Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.     

Ovarian follicular development in cattle selected for twin ovulations and births

Comparisons of numbers of antral ovarian follicles and corpora lutea (CL), of blood hormone concentrations, and of follicular fluid steroid concentrations and IGFBP activity were conducted between cows selected (twinner) and unselected (control) for twin births to elucidate genetic differences in the regulation of ovarian follicular development. Ovarian follicular development was synchronized among cows by a single i.m. injection of PGF2alpha on d 18 of the estrous cycle; six cows per population were slaughtered at 0, 24, 48, and 72 h after PGF2alpha. Jugular vein blood was collected from each animal at PGF2alpha injection and at 24-h intervals until slaughter. Ovaries of twinner cows contained more small (< or = 5 mm in diameter, P < 0.05), medium (5.1 to 9.9 mm, P < 0.05), and large (> or = 10.0 mm, P < 0.01) follicles and more (P < 0.01) CL than ovaries of controls. Follicular fluid concentrations of estradiol, androstenedione, testosterone, and progesterone reflected the stage of follicular development and were similar for twinner and control follicles at the same stage. Earlier initiation of follicular development and/or selection of twin-dominant follicles in some twinner cows resulted in greater concentrations of estradiol in plasma at 0, 24, and 48 h and of estradiol, androstenedione, and testosterone in follicular fluid of large follicles at 0 h after PGF2alpha for twinner vs. control cows (follicular status x time x population, P < 0.01). Binding activities of IGFBP-5 and -4 were absent or reduced (P < 0.01) in follicular fluid of developing medium and large estro-gen-active (estradiol:progesterone ratio > 1) follicles but increased with atresia. Only preovulatory Graafian follicles lacked IGFBP-2 binding, suggesting a possible role for IGFBP-2 in selection of the dominant follicle. Concentrations of IGF-I were twofold greater (P < 0.01), but GH (P = 0.10) and cholesterol (P < 0.05) were less in blood of twinners. Three generations of selection of cattle for twin ovulations and births enhanced ovarian follicular development as manifested by increased numbers of follicles within a follicular wave and subsequent selection of twin dominant follicles. Because gonadotropin secretion and ovarian steroidogenesis were similar for control and twinner cattle, enhanced follicular development in twinners may result from decreased inhibition by the dominant follicle(s), increased ovarian sensitivity to gonadotropins, and/or increased intragonadal stimulation, possibly by increased IGF-I.     

The effect of selenium supplementation on immunity, and the establishment of an experimental Haemonchus contortus infection, in weaner Merino sheep fed a low selenium diet

SUMMARY: Immunity in 12 weaner Merino sheep fed a low selenium (Se) diet (low Se sheep) was compared with that in 10 matching sheep fed the same diet but each given an intraruminal Se pellet (high Se sheep), while the sheep were housed in individual, sheltered pens. All sheep were challenged with killed Brucella abortus cells (days 0 and 28), rabbit red blood cells (days 0, 7 and 28) and corynebaclerium pseudotuberculosis toxoid (days 0 and 28), and serum antibody titres were measured weekly for 8 weeks from day 0. The sheep were then experimentally infected with Haemonchus conforfus, and slaughtered 8 weeks later. The mean antibody titre to B. abortus, measured by 4 different tests, was significantly higher in the high Se sheep on occasions during the primary immune response phase (Rose Bengal test - day 21 (p < 0.05), day 28 (p < 0.025); complement fixation - day 7 (p < 0.05); enzyme-llnked immunosorbent assay - day 14 (p < 0.01); serum agglutination - no differences), but not during the secondary phase. The mean antibody titre to rabbit red blood cells, measured by haemagglutination test, was marginally higher in the high Se sheep on day 49 (p = 0.049). The mean antibody titre to C. pseudotuberculois, measured by enzyme-linked immunosorbent assay, was not significantly different between the groups at any time during the trial. In addition, the mean invitro responsiveness of peripheral blood lymphocytes to stimulation with phytohaemagglutinin in the high Se sheep was significantly greater than that in 10 sheep from the low Se group on day 22 (p < 0.01), but not day 50. However, there were no significant differences in the mean number of sheep in which the infection with H. contortus established, time to first shedding of eggs in faeces, abomasal worm burdens at necropsy, or inflammatory response in the abomasal mucosa in the sheep in each group. The results showed that the low Se sheep produced strong overall immune responses that were largely comparable to those in the high Se sheep.     

Genetic Diversity Based on AIIozyme Alleles of Chinese Cultivated Rice

Genetic diversity was analyzed with 6 632 core rice cultivars selected from 60 282 Chinese rice accessions on the basis of 12 allozyme loci, Pgil, Pgi2, Amp1, Amp2, Amp3, Amp4, Sdhl, Adhl, Estl, Est2, Est5 and Est9, by starch gel electrophoresis. Among the materials examined, 52 alleles at 12 polymorphic loci were identified, which occupied 96.3% of 54 alleles found in cultivated germplasm of O. sativa L. The number of alleles per locus ranged from 2 to 7 with an average of 4.33. The gene diversity （He） each locus varied considerably from 0.017 for Amp4 to 0.583 for Est2 with an average gene diversity （Ht） 0.271, mid Shannon-Wiener index from 0.055 to 0.946 with an average of 0.468. The degree of polymorphism （DP） was in a range from 0.9 to 46.9% with an average of 21.4%. It was found that the genetic diversity in japonica （Keng） subspecies was lower in terms of allele＇s number, Ht and S-W index, being 91.8, 66.2 and 75.7% of indica （Hsien） one, respectively. Significant genetic differentiation between indica and japonica rice has been appeared in the loci Pgil, Amp2, Pgi2, and Est2, with higher average coefficient of genetic differentiation （Gst） 0.635, 0.626, 0.322 and 0.282, respectively. Except less allele number per locus （3.33） for modern cultivars, being 76.9% of landraces, the Ht and S-W index showed in similar between the modem cultivars and the landraces detected. In terms of allozyme, the rice cultivars in the Southwest Plateau and Central China have richer genetic diversity. The present study reveals again that Chinese cultivated rice germplasm has rich genetic diversity, showed by the allozyme allele variation.     

Observation of Interference Between Two Bose Condensates

Interference between two freely expanding Bose-Einstein condensates has been observed. Two condensates separated by approximately 40 micrometers were created by evaporatively cooling sodium atoms in a double-well potential formed by magnetic and optical forces. High-contrast matter-wave interference fringes with a period of approximately 15 micrometers were observed after switching off the potential and letting the condensates expand for 40 milliseconds and overlap. This demonstrates that Bose condensed atoms are "laser-like"; that is, they are coherent and show long-range correlations. These results have direct implications for the atom laser and the Josephson effect for atoms.     

Quantification of insulin-like growth factor binding protein mRNA using real-time PCR in bovine granulosa and theca cells: effect of estradiol, insulin, and gonadotropins

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1-5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased (P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased (P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased (P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased (P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect (P>0.10). Estradiol (3 and 300 ng/ml) decreased (P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased (P<0.05) IGFBP-4 expression, but had no effect (P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased (P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells.     

Comparison of sevoflurane and isoflurane anesthetic index in unpremedicated dogs

Anesthetic respiratory effects of sevoflurane (SEVO) were compared with isoflurane (ISO) in unpremedicated dogs. Minimum alveolar concentration (MAC), apneic concentration (AC), and anesthetic index (AI) of SEVO and ISO were determined in eight 1‐year‐old healthy dogs, weighing 19 ± 3 kg (mean ± SEM) in a randomized complete block multiple cross‐over design. Dogs were mask‐induced with either SEVO or ISO in 100% oxygen. Following endotracheal intubation, dogs were instrumented, mechanically ventilated, and MAC was determined using a tail‐clamp method. Next, spontaneous ventilation was re‐established, and anesthetic concentration was increased to determine the AC. Throughout the anesthetic event, heart rate (HR), systolic blood pressure (SAP), mean blood pressure (MAP), diastolic blood pressure (DAP), respiratory rate (RR), end‐tidal carbon dioxide (Pe ′CO₂), and oxyhemoglobin saturation (SpO₂) were recorded at 3‐minute intervals. Following AC determination, AI was calculated as AC/MAC, and dogs were allowed to recover. Each dog was anesthetized four times (twice with ISO and SEVO each) at 1‐week intervals. All data were analyzed using the two‐way anova . Multiple comparisons were performed between ISO and SEVO treatments. Statistical significance was set at p < 0.05. Significant differences were noted between agents for MAC (SEVO, 2.13 ± 0.10%; ISO, 1.38 ± 0.14%; p < 0.0001), AC (SEVO, 7.34 ± 0.13%; ISO, 3.60 ± 0.13%; p < 0.0001), and AI (SEVO, 3.46 ± 0.22; ISO, 2.63 ± 0.14; p = 0.0002). Physiologic parameters were compared between SEVO and ISO at 1MAC, 2MAC, 3MAC, and AC. No differences were noted between SEVO and ISO treatments for cardiovascular parameters (HR, SAP, MAP, DAP). Significant differences were noted, favoring SEVO, for all respiratory parameters (RR, Pe ′CO₂, SpO₂) at increasing MAC multiples. Additionally, regression analysis was conducted for physiologic variable data points. Analysis of Pe ′CO₂ data points demonstrated a significant slope difference of ?6.47 ± 1.02 (B_SEVO ? B_ISO; p < 0.0001; r² = 0.6042) favoring SEVO. While expected dose‐related ventilatory depression was noted for both agents, all the respiratory parameters for SEVO demonstrated less respiratory depression than ISO at equipotent doses. These results indicated that SEVO caused less dose‐dependent ventilatory depression than ISO, having a significantly higher AI and causing less detrimental change in pulmonary parameters at increasing levels of MAC.     

Proteolysis of insulin-like growth factor binding proteins during preovulatory follicular development in cattle

The present study was conducted to evaluate changes in follicular fluid (FF) insulin-like growth factor binding protein (IGFBP) proteolytic activity and levels of steroids and IGFBP during follicular development in cattle. Estrous cycles of cows were synchronized with two injections of prostaglandin F2alpha (PGF) 11 d apart and follicular growth monitored via daily rectal ultrasonography in order to identify the dominant follicle. All cows were ovariectomized 48 hr after the second injection of PGF. Follicular fluid was collected individually for all follicles > 5 mm and pooled for small (1 to 5 mm) follicles. Follicular fluid estradiol and androstenedione levels were greater (P < 0.05) and progesterone and IGFBP-3 levels not different (P > 0.10) in large dominant than in small (1 to 5 mm) or large (>5 mm) subordinate follicles, whereas IGFBP-2, -4 and -5 levels were less (P < 0.05) in large dominant than in small or large subordinate follicles. To evaluate proteolysis of IGFBPs, FF was incubated with recombinant human (125) I-labeled IGFBP-2, -3, -4, and -5 and proteins separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of (125)I-lableled IGFBP-2 or -3. However, cleavage of (125)I-labeled IGFBP-4 and -5 by FF from large dominant follicles was greater (P < 0.05) than by FF from small or large subordinate follicles indicating that a protease to IGFBP-4 and -5 exists in estrogen dominant follicles. We conclude that lower levels of IGFBP-2 in estrogen dominant follicles of cattle are not due to increased proteolysis, whereas decreases in IGFBP-4 and -5 levels are likely due, in part, to increased protease activity. Changes in IGFBP may alter levels of bioavailable IGFs that stimulate steroidogenesis and mitogenesis in developing bovine follicles.