影响猪精子介导基因转移效果的因素研究

为优化猪精子载体法技术参数,利用荧光定量PCR、荧光显微镜检测和精液常规检测方法,分析不同DNA转染剂、不同孵育温度和不同形态DNA对猪精子转染外源DNA的影响。结果表明,PEI和TransFast转染剂能极显著提高猪精子转染效果,且PEI优于TransFast,而NanoFect转染剂包裹DNA不能转染精子。随着转染温度的升高,精子的活力和活率均明显下降,而转染率和内化外源DNA量基本保持稳定,而吸附外源DNA量呈下降趋势。环状DNA和线性化DNA在与精子共孵育后,两者的精子活力、活率、转染率和内化外源DNA量差异均不显著,精子吸附线性化DNA量极显著高于环状DNA量。综合分析表明,外源DNA形态对猪精子转染效果无显著影响;PEI和TransFast能够显著提高猪精子转染效果;共孵育温度以17℃为最佳,本结果为开展精子载体法制备转基因猪研究提供了基础试验依据。     

获能液和DMSO对猪精子获能效率的影响

为了探讨获能液和二甲基亚砜(DMSO)对猪精子获能效率的影响,试验采用SPSS 14.0软件进行分析,各处理平均值采用邓肯氏多重比较。结果表明:CM获能液获能3小时时超激活运动率为57.9%;mTBM获能液获能4小时时超激活运动率为55.7%;0.1%DMSO获能液获能30分钟时精子活率为70.20%,顶体反应率为70.44%,与其他组相比差异显著(P<0.05)。     

猪精子结合和内化外源基因的影响因素研究

精子介导基因转移(SMGT)是目前转基因动物研究中简单而高效的方法之一,其中精子结合和内化外源基因的效率是精子介导转基因成功的关键.本试验以DIG标记的线性化EGFP作为示踪基因来检测猪精子结合和内化外源基因的影响因素,结果表明:猪精子能够自发结合外源基因,结合部位主要在精子顶体后区.精子结合外源DNA的阳性率随共孵育时间延长而增加,在37℃或39℃时孵育60 min后,阳性率不再增加;在17℃时孵育90min后,阳性率不再增加.在检查的15只公猪样本中,精子与外源DNA的结合率为6.57%～35.81%,内化率为2.99%～24.66%,个体间差异显著(P<0.01).精浆能够强烈抑制外源基因的结合和内化,脂质体及DMSO能够显著提高转染效率.死精子能够结合外源基因,但不能将其内化;反复冻融导致质膜破裂的精子对外源基因有更高的结合率,且不受个体影响.     

精子介导法制备GHRH转基因猪的研究

以eGFP为报告基因,生长激素释放激素(GHRH)为目的基因,利用精子介导的基因转移方法,将构建慢病毒载体质粒LV-EGFP-GHRH直接和猪精子孵育,再通过体外人工受精建立GHRH转基因猪模型.经PCR检 测,52头后代中发现7头阳性个体,转基因效率为7.42%.本研究探索了应用精子介导的转基因方法将GHRH转移到动物体内,为猪的转基因育种提供了新的依据.     

猪精子不同处理方式对其IVF的影响

猪体外受精(in vitro fertilization,IVF)技术效率的不稳定性限制了该技术在畜牧生产中的应用。本研究比较了精子获能前后的状态和活精子数之间的关系;精子获能后的浓度和状态对IVF后胚胎卵裂率、囊胚率的影响;不同个体来源的精子混合后对IVF卵裂率、囊胚率的影响。结果表明:精子获能前后,活精子数无显著性差异(P>0.05);检测精子的浓度与状态与卵裂率、囊胚率无直接关联;利用混合精子进行IVF,单一精子所得到的IVF囊胚率分别是A17.00%、B 12.24%、C 33.00%、D 4.81%、E 10.00%、F 22.00%,混合精子的囊胚率分别是A+B 77.00%、C+D 67.01%、E+F 35.50%,混合精子和单一精子的囊胚率差异不显著。     

菟丝子提取物对猪精子冷冻效果的影响

在猪精液冷冻液中添加不同浓度的菟丝子提取物,研究冻后精子的形态正常率、质膜完整率、SOD活性及MDA含量,探讨菟丝子提取物对猪精液冷冻保存效果。结果表明,冷冻-解冻后,猪精子形态正常率、质膜完整率和SOD活性均显著降低,MDA含量显著高于对照组(P<0.05)。各冷冻组相比,菟丝子中剂量组精子形态正常率、膜完整率和SOD活性均高于其他冷冻组,MDA含量低于各冷冻组,但各指标仅表示与菟丝子低剂量组存在显著差异(P<0.05)。试验表明,适量的菟丝子提取物可对抗冷冻造成的膜损伤,提高精子SOD活性,降低MDA含量。     

弱酸对猪精子活率及产仔性别的影响

分别向猪精液中加入醋权、乳酸、草酸、柠檬酸,改变了精液的ＰＨ值,并测定精子的活率,当精液的ＰＨ值达到６．５,精子沃经达到６０％时,将此褒庇人发情母猪人工配种,结果当每ｍｌ精液中加入１％的醋酸０．１５ｍｌ（ｐＨ６．５）时,对母猪的产仔数影响不显著（Ｐ〉０．０５）,但与对照组相比可显著地提高仔母猪的比例（Ｐ〈０．０５）。     

Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transfer

REASON FOR PERFORMING STUDY: Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. OBJECTIVES: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. METHODS: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also transfected with fluorescently-labelled DNA (Alexa647-pEGFP) and changes in sperm viability and DNA uptake were assessed. Mares were inseminated with pEGFP-transfected spermatozoa and embryos recovered. Expression of pEGFP was assessed by epifluorescence microscopy of embryos, and the presence of pEGFP DNA and mRNA was assessed by PCR and RT-PCR, respectively. RESULTS: Liposome-mediated transfection increased the incorporation of 32P-pEGFP into spermatozoa compared to controls. Flow cytometric evaluation of spermatozoa after transfection with Alexa647-pEGFP revealed a linear increase in the proportion of live, Alexa647+ spermatozoa with increasing DNA concentrations. After insemination with transfected spermatozoa, 8 embryos were recovered. There was no evidence of EGFP expression in the recovered embryos; however, PCR analysis revealed evidence of the pEGFP transgene in 2 of 5 embryos analysed. CONCLUSIONS: The incorporation of exogenous DNA by equine spermatozoa was enhanced by liposome-mediated transfection and this did not adversely affect sperm viability, acrosomal integrity or fertility. Although the EGFP transgene was detected in a proportion of Day 7-10 embryos, there was no evidence of expression of EGFP in these embryos. POTENTIAL RELEVANCE: Sperm-mediated gene transfer offers a potential technique for the generation of transgenic equids.     

Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI

Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors.     

Effects of sperm pretreatment on efficiency of ICSI-mediated gene transfer in pigs

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.     

野猪SLA—DQB外显子2的核苷酸多态性分析

本试验采用DNA测序技术测定了15头野猪235bp的SLA—DQB基因外显子2序列．结合GeneBank中报道的另3头野猪和18头家猪共36个个体的相应序列,重点对野猪SLA—DQB外显子2进行核苷酸多态性分析。结果表明野猪SLA—DQB外显子2具有较丰富的核苷酸多态性;在家猪品种改良中,野猪具有潜在的遗传价值。     

Reproductive efficiency of a new modified boar semen extender for liquid storage

In the present study the Authors developed a new modified boar semen extender for short-term liquid storage, based on the use of amikacin sulphate and fructose rather than gentamicin and glucose. The new extender (ME-S) was evaluated and compared in vitro to commercial ones (CRONOS^™, TRIXcell^™) and to a modified extender designated for long term storage (ME-L) for progressive motility. Progressive motility was not different (P>0.05) among extenders until 120 h of storage, as differences among extenders became significant (P<0.05) at 144 and 166 h. Motility data across time were better for ME-S than TRIXcell^™ (P<0.05). No differences were observed about the morphology and membrane integrity (ORT) among the new extender (ME-S) and the commercial ones. Following the results of the in vitro comparison, an artificial insemination field trial was performed for reproductive efficacy. In this trial ME-L was not used because it was not completely reliable yet. A total of 1011 sows were bred: 506 with ME-S and 505 with a commercial one (CRONOS^™). The pregnancy rate for ME-S was 93.68% (474 pregnant sows), as the commercial extender resulted in 452 pregnancies (89.5%). The statistical comparison was significant (P<0.05) and the number of live piglets born showed an increase of 52.     

微量元素硒在公猪营养和繁殖中的生理作用

硒通过参与硒蛋白的合成在猪营养中发挥重要作用.硒蛋白是调节体内抗氧化系统的中心,其中谷胱甘肽过氧化物酶是猪抗氧化功能研究最多的硒蛋白.但其他硒蛋白在公猪精液生产和维持精液质量中也有重要作用.公猪精液的特点是含有大量易氧化的长链多不饱和脂肪酸,需要有效的抗氧化防御.猪对硒的需求因环境和其他条件的不同而不同,同时处于繁殖期...     

Assessment of storage effects in liquid preserved boar semen

Fertility of extended boar semen declines within the first 72 h of storage in vitro. Standard semen assessment, such as motility and membrane integrity, allows detection of lethal damage of spermatozoa. However, conventional sperm assessment often lacks standardization and does not allow identification of sub-lethal changes of sperm quality during the initial 72 h of storage. In the present brief review, recent strategies for quality assessment of liquid preserved boar semen are discussed and basic implications for experiments designed to detect storage effects are given.     

NTF3基因与公猪繁殖性能相关性研究进展

神经营养因子3(neurotrophin 3,NTF3)是一种由靶细胞分泌合成并经神经突起逆向转运至胞体而发挥作用的小分子蛋白质,主要在神经系统的发育和生理功能维持中发挥重要作用。但近年来的研究表明,NTF3及其受体与睾丸的发育、成熟和精子发生过程等密切相关,同时,NTF3基因对公猪性机能发育、性成熟、生精调节等方面具有重要调控功能。本文简介NTF3的分子结构、分布、受体及作用机制、生物学功能,重点阐述NTF3基因的启动子、转录因子及其与公猪繁殖性能相关性的研究状况,以期为今后开展更多、更深入的科学研究提供参考。     

Expression of lactoferrin in the boar epididymis: effects of reduced estrogen

Lactoferrin is regulated by estrogen in the female reproductive tract and evidence in immature mice suggests that it may be estrogen regulated in males as well. The estrogen regulation of lactoferrin in the epididymis of the boar, a high estrogen-producing male, is unknown. This study was designed to test the hypothesis that lactoferrin expression in the boar epididymis is regulated by estrogen. Twenty-one littermate pairs of boars were treated with vehicle or Letrozole, an aromatase inhibitor, from 1 week of age until castration at 2 through 8 months. Epididymal tissue was collected at castration and fixed for immunolocalization of lactoferrin. Epididymal and testicular tissues were also collected from five mature boars (1-2.5 years) and fixed for immunocytochemistry (ICC). Lactoferrin was localized in the principal cell cytoplasm of the caput, corpus and cauda of developing boars but only in the corpus and cauda of mature boars. Basal cells were negative for lactoferrin. Sperm in the corpus and cauda was also positive for lactoferrin. The efferent ducts and testes were negative for lactoferrin. Intensity of lactoferrin immunostaining increased with age in the corpus and cauda regardless of treatment. Reduced endogenous estrogen in the epididymis during development did not affect the intensity of immunostaining between control and Letrozole-treated animals. Lactoferrin expression in the epididymis of the developing boar does not appear to be regulated by estrogen.