Fertilizing Ability of Electro-Ejaculated Cryopreserved Semen in the Domestic Cat

Semen collection and AI in the cat are still not routine procedures. The correlation between semen quality and fertility under natural conditions is a relatively unknown field in the cat. In the present study, functional in vitro tests, such as the ability to bind and penetrate the zona pellucida or to fertilize in vitro, were used to determine fertilizing ability of sperm cryopreserved with a practical and efficient freezing protocol previously developed in our laboratory. Semen was collected by electroejaculation, evaluated for motility and diluted with Tris-glucose-citrate egg-yolk extender supplemented with Equex STM paste (0.5% v/v). After equilibration and loading into 0.25 ml straws, semen was frozen at 3.85 degrees C/min. Frozen-thawed semen was co-cultured with in vitro matured cat oocytes. Penetration rate was recorded 30 h after in vitro fertilization and cleaved zygotes were cultured in vitro until day 7. A correlation was found between sperm motility index (SMI) after thawing and semen fertilizing ability (p<0.05). In conclusion, it was demonstrated that the post-thaw motility quality, expressed as SMI, of spermatozoa frozen using the protocol mentioned above can be considered an index of the sperm ability to penetrate in vitro matured oocytes.     

Assessing in vivo Fertilizing Capacity of Liquid-Preserved Boar Semen According to the 'Hanover Gilt Model'

The goal of this study was to determine the ability of the Hanover gilt model to assess in vivo fertilizing capacity of preserved sperm and to consider whether any modifications to this model were needed. This model evaluates the fertilizing capacity of semen based on the fertilization rate, the rate of normal embryos and the accessory sperm count of 3–5‐day embryos. Its distinguishing characteristics are the use of one‐time insemination of sperm in reduced numbers, of spontaneously ovulating gilts and of ovulation detection through ultrasound examination of ovaries. Reduced sperm numbers allow for an accurate evaluation of the fertilizing potential of different semen treatments, thereby avoiding the compensatory effect of doses calibrated to maximize fertility. The model's usefulness was assessed in a trial run designed to compare the fertilizing capacity of liquid boar semen diluted into two different extenders. The diluent, the boar and the backflow, had no significant effect on any of the parameters studied. Gilts inseminated less than 24 h before ovulation had a significantly higher (p < 0.01) fertilization rate and accessory sperm cell count (p < 0.05) than those inseminated more than 24 h before ovulation. Very good/good embryos from homogeneous litters (only very good/good embryos were present) had a significantly higher (p < 0.01) accessory sperm count than those from heterogeneous litters (at least one embryo was of a different quality and/or oocytes were present). Both very good/good and degenerated/retarded embryos from heterogeneous litters had low accessory sperm numbers. This suggests that accessory sperm count is significantly related to the quality of the litter, but not to the quality of the embryo within gilts. It can be concluded that the Hanover gilt model is sensitive enough to show fertility differences (in this study, those associated with in vivo ageing of semen), while using relatively few gilts and little time.     

In vitro Fertilizing Capacity of Deep Frozen Boar Semen Packaged in 0.5 and 5 ml Straws

The aim of this study was to evaluate the straw size effect used for freezing on the in vitro fertilizing capacity. Twenty-one ejaculates from seven fertile boars were frozen under controlled conditions in 0.5 and 5 ml straws. Thawed semen was compared to fresh semen. For fresh and thawed semen in 0.5 and 5 ml straws, the results were: 92.18, 77.38 and 79.04% sperm penetration; 80.68, 66.89 and 69.33% monospermy; 11.51, 10.49 and 9.74% polyspermy; 86.19, 47.14 and 47.02% motility and 75.52, 48.19 and 46.81% normal apical ridge (NAR), respectively. Analysis of variance and test of multiple comparisons showed that under the conditions employed, penetration, monospermy, motility and NAR were significantly reduced by freezing–thawing, but polyspermy was much less affected. The results obtained suggest that frozen boar semen is adequate for in vitro fertilization. In addition freezing in 5 ml straws did not have any detrimental effect on either penetration, monospermy, polyspermy, motility and NAR, in comparison with freezing in 0.5 ml straws.     

Cryo-scanning Electron Microscopy Discloses Differences in Dehydration of Frozen Boar Semen Stored in Large Containers

In general, freezing in flat plastic polyethylene terephthalate (PET) bags (FlatPacks) at 50°C/min gives better post-thaw viability, in terms of sperm motility and membrane integrity, than does freezing in plastic maxi-straws, probably owing to differences in cryobiology. To test the hypothesis that this better survival post-thaw relates to the degree of sperm dehydration during freezing, the present study investigated the structure of boar semen in a frozen state using cryo-scanning electron microscopy (cryo-SEM) to compare two different packages (FlatPacks and maxi-straws) for single artificial insemination (AI) doses, and three different freezing rates. The semen was split-sample frozen in maxi-straws or FlatPacks (both holding 5 ml) using 3% glycerol as cryoprotectant. Three freezing rates were applied from −5°C to −100°C, namely 2°C/min, 50°C/min and 1200°C/min, the lattermost by plunging the samples into liquid nitrogen (LN₂). The samples were thereafter fractured into LN₂ and larger areas of extra-cellular, unbound frozen water ('ice lakes') were measured to determine the degree of dehydration of the spermatozoa. These areas decreased in size with an increase in cooling rate, the differences in size being more dramatic for maxi-straws than for FlatPacks. Size of ice lakes was also influenced by location within package in relation to cooling rate, the central values being always smaller in maxi-straws than in Flatpacks (p < 0.05 at 2°C/min and 50°C/min) but not at 1200°C/min, which suggested the FlatPack allows for more homogenous freezing of boar semen.     

In vitro Evaluation of in vivo Fertilizing Ability of Frozen Rabbit Semen

The present work studied different spermatozoa parameters and the ability of frozen rabbit spermatozoa to fertilize, in vitro, in vivo‐matured oocytes, as a test to predict their in vivo fertility and prolificacy. Semen from rabbit bucks was frozen using two freezing protocols [in a freezer at ?30°C or in liquid nitrogen vapour (LNV)]. For the in vivo trial, females were inseminated with frozen‐thawed spermatozoa. Oocytes used for in vitro testing were recovered 14 h after ovulation induction from donors and co‐incubated with 2 × 10⁶ frozen‐thawed spermatozoa during 4 h at 37°C in Tyrode's medium under an atmosphere of 5% CO₂ in air with maximal humidity. After co‐incubation period, presumptive zygotes were cultured in TCM199 supplemented with 20% foetal bovine serum (FBS), under the same conditions described above. Although no statistical differences were observed between freezing protocols in seminal parameters [motility rate: 40 and 35%, VCL: 35 and 46 μm/s, amplitude of lateral head displacement (ALH): 1.7 and 2.4 μm, for semen frozen at ?30°C and in LNV, respectively], significant differences were noted in the fertilizing ability in vivo and in vitro. Semen frozen at ?30°C showed the highest fertilizing ability in vitro (26.7% vs 6.2 and 8.7% for semen frozen at ?30°C, in LNV and fresh semen, respectively) and the lowest fertility rate in vivo (21.7% vs 64.2% and 70.6% for semen frozen at ?30°C, in LNV and fresh semen, respectively). Sperm frozen at ?30°C seemed to be more capacitated.     

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.     

In vitro Evaluation of the Quality and Fertilizing Capacity of Boar Semen Frozen in 0.25 ml straws

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.     

Analysis of In vitro Fertilizing Capacity to Evaluate the Freezing Procedures of Boar Semen and to Predict the Subsequent Fertility

A porcine in vitro fertilization (IVF) system and seminal quality parameters of frozen–thawed boar semen were used to assess the effectiveness of two different thawing rates of frozen boar semen, and to address the question of whether differences between fertility of ejaculates could be predicted in a limited field trial. In the first experiment, two thawing procedures were analysed (37°C, 30 s; 50°C, 12 s) and no differences in sperm quality were found. However, when the procedure was 50°C, 12 s the IVF results showed a higher number of sperm per penetrated oocyte and a near 10 points higher rate of pronuclear formation. In the second experiment, the fertility results obtained in the limited field trial show to be efficient enough for application in a commercial use, especially for three of the employed boars (fertility ≥80%). In this limited study, the conventional seminal parameters are not accurate enough to discriminate good and bad boars in relation to fertility. On the contrary, parameters of in vitro penetrability are more precise to predict subsequent fertilities. As conclusion, the IVF fertilization system seems to be a good tool to evaluate the quality of frozen–thawed boar semen previous to its commercial way, to verify the bank semen storage quality and a good way to assay new sperm freezing procedures, as it is the more precise evaluating method in estimating the potential fertilizing ability.     

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.     

猪精液中五种病毒的初步检测

为探明猪精液在疫病传播中的作用,应用PCR和RT-PCR技术分别从发病猪场和未发病猪场采集种公猪精液进行猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV-2)和牛病毒性腹泻病毒(BVDV)的检测。结果发现,发病猪场的种公猪精液中CSFV、PRRSV、PRV、PCV-2的感染率分别为30.0%、50.0%、4.0%和30.0%,而未发病猪场的感染率分别为4.0%、20.0%、0.0%和8.0%,这表明精液是多种猪病原传播的良好媒介,提示种公猪对于疫病的发生和传播起着重要作用。     

猪精液冷冻技术研究进展

本文综述了猪冷冻精液的国内外发展简史，对冷冻保存机理进行了简要阐述，并从冷冻保存稀释液、冷冻保护剂及其浓度、诱发结晶和解冻等方面阐述了影响猪冷冻精液的因素，指出了目前在猪精液冷冻理论和实践中的存在问题，并针对这些问题提出了一定的解决办法。     

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。     

猪精液液态保存的研究进展

猪的人工授精主要使用液态保存精液 ,此法保存精子的存活率高、功能受损较小、授精繁殖率较高且操作简便。体外液态保存猪精液时 ,不仅要注意猪精子对温度变化敏感的特性 ,还要考虑酸碱度、离子的种类和强度、渗透压、过氧化损伤和微生物污染等对精子的影响 ,要向稀释液中添加各种保护成分 ,维持精子的存活和功能。目前主要是比较和改进各种稀释液 ,建立准确的精子质量评价方法 ,提高受精率     

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。     

猪精液冷冻技术研究进展

本文综述了猪冷冻精液的国内外发展简史,对冷冻保存机理进行了简要阐述,并从冷冻保存稀释液、冷冻保护剂及其浓度、诱发结晶和解冻等方面阐述了影响猪冷冻精液的因素,指出了目前在猪精液冷冻理论和实践中的存在问题,并针对这些问题提出了一定的解决办法.     

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。     

Fertility Results of Artificial Inseminations Performed with Liquid Boar Semen Stored in X-CellTM vs BTS Extender

The objective of the present field study was to compare the fertility results for boar semen diluted in X-cell stored up to 4-5 days before artificial insemination (AI) with semen diluted in Beltsville thawing solution (BTS) used for AI following 2-3 days of storage (where the first day being the collection day). A total number of 2601 double inseminations in Norwegian herds were included in this two-trial study. All the boars used in the study were mature cross-bred Norwegian Landrace x Duroc (LD), which were routinely used for AI in Norway. The inseminated gilts and sows were Norwegian Landrace x Yorkshire (LY). The AI doses contained 2.5 billion spermatozoa, and consisted of a mixture of semen from three, occasionally four, boars (i.e. heterospermic semen). Fertility was measured in terms of the likelihood of farrowing and subsequent litter size. The fertility of the semen in both of the extenders was satisfactory and no significant differences were found either in semen stored 4-5 days in X-cell compared with 2-3 days in BTS or in semen stored 2-3 days in X-cell compared with 2-3 days in BTS. The storage capability findings for the long-term extender X-cell could significantly simplify the practical issues of semen production and the distribution of AI doses containing 2.5 billion spermatozoa. However, in pig production systems where all semen is used within 2-3 days, the short-term extender BTS is as good as the more expensive extender X-cell.     

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）。