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1.
钙离子载体及咖啡因对绵羊精子获能的影响 总被引：1，自引：0，他引：1
通过添加不同浓度(0.05、0.1、0.2、0.5、1、5、10μmol/L)的钙离子载体(A23187,IA)及2 mmol/L咖啡因,探讨不同作用时间其对绵羊精子体外获能的影响。结果发现,IA浓度越高,精子的体外获能率越高,顶体反应率越低,活力越低,死精子就越多;而延长作用时间对获能、顶体反应的影响不明显。IA和咖啡因共同作用后,咖啡因能够促进IA诱导顶体反应的发生。建议用IA诱导绵羊精子获能时,其浓度以0.05~0.2μmol/L为宜,作用时间1 min。 相似文献
2.
《黑龙江畜牧兽医》2016,(23)
为了探讨不同浓度肝素对塔里木马鹿精子体外获能的影响,试验随机取塔里木马鹿冻融精子,添加到SP-TALIP获能液中并添加不同浓度(0,10,20,50,100μg/m L)的肝素,在显微镜下检测0,2,4,6,8小时时的精子活力,试验采用金霉素(CTC)染色法检测精子获能率及顶体反应率等指标,探讨不同浓度肝素对塔里木马鹿精子活力、存活时间、获能率、顶体反应率的影响。结果表明:精子活力随肝素浓度升高而呈下降趋势,4 h后10μg/m L和20μg/m L肝素组精子活力显著高于其他组(P0.05),其中20μg/m L肝素组精子存活时间最长,显著高于其他组(P0.05)。随着培养时间的延长,各试验组精子获能率与对照组相比明显提高,2小时、4小时时20μg/m L肝素组精子获能率显著高于其他组(P0.05);各试验组精子顶体反应率与对照组相比有升高趋势。说明获能液中添加20μg/m L肝素可有效诱导塔里木马鹿精子体外获能。 相似文献
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为比较不同发情阶段和不同浓度的绵羊血清在绵羊体外受精过程中对绵羊精子体外获能及存活时间、受精效果的影响,试验以绵羊输卵管合成液(SOF)为基础获能液,在其中分别添加发情当天、发情结束和发情间期的绵羊血清,血清浓度分别为0%、2%、5%、10%和20%.利用金霉素荧光染色(CTC,chlortetracycline)法及体外受精2种方法检测不同血清及其浓度对绵羊精子获能的影响.结果表明:1)当在获能液中添加发情当天绵羊血清时,在10%和20%血清浓度下作用45 min的获能效果最好,其获能而且未完成顶体反应的精子率(45.28%和44.52%)显著高于0%、2%和5%血清浓度组(30.14%、28.71%和35.49%,P<0.05);2)当在获能液中添加发情结束时或发情间期的绵羊血清时,在含不同浓度的这2种血清的获能液中作用45 min后,各浓度组引起的获能精子百分率变化均不显著(P>0.05);3)在含20%发情当天绵羊血清的获能液中,精子的存活时间(18 h)显著高于含20%发情结束和发情间期的绵羊血清的获能液(11 h;11 h);4)在不同血清诱导获能后进行的体外受精的试验中,添加发情当天绵羊血清组的卵裂率和囊胚率(62.5%和26.25%)均显著高于发情结束时血清组(42.22%和11.11%,P<0.05)和发情间期血清组(37.36%和15.38%,P<005).以上结果表明:在进行绵羊体外受精试验中,采用20%发情当天绵羊血清作用45 min比采用发情结束和发情间期的绵羊血清能更好地诱导精子获能. 相似文献
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《当代畜牧》2016,(18)
笔者通过试验研究不同浓度还原型谷胱甘肽(glutathione,GSH)对精子获能的影响,以及与获能有关的关键基因在获能后相对表达量的差异。笔者在获能液中加入不同浓度GSH(0 mg/m L、5 mg/m L、10 mg/m L、15 mg/m L、20 mg/m L),并将其分别与精子悬液一同培养,以孕酮诱导顶体反应,通过涂片染色镜检得到顶体发生率;另外,试验中采用实时荧光定量PCR来得到获能相关基因蛋白激酶A催化亚单位β(PRKACB)和ACP6(acid phosphatase 6,lysophosphatidic)在获能后的表达。结果表明,获能液中添加GSH对兔精子获能有一定的影响。 相似文献
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为探讨钙离子浓度对绵羊精子获能的影响,将绵羊精子在不同钙离子浓度的绵羊输卵管合成液（SOF液）中培养,并用钙离子载体A23187进行诱导。结果发现,不同钙离子浓度对绵羊精子的影响差别不明显,经A23187诱导后,1.7 mmol/L组和2 mmol/L组的顶体反应率（AR）与其他组有显著差异（P<0.05）,1.7 mmol/L浓度中,绵羊精子存活时间为16 h,2 mmol/L浓度中的存活时间为10 h,因此认为绵羊精子获能的适宜钙离子浓度为1.7 mmol/L,且获能过程中钙离子的存在是必要的。 相似文献
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为比较不同获能方法对绵羊精子的获能效果，新鲜绵羊精液分别经肝素上游法(swim-up)、钙离子载体(IA，A23187)诱导法、Percoll离心法和Percoll离心后肝素孵育法进行精子获能的诱导后与体外成熟的绵羊卵母细胞进行体外受精，并利用金霉素荧光染色（chlortetracycline，CTC）法结合各自相应的体外受精结果对其获能效果进行检验。结果显示上游法处理后精子的获能比例和受精后的卵裂率均高于其它组, 说明肝素上游法可以较好的诱导绵羊精子获能。 相似文献
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为研究获能液中添加咖啡因和亚牛磺酸对牛精子功能的影响,本研究将荷斯坦牛冻精解冻后分别添加在含不同浓度咖啡因(0、2.5、5.0、7.5mmol/L)或亚牛磺酸(0、5、10、20、40μmol/L)的获能处理液中,且每个处理组加入约200μL的精液,在CO_2培养箱里经上游处理45min,以评估咖啡因和亚牛磺酸对牛精子活力、顶体及质膜完整性的影响,进而探讨在获能液中亚牛磺酸替代咖啡因的效果。结果显示,添加2.5、5.0mmol/L咖啡因组经上游法获能处理后的牛精子活力和顶体完整率均显著高于对照组(P0.05),且2.5mmol/L咖啡因组精子活力最高;添加10、20μmol/L亚牛磺酸组经上游法获能处理后的牛精子活力、顶体完整率和质膜完整率均显著高于对照组(P0.05),且20μmol/L亚牛磺酸组的精子功能参数值最高;将筛选的最佳浓度2.5 mmol/L咖啡因和20μmol/L亚牛磺酸采用同样的方法处理,发现20μmol/L亚牛磺酸组的精子顶体完整率和质膜完整率均显著高于2.5mmol/L咖啡因组和对照组(P0.05)。因此,20μmol/L亚牛磺酸可以替代2.5mmol/L咖啡因用于体外受精体系中的精子获能处理,有助于提高精子功能参数。 相似文献
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β-谷甾醇、淫羊藿苷对猪精子体外获能效果的影响 总被引：2，自引：0，他引：2
探讨中药单体成分β-谷甾醇(β-sitosterol)、淫羊藿苷(Icariin,Ica)对猪精子体外获能的影响,以期提高体外受精效果.以mTBM+肝素获能液为对照组,分别添加β-谷甾醇、淫羊藿苷的低、中、高3个剂量为试验组,利用金霉素荧光染色(chlortetracycline,CTC)法结合体外受精检验体外获能效果.在mTBM+肝素中添加β-谷甾醇2(中剂量0.08 mg/L)、淫羊藿苷2(中剂量17 mg/L)的B型精子率(获能精子)显著高于对照组和其他剂量组(P＜0.05),F型精子率(未获能精子)显著低于对照组和其他剂量组(P＜0.05),AR型精子率(发生顶体反应精子)与对照组和其他剂量组差异不显著(P0.05),卵裂率和囊胚率均极显著高于对照组和其他剂量组(P＜0.01);表明中药单体成分β-谷甾醇、淫羊藿苷能够显著促进猪精子体外获能效果. 相似文献
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为探讨褪黑素、亚牛磺酸、左旋肉毒碱三种抗氧化剂对牛精子体外获能效果的影响,将荷斯坦牛细管冻精解冻后,各取200μL精液分别添加在含10~(-4) M褪黑素(MLT)、20μM亚牛磺酸(HT)、10 mM左旋肉毒碱(LC)的获能液中,经上游处理45 min,采用目测法、金霉素(CTC)染色法评估三种抗氧化剂对牛精子活力、精子获能状态的影响。结果表明:添加10~(-4) M MLT、20μM HT、10 mM LC经上游法获能处理后的牛精子活力(83.67%、80.65%、75.00%vs 66.67%)均显著高于对照组(P0.05),而顶体反应率(9.87%、10.09%、14.30%vs 18.73%)均显著低于对照组(P0.05)。另外,添加10 mM LC、10~(-4) M MLT组获能处理后B型精子所占比率(37.26%、29.84%vs 16.26%)均显著高于对照组(P0.05),且10 mM LC组精子获能率最高;而20μM HT组与对照组无明显差异(22.80%vs 16.26%,P0.05)。因此,添加10 mM LC、10~(-4) M MLT可改善精子体外获能效果,有助于提高体外受精的受精率及早期胚胎的质量。 相似文献
11.
Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa 
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下载免费PDF全文 E Breininger D Dubois VE Pereyra PC Rodriguez MM Satorre PD Cetica 《Reproduction in domestic animals》2017,52(5):731-740
The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa. 相似文献
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Mirshokraei P Hassanpour H Mehdizadeh A Akhavan Taheri M 《Research in veterinary science》2011,91(2):281-284
To evaluate effects of different concentrations of pentoxifylline, as phosphodiesterase inhibitor, on quality of motility, capacitation and acrosome reaction, Ejaculated spermatozoa were collected from crossbred dogs. The sperm were incubated at concentrations of 0.1, 1, 10 and 100 mM pentoxifylline for 2 h. Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Capacitation and acrosome reaction were also evaluated by chlortetracycline fluorescence staining. SMI as quality index of sperm was significantly increased in concentrations of 10 and 100 mM pentoxifylline during 1 and 2 h compared to control. The number of capacitated or acrosome reacted spermatozoa significantly (P < 0.05) were higher than controls at high concentrations of pentoxifylline (10 and 100 mM) during 1 and 2 h. In conclusion, high concentration of pentoxifylline is able to induce capacitation and acrosome reaction and improves quality of motility in canine ejaculated spermatozoa. 相似文献
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A‐S Bergqvist A Johannisson L Bäckgren A‐M Dalin H Rodriguez‐Martinez JM Morrell 《Reproduction in domestic animals》2011,46(1):e74-e78
This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation‐like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation‐like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation‐like status between colloid‐selected and non‐selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome‐reacted spermatozoa. In conclusion, SLC through Androcoll?‐E does not adversely affect the capacitation‐like status of stallion spermatozoa, although it did increase with time during cold storage. 相似文献
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The aim of this work was to study the effect of progesterone (P4) on capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa in vitro. Spermatozoa were incubated (0-180 min) in capacitation medium supplemented with 0, 0.1, 1.0 and 10.0 microg/ml of P4. At different time intervals aliquots were taken to determine sperm plasma membrane lipid destabilization, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment aimed to study the effects of P4, as potential inducer of AR in heparin-capacitated spermatozoa. The acrosomal status and viability of spermatozoa were evaluated under an epifluorescence microscope using Ethidium homodimer/peanut agglutinin fluorescein isothiocyanate staining method. Plasma membrane scrambling in spermatozoa was assessed by a flow cytometer, using merocyanine staining. The results show that P4 at the concentrations used had no negative effects on sperm viability. Progesterone significantly enhanced sperm capacitation (p < 0.001), but had no effect on plasma membrane lipid stability (p > 0.05) and did not significantly increase the AR of heparin-capacitated spermatozoa (p > 0.05). Progesterone displayed its effects in a dose-dependent manner with a maximum effect of 10 microg/ml P4 at 180 min of incubation. The results demonstrate that in cryopreserved bovine semen, P4 acts as capacitating, but not as an AR-inducing agent. 相似文献
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为研究获能液中添加咖啡因和亚牛磺酸对牛精子功能的影响,本研究将荷斯坦牛冻精解冻后分别添加在含不同浓度咖啡因（0、2.5、5.0、7.5 mmol/L）或亚牛磺酸（0、5、10、20、40 μmol/L）的获能处理液中,且每个处理组加入约200 μL的精液,在CO2培养箱里经上游处理45 min,以评估咖啡因和亚牛磺酸对牛精子活力、顶体及质膜完整性的影响,进而探讨在获能液中亚牛磺酸替代咖啡因的效果。结果显示,添加2.5、5.0 mmol/L咖啡因组经上游法获能处理后的牛精子活力和顶体完整率均显著高于对照组（P<0.05）,且2.5 mmol/L咖啡因组精子活力最高;添加10、20 μmol/L亚牛磺酸组经上游法获能处理后的牛精子活力、顶体完整率和质膜完整率均显著高于对照组（P<0.05）,且20 μmol/L亚牛磺酸组的精子功能参数值最高;将筛选的最佳浓度2.5 mmol/L咖啡因和20 μmol/L亚牛磺酸采用同样的方法处理,发现20 μmol/L亚牛磺酸组的精子顶体完整率和质膜完整率均显著高于2.5 mmol/L咖啡因组和对照组（P<0.05）。因此,20 μmol/L亚牛磺酸可以替代2.5 mmol/L咖啡因用于体外受精体系中的精子获能处理,有助于提高精子功能参数。 相似文献
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The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane‐associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2′,5′‐dideoxyadenosine was used as a membrane‐associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin‐capacitated spermatozoa was significantly higher than in hyaluronic acid‐treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane‐associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane‐associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin‐capacitated spermatozoa. 相似文献
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Superoxide anion radical, produced in low quantities, plays a positive role in sperm function. Spermatozoa produce superoxide anion radical during posttesticular development, which shows an abrupt increase during capacitation. The NAD phosphate oxidase (NOX) family members NOX2 and NOX5 are the 2 enzymes implicated in superoxide production in spermatozoa. We examined the organization of NOX2 in goat spermatozoa during epididymal maturation, capacitation, and acrosome reaction. Spermatozoa from testis, caput epididymidis, corpus epididymidis, and cauda epididymidis possessed components of the phagocytic oxidase (PHOX; i.e., gp91phox, p22phox, p67phox, p47phox, p40phox), and ras-related C3 botulinum toxin substrate 1/2 (Rac1/2) on spermatozoa, and their concentrations did not show significant alterations during epididymal maturation. During capacitation in vitro, p22phox underwent Thr-phosphorylation, which resulted in a mobility shift of the corresponding band toward greater molecular mass. The Rac1/2 also showed a mobility shift from 32 to 23 kDa during capacitation. During progesterone-induced acrosome reaction, the spermatozoa experienced a total loss of p22phox and p47phox. The p47phox, but not p22phox, was detected in the exocytic vesicles of the acrosome. The Thr-phosphorylated form of p22phox was ubiquitinated and degraded through proteasome-mediated pathways in goat sperm cell lysates. Thus, Thr phosphorylation of p22phox acts as a regulatory switch in goat spermatozoa that transiently activates the NOX2 system during capacitation and subsequently directs it for degradation through the ubiiquitin-proteasomal pathway during progesterone-induced acrosome reaction. 相似文献