Absence of a Formate-Induced Release of Bicarbonate from Photosystem II

Formate has been proposed to inhibit electron flow in photosystem II by replacing endogenous bound bicarbonate on the reaction center complex. A mass spectrometer was used to measure directly the CO₂/HCO₃⁻ released when maize thylakoids, showing normal rates of electron flow, were treated with formate. Although the formate inhibited electron flow by 95%, no release (displacement) of CO₂/HCO₃⁻ was detected. This is consistent with the concept that membrane-bound HCO₃⁻ is not a requirement for normal rates of electron flow through photosystem II. Moreover, formate and other monovalent anions do not inhibit electron flow by removing bound HCO₃⁻ but by binding to empty sites. The “bicarbonate effect” is a reversal, by high concentrations of exogenous bicarbonate, of anion inhibition of photosystem II.     

Leghemoglobin in Lupin Plants (Lupinus albus cv Multolupa)

Leghemoglobin was localized by immunogold techniques in nodules of Lupinus albus cv Multolupa inoculated with Bradyrhizobium sp. (Lupinus) strain ISLU 16. The protein localization was performed in nodules embedded in Spurr's and Araldite epoxy resins and Lowycryl K4M. A very good preservation of both the ultrastructure and antigenicity was obtained with the tissues embedded in Araldite following glutaraldehyde fixation and unpostfixed in osmium tetroxide. Lupin leghemoglobin is a stable and abundant protein which allows a conventional method to be safely used for localization of leghemoglobin. Labeling of leghemoglobin was specifically confined to the cytosol matrix and nuclei. Gold particles were never observed in the peribacteroidal spaces nor in the cytoplasmic organelles of the infected cells. Decrease of leghemoglobin was observed when the plants were grown with 10.7 micromolar and 21.4 micromolar of nitrate.     

Cytochemical localization of calcium in renal sac nephrocytes of Helix aspersa

In renal sac nephrocytes of Helix aspersa, intracellular calcium has been localized using the oxalate-pyroantimonate (OPA) and phosphate-pyroantimonate (PPA) methods. Pyroantimonate precipitates are preferentially localized in the excretory spherule and in vesicles located in the basal and lateral regions of the nephrocyte. Such vesicles appear to release their content into the excretory vacuole. Calcium may interact with diverse types of molecules present in the excretory vacuole, thus favouring stabilization and packaging of the excretory spherule.     

Electrofusion parameters for mouse two-cell embryos

We studied electrofusion of mouse two-cell embryos in order to define parameters which would result in a high yield of fused embryos. Various cell alignment times (from <10 to >60 s) and alternating current percentages (2 to 100%) were examined. The fusion parameters tested were the number of fusion pulses (1-9), pulse length (30-90 mus) and pulse strength (0.50-1.79 kV/cm). Furthermore different combinations of these three parameters were tested. In addition the influence of several embryo culture media on the fusion rates was examined. The results show that the fusion rate of the embryos increases with shorter alignment and higher percentages of the alternating current. The highest fusion rate (95%) was obtained by use of one pulse with a duration of 70 mus and a field strength of 0.60-0.79 kV/cm. The survival rate of the embryos was best if Whitten Medium was used before and after the fusion pulses. The fusion of two-cell stages results in tetraploid embryos which can serve as models for studies in polyploid cells.     

The use of synthetic gonadotropin releasing hormone treatment in the collection of sheep embryos

Gonadotropin releasing hormone (GnRH) treatment was examined as a means of improving the efficacy of embryo collection in the sheep following intrauterine insemination of frozen-thawed semen. In summary, treatment consistently improved fertilization rates and the number of fertilized ova collected per ewe was enhanced compared with untreated ewes. The yield of fertilized ova in ewes treated with follicle stimulating hormone (FSH) was maximized by administering GnRH 36 h after progestagen treatment; 24 h was the preferred time in ewes treated with pregnant mare serum gonadotropin (PMSG). There was a significant (P < 0.001) increase in the percentage of unfertilized ova in the former treatment when GnRH was given at 24 h. An examination of the time of insemination (0, 6, 12 and 18 h before the median time of ovulation) indicated that fertilization rates were highest when insemination occurred at 6 h in both GnRH-treated ewes and in untreated ewes. In GnRH-treated ewes, the recovery of ova was lowest when insemination occurred at the time of ovulation. The number of motile frozen-thawed spermatozoa required for fertilization following treatment was estimated to be approximately 20 x 10(6) per uterine horn. GnRH-treatment also improved the yield of fertilized ova in sheep that were naturally mated, although this yield was lower than that obtained with intrauterine insemination of frozen-thawed semen. It is concluded that fertilization failure, a major problem in sheep embryo collection, can be eliminated through judicious use of GnRH treatment and properly timed intrauterine insemination.     

Induction of ovulation in gilts with cloprostenol

Prepuberal gilts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. In this model, ovulation occurred at 42 +/- 2 h post hCG treatment. When 500 mug of cloprostenol was injected at 34 and of 36 h after hCG injection, 78% of the preovulatory follicles ovulated by 38 h compared with 0% in the control gilts. In addition, plasma progesterone concentrations were significantly higher in the cloprostenol-treated group than in the control group (P<0.01) at 38 h, indicating luteinization along with premature ovulation. These results suggest that prostaglandin F(2)alpha (PGF(2)alpha) or an analog can be used to advance, synchronize or induce ovulation in gilts.     

Fertility of Bos indicus and Bos indicus x Bos taurus crossbreed cattle after estrus synchronization

Ninety-five cows (79 Boran and 16 Boran-Brahman crossbreeds) and 107 heifers (55 Boran and 52 Boran x Friesian F1 crossbreeds) were used to determine estrus response, estrus response interval and pregnancy rate following synchronization with prostaglandin (PGF(2)alpha), a progesterone-releasing intravaginal device (PRID) and Synchro-mate B (SMB). The proportion of cattle responding to synchronization treatment was 62.5, 43.5 and 57.7% for cows and 85.7, 68.0 and 81.5% for heifers using PGF(2)alpha, PRID and SMB, respectively. The overall mean response was 59 and 81.8% for cows and heifers, respectively. The estrus response of the control animals over a 45-d breeding period was 72.7 and 90% for cows and heifers, respectively. The estrus response interval for cows was 31.8, 22.1 and 18.0 h and it was 51.1, 38.0 and 21.6 h for heifers with PGF(2)alpha, PRID and SMB treatment, respectively. Mean pregnancy rate for cows was 50.0, 34.8, 46.2 and 68.8% and for heifers it was 60.7, 40.0, 55.6 and 77.8% in the PGF(2)alpha, PRID, SMB and control groups, respectively. Based on these findings, it was concluded that both PGF(2)alpha and SMB produce a satisfactory estrus response and pregnancy rate in the cattle studied.     

The effects of estrous cow serum on the in vitro maturation and fertilization of the bovine follicular oocyte

The effect of serum obtained from a cow at the time of standing estrus (serum A), at ovulation (serum B), and at 24 h after ovulation (serum C) on the in vitro maturation and fertilization of bovine oocytes was examined. Of 144 (Group A), 159 (Group B), and 158 (Group C) oocytes, 77 (53.4%), 82 (51.6%) and 82 (51.9%) oocytes were characterized by expansion of cumulus cells, respectively. There was no significant difference in the effect of the three types of cow serum on the cumulus expansion (P < 0.05). Of 461 oocytes, 316 oocytes were cultured with sperm for fertilization, and 145 oocytes were cultured without sperm for evidence of parthenogenetic development. Of 56 (Group A), 56 (Group B), and 62 (Group C) oocytes with expanded cumulus cells, 19 (33.9%), 7 (12.5%), and 11 (17.7%) oocytes were cleaved, respectively, after exposed to the sperm for 24 h. There was a significant difference in the effect of the three types of cow serum on the fertilization rate (P < 0.05). A total of 145 oocytes was cultured in the absence of sperm and no evidence of parthenogenetic division was observed. The effect of the three types of serum obtained from the cow on the maturation of oocytes was not significant, but a significant difference did exist in the fertilization rate of oocytes. Cow serum obtained at the time of standing estrus had a beneficial effect on the fertilization rate of oocytes in vitro.     

The efficacy of estrus detection and fertility following synchronization with PGF2a or synchro - mate- B in Zebu cattle

Two experiments were carried out to assess the efficacy of estrus detection and fertility in Zebu cattle after synchronization with prostaglandin F2a or a progestagen. The first experiment compared estrus detection rates and fertility following insemination in 42 cows previously synchronized with either 25 mg of PGF2a or with a 6 mg of Norgestomet implant plus 5 mg i.m. of estradiol valerate (SMB). Differences were observed in the percentage of cows detected in estrus (54 vs 95%, respectively, P < 0.05), but not in fertility at the first synchronized estrus (26 vs 15%), nor in the detection rate and fertility at the subsequent estrous period (38 v 47%). The second experiment evaluated the efficacy of estrus detection at different time intervals in 30 cows, comparing estrus synchronized with PGF2a with the subsequent estrous period. The observation periods were continuous, day and night, for 100 h both after PGF2a treatment and from Day 18 of the treatment cycle (Period 1). In addition, the animals were administered PGF2a again on Day 10 of the second cycle and observed continuously from 0600 to 1800 h, and from Day 18 of the treatment cycle (Period 2). Finally, the same treatment regimen was used except that the observation was between 0600 to 0700 h and 1800 to 1900 h (Period 3). No differences were obtained in the percentage of cows detected in estrus in the synchronized and nonsynchronized groups (average 75%); however, accuracy in the detection of estrus in Period 3 differed in the nonsynchronized and synchronized estrus groups by 40% (P < 0.05) compared with the other two, more intense observation periods.     

Recycle use of immobilized glucoamylase by tangential flow filtration unit

Various properties of glucoamylase immobilized onto corn stover supporting material and separation of immobilized enzyme by tangential flow filtration unit were studied. Optimum pH and temperature of immobilized enzyme were 3.5 and 60 degrees C, respectively. Enzyme stability was studied in a packed-bed column. The starch conversion rate was attained at 81% for 15 days; after that, the hydrolysis rate gradually decreased. Size of supporting material proved to be an important factor, with higher activity and good loading yield resulting from smaller supporting material. Glucoamylase immobilized onto supporting material less than 44 mum was used for hydrolysis of 10% soluble starch at pH 3.5 and 40 degrees C for 3 h. Then immobilized glucoamylase was separated from the product by means of a tangential flow filtration unit using a 0.2-mum pore size Nylon 66 membrane filter. This operation was continued until 180 ml filtrate was obtained from a 260-mL starting volume. Then, the next batch was started by adding 180 mL starch substrate into the reactor. The batchwise experiments were repeated 20 times. The average filtration rate of each batch was determined and found to sharply decline during the first four batches. Thereafter, it gradually decreased from batch to batch. The cause of decreasing filtration rate appeared to be due to retrogradation of starch. The percentage of starch hydrolysis within 20 batches was in the range 89-96%. The filtration rate becomes higher if the hydrolyzation time is extended to 14 h. Resistance to filtration was also investigated. Almost all of the total resistance is related to insoluble materials, with the significant part of this from the resistance due to insoluble materials deposited on a surface of membrane and boundary layer resistance. Using a microscopic method, no microorganisms were found in the filtrate.