG axis.