第四讲猪人工授精

第八节精液的稀释一、精液稀释的目的猪的精液如果不经过稀释,在体外最多保存半小时,活力很快下降,而且很快失去受精能力。这是因为精清促进精子运动,缩短精子体外保存时间(见图1)。精液的稀释就是用稀释液降低精子的密度,为精子提供营养、缓冲物质,并维持适当的渗透压和pH,以利于精液的保存。因此要求原精液应尽快稀释。     

提高肉用种鸡人工授精效果的研究Ⅴ：肉用种鸡精液稀释液的筛选

试验用STJX、BPSE、638334、Lake家禽精液稀释液,用狄高肉用种公鸡精液按1:1进行稀释,观察鲜精稀释后立即授精;在0-2℃温度下精子存活时间;贮存5小时授精试验,在此基础上,选用STJX和Lake稀释液在0-2℃温度下贮存24     

猪精液离心低温保存技术的探讨

<正> 精子在体外,精清并不能使精子的生活力保持长久,为提高其生活力,我们对猪精液通过离心清除部分精清,然后稀释,用低温保存的方法进行试验。 一、稀释液的制备:采用2种稀释液,其配制比例如表1:     

常温稀释液不同温度保存蓝狐精液的效果研究

为了研究常温稀释液不同温度保存蓝狐精液的效果,试验采用东林Ⅱ号狐常温精液稀释液对8只引进芬兰种蓝狐的精液稀释后,分别在36℃(体温)、25℃(常温)、4℃(低温)条件下对精子的存活时间进行检测。结果表明:4℃条件下,有效生存指数平均值为17.21;25℃条件下,有效生存指数平均值为6.19;36℃条件下,有效生存指数平均值为3.94。36℃精子保存时间不宜超过3 h,25℃精子保存时间不宜超过5 h,4℃精子保存时间不宜超过20.5 h。     

“靓精”对稀释猪精液保存质量的影响

<正>种公猪精液在采集、稀释液配制和精液稀释过程中,会因外界环境或操作疏忽而使精液或稀释液受到某些病原微生物的污染,影响稀释精液的质量和保存时间。"靓精"为一种精液净化保护剂,含5%聚维酮碘,能抑制或杀灭病原微生物,减少病原微生物对精子质量的影响,提高精子活力,延长保存时间。本试验在公猪精液中加入靓精,通过检测稀释精液在保存期间的精子活力和精子畸形率,观察靓精对稀释精液保存质量的影响。     

鸡精液稀释打击影响因素及其应用价值分析

精液稀释打击研究在种鸡场精液稀释、公鸡精液大批量镜检以及精液冷冻中有很好的应用前景。研究比较了不同稀释液、不同稀释比例与精液是否冷藏等条件下精液稀释后的活力,以及精液与稀释液不同添加顺序1∶1稀释后人工授精的受精率与孵化率。结果表明:1鸡精液在先冷藏再等温稀释的情况下,高倍稀释的鸡精子活力显著低于低倍稀释(P0.05),而在先等温稀释后冷藏的情况下,高倍稀释与低倍稀释的鸡精子活力差异不显著(P0.05);2在体温预热稀释液的前提下,先添加稀释液再采精、先采精再添加稀释液以及原精组,各组之间受精率与孵化率没有显著差异(P0.05)。因此,鸡精液稀释打击需根据具体情况而定,在采集后立即等温稀释的稀释打击最小,可用于鸡精液稀释、镜检与冷冻等研究领域。     

羊鲜精液保存有妙招

正虽然不同条件下精液保存所用的稀释液不同,但都要求等温稀释,即稀释液的温度与精液的温度相同或相近,而且要尽快稀释,以避免精子受刺激而死亡。稀释时,先将精液吸至经清洗、消毒的暗色小瓶内,再将准备好(经水浴或恒温箱保存加温)的稀释液沿瓶壁缓缓加入,然后轻轻摇动混匀。通常根据精子活力和密度稀释精     

“靓精”对山羊精液液态低温保存效果的影响

为探究山羊精液液态低温保存过程中添加不同稀释倍数“靓精”对保存效果的影响。实验以稀释液中未添加靓精为对照组,在稀释液中分别添加稀释50倍、100倍、200倍、400倍的靓精为实验组,经37℃孵育后持续检测精子活力、活率。结果表明,精液孵育后,与未添加靓精对照组相比,稀释液中添加靓精的实验组精子保存时间、活率及活力均得到提高,其中添加稀释200倍靓精组,精子保存时间最长,精子活率和活力均优于其他实验组。表明稀释液中添加靓精有助于延长山羊精子保存时间,提高精子质量。     

波尔山羊冷冻精液稀释液及稀释倍数研究

通过研究波尔山羊冷冻精液稀释液和稀释倍数,以筛选最佳稀释液配方、确定适宜的稀释倍数、提高波尔山羊精液冷冻效果。结果表明,4号稀释液解冻后精子活率高于1号、2号、3号稀释液解冻后精子的活率,差异极显著( P<0 .0 1 ) ;4号稀释液解冻后精子存活时间为61 .2 6h,长于1号、2号稀释液精子存活时间( P<0 .0 1 ) ,短于3号稀释液精子存活时间,但差异不显著( P>0 .0 5 ) ,说明4号稀释液冷冻效果最好。精液稀释3倍、4倍、5倍解冻后精子活率分别为0 .5 0 2、0 .5 0 1、0 .495 ( P>0 .0 5 ) ,均高于稀释6倍、7倍、8倍的精子活率( P<0 .0 1 ) ,表明波尔山羊冷冻精液稀释倍数以3～5倍为宜。     

不同稀释方法对小型宠物犬精液冷冻效果影响

为了研究Tris-果糖稀释液在不同稀释方法下对小型宠物犬精液冷冻的效果,选取常见的3个小型宠物犬品种共计6只犬进行试验,精液稀释过程分别采用一步稀释法和两步稀释法。结果表明:冷冻精液解冻后各组犬精液的同一剂型间的精子活力差异均不显著(P0.05);一步稀释法中的颗粒冻精与0.25 mL细管冻精和0.50 mL细管冻精相比,解冻后精子活力差异均显著(P0.05),而0.25 mL细管冻精与0.50 mL细管冻精的解冻后精子活力差异极显著(P0.01);采用两步稀释法的颗粒冻精、0.25mL细管冻精及0.50 mL细管冻精的解冻后精子活力组间差异均不显著(P0.05)。用Tris-果糖稀释液稀释精液时,使用两步稀释法对不同冻精剂型的精子活力影响不明显。     

Distinct Effects of Boar Seminal Plasma Fractions Exhibiting Different Protein Profiles on the Functionality of Highly Diluted Boar Spermatozoa

The aim of this study was to evaluate how different protein profiles of seminal plasma (SP) fractions affect sperm functionality in vitro. Ejaculates from three boars were separated into six fractions. The fractions differed from each other in their sperm content, in their total SP protein content, and their spermadhesin PSP-I/PSP-II and heparin-binding protein (HBP) concentrations. Spermatozoa were mainly recovered in fraction 2 (sperm-rich fraction, >1800 × 10⁶ spermatozoa/ml), whereas the pre-sperm fraction 1 and the post-sperm fractions 4–6 contained low numbers of spermatozoa (<500 × 10⁶/ml). Except in fraction 2, the total SP protein concentration and the concentration of both, spermadhesin PSP-I/PSP-II and the HBPs increased with fraction order. Distinct time-dependent effects were observed on motility characteristics and membrane integrity of highly diluted boar spermatozoa upon incubation with a 10% dilution of the SP from each fraction. The highest sperm viability was recorded after exposure for 5 h to fraction 2, followed by fractions 1 and 3. The percentages of motile spermatozoa also differed significantly among fractions after 5 h of incubation. Spermatozoa incubated with SP of fractions 1–3 showed the highest percentage motility. We conclude that different SP fractions exert distinct effects on the functionality of highly diluted boar spermatozoa. Fractions 1–3 appear to promote sperm survival, whereas fractions 4–6 seem to be harmful for preserving the physiological functions of highly diluted boar spermatozoa.     

蓝狐睾丸几种化学成分的检测

通过放射免疫测定技术及激光速率散射比浊法对蓝狐睾丸中某些化学成分的测定分析,得出与蓝狐生殖有关的睾酮(T)、孕酮(P)、雌二醇(E2 )、生长激素(GH)、人绒毛膜促性腺激素(HCG) ,其含量分别为( 83.3 0±1 1 2 .5 8)ng/g、( 3 0 .1 6±5. 69)ng/g、( 2 41 . 3 5±3 5 .2 1 )pg/g、( 42 .2 2±5 .78)ng/g、( 2 5 9.90±3 9.1 8) μg/g;与生命活动有关的三碘甲状腺原氨酸(T3)、四碘甲状腺原氨酸(T4)、生物信使cAMP、cGMP,以及免疫球蛋白IgA、IgG、IgM等成分,其含量分别为( 0 .1 1±0.0 2 )mg/g、( 0.1 0±0 .0 2 )mg/g、( 0.0 6±0.0 1 )mg/g,为此产品的开发利用提供理论依据     

Upregulation of CRISP-3 and kallikrein in stallion seminal plasma is associated with poor tolerance of cooled storage

For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expression in SP with either positive or negative effects on sperm survival during storage was studied using two-dimensional differential gel electrophoresis and liquid chromatography–mass spectrometry. Lower sperm chromatin integrity was associated with upregulation of the proteins kallikrein, CRISP-3 and HSP-1, while higher chromatin integrity was associated with upregulation of TIMP-2. In the sperm-rich fractions, kallikrein and CRISP-3 differed significantly between SP samples with differing effects on sperm chromatin integrity. In the sperm-poor fractions, TIMP-2 and HSP-1 differed significantly between the two SP groups. Differences in the seminal plasma proteome are associated with sperm longevity during cooled storage.     

Effect on fertility of low numbers of fowl spermatozoa inseminated in aqueous diluent or semen components of the fowl and turkey

A study was undertaken to compare the effect on fertility in the fowl of aqueous medium, natural homologous seminal plasma, heterologous turkey seminal plasma and whole turkey semen when whole fowl semen was excessively diluted with these media and inseminated fresh. High dilution with fowl seminal plasma resulted in the best fertility. Dilution with the turkey semen components produced fertility no different from that with aqueous diluent when the dose of spermatozoa was 5 X 5 or 10 X 10(6). The results of this study confirm that 5 X 5 to 10 X 10(6) good quality spermatozoa are sufficient to produce acceptable fertility in weekly inseminations of fresh semen. This enables good quality semen to be highly diluted. However, at high dilution rates there is a need to reconsider the composition of semen diluents, with respect to simulating as yet unknown properties provided by factor(s) in homologous ductus deferens seminal plasma.     

蓝狐阴茎化学成分的研究

应用放射免疫测定技术及激光速率散射比浊法对蓝狐阴茎中某些化学成分进行测定分析 ,得出与蓝狐生殖有关的睾酮 (T)、孕酮 (P)、17β -雌二醇 (E2 )、生长激素 (GH)、人绒毛膜促性腺激素 (HCG) ,其含量分别为 (2 0 5 0± 3 6 5 )ng/g、(0 6 7± 0 12 )ng/g、(99 2 5± 9 87)pg/g、(18 2 1± 2 98)ng/g、(4 5 6 3± 5 2 9)mIU/g ,得出与蓝狐生命活动有关的三碘甲状腺原氨酸 (T3 )、四碘甲状腺原氨酸 (T4)、生物信使cAMP、cGMP及免疫球蛋白IgA、IgG、IgM等成分含量分别为 (1 17± 0 17)ng/g、(2 6 13± 3 6 6 )ng/g、(2 1 39± 2 89)ng/g、(2 7 75±3 18)ng/g、(0 0 8± 0 0 1)mg/g、(0 0 7± 0 0 2 )mg/g、(0 0 4± 0 0 1)mg/g。     

Ram seminal plasma and fertility: results from an ongoing field study

The effects of partial replacement of ram semen diluent with ram seminal plasma on the fertility of ewes were studied. Crossbred Chios ewes (n = 152) were assigned to six groups. The oestrous cycles of the ewes were synchronised at the peak (Groups A, B, C and D) and at the end (Groups E and F) of the breeding season by means of intravaginal sponges impregnated with fluorogestone acetate (FGA) for 14 days. Four hundred IU of PMSG were injected intramuscularly at the time of sponge removal. Ewes of Groups A, C and E were artificially inseminated with ram semen diluted with skim milk extender, while those of Groups B, D and F with ram semen diluted with 50% skim milk and 50% ram seminal plasma. The addition of ram seminal plasma induced a significant increase (P < 0.05) in litter size in Groups B and D when compared with that of Groups A and C (1.85 and 1.88 vs. 1.39 and 1.52, respectively). This increase was not significant when insemination was performed at the end of the breeding season (2.0 vs. 1.4). These results indicate that the addition of seminal plasma can influence the fertility of ewes or the fertilising capacity of extended ram semen to some extent.     

Measurement of Alkaline Phosphatase in Canine Seminal Plasma – An Update

In dogs, diagnosis of incomplete ejaculation and azoospermia can be made by measuring the activity of the enzyme alkaline phosphatase (AP) in seminal plasma. However, even though upper cut‐off value of 5000 IU / l is given in the literature, results by different assays may vary considerably. Furthermore, no data exist concerning the stability of the enzyme during storage of frozen seminal plasma, and no recommendations for pre‐analytic dilutions can be found. During the present study, we compared results from a conventional large scale wet chemistry analyzer to a widely used dry chemistry point of care system (POC) and established a best practice for pre‐analytical dilutions. Furthermore, stability of enzyme activities in seminal plasma during storage at ?18°C for 24 h was evaluated. The average activity of AP in the 2nd fraction of normal ejaculates measured by Reflotron® was 107 328 IU / l. After 24 h of frozen storage, activities did not differ significantly (96 844 IU / l, p > 0.05). Fresh and frozen samples were analysed in parallel by the POC and conventional chemistry analyser, and the results compared that did not reveal a significant difference (p > 0.05). A dilution of seminal plasma with physiologic saline 1 : 100 prior to analysis was sufficient for the qualitative information whether AP activity is below or above 5000 IU / l. Present data show that AP measurement by a POC dry chemistry system is sufficiently accurate in diluted seminal plasma for the diagnosis of azoospermia and that seminal plasma can be stored frozen for 24 h before analysis.     

European Eel Sperm Diluent for Short‐term Storage

The sperm of European eel shows a high density and the time of spermatozoa motility is very short after activation with sea water. These characteristics make difficult the sperm handling and its quality assessment. Several diluents were previously described for the Japanese eel obtaining over 3 weeks’ conservation times under refrigeration, but they rendered bad results in the European species. In the present study, several diluents were developed taking as basis the P1 medium, and using different dilution ratios (1 : 50, 1 : 100) and two pH (6.5, 8.5). The effect of the addition of bovine serum albumin (BSA, 2% w/v) was also evaluated. At 24 h, undiluted samples already showed significant lower motility and viability than sperm samples diluted in the different media. The results for diluents with pH 6.5 and 8.5 were different. Spermatozoa diluted in media at pH 6.5 cannot be activated at 24 h, while samples diluted in the diluents with pH 8.5 and added with BSA did not show significant differences with respect to the fresh sperm motility until 48 h. The viability (percentage of alive cells) did not show differences until 1 week, independent of the dilution ratio. After 1 week, the motility was approximately 30% in the media containing BSA, which presented no differences for head size of the spermatozoa (perimeter and area) until 72 h and 1 week, respectively. In conclusion, the combination of one medium having similar physico‐chemical characteristics to the seminal plasma, including pH 8.5, and supplemented with BSA can be used in different dilution ratios for the sperm’s short‐term storage, preserving its motility capacity.     

Effects of final dilution rate, sperm concentration and times for cooling and glycerol equilibration on post-thaw characteristics of canine spermatozoa

This study re-evaluated a protocol for cryopreservation of canine semen. Semen from 4 beagle dogs was pooled, concentrated by centrifugation and adjusted to increasing sperm concentrations by adding back seminal plasma. The prepared or original semen was diluted with an extender (Egg yolk-Tris-citrate-glucose) and cooled to 4 degrees C (cooling), followed by a second dilution with the same extender including glycerol, equilibrated at 4 degrees C (equilibration), then stored in liquid nitrogen. The semen was diluted for frozen samples having a fixed sperm concentration with increasing dilution rates or for those having the reverse combinations. Various dilution rates of 2.5-10 folds or sperm concentrations of 0.25-2.5 x 10(8)/ml had no significant effect on post-thaw sperm characteristics. When cooling was done for different times (0-26 hr) with glycerol equilibration for 1 hr, post-thaw characteristics were better at 2 and 3 hr of cooling, while various times for equilibration (0-4 hr) with cooling for 3 hr had no effect. These results suggest that different dilution rates and sperm concentrations within the ranges tested may not affect the post-thaw sperm characterisitics and that sufficient time for cooling may be essential but a specific equilibration time may not necessarily be required.     

Liquid Storage of Goat Semen in Chemically Defined Extenders

The suitability of certain commercial and self‐made chemically defined extenders for liquid storage of goat semen was tested and the effects of storage temperatures, dilution rates and sperm washing and pH of extenders on the goat sperm during liquid storage were observed. Semen was collected from nine goat bucks of the Lubei White and Boer breeds using an artificial vagina. Each ejaculate after initial evaluation was diluted with a specific extender, cooled and stored at a desired temperature. Stored semen was evaluated for sperm motility and other parameters every 24 or 48 h of storage. The ranking order of the existing milk‐ and yolk‐free extenders in sustaining goat sperm motility was Androhep > Zorlesco > Beltsville thawing solution > the Tris–glucose medium. The new extender (mZA) which was formulated based on Zorlesco and Androhep was more suitable for goat sperm than Androhep. The mZAP extender with Bovine Serum Albumin (BSA) replaced with polyvinyl alcohol (PVA) worked as efficiently as the mZA in maintaining sperm motility, membrane integrity, acrosome intactness and capacitation status. Goat sperm motility was best maintained at 5°C during liquid preservation, but decreased significantly as the temperature increased. When semen was sixfold diluted, sperm motility was maintained longer (p < 0.05) after centrifugation, but sperm motility did not differ between the centrifuged and non‐centrifuged groups when semen was 11‐fold diluted. When the extender pH was adjusted from 6.6 to 6.04, the efficiency increased significantly in both Androhep and mZAP. A forward sperm motility of 34% was maintained for 9 days when buck semen was 11‐fold diluted and stored at 5°C in mZAP, with pH adjusted to 6.04. It is concluded that for liquid storage of buck semen, the mZA extender was more suitable than other extenders; BSA can be replaced with PVA in mZA; centrifugation to remove seminal plasma can be omitted by adequate dilution; and the storage temperature and pH of extenders affected sperm motility significantly.