猪精液的冷冻保存技术

综述了国内外猪冷冻精液的发展简史及研究成果,对猪精液的冷冻保存机理进行了简要阐述,并从稀释液、冷冻保护剂及其浓度、冷冻前处理、冷冻剂型、诱发结晶、冷冻速率、解冻等方面阐述了影响猪精液冷冻的因素,指出了目前在猪精液冷冻理论和实践中存在的问题,并提出了解决办法.     

猪精液超低温冷冻保存研究进展

猪人工授精(AI)技术已经广泛应用于畜牧业生产,但精液来源基本是17℃保存的新鲜猪精液,不利于精液长期保存和猪种资源保存。超低温冷冻保存技术是猪精液长期保存最有效的方法,冷冻精液可以实现不同地区、不同国家优良种猪精液间的交流,从而大大提高猪种资源的利用率,但冷冻解冻后的精液质量较差,受胎率、窝产仔数远低于鲜精,极大地限制了猪精液冷冻保存技术的广泛应用。本文综述了猪精液冷冻保存研究概况、猪精液冷冻损伤机理和猪精液冷冻保存影响因素,以期为猪精液超低温冷冻保存研究提供新的思路和方法。     

抗氧化剂在公猪精液冷冻保存中的应用

近几年来,在公猪精液冷冻稀释液中添加抗氧化剂以提高冷冻精液质量的研究受到广泛的关注。添加谷胱甘肽、超氧化物歧化酶和过氧化物酶、维生素E、L-半胱氨酸和L-谷氨酰胺、中草药成分等抗氧化剂可以有效地防止氧化损伤,提高解冻后精子活率、精子成活力、质膜与顶体完整性等,进而提高冷冻保存精液的受精能力。作者综述了抗氧化剂的抗精子冷冻损伤的作用机制及不同抗氧化剂的应用效果,并对其在公猪精液冷冻保存中的应用前景进行了展望。     

海藻糖对猪精液冷冻保存效果的影响

在传统的Tris-柠檬酸-葡萄糖稀释液基础上，分别添加25％、50％、75％、100％的海藻糖，研究不同浓度海藻糖对猪精液冷冻后精子质量的影响。结果表明，海藻糖相对于对照TCG稀释液能够显著改善和提高猪精液的冷冻效果，其最佳添加浓度为25％，冷冻-解冻后猪精子活力、活率、线粒体活性、质膜完整性以及顶体完整率均显著提高（P〈0．05），分别达到41．38％、46．34％、44．56％、43．51％和64．09％。海藻糖可以明显抑制精子获能，获能处理前精子获能率仅为3．68％，而获能处理后达到41．82％，有利于促进精子获能。精液稀释液中甘油的适宜添加浓度为2％，海藻糖只有与甘油共同作用，才能在冷冻-解冻过程更加有效地保护精子。猪精子活力、活率、线粒体活性、质膜完整率、顶体完整率等之间存在极显著的正相关关系（P〈0．01），而与获能处理前精子的获能率存在显著的负相关关系（P〈0．05）。     

聚乙烯吡咯烷酮对公猪精液冷冻的影响

试验旨在评价聚乙烯吡咯烷酮（polyvinylpyrrolidone,PVP）对公猪精液冷冻的影响。试验分为5组,分别为对照组（不添加PVP）和PVP处理组（在冷冻基础液中分别加入0.25%、0.50%、1.00%、2.00% PVP）。采用手握法采集松辽黑猪精液,用5种冷冻基础液稀释,在25 ℃平衡1 h,17 ℃平衡2 h,4 ℃平衡3 h后灌装于0.5 mL细管中,在液氮上方3 cm处熏蒸10 min,保存在液氮罐中30 d后进行检测。样本解冻后分别检测精子活力、质膜完整性、顶体完整性、线粒体活性、DNA完整性、过氧化氢酶（catalase,CAT）活性、超氧化物歧化酶（superoxide dismutase,SOD）活性、谷胱甘肽过氧化物酶（glutathioneperoxidase,GSH-Px）活性、活性氧簇（reactive oxygen species,ROS）水平及丙二醛（malondialdehyde,MDA）水平。结果显示,与对照组相比,冷冻基础液中添加0.50% PVP显著提高冻融后精子的活力、质膜完整性、顶体完整性、线粒体活性、CAT活性、SOD活性、GSH-Px活性（P<0.05）,显著降低精子ROS和MDA水平（P<0.05）;与对照组相比,添加PVP有利于提高DNA完整性,但差异不显著（P>0.05）。因此,猪精液冷冻基础液中添加PVP可改善冻融后精子质量,添加0.50%效果最佳。     

二甲基甲酰胺对猪精液冷冻保存效果的影响

用二甲基甲酰胺(DMF)完全替代甘油,比较不同平衡时间和不同DMF添加量对猪精液冷冻保护效果的影响。结果表明,DMF能完全替代甘油,获得较好的冷冻保护效果。最佳平衡时间为90 min,解冻后精子活力为(44.57±0.72)%,显著高于对照组和其他组(P0.05)。当DMF添加量为5%时,冻后精子活力、活率、线粒体活性、顶体完整率和质膜完整率分别为(49.91±0.39)%(、46.51±0.26)%、(47.51±0.52)%(、49.84±0.56)%、(46.30±1.61)%,均显著高于2%、3%、6%DMF添加组(P0.05),但与4%DMF添加组相比,冻后精子活力、活率和质膜完整率差异不显著(P0.05)。本试验结果表明,DMF最适添加量为5%。     

抗氧化剂在猪精液冷冻保存技术中的应用

近年来,在猪精液冷冻保存技术中应用抗氧化剂以提高冷冻精液质量的研究受到了广泛关注。国内外相继报道,添加甲基黄嘌呤、丁羟甲苯、谷胱甘肽、超氧化物歧化酶和过氧化物酶、维生素E及褪黑素等抗氧化剂可以有效改善冷冻保存猪精液的精子运动学参数,保护精子质膜、顶体和DNA完整性,提高冷冻-解冻后精子的受精能力。为了更好地了解抗氧化剂的抗精子冷冻损伤的作用机制、客观地评价不同抗氧化剂的应用效果及展望其在猪精液冷冻保存技术中的应用前景特综述如下。     

猪精液细管法冷冻保存技术的研究

为研制更为简易、高效的冷冻稀释液配方及冷冻程序,充分发挥优良种公猪的生产潜力,本实验采用细管法对种公猪精液冷冻保存技术进行了研究,比较了4种基础液配方稀释液冷冻效果,基础液I:葡萄糖、蔗糖、柠檬酸钠;基础液II:葡萄糖、蔗糖;基础液III:葡萄糖、乳糖、柠檬酸钠;基础液IV:葡萄糖、乳糖。结果表明:采用一步法稀释和IV液冷冻保存猪精液,其解冻后精子活率(0.501)极显著(P<0.01)高于I液(0.359)和III液(0.359),显著(P<0.05)高于II液(0.476),II液显著(P<0.05)高于I液和III液;解冻后IV液的精子顶体完整率(26.9%)显著(P<0.05)高于I液(22.4%)、II液(24.2%)和III液(22.5%),IV液冷冻解冻后精液的精子活率,在室温(23±2)℃下4h内能够保持0.30以上。     

Cryopreservation of Piau‐Breed Wild Boar Sperm: Assessment of Cooling Curves and Centrifugation Regimes

This study aimed to assess the effects of different cooling curves and centrifugation regimes used in cryopreservation protocols on the post‐thaw viability of Piau‐breed wild boar (Sus scrofa) sperm using in vitro assessment tests. Two centrifugations (800  g for 10 min and 2400  g for 3 min) and two cooling curves (conventional cooling using nitrogen vapour – freezing 1 and automated cooling using a programmed freezing machine – freezing 2) were tested. Therefore, the treatments were divided into M3 – centrifugation at 2400  g for 3 min and freezing 2; M10 – centrifugation at 800  g for 10 min and freezing 2; R3 – centrifugation at 2400  g for 3 min and freezing 1; and R10 – centrifugation at 800  g for 10 min and freezing 1. No significant differences (p > 0.05) between treatments occurred post‐thawing regarding the total sperm motility means recorded. The mean values of the different treatments were not different from each other regarding the supravital staining (SV), hypo‐osmotic test (HO), sperm–egg binding assay or sperm morphology. This study showed that both the cooling curve and the centrifugation regime affected the quality of post‐thaw sperm, and centrifugation for shorter times and cooling curves using automated cooling are the most suitable for minimizing sperm injury.     

冷冻保护剂对猪精液的冷冻保护作用

论文通过阐述猪精液冷冻保护过程中冷冻保护剂与精子之间的相互作用,揭示渗透性保护剂和非渗透性保护剂的作用机制,指出目前猪精液冷冻保存的局限性,同时展望了猪精液冻存的研究趋势,为猪精液的冷冻保存研究和应用提供参考.     

Cryopreservation of Boar Semen: I: A literature review

The present review summarizes information concerning the methods available to cryopreserve boar semen, covering the historical background, cryobiology and cryoprotecting considerations, technological developments and recent advances in cryopreservation methodologies. Successful methods for cryopreservation of boar semen have not been achieved despite numerous efforts world wide. Improvements in semen preservation technologies have been deterred by lack of in vitro methods that can accurately predict in vivo fertilizing capacity of frozen boar semen. The cell membrane is of crucial importance with regard to freeze-thaw survival of spermatozoa. It is important to optimize the survival of the plasma membrane as this is a non homogenous entity both in structure and function. The boar sperm membrane exhibits extreme sensitivity to freezing treatment. Freezing and thawing results in considerable changes in electrolyte dynamics and damages have mainly been associated with alterations in the head membranes especially at thawing. To date fruitless efforts have been carried out to find a cryoprotectant for the spermatozoa membranes and glycerol still continues to be used despite its harmful effects to the membranes.     

稀释液中添加N-乙酰半胱氨酸对猪精液冷冻保存效果的影响

本文以手握法采集的猪精液为试验材料,以解冻后精子的活率、顶体完整率、低渗膨胀率、运动学参数(VAP、LIN、ALH、BCF)和膜脂质过氧化反应为评定标准,旨在探讨N-乙酰半胱氨酸对猪精液冷冻保存效果的影响。结果表明,在冷冻稀释液中添加1mmol/L的N-乙酰半胱氨酸可以显著地提高解冻后精子的活率和顶体完整率,降低精子的低渗膨胀百分率。在运动学参数方面,只有VAP显著升高,而对其它运动学参数没有影响。在冷冻稀释液中添加N-乙酰半胱氨酸对膜脂质过氧化反应没有影响。     

Validation of the FACSCount AF System for Determination of Sperm Concentration in Boar Semen

A flow cytometric method has been developed for rapid determination of sperm concentration in semen from various mammalian species. * * Patent Pending, Int. Publication Number WO/00/54026.
All cells containing DNA are stained with SYBR‐14 or propidium iodide (PI) and sperm concentration is determined in relation to an internal standard of fluorescent microspheres (beads). Satisfactory staining can be achieved within 2–3 min and the following flow cytometric analysis on the FACSCount AF System rapidly provides the user with a precise and accurate assessment of the sperm concentration. In this study, the FACSCount AF System and Sperm Counting Reagent (BD Biosciences) was compared with microscopic counting using a Bürker–Türk haemocytometer. In addition, sperm concentration was determined using the Corning 254 spectrophotometer which is used routinely by Danish artificial insemination stations for boars. The results show that the agreement between flow cytometry and microscopic counting is very high. The slope for the regression line was 1.12 (SE = 0.03) with an estimated intercept with the Y‐axis of 22 × 10⁶sperm/ml (SE = 10 × 10⁶ sperm/ml) and an estimated error of the model of 10 × 10⁶ sperm/ml. For the spectrophotometer, the slope of the regression line was 1.09 (SE = 0.07) with an estimated intercept of 137 × 10⁶ sperm/ml (SE = 25 × 10⁶ sperm/ml). The average error made by the spectrophotometer was 55 × 10⁶ sperm/ml. In addition, the results obtained using flow cytometry was highly repeatable (CV = 2.7%) in comparison with the spectrophotometric method (CV = 6.3%). These results indicate that the FACSCount AF System is a valuable tool for precise and accurate assessment of sperm concentration in boar semen and that use of this system may lead to production of more uniform insemination doses containing a specific number of sperm per dose.     

冷冻速率和解冻温度对猪精液冷冻效果的影响

为优化冷冻和解冻方法,提高冷冻效果,本试验比较了不同冷冻速率(-100 ℃ 10 min、-120 ℃ 10 min、-140 ℃ 10 min)和不同解冻温度(37 ℃ 30 s、45 ℃ 30 s、52 ℃ 30 s、60 ℃ 30 s)对猪精液冷冻效果的影响。结果表明,采用-120 ℃熏蒸10 min,解冻后精子活力为0.36,质膜完整率和顶体完整率也优于其他2组,且差异显著(P<0.05)。采用37 ℃ 30 s方法解冻,精子活力、质膜完整率显著高于其他3组,顶体完整率也高于其他3组,但差异不显著(P>0.05),其畸形率最低和60 ℃ 30 s组差异明显(P<0.05),但与45 ℃ 30 s组和52 ℃ 30 s组差异不显著(P>0.05)。因此,采用-120 ℃平衡10 min冷冻,37 ℃ 30 s水浴解冻方法更为适合0.25 mL细管猪冻精解冻。     

不同浓度芦丁和冷冻速率对猪精子冷冻保存效果的研究

【目的】 探讨冷冻稀释液中添加不同浓度芦丁和不同冷冻速率对杜洛克公猪精子冷冻保存效果的影响及其二者的互作关系,以期为指导生产实践和提高优良种猪利用率提供依据。【方法】 试验分为6组,分别为空白对照组（冷冻稀释液Ⅰ液）和试验组（分别在冷冻稀释液Ⅰ液中添加0.2、0.4、0.6、0.8和1.0 mmol/L芦丁）,在距离液氮面上1 cm （快冷冻）和3 cm （慢冷冻）处分别进行冷冻。在液氮中保存30 d后,检测冷冻-解冻精子的运动参数、质膜完整率（MI）、顶体完整率（AI）、DNA完整率、线粒体膜电位（MMP）、活性氧（ROS）水平、丙二醛（MDA）和ATP含量,以及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）的活性来评价解冻后的精液品质。【结果】 冷冻速率方面,在相同浓度芦丁冷冻液中,慢冷冻效果均好于快冷冻效果,差异显著（P<0.05）。芦丁浓度方面,快冷冻和慢冷冻组均以添加0.6 mmol/L芦丁的效果最好（P<0.05）,添加0.8 mmol/L芦丁的效果次之。二者互作方面,冷冻速率与芦丁浓度间存在交互作用,其中慢冷冻×0.6 mmol/L芦丁组合的精子的运动参数、质膜完整率、顶体完整率、DNA完整率、线粒体膜电位、ATP含量均显著高于其他组合试验组（P<0.05）,ROS水平显著低于其他组合试验组（P<0.05）,SOD、CAT和GSH-Px的活性较其他组合试验组均显著提高（P<0.05）。【结论】 不同芦丁浓度与不同冷冻速率间存在互作效应,其中在冷冻稀释液中添加0.6 mmol/L芦丁慢冷冻猪精子效果最好。     

Glycerol,Methyl‐Formamide and Dimethyl‐Formamide in Canine Semen Cryopreservation

The aim of the present work was to compare the efficiency of methyl‐formamide (MF), dimethyl‐formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk–TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo‐osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 ± 5.4%, 61.0 ± 7.4%, 2.9 ± 0.5 and 57.1 ± 5.0% for GL; 59.0 ± 8.9%, 50.0 ± 10.0%, 2.5 ± 0.7 and 66.9 ± 7.7% for MF; and 44.0 ± 21.0%, 37.0 ± 19.8%, 2.1 ± 0.6 and 61.1 ± 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 ± 12.4%, 35.8 ± 18.4% and 34.4 ± 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.     

Evaluation of the Karras Spermdensimeter in Relation to the Neubauer Counting Chamber for Sperm Concentration Measurement in Boar Semen

The correct measurement of spermatic concentration in boar semen is one of the basic tasks routinely performed at the Swine Artificial Insemination Center (CIAS). This article explains two experiments whose objectives were: Experiment 1: This trial had two objectives: (1) to evaluate the precision of the spermdensimeter (SPM) with relation to the Neubauer count chamber (CN) which is considered the standard technique in the field and (2) to correct the scale on the SPM table. Experiment 2: This trial also had two objectives: (1) to evaluate the time and repetitions required in the SPM and (2) to compare the SPM adjusted table produced in Expt 1 with the SPM standard table. The methods in Expt 1 were used to measure total spermatic concentration in 141 boar ejaculates. The average spermatic concentrations (10(6) spermatozoids/ml) and average dose number were 229.1 and 22.6 for CN, and 283.6 and 28.0 for SPM. These results indicate that the SPM has a tendency to overestimate spermatic concentration with relation to the CN. The SPM produced a smaller coefficient of variation and needed lesser examination time than the CN (p < 0.05). The SPM adjusted table created in Expt 1 produced similar results as the SPM and CN standard tables (2.96 billion live spermatozoids/dose vs 2.36 billion live spermatozoids/dose). Incorporation and evaluation of supplemental data from other CIAS regarding the use of the SPM adjusted table in the field entitled 'Adjusted Table for Karras Spermdensimeter LPS-FMVZ-USP' would be valuable to prove its efficiency.