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1.
卵母细胞胞浆内单精子注射(ICSI)技术作为辅助受精的一种手段,自从出现以来,就显示出广泛的应用前景.ICSI将体外受精和胚胎的显微操作技术相结合,大大降低对精子质量的要求,在畜牧业生产实践和哺乳动物生殖生理的基础研究中都有着十分重要的意义.然而,ICSI过程中的每一个环节都可能影响到其效果,最主要和最根本的影响因素是精子和卵子本身的状态.论文就精子有关方面的因素,简要综述了精子顶体与核周鞘、细胞骨架与中心粒、DTT处理和卵母细胞激活因子(SOAF)等对ICSI的影响. 相似文献
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Nakai M Kashiwazaki N Ito J Maedomari N Ozawa M Shino M Noguchi J Kaneko H Kikuchi K 《The Journal of reproduction and development》2011,57(2):183-187
In intracytoplasmic sperm injection (ICSI) technique, a sperm was injected into ooplasm directly using a glass pipette. The fertilization physiology in ICSI is considered quite different from that of the natural fertilization. The different mechanisms for fertilization may be the causes of various results in ICSI. In this paper, we focus on the state of sperm membranes, nuclear or DNA integrity during ICSI procedure and discuss the influence of these factors on fertilization and embryonic development. We also introduce some examples in application of ICSI for new technologies in pigs. 相似文献
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Toshinori OIKAWA Tomoko ITAHASHI Takashi NUMABE 《The Journal of reproduction and development》2016,62(1):11-16
The purpose of this study was to determine whether dithiothreitol (DTT) treatment of sperm and ethanol
activation improve embryo production by intracytoplasmic sperm injection (ICSI). Further, we compared ICSI
with standard in vitro fertilization (IVF) in oocytes obtained from cattle. We demonstrated
that DTT reduced the disulfide bond in the bovine sperm head. Using oocytes obtained from a slaughterhouse,
ICSI-DTT treatment without ethanol showed the highest rate of blastocyst formation. We applied these results
to fertilization using ovum pick-up (OPU). Eleven Japanese black cattle served as donors for OPU plus standard
IVF (OPU-IVF). Of them, four donors with low embryo development rates were selected to determine whether
embryo development was enhanced by OPU plus ICSI (OPU-ICSI). We assessed effects on embryo development
following IVF and ICSI in oocytes obtained using OPU. Blastocyst rates were significantly higher for OPU-ICSI
than for OPU-IVF. Our results suggest that OPU-ICSI improves the blastocyst development rate in donors with
low embryo production compared with the standard OPU-IVF. 相似文献
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Artificial insemination (AI) is the oldest and currently most common technique in the assisted reproduction of animals and humans. The introduction of AI in farm animals was forced by sanitary reasons and the first large-scale applications with a commercial goal were performed in cattle in the late 1930s of last century. After the Second World War, cryopreservation of semen facilitated distribution and AI was mainly performed for economic reasons, especially in dairy cattle industry. In humans however, AI was initially performed in cases of physiological and psychological sexual dysfunction, but later on also in cases of infertility caused by immunological problems. Currently, the most common indications for intra-uterine insemination (IUI) in humans are unexplained infertility and male subfertility. In these cases, IUI is considered as the treatment of the first choice, before more invasive techniques such as in vitro fertilization (IVF) and intracytoplasmatic sperm injection (ICSI) are used. In contrast with humans, the quantity and quality of semen produced by farm animals is much higher and permits dilution and production of several insemination doses per ejaculate. However, with the introduction of sex-sorted semen in farm animals, the same problem of low-quality semen as in humans has arisen. In cattle, pigs and horses, conventional insemination with low numbers of sex-sorted spermatozoa results in a significant decrease in fertility. To improve the fertility rates with this semen, new insemination techniques have been developed in order to deposit spermatozoa closer to the site of fertilization. In sows and mares the advantage of utero-tubal junction (UTJ) insemination has already been proven; however, in cattle it is still under investigation. In this review, the differences and similarities in the application of AI between animals and humans are discussed and as AI in farm animals is most successful in cattle, the situation in this species is elaborated the most. 相似文献
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试验旨在探讨单精子胞浆内注射（intracytoplasmic sperm injection,ICSI）法生产猪体外受精卵和应用猪ICSI受精卵进行胞质注射生产转基因胚胎的可行性。首先对比了猪体外受精（in vitro fertilization,IVF）受精卵与ICSI受精卵的胚胎发育效率;然后观察了猪ICSI受精卵的双原核形成时间及效率,对精子注射到胞质后6~18 h分6个时间段进行地衣红染色,对比精子进入卵胞质后的状态及原核形成;最后对猪IVF受精卵受精后8~10 h及ICSI受精卵受精后12~14 h进行EGFP-N1质粒（20 ng/μL）胞质注射,观察胚胎发育效率及转基因效率。结果表明,ICSI受精卵的胚胎发育率（卵裂率89.4%和67.9%、囊胚率36.5%和16.1%）显著优于IVF组（P<0.05）,适合用于猪的体外受精卵试验;猪ICSI受精卵双原核在精子注射到卵胞质后12~14 h形成,双原核形成率为54.90%,显著高于其余5个试验组（P<0.05）;ICSI受精卵胞质注射组胚胎卵裂率（86.2%和66.3%）、囊胚率（30.0%和13.6%）及转基因效率（18.5%和0）均显著高于IVF受精卵胞质注射试验组（P<0.05）。本试验结果为采用ICSI受精卵进行胞质注射生产转基因猪的研究奠定了基础。 相似文献
9.
本试验探讨了不同辅助激活方法(Calciumionophore A23187激活、Calciumionophore A23187+6-DMAP联合激活和电激活)、不同精子预处理方法(液氮冻融处理和0.1%Triton X-100处理)和在添加半胱氨酸的胚胎培养液中培养不同时间(0 h、4 h、12 h和168 h)对猪卵母细胞内单精子注射(ICSI)胚胎体外发育的影响。结果显示:与无辅助激活相比,A23187+6-DMAP联合激活和电激活均能显著提高ICSI卵母细胞的激活率、卵裂率和囊胚率(P0.05),A23187+6-DMAP联合激活能显著提高ICSI卵母细胞的受精率(P0.05)。液氮冻融精子组ICSI卵母细胞的雄原核形成率显著高于活精子组(P0.05)。在添加半胱氨酸的胚胎培养液中培养4 h的ICSI卵母细胞受精率、雄原核形成率和囊胚率显著高于0 h组(P0.05)。以上结果表明,猪卵母细胞在ICSI后需要辅助激活来启动胚胎顺利发育,A23187+6-DMAP激活效果较好。液氮冻融精子可以促进ICSI后雄原核的形成。半胱氨酸处理4 h对猪ICSI卵母细胞受精和发育均有促进作用。 相似文献
10.
LG Devito CB Fernandes IDP Blanco PM Tsuribe FC Landim‐Alvarenga 《Reproduction in domestic animals》2010,45(4):654-658
Intracytoplasmic sperm injection (ICSI) consists of the introduction, by micromanipulation, of a single sperm into the cytoplasm of a mature egg. This technique is particularly advantageous when only a few sperm are available for fertilization, representing an important tool in preserving genetic material, especially from poorly fertile males. The results from ICSI in cattle are very often unsatisfactory and difficult to reproduce. Thus, the goal of this study was to evaluate the effect of the use of a Piezo drill (PD) and oocyte activation with ionomycin + roscovitine (I + R) during ICSI in cattle oocytes. After in vitro maturation (24 h), cumulus complex oocytes were divided into four groups: G1 – the ICSI was performed without the use of a PD and the oocyte was activated with I + R; G2 – the ICSI was performed with the use of the PD and activation with I + R; G3 – the ICSI was performed with the use of the PD, but without activation and G4 – parthenogenetic control, treated with I + R, but without sperm injection. The presumptive zygotes were cultured for 7 days and evaluated on day 3 for cleavage rate and on day 7 for blastocyst formation. Embryo production by standard in vitro fertilization in the laboratory was 78% for cleavage (117/150) and 35% for blastocyst formation (41/150). The cleavage rates obtained in G1, G2 and G4 were similar (66.7%, 71.6% and 66.3%, respectively), demonstrating the beneficial effect of oocyte activation. However, in G3, despite the presence of the sperm and the electric stimulation of a PD, the cleavage rates were significantly lower (17.5%) compared with the groups that used chemical activation, even in the absence of sperm (G4). Despite the beneficial effects of activation, this stimulus alone, or in the absence of the PD, was not sufficient for adequate morulae formation (13.4%, 37.9%, 0.0% and 13.5% for G1, G2, G3 and G4, respectively). Only in G2, when the PD was used followed by artificial activation, blastocysts were obtained (14.7%). These results indicate that cattle oocytes must be activated after ICSI to produce viable embryos. 相似文献
11.
单精注射法生产转基因小鼠的研究 总被引：1，自引：0，他引：1
精子胞质内显微受精技术（ICSI）作为辅助受精的一种手段,将体外受精研究和胚胎的显微操作技术结合起来,比以往对精子质量的要求大大降低,使其无论在畜牧业生产实践还是在哺乳动物的生殖生理基础研究中都有着十分重要的意义。本研究用小鼠精子及小鼠精子与GFP基因孵育后对小鼠卵母细胞进行ICSI,获得子代鼠。提取鼠尾基因组DNA,应用PCR、Southern blot进行整合检测。在发育至成年的11只小鼠中经PCR和Southernblot检测到3只阳性（27.3%）。结果表明,利用ICSI技术可以高效地生产转基因小鼠。 相似文献
12.
In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection
Matsukawa K Akagi S Adachi N Sato F Hasegawa T Takahashi S 《The Journal of reproduction and development》2007,53(4):877-885
In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing. 相似文献
13.
K Kikuchi N Kashiwazaki T Nagai M Nakai T Somfai J Noguchi H Kaneko 《Reproduction in domestic animals》2008,43(S2):401-406
In vitro fertilization (IVF) of in vitro matured (IVM) oocytes in pigs has become the most popular method of studying gametogenesis and embryogenesis in this species. Furthermore, because of recent advances in in vitro culture (IVC) of IVM–IVF embryos, in vitro production (IVP) of embryos now enables us to generate viable embryos as successfully as for in vivo -derived embryos and with less cost and in less time. These technologies contribute not only to developments in reproductive physiology and agriculture but also to the conservation of porcine genetic resources and the production of cloned or genetically modified pigs. However, in IVP, there still remains the problem of abnormal ploidy, which is caused by performing procedures under non-physiological conditions. In recent years, unique technologies such as intracytoplasmic sperm injection (ICSI) or xenografting of gonadal tissue into immunodeficient experimental animals have been developed to help conserve gamete resources. These technologies combined with IVP are expected to be useful for the conservation of gametes from important genetic resources. Here, we discuss the developmental ability and normality of porcine IVP embryos and also the utilization of ICSI and xenografting in advancing biotechnology in pigs. 相似文献
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Michiko NAKAI Manabu OZAWA Naoki MAEDOMARI Junko NOGUCHI Hiroyuki KANEKO Junya ITO Akira ONISHI Naomi KASHIWAZAKI Kazuhiro KIKUCHI 《The Journal of reproduction and development》2014,60(3):256-259
In pigs, the embryonic developmental ability after intracytoplasmic sperm injection (ICSI) is inferior to that resulting from
in vitro fertilization (IVF). We evaluated the timing of cell division up to blastocyst formation on
embryonic development after ICSI using either whole sperm (w-ICSI) or the sperm head alone (h-ICSI) and IVF as a control. At
10 h after ICSI or IVF, we selected only zygotes, and each of the zygotes/embryos was evaluated for cleavage every 24 h until
168 h. We then observed a delay in the 1st and 2nd cleavages of h-ICSI embryos and also in blastocoele formation by w-ICSI
embryos in comparison with IVF embryos. The rate of blastocyst formation and the quality of blastocysts in both ICSI groups
were inferior to those in the IVF group. In conclusion, the delay in cleavage of porcine ICSI embryos shows poorer embryonic
development. 相似文献
16.
Yoo JG Hur CG Park MR Park JY Hwang KC Kim JH Kim JH Cho SK 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2012,74(4):429-434
The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P<0.05). The cleavage rate was significantly higher in EST activated ICSI group (78.6%) than in the IVF and non-activated ICSI groups (51.8 and 46.0%, respectively, P<0.05), as was the proportion of oocytes that developed to the blastocyst stage at day 7 (18.9 vs. 11.6 and 9.1%, respectively, P<0.05). Diploid blastocysts were observed in 52.4, 63.0, and 65.2% of oocytes in the IVF, activated, and non-activated ICSI groups, respectively. Eight out of 23 gilts (34.8%) were confirmed to be pregnant in activated ICSI groups, but none of these pregnancies were carried to term. These results show that oocyte activation after ICSI is effective in elevating the cleavage rate and blastocyst development, while ensuring normal chromosome composition. Further research is needed to determine the pregnancy maintenance requirements for ICSI-embryos in pigs. 相似文献
17.
The Development and Application of the Modern Reproductive Technologies to Horse Breeding 总被引：5，自引：0，他引：5
WR Allen 《Reproduction in domestic animals》2005,40(4):310-329
Although the horse was probably the first animal to experience and benefit from artificial insemination, it trailed the field somewhat with regard to the application of embryo transfer and other oocyte and embryo-related modern breeding technologies. But with a late run it is now back in mid-field and gaining fast on the other large domestic species in the application of the many technological advances of the past 20 years to sound breeding practice. Improvements in extenders and cryoprotectants have resulted in a veritable upsurge in the transport and insemination of cooled and frozen stallion semen, and parallel improvements in ovulation induction and synchrony, exogenous gonadotrophic stimulation of multiple fertile ovulations and simplified, more efficient methods for non-surgical transfer of embryos to recipient mares, coupled with relaxation of breed society registration restrictions, have together contributed to a similar upsurge in the application of embryo transfer to all breeds and athletic types of horses worldwide, with the continuing and notable exception of the Thoroughbred. Although conventional in vitro fertilization remains something of an unjumped fence in equids, other modern breeding technologies like hysteroscopic low-dose insemination, fluorescence-activated sex sorting of stallion spermatozoa, between-species embryo transfer, embryo freezing and bisection, transvaginal ultrasound-guided oocyte collection, intracytoplasmic sperm injection for fertilization (ICSI), gamete intrafallopian transfer (GIFT) and now nuclear transfer (cloning), have all been applied to equids with encouraging success. Cloning, especially, holds enormous promise for the Sporthorse industry to re-create champion geldings in stallion form for breeding purposes. 相似文献
18.
Cheng WM Wu ZH Zhang X Zhu YB Pang YW Guo M Wang D Tian JH 《Reproduction in domestic animals》2012,47(4):609-614
To improve the activation protocol for in vitro-maturated porcine oocytes after intracytoplasmic sperm injection (ICSI), we examined the combined effect of U0126, a specific inhibitor of mitogen-activated protein kinase kinases, and an electrical pulse on pronuclear formation and developmental competence. Two approaches were tested: (i) 6-h treatment of ICSI oocytes with U0126 applied at different intervals (0, 2, 3 or 4 h) after the electrical pulse and (ii) treatment of ICSI oocytes with U0126 applied 4 h after the electrical pulse over an additional 4, 6 or 8 h. Another protein kinase inhibitor, 6-dimethylaminopurine, was used as a chemical activator in control experiments. The highest rates of diploid embryo formation, normal fertilization and blastocyst formation were observed after 6 h of exposure to U0126 starting 4 h after the electrical pulse. Therefore, U0126 can be used as an activating agent for porcine oocytes fertilized by ICSI. 相似文献
19.
Fujinami N Hosoi Y Kato H Matsumoto K Saeki K Iritani A 《The Journal of reproduction and development》2004,50(2):171-178
Developmental potential of bovine embryos that are not artificially activated after intracytoplasmic sperm injection (ICSI) is generally very low. In this study, we investigated effects of artificial activation with ethanol on kinetics of maturation promoting factor (MPF) activity (p34(cdc2) kinase activity) and development of bovine oocytes following ICSI. Treatment of oocytes with ethanol at 4 h after ICSI improved their first cleavage and further preimplantation development (51% vs. 13%, 14% vs. 4%: treatment with vs. without ethanol, respectively). MPF activity of oocytes was lowered until at least 2 h after ICSI. In oocytes without activation after ICSI, MPF activity temporarily elevated at 6 h after ICSI, whereas this phenomena was not observed in the oocytes treated with ethanol. Furthermore, MPF activity was elevated 20 h after ICSI in oocytes activated with ethanol, whereas this elevation of MPF activity was not shown in oocytes without activation. These results indicate that the stimulus of sperm was sufficient to lower MPF activity of oocytes following ICSI, and moreover the activation treatment of bovine oocytes with ethanol after ICSI served to maintain the low levels of MPF activity until the next cell cycle started. 相似文献
20.
R. Cochran M. Meintjes B. Reggio D. Hylan J. Carter C. Pinto D. Paccamonti R.A. Godke 《Journal of Equine Veterinary Science》1998,18(11):736-740
Recently, in vitro fertilization (IVF) in the horse has met with less than anticipated results. Various problems associated with equine IVF include: (1) the inability to collect large numbers of good quality oocytes, (2) the alteration of the zona pellucida associated with in vitro maturation of equine oocytes, and (3) the improper preparation of equine sperm cells for IVF of these oocytes. Therefore, this study was conducted to achieve fertilization via sperm injection of equine oocytes and to produce live offspring from this IVF procedure. Oocytes were collected by transvaginal ultrasound-guided oocyte retrieval procedures from early pregnant mares of mixed breeds (day 14 to day 70 of pregnancy) and were matured in vitro and subjected to intracytoplasmic sperm injection (ICSI). Injected oocytes were then cultured for 48 hours in either TCM-199 or P-1 medium (glucose and phosphate-free medium) supplemented with 15% fetal bovine serum. Cleavage rates for embryos cultured in the two culture media were different (47% vs. 63% in TCM-199 and P-1, respectively). Also, four Grade 1 embryos were surgically transferred into the oviducts of four recipient mares (one embryo/mare) at 48 hours post-ICSI, with three pregnancies (75%) developing as ultrasonically demonstrated by the presence of an embryonic vesicle in the uterine body by day 16 post-ICSI. On June 23rd one live filly was born after 328 days of gestation and subsequently, a second healthy filly was born after 319 days of gestation. To our knowledge, this is the first report of live foals resulting from in vitro fertilization (via ICSI) of in vitro matured oocytes recovered from pregnant mares using an efficient, repeatable transvaginal ultrasound-guided procedure. 相似文献