Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage

The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer.     

Transmission of bovine brucellosis from dam to offspring

Transmission of bovine brucellosis from dam to offspring under natural circumstances was demonstrated in a 2 1/2-year study of an infected cow and her calf. The cow delivered a full-term heifer calf after her first gestation. The calf remained with its dam until 7 months of age, then was placed in isolation until bred. During her first gestation, the second-generation heifer became seropositive for Brucella abortus. She later gave birth to a calf with B abortus infection, as determined by isolation of B abortus biotype 1 from stomach contents, heart blood, lung, and spleen of the calf. The same organism was isolated from the placenta and milk of the dam.     

Different factors affect developmental competence and cryotolerance in in vitro produced bovine embryo

The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.     

第X期至“蚊虫珠”期鸡胚的易壳培养

将发育至第X期的鸡胚转和代用蛋壳内，分为4组分别添加蛋清至满或空余一定气室，正立或倒置孵化，观察鸡胚发育至“蚊虫珠”期胚胎的比较。结果发现鸡胚仅在代用蛋壳气室充盈无气泡、并与壳膜充分接触量，85%（34/40）的鸡胚能发育至“蚊虫珠”期。     

Consequences for the bovine embryo of being derived from a spermatozoon subjected to oxidative stress

Objective To determine whether oxidative damage of ejaculated frozen–thawed sperm prior to oocyte insemination in vitro affects the competence of the resultant embryo to develop to the blastocyst stage. Method Extended frozen semen from bulls was thawed, subjected to Percoll gradient purification to obtain motile spermatozoa and mixed with medium containing the pro-oxidants menadione or tert-butyl hydroperoxide. After 3 h at 38.5°C, the sperm were washed and used to inseminate oocytes in vitro. Embryo development proceeded until 8 days after insemination. Results Treatment of sperm with 15 or 30 µmol/L menadione reduced the proportions of oocytes that cleaved and those that developed to the blastocyst stage; 30 µmol/L menadione reduced the proportion of cleaved embryos that developed to the blastocyst stage at day 8 after insemination. Oocytes inseminated with sperm treated with 150 or 300 µmol/L tert-butyl hydroperoxide had lower proportions of cleavage and blastocyst development, and the proportion of cleaved embryos becoming blastocysts was also reduced. Conclusion Oxidative damage to ejaculated sperm can compromise the ability of the sperm to cause oocyte cleavage and leads to formation of embryos with reduced competence for development.     

不同型牛血清对家兔原核胚序贯培养体外发育的影响

采用不同型血清分别添加到TCM199和mTCM199培养液、RPMI1640和mRPMI1640培养液中，对兔原核期受精卵进行了体外序贯培养，并对各组间不同时期发育率进行了分析比较。结果显示：体外培养至第72h时，3个体外序贯培养体系间8-细胞胚率、桑椹胚率差异不显著（P〉0．05），当体外培养至囊胚时，100mL／L NBS＋TCM199（mTCM199）培养体系、100mL／L NBS＋RPMI1640（mRPMI1640）培养体系的囊胚率均显著低于100mL／L FBS＋RPMI1640（mRPMI1640）培养体系的囊胚率（三组的囊胚率依次是27．3％、35．9%、97．2%，P〈0．01）。但前两组之间差异不显著。结果表明，不同型血清对兔早期胚胎体外正常发育具有很大影响，序贯培养中添加国产NBS的培养液的培养效果明显低于添加进口FBS的培养液的培养效果。     

Consequences for the bovine embryo of being derived from a spermatozoon subjected to post-ejaculatory aging and heat shock: development to the blastocyst stage and sex ratio

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.     

Full-term development of offspring using round spermatids produced ectopically from fetal male germ cells

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells, which originate from primordial germ cells in the early embryo. Previously, we reported that the transplantation of fetal male gonadal tissue into the recipient testis was effective obtaining functional sperm. This transplantation technique is a promising new approach for the preservation of testicular function in a mutant animal with embryonic lethality. In the present study, we examined whether spermatogenesis from fetal male germ cells is induced under ectopic conditions in male and female recipients. Nine to 10 weeks after the transplantation of male gonads prepared from embryos at 12.5 or 16.5 days post gestation, male germ cell differentiation occurred under the skin of male and female recipient nude mice. Histological analyses revealed that grafted gonads contained haploid germ cells such as round or elongated spermatids. Furthermore, we succeeded in obtaining normal progeny by injecting the ectopically produced round spermatids into the cytoplasm of oocytes, even when the male germ cells had been generated in female recipients. These results indicate that the transplantation of fetal male gonads under the skin of recipient mice is a useful technique for obtaining functional male gametes.     

Secretory virulence factors produced by Staphylococcus aureus isolates obtained from mastitic bovine milk--effect on bovine polymorphonuclear neutrophils

The aim of the research was to test whether exogenic virulence factors secreted by Staphylococcus aureus isolates are involved in mechanisms that allow the bacteria to modulate and evade phagocytosis by bovine polymorphonuclear neutrophils. The research was based on the comparison of the effects of supernatants, prepared from cultures of 30 S. aureus isolates, on the functional properties of bovine neutrophils in vitro. S. aureus isolates were collected from milk samples from cows with clinical mastitis. Supernatants, which were used to treat leukocytes, were prepared from 18 h S. aureus cultures. Exogenic virulence factors secreted by S. aureus isolates significantly influenced the phagocytosis parameters evaluated. Depending on their leukotoxic or superantigenic properties, supernatants could affect the ingestion process, and also showed an influence on the digestion efficiency and phagocytosis carried out by bovine polymorphonuclear neutrophils in vitro.     

Production of offspring from one-day-old oocytes stored at room temperature

To determine the feasibility of preserving oocytes without freezing, we stored mouse oocytes in several media at different temperatures for one day. Confocal microscopy of the metaphase-II spindle in these stored oocytes revealed gross abnormalities in both the spindle and the arrangement of chromosomes. The abnormal spindles could not be rescued by transplanting the aged spindle-chromosome complex into a fresh enucleated oocyte. A diploid parthenogenetic development showed that some of the oocytes stored at room temperature could still develop into blastocysts (10-57%). However, oocytes stored in a refrigerator (5%) or incubator (0%) lost the potential almost entirely. Fertilization of room-temperature-preserved oocytes with fresh spermatozoa by ICSI or IVF resulted in, respectively, 4 and 10%, full-term births. These results suggest that when oocytes are stored at room temperature for one day, most have irreversible damage not only to their cytoplasm but also to the spindle. However, since at least a few percent of stored oocytes retained the potential for full-term development, it may be possible to overcome these problems and develop a simple method for preserving mammalian oocytes without freezing.     

牛体外受精胚胎移植条件的研究

通过体外受精技术和胚胎移植技术充分利用现有优秀种公牛资源,大量生产优质胚胎,可以充分发挥良种奶牛的繁殖潜力,加快良种奶牛繁殖速度[1].因此开展体外胚胎生产的研究,探索出一套简单有效的体外胚胎生产技术,找到适合于体外胚胎的移植方法和时机,则无论对于目前养牛业发展还是对纯粹的科学研究都是非常必要的.本研究对奶牛胚胎移植做了全面的研究和探索,希望能形成一套简单规范的体外胚胎生产和移植的技术,以满足科研和生产的需求.     

A case of an embryo transfer calf infected with bovine leukemia virus from the recipient cow

A case was discovered where the embryo transfer (ET) calf had been infected with bovine leukemia virus (BLV) from the recipient cow. The embryo was transferred from the BLV-uninfected donor cow to the recipient cow. However, the BLV test had not been performed to the recipient cow before ET was performed. The ET calf was raised in a calf hatch from birth to 1-month old and was given the recipient cow's colostrum and milk artificially. The ET calf was raised with the two other calves from a 1-month old to a 6-month old. The BLV test was performed to the ET calf by agar gel precipitation (AGP) and passive haemagglutination (PHA) assay when the ET calf was 6 months old. Because the ET calf was positive, the BLV test was performed to the recipient cow, the two other calves raised with the ET calf and the two dams of the two other calves. Because the recipient cow only was positive at the time of the first test, we judged that the ET calf had been infected with BLV from the recipient cow. The importance of the BLV test being carried out on the recipient cow for the prevention of enzootic bovine leukemia in a case of ET was recognised.     

Effect of linoleic acid-albumin on post-thaw survival of in vitro-produced bovine embryos at the 16-cell stage

The effect of addition of linoleic acid-albumin (LAA) to culture medium before freezing on the survival rate of bovine 16-cell embryos after freezing-thawing was investigated. Embryos were incubated in CR1aa containing LAA (0.25 mg/ml) for 4 days after insemination. A conventional slow cooling method was used, in which embryos were cooled at a rate of 0.3 degrees C/min to -30 degrees C in medium supplemented with 1.5 M ethylene glycol and 0.2 M trehalose. The developmental rate to the blastocyst stage of thawed embryos that had been cultured with LAA-containing medium before freezing was higher than that of these cultured without LAA (P<0.05). However, with fresh, non-frozen, embryos that were incubated under the same culture conditions (with and without LAA), no such difference was found.     

A new method for bovine embryo production: a potential alternative to superovulation

Eight cows were used to study the feasibility of transvaginal ultrasound-guided puncture of follicles as a method for the collection of immature oocytes for embryo production in vitro. In six trials at intervals of seven days, 104 oocytes were collected. After in vitro maturation and fertilisation the 104 oocytes were transferred to the oviducts of sheep. Six days later, 75 oocytes were recovered by flushing the oviducts. Twenty-four per cent of the recovered oocytes/embryos had developed into transferable and viable morulae and, or, blastocysts. The data show that this non-surgical and repeated collection of immature oocytes can be used successfully for the in vitro production of bovine embryos. The procedure may produce yields of embryos comparable to those obtainable by conventional superovulation procedures.     

Production of offspring from vacuum-dried mouse spermatozoa and assessing the effect of drying conditions on sperm DNA and embryo development

Freeze-dried sperm (FD sperm) are of great value because they can be stored at room temperature for long periods of time, However, the birth rate of offspring derived from FD sperm is low and the step in the freeze-drying process particularly responsible for low offspring production remains unknown. In this study, we determined whether the drying process was responsible for the low success rate of offspring by producing vacuum-dried sperm (VD sperm), using mouse spermatozoa dried in a vacuum without being frozen. Transfer of embryos fertilized with VD sperm to recipients resulted in the production of several successful offspring. However, the success rate was slightly lower than that of FD sperm. The volume, temperature, and viscosity of the medium were optimized to improve the birth rate. The results obtained from a comet assay indicated that decreasing the drying rate reduced the extent of DNA damage in VD sperm. Furthermore, even though the rate of blastocyst formation increased upon fertilization with VD sperm, full-term development was not improved. Analysis of chromosomal damage at the two-cell stage through an abnormal chromosome segregation (ACS) assay revealed that reduction in the drying rate failed to prevent chromosomal damage. These results indicate that the lower birth rate of offspring from FD sperm may result from the drying process rather than the freezing process.     

Studies of embryo transfer from cattle clinically affected by bovine spongiform encephalopathy (BSE)

Semen from 13 bulls, eight with clinical bovine spongiform encephalopathy (BSE), was used to artificially inseminate (AI) 167 cows with clinical BSE, and their resultant embryos were collected non-surgically seven days after AI. The viable and non-viable embryos with intact zonae pellucidae were washed 10 times (as recommended by the International Embryo Transfer Society) then frozen. Later, 587 of the viable embryos were transferred singly into 347 recipient heifers imported from New Zealand, and 266 live offspring were born of which 54.1 per cent had a BSE-positive sire and a BSE-positive dam. The recipients were monitored for clinical signs of BSE for seven years after the transfer, and the offspring were monitored for seven years after birth. Twenty-seven of the recipients and 20 offspring died while being monitored but none showed signs of BSE. Their brains, and the brains of the recipients and offspring killed after seven years, were examined for BSE by histopathology, PrP immunohistochemistry, and by electron microscopy for scrapie-associated fibrils. They were all negative. In addition, 1020 non-viable embryos were sonicated and injected intracerebrally into susceptible mice (20 embryos per mouse) which were monitored for up to 700 days, after which their brains were examined for spongiform lesions. They were all negative. It is concluded that embryos are unlikely to carry BSE infectivity even if they have been collected at the end-stage of the disease, when the risk of maternal transmission is believed to be highest.