不同甘油浓度与平衡时间对食蟹猴精液冷冻效果的影响

探讨了不同甘油浓度(3%、5%、7%、11%)和不同平衡时间(30、60、90、120min)对食蟹猴(Macaca fascicularis)精液冷冻效果的影响,以建立和优化食蟹猴精液冷冻的程序。参照TTE稀释液成分组成改良型TTE,冷冻前和解冻后均检测精子的活力、畸形率、质膜完整性、顶体完整率。结果显示,平衡时间为30min时精子的冷冻解冻后活力、复苏率均高于平衡时间90min和120min组,差异显著(P<0.05),比60min组稍好;甘油浓度为3%、5%组的精子冷冻解冻后活力及复苏率均高于甘油浓度11%组,差异显著(P<0.05),比7%组好;不同甘油浓度各组间以及不同平衡时间各组间畸形率、质膜完整性、顶体完整率差异不显著(P<0.05)。由此得出如下结论,在食蟹猴精液冷冻中,在改良TTE中加入3%~5%的甘油且平衡30min可以获得较好效果,精子冻后活率和复苏率达到45%和62%。     

大熊猫冷冻精液解冻速度和解冻液中添加Pentyoxyfilline对其解冻后活力的影响

建立大熊猫的精子库,进行远距离圈养大熊猫种群间的人工授精和遗传物质的转运,维持遗传多样性,是目前大熊猫遗传管理的优先方法。要成为最有效的工具,精子库保存的精子解冻后的活力必须很好。本文对大熊猫冷冻精液的解冻速度和解冻液中添加化学激活剂Pentyoxyfilline(PF)后的精子活力进行了试验。试验用的精液采自11只成年大熊猫,精液冷冻速度为每分钟-40℃～-100℃。试验Ⅰ:将冷冻精液放入3种不同温度的水浴中解冻:(1)22℃(慢速解冻);(2)37℃(中速解冻)(3)50℃(快速解冻)。将冷冻前精子活力(78 1±2 9%)和解冻后的平均精子活力进行比较,快速解冻后的精子活力(57 5±5 4%)显著地降低(P<0 05),而中速解冻的精子活力(67 5±3 1%)和慢速解冻的精子活力(73 33±2 1%)与冷冻前的活力接近。试验Ⅱ:使用中速解冻方法解冻精液后,分别加入最终浓度为0mM、1mM、5mM和10mM的PF,然后分别保温15min和24h。在PF(0mM、1mM、5mM和10mM)中分别孵育15min的解冻精子活力,运动状态,活率和顶体正常率在试验期的90min内都很相似(P>0 05)。在1mMPF中孵育24h的精子活力没有变化(P>0 05)。在5mM和10mMPF中孵育过的精子活力(5mM:24 0±4 7%;10mM19 5±3 6%)比没有加PF的对照组的精子活力(38 3±5 2%)显著地低(P<0 05)。而且,在10mM     

激光辐射奶牛冷冻精液对精子诱变效应的研究

为了探索激光辐射精子的诱变效应,本试验采用ＴＳＴ（Ｔｒｉｐｌｅ Ｓｔａｉｎ Ｔｅｃｈｎｉｑｕｅ,即精子三重染色法）、ＳＰＡ（Ｓｐｅｒｍ Ｐｅｎｅｔｒａｉｔｉｏｎ Ａｓｓａｙ,即精子穿透分析法和ＣＡＳ（Ｃｈｒｏｍｏｓｏｍａｌ Ａｎｅｕｐｌｏｉｄｙ ｉｎ Ｓｐｅｒｍａｔｏｚｏａ,即精子单倍染色体制备技术）,三项有关精子的现代分析技术,对激光辐射精子的受精学、遗传学效应进行了研究。结果表明：Ｈｅ－Ｎｅ激     

两种冷冻保护剂和冷冻程序对兔精子冷冻效果的比较

目的比较不同冷冻保护剂和冷冻程序对兔精子冷冻保护的影响,以期提高兔精子冷冻保存的效果和效率。方法用三步降温法（程序Ⅰ）和两步降温法（程序Ⅱ）两种冷冻程序与终浓度分别为2%,3%,4%,5%的甘油和乙酰胺两种冷冻保护剂配合进行精液冷冻保存,统计精子复苏率。结果使用程序Ⅱ添加3%乙酰胺的冷冻保护剂实验组的精子复苏率较高,同其它组比较差异有显著性意义（P〈0.05）;程序Ⅱ比程序Ⅰ节省约70%的时间,同种浓度冷冻保护剂的不同冷冻程序组之间精子复苏率差异无显著性意义（P〉0.05）。结论程序Ⅱ与3%乙酰胺配合可以取得良好的冷冻保存效果;用程序Ⅱ进行兔精液冷冻保存可以大幅缩短操作时间。     

白鹤冷冻精液人工授精实验

为了探索鹤类精液冷冻保存和使用技术,2003～2005年,进行了白鹤(Grus leucogeranus)的冷冻精液保存及人工授精实验。使用Beltsville家禽精液稀释液作为白鹤精液稀释液,12%的二甲基亚砜(DMSO)为冷冻液。精液样本冷冻经过三个阶段的降温,最后保存在液氮中。成功保存了编号93001雄性白鹤精液36 支（0.2 ml/支）。冷冻精液在0~4℃冰水中解冻3~5 min,解冻后白鹤精液精子活率为29.3%±15.5%（n=16）,2004和2005年分别为92101号雌鹤产的两窝卵进行人工授精实验,2年共产卵5枚,其中1枚卵受精并成功孵化。实验发现在雌鹤产卵前一周和产卵期间每天输精,并增加每次输精量,同时在产完1枚卵后4 h内完成一次输精,效果最佳。     

猪精液冷冻影响因素的研究进展

由于在经济生产中的重要意义,猪精液冷冻保存技术已成为研究的焦点。文章综述了国内外猪精液冷冻技术的研究成果,主要阐述精液的冷冻保存原理,并从冷冻稀释液和冷冻保护剂、冷冻方法和解冻方法等方面阐述了猪精液冷冻的影响因素。     

藏酋猴精液深低温冻存的研究──不同的冷冻程序、冷冻稀释保存液冻存效果的比较

以存活率（ＳＲ）、复苏运动度（ＲＭ）、ＳＰＡ为精子活力检测指标,对四种冷冻程序ＰＳＦ、Ｈ３Ｐ、ＬＰ、ＭＤＰ和卵黄的、无卵黄的冷冻稀释保存液进行了藏酋猴（Ｍａｃａｃａ ｔｈｉｂｅｔａｎａ）精液冻存比较研究。 ＰＳＦ的ＳＲ为９０．１±１．９％,ＲＭ为６３．８±２．８％,显著高于其他三种冷冻程序（Ｐ＜０．０１）。卵黄冷冻稀释保存液（ＭＤＭ）的ＳＲ（９５． ４±１．３％）和ＲＭ（８８．０±１０．２％）显著高于无卵黄防冻液（ＦＣＳ－Ｇ／ＴＨ）的ＳＲ（９０．１±１．９％）和ＲＭ（６３．６±２．８％）,但前者在复苏一小时后,ＲＭ（１２．５±１．６％）明显低于后者的ＲＭ（５４． ７±２． ２％）。用 ＦＣＳ～Ｇ／ＴＨ冻存的精液经 ＳＰＡ检测,其穿透率为新鲜精子的 ５１． ９％。结果提示：１）慢速降温冷冻程序适于藏酋猴精液冻存;２）卵黄冷冻稀释保存液能较好地保存藏酋猴精液的活动度;而无卵黄防冻液则有利于延长其冷冻精子的运动寿命。这可能与两者的稀释液及所含的脂蛋白不同有关。     

病理冷冻切片组织速冻与解冻方法的优化

目的优化病理冷冻切片组织速冻与解冻的方法,以期提高冷冻组织制片的效率和切片质量。方法随机抽取50例术中快速冷冻切片诊断为乳腺浸润性导管癌病例,每例取3块组织,分3组进行速冻处理:不加盖冰锤组,冰冻切片机配置冰锤组,专利冰锤组。分析比较3组的速冻时间和冷冻切片HE染色质量。将冷冻制片后剩余的组织用不同的方法分两组进行解冻处理:固定剂融冰组,流水融冰组;将解冻处理后的组织与新鲜标本直接固定的组织行人表皮因子受体(HER-2)免疫组织化学染色,比较3组在免疫组织化学染色效果方面的差异。结果运用专利冰锤组件速冻,冷冻速度快,运用专利冰锤组件制出的冷冻切片组织结构形态良好,冰晶、褶皱现象少。三组不同方式处理的组织HER-2免疫组织化学染色阳性率一致,但组织新鲜固定组和固定液融冰组在阳性细胞定位和阳性染色强度方面均强于流水融冰组。结论推荐运用专利冰锤组件速冻组织,速度快且冷冻切片质量佳;冷冻制片后剩余组织的解冻处理,推荐在固定液中解冻,边溶化边退冰,最大限度的保存组织抗原以利于后续的工作。     

冷冻—解冻猪精子的超微结构观察

自从50年代初Polge发现甘油有卓越的防冻作用以后,动物精液冷冻技术发展很快。现在,奶牛和羊冷冻精液已广泛应用于人工授精和体外受精中,效果与鲜精接近,而猪冷冻精液在人工授精后虽也能产仔,但妊娠率与每窝产仔数均下降(Johnson等,1981);体外受精率可达85％以上,但精子浓度需用5—6×10_7精子/ml,比鲜精用量提高25—100倍(Wang等,1991)。顶体形态正常精子的减少是造成冷冻精液受精率低的重要原因之一(Pursel,1979),用光镜不能清楚地观察顶体的形态变化,但运用电镜技术对猪冷冻-解冻精子的顶体形态的超微结构研究报道不多(Courtens等,1985;Hashizume,1990)。针对猪冷冻—解冻精子体内/体外受精能力降低的问题,我们用透射电镜对冷冻的猪精子解冻0 h及4 h后精子顶体与头部质膜的超微结构变化进行了观察。     

Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage

An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal plasma. Percent total sperm motility (TMOT) and progressively motile (PMOT) was reduced (P < 0.05) in the SP50NC treatment after 24, 48, 72, and 96 h of storage, and TMOT did not differ (P > 0.05) in the SP10C, SP50C, SP10NC groups after the same storage periods. The % COMP-_αt did not differ (P > 0.05) among treatments at any time period. Percent membrane intact sperm (SMI) was reduced in SP50NC, as compared to SP10C at 48, 72, and 96 h (P < 0.05). Daily centrifugation and resuspension of sperm exposed to 50% seminal plasma for the first 24 h (SP50C) yielded similar TMOT, PMOT, VCL, SMI, % COMP-_αt (P > 0.05) to Groups SP10NC and SP10C after 96 h of storage. Daily centrifugation and resuspension of cool-stored equine semen in fresh extender may be a method to increase sperm longevity.     

Effect of cryopreservation protocol on postthaw characteristics of stallion sperm

Three ejaculates from each of eight stallions were subjected to cryopreservation in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Semen was exposed to a fast prefreeze cooling rate (FAST; semen immediately subjected to cryopreservation) or a slow prefreeze cooling rate (SLOW; semen pre-cooled at a controlled rate for 80 min prior to cryopreservation). Postthaw semen was diluted in initial freezing medium (FM) or INRA 96 (IMV Technologies, L'Aigle, France) prior to analysis of 10 experimental end points: total motility (MOT; %), progressive motility (PMOT; %), curvilinear velocity (VCL; μm/s), linearity (LIN; %), intact acrosomal and plasma membranes (AIMI; %), intact acrosomal membranes (AI; %), intact plasma membranes (MI; %), and DNA quality. Eight of 10 experimental endpoints (MOT, PMOT, average-path velocity [VAP], mean straight-line velocity [VSL], LIN AIMI, AI, and MI) were affected by extender type, with egg yolk-based extender yielding higher values than milk/egg yolk-based extender (P < 0.05). Exposure of extended semen to a slow prefreeze cooling period resulted in increased values for six of eight endpoints (MOT, PMOT, VCL, AIMI, AI, and MI), as compared with a fast prefreeze cooling period (P < 0.05). As a postthaw diluent, INRA 96 yielded higher mean values than FM for MOT, PMOT, VCL, average-path velocity, and mean straight-line velocity (P < 0.05). Treatment group FM yielded slightly higher values than INRA 96 for LIN and MI (P < 0.05). In conclusion, a slow prefreeze cooling rate was superior to a fast prefreeze cooling rate, regardless of freezing extender used, and INRA 96 served as a satisfactory postthaw diluent prior to semen analysis.     

Factors affecting sperm recovery rates and survival after centrifugation of equine semen

Conventional centrifugation protocols result in important sperm losses during removal of the supernatant. In this study, the effect of centrifugation force (400 or 900 × g), duration (5 or 10 min), and column height (20 or 40 mL; Experiment 1); sperm concentration (25, 50, and 100 × 10⁶/mL; Experiment 2), and centrifugation medium (EZ-Mixin CST [Animal Reproduction Systems, Chino, CA, USA], INRA96 [IMV Technologies, Maple Grove, MN, USA], or VMDZ [Partnar Animal Health, Port Huron, MI, USA]; Experiment 3) on sperm recovery and survival after centrifugation and cooling and storage were evaluated. Overall, sperm survival was not affected by the combination of centrifugation protocol and cooling. Total sperm yield was highest after centrifugation for 10 min at 400 × g in 20-mL columns (95.6 ± 5%, mean ± SD) or 900 × g in 20-mL (99.2 ± 0.8%) or 40-mL (91.4 ± 4.5%) columns, and at 900 × g for 5 min in 20-mL columns (93.8 ± 8.9%; P < 0.0001). Total (TMY) and progressively motile sperm yield followed a similar pattern (P < 0.0001). Sperm yields were not significantly different among samples centrifuged at various sperm concentrations. However, centrifugation at 100 × 10⁶/mL resulted in significantly lower total sperm yield (83.8 ± 10.7%) and TMY (81.7 ± 6.8%) compared with noncentrifuged semen. Centrifugation in VMDZ resulted in significantly lower TMY (69.3 ± 22.6%), progressively motile sperm yield (63.5 ± 18.2%), viable yield (60.9 ± 36.5%), and survival of progressively motile sperm after cooling (21 ± 10.8%) compared with noncentrifuged semen. In conclusion, centrifuging volumes of ≤ 20 mL minimized sperm losses with conventional protocols. With 40-mL columns, it may be recommended to increase the centrifugal force to 900 × g for 10 min and dilute the semen to a sperm concentration of 25 to 50 × 10⁶/mL in a milk- or fractionated milk-based medium. The semen extender VMDZ did not seem well suited for centrifugation of equine semen.     

Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri)

Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at −172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability.     

Sperm viability, motility and morphology in emus (Dromaius novaehollandiae) are independent of the ambient collection temperature but are influenced by storage temperature

A protocol for storage of emu semen >6 h has not yet been optimized. The objective was to determine: a) whether sperm quality was adversely affected by sudden exposure to low temperatures (5, 10 and 20 °C) during collection; and b) the effects of three storage temperatures (5, 10 and 20 °C) on survival of emu sperm. In two experiments, each repeated three times on alternate days, ejaculates were diluted 1:1 with precooled (5, 10, or 20°C) UWA-E3 diluent and stored for up to 48 h. Collection temperature, or interaction with either the storage time or storage temperature, had no significant effect on sperm viability, motility, or morphology. Mass Motility Score (2.91-3.27 ± 0.26, mean ± SEM), and percentages of live (72.4-76.2 ± 2.4) and morphologically normal sperm (63.3-64.5 ± 2.3) were comparable among collection temperatures. Conversely, storage temperature and storage time affected (P < 0.05) sperm viability, motility, and morphology. After storage for 48 h, percentages of viable, normal, and motile sperm were higher (P < 0.001) at 5 °C (58.7% ± 1.1, 44.7% ± 1.3, and 50.7% ± 4.9, respectively) and 10 °C (62.6% ± 1.1, 54.1% ± 1.3, and 60.4% ± 4.9) than at 20 °C (27.6% ± 1.1, 20.1% ± 1.3, and 25.9% ± 4.9). Beyond 6 h of storage, the percentage of abnormal sperm was higher (P < 0.001) for storage at 5 °C compared to 10 and 20 °C. After 48 h, bacterial counts were considerably higher at 20 °C compared to 5 and 10 °C (P < 0.001). The pH of stored sperm suspension remained unaffected at 5 and 10 °C, but at 20 °C declined to 6.5 ± 0.03 after 24 h (P < 0.05) and to 6.0 ± 0.03 after 48 h (P < 0.001). We concluded that emu semen could be collected at low ambient temperatures (5-20 °C) without compromising its in vitro storage duration and that semen quality during storage for 48 h was better if it was stored at 10 °C than at 5 or 20 °C.     

小鼠卵巢组织的超速冻存法研究

目的　本实验通过对小鼠卵巢组织进行冻存研究 ,掌握卵巢的低温生物学特性 ,摸索出一种简便有效的组织器官冻存法 ,为卵巢移植及器官冷冻提供有用的技术方法。方法　通过对小鼠卵巢组织进行慢速程序法与快速液氮蒸汽法冻存 ,比较分析了不同方法所需保护剂种类、浓度、渗透平衡时间。采用对解冻后卵巢组织超微结构观察、组织化学染色、激素测定及自体、异体移植后动情期的恢复作为评价指标。结果与结论　通过上述实验表明用同种冷冻保护剂 ,液氮蒸汽法冻存的卵巢组织超微结构保存良好 ;组织化学染色示其活性与程序法冻存组织相同 ;自体、异体移植后 ,小鼠动情周期的恢复率及血清雌二醇水平各项指标均与慢速程序法冷冻无显著性差异     

The impact of cushioned centrifugation protocols on semen quality of stallions

The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/mL. Sperm recovery rate among centrifugation treatment groups was compared. Motion characteristics, plasma membrane intactness (SMI), and DNA quality (COMPαt) of sperm were compared among treatment groups and uncentrifuged controls immediately following centrifugation (Time 0 h) and following 24 h of cooled storage (Time 24 h). Centrifugation treatment did not affect sperm recovery rate (P > 0.05). At Time 0 h, no differences in experimental end points were detected between cushion-fluid volumes tested (P > 0.05). Values for percent total sperm motility, percent progressive sperm motility, and track straightness were similar between sperm-number treatments subjected to centrifugation (P > 0.05). At Time 24 h, values for all experimental endpoints were similar between centrifugation treatments for cushion volume per tube, and between centrifugation treatments for sperm number per tube (P > 0.05). Centrifugation treatments and control treatments were similar for five of six variables tested (P > 0.05). Sperm storage concentrations of 25 × 10⁶ and 250 × 10⁶/mL yielded similar values for percent total sperm motility, percent progressive sperm motility, percent SMI, and percent COMPαt (P > 0.05). A storage concentration of 250 × 10⁶ sperm/ml yielded higher values for curvilinear velocity, and lower values for straightness, than all other groups (P < 0.05). In conclusion, centrifugation with as little as 1 ml of cushion fluid and a sperm number of up to 3 × 10⁹ sperm in 50-ml conical-bottom centrifuge tubes had no detrimental effect on initial or cool-stored sperm quality. Additionally, storage of centrifuged sperm at a concentration of 250 × 10⁶/mL with 20% seminal plasma (v/v) did not have a detrimental effect on percentages of motile or progressively motile sperm, or sperm DNA quality.     

Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm

The effects of cryopreservation on the frequency and type of chromosomal abnormalities in human sperm were investigated. Employing a technique that enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 10 normal men were examined before and after freezing in liquid nitrogen. A total of 1,960 sperm karyotypes were analyzed, 1,132 before freezing and 828 after freezing. There was no significant difference in the frequency of structural chromosomal anomalies (10.5% prefreeze vs. 8.5% postfreeze), but there was a significant decrease in the frequency of numerical abnormalities (5.2% prefreeze vs. 3.0% postfreeze). However, there was a large excess of hypohaploid complements compared with hyperhaploid complements, suggesting that the hypohaploid complements were caused by technical artefact. A conservative estimate of aneuploidy, derived by doubling the hyperhaploid frequencies, did not differ before (0.4%) and after (0.4%) freezing. There was no evidence for interdonor variability in response to sperm cryopreservation for total chromosomal abnormalities, structural abnormalities, and sex ratios. The sex ratios were also not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the frequencies of chromosomal abnormalities or alter the sex ratio in human sperm, provided that an adequate cryoprotective buffer and freezing system is employed.