Effect of insulin,transferrin and selenium and epidermal growth factor on development of buffalo oocytes to the blastocyst stage in vitro in serum-free,semidefined media

The in vitro development of buffalo oocytes up to the blastocyst stage was studied in serum-free, semidefined media containing bovine serum albumin, follicle-stimulating hormone (FSH), insulin, transferrin and selenium (ITS) and epidermal growth factor (EGF). In experiment 1, oocytes aspirated from abattoir-derived ovaries were cultured in eight serum-free, semidefined culture media containing different combinations of these four factors. In experiment 2, the maturation of buffalo oocytes and the development of the embryos were compared in a complex co-culture system and in the serum-free, semidefined media. Supplementation with FSH and EGF significantly (P < 0.05) increased the maturation rates of buffalo oocytes, and the yield of blastocysts was higher (P < 0.05) in media containing EGF and ITS. The yield of blastocysts was lower in the serum-free semidefined media (P < 0.05) than in the complex co-culture system.     

In Vitro Maturation of Dromedary (Camelus dromedarius) Oocytes: Effect of Different Protein Supplementations and Epidermal Growth Factor*

The present experiment was aimed to compare the effect of different protein supplementation sources, foetal calf serum (FCS), oestrous dromedary serum (EDS) and BSA, in experiment 1, and the effect of different concentrations of epidermal growth factor (EGF), in experiment 2, on in vitro nuclear maturation of the dromedary oocytes. Cumulus oocyte complexes (COCs) were harvested from the ovaries collected from a local slaughterhouse by aspirating the visible follicles in PBS supplemented with 5% FCS. Pooled COCs were randomly distributed to 4‐well culture plates containing 500 μl of the maturation medium and cultured at 38.5°C in an atmosphere of 5% CO₂ in air for 32–36 h. The basic maturation medium consisted of TCM‐199 supplemented with 0.1 mg/ml L‐glutamine, 0.8 mg/ml sodium bicarbonate, 0.25 mg/ml pyruvate, 50 μg/ml gentamicin, 10 μg/ml bFSH, 10 μg/ml bLH and 1 μg/ml estradiol. In experiment 1, this medium was supplemented with 10% FCS, 10% EDS or 0.4% BSA, whereas in experiment 2, it was supplemented with 0.4% BSA and 0, 10, 20 or 50 ng/ml of EGF. The oocytes were fixed, stained with 1% aceto‐orcein stain and their nuclear status was evaluated. Oocytes were classified as germinal vesicle, diakinesis, metaphase‐I, anaphase‐I (A‐I), metaphase‐II (M‐II) and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. There was no difference (p > 0.05) observed in the proportion of oocytes reaching M‐II stage between the media supplemented with FCS (71.5 ± 4.8), EDS (72.8 ± 2.9) and BSA (72.7 ± 6.2). In experiment 2, a higher proportion (p < 0.05) of oocytes reached M‐II stage when the medium was supplemented with 20 ng/ml of EGF (81.4 ± 3.2) when compared with the media supplemented with 10 ng/ml (66.9 ± 4.1) and control (67.2 ± 7.1) groups. It may be concluded that the maturation media for dromedary camel oocytes can be supplemented with any of the three protein sources, i.e. FCS, EDS and BSA without any significant differences on the maturation rates. Also, a supplementation of 20 ng/ml of EGF in the maturation medium seems to be optimal and improves the nuclear maturation of dromedary camel oocytes.     

Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro

The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp. 1) or alpha-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.     

Regulation of in vitro growth of preantral follicles by growth factors in goats

Goat preantral follicles were cultured to investigate the effects of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the in vitro growth and viability of oocytes. Preantral follicles were isolated mechanically and enzymatically (using collagenase and DNase) from prepuberal goat ovaries. The working medium was composed of Defined Eagle's Minimum Essential Medium (DMEM) supplemented with HEPES (20 mM), 10% fetal calf serum (FCS), hypoxanthine (2 mM), dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) (2 mM), penicillin (75 ng/ml) and streptomycin (50 ng/ml). The culture medium consisted of the working medium with follicle stimulating hormone (FSH) (100 ng/ml) and hydrocortisone (40 ng/ml) added. In the experiment, goat preantral follicles were cultured for 9 days in the culture medium and in the culture medium supplemented with either IGF-I (100 ng/ml), EGF (50 ng/ml), bFGF (50 ng/ml) or IGF-I (100 ng/ml)+EGF (50 ng/ml). The results indicated that IGF-I (100 ng/ml) effectively maintained the survival of oocytes and promoted their growth; EGF (50 ng/ml) enhanced the survival rate of oocytes but had a negative effect on oocyte growth; bFGF (50 ng/ml) stimulated oocyte survival but had no obvious effect on their growth while IGF-I (100 ng/ml) and EGF (50 ng/ml) in combination had a greater effect on both survival and growth rate of oocytes than IGF-I or EGF alone. The supplementation of IGF-1 and EGF to the culture medium is recommended in the culture of goat preantral follicles.     

Development of a culture system for bovine granulosa cells: effects of growth hormone, estradiol, and gonadotropins on cell proliferation, steroidogenesis, and protein synthesis

The objectives of the present studies were 1) to develop a culture system that has the positive effect of serum on granulosa cell attachment and allows subsequent expression of hormonal effects in serum-free medium and 2) to determine the effect of insulin, epidermal growth factor (EGF), estradiol (E2), and growth hormone (GH) on growth, steroidogenesis, and(or) protein synthesis of bovine granulosa cells. Cells from small (1 to 5 mm) and large (greater than 8 mm) follicles were collected from cattle and cultured for either 4 or 6 d. When cells from small follicles were cultured, insulin (5 micrograms/ml) increased (P less than .05) cell numbers (cells x 10(5)/well) severalfold compared with controls. Alone, EGF (10 ng/ml), FSH (200 ng/ml), LH (200 ng/ml), E2 (2 micrograms/ml), or GH (0 to 1,000 ng/ml) had no effect on cell numbers. However, when included with insulin, 30, 100, and 300 ng/ml of GH increased (P less than .05) granulosa cell numbers on d 4 of culture. Insulin alone increased (P less than .05) progesterone production (ng.10(5) cells-1.24 h-1) by severalfold on d 4, but EGF, FSH, LH, or GH alone had no effect and E2 inhibited progesterone production. In the presence of insulin, FSH and GH (100 ng/ml) increased (P less than .05) progesterone production on d 4 of culture, whereas EGF (10 ng/ml) elicited a decrease (P less than .05) in production. In cells from both sizes of follicles, GH (300 ng/ml) increased synthesis of cellular proteins (greater than 10 kDa). In cells from only large follicles, LH (200 ng/ml) decreased synthesis and secretion of proteins (greater than or equal to 3.5 kDa). These results support the hypothesis that GH may have direct effects on bovine ovarian function.     

Isolation and characterization of equine microvascular endothelial cells in vitro

The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobblestone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors, Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.     

Modeling growth factor activity during proinflammatory stress: methodological considerations in assessing cytokine modulation of IGF binding proteins released by cultured bovine kidney epithelial cells

The present research was conducted to model potential mechanisms through which IGFBPs might be affected by a key proinflammatory response initiating cytokine tumor necrosis factor (TNF-)-. Madin–Darby bovine kidney epithelial (MDBK) cells, known to release IGFBPs in response to several stimuli, were grown under several conditions and challenged with forskolin (F) or recombinant TNF- for 24 h. Forskolin increased IGFBP-3 gene expression and media content of BP-3 protein. TNF- increased basal and augmented F-mediated IGFBP-3 gene expression. However, TNF- effects on the measurable media content of IGFBPs were influenced by culture conditions; in the absence of added protease inhibitors (PIs) or sufficient media albumin concentration (high BSA, 1 mg/ml), the effect of TNF- was to decrease (P < 0.02) measurable IGFBPs. In the presence of PI and high BSA, media IGFBP-3 levels were shown to be increased by TNF- consistent with the gene expression data. Changes in media IGFBP-3 protease activity were examined further to explain the observed effects of TNF- on production and destruction of IGFBPs in media. When recombinant human IGFBP-3 (500 ng/ml) was added to PI-free, low BSA 100 μg/ml) media from TNF-treated MDBK cells, less than 10% of the BP-3 was recognizable by Western blot in 30 min; conversely, inclusion of High BSA and PI in media resulted in attenuation of the protease effect on the IGFBPs. The data suggest that the MDBK model of cellular response to proinflammatory stimulus is affected by culture conditions and that TNF- affects media content of IGFBPs through effects on IGFBP gene expression coupled with degradation of IGFBPs via enhanced proteolytic enzyme release.     

Intracytoplasmic Glutathione Levels in Heifer Oocytes Cultured in different Maturation Media and its Effect on Embryo Development

The present study was carried out to study the effect of different maturation media on embryo development of heifer oocytes and on their glutathione (GSH) synthesis during in vitro maturation (IVM). Immature heifer oocytes were matured in parallel in one of four maturation media: (i) Tissue Culture Medium (TCM)-199 supplemented with 10 ng/ml of epidermal growth factor (EGF); (i) TCM-199 supplemented with 10 ng/ml of EGF plus 1 microg/ml of FSH; (iii) TCM-199 supplemented with 10% of foetal bovine serum (FBS) and (iv) TCM-199 supplemented with 10% of FBS plus 1 microg/ml of FSH. Cow oocytes were used as control and were matured in TCM-199 supplemented with 10 ng/ml of EGF. No differences were observed in blastocyst rate among the different heifer oocyte groups (8.8, 7.5. 8.4 and 6.8%, respectively) however, the percentage of blastocysts obtained from cow oocytes was significantly higher (30%; p < 0.01) than those obtained from heifer oocytes. De novo GSH synthesis during oocyte maturation of heifer and cow oocytes was detected. No significant differences in intracytoplasmic GSH levels were observed among the experimental heifer oocyte groups or between heifer and cow oocytes both before and after IVM. In conclusion, the blastocyst yield obtained from heifer oocytes was lower than that from cow oocytes and this fact could not be explained by significant differences in intracytoplasmic GSH contents of oocytes before or after IVM.     

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.     

Stimulating effects of androgen on proliferation of cultured ovarian germ cells through androgenic and estrogenic actions in embryonic chickens

The effect of androgen on germ cell proliferation was evaluated by a chicken ovarian germ–somatic cell co-culture model and the mechanisms were explored. Ovarian cells were dispersed from 18-day-old embryos, cultured in serum-free McCoy's 5A medium and challenged with testosterone (T) alone or in combinations with androgen receptor antagonist Flutamide, estrogen receptor antagonist Tamoxifen or aromatase inhibitor Letrozole for 48 h. Germ cells were identified by c-kit immunocytochemistry. The number of germ cells was counted and the proliferating cells were identified by immunocytochemistry of proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results showed that T (10⁻⁷ to 10⁻⁶ M) significantly increased the number of germ cells (P < 0.05) and this stimulating effect was inhibited by Flutamide (10–1000 ng/ml), Tamoxifen (10–1000 ng/ml) or Letrozole (10⁻⁹ to 10⁻⁷ M) in a dose-dependent manner. Furthermore, PCNA-LI of germ cells displayed similar changes with the numbers of germ cells. These results indicated that T-stimulated proliferation of cultured ovarian germ cells through both, androgenic and estrogenic actions in embryonic chickens.     

水牛卵巢颗粒细胞的分离培养及凋亡测定

研究主要是通过比较水牛卵巢颗粒细胞在不同培养基中的生长状态,并对细胞的增殖、核型及凋亡情况进行检测,以了解水牛卵巢颗粒细胞体外生长特性,建立水牛卵巢颗粒细胞的体外培养体系。结果发现,分离获得的水牛卵巢颗粒细胞存活率约为60%;采用DMEM培养基,颗粒细胞的生长速度和生长状态优于TCM-199和DMEM/F12培养基;细胞培养24 h后开始零星增殖,3~5 d增殖速度达到高峰;第1、3、5、7代颗粒细胞正常核型比率差异不显著,均在85%以上;第1代颗粒细胞的凋亡率与第5代差异显著(P<0.05),与第7代差异极显著(P<0.01)。结果表明,DMEM培养基更适宜用于水牛颗粒细胞的体外培养;水牛颗粒细胞能稳定地进行传代培养,染色体的数目不会发生明显改变,但细胞凋亡率会随着培养代数的增加而明显升高。     

Effect of Growth Factors on Bovine Blastocyst Development in a Serum-Free Medium

Shamsuddin, M.: Effect of growth factors on bovine blastocyst development in a serum-free medium. Acta vet. scand. 1994, 35, 141-147. - To investigate the effect of growth factors on pre-implantation development, bovine zygotes, produced by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes, were cultured in a serum-free medium to which the following growth factors were added one at a time: epidermal growth factor (EGF), acidic fibroblast growth factor (a-FGF), insulin-like growth factor-II (IGF-II), platelet-derived growth factor from human platelets (PDGF), and platelet-derived growth factor-AB, human, recombinant (PDGF-AB). All growth factors were added at a dose of either 10 or 50 ng/ml, except PDGF which was added at a dose of either 5 or 15 ng/ml. The control medium was TCM 199 supplemented with sodium pyruvate (0.25 mmol/1), BSA (10 mg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), and sodium selenite (5 ng/ml). Embryos were cultured for 8 days (day of insemination = Day 0). The mean percentages of first cleavage on Day 2 varied from 67% to 86% and the differences between the 2 doses, or between the control and growth factor- treated groups were not significant (p≥0.13). The effects of the two doses on subsequent development up to the blastocyst stage did not differ either (p≥0.12). There was no stimulatory effect of any of the used exogenous growth factors on embryo development up to the morula or blastocyst stage on Day 7, or blastocyst stage on Day 8. Moreover, medium supplemented with PDGF had fewer blastocysts than the control (p≤0.03). The results indicate that growth factor supplementation may not necessarily increase the yield of blastocysts from bovine IVM-IVF oocytes in a serum-free medium.     

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.     

大豆黄酮对伊莎鸡卵泡发育及其P450arom基因表达的影响

实验探讨了大豆黄酮(DAI)对伊莎鸡卵泡发育及其芳香化酶(P450arom)mRNA表达的影响。实验选取16只产蛋后期伊莎鸡,等分为对照组和DAI处理组。对照组饲喂基础日粮,实验组在基础日粮中添加10 mg/kgDAI。实验持续7周后,分离排卵前卵泡(F1、F2、F3……)的颗粒层及小黄卵泡和大白卵泡,通过RT-PCR法检测P450arom mRNA表达的相对丰度。结果表明:DAI明显提高了伊莎鸡小黄卵泡和大白卵泡的数量,P450arommRNA在伊莎鸡不同发育阶段卵泡中的表达存在差异,部分卵泡P450arom mRNA表达的相对丰度显著增加。因此,在产蛋后期伊莎鸡基础日粮中添加DAI可增加不同发育阶段卵泡的数目,上调部分卵泡中与发育相关的基因表达以促进卵泡发育。     

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.     

Equine oocyte maturation with epidermal growth factor

Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species. This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods. Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15-25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM-199 supplemented with 50 ng/ml EGF) and EMS Group (TCM 199 supplemented with 10% v/v oestrous mare serum). Each group was divided further into 3 treatments with tyrphostin A-47, a specific tyrosine kinase inhibitor, at 0, 10(-4) and 10(-6) mmol/l. Maturation was determined as the percentage of oocytes reaching metaphase II stage at the end of the culture period. Immunohistochemical detection of EGF-receptor (EGFR) was performed using a streptoavidin-biotin method. The recovery rate and oocyte retrieval were 84.6% (recovered oocytes/follicles aspirated) and 6.55 (oocytes/mare), respectively. Treatment with EGF significantly (P<0.05) increased the incidence of metaphase II stage compared with the control group (69.4 vs. 26.9% in controls, respectively). The specific-tyrosine kinase inhibitor A-47 was effective in suppressing EGF-effect on EGF-cultured oocytes; no significant differences were observed in EMS-supplemented oocytes when cultured with A-47. EGF-receptor was localised in follicles, with localisation being more prominent in the cumulus than in mural granulosa cells. This finding, together with the increase of oocyte nuclear maturation rate when using EGF in culture media and the inhibition of maturation by tyrphostin A-47, suggests a physiological role for EGF in the regulation of equine oocyte maturation. The results should help successful development of assisted reproductive technology in the horse.     

野牦牛成纤维细胞的分离培养与传代

为了提高野牦牛成纤维细胞分离培养效果,研究了组织保存方法、原代培养方法、培养液类型、添加细胞生长因子和冷冻保存液配方对野牦牛体细胞分离培养与传代的作用。结果表明,PBS、D/F12和TCM199适合野牦牛皮肤组织的保存,D/F12和TCM199培养液适合组织块培养,组织块成活率达到（86.29±4.62）%,组织块法适合野牦牛成纤维细胞的原代培养;D/F12培养液培养野牦牛成纤维细胞效果较好,群体倍增时间和平台期密度分别为38.47 h和2.075×10⁶/mL,正常二倍体核型百分率为84.33%;表皮生长因子（EGF）和碱性成纤维细胞因子（bFGF）产生了较明显的促增殖作用,平台期细胞密度明显增加;2种冷冻保存液（D/F12添加胎牛血清和二甲亚砜及胎牛血清添加二甲亚砜）均能用于野牦牛细胞冷冻,细胞存活率分别是87.33%和89.00%。     

Bovine costimulator. I. Production kinetics,partial purification,and quantification in serum-free Iscove's medium

Bovine peripheral blood leukocytes were examined for blast transformation in response to T-cell lectins in serum-containing RPMI 1640 medium and serum-free Iscove's medium. Phytohemagglutinin-induced blastogenesis was significantly greater in Iscove's medium than in RPMI containing ten percent fetal calf serum. Concanavalin A-induced blast transformation was equivalent in both media. However, the kinetics of lectin response and the quantity of lectin required for optimum blastogenesis was considerably different in the two culture media. Concanavalin A-induced blast transformation of bovine thymocytes in Iscove's medium revealed that at a concentration of 10⁶ cells/ml, inconsequential blastogenesis ensued; but at 10⁷ cells/ml blast transformation was significant and dose-dependent. Therefore, conditioned media from concanavalin A-stimulated bovine peripheral blood leukocytes, prepared in serum-free Iscove's medium, were assayed for costimulator activity using bovine thymocytes at 10⁶ cells/ml in Iscove's medium as indicator cells. Both optimum lectin requirements and cell concentrations for production of costimulator activity were found. Conditioned medium, generated with the total exclusion of serum and with optimal costimulator activity, was fractionated via gel exclusion chromatography. A quantitative assay was described, and results indicated that bovine costimulator had an approximate molecular weight of 20,000 daltons.     

A chemically defined medium for the growth of Cowdria ruminantium

Chemically defined media, termed SFMC-23 and SFMC-36, were devised for the in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants. Both media were based on Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12) containing various supplements. Medium SFMC-23 and SFMC-36 supported the long-term growth of the Welgevonden stock of C. ruminantium for a total of 55 and 28 passages, respectively, with regular passage intervals of 3 days. Using SFMC-23, split ratios varied from 5-10, depending on which host cell line was used. Other stocks of C. ruminantium (Sankat, Blaauwkrantz, Senegal) were successfully propagated for a test period of ten passages.