Effects of porcine zona pellucida immunocontraceptives in zoo felids.

Methods of contraception are necessary for management of zoo felids; however, the most commonly used contraceptive (melengestrol acetate implant) is associated with serious adverse reactions with long-term use. Porcine zona pellucida (pZP) vaccines are promising as contraceptives, but their safety in zoo felids has not been tested. pZP vaccine was administered to 27 female felids representing 10 species, including African lion (Panthera leo), Asian leopard (P. pardus), jaguar (P. onca), tiger (P. tigris), snow leopard (P. uncia), cougar (Felis concolor), Siberian lynx (F. lynx), Canada lynx (F. canadensis), serval (F. serval), and bobcat (F. rufus), in 15 facilities. Over 6 wk, each animal received three i.m. injections of 65 microg pZP with Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, or carbopol as the adjuvant. Behavioral signs of estrus were seen in 14 of the vaccinated felids. An unacceptably high incidence of adverse reactions was seen including injection site swelling, lameness, limb swelling, or abscessation (or all) in five felids after injection with FCA as the initial adjuvant. Adverse behavioral signs, including increased irritability and aggression, were seen in four felids. Six of the felids were assayed for antibodies against pZP during the 12 mo after vaccination; all showed antibody production. Antibody levels appeared to peak 1-4 mo after vaccination began, although elevated antibody levels persisted in two animals for > 12 mo after the first injection. All vaccinated felids were ovariohysterectomized 3-13 mo after vaccination. Folliculogenesis was present in all treated animals, and there was no histopathologic evidence of inflammatory damage to ovaries. Contraceptive efficacy was not specifically evaluated in this study; however, two of the three felids housed with an intact male became pregnant during the study, one of which gave birth to healthy cubs.     

Role of acidification elicited by sialylation and sulfation of zona glycoproteins during oocyte maturation in porcine sperm-zona pellucida interactions

The porcine zona pellucida (ZP) undergoes biochemical changes during the final phase of maturation prior to fertilization. The present study was conducted to elucidate whether the acidification of ZP glycoproteins during porcine oocyte maturation influences sperm-ZP interactions. Two-dimensional gel electrophoresis clearly demonstrated that ZP acidification occurred in accordance with the sialylation and sulfation of ZP glycoproteins in oocytes matured for 44 h. The increases in the incidences of sperm penetration and polyspermy with the progress of the IVM culture period were significantly suppressed by ZP desialylation on treatment with neuraminidase as a consequence of reductions in the number of sperm bound to ZPs and the acrosome reaction (AR) in ZP-bound sperm (P<0.05). In contrast, the blocking of ZP sulfation by NaClO(3) treatment during IVM markedly reduced the incidence of polyspermy with no inhibitory effect on penetration, but the number of sperm bound to ZPs and the rate of AR-inducing sperm were decreased to the same level as in desialylated oocytes. The results indicate that ZP sulfation influences sperm-ZP interactions in a ZP sialylation-independent manner. Moreover, sialylation and sulfation were not associated with a protective proteolytic modification of the ZP matrix before fertilization. These findings suggest that ZP acidification elicited by the sialylation and sulfation of ZP glycoproteins during oocyte maturation contributes to the porcine ZP acquiring the capacity to accept sperm.     

The involvement of N‐glycosylation of zona glycoproteins during meiotic maturation in sperm–zona pellucida interactions of porcine denuded oocytes

The present study was conducted to delineate whether N‐glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation and whether this N‐glycosylation played a role in sperm–ZP interactions of porcine cumulus denuded oocytes (DOs). After mechanical removal of cumulus cells from cumulus oocyte complexes (COCs), DOs were cultured for 44 h in in vitro maturation (IVM) culture. The experiments were carried out to determine the effects of tunicamycin, a specific N‐glycosylation inhibitor, for various intervals during IVM on sperm–ZP interactions in porcine DOs. The results determined that DOs could induce meiotic maturation, although the maturation rate of DOs was earlier than that of COCs. In addition, N‐glycosylation of ZP glycoproteins occurred during meiotic maturation and was crucial in sperm–ZP interactions, was responsible for sperm penetration, sperm binding to ZP and induction of acrosome reaction in ZP‐bound sperm. However, the inhibition of N‐glycosylation by tunicamycin during IVM did not influence ZP hardness and male pronuclear formation, indicating that this N‐glycosylation was involved in the initial stage of fertilization. We conclude that 24–44 h of N‐glycosylation of ZP glycoproteins during meiotic maturation was crucial in sperm penetration and sperm binding to ZP and the induction of acrosome reaction in sperm bound to ZP of porcine DOs.     

The importance of the zona pellucida for disease control in livestock by embryo transfer

From the point of view of disease risk, the movement of livestock by embryo transfer is undoubtedly much safer for trading than the movement of live animals or semen. Nevertheless, strict governmental control by veterinary certification of health of embryos is still vital. In cattle, sheep and pigs, unlike laboratory species such as the mouse, infectious agents do not appear to pass through the zona pellucida (ZP) into the embryo proper. Some agents do, however, adhere firmly to the outer surface of the ZP, especially onto those of the pig. Disease risks associated with the inadvertent transmission of infectious agents when embryos are moved are, therefore, intimately connected with the nature and properties of the ZP. This article reviews current knowledge on the physical and adhesive properties of the ZP and discusses how risks associated with the possible presence of infection on its surface can be minimized. Further research is urgently needed so that realistic but safe veterinary certification of the health of embryos for international trade can be devised.     

Transfer of bovine demi-embryos with and without the zona pellucida

Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.     

Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses

A multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect multiple viral infections of swine. Specific primers for each of four common DNA viruses, namely, pseudorabies virus (PRV), porcine circovirus type I (PCV1), porcine circovirus type II (PCV2), and porcine parvovirus (PPV), were used for testing procedure. The assay was shown to be highly sensitive in that as little as 10(-4) ng of each of the respective amplicons (approximately equal to 10,000 molecules) was detected when a composite of all four viruses (including both field and gene-deleted permutations of PRV) was tested as a single sample. It was also effective for detecting one or more of these same viruses in various combinations in specimens including lymph nodes, lungs, spleens, and tonsils collected from clinically ill pigs, and in specimens in spleen collected from aborted fetuses. The relative efficiency (compared to performing separate assays for each virus) and apparent sensitivity of mPCR suggest its potential application for routine molecular diagnostic purposes.     

Effect in vitro of bovine viral diarrhea virus on bovine embryos with the zona pellucida intact,damaged and removed

The in vitro effect of bovine viral diarrhea virus (BVDV) on the survival of day 7 to day 7.5 bovine embryos collected from superovulated donors was studied. Fifty-four experimental embryos with the zona pellucida (ZP) intact, damaged or removed were exposed to 1×10⁴ TCD₅₀/ml of the NADL cytopathic strain of BVDC at 37°C for 24 hrs and compared to 36 control embryos that were cultured for 24 hr. Seven embryos with the ZP-removed were similarly exposed for 48 hrs and compared to five control embryos. The overall survival rate was 68% for embryos exposed to BVDV for 24 hrs and 77% for embryos not exposed (P>0.05). Extended exposure of the embryos with the ZP removed to virus for 48 hrs did not affect their survival rate compared to controls. Damage to the ZP by cracking or total removal of the ZP by micromanipulation or acidic Tyrode's solution had no effect on subsequent embryonic survival in the presence of BVDV. It was concluded that exposure to BVDV in vitro is not cytopathic for morula and blastocyst stage bovine embryos over a 48 hr period, even when they are not protected by the ZP.     

猪细小病毒-伪狂犬病病毒二联油乳剂灭活疫苗的研制

猪细小病毒(Porcine paruovirus，PPV)和伪狂犬病病毒(Pseudorabies virus，PRV)均是导致母猪发生繁殖障碍性疾病的主要病原，它们在世界范围内广泛分布，造成了巨大的经济损失，严重制约了养猪业的健康发展，已经成为猪病研究的热点。     

多重PCR检测猪细小病毒和猪伪狂犬病病毒的研究

根据GenBank上已发表的猪细小病毒（Porcine parvovirus，PPV）的VP2基因序列和猪伪狂犬病病毒（Porcine pseudorabies virus，PRV）的gH基因序列，设计合成了两对特异引物，分别建立了PPV和PRV的单项PCR诊断方法，通过对扩增条件的筛选，最终成功地建立了PPV和PRV的复合PCR诊断方法，即利用一次PCR反应，可同时扩增PPV的751bp和PRV355bp的特异性片段，而扩增猪圆环病毒Ⅱ型（PCV．2）及相应的培养细胞（PK-15）核酸结果均为阴性，对PPV和PRV的最低检出量分别为100Pg和10Pg的DNA。该方法适合对PPV和PRV的联合检测和鉴别诊断。     

猪细小病毒与猪伪狂犬病毒二重SYBR GreenⅠ实时荧光PCR方法的建立与检测

本研究旨在建立一种能快速、灵敏、同时检测出猪细小病毒与猪伪狂犬病毒的基于SYBR GreenⅠ实时荧光定量PCR方法。参照GenBank中登录的相关基因序列,设计了2对引物分别用于扩增PRV gH基因与PPV NS1基因的部分片段。将测序正确的PRV gH基因与PPV NS1基因片段克隆入pGEM-T Easy载体,转化大肠杆菌DH5α,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBR GreenⅠ荧光定量PCR标准曲线和熔解曲线,并对其灵敏性、特异性和重复性进行验证。结果表明,猪细小病毒与猪伪狂犬病毒荧光定量PCR的标准曲线的Tm值分别为80.9℃和86.5℃,熔解曲线特异,灵敏度分别可达248拷贝/μL和160拷贝/μL,是普通PCR检测方法的100倍。本次建立的猪细小病毒与猪伪狂犬病毒荧光定量PCR检测方法实现了2种病毒的同时检测,能够对PRV、PPV混合感染的临床病料进行快速诊断。     

Pseudorabies virus infections in explants of porcine nasal mucosa.

The spread of infection and the morphogenesis of three pseudorabies virus strains were studied in explants of porcine nasal mucosa. Virulent NIA-3 virus was compared with a deletion mutant 2.4N3A, and with a non-virulent Bartha virus. All three virus strains infected nasal epithelial cells. NIA-3 virus particles were enveloped mainly at the inner nuclear membrane; the virus rapidly invaded the stroma, causing widespread necrosis. In contrast, 2.4N3A virus particles were enveloped mainly at the endoplasmic reticulum and the infection spread more slowly. Bartha virus particles were enveloped mainly at the endoplasmic reticulum; the infection spread slowly and remained restricted to the epithelial cells. In situ DNA hybridisation showed an accumulation of Bartha virus DNA in the nucleus 24 hours after inoculation. In nasal mucosa viral virulence seemed directly related to the speed of replication and release of virus from infected cells.     

Isolation of transmissible Gastroenteritis virus, pseudorabies virus, and porcine enterovirus from pharyngeal swabs taken from market-weight-swine

Pharyngeal swab samples were collected at a central Iowa abattoir from 6,010 market-weight slaughter hogs from September 1, 1979 to August 31, 1980. The swab samples were examined for cytopathic viruses by inoculation of monolayer cultures of continuous line of swine testicular cells. Of the 6,010 swab samples tested, transmissible gastroenteritis virus was isolated from 91 (1,51%), pseudorabies virus was isolated from 431 (7.17%), and porcine enterovirus was isolated from 21 (0.35%). Although all 3 viruses were identified throughout the year, transmissible gastroenteritis and pseudorabies viruses were found more frequently during the winter and early spring. In contrast, porcine enterovirus was detected more frequently during the spring and summer.     

多重PCR同时检测猪圆环病毒2型,猪细小病毒,猪繁殖与呼吸综合征病毒和猪瘟病毒

本试验建立一种可同时检测猪圆环病毒2型(PCV-2),猪细小病毒(PPV),猪繁殖与呼吸综合征病毒(PRRSV),猪瘟病毒(CSFV)4种病毒的多重PCR方法.对于每一种特定的病毒,用4对寡核苷酸引物均能特异扩增其目的片段.以含有病毒目的片段的质粒为模板,测定了多重PCR的检测灵敏度,PRRSV和CSFV检测最低限是48 pg,而PPV和PCV-2为0.48 pg.利用建立的多重PCR方法对具有产自有繁殖障碍母猪的仔猪或具有呼吸障碍症状的76个仔猪样本进行检测.检出了4种病毒的存在,其中26个样本(34.2%)同时感染了2种以上病毒.结果表明多重PCR方法检测猪混合感染的病毒,是一种快速、灵敏、低成本、高效率的病原学诊断工具.     

多重PCR／RT—PCR技术检测PRRSV、PPV、PRV和PCV-2

根椐GenBank中已发表的猪蓝耳病毒(PRRSV)、猪细小病毒(PPV)、伪狂犬病毒(PRV)和猪圆环病毒Ⅱ型(PCV-2)等4种病毒基因序列,对各病毒基因区进行同源性分析,确定PRRSV M和N、PPV VP2、PRV gD、PCV-2ORF2基因的保守区为各病毒的诊断靶序列.在建立各病毒单项PCR技术的基础上,优化多重PCR反应条件,建立了4种病毒的四重PCR技术,可同时扩增PRRSV的660 bp(北美株),PPV的313 bp,PRV的217 bp,PCV-2的447 bp的特异性片段.用82份临床病料对本研究多重PCR技术和单项PCR/RT-PCR技术进行对比验证,结果显示,两者的总符合率为93%以上.表明建立的多重PCR检测方法,具有特异、快速、准确的特点,可用于对这4种病毒的同时检测和鉴别诊断.     

Effect of the size of zona pellucida opening on hatching in the common marmoset monkey (Callithrix jacchus) embryo

The use of the common marmoset monkey in biomedical research has increased recently, and further attention has been devoted to this model after the successful production of transgenic marmosets. To extend genetic engineering approaches to widespread biomedical research fields, efficient prolonged in vitro culturing of embryo development is necessary. We aimed to evaluate the effects of the size of the zona pellucida opening on promoting the hatching process in the marmoset embryo. Piezo‐microdrilling of a 6‐μm opening in eight embryos resulted in four partially hatched embryos and one hatched embryo after 5 days of culture. Piezo‐microdrilling a 20‐μm opening in 11 embryos resulted in nine partial hatchings and no hatched embryos. Piezo‐scraping an 80‐μm opening in six embryos resulted in no partially hatched embryos and five hatched embryos. These results suggest that an 80‐μm opening, rather than 6‐μm or 20‐μm openings, is suitable to complete the hatching process in the marmoset embryo.     

表达猪细小病毒VP2蛋白和猪IL-18重组伪狂犬病毒的构建

为了获得表达猪细小病毒(PPV)VP2蛋白和细胞因子猪白细胞介素-18(IL-18)的重组猪伪狂犬病毒(PRV)。将PPV VP2基因和猪IL-18基因分别插入到PRV转移质粒PG中,得到重组质粒PG18-VP2。利用脂质体转染法将重组转移质粒PG18-VP2与猪PRV弱毒株DNA共转染猪睾丸(ST)细胞,以EGFP荧光标记,通过5轮蚀斑筛选纯化,成功获得表达PPV VP2蛋白和猪IL-18且带EGFP标记的重组伪狂犬病毒IL18-VP2-rPRV。用重组病毒感染ST细胞,12h后在荧光显微镜下可见明亮的绿色荧光;通过RT-PCR证实感染细胞中含有PPV VP2和猪IL-18mRNA;Western-blot试验结果显示,重组病毒能表达具有生物活性的PPV VP2蛋白和猪IL-18。重组病毒经连续20次传代后感染细胞仍能发出绿色荧光,PCR检测表明VP2及IL-18基因在重组病毒中能稳定遗传。为进一步研究表达PPV VP2蛋白和猪IL-18的重组猪伪狂犬病毒的免疫效力奠定了基础。     

Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro

The purpose of this study was to investigate the adherence of bovine viral diarrhea virus (BVDV) to bovine mature, or immature, cumulus-free oocytes and to in vitro fertilized embryos, maintained in vitro in a ligated bovine oviduct to allow for the hardening of the zona pellucida. Incubation of the oocytes and embryos in the oviduct for 5 h caused hardening of the zona pellucida as measured by resistance to pronase digestion (which increased from approximately 3 min to 7 h; P >0.001). However, there was no difference between the number of infected oocytes and embryos (n = 965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P > 0.05). It was concluded that the modification of the proteolytic resistance properties of the zona pellucida during in vitro oviductal incubation did not influence the adherence of BVDV to zona pellucida of oocytes or in vitro fertilized embryos.     

Laboratory evaluation of selected disinfectants as virucidal agents against porcine parvovirus, pseudorabies virus, and transmissible gastroenteritis virus

Of a variety of disinfectants evaluated, only sodium hypochlorite and sodium hydroxide inactivated porcine parvovirus (PPV) after a 5-minute incubation period. After the same incubation time, pseudorabies and transmissible gastroenteritis viruses were inactivated by all of the disinfectants tested. When the incubation time was increased to 20 minutes, 2% glutaraldehyde and a double-strength concentration of a commercial formaldehyde preparation also inactivated PPV. Formaldehyde vapor and ultraviolet radiations inactivated PPV also, but relatively long exposure times were required.