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.     

小鼠卵母细胞冷冻技术研究

采用PBS作为冷冻基础液,分别用甘油和二甲基亚砜(DMSO)作为冷冻保护液,在程序化冷冻保存和玻璃化冷冻保存条件下,研究小鼠生发泡期(GV期)卵母细胞的抗冻能力。结果表明,2种冷冻方法对小鼠GV期卵母细胞解冻后形态正常率和存活率无显著影响(P>0.05)。冷冻保护剂种类对小鼠GV期卵母细胞解冻后形态正常率无显著影响(P>0.05);但对存活率有显著影响,玻璃化冷冻采用二甲基亚砜作为冷冻保护液效果极显著优于甘油(P<0.01)。以冷冻效果较好的二甲基亚砜作为冷冻保护液,采用玻璃化冷冻不同发育阶段(GV期和MⅡ期)的小鼠卵母细胞,解冻后形态正常率无显著差异(P>0.05),但存活率GV期要显著优于MⅡ期卵母细胞(P<0.05)。     

家畜卵母细胞冷冻保存的研究进展

本文阐述了卵母细胞冷冻保存对体外受精、转基因动物研究及人类医疗方面的意义,论述了卵母细胞冷冻保存的发展过程、方法与机理及冷冻保护剂种类,通过揭示冷冻、解冻对细胞的作用,进一步探讨了各种冷冻保护剂及冻存方法对保持细胞生命力的功效。在当前卵母细胞冷冻研究中,玻璃化冷冻法发展迅速,并出现超快速玻璃化法。     

Factors affecting cellular outgrowth from porcine inner cell masses in vitro

During early embryonic development, endodermal cells leave the inner cell mass (ICM) and migrate over an extracellular matrix located on the blastocoelic side of the trophectoderm to form extraembryonic endoderm. Two experiments were conducted to evaluate factors supporting porcine endodermal cell migration in vitro. In Exp. 1, porcine ICM were cultured on matrices of collagen IV, fibronectin, or laminin. Percentages of ICM generating cellular outgrowth on fibronectin (5/11; 45%) and laminin (4/10; 40%) were similar (P > 0.10); however, collagen IV (0/10; 0%) failed (P < 0.05) to support cellular outgrowth. Inner cell mass and outgrowth areas and numbers of cells in outgrowths were similar (P > 0.10) for fibronectin and laminin, and increased (P < 0.05) with time in culture. In Exp. 2, ICM were cultured on fibronectin or laminin in medium containing 0 or 500 microg/mL of the inhibitory tripeptide, arg-gly-asp (RGD), or on laminin in medium containing 0 or 10 microg/mL recombinant human tissue inhibitor of matrix metalloproteinases-2 (rhTIMP-2). Inner cell mass and outgrowth areas and numbers of cells in the outgrowths for ICM cultured on fibronectin did not differ (P > 0.10) due to the presence of RGD. Inner cell masses cultured on laminin in medium containing 500 microg/mL RGD had fewer cells in the outgrowths and slower rates of cell migration compared with 0 microg/mL (P < 0.05). No differences (P > 0.10) in ICM and outgrowth areas and numbers of cells in the outgrowths were observed for ICM cultured on laminin in medium containing 0 or 10 microg/mL rhTIMP-2. Both fibronectin and laminin supported porcine ICM outgrowth in vitro; however, because outgrowth on fibronectin was not inhibited by RGD, endodermal cells must express an integrin that recognizes an alternative sequence in fibronectin. Cell migration on laminin was inhibited by RGD, suggesting either RGD competes with laminin for binding sites on endodermal cells or binding RGD alters endodermal cell migration on laminin. Because rhTIMP-2 had no effect on cell outgrowth, porcine ICM do not appear to be responsive to the proliferative effects of rhTIMP-2.     

Factors affecting premature chromosome condensation of cumulus cell nuclei injected into rat oocytes

To date, production of cloned rats by somatic cell nuclear transfer (NT) has not yet been successful. Inducing premature chromosome condensation (PCC) of injected cell nuclei in recipient cytoplasm is considered essential for successful mouse cloning by the Honolulu method. In the present study, some factors affecting PCC of rat cumulus cell nuclei injected into rat oocytes were examined. Wistar female rats (young: 4 to 5-week-old, mature: > or =10-week-old) were superovulated by injections of eCG and hCG, and oocytes recovered 14 or 17 h after hCG injection were received with cumulus cell nuclei using piezo-driven micromanipulator. When the oocytes were recovered 14 h post-hCG injection from young rats and the nuclear injection into oocytes was completed within 45 min, PCC was observed in 44-49% of NT oocytes. In the case of oocytes from mature rats, PCC occurred in 11-19% of the NT oocytes. Oocytes recovered 17 h post-hCG injection did not support PCC of the injected nuclei (0-7%) regardless of the donor age. Treatment of oocytes with a neutral cysteine protease inhibitor, N-acetylleucylleucylnorleucinal, slightly increased the incidence of PCC (48 vs 37%). Comparison of rat strains for oocyte donors indicated that proportions of NT oocytes undergoing PCC in Wistar and LEW oocytes (41-46%) were higher than those in Donryu and F344 oocytes (17-25%). Thus, ability of rat oocytes to promote PCC of the injected nuclei is dependent on the characteristics of oocytes, such as age or strain of donor rats, and timing of oocyte recovery.     

小鼠未成熟卵母细胞OPS法冷冻保存技术的研究

试验用OPS法对小鼠未成熟卵母细胞进行玻璃化冷冻保存研究,并对解冻后小鼠未成熟卵母细胞的成熟情况进行观察。结果表明:小鼠未成熟卵母细胞在10%EG、10%DMSO前处理后,在EFS30、EFS40、EDFS30和EDFS40中平衡15～45s进行冷冻保存,使卵母细胞解冻后的形态正常率最高达92.4%,与对照组无显著差异(P>0.05),成熟率达40.5%。     

水牛卵母细胞玻璃化冷冻保存

以水牛MII期的卵母细胞为材料,利用玻璃化冷冻液EDS33(16.5%EG+16.5%DMSO+sucrose)对水牛MII期的卵母细胞进行两步法(玻璃毛细管(GMP)和拉细的开口塑料细管(OPS))玻璃化冷冻保存,即卵母细胞首先放入预平衡液(7.5%EG+7.5%DMSO+sucrose)中平衡3 min,再移入玻璃化冷冻液中30 s后装管直接投入液氮.解冻是在蔗糖浓度逐渐降低的解冻液中进行的.解冻后存活的卵母细胞孤雌激活,通过囊胚发育率作为评定卵母细胞冷冻效果的指标.结果发现,GMP和OPS法冷冻保存的水牛卵母细胞解冻后的存活率(分别为96.80%和97.41%)与对照组卵母细胞的存活率(100%)3者之间差异均不显著(P>0.05).GMP法和OPS法冷冻的水牛卵母细胞激活后的胚胎分裂率和囊胚发育率2者均明显低于对照组(分别为30.58%和28.32% vs50.94%,10.81%和9.38% vs 29.63%,P<0.05),而这2种方法冷冻的水牛卵母细胞激活后的分裂率和囊胚发育率差异均不显著(P>0.05).这表明GMP和OPS玻璃化冷冻方法可以用于水牛卵母细胞的冷冻,并且玻璃化冷冻的卵母细胞能继续分裂并发育到囊胚.     

Factors affecting the transfer of porcine parvovirus antibodies from sow to piglets

The aim of the study was to investigate the effects of a number of environmental, behavioural and biological factors on passive immunization of piglets as assessed by transfer of porcine parvovirus (PPV) antibodies (ab) from the colostrum of PPV vaccinated mothers to the serum of the piglets. Twenty primiparous sows were housed in pens with peat, straw and branches for nest building. Half the sows were prevented from achieving feedback from a completed farrowing nest by repeated removal of the nest from 10 to 12 h after nest building had begun, whereas the other half kept their nests. Sow serum PPV-ab titres were positively related to colostrum PPV-ab titres at birth of the first piglet (BFP) (P < 0.001). Litter average piglet PPV-ab titre was positively related to both sow serum and colostrum PPV-ab titres (both P < 0.001). In addition, in the individual piglets. PPV-ab titres were reduced as time from BFP to birth and time from birth to first sucking increased and time spent sucking decreased (all P < 0.01). There were no effects of treatment, time spent in lateral recumbency by the sow, number of times the sow stood or piglet weight on day 1 on piglet serum PPV-ab titres. Preventing prolonged farrowing, while at the same time ensuring the piglets' access to the udder, is important for transfer of maternal immunity. Measurements of specific antibodies in sow serum during the periparturient period and in piglet serum at 28 days of age may provide a practical tool for evaluating transfer of maternal immunity from sow to piglets.     

牛卵泡卵母细胞的体外成熟和冷冻保存

在简化牛卵母细胞体外成熟的基础上,观察了保护剂、平衡温度、预平衡方法、解冻方法以及冷冻方法对牛卵泡卵母细胞冷冻的影响。结果表明:在体外成熟培养液中添加26.2 mmol/L的NaHCO3和对屠宰场卵巢进行选择更能促进牛卵母细胞的体外成熟;无论是程序冷冻还是玻璃化冷冻,乙二醇(EG)与甘油(GLY)相比更能促进牛卵泡卵母细胞的冷冻效果;在程序冷冻中,冷冻液中添加0.1 mol/L蔗糖比添加0.3mol/L的蔗糖更能促进牛卵泡卵母细胞的冷冻;在玻璃化冷冻中,37℃和25℃的平衡温度比4℃更适合牛卵泡卵母细胞的冷冻;在10%、5%、1%的预平衡浓度之间,5%的预平衡浓度即可达到预平衡的效果;在解冻时,多步脱除保护剂更能保护冷冻的卵母细胞;比较了几种最小样本量(minimum size sample,MSS)玻璃化冷冻方法,解冻成熟培养后的成熟率,拉细毛细玻璃管冷冻法为(41.67±3.19)%,极显著高于未拉细毛细玻璃管冷冻法(glassmicropipette,GMP)的(30.19±1.93)%和固体表面冷冻法(solid surface vitrification,SSV)的(28.33±2.89)%(P<0.01)。     

小鼠卵母细胞SSV法冷冻保存技术的研究

试验用2种前处理液(10%EG和10%EG 10%DMSO)和4种玻璃化冷冻液(EFS30、EFS40、EDFS30和EDFS40)对小鼠卵母细胞进行玻璃化(SSV)法冷冻保存,研究小鼠卵母细胞冷冻后的发育潜力。结果表明:小鼠未成熟卵母细胞形态正常率最高可达92.3%,成熟率达57.2%;成熟卵母细胞形态正常率最高可达91.5%。     

毛细玻管玻璃化冷冻法冷冻保存牛卵母细胞的研究

为了探讨毛细玻管玻璃化冷冻法(GMP)对牛卵母细胞冷冻效果的影响,试验采用毛细玻管玻璃化冷冻法冷冻不同发育阶段的牛卵母细胞,并且对不同预处理方式和玻璃化时间对玻璃化冷冻牛卵母细胞解冻后形态及体外受精卵裂率的影响进行了比较.结果表明:采用毛细玻管玻璃化冷冻法冷冻GV期和体外培养6 h、18 h、22 h各发育阶段的卵母细胞,解冻后体外受精后卵裂率分别是36.67%、40.98%、41.27%、52.38%,前三者之间差异不显著(P>0.05),前三者与培养22 h卵母细胞(52.38%)和对照细胞(68.42%)差异极显著(P<0.01);采用不同浓度的稀溶液预处理卵母细胞,3%乙二醇稀溶液预处理玻璃化前牛卵母细胞可分别获得较高的卵裂率(36.96%);卵母细胞在玻璃化溶液中暴露时间越长受到损伤越严重,暴露30 s能获得最好的玻璃化冷冻效果.     

抗氧化剂在猪精液冷冻保存中的作用

随着冷冻精液技术的发展,抗氧化剂作为精液稀释液中必不可少的一类成分,在降低精子的氧化损伤、提高精子活力质量上起到了关键的作用。文章对近些年来国内科研团队所使用的抗氧化剂进行综述,总结其对精液品质提升改善的能力,为猪精液冷冻技术的发展提供理论依据和参考。     

猪卵母细胞体外受精条件的优化

猪卵母细胞体外受精(in vitro fertilization,IVF)技术为受精和胚胎早期发育机理等基础理论的研究及生产实践提供了重要的研究手段。虽然猪IVF的研究已经取得了一些进展,但其囊胚发育率仍然较低,亟需建立更为稳定、高效的体外受精方法。本试验通过比较精子浓度,精子获能处理,不同精卵共孵育培养体系以及在卵母细胞成熟过程中添加不同激素等影响猪卵母细胞体外受精胚胎发育能力的因素,以求找到最佳的猪卵母细胞体外受精体系。结果显示:在精子浓度为1×10~5~1×10~7/mL,5×10~6/mL的精子浓度显著提高体外受精效率(P<0.05);以茶碱、咖啡及咖啡因联合使用肝素作为获能物质处理精子,发现2.5 mmol/L茶碱处理组体外受精效率显著提高(P<0.05);通过比较不同的受精体系,发现使用40个卵/500μL体系显著提高体外受精效率(P<0.05);在卵母细胞成熟液中添加不同激素组合,发现添加10IU/mL PMSG,10IU/mL HCG和2.5IU/mL FSH激素组合,体外受精效率显著提高(P<0.05)。本试验结果表明,在M199中添加10IU/mL PMSG,10IU/mL HCG和2.5IU/mL FSH体外成熟培养卵母细胞,以5×10~6/mL精子浓度,2.5mmol/L茶碱作为获能物质处理精子,40个卵/500μL共孵育的IVF体系效果最佳,其卵裂率为(61.33±0.77)%,囊胚率为(28.33±1.08)%。本试验将为深入研究猪IVF提供理论参考。     

玻璃化冷冻对猪卵母细胞超微结构的影响

本研究以GV期和MII期卵母细胞作为试验材料,通过透射电镜方法观察卵母细胞冷冻前后的超微结构变化,结果发现,透射电镜下观察卵母细胞发现,GV期卵母细胞与透明带连接紧密,微绒毛伸入透明带中,皮质区分布大量的线粒体。脂滴分为两种,一种为灰色脂滴,一种为深色脂滴。MII期卵母细胞排出第一极体,质膜下分布大量的皮质颗粒。微绒毛缩短成矮柱状,线粒体常聚集存在,卵周隙出现。冻后GV期卵母细胞微绒毛消失,线粒体肿胀,线粒体内有囊泡样结构,包围脂滴的内质网不完整,胞质内溶解为絮状,未见高尔基体。冻后MII期卵母细胞透明带损伤、微绒毛、细胞膜损伤甚至消失、极少量皮质颗粒分布于皮质区。脂滴部分溶解,相互连接,脂滴周围伴随大量肿胀呈圆形的线粒体,嵴不明显,内呈囊泡样,未见到高尔基复合体结构。     

猪卵母细胞与猪、水牛及食蟹猴精子的胞质内显微受精

为探讨猪精子、水牛精子及食蟹猴精子分别注入到猪卵母细胞后原核形成及早期胚胎发育情况,利用屠宰场收集的猪卵母细胞,经体外成熟44～48h后,进行胞质内显微受精(ICSI)操作。试验1:体外培养18～19h,用Ho-echst33342荧光染色,检查原核形成情况。试验2:进行体外培养,ICSI后2d检查分裂率,7d记录囊胚率。试验1结果显示:在原核形成率上,猪同种显微受精,原核形成率(50.10%)显著高于注入水牛精子(37.06%)及食蟹猴精子(37.48%),且差异显著(P0.05),注入水牛精子和食蟹猴精子间差异不显著。试验2结果显示:猪同种显微受精所得的分裂率(86.58%)和囊胚率(29.93%)与水牛精子注入(70.98%,18.48%)、食蟹猴精子注入(78.69%,16.92%)差异显著(P0.05)。结果表明,水牛精子、食蟹猴精子分别注入到猪卵母细胞后,观察到雌雄原核和精子解聚;水牛精子注入猪卵母细胞与食蟹猴精子注入猪卵母细胞,经体外培养发育到囊胚。     

Factors affecting measurements of glucose metabolism and lipolytic rates in porcine adipose tissue slices in vitro

Porcine adipose tissue glucose metabolism and lipolytic rates have been measured for many years by numerous investigators. However, there is little or no documented indication of the effects of variation in tissue handling procedures or variations in incubation medium components on metabolic rates. We have systematically varied conditions to provide such documentation for these much used techniques. The temperature (18 to 38 C) of tissue during transport had little effect. The medium for tissue transport probably should be buffered. Use of Hepes buffer at greater than 10 or 25 mM in incubation media inhibited glucose metabolism and lipolysis. Calcium ion effects on glucose metabolism or lipolysis could not be demonstrated. Dimethyl sulfoxide should not be used routinely. Ascorbate at .56 mM did not inhibit glucose metabolism or lipolysis. Glucose metabolism was increased by glucose concentration to about 5 mM and not inhibited at higher concentrations; we recommend 10 or 20 mM glucose to ensure maximal rates. Insulin stimulated glucose metabolism but effects were slight, not related to insulin concentration and not consistently observed. Addition of some albumin preparations did not allow expression of insulin stimulation; we recommend albumin be omitted or, if included, carefully monitored. Lipolytic rates were dependent on albumin concentration, but rates were similar with all albumin preparations. Insulin markedly inhibited hormone-stimulated but not basal lipolysis. Adenosine, an inhibitor of lipolysis, did not affect glucose metabolism rates. An artificial oxygen carrier did not increase anabolic activity. Incubation in serum increased rates of glucose metabolism relative to lipolysis so that refinement of the incubation might lead to greater anabolic than catabolic rates in vitro to reflect the status of adipose tissue in growing pigs in vivo. Tissue handling and incubation conditions can markedly affect metabolic rates, and should be understood and controlled.     

猪MII期卵母细胞的玻璃化冷冻的初步研究

试验采用3种冷冻载体(麦管、开放式拉长麦管和半麦管)对猪卵母细胞玻璃化冷冻后发育能力的影响进行研究,以探讨采用自制半麦管作为载体对猪MII期卵母细胞玻璃化冷冻后发育能力的影响.结果发现,采用半麦管法、OPS法玻璃化冷冻的猪卵母细胞的形态完整率、存活率、卵裂率分别为90.85%、60.07%、20.43%和84.15%、55.71%、14.71%,无显著差异(P＞0.05),但采用半麦管法的结果好于OPS法;与麦管法的形态完整率、存活率和卵裂率(分别为60.06%,39.64%和0)均有显著性差异(＜0.05).结论为采用半麦管法玻璃化冷冻猪卵母细胞可以稍微提高其冻后发育能力.     

冷冻方法和冷冻保护液对山羊卵母细胞发育的影响

比较了不同冷冻方法和冷冻保护液对山羊卵母细胞冷冻-解冻后体外受精和激活后的发育效果.结果表明,开放式拉长塑料细管(OPS)法的效果好于玻璃化细管法和常规冷冻法;在OPS法中,20%EG 20%DMSO对卵母细胞的冷冻保护效果好于EDFS40,而在玻璃化细管法冷冻中,则是EDFS40好于20%EG 20%DMSO.对于常规冷冻法而言,则是1.5mol/L EG的冷冻效果好于1.5mol/L PROH.     

Effect of relaxin on in vitro fertilization of porcine oocytes

Porcine relaxin is a peptide hormone belonging to the insulin super family that has a variety of biological functions. The present experiment was designed to investigate the effects of relaxin on sperm function and on in vitro fertilization (IVF) of porcine oocytes. Porcine spermatozoa were washed, swum-up, and incubated for 1-4 h in mTALP medium supplemented with 0, 20 or 50 ng/ml porcine relaxin. Motility was determined by observing the type of forward movement of the spermatozoa, and acrosome status was evaluated by applying the triple staining technique. Immature oocytes were aspirated from antral follicles and matured in IVM medium (modified NCSU-37). Matured oocytes were co-cultured with spermatozoa in IVF medium (mTALP) supplemented with 0, 5, 10, 15 or 20 ng/ml relaxin. After 6 h of sperm-oocyte co-incubation, putative zygotes were cultured for 18 h in oocyte culture medium NCSU-37 and then assessed for the rates of monospermy, polyspermy, and male pronucleus formation after acetic orcein staining. Relaxin improved (P<0.05) sperm motility and increased the percentage of acrosome-reacted live spermatozoa during 1-4 h of incubation, although viability was not significantly improved. Significantly (P<0.05) the highest percentage of monospermic (31.7%) and lowest percentage of polyspermic (16.5%) fertilization was achieved from the sperm-oocyte co-culture group treated with 20 ng/ml relaxin as compared to other groups. The percentage of male pronucleus formation was significantly (P<0.05) greater in the 20 ng/ml relaxin-treated sperm-oocyte co-culture group than in the other groups. These results indicate that supplementation with relaxin is capable of improving sperm function and fertilization of porcine oocytes in vitro.