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1.
Transfer and low temperature storage of embryos are now proven techniques for a number of mammalian species. These techniques are useful in control of disease and in saving genotypes from infected animals. The place of embryos in the epidemiology of disease depends upon whether the causative organism can gain entry to the oocyte before or at fertilisation and on whether the young embryo can be invaded by organisms in the uterine environment. There is little evidence that important live-stock diseases are transmitted via gametes. The zona pellucida surrounding the embryo is an effective barrier against a number of important disease organisms; in some cases the embryo is susceptible once it has hatched from the zona pellucida. It is important therefore in considering the use of embryos in disease control, to ensure that virus is not attached to the surface of the zona pellucida from where it can infect the recipient and/or the embryo after hatching. Washing procedures have been devised together with the use of enzymes and antisera to remove virus from the surface of embryos. Some viruses enter pores and sperm tracks in the zona and removal of these may present a problem. African swine fever virus has been shown to resist removal by treatment with enzymes. There are no guidelines as to the likely interaction between a certain virus and embryos. Therefore each virus of interest must be tested to determine whether it can be transmitted via washed embryos. Nevertheless there are numerous instances of the use of embryo transfer to eradicate a specific disease or to save valuable genetic material from infected animals without transmitting disease.  相似文献   

2.
The presence of the zona pellucida has been perceived as a requirement for the oviductal transfer of cloned embryos at early stages of development while protecting the embryo from an immune system response. We hypothesized that steroid hormone therapy could reduce a potential cellular immune response after the transfer of zona‐free cloned embryos into the oviduct of recipient female goats. In Experiment 1, seven does were used to study the systemic immunosuppressant effect of the methylprednisolone administration (for 3 days) on blood cell counts. Whole blood was collected prior to treatment with methyprednisolone and then on Days 1, 2, 3, 4, 7, 14, 21 and 28 after the first dose of methylprednisolone for the analysis of haematological parameters. Methylprednisolone treatment significantly reduced circulating white blood cells and neutrophils in comparison with pre‐treatment levels, demonstrating a systemic immunosuppressant effect. In Experiment 2, a group of 58 does were used as recipient females to study the effect of administration of methylprednisolone for 3 days on the establishment of pregnancies after the transfer of zona‐free cloned embryos into the oviducts. No effects on pregnancy rates on Day 30 were observed regarding the distinct treatment groups (control vs. methylprednisolone), the source of oocytes (in vivo‐ vs in vitro‐matured) or the presence or absence of the zona pellucida in embryos. In summary, methylprednisolone was effective at inducing a systemic immunosuppressed state in goats, but the treatment prior to embryo transfer did not affect pregnancy rates. Moreover, pregnancy rates were similar between zona‐free and zona‐intact goat cloned embryos.  相似文献   

3.
The advantages of transporting mammalian preimplantation embryos rather than postnatal living animals include reduced costs of transportation and rapid dissemination of genetic material between countries. However, the risk of transmission of diseases through the embryos must be considered. The disease control potential of embryos will depend on proper handling and washing, and the integrity of zona pellucida. Researches on embryo-pathogen interactions have shown that some pathogens are carried through the gametes and others could not infect the gametes. Some pathogens were found to adhere to the zona pellucida and others could penetrate the zona pellucida. To date, data presented appear to suggest no concrete model guidelines for embryo-pathogen interaction. The interaction seems to depend on the species and the pathogen involved.  相似文献   

4.
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.  相似文献   

5.
Cattle embryos at the compact morula and blastocyst stages were bisected by two different methods. In some embryos the embryo bisection was performed outside of the zona pellucida and both halves were replaced into it. Other embryos were bisected within the zona pellucida and no pipetting of the halves was required. They were transferred to synchronized recipient cows and the pregnancy rates and twinning rates were evaluated.  相似文献   

6.
To investigate the susceptibility of early bovine embryos to noncytopathogenic bovine viral diarrhea virus (NCP BVDV), 2- and 4-cell embryos produced in vitro from which zona pellucida had been removed by pronase treatment, and hatched blastocysts were exposed to 10(6) TCID50/m/ of NCP BVDV No. 12 strain. The virus was detected in all embryo samples immediately prior to cultivation but not in the medium. After 24-hr culture, the virus was isolated from four media and two embryo samples in four experiments in the blastocyst group, and the viral antigen was demonstrated in the cytoplasm of the embryo cells by the immunofluorescent technique. By contrast, no virus was recovered from, or viral antigen detected in samples from the 2- and 4-cell embryo group in any of the experiments, even though they were exposed to the virus after removal of the zona pellucida. These findings suggest that 2- and 4-cell embryos are unlikely to be susceptible to NCP BVDV, but that blastocysts are capable of being infected with the virus. hatched blastocyst, noncytopathogenic bovine viral diarrhea virus.  相似文献   

7.
The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
试验旨在探究腺病毒感染水牛颗粒细胞和胚胎的最佳条件,以提高转基因水牛胚胎的生产效率。以水牛颗粒细胞和体外发育的胚胎为研究对象,分别用0、1×10~0、1×10-1、1×10-2、1×10-3、1×10-4、1×10-5 GFU/mL腺病毒感染水牛颗粒细胞,获得最佳感染浓度后,在最佳浓度条件下分别感染24、48、72、96 h,摸索最佳感染时间,用倒置荧光显微镜观察试验结果。利用腺病毒感染水牛颗粒细胞的最佳浓度和时间分别感染2细胞和4细胞期胚胎,对胚胎的最佳感染条件进行摸索,分析胚胎分裂率、囊胚率和转染囊胚率。结果显示,1×10-2 GFU/mL浓度和48 h感染时间可获得水牛颗粒细胞的最佳腺病毒感染效率。腺病毒感染2细胞和4细胞期无透明带和非完整透明带胚胎后胚胎发绿光,而感染完整透明带胚胎后不发光,2细胞期胚胎感染后停止发育,4细胞期开始感染的非完整透明带胚胎可继续发育至囊胚。于4细胞期分别感染完整透明带组、非完整透明带组和无透明带组水牛胚胎,结果显示,非完整透明带转染组在囊胚率和囊胚转染效率上均优于其他组。综上,非完整透明带,1×10-2 GFU/mL和48 h为感染浓度和时间,4细胞期为感染起始期的腺病毒介导的水牛转基因胚胎生产方法能够实现目标基因在水牛胚胎中的高效表达,从而达到提高转基因水牛胚胎生产效率的目的。  相似文献   

9.
The relationship of plasminogen activator (PA) production to cell stage, cell number and changes in overall diameter and zona pellucida thickness for bovine embryos developing in vitro was determined. Late morulae to blastocysts (n = 80) were collected nonsurgically from naturally mated, estrous-synchronized, superovulated crossbred beef cows. Embryos were cultured, one embryo per 25-microliters microdrop, for 6 d. At 24-h intervals, embryos were evaluated for stage of development and transferred to fresh microdrops; media were recovered for PA analysis. In addition, embryo diameter and zona pellucida thickness were measured with an ocular micrometer. Plasminogen activator production was determined using a caseinolytic assay with urokinase as the standard. Changes in diameter, zona pellucida thickness and PA production per 24-h interval for each embryo were plotted, and the graphs were cut out and weighed. Sixty-one embryos (76%) completed the hatching process. Total PA production was correlated positively (P less than .005) to embryonic size (r = .40), developmental stage (r = .35) and cell number (r = .35) and negatively, but weakly, correlated to zona pellucida thickness (r = -.13; P = .267). Hatched embryos produced more total PA than embryos that did not hatch (.140 +/- .011 vs .070 +/- .019 g; P less than .01). These results suggest that as embryonic size and cell number increase and development progresses, bovine embryos liberate more PA.  相似文献   

10.
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.  相似文献   

11.
The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time‐lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time‐lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non‐viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro‐handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre‐implantation developmental kinetics should be observed.  相似文献   

12.
Porcine oocytes and pre-implantation embryos from the same, as well as from different animals, have an extremely heterogeneous morphology of the zona pellucida (ZP) surface, as shown by scanning electron microscopy. For years, it has been believed that this heterogeneous morphology plays an important role in the sperm-oocyte interaction. The aim of this study was to analyse the zona morphology and sperm-binding patterns on the porcine ZP. Oocytes were divided into four categories: immature, matured in vivo, or matured in vitro over a time period of 24 or 48 h. The zona morphology of early embryos grown in vivo or in vitro was also investigated. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between the different kinds of maturation in terms of these zona types. All oocytes exhibited extremely heterogeneous zona morphologies, with no clear trend. During subsequent in vivo embryo development, the zona surface changes from a porous structure to one with a compact surface, while the morphology of in vitro embryos remained compact at all stages of development. The analysis of the number and distribution patterns of spermatozoa trapped in the ZP revealed extremely variable patterns, regardless of the zona morphology. Differences were only present if sorted or unsorted spermatozoa were used for insemination. Regardless of the number of inseminated spermatozoa after sorting, only a few (1-2) could be detected on the ZP. Whether oocytes were matured in vivo or in vitro was not a relevant factor. Unsorted spermatozoa bound in higher numbers than sorted ones. The number was directly dependent on the number of spermatozoa used for insemination.  相似文献   

13.
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.  相似文献   

14.
The association of bovine immunodeficiency virus (BIV) with embryos derived by in vitro fertilization from oocytes of experimentally infected heifers or oocytes/embryos exposed to the virus in vitro was investigated. Using a nested-PCR assay, proviral DNA of BIV was not detected in follicular fluid or in embryos derived from BIV-infected donors. In vitro exposure of oocytes to BIV during maturation or insemination with BIV-infected semen resulted in zona pellucida-intact embryos testing negative for BIV provirus. However, exposure of zona pellucida-free day-7 embryos to the virus resulted in a positive BIV assay for 28% of the batches of embryos, suggesting that the zona pellucida has a role in protecting against BIV infection. The presence of BIV in the IVF system had no apparent effect on the development of bovine embryos to the blastocyst stage.  相似文献   

15.
透明带的切割是哺乳动物早期胚胎显微操作技术中的一个重要环节，本文以小鼠为例介绍一种新的透明带切割方法。  相似文献   

16.
While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.  相似文献   

17.
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18.
Transfer of bovine demi-embryos with and without the zona pellucida   总被引：1，自引：0，他引：1  
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.  相似文献   

19.
The ability of seven cytopathic strains of bovine viruses to adhere to the zona pellucida of six-to-eight day-old bovine embryos were compared. Embryos were exposed to virus by placing them either in virus suspensions or by culturing them on infected bovine turbinate cultures for 18-24 h. After exposure to bovine virus diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBV), bluetongue virus (BTV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), parainfluenza 3 virus (PI3), or bovine enterovirus virus (BEV), the embryos were tested for virus by culture in bovine turbinate cells and by morphological examination using electron microscopy (EM). A special technique to minimize loss of embryos processed for EM was developed. More embryos had viral particles on the surface of the zona pellucida after exposure to 18-24 hour infected cell cultures than did embryos exposed to viral culture suspensions. The most dramatic finding was that BTV adhered in large numbers to the surface of the zona pellucida of exposed embryos. IBRV, PRV, and VSV comprised an intermediate group, with virions occasionally detected on the surface of exposed embryos after 5 washes. Therefore, extensive washing is required. The PI3 and BEV were easily removed from embryo-exposed virus by washing. BVD was difficult to identify morphologically, making assessment by EM unreliable. There was no evidence that any one of the seven viruses penetrated the intact zona pellucida. Using a micromanipulator, 42 embryos were also directly inoculated through the zona pellucida with +/- 50 picoliters of virus inoculum or medium.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

20.
The objective of the present study was to clarify the possible role of the zona pellucida (ZP) in early development of rat embryos and to determine the effect of glycosaminoglycans on the development of ZP-free 8-cell embryos before or after embryo transfer at the blastocyst stage. Eight-cell embryos were divided into three groups comprised of, 1) intact controls, 2) embryos with the ZP was removed with acidic solution and 3) pairs of ZP-free 8-cell embryos aggregated in a small hollow. These embryos were cultured in a chemically defined mR1ECM for 24 h. Developmental ability to the blastocyst stage and mean cell number in the blastocyst was lower in ZP-free embryos than in intact controls. When these blastocysts were transferred, the farrowing rate and efficiency of embryos developed to term were also lower in ZP-free embryos, but not in the aggregated ones. Supplementation with hyaluronan (HA; 63-250 μg/ml) or heparan sulfate proteoglycan (HS; 15 μg/ml) significantly improved blastocyst formation of ZP-free embryos and the cell number in the blastocyst by reducing the incidence of apoptosis. However, there were no beneficial effects of HA or HS on farrowing and newborn rates after transfer of the blastocysts. In conclusion, the ZP plays roles in maintaining successful development of early rat embryos at least from the 8-cell stage not only to the blastocyst stage but also to posttransfer stages. Glycosaminoglycans, such as HA or HS, appear to contribute to successful cleavage during early development to the blastocyst stage but may be insufficient to maintain the posttransfer survival of ZP-free embryos.  相似文献   

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