Bovine Serum Albumin In Saline Research Articles

Evaluation of Calcium-Free Tyrode's Sperm Capacitation Medium for Use in Bovine In Vitro Fertilization

Our objective was to determine if a bovine sperm capacitation technique, developed with zona-free hamster oocytes, could be used for the in vitro fertilization of in vitro matured bovine zona-intact oocytes. Bovine cumulus-enclosed primary oocytes from 2- to 5-mm follicles were matured in tissue culture Medium 199 containing Earle's salts and bicarbonate and supplemented with 10% fetal calf serum, FSH (10µg/ml),and estradiol-17β (1.5µg/ml) for 24h at 37°C under paraffin oil. Ejaculated bovine sperm, washed thrice in bovine serum albumin-saline (pH 7.6) and capacitated for 4h in Ca++-free Tyrode's medium (pH 7.6), were diluted to 2×106 sperm/ml in Medium 199 supplemented with 10% fetal calf serum. Oocytes were added (10/500 µ1 droplet) to this medium containing the capacitated sperm, freeze-thawed killed sperm, or no sperm and incubated for 8h before transfer to fresh medium and then incubated for 40h. At the end of each incubation, a portion of the oocytes were stained and evaluated for development or fertilization. After 24h of culture, 49% of the oocytes had matured (metaphase II). Fertilization rates were 55.6% after exposure of all oocytes to Ca++-free Tyrode's capacitated sperm and 82.5% if only metaphase II oocytes were selected. The parthenogenetic controls were negative (1.4% and 0%). Therefore, the Ca++-free Tyrode's sperm capacitation technique can be used for bovine in vitro fertilization studies.

Induction of Bovine Sperm Capacitation by TEST-Yolk Semen Extender

Ejaculated bull semen was diluted 1:10 in the TEST-yolk buffer, cooled slowly to 4°C, and stored for up to 48h. Aliquots were taken at 0, 4, 8, 16, 24, and 48h and washed once or three times in bovine serum albumin-saline and the sperm pellets resuspended in this saline. Fertilization of zona-free hamster oocytes was used to assess sperm capacitation. Motility differed between samples washed once or three times (53.7 vs. 21.7%). Motility was highest at 4h storage but did not differ between 16, 24, or 48h of storage. More sperm without intact acrosomes were found at 4h than at 0h, but the percentage did not change further until after 24h. Penetration of oocytes was not different between sperm washed once or three times (28.5 vs. 26.9%). No penetration occurred at 0h, and highest penetration rates occurred at 4 and 8h of storage (32.1 and 33.4%). Penetration rates at 16, 24, and 48h were not different (25.3, 25.2, 22.5%). In conclusion, storage of bull sperm in TEST-yolk buffer for 4 to 48h resulted in capacitation. Even though capacitation was induced by 4h, at least 71% of the sperm population had not undergone an acrosome reaction by 48h of storage. This may explain why penetrability was maintained over this period.

Identification of the Capacitating Agent for Bovine Sperm in Egg Yolk-TEST Semen Extender

Bovine sperm can be capacitated in egg yolk-TEST buffer. To determine what constituent of the buffer was responsible, ejaculated semen was diluted 1:10 at 37°C with the following 20% egg yolk (vol/vol)-containing buffers: TES-Tris, TES-tetramethylammonium hydroxide, taurine-Tris, citric acid-Tris, citrate, egg yolk salts, egg yolk proteins Tris, and citrate-taurine. Buffers were pH 7.6 and 321 to 325 mOsmol/kg. Extended semen was cooled slowly to 4°C and stored 8h. Sperm taken at 0 and 8h were washed in pH 7.6 bovine serum albumin-saline and assessed for motility and capacitation using zona-free hamster eggs. Sperm motilities at 0 and 8h were similar (60 to 73%) in all extenders except citric acid-Tris (54%) and egg yolk proteins Tris (15%). Bull sperm, stored 8h in egg yolk-TEST, became capacitated. Because sperm storage in egg yolk-citrate did not result in penetration, both egg yolk and citrate were ruled out as capacitating agents. Capacitating activity resided in the TES and Tris molecules. The TES molecule contains a Tris component and this capacitated bull sperm. The TES molecule also contains a taurine component. However, taurine was not a capacitating agent for bull sperm. In conclusion, both TES- and Tris-containing buffers, alone or together (TEST), were equally effective in capacitating bull sperm.

Effect of Washing and Capacitating Media pH on Bull Sperm Motility, Acrosome Integrity, and Ability to Penetrate Zona-Free Hamster Oocytes

Bovine-ejaculated sperm were washed thrice in bovine serum albumin-saline media, pH 7.2 to 8.4, and incubated at 37°C in Ca++-free Tyrode's media, pH 7.2 to 8.4, for 0, 2, 4, 6, and 8h. Motility was highest when sperm were washed in pH 7.2 medium and incubated in pH 8.0 or 8.4 media. Motility remained above 50% until 8h. Washing in pH 7.6, 8.0, or 8.4 media induced more acrosome reactions after incubation than washing at pH 7.2. Percentage of acrosome-reacted sperm increased at each successive time period. Sperm penetrated more oocytes at 4, 6, and 8h when wash medium pH was fixed at 7.2 and capacitating media pH was raised at .4 unit increments from 7.2 to 8.4. When sperm were washed in pH 7.2 medium, the postincubation penetration rates peaked at 8h. With wash media of pH 7.6, 8.0, or 8.4, the postincubation penetration rates peaked at 4h and then gradually declined. In conclusion, the most effective system for capacitating bull sperm was a pH 7.6 wash followed by capacitation in pH 7.6 medium for 4 to 8h and this system resulted in the highest penetration rates. Wash media pH hastened capacitation but was not a capacitating agent.

Paradoxical relationship between atriopeptin plasma levels and diuresis-natriuresis induced by acute volume expansion.

Surgical removal of one or both atrial appendages was employed in rats to reduce the intrinsic stores of atriopeptin (AP). In conscious rats (with intact baroreceptor reflexes), bilateral or unilateral atrial appendectomy suppressed the diuresis and natriuresis produced by acute volume expansion. Surprisingly, volume expansion (with 4% bovine serum albumin in saline at 1.5 ml/kg per min for 15 min) did not result in an increase in plasma AP immunoreactivity (APir) in control or atrial-appendectomized conscious rats. Previous studies demonstrated that acute volume expansion in anesthetized animals caused increased plasma APir. Indeed, we found that volume expansion causes comparable diuresis-natriuresis in conscious and chloral hydrate-anesthetized rats, but only the latter group exhibits an increase in plasma APir. Brattleboro rats, which are deficient in vasopressin, exhibit the same response as Long-Evans controls in that acute volume expansion in conscious animals produces a pronounced diuresis and natriuresis but no APir release, but when these same animals are anesthetized, there is a simultaneous induction of diuresis-natriuresis and APir release by volume expansion. Plasma AP does not increase in conscious rats despite a large volume load, 30-40% of the total blood volume given in 15 min, and the natriuresis-diuresis appears to also be independent of vasopressin. On the other hand, the diuresis induced by acute volume expansion in anesthetized rats seems dependent on the elevated APir, since rats made autoimmune to AP (which are nonresponsive to exogenous AP infusions) exhibit a diuresis in conscious but not anesthetized rats. We therefore conclude that the participation of AP in volume homeostasis is more likely in pathophysiological states and that another mechanism or possibly another atrial factor mediates the diuresis-natriuresis induced by volume expansion in conscious rats.

Factors affecting massive postmortem bronchoconstriction in guinea pig lungs.

To examine endogenous factors affecting the development of the massive bronchoconstriction in the postmortem guinea pig lung, 58 anesthetized open-chest animals were divided into three groups: 1) exsanguination only (n = 13), 2) pulmonary perfusion with 5% dextran and 1% bovine serum albumin (BSA) in Tyrode's solution (Ca2+ perfusate) (n = 21), and 3) pulmonary perfusion with 5% dextran and 1% BSA in saline (Ca2+-free perfusate) (n = 24). These groups were further divided into several subgroups according to treatments: 1) substance P depletion by chronic administration of capsaicin, 2) acute capsaicin treatment to release substance P, 3) dazoxiben treatment to block endogenous synthesis of thromboxane A2, 4) diethylcarbamazine treatment to eliminate leukotriene (LT) synthesis, and 5) FPL 55712 treatment to antagonize actions of LT. Vital capacity from the deflation pressure-volume (PV) curve of the lung was used as the indicator of bronchoconstriction. Most PV curves were performed for 30 min following exsanguination or artificial perfusion. Ca2+-free perfusate enhanced the airway spasm at 5-10 min, but the spasm disappeared gradually after 10 min. Substance P depletion significantly decreased (P less than 0.01) the bronchial constriction at 20-30 min, whereas substance P release induced severe airway spasm (P less than 0.01) during the entire study. In addition, FPL 55712 reduced the bronchospasm (P less than 0.05) in Ca2+ perfusate at 30 min. Thus Ca2+ and several endogenous mediators may be involved with the airway spasm of the postmortem guinea pig lung.

Studies on Phaseolus vulgaris phytohemagglutinin : Structural requirements for simple sugars to inhibit the agglutination of human group A erythrocytes

Gilles Dupuis and Benoit Leclair Ddpartement de Biochimie, Universitd de Sherbrooke, Sherbrooke, Qudbec, JI H 5N4 Canada Received 7 May 1982 Phytohemagglutinin specificity Erythroagglutination Haptenic sugars 1. INTRODUCTION Lectins are proteins which can act as cell agglu- tinins and, in some cases, can induce lymphocyte transformation [1-3]. These properties are related to their ability to act as sugar-recognizing proteins. A classification of lectins, based on their sugar spe- cificity, has been proposed [4]. Phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris (red kid- ney beans), possesses a large sugar binding site which enables recognition of complex oligosaccha- ridic structures at the cell surface [5-7]. There are conflicting reports concerning the ability of PHA to specifically recognize monosaccharides [5-14]. Furthermore, it has been claimed that the lectin does not show specificity towards agglutination of human erythrocytes [4,12]. Here, we have investigated the specificity of PHA towards agglutination of the major human erythro- cyte blood groups using the technique in [15]. Our results show that PHA agglutinates preferentially human erythrocytes of group A. A series of mono- saccharides have been used to assess the structural requirements for monosaccharides to inhibit the agglutination reaction. Results show that the lectin can distinguish between opposite configuration or substitution at positions 2 and 4 of the pyranose ring. Abbreviations: PHA phytohemagglutinin; PBSA, phos- phate-buffered saline-bovine serum albumin 2. MATERIALS AND METHODS Phytohemagglutinin prepared according to [16], was purified on a Sepharose-thyroglobulin column as in [17]. The lectin appeared to be homogeneous by polyacrylamide gel electrophoretic analysis at pH 9.3 and 413. Mono-, di-, tri- and tetrasaccharides were obtained from Sigma (St Louis MO) or from PL-Biochemicals (Milwaukee WI). l-Amino-l-de- oxy-D-galactopyranose was prepared as in [18]. Blood obtained by venipuncture was centrifuged (10 min/600 x g), plasma and buffy coat were removed and the erythrocyte suspension was dilu- ted (1:10) with Dulbecco's phosphate-buffered sa- line containing 0.5% (w/v) bovine serum albumin (PBSA) and centrifuged. An additional wash with PBSA was performed and the red blood cells were diluted to 2 x 106/ml. Agglutination assays were performed according to [15]. Various amounts of PHA (1 mg/ml) in phosphate-buffered saline were deposited, in du- plicate, in the wells of a 'tissue cluster' plastic box (Costar, Cambridge MA) and the volume was completed to 0.1 ml with PBSA. Erythrocytes (2 x 105 cells) in PBSA (0.1 ml) where then added. The box was gently agitated (wrist-action shaker) over- night, at 4°C. The cells were carefully aspirated with a short (14.5 cm) Pasteur pipette and deposited into a spectrophotometric cuvette. Volume was completed to 1 ml by addition of PBSA (0.8 ml). Readings were performed at 550 nm. An interval of -3 s elapsed between dilution and absorbance readings. For inhibition of agglutination, PHA (8-10 #g) was added to individual wells of the tissue Published by Elsevier Biomedical Press 00145793/82/0000-0000/$2.75 © 1982 Federation of European Biochemical Societies 29

Persistence of human rhinovirus infectivity under diverse environmental conditions.

The persistence of human rhinovirus type 2 and type 14 infectivity was studied under various laboratory conditions designed to mimic those commonly found in the environment. The effects of temperature, ionic strength, protein content, and evaporation were compared. Both viruses were stable (less than 0.3-log decrease in titer) at 6 and 23 degrees c for 24 h in the liquid state regardless of salt or protein additives; a titer decrease of less than 1.0 log was noted at 37 degrees C. However, evaporation at 37 degrees C reduced virus infectivity by 3.2 to 4.5 logs in buffered water, an effect which could be significantly lessened by the addition of bovine serum albumin in saline (2.0- to 2.9-log decrease in titer). These studies support and extend observations by others that the human rhinoviruses retain sufficient infectivity after drying on hard surfaces to permit their transmission to susceptible persons upon contact.

Induction and Mechanism of Tolerance to Bovine Serum Albumin in Mice Given Total Lymphoid Irradiation (TLI)

Abstract BALB/c mice given total lymphoid irradiation (TLI) were injected i.p. with bovine serum albumin (BSA) in saline, and challenged with DNP-BSA in complete Freund's adjuvant 6 weeks later. The latter animals made no anti-DNP antibody response as measured by a modified Farr assay, but made a normal anti-DNP response after challenge with DNP-BGG in adjuvant. Normal mice or mice given whole body irradiation were not tolerized by the i.p. injection of BSA in saline. Spleen cells from unresponsive mice (TLI + BSA in saline) suppressed the adoptive secondary anti-DNP response of sublethally irradiated syngeneic hosts given BSA-primed T cells, DNP-BSA-primed B cells, and DNP-BSA in saline. The suppressor cells were antigen specific, and were inactivated by in vitro treatment with anti-Thy 1.2 antiserum and complement. The findings suggest that soluble antigens administered to mice after TLI evoke a state of tolerance that is maintained by antigen-specific suppressor T cells. A similar mechanism may be involved in the maintenance of tolerance to allografts. These findings may have important clinical implications for patients treated with TLI for lymphoid malignancies.

Selective detection of humoral antibodies in bronchial asthma using fluorescent antibody technique.

In patients with asthma, various types of antibodies can be differentiated according to their chemical nature and biological activity. For example, electrophoresis shows that reagin is in the y-1A fraction and blocking antibody in the 72-fraction. The former participates in anaphylaxis, and the latter suppresses anaphylaxis. Although they both combine with antigen, their mechanisni of combining may differ somewhat. On the basis of these characteristics, a new plan for the selective detection of these antibodies was outlined and is described in this report. Sensitized erythrocytes can be used for thc purpose of giving form to soluble allergens and of concentrating the allergens on a cell surface or at cell boundaries. These sensitized erythrocytes presumably become surrounded by antibody when they come into contact with the serum of an asthmatic patient, and the antigenantibody complexes should be visible when the fluorescent antibody technique is eniploycd with the use of labelled anti-human 7-globulin. It was hypothesized that with the use of fluorescent anti-human 7-1A globulin reagins could be detected specifically, and with the use of fluorescent antihuman y2-globulin, blocking antibody. The first step was to show that asthma antibodies can be demonstrated on sensitized erythrocytes with fluorescent antibody technique. Procedure Antigen: Commercial housc dust extract was used mainly. Sensitized erythrocytes: Human erythrocytes of blood type were used. A medium containing tannic acid or bidiazotized benzidine (BDB) was used to sensitize the erythrocytes by Boyden'sl and by Stavitsky's2 methods as previously described. Antigen-antibody reaction on smear preparations: Sensitized erythrocytes were resuspended in 0.2% bovine serum albumin saline solution, and smeared on thin glass slides. After quick drying in front of an electric fan, smears were fixed in absolute alcohol at -70°C, then covered with a patient's serum, sealed with a cover glass to prevent drying. The preparation was left in a moist chamber at 0 5°C for several hours. After prolonged and careful washing, the preparation was fixed again in absolute alcohol at -70°C.