Effects of Hormone Factors on the in vitro Culture Flowering Induction of Dendrobium officinate Kimura et Migo

[Objective]The aim was to select the suitable hormone factors for flowering induction in vitro culture of Dendrobium officinate Kimura et Migo.[Method]The test-tube plantlets from the stems of Dendrobium officinate Kimura et Migo were used as the experimental materials and MS medium as the basic medium.Comparative tests have been done between single-factor hormone treatments（different concentrations of PP333 or TDZ） and multi-factor hormone treatments（different combinations of PP333,TDZ,6-BA and NAA） to research the effects of hormone factors on the flowering induction of the plantlets.[Result]Among the single-factor hormone treatments,the suitable concentration and the rate of flower buds formation of PP333 treatment were 0.2 mg/L and 8.5%,the that of TDZ treatment were 0.06 mg/L and 15.5%;the effects of multi-factor hormone treatments on the flowering induction were ordered as follow：（PP333 ＋ 6-BA ＋ NAA ＋ TDZ）〉 （PP333 ＋ 6-BA ＋ NAA）〉 （PP333 ＋ 6-BA） and（PP333 ＋ NAA） ;the most suitable treatment was PP333 0.3 mg/L ＋ 6-BA 0.5 mg/L ＋ NAA 0.5 mg/L ＋ TDZ 0.06 mg/L,the rate of flower bud formation and the rate of the blossomed flower were respectly reached to 80.4% and 90.3%.[Conclusion]PP333 and TDZ showed the important effect on the flowering induction in vitro culture of Dendrobium officinate Kimura et Migo.The effect of TDZ was better than that of PP333.It is much more conducive to the flower bud formation,when using appropriate concentration of TDZ combined with other hormones properly.

In vitro Culture of Unfertilized Ovary of Strawberry(Fragaria spp.)

[Objective] This study was to investigate the in vitro culture of unfertilized ovary of strawberry.[Methods] Employing single factor experiment,we investigated six key factors including genotype,extrinsic hormone,sucrose concentration,low temperature pretreatment,growth environment and development status,and illumination condition on induction of gynogenesis in vitro of unfertilized ovary,on the induction of gynogenesis in vitro of unfertilized ovary.[Results] The optimal conditions for in vitro culture of unfertilized ovary of strawberry were as follows：the primary flower buds cultured on bare land as explants,selection of appropriate genotype,2,4-D as external hormone,sucrose at the concentration of 6%,low temperature pretreatment for 48 hours and dark culture under alternated temperature.[Conclusion] The research provided reference for ploidy breeding in strawberry.

Toxicity Evaluation of in vitro Cultures of Freshwater Cyanobacterium Microcystis aeruginosa:Ⅰ.Hepatotoxic and Histopathological Effects in Rats

Laboratory cultures of freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa PCC 7806 was cvaluated for its hepatotoxic effects in rats. The lyophilized cell extract injected intraperitoneally at 1 and 2 LD50 (15.8 and 31.6 mg/kg, respectively) produced significant increase in liver-specific enzymes viz. plasma alkaline phosphatase,γ-glutamyl transferase, lactate dehydrogenase with a concomitant decrease in hepatic glutamic pyruvic transaminase. A corresponding increase in liver body weight index and histopathological changes in liver (degeneration of hepatocytes, congestion and hemorrhage etc.) are indicative of a dose and time dependent hepatotoxic nature of the algal extract

In vitro culture of immature embryos from Koelreuteria bipinnata var. integrifoliola

For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that： 1） germination rate of grade 3 embryos in immature seeds with 0.6-0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4-0.6 cm diameter was 77,8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2） The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3） The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata vat. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.

Normal and Degenerated Rabbit Nucleus Pulposus Cells in in vitro Cultures: A Biological Comparison

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus （NP） cells in vitro in order to provide seed cells for intervertebral disc （IVD） tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models ofintervertebral disc degeneration （IDD）. Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix （ECM）-related genes （aggrecan and type II col- lagen） were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding （P〈0.05）. The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance （.4） value at passages 4-7 was obviously decreased as compared with that at passage 1 （P〈0.05）. Cell cycle analysis showed that the proportion of normal NP cells at G1 phase was 65.4%-3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase With the value being 77.6%-4.8%. The degen- erated NP cells were predominantly arrested at Gt phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.

Direct Regeneration of Plants Derived from in vitro Cultured Shoot Tips and Leaves of Poplar （Populus×euramericana ＇Neva＇）

The purpose of the present study was to establish a regeneration procedure for Populus × euramericana ＇Neva＇ by using in vitro shoots tips and leaves. For sterilization, 0.1% （w/v） mercuric chloride （HgCl2） solution for 8 to 10 min was the optimal treatment for this poplar cultivation. The effects of benzyladenine （BA） and α-naphthaleneacetic acid （NAA） added to Murashige and Skoog （MS） medium were tested on organogenesis. The highest regeneration rate and numbers of shoots/explant from shoot tips （96.7%, 9.8） and leaves （90.0%, 8.7） were obtained on the half-strength MS medium supplemented with 0.5 mg/L BA and 0.1 mg/L NAA. The optimal medium for in vitro rooting of shoots was on a half-strength MS medium containing 1 mg/L indolebutyric acid （IBA） with the highest rooting frequency （93.3%） and numbers of roots/explant （8.2）. For acclimatization, in vitro rooted plantlets were transferred to plastic cups containing vermiculite and peat （1： 1）. After acclimatization, transplanted plantlets grew well in a shade house. Therefore, we believe that this efficient plant regeneration protocol especially by leaf explants is very important for in vitro clonal propagation of Populus×euramericana ＇Neva＇.

Establishment of in vitro culture of Populus euphratica Olivier

The purpose of our study was to establish a regeneration system for micropropagation of Populus euphratica Olivier. On the basis of an analysis of plant leaf mineral nutrients, a special medium was proposed, called MP2. In optimizing media for in vitro plant cultures including MS, B5 and MP2 media we employed hormones, auxin IAA, cytokine benzyladenine （BAP） and gibberellic acid （GA） in our factorial experiments on media. Adventitious shoots were derived from cuttings of adult plants taken from Xingii- ang, west China, on selected media with MP2^＋ 0.5 mg·L^-1BA＋0.1 mg·L^-1 NAA. The shoots were elongated on a medium with 0.25 mg·L^-1 BAP, 0.1 mg·L^-1NAA and 2 mg·L^-1 GA and were then rooted on a medium with 0.2-0.5 mg·L^-1 IBA. All the media were incorporated with 30 g·L^-1 sucrose and an adjusted pH at 6.3.

Mutated Clones of Caladium Humboldtii ＇Phraya Savet＇ from in vitro Culture and Occurrence of Variants from Somatic Hybridization between Two Caladium Species

Variations in Caladium humboldtii cv. ＇Phraya Savet＇ from in vitro culture were observed. Callus and small shoots were induced from unexpanded leaf segments cultured on modified MS medium supplemented with 2.69 p.M l-Naphthalene acetic acid （NAA） and 17.76 μM N6-Benzyladenine （BA）. Shoots were transferred onto modified MS medium supplemented with 8.88 μM BA for shoot multiplication. Subsequently, roots were induced on MS without growth regulator. The regenerated plantlets were vigorously grown in glasshouse conditions. From 4 morphological groups （leaf color ratio, leaf color, petiole and leaf pattern）, the regenerated ＇Phraya Savet＇ caladium plants were divided into 6 types. The occurrence of variants was 52%. Most of the mutated plants were observed from only leaf color ratio （green ： white） and leaf shape. The most significance for commercial value from mutated clones was round leaf obtaining 20%. Characteristics of new clones from somatic hybridization between C humboldtii cv. ＇Phraya Savet＇ and C. bicolor cv. ＇Suvarnabhum＇ using thin cell layer technique from in vitro calli were investigated. Each thin cell layer of induced callus, about 0.5 - 1 mm thick, from both caladiums was alternately laid on the top of each other for 8 layers. Subsequently, the combination of thin cell layers was cultured and the regenerated plantlets were grown in glasshouse conditions. From 3 morphological groups （leaf pattern, leaf color and petiole）, the regenerated caladium plants were found dissimilarly to both original caladiums at 85 percent with 8 types of different characters. Somatic hybridization between C. humboldtii cv. ＇Phraya Savet＇ and C. bicolor cv. ＇Suvarnabhum＇ gave rise to a number of most hybrids with all conserving C. bicolor characters.

Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND： Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE ： To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN： A comparative experiment.SETTING： Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats （male or female） of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College （certification number was 021-97-03）. METHODS：① Preparation of cerebral cortical neurons of rats： The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons： After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages （1-20 g/L） was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③Grouping： After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours.④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES： ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons;② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining;③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④DNA agarose gel electrophoresis ladder-like strap appeared or not;⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS：① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons （P 〉 0.05）. ②The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [（13.00±4.52）%,（12.72±2.15）%, （11.80±1.18）%,（38.03±1.05）%, P 〈 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture （36.77±1.45）%, （36.60±1.61）%, （36.37±2.02）%, （38.03±1.05）%, P 〉 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L （P 〈 0.01）. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation （P 〈 0.01）. ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia;⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased （P 〈 0.01）, and those of Bax protein and Caspase-3 protein were reduced （P 〈 0.01）, and the ratio of Bcl-2/Bax was increased （P 〈 0.01）. CONCLUSION： Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

Preparation, cryopreservation and in vitro culture of spermatogonial stem cells of new born calves

The experiment was designed to study the preparation, cryopreservation and culture of calf spermatogonial stem cells in order to provide some data for theoretical research and practical use of calf spermatogonial stem cells. As for enzymolysis of testis tissue, two digestive method A and B. The cryopreservation cooling procedure was that spermatogonial stem cells was equilibrated at 4℃ for lh, at -20℃ for lh, at -40℃ for 2h, at -80℃ overnight and then stored in liquid nitrogen. The different concentration of three eryoprotectants were compared. The results indicated that both method A and method B achieved the same high motility rates of cells with method B needing time longer, calf spermatogonial stem cells could be well cryopreserved by the stage cooling procedure. Satisfactory cryopreservative results were gotten by adding 10% DMSO or 10% EG to cryopreservative medium, the former was better and adding sucrose could improved cryopreservative effect. The culture behaviors of spermatogonial stem cells kept the same before and after cryopreservation. It was concluded that an effective method was verified for preparation, cryopreservation and culture of new calf spermatogonial stem cells.

In vitro Culture of Early Rabbit Embryos Injected with Foreign Gene and Its Retention

Fertilized rabbit eggs injected with SMTPGH gene were cultured in vitro and retention of theinjected gene was studied using PCR technique and nonradioactive labelling methed. In a mediumof TC199 + 10% FCS, three quarters of the fertilized eggs developed to the blastocyst stage. Noapparent change of the injected gene was found before the 8-cell stage, after which it was eitherintegrated into the chromosome of the host or lost gradually. But finally, the retention rate of theinjected gene should be equal to its integration rate.

Quantitative detection and comparison of sulfate glycosaminoglycans content in extracellular matrix of in vitro cultured epiphyseal, articular and rib chondrocytes

Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans ( GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of epiphyseal, articular and rib chondrocytes. Methods Sulfate GAG content in extracellular matrix of three chondrocytes was measured by the modified dimethylmethylene blue (DMB) method. The changes of the toluidine blue (TB) stain of chondrocytes were observed by light microscope. Results Primary chondrocytes had the highest content of sulfate GAG in the extracellular matrix, ie, epiphyseal chondrocytes reached ( 70. 12 ± 7. 72 )μg/cm2, articular chondrocytes (92.00 ± 10.15) μg/cm2 and rib chondrocytes (80.61 ± 11. 40) μg/cm2, respectively. On the third pasage chondrocytes, epiphyceal chondrocytes decreased to (53.27 ± 9. 50 ) μg/cm2, articular chondrocytes to (63.88 ± 11.92) μg/cm2 and rib chondrocytes to (58.94 ±8.21) μg/cm2, respectively. The change of TB in every passage

In vitro Culture of Bonamia ostreae and Its Identification

In recent years,bonamiosis has frequently occurred in European areas,which has caused the death of oyster in a wide range and brought enormous economic losses to the breeding industry of oyster. Nowadays,the study on Bonamia sp. is still in the elementary phase. The technology of pathogen’s culture in vitro is the basis for further study on the pathogenesis of Bonamia sp.,its interaction with hosts and the prevention and control of the related disease. In this study,total tissues of oyster identified by PCR were used as culture media to in vitro culture. After one month,they were identified by the method of in situ hybridization. It was found that the results of in situ hybridization were accordant with PCR results. And Bonamia ostreae were detected by in situ hybridization after B. ostreae were cultured for one month. We successfully established a simple and feasible method for in vitro culturing B. ostreae.

In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration

Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors （GFs）, cytokines, transcription factors （TFs）, hormones, oxidative stress products, metabolic net- works, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentia- tion, causing them to lose hepatocyte function. For this mason, most studies focus on inducing stem cells, such as embryonic stem cells （ESCs）, induced pluripotent stem cells （iPSCs）, hepatic progenitor cells （HPCs）, and mesenchymal stem cells （MSCs）, to differentiate into hepatocyte-like cells （HLCs） in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to main- tain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regenera- tion. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

Culture Experiment in vitro of Eperythrozoon of Mustela lutreola

[Objective] The research aimed to make the culture experiment in vitro of eperythrozoon of Mustela lutreola. [Method] 30% newborn bovine serum or Mustela lutreola was added in RPMI-1640, DMEM, M-199 and HL culture solutions. Epery-throzoon of M. lutreola was cultured in vitro under the conditions of 37 ℃, 5% CO2. The culture solutions were replaced every 24 hours. Appropriate amount of healthy erythrocytes were supplemented to make subculture. [Result] The infection rate and infection intensity of eperythrozoon in RPMI-1640 and HL culture solutions were higher than that in DMEM and M-199 culture solutions. The infection rate and infec-tion intensity of eperythrozoon in RPMI-1640 and HL culture solutions with adding 30% serum of M. lutreola were higher than that with adding 30% newborn bovine serum. And the infection rate and infection intensity of eperythrozoon in RPMI-1640 culture solution were slightly higher than that in HL culture solution. The infection rate was over 80% after culturing 12 hours. 23 generations of subculture in vitro was made. [Conclusion] The research provided an effective approach for screening out the drugs that could effectively control eperythrozoon of M. lutreola, preparing diagnostic antigen and producing the relevant vaccine.

Effect of pre-culture on virus elimination from in vitro apple by thermotherapy coupled with shoot tip culture

We evaluated the role of pre-culture on survival rate of in vitro apple plants treated by thermotherapy. Two apple cultivars, Malusxdomestica cv. Pink Lady and Huafu, were used in the experiment and both have widely grown in China and infected with Apple chlorotic leafspot virus （ACLSV） and Apple stem grooving virus （ASGV）. Results in growth and virus titer of apple plants did not exhibit clear trends during five different periods of pre-culture. Whilst, pre-culture increased the survival rate of the two cultivars during thermotherapy. The survival rate of plants pre-cultured for 13 d （P-13d） was 14 and 51% higher than that of P-ld plants for Pink Lady and Huafu, respectively. Moreover, pre-culture positively influenced regeneration of Huafu plants. The average survival rate of plants regenerated from P-ld and P-4d was 20% lower than that regenerated from P-7d, P-10d, and P-13d. The efficiency of virus eradication was determined by reverse-transcription PCR with two primer pairs for each virus, and the detection results showed that pre-culture scarcely affected apple virus elimination. Despite the fact that the two viruses were hardly detected at 5 d of thermotherapy, no virus-free plants were found in the two cultivars of regenerated apple plantlets after 30-d treatment.

In vitro culture of mammalian inner ear hair cells

Auditory function in vertebrates depends on the transduction of sound vibrations into electrical signals by inner ear hair cells.In general,hearing loss resulting from hair cell damage is irreversible because the human ear has been considered to be incapable of regenerating or repairing these sensory elements following severe injury.Therefore,regeneration and protection of inner ear hair cells have become an exciting,rapidly evolving field of research during the last decade.However,mammalian auditory hair cells are few in number,experimentally inaccessible,and barely proliferate postnatally in vitro.Various in vitro primary culture systems of inner ear hair cells have been established by different groups,although many challenges remain unresolved.Here,we briefly explain the structure of the inner ear,summarize the published methods of in vitro hair cell cultures,and propose a feasible protocol for culturing these cells,which gave satisfactory results in our study.A better understanding of in vitro hair cell cultures will substantially facilitate research involving auditory functions,drug development,and the isolation of critical molecules involved in hair cell biology.

In vitro long-term culture and differentiation of mouse spermatogonia stem cells

The studies from 7 or 8 days old mice were used to prepare the spermatogonial stem cells. The isolation and purification of spermatogonial stem cells were done by means of the Percoll discontinual density gradient concentration. The cells of the 3rd band were collected and cultured in vitro in DMEM supplemented with 2.5% or 10% fetal bovine serum （FBS）. The results showed that with the feeder layer and 2.5% serum, the spermatogonial stem cells could proliferate, differentiate last more than 4 months.

Echinococcus Granulosus: Suitable in vitro Protoscolices Culture Density

The present study is to determine the suitable protoscolices （PSCs） density for long-time culturing in vitro. The PSCs were divided into eight groups with different densities and the viability tests were carried out with 0.1% methylene blue staining. Then the infection ability of cultured PSCs was assessed by the mean cyst weight of mice inoculated intraperitoneally with PSCs after 8 months post-infection.

Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 us, with a blastocyst development rate of (20.12 ± 8.18) % (P > 0.05). No significant difference was found between treatments of multiple pulses and a single pulse (P > 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35) % ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) % , P > 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 + 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79, P<0.01). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2(5%CO2: 7%O2:88%N2) also showed no significant difference from those in high O2(5%CO2 in air) [(20.78 ± 8. 80)% and 17.0016.12 vs. (25.30 ± 7.55)% and 18.0116.79, P>0.05]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.