慢病毒与慢病毒病研究进展

人的艾滋病与动物慢病毒病的相关性和相似性,使慢病毒和慢病毒病成为医学和兽医学领域最为关注的问题之一.作者就慢病毒的病原特性、致病机制及相关几种常见动物慢病毒病作一论述.     

慢病毒载体的研究进展及应用

慢病毒载体是近年来受到广泛关注的一种逆转录病毒载体,具有更安全、转移效率高、可将目的基因整合入宿主基因组和可感染非分裂期细胞等优点,因此有望成为理想的基因转移载体,并在临床和生产实践中广泛应用。作者主要以HIV-1为代表对慢病毒载体的构建及其在基因治疗和转基因动物生产中的应用作一综述。     

转基因动物中慢病毒载体包装的优化

慢病毒载体作为基因治疗载体发展起来,现已用于转基因动物制备.本实验用磷酸钙沉淀法将转移质粒(pCCL-hCMV-eGFP)、包装质粒(pCMVΔR8.9)、包膜蛋白质粒(pMDG(VSV-G))三质粒共转染来源于人胚肾细胞系的包装细胞-293T,转染48 h后收集病毒上清,采用6孔板培养感染细胞,荧光显微镜计数EGFP阳性细胞法测定病毒滴度.载体滴度达2×107 TU/ml左右.     

慢病毒载体法制备转基因动物研究进展

慢病毒能够感染分裂细胞和非分裂细胞,因而被发展成为重要的转基因载体,已成为制备转基因动物的一种工具,转基因效率明显提高。该文介绍了制备转基因动物的技术方法,比较了慢病毒载体制备转基因动物的特点和优势,介绍了慢病毒载体安全设计的发展,并将近年来国内外利用慢病毒载体法制备转基因动物的研究进行了概述。     

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.     

猪圆环病毒的致病作用与防制措施

猪圆环病毒(PCV)有两个血清型,PCV1和PCV2。其中PCV1无致病性,感染猪不出现临床症状,而PCV2可产生多种致病作用。自2001年我国部分地区发生猪圆环病毒病之后,去年,不少规模化养猪场呈暴发流行。1PCV2的致病作用1.1引起免疫抑制研究表明,PCV2具有免疫抑制性。感染PCV2的猪一般都发生免疫缺陷,主要是淋巴细胞缺损如淋巴器官中的T细胞和B细胞数量减少,淋巴组织中巨噬细胞浸润,外周血和淋巴组织中的巨噬细胞、单核细胞数量升高。因此,病猪对疫苗接种没有免疫应答,对低致病性或减弱疫苗的微生物可引发疫病,重复发病对治疗无应答性。1.…     

猪精子冷冻损伤的研究

冷冻保存后的猪精子，结构受到损伤，妊娠率下降。其超微结构变化与正常鲜精的顶体反应非常相似，包括质膜膨胀碎裂，顶体外膜泡状化。我们提议上述变化称为假顶体反应。     

卵黄对精子保护作用的机制

在过去的60年里,冷冻精液稀释液的配方发生了很大的变化,但是其中的抗冻剂却始终是卵黄、奶及甘油.由于卵黄在保存、降温、冷冻过程中对精子的保护作用机制仍是不解之谜,因而目前尚无一种有效的可避免污染的化合物来替代他们.     

猪单精子显微注射技术的研究进展

猪单精子显微注射(ICSI)技术还未完全建立,目前的研究主要集中在注射前精子处理和ICSI卵母细胞的激活处理对ICSI卵母细胞发育的影响上。与其他哺乳动物相比,猪的ICSI生产效率依然很低,提高猪ICSI效率的方法还很少。本文简要介绍猪ICSI技术研究概况、影响因素、应用前景及其存在问题。     

Factors affecting porcine sperm mediated gene transfer

“Sperm mediated gene transfer” (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene–spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2 h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine AI a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by AI was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa.     

Factors affecting porcine sperm mediated gene transfer

“Sperm mediated gene transfer” (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene–spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2 h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine AI a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by AI was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa.     

精子不同处理和胰岛素不同浓度对猪卵母细胞胞质内单精子显微受精的影响

探讨了分别使用新鲜、冷冻和超声波断尾精子以及在胚胎培养液中分别添加不同浓度胰岛素对猪卵母细胞胞质内单精子显微受精(ICSI)胚胎早期发育的影响.结果:(1)使用冷冻解冻精子与新鲜精子相比对猪卵母细胞ICSI后的卵裂率和囊胚率均无显著影响(P＞0.05);2)精子断尾与否对猪卵母细胞ICSI后的分裂率和囊胚率没有显著影响(P＞0.05);3)在胚胎培养液中添加5 mg/L胰岛素与对照组相比可显著提高猪ICSI胚胎的囊胚发育率(18.22% vs 3.60%,P＜0.05).     

Relationship between sperm quality traits and field-fertility of porcine semen

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 × 10⁸ sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 × 10⁶ sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 × 10⁹ spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2~76.5%) and SCI (0.16~4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity.     

The relationship of porcine sperm zona-binding ability to fertility

Several laboratory assays have been designed to assess the fertility potential of a semen sample before insemination, but none have been consistent and accurate predictors of fertility. To determine whether zona-binding ability may be a useful fertility predictor, we validated and used an in vitro competitive assay to measure the ability of porcine sperm to bind to the zona pellucida. The zona-binding ability of sperm from 11 boars that exhibited a broad range in average litter size and farrowing rate was determined. Sperm from each boar were compared directly with sperm from eight other boars in a systematic, pairwise fashion. Sperm from two semen samples were labeled with fluorophores at concentrations that did not affect motility or zona-binding ability. An equal number of labeled sperm from each boar was coincubated with homologous oocytes. Least squares means from analysis of variance were used to rank boars based on zona-binding ability. The competitive assay was effective in establishing a ranking of the boars (R2 = 0.62). Furthermore, there was a correlation between zona-binding ability and fertility when estimated by average litter size (r = 0.64, P < 0.05) but not when estimated by farrowing rate (r = -0.28). The explanation for this difference was that litter size and farrowing rate were poorly correlated (r = 0.14). In conclusion, a competitive zona-binding assay distinguished boars that sired either small or large litters. Competitive zona-binding ability may be useful for identifying boars with reduced fertility that produce smaller litters following insemination.     

猪精子冷冻损伤与保护剂的研究进展

猪精液冷冻保存技术主要是利用液氮(-196℃)作为冷源,在超低温的环境下使精子生理代谢完全暂停,实现长期保存的目的.猪精子在低温环境主要损伤是细胞内结冰晶造成的机械损伤、游离的活性氧造成的化学损伤,对这两种损伤机制研究是国内外科研工作者们的研究热点.文章主要简述猪精液冷冻技术发展简况,精子在冷冻过程中受损伤机制和现阶段...     

Experimental evidence for the influence of cumulus-oocyte-complexes on sperm release from the porcine oviductal sperm reservoir

In the pig, a temporal relationship is suggested between sperm release from the sperm reservoir (SR) and ovulation, but the mechanism(s) is still under discussion. In two experiments, the influence of transferred ova on the release of SR-spermatozoa at ovulation and the effect of supplementation with non-sulfated glycosaminoglycan hyaluronan (HA) on embryo development and the number of accessory spermatozoa, respectively, were examined. PMSG/hCG primed ovectomized gilts that had previously received endoscopic low-dose insemination into the cranial uterine horn were used as an experimental model. After salpingectomy, tubal segments (ampulla, cranial, and caudal isthmus) were flushed and sperm numbers or respective accessory spermatozoa were counted. In Experiment 1, the distribution of the sperm population was altered in the presence of cumulus-oocyte-complexes (COCs). A higher proportion of spermatozoa was found after transfer of COCs into one oviduct in the ampulla and cranial isthmus segments compared with the controls (17.5 vs. 4.9%, p<0.05). In Experiment 2, the quality of the transferred ova and treatment influenced the presence of accessory spermatozoa. Transfer of COCs together with HA increased (p<0.05) the number of accessory spermatozoa compared with the other treatment groups and was similar to those in the "undisturbed" controls. No modifications were obtained regarding mean blastomere numbers (2.6 +/- 0.2 to 3.1 +/- 0.2). In summary, this study was demonstrated that cumulus-oocyte-complexes may be involved in triggering sperm release from the pig oviductal SR and that HA might be related to sperm release.     

The porcine sperm reservoir in relation to the function of hyaluronan

The oviduct plays a role in successful animal reproduction not only in spermatozoa and ova transport to the fertilization site but also by affording a microenvironment for fertilization and early embryonic development. The sperm reservoir (SR) is restricted in the uterotubal junction (UTJ) and caudal isthmus. Billions of porcine spermatozoa are distributed to the female reproductive tract during/after insemination, and small amounts of them are stored for about 36–40 hours in the SR, which maintains sperm viability in the pre-ovulation period through its surface epithelium and production of fluid. The SR regulates the release of spermatozoa so that only a small population moves towards the fertilization site (ampulla) to decrease polyspermy. This review attempts to provide information about the structure and function of the porcine SR, its intraluminal content (hyaluronan, HA), and the influences of HA on porcine spermatozoa in vivo. In pigs, the spermatozoa are stored in a mucous-like fluid within the UTJ and caudal isthmus in the pre-ovulation period. The oviduct fluid contains sulfated glycosaminoglycans (GAGs) and non-sulfated GAGs, i.e., HA. It is interesting to note that HA is synthesized by hyaluronan synthase-3 (HAS-3), and its receptor, CD44, is found in the epithelium of the porcine SR site. Additionally, sperm capacitation does not occur in vivo in the SR during the pre- and peri-ovulation periods, but spermatozoa in the SR will attempt to capacitate if exposed to bicarbonate. However, capacitation in the SR will rise in the post-ovulation period, indicating the role of HA in modulating sperm capacitation after ovulation. All data support the understanding that the porcine SR ensures the viability of fertile spermatozoa and maintains the non-capacitated status during the pre-ovulation period. This basic knowledge about the SR is believed to be useful to advance sperm preparation procedures for in vitro fertilization (IVF) and improve the preservation process of porcine semen.