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1.
Adenosine triphosphate (ATP) levels in sperm from steelhead trout (Oncorhynchus mykiss) were found to be 12.0 ± 1.4 pmol ATP per 106 sperm cells. Sperm were stored at 0–2 °C for up to 72 h under 100 and 21% O2, and 100% N2. The sperm ATP content of samples maintained under 100 and 21% O2 both decreased to about 70 and 50% of initial values after 24 and 72 h, respectively. ATP levels of sperm stored under 100% N2 decreased to 20% of initial values after only 4 h and to less than 10% of initial values after 24 h; they remained unchanged throughout the following 48 h. These low levels, however, were reversible. Following 4 h incubation under 100% N2, sperm ATP levels rose upon exposure to and maintenance under 100% O2, increasing to 50% of initial values after a total of 72 h. ATP levels in sperm maintained with cyanide for 4 h decreased to less than 5% of initial values. The magnitude of the change in sperm ATP levels after 24 h in samples maintained under 100% O2 correlated positively with the initial sperm ATP levels. Sperm ATP levels, motility, and fertility were all correlated positively in a subset of samples examined. The initial percent of living sperm (sperm viability) was 97.5 ± 0.5% and was unaltered through 24 h under all O2 tensions. Sperm viability remained unchanged through 72 h under 21% O2 and decreased by 10 and 30% by 72 h under 100% O2 and 100% N2, respectively. Therefore, reductions in sperm ATP levels could not be attributed to cell death as viability decreased only modestly over these durations. Spermatocrit values were unaltered throughout the 72 h incubation for samples maintained under 100 and 21% O2, however, a 95% increase in spermatocrit occurred after 72 h in samples maintained under 100% N2. These data indicate that intracellular ATP in steelhead trout sperm can be modulated by ambient oxygen without significant loss of viability and that this ATP has a probable role in cell motility and fertility, and sperm packing and cell flexibility.  相似文献   

2.
The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n?=?5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg?1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.  相似文献   

3.
The composition of seminal plasma and metabolism of sperm of the cyprinid fishAlburnus alburnus were investigated. Statistically significant correlations were found between motility parameters and seminal fluid osmolality, pH, Na+, K+ and protein levels (negative correlations: % immotile spermatozoa-Na+, K+; positive correlations: % motile spermatozoa-osmolality, pH, Na+, K+, protein; % linear motile spermatozoa-pH protein; swimming velocity of spermatozoa-pH, Na+, protein). Spermatozoan motility and ATP metabolism and glycolysis were correlated as indicated by measurement of ATPase, pyruvate kinase, adenylate kinase and lactate dehydrogenase activity. The physiological meanings of these correlations and their possible significance for quality control of semen are discussed.Abbreviations used ACP acid phosphatase - ADP adenosine diphosphate - AK adenylate kinase - ALP alkaline phosphatase - ASPAT aspartate aminotransferase - ATP adenosine triphosphate - ATPase magnesium dependent adenosine triphosphatase - CRPO creatine phosphate - -GLU \-D-glucuronidase - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PK pyruvate kinase  相似文献   

4.
The motility and fertilizing ability of the Persian sturgeon, Acipenser persicus, spermatozoa were investigated. Optimum ionic content (Na+, K+, Ca2+ and Mg2+) and pH of activation solution as well as the optimum dilution rate were determined. The results show optimum motility characteristics of spermatozoa in buffered solutions containing 25, 0.2, 3 and 10 mM L?1 Na+, K+, Ca2+ and Mg2+, respectively, at dilution rate 1:50 and pH 8.0. To test the fertilizing ability of sperm, two buffered saline solutions were used as activation solution of sperm motility. The present study indicated (1) spermatozoa motility is one of key factors that influence on fertilizing ability of sperm, (2) a high fertilizing ability of sperm is obtained after dilution in saline solutions rather than in freshwater and (3) a maximum fertilization rate occurs in buffered saline solution containing 0.2 mM L?1 K+. There is also a good correlation between biochemical characteristics of seminal plasma and fertilizing ability of sperm.  相似文献   

5.
The buffering capacity of air-equilibrated seminal plasma from the chinook salmon Oncorhynchus tshawytscha and steelhead Oncorhynchus mykiss were determined. Buffering capacities were significantly less at pH values at and above 7.3 than over the range 6.0 to 7.0 and were particularly low between pH values of approximately 7.5 to 8.5. The potential of chinook salmon sperm to become motile upon addition to water was highly sensitive to pH. Only very low levels of motility were demonstrable when sperm had been maintained at pH values at or below 7.5 prior to water exposure while maximum motility was noted when sperm had been maintained at pH values of about 8.0 and above. Thus, the potential for motility appeared especially sensitive to that pH range over which seminal plasma exhibited its lowest buffering capacity. Buffering capacity, protein content and inorganic phosphate content of chinook salmon and steelhead seminal plasmas were less than those of corresponding blood plasma or ovarian fluid suggesting that seminal plasma is unique among salmonid body fluids in its composition and low buffering capacity. This low buffering capacity is likely to facilitate the role of the sperm duct epithelium in semen pH regulation and thus, in the sperm's acquisition of the potential for motility upon exposure to water. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

6.
This study examined the pH sensitivity of steelhead, Oncorhynchus mykiss (Walbaum), sperm motility relative to the composition of incubation and activation media. The percentage of sperm that initiated motility following incubation in a sperm immobilizing solution (SI) titrated to different pH values and subsequent activation by dilution in buffered swimming medium (SM) at pH 8.5 or 50% ovarian fluid (OF) showed little or no pH sensitivity; sperm diluted in de‐ionized water (DI) showed no motility after incubation at any pH. In contrast, motility of sperm diluted in tap water (TAP) was highly sensitive to the pH of the incubation medium. Sperm incubated with buffered seminal plasma at high, but not low pH demonstrated high percent motility when diluted with DI. Sperm incubated in low‐pH SI demonstrated high motility only when diluted into high‐pH SM. The effects of the composition of incubation and activation media on sperm motility were generally reflected in comparable effects on fertility. Therefore, these data indicate that the pH sensitivity of sperm motility and fertility depends on the composition of commonly used incubation as well as activation media.  相似文献   

7.
Compared to control fish, gonadotropin releasing hormone-analogue (GnRHa) treatment delivered either by microspheres or cholesterol pellets successfully increased sperm production (cells kg–1) and milt volume (ml kg–1) in mature yellowtail flounder Pleuronectes ferrugineus during the spawning season. Spermatocrit decreased in both treated and control groups between 12 and 29 days post-implantation, indicating a seasonal decrease in sperm concentration, rather than an effect of the GnRHa treatments.Plasma levels of testosterone, 11-ketotestoterone and 17,20dihydroxy-4-pregnen-3-one (1720P) showed no clear pattern either across treatments or over days, however this does not exclude the possiblity that GnRHa had its effect on milt volumes via the stimulation of steroid production since the sampling protocol did not allow for the rapid clearance of steroids from the plasma. GnRHa treatment did not have a negative effect on sperm fertilizing ability, percentage hatch or larval morphology. Sperm motility and seminal plasma pH were increased by GnRHa treatment.  相似文献   

8.
Mediators and mechanisms responsible for the inhibitory modulation of trout intestinal smooth muscle were examined using a series of putative mediators and substances known to modulate neurotransmission in mammalian systems. Frequency response relationships to transmural stimulation and concentration response relationships to 5-hydroxytryptamine, carbachol, and substance P were established on paired segments of rainbow trout intestinein vitro in the presence and absence of putative modulatory agents. Modulation of neurally-mediated contractions of trout intestine was achieved with dibutyryl cyclic AMP and forskolin, agents that increase intracellular levels of cyclic AMP. The effect appears to be at the level of the smooth muscle, since the adenylate cyclase activator, forskolin, inhibited muscarinic and serotoninergic contractions as well as transmurally stimulated contractions. Substance P-induced contractions were unaffected by forskolin. The endogenous agonists/neurotransmitters which would increase cyclic AMP levels in rainbow trout intestinal smooth muscle are as yet unknown. The effects do not appear to be modulated by vasoactive intestinal peptide (VIP), calcitonin, calcitonin gene-related peptide (CGRP), or agents that activate -adrenoceptors. Prostaglandin E2 (PGE2) and 2-adrenergenic agonists are possible agents which will decrease contractility of the smooth muscle. They were only active in the proximal intestine and on transmurally stimulated contractions. The effects of both PGE2 and 2-agonists appear to be prejunctional, decreasing release of contractile neurotransmitters in the enteric nervous system.  相似文献   

9.
Extremely low levels of the maturation inducing steroid (MIS) 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP) were found in the ooplasm and ovarian follicle membranes of Atlantic salmonSalmo salar ouananiche, a finding that is at variance with the elevated blood levels of the steroid. The uptake of MIS at physiological concentrations into brook trout follicles occurred by passive diffusion. Uptake of the steroid into the ovarian follicle membrane, consisting of zona radiata and the attached follicle cells, deviated from linearity in a double reciprocal plot. These results suggest that 17,20-DHP is binding to a receptor-like protein in the ovarian follicle or the zona radiata membrane surrounding the oocyte, and extend our previous demonstration of 17,20-DHP receptor-like activity in the zona radiata membrane of the late stage brook trout oocytes. An oocyte cytoplasmic receptor gave subunits on SDS PAGE that were similar to the membrane and cytosol receptors previously described.  相似文献   

10.
Abstract Four parameters were examined in order to define sperm quality in turbot Scophthalmus maximus L., sperm: (1) sperm motility, measured by direct counts of the number of active spermatozoa, expressed as % of total spermatozoa; (2) retention of motility after activation, measured by direct counts, 0–60min after activation, expressed as a % of the initial level of activity; (3) resistance to thermal stress, measured as change in retention of motility, and (4) adenosine phosphate (ATP) concentration, determined for samples of non-activated sperm. The proportion of motile spermatozoa at activation ranged from 34·8% to 97·6% (mean 76·3%) for the individual males tested. Turbot sperm retained on average 52% (range 27–90%) of its initial activity one hour after activation. Sperm samples which were stressed by cooling to –27°C retained only 8·6% (range 0–25%) of initial activity, compared to control samples which retained 49% (range 38–63%) of initial activity. The retention of motility after activation was not significantly related to the initial motility or the levels of ATP. Concentrations of ATP in turbot sperm (mean 0·46mg ATP/106 spermatozoa, equivalent to 9·2nmol ATP/108 spermatozoa) were comparable to those measured in mammals.  相似文献   

11.
Vitellogenin (Vtg) was purified from the plasma of 17-estradiol (E2)-injected male greenback flounder,Rhombosolea tapirina. The molecular weight of the native Vtg was estimated by gel filtration as 540 kD. SDS-PAGE and Western blotting analyses indicated that this protein consisted of three bands with molecular weights of 155, 104, 79 kD, respectively. A polyclonal antibody against the highest molecular weight band of putative Vtg was generated in sheep and an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA) was developed. The assay was validatedfor plasma Vtg measurement in greenback flounder. Serial dilutions of plasma from vitellogenic females parallelled the standard Vtg curve, whereas no cross-reaction was observed with the plasma of males in the ELISA. The Vtg ELISA was used to assess the induction of Vtg by E2 in vivo in males. The induction of Vtg in greenback flounder showed a time- and dose-dependent response as in other species. In E2-treated fish, detectable levels of Vtg were first found at 48 h, and reached a peak at 96 h post-injection. Plasma levels of Vtg increased as the E2 dose increased with a threshold of 0.1 mg kg–1.  相似文献   

12.
The effects of sub-lethal doses of dichlorvos and formalin, antimicrobial/parasitic agents used in aquaculture, on lipid composition and metabolism of rainbow trout skin cells in primary culture were investigated. [1-14C]Stearic (18:0), [1-14C]lin 18:2n-6) and [1-14C]linolenic (18:3n-3) acids were used as tracers to determine effects on fatty acid incorporation and metabolism. Formalin increased cell numbers and reduced the lipid content of the cells and the incorporation of radioactive fatty acids. The effects of dichlorvos were qualitatively similar but quantitatively less. Formalin induced relatively small but significant changes in lipid class composition including a decreased proportion of phosphatidycholine with increased proportions of sphatidylethanolamine and phosphatidylserine. Dichlorvos had no significant effect on lipid class compositions. The trout primary skin cells expressed substantial 9, 6 and 5 fatty acyl desaturase activities. Although, as expected, the cells were m active towards [1-14C]18:3n-3, the cells were unusually active towards [1-14C]18:2n-6. Both dichlorvos and, especially, formalin appeared to significantly inhibit 9 and 6 desaturation. Changes in the distribution of radioactivity between individual spholipid classes was also influenced by formalin and dichlorvos, and this may be related to changes in desaturase activity. This study has shown that topically active agents used in aquaculture, formalin and dichlorvos, had a range of effects on the rainbow trout skin cell cultures that may affect cell proliferation and lipid and fatty acid metabolism. Both agents significantly inhibited desaturation of fatty acids, particularly of 18:2n-6 to 20:4n-6 and, as 20:4n-6 is a major eicosanoid precursor ish and considering the importance of eicosanoids in the biochemistry of skin, it is suggested that these agents may have direct effects on fish skin that could have important consequences for fish health in general.  相似文献   

13.
Adenosine triphosphate (ATP) levels in sperm from chinook salmon (Oncorhynchus tshawytscha) were found to be 12.1±1.9 pmol ATP per 106 sperm cells (mean±SEM, n=18). Sperm were stored at 0–2 °C for up to 72 h under 100, 21, and 0% O2. Changes in sperm ATP content of samples maintained under 100 and 21% O2 were indistinguishable, decreasing to 50% of initial values after 72 h. ATP levels of sperm stored under 0% O2 decreased to 10% of initial values within 8 h and remained unchanged throughout the following 64 h. The initial percent of living sperm (sperm viability) was 94.7±1.7% (mean±SEM, n=12) and was unaltered at 24 h under all O2 tensions. At 72 h, sperm viability remained unchanged under 21% O2 and had decreased by 20% under 100% O2 and by 30% under 0% O2. Therefore, reductions in sperm ATP levels could not be attributed to cell lysis, as viability decreased only modestly over these durations. Fertilizing ability correlated positively with sperm ATP levels. Spermatocrit values increased by 20% for the samples maintained under 100 and 21% O2 after 72 h. However, a 20% increase in spermatocrit occurred within 24 h in samples maintained under 0% O2, rising 70% after 72 h under 0% O2. These data indicate that chinook sperm ATP (1) can be altered and manipulated without significant loss of viability, (2) is positively associated with fertilization, and (3) probably plays a role in sperm packing and cell flexibility.  相似文献   

14.
Effects of NH3 concentration in sea water and pH of sea water on the motility of spermatozoa obtained from testes were examined in the Japanese pearl oyster Percent motility at 30 s after dilution increased with increasing NH3 concentration in sea water from 0.75–2.0 mM. When spermatozoa were diluted with sea water containing 0.75 mM NH3, which is widely used as the insemination fluid in the hatchery of this species, the percent motility increased with time elapsed after dilution, and peaked at 5 min. For spermatozoa diluted with sea water containing 2.0 mM NH3, the percent motility increased rapidly and peaked at 30 s. The pH of sea water increased with increasing NH3 concentration from 8.2 (0 mM NH3) to 9.9 (5.0 mM NH3). When spermatozoa were diluted with artificial sea water at various pH (buffered without NH3 at 6.0–10.0), only spermatozoa diluted with artificial sea water of pH 10.0 were motile, and the percent was considerably lower than those in ammonical sea water. These results indicate that sea water containing 2.0 mM NH3 is a suitable solution for evaluating sperm motility, and that NH3 and/or ammonium ions may activate sperm motility in this species.  相似文献   

15.
The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.  相似文献   

16.
All evidence to date suggest that sperm motility is the primary determinant of fertilization success in externally fertilizing fish species. Ovarian fluid, which comprises 10–30% of the total egg volume in salmonids, enhances sperm motility with respect to swimming speed, trajectory and the duration of movement. It was recently demonstrated that there is individual variability in sperm motility enhancing potential of ovarian fluid of particular females. In the present study we examined the effect of particular ovarian fluids collected from 31 females on the sperm motility parameters of one male of rainbow trout (Oncorhynchus mykiss) using computer-assisted sperm analysis (CASA). During our experiment we also monitored the pH of ovarian fluid. We found that particular fluids differed in the ability to activate spermatozoa; sperm remained immotile in four fluids and exhibited 50–100% motility in 27 samples. The percentage of motile sperm, velocity and duration of movement positively correlated with ovarian fluid pH (r2 = 0.34–0.62). These data strongly suggest that the pH of the ovarian fluid is the primary determinant of sperm motility in rainbow trout under natural conditions of fertilization.  相似文献   

17.
We investigated the effects of open- and closed-system temperature changes on the O2 affinity of Atlantic bluefin tuna (Thunnus thynnus) blood using in vitro methods essentially identical to those previously employed on tropical tuna species. Bluefin tuna blood has a general O2 affinity (P 50 = 2.6–3.1 kPa or 19–23 mm Hg at 0.5% CO2) similar to that of skipjack tuna, yellowfin tuna, and kawakawa blood (P 50 = 2.8–3.1 kPa at 0.5% CO2) but significantly above that of bigeye tuna blood (P 50 = 1.6–2.0 kPa at 0.5% CO2). We therefore hypothesize that bluefin tuna are less tolerant of hypoxia than bigeye tuna. Further, we found the P 50 of bluefin tuna blood to be slightly reduced by a 10°C open-system temperature increase (e.g., from 4.83 kPa at 15°C to 3.95 kPa at 25°C) and to be completely unaffected by a 10°C closed-system temperature change. Bluefin tuna blood, therefore, had a significantly reduced Bohr effect when subjected to the inevitable changes in P CO 2 and plasma pH that accompany closed-system temperature shifts (0.04–0.09 Δlog P50ΔpH−1) compared with the effects of changes in plasma pH accomplished by changing P CO 2 alone (0.81–0.94 Δlog P50 Δ pH−1). This response is similar to that of skipjack tuna blood, but different from yellowfin or bigeye tuna blood. During closed-system temperature changes at oxygen levels above P 50, however, bluefin tuna blood showed a reversed temperature effect (i.e., P O 2 decreased in response to an increase in temperature). Unlike in other tuna species, temperature effects on O2 affinity of bluefin tuna whole blood were similar to those previously reported for hemoglobin solutions, suggesting that red cell-mediated ligand changes are not involved.  相似文献   

18.
Colisa lalia (Ham.) has been used as a model for the development of techniques for induced spawning that are applicable to small teleosts where ovulation requires prolonged exposure to suitable breeding conditions.Human chorionic gonadotrophin (hCG; 4 IU per fish) induced ovulation within 24 hours, whereas homologous pituitary extracts were relatively ineffective. When administered in saline ( 20 g per fish per injection), des-Gly10,[D-Ala6]- LHRH N-ethylamide (LHRHa) was ineffective, but it stimulated ovulation in a proportion of fish when administered ( 1.6 g per fish) as an emulsion in Freund's incomplete adjuvant (FIA). Together, these results suggest that ovulation requires the synthesis as well as the secretion of gonadotrophin in C. lalia.Long-term treatment with thyroid hormones appeared to enhance the ovulatory response to LHRHa in FIA, possibly by effects on the ovary; whereas the various steroids tested were ineffective at the dosages used  相似文献   

19.
Heavy metals are highly toxic elements that are present in the environment, especially in water. Mercury chloride (HgCl2) stands out among these compounds because of its strong ability to induce damage to any tissue with which it comes into contact. The gametes of spawning aquatic animals, such as fish, are susceptible to such damage. Thus, our objective was to evaluate the toxic potential of HgCl2 in the capacitation and activation of Rhamdia quelen sperm. Semen was collected from seven males and activated in 58 mM sodium chloride (NaCl) containing 0 (control), 4?10, 7?10, 7?9, and 7?8 M HgCl2. The evaluated variables included motility, vigor, motility time, morphology, membrane integrity, membrane fluidity, mitochondrial functionality, production of reactive oxygen species (ROS), and DNA fragmentation. All evaluated HgCl2 concentrations increased primary pathologies and reduced motility, vigor and motility time. Damage to membrane integrity and fluidity began occurring at a concentration of 7?10 M HgCl2. These results indicate that HgCl2 has a toxic effect on different sites of fish spermatozoa and that sperm motility decreases after exposure to HgCl2, impairing sperm capacitation and activation.  相似文献   

20.
Gastric acid secretion from isolated cod stomach mucosa was measured using a pH-static titration method. A basal acid secretion rate (BASR) of 6.0±0.6 nEqH+min–1cm–1 was measured when using 0.9% NaCl as luminal solution. There was a dose-dependent increase in response to histamine between 0.12 and 0.20 M (EC50=0.15 M), above which gastric acid secretion plateaued at 13.5±1.8 nEqH+min–1cm–1. Ranitidine, a H2-receptor antagonist, completely blocked the stimulatory effect of histamine and reduced the BASR. The H1-receptor antagonist, clemastine, did not inhibit the response to histamine. Acid secretion rates decreased significantly when the pH of the luminal side of the mucosa was lowered from pH 5.75 to pH 4.50, indicating that a negative feedback mechanism was operating. Histological staining showed that oxynticopeptic cells were uniformly distributed throughout the cardiac stomach.It is concluded that the acid secretion in the isolated stomach mucosa of cod can be measuredin vitro with a pH-static titration method. The method was used to demonstrate that the BASR is downregulated by a decrease in pH. Furthermore, we conclude that the histamine receptor in the cod stomach mucosa resembles the mammalian H2-receptor and that histamine is secreted under basal conditions.  相似文献   

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