Liquid culture of the entomopathogenic nematode,Steinernema colombiense: evolution of the nematode concentration,particles volume fraction,rheological properties and surface tension

ABSTRACT

The rheological properties and surface tension (σ) of culture broths with suspended particles are factors that affect the performance of fermentation processes. This article presents the first report concerning the evolution of suspended particles, rheological properties and σ of culture broths during the liquid culture of Steinernema colombiense and its symbiont bacterium, in a medium containing 1.25% (w/v) egg yolk. Cultures started with 1087?infective-juveniles/mL and finished with 53,583?individuals/mL after 10 days. Culture broths were moderately concentrated suspensions with total particles volume fraction in the range 0.08–0.14. The suspended particles were nematodes of different sizes depending on their developmental stage, and egg yolk particles. Rheological properties corresponded to pseudoplastic fluids with flow behaviour index values ?0.3 (dimensionless), greatly determined by the symbiotic bacterium activity. After nematode inoculation, the apparent viscosity values (η_a; Pa s) for the whole culture broth and its supernatants were in the ranges 0.010–0.018 and 0.008–0.015, respectively. Concerning σ, their values were in the range 31.22–37.74?mN/m, probably determined by the contents of egg yolk.     

Heterologous expression of azurin from Pseudomonas aeruginosa in food-grade Lactococcus lactis

Abstract

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3?h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10?mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.     

乳杆菌发酵上清对耐药白假丝酵母生物膜的作用

目的 研究德氏乳杆菌(Lactobacillus delbrueckii,L.delbrueckii)和发酵乳杆菌(Lactobacillus fermentum,L.fermentum)发酵上清液对3株耐氟康唑白假丝酵母临床分离株CA3、CA6、CA8生物膜形成和分散的作用。 方法 通过MIC试验,确认3株白假丝酵母临床分离株对氟康唑耐药;用96孔板构建体外白假丝酵母生物膜;用棋盘法分别检测L.delbrueckii、L.fermentum发酵上清液与氟康唑联用对3株白假丝酵母的作用;用XTT法对生物膜形成量进行定量分析;检测L.delbrueckii、L.fermentum发酵上清液与氟康唑联用对白假丝酵母时间-生长曲线的影响;显微镜拍照检测L.delbrueckii、L.fermentum发酵上清液单独和氟康唑联用对白假丝酵母生物膜形态的影响;平板培养法检测L.fermentum发酵上清液对白假丝酵母黏附作用的影响。 结果 CA3、CA6、CA8三株临床分离菌株对氟康唑耐药,MIC浓度均为8 μg/mL。L.delbrueckii与L.fermentum发酵上清液与氟康唑联用对3株耐药型白假丝酵母的生物膜形成与分散均未表现出协同作用,且发酵上清液与氟康唑联用效果不如发酵上清液单独处理效果好,L.fermentum发酵上清液对生物膜的分散作用较L.delbrueckii发酵上清液强。L.fermentum发酵上清液抑制白假丝酵母的初始黏附。 结论 L.delbrueckii、L.fermentum发酵上清液单独应用时均对耐氟康唑白假丝酵母生物膜的形成与分散有干预作用,与氟康唑联用时未表现出协同作用,抑制耐药白假丝酵母生物膜的作用可能与抑制菌丝形成和起始黏附有关。     

Physiologic studies of snail-schistosome interactions and potential for improvement of in vitro culture of schistosomes

Summary Despite extensive efforts to develop suitable media for rearing the intramolluscan stages of schistosomes, successful in vitro culture of these parasites remains elusive. Recent³¹P NMR studies demonstrated that the levels of free phospholipids, particularly phosphatidylcholine, in the digestive gland of the snail,Biomphalaria glabrata, were dramatically reduced when the host was infected withSchistosoma mansoni. It was speculated that absorption of host phosphatides may be an important source of membrane phospholipid precursors and fatty acids for developing sporocysts and cercariae. During the present investigations,B. glabrata was maintained on a high fat diet of egg yolk, and the lipid composition of control uninfected and infected snails examined by³¹P and¹³C NMR. In addition, the levels of host hemolymph metabolites, including glucose and urea, considered as indicators of parasite nutrient uptake, were monitored. The lipid level of snails fed egg yolk was greatly increased, and hosts developed patent infections in approximately half the time of infected snails maintained on lettuce. The composition of the free phospholipids accumulated in the tissues ofB. glabrata fed egg yolk were the same as those previously reported in the cercarial stage ofS. mansoni. Moreover, the fatty acids ofS. mansoni and those reported here in the neutral lipids and free phosphatides in the host tissues were similar. Uninfected snails maintained on lettuce had higher hemolymph levels of glucose than those reared on egg yolk, and infected hosts on egg yolk had significantly lower levels of hemolymph urea. β-hydroxybutyrate was the principal hemolymph metabolite in snails fed egg yolk, but was not detected in snails maintained on lettuce. The level of β-hydroxybutyrate in the hemolymph of snails on egg yolk was significantly reduced by infection. The results indicated that the pattern of host hemolymph nutrient utilization by larval schistosomes may be markedly altered by host diet, and it was concluded that host lipids may directly and indirectly be important nutrients for developing schistosomes. Future studies on in vitro culture of the intramolluscan stages of schistosomes should emphasize the potential role of lipids and attempt to define the nutritive value of those medium components that presently supply lipids in culture media, most notably serum.     

Macrophage supernatants have both stimulatory and suppressive effects on mesangial cell proliferation

Macrophages may modulate mesangial expansion following renal injury via secretory products. We undertook the present study to determine the effects of macrophages supernatants on mesangial cell proliferation. Macrophage supernatants collected in serum-free media after 24 hours caused significantly enhanced mesangial cell proliferation in long-term culture at concentration up to 50% but caused suppression at higher concentration (control, 122,000 ± 14,000 cells/well: 50% supernatant, 188,000 ± 15,100 cells/well, p < 0.02 compared to control, n = 4; 80% supernatant, 52,000 ± 3,500 cells/well, P < 0.01 compared to control, n = 4). In short-term culture [³H] thymidine incorporation, a measure of DNA synthesis, was significantly enhanced compared to control at supernatant concentrations up to 30% (30% supernatant, 4,120 ± 310 cpm/well; control, 3,210 ± 97 cpm/well, P < 0.5, n = 4), but uptake was reduced at high concentration (80% supernatant, 2,900 ± 74 cpm/well; control, 3,210 ± 97 cpm/well, P < 0.05, n= 4) When macrophages supernatants were collected after 48 hours incubation and incubated with mesangial cells, mesangial cell thymidine uptake was significantly suppressed compared to control (48-hours supernatant, 4,060 ± 260 cpm/well; control, 5,890 ± 270 cpm/well, P < 0.01, n = 4) and comapared to 24-hour supernatants, which enhanced uptake (24-hour supernatant, 8,080 ± 340 cpm/well; control, 5,890 ± 270 cpm/well, P < 0.01, n =4). Our results suggest that macrophages supernatants can directly enhance mesangial cell proliferation in vitro in both short-term and long-term culture, though this effect is lost at high concentrations of supernatant. These data lend support to the potential role of the macrophage in mediating mesangial expansion following renal injury. © 1993 Wiley-Liss, Inc.     

The in vitro antibiofilm activity of selected marine bacterial culture supernatants against Vibrio spp.

The aim of the work is to investigate the effect of marine bacterial culture supernatants on biofilm formation of Vibrio spp., a major menace in aquaculture industries. Vibrio spp. biofilm cause life-threatening infections in humans and animals. Forty-three marine bacterial culture supernatants were screened against the hydrophobicity index, initial attachment and biofilm formation in Vibrio spp. Twelve culture supernatants showed antibiofilm activity. The bacterial culture supernatants S8-07 (Bacillus pumilus) and S6-01 (B. indicus) inhibited the initial attachment, biofilm formation and dispersed the mature biofilm at 5% v/v concentration without inhibiting the growth. Analysis by light microscopy and confocal laser scanning microscopy showed that the architecture of the biofilm was destroyed by bacterial supernatants when compared to the control. The bacterial supernatants also reduce the surface hydrophobicity of Vibrio spp. which is one of the important requirements for biofilm formation. Further characterization of antibiofilm activity in S8-07 culture supernatant confirmed that it is an enzymatic activity and the size is more than 10 kDa and in S6-01, it is a heat-stable, non-protein compound. Furthermore, both the supernatants failed to show any biosurfactant activity. The culture supernatants of S8-07 and S6-01 with promising antibiofilm property have potential for application in medicine and marine aquaculture.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Liquid storage of Asian elephant (Elephas maximus) sperm at 4 degrees C

The Asian elephant (Elephas maximus) population in the wild has been in decline for several decades and breeding in captivity has not been self-sustaining. The use of artificial insemination (AI) can help overcome many of the difficulties associated with breeding elephants in captivity; however, the ability to store semen for extended periods of time is critical to the successful application of AI to elephants. The objective of the present study was to assess the effects of four different semen extenders and the presence of egg yolk on the viability and motility of Asian elephant semen stored at 4 °C. High quality ejaculates (n=4) were collected from two Asian elephant bulls by rectal massage. Aliquots of each ejaculate were extended in four different diluents (Beltsville thawing solution (BTS); Tris–citric acid (TCA)/fructose-based; Beltsville F5 (BF5); dextrose-supplemented phosphate-buffered saline (PBS)) with or without egg yolk then cooled and stored at 4 °C. The percentages of viable (viability) and motile (motility) sperm were evaluated at 8, 24 and 48 h following collection. The addition of egg yolk significantly reduced the percentage loss in viability from initial collection to 48 h compared to extenders without egg yolk (17.0±8.2 versus 32.6±8.9 decline in percent viable sperm in the population, respectively; P<0.05). Extender and egg yolk affected (P<0.005) total motility and percent progressively motile sperm at all evaluation times during incubation. TCA + egg yolk maintained higher (P<0.05) levels of progressive motility compared to other extenders supplemented with egg yolk. These results indicate that Asian elephant semen extended in TCA diluent supplemented with egg yolk can maintain at least 50% viability and motility when stored at 4 °C for 48 h.     

A comparison of the protective action of chicken and quail egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa

Egg yolk-based diluents provide adequate cryoprotection for the sperm of several mammalian species. Traditionally, chicken egg yolk has been used as additive for the freeze preservation of spermatozoa because of its wide availability. Variations in the chemical composition of the egg yolk of different avian species appear to influence the protection afforded during cooling, freezing, and thawing. The aim of the present study was to assess the use of quail egg yolk as a novel additive for the epididymal spermatozoa of a threatened wild ruminant species—the Spanish ibex—and to compare its efficacy with chicken egg yolk. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w v⁻¹), 2.2% citric acid (w v⁻¹), 0.6% glucose (w v⁻¹), 5% glycerol (v v⁻¹), and 6% egg yolk (v v⁻¹). Sperm masses from the right epididymes were diluted with TCG-6% chicken egg yolk medium, while those from the left were diluted with TCG-6% quail egg yolk. The thawed spermatozoa preserved with TCG-6% quail egg yolk extender exhibited lower motility (P < 0.001), membrane integrity (P < 0.001), and viability (P < 0.01) than those diluted with the TCG-6% chicken egg yolk extender. The fertility of spermatozoa frozen in TCG-6% chicken egg yolk tended to be higher than in those frozen with TCG-6% quail egg yolk (63.3% vs 36.4%, P = 0.19). These results show that quail egg yolk offers no advantages over chicken egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.     

Distribution and phospholipase activity of Candida species in different denture stomatitis types

The aim of this study was to evaluate the correlation between frequency and phospholipase activity of Candida species and denture stomatitis according to Newton’s classification. Seventy-five complete denture wearers were evaluated for the presence of yeasts on the palatal mucosa by culture method. In addition, the number of yeast isolates producing phospholipase and amount of this enzyme were determined using egg yolk agar plate method. According to Newton’s classification, 25 denture wearers were with healthy palatal mucosa while 50 were with any types of denture stomatitis. The frequency of yeasts was linked to whether subjects had Type II or Type III, but not Type I denture stomatitis. Candida albicans was the most frequently isolated species in denture wearers with and without clinical signs of denture stomatitis and it was the only species produced phospholipase. Although the amount of phospholipase produced by the C. albicans isolates from denture wearers in control and Type II and III DS groups was not significantly different, there was statistically significant difference in the number of C. albicans isolates producing phospholipase between patients with and without clinical signs of DS.     

Formation of Protoplasts from Cultured Tobacco Cells and Arabidopsis thaliana by the Action of Cellulosomes and Pectate Lyase from Clostridium cellulovorans

The crude culture supernatants from Clostridium cellulovorans were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and Arabidopsis thaliana. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from C. cellulovorans was more effective than those of commercial enzymes based on protein content.     

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Vibrio mimicus are the reservoirs of the heat-stable enterotoxin gene (nag-st) among species of the genus Vibrio

Using a 0.27 kb DNA probe specific for the heat-stable enterotoxin gene (nag-st) of Vibrio cholerae non-O1, 1109 strains representing 17 species of the genus Vibrio, isolated from clinical and environmental sources were examined. The nag-st gene was preponderantly associated with strains classified as V. mimicus; 16.8% of these strains hybridized. It was more frequent in the clinical isolates (22.6%) than in the environmental isolates (13.7%). The incidence of nag-st gene-positive strains of V. mimicus isolated from different countries was uniformly high and ranged between 8.7% (Bangladesh) and 57.1% (environmental strains from USA). The incidence of the nag-st gene was much lower among strains of V. cholerae non-O1 (3.6%). Probe-positive and-negative strains of V. mimicus and V. cholerae non-O1 were used to evaluate the performance of the conventional suckling mouse assay for detection of the NAG-ST enterotoxin. Of the 31 probe-positive strains, only five (16.1%) yielded a positive fluid accumulation ratio (FA ratio) when neat heated culture supernatant was used to perform the suckling mouse assay. All the 31 probe-positive strains gave a positive FA ratio when 20-fold concentrated and heated culture supernatants of the strains were used to perform the suckling mouse assay. The need to concentrate (by at least 20-fold) the culture supernatant of strains of V. mimicus and V. Cholerae non-O1 was identified as an important step to obtain consistent results when using the suckling mouse assay for detection of NAG-ST.P. Yuan, A. Ogawa and T. Takeda are with the Department of Infectious Disease Research, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154, Japan; P. Yuan is also with the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. T. Ramamurthy and G.B. Nair are with the National Institute of Cholera and Enteric Diseases, Calcutta, India. T. Shimada is with the National Institute of Health, Tokyo 141, Japan. S. Shinoda is with the Faculty of Pharmaceutical Sciences, Okayama University, Japan.     

Production of proteinase and phospholipase by Paracoccidioides brasiliensis

We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis. Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 °C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 °C. After 30 days net enzyme activity was visualized and quantitavely evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it. Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P. brasiliensis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Serum and Egg Antibody Responses in Chickens to Escherichia coli

The effects of Freund’s adjuvants on antibody production in chickens against E. coli whole cells were examined. The levels of anti-E. coli IgG antibodies in serum were higher when Freund’s complete (FCA) or incomplete adjuvant (FIA) was administered than that without adjuvant. Production of antibodies recognizing E. coli cells and their lipopolysaccharide was enhanced by FIA, while both FIA and FCA enhanced production of antibodies recognizing outer membrane components. In contrast, serum IgM antibody levels were higher when no adjuvant was used. Anti-E. coli IgG antibodies in serum were efficiently transferred to egg yolk, giving antibody activity in egg yolk similar to that in serum. However, anti-E. coli IgM antibodies were not detected in the egg, suggesting that egg (white) IgM was not influenced by antigenic stimulation of the humoral immune system. Antimicrobial activity of the egg yolk IgG was highest when the bacteria antigen was injected with FIA.     

Isolation and Partial Characterization of Bacteriophages of the Phytopathogen Pseudomonas syringae

Bacteriophages isolated from culture supernatants of Pseudomonas syringae pv. syringae and from sewage were identified. The DNA from each phage was isolated and digested with the restriction endonuclease EcoRI. Eight isolates were determined to be different, with two phage isolates from sewage having restriction patterns identical to two phages from culture supernatants. The sizes of the phage DNA ranged from 24 to49 kilobases for isolates from sewage and from 39 to 52.5 kilobases for the isolates from culture supernatants. Buoyant densities of phage particles in CsCl varied from 1.498 to 1.507 g/cm³ for isolates from sewage and from 1.506 to 1.516 g/cm³ for isolates from culture supernatants. Electron microscopy revealed four morphological types. Based on plaque-forming ability of culture supernatants, 31 out of 47 strains of P. syringae are probably lysogenic.     

Comparison between aseric and seric culture-derived exoantigens of Babesia divergens in their ability to induce immunoprotection in gerbils

Babesia divergens Rouen 1987 was cultivated with a high percentage of parasitized erythrocytes (30–40%) in either RPMI 1640 supplemented by 10% human serum or in a serum-free medium consisting of RPMI 1640 supplemented with 5 g/l Albumax I®. Analysis of serum and Albumax culture supernatants, using polyacrylamide gel electrophoresis, revealed the presence of at least 10 parasitic exoantigens of B. divergens with molecular weights ranging between 27 and 200 kDa. The gerbils were injected twice, at 3-week intervals, with Albumax culture supernatants or seric culture supernatants. The vaccine doses ranged from 3 μl to 1.5 ml. The highest immunofluorescent antibody titers in gerbils (in 42 days) were obtained using Albumax supernatant and Quil A saponin as adjuvant. Analysis of the gerbil humoral response by immunoprecipitation showed that only three exoantigens were immunodominant: 92 kDa, 50 kDa and 37 kDa proteins. The gerbils were challenged 3 weeks after the last vaccine injection and the maximum protection was observed with vaccine doses ranging from 30 μl to 1.5 ml of culture supernatant and Quil A adjuvant. Albumax medium-derived antigens potentiated better protection at lower dose rates than that of serous medium-derived antigens (for example the gerbil mortality was 0% when they are immunized with 30 μl of Albumax supernatant and 100% with 30 μl of seric supernatant).     

Assembly of double-shelled rotaviruslike particles by simultaneous expression of recombinant VP6 and VP7 proteins.

Simultaneous coinfection of Spodoptera frugiperda cells with baculovirus recombinants containing the genes for VP6 and VP7 of bovine rotavirus strain C486 resulted in the production of spherical particles resembling smooth, double-shelled rotavirus. These particles were predominantly located in the cell culture supernatant instead of being cell associated. Pretreatment of infected-cell culture supernatants with CaCl2, prior to particle purification, was found to enhance the smooth appearance of the particles. The authenticity of the proteins making up the particles was demonstrated by their reactivity with antiserum specific for double-shelled C486 rotavirus.     

Culture of equine embryos in media containing egg yolk, mare's milk and saline: Preliminary results

A medium containing egg yolk, mare's milk and/or modified PBS was used to culture Day-8 to 8.5 equine blastocysts. Twenty-one variants of the medium containing different concentrations of the 3 components were prepared. Embryos were recovered nonsurgically and placed into the media at 37 degrees C for 24 h. A total of 45 embryos was cultured; of these 7 died in culture and 13 showed inadequate development at the onset, while 25 continued to grow in the media. It was established that embryos grew best in media containing 20 to 60% yolk, 20 to 60% mare's milk and/or 20 to 60% PBS. It was found experimentally that egg yolk was the main component of the media, while mare's milk and PBS were interchangeable. Two mares became pregnant after transfer of 1 cultured blastocyst per mare. One of the mares lost the fetus at 9 mo, while the other carried the fetus to term and foaled normally.