胞质内精子注射及其影响因素的研究

作者综述了胞质内精子注射(ICSI)技术的原理及其在人及其他动物上的研究应用，阐述卵子、精子操作技术等对ICSI结局的影响，指出目前存在的问题和应用前景。     

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。     

牛卵母细胞胞浆内单精子注射的研究进展

本文叙述了牛卵胞浆内单精子注射(intracytoplasmic sperm injection ICSI)的研究进展。主要分析了前期处理精子对ICSI的影响,比较了各种激活处理对ICSI的提高效果,展望了ICSI的应用前景。     

牛的体外受精技术研究进展

本文阐述了体外受精技术在牛胚胎工程和育种研究中的意义，介绍了牛体外受精技术的研究历史和目前的研究进展，对牛体外受精技术的技术指标现状及经济效益进行了评估，并就牛的体外受精技术当前研究存在的问题作了评述，对其前景做了展望。     

基因扩增技术在牛早期胚胎性别鉴定中的应用

家畜早期胚胎性别鉴定是近年来胚胎工程领域研究的重点，而基因扩增技术在胚胎性别鉴定的应用为实现家畜性别的人为控制带来了新的希望。目前用于牛胚胎性别鉴定的基因扩增方法主要有PCR法和LAMP法等。文章对SRY基因、动物性别决定的生物学机理和用于牛早期胚胎性别鉴定的基因扩增技术作一简要综述。     

影响胞质内显微受精的因素

胞质内显微受精(ICSI)技术是20世纪80年代兴起的一项辅助受精技术,对育种和治疗男性不育有着重要的意义,文章对影响ICSI的因素和目前存在的问题及争议进行了系统的阐述。     

单精注射法生产转GFP基因猪胚胎的研究

卵胞质内单精子注射(ICSI)的出现为治疗人类男性不育提供了新的途径。作为一种家畜胚胎工程新技术，ICSI可以解决猪的多精子受精问题。本研究用冷冻/解冻的猪精子与GFP基因孵育后对猪IVM卵母细胞进行ICSI，通过对猪ICSI卵母细胞发育能力和基因表达效率的分析，初步研究以精子为载体的转基因与卵细胞质内单精注射相结合的技术(SMGT-ICSI)生产转基因猪胚胎的技术路线的可行性。用GFP基因转染的精子注射后化学激活的ICSI卵母细胞基因表达率显著高于电激活处理的ICSI卵母细胞的基因表达率。用精子头部注射的猪卵母细胞和用完整精子注射的猪卵母细胞总基因表达效率无显著差异(P>0.05)。结果表明，用冷冻/解冻后的猪精子或精子头部与外源DNA孵育后通过ICSI方法生产转基因胚胎是可行的。     

牛胚胎移植技术现状与前景

牛胚胎移植技术现状与前景（日）ＡｋｉｒａＨａｎａｄａ１８９０年ＷａｌｔｅｒＨｅａＰ利用胚胎移植技术从一家兔成功地获得２只仔兔，至今已有１００多年的历史，已用胚胎移植法成功地从马、猪、绵羊和山羊分娩出仔畜。不过，目前该技术在生产上仅用于牛，可以利用这些...     

玻璃化冷冻牛卵母细胞单精子注射后体外发育能力的研究

实验研究了不同成熟培养时间的牛卵母细胞玻璃化冷冻及胞质内单精子注射(ICSI)后的受精效果。结果表明:成熟后的新鲜牛卵母细胞按照ICSI注射方法穿刺而不注射精子组与未经穿刺的对照组相比,孤雌激活后的卵裂率、囊胚发育率及囊胚细胞数无显著差异(P>0.05);成熟培养16h(MⅠ)和23h(MⅡ)卵母细胞冷冻解冻后形态正常率均显著低于新鲜对照组(76.66%、87.33%vs100.0%)(P<0.05),冷冻解冻后二者分别成熟培养至24h,ICSI后胚胎的囊胚发育率(5.29%、14.41%)显著低于新鲜对照组(24.40%)(P<0.05);成熟培养23h与成熟培养16h的卵母细胞冷冻解冻后形态正常率及ICSI后囊胚发育率(14.41%vs5.29%)均有显著性差异(P<0.05)。实验证明,ICSI操作不会影响卵母细胞发育潜力;玻璃化冷冻影响卵母细胞解冻后形态正常率以及ICSI后胚胎的发育能力;成熟培养23h比16h的卵母细胞冷冻保存后经ICSI的胚胎发育潜力高。     

牛的体外受精技术研究进展

本文阐述了体外受精技术在牛胚胎工程和育种研究中的意义,介绍了牛体外受精技术的研究历史和目前的研究进展,对牛IVF技术的技术指标现状及经济效益进行了评估,并就牛的体外受精技术当前研究存在的问题作了评述,对其前景做了展望。     

Application study of intracytoplasmic sperm injection for golden hamster and cattle production

This paper describes several technical improvements and our results in hamster intracytoplasmic sperm injection (ICSI), hamster round spermatid injection (ROSI) and bovine ICSI. The hamster is the mammalian species in which ICSI was first tried to produce fertilized oocytes. However, until recently, no live offspring following ICSI have ever been obtained. We reported the birth of live offspring following hamster ICSI. Improved points to success were 1) performing hamster ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under microscope with a red light source and 2) injecting sperm heads without acrosomes. Under controlled illumination, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally, cleaved, and developed into morulae. Nine live offspring (19%) were born by transfer of hamster ICSI-derived embryos. Furthermore, we reported the birth of live offspring following hamster ROSI. About 70% of oocytes injected with round spermatids broken before injection were fertilized normally and about half of them developed to morulae and blastocysts. Three (5%) live young were born by transfer of hamster ROSI-derived embryos. On the other hand, in cattle, the main improvements were 1) injection of spermatozoa immobilized by scoring their tail just before injection into oocytes, and 2) additional ethanol activation 4 h after ICSI. About 70% of oocytes injected were activated 4 h after ICSI, and about 30% of them developed to blastocysts. Twenty-four live calves (39%) were born by non-surgical transfer of ICSI-derived embryos. Those results shows that, at present, live offspring are able to be obtained following hamster ICSI, ROSI and bovine ICSI, but further improvement is required due to higher production efficiency of offspring.     

Activation with ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of MPF activity

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.     

High survival of bovine mature oocytes after nylon mesh vitrification,as assessed by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is an alternative technique to in vitro fertilization (IVF) for producing transferable blastocysts, especially in combination with cryopreserved oocytes, when the IVF system does not work sufficiently. The present study was conducted to directly compare the efficacy of producing bovine blastocysts by ICSI and IVF from vitrified-warmed and fresh oocytes. Denuded oocytes with a detectable first polar body were vitrified-warmed using a nylon mesh device. In the non-vitrified control group, blastocyst yields 8 days after IVF and ICSI were 32.0 and 26.8%, respectively. Oocyte vitrification and subsequent IVF resulted in an impaired blastocyst yield (15.0%); however, such a loss of efficacy due to vitrification was not observed in the ICSI group (blastocyst yield, 25.2%). The alignment of cortical granules beneath the oolemma was comparable between the fresh control and vitrified-warmed oocytes. Here, we report the high survival of vitrified-warmed bovine oocytes, as assessed by ICSI.     

A retrospective study of oesophageal endoscopy in cattle - oesophagoscopy for diagnosis of mucosal disease

Oesophageal endoscopy in cattle has been rarely reported in the literature. It has, however, been used for many years as an aid to the diagnosis and treatment of illnesses of the upper gastrointestinal tract in small animals and horses. We have assessed the potential value of oesophagoscopy on 120 bovine patients presented at the 2nd Medical Clinic for Ruminants and Swine at the University of Veterinary Medicine, Vienna, over a period of three years. In the course of the study, cattle exhibiting symptoms such as salivation, regurgitation or swelling in the neck region were referred for endoscopic examination.The main indication was to confirm or exclude bovine virus diarrhoea virus infection in calves, in which typical erosions were visible endoscopically on the mucosa of the oesophagus in infected animals. In addition, endoscopy proved to be useful for the diagnosis of diverticula, ruptures and inflammation of the oesophagus. Oesophagoscopy can be considered to be a valuable supplementary aid to clinical examination and, in many cases, can facilitate diagnosis.     

Two cases of reciprocal chromosomal translocation (4; 7)(p+; q-) (2; 8)(q-; q+) in piglets produced by ICSI

In this study, the karyotypes of 14 piglets from four different litters produced by intracytoplasmic sperm injection (ICSI) and embryo transfer were analysed. The chromosome analysis was based on a classical cytogenetic examination following the standard protocols of lymphocyte cultures. Two cases of reciprocal translocation [(4; 7)(p+; q-) and (2; 8)(q-; q+)] were detected in two female transgenic piglets. These animals showed neither anatomical nor physiological alterations and had normal growth. To our knowledge, this is the first karyotype study of piglets produced by ICSI.     

Cattle industry and zoonotic risk

The intensive farming of dairy and beef cattle has elicited a decrease in the herds and an increase in the number of animals per herd. The high concentration of cattle and the movement of the animals among herds has led to an increase in the health risks. In this context we have to consider the role of microbial agents of zoonoses, such as bacteria, parasites, and in some cases viruses. Notably, foodstuffs, such as meat, milk and dairy products, are the main sources of zoonoses of bovine origin. In particular, raw milk must be considered at high risk for trasmission of pathogens from cattle to humans. The European Regulation concerning food safety provides specific requirements for animal products and in bovine health management. Given the direct responsibility of the producer, the adoption of a self-regulation regimen on animal health, dairy and meat products must be planned by farmers.     

Cattle industry and zoonotic risk

The intensive farming of dairy and beef cattle has elicited a decrease in the herds and an increase in the number of animals per herd. The high concentration of cattle and the movement of the animals among herds has led to an increase in the health risks. In this context we have to consider the role of microbial agents of zoonoses, such as bacteria, parasites, and in some cases viruses. Notably, foodstuffs, such as meat, milk and dairy products, are the main sources of zoonoses of bovine origin. In particular, raw milk must be considered at high risk for trasmission of pathogens from cattle to humans. The European Regulation concerning food safety provides specific requirements for animal products and in bovine health management. Given the direct responsibility of the producer, the adoption of a self-regulation regimen on animal health, dairy and meat products must be planned by farmers.