Advancements in cryopreservation of domestic animal embryos.

The development of embryo freezing technologies revolutionized cattle breeding. Since then, advancements in cryobiology, cell biology, and domestic animal embryology have enabled the development of embryo preservation methodologies for our other domestic animal species, including sheep and goats. Recently, technologies have been developed to cryopreserve pig embryos, notorious for their extreme sensitivity to cooling; horse embryo cryopreservation is in its infancy. While cryopreservation can enhance the utilization of in vitro embryo production technologies, cryosurvival of in vitro-produced (IVP) or micromanipulated embryos is less than that of in vivo-derived embryos. This review outlines recent efforts in livestock embryo cryopreservation. In the near future, use of preserved embryos could be a routine breeding alternative for all livestock producers providing 1) preservation methods for maternal germplasm, 2) global genetic transport, 3) increased selection pressure within herds, 4) breeding line regeneration or proliferation, and 5) methodology for genetic rescue.     

Non-surgical transfer of deep-frozen bovine embryos

Preservation of cattle embryos by methods of deep-freezing has recently been established (1, 11, 12) and provides a valuable addition to the possibilities of controlled breeding by embryo transfer in cattle.Already long distance transport of frozen embryos has been demonstrated (2, 6) and adopted by some commercial interests. However, in all publications to date, embryos have been transferred to recipients by surgical methods, even though non-surgical methods of embryo recovery and transfer would be preferred for commercial embryo transfer.The purpose of the present experiments was to utilize non-surgical methods of embryo recovery and transfer of deep-frozen cattle embryos to demonstrate the feasibility of the procedure for a farm service to interested breeders. The particular advantage of non-surgical embryo transfer methods is that neither the donor nor recipient need to leave the farm. Embryo preservation by freezing obviates the necessity for synchronization of recipients for immediate transfer from the donor and allows considerable freedom in the choice of the recipients and the timing of embryo transfers.     

Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification

Pig embryos suffer severe sensitivity to hypothermic conditions, which limits their ability to withstand conventional cryopreservation. Research has focused on high lipid content of pig embryos and its role in hypothermic sensitivity, while little research has been conducted on structural damage. Documenting cytoskeletal disruption provides information on embryonic sensitivity and cellular response to cryopreservation. The objectives of this study were to document microfilament (MF) alterations during swine embryo vitrification, to utilize an MF inhibitor during cryopreservation to stabilize MF, and to determine the developmental competence of cytoskeletal-stabilized and vitrified pig embryos. Vitrified morulae/early blastocysts displayed MF disruptions and lacked developmental competence after cryopreservation; hatched blastocysts displayed variable MF disruption and developmental competence. Cytochalasin-b did not improve morula/early blastocyst viability after vitrification; however, it significantly (P < 0.05) improved survival and development of expanded and hatched blastocysts. After embryo transfer, we achieved pregnancy rates of almost 60%, and litter sizes improved from 5 to 7.25 piglets per litter. This study shows that the pig embryo cytoskeleton can be affected by vitrification and that MF depolymerization prior to vitrification improves blastocyst developmental competence after cryopreservation. After transfer, vitrified embryos can produce live, healthy piglets that grow normally and when mature are of excellent fecundity.     

In vitro technologies related to pig embryo transfer

Embryo transfer in swine (ETS) has been used for commercial and breeding application only to a limited extent. However this technique is an essential prerequisite for the application of new reproductive techniques in pigs. This paper will give an overview on steps of pig embryo transfer including selection and stimulation of donor sows, recovery of embryos, embryo handling and the transfer of recovered embryos into recipients. Furthermore the current status and further application of ET related in vitro technologies in pig production are described.     

Comparison of slow- and rapid-cooling protocols for early-cleavage-stage Macaca fascicularis embryos

Cryostorage of nonhuman primate embryos by time-consuming slow-cooling methods is often limited to early cleavage stages. Effective rapid-cooling methods have been developed for many species and represent valuable tools for laboratory- and field-based studies of nonhuman primate reproductive biology. However, few rapid-cooling protocols have been applied to nonhuman primate embryos in terms of comparing various developmental stages. Here we compare slow cooling vs. two- and three-step rapid cooling of two-, four-, and eight-cell Macaca fascicularis (Mf) embryos. Rapid cooling was conducted in open pulled straws (OPS) using cooling solutions containing reduced quantities of ethylene glycol (EG) and supplemented with either of two high-molecular-weight polymers, ficoll and dextran. The survival of the slow-cooled embryos, but not the rapid-cooled embryos, was independent of embryonic stage at cryostorage. Slow cooling was associated with greater cell survival (82%) post thaw compared to warming following rapid cooling (18-29%). Slow cooling resulted in a high proportion of embryo survival (18/20; 90%) and cleavage (15/18; 83%) post thaw. Rapid cooling resulted in significantly reduced percentages of embryo survival (26-32%) and embryo cleavage in culture (29-38%) after warming. Conventional slow cooling was more effective than the rapid-cooling protocols employed in this study for cryopreservation of early-cleavage-stage Mf embryos.     

Use of assisted reproduction for the improvement of milk production in dairy camels (Camelus dromedarius)

Despite their production potential and ability to survive on marginal resources in extreme conditions, dromedaries have not been exploited as an important food source. Camels have not been specifically selected for milk production, and genetic improvement has been negligible. High individual variation in milk production both within the population and within breeds provides a good base for selection and genetic progress. In this paper, we discuss the possibilities and constraints of selective breeding for milk production in camels, and include a summary of the use of embryo transfer at the world's first camel dairy farm. Embryo transfer is an integral part of the breeding strategy at the camel dairy farm because it increases selection intensity and decreases the generation interval. Using high milk-producing camels as donors and low producing camels as recipients, 146 embryos were recovered (6.1 ± 1.0 embryos/donor; range: 0–18). Embryos were transferred non-surgically into 111 recipients (83 single and 28 twin embryo transfers). Pregnancy rate at 21 days and 5 months was 55% (61/111) and 45% (50/111), respectively. Finally, a total of 46 recipients delivered a live calf. These results document the utility of embryo transfer using high milk producing dromedaries as donors.     

Developmental competence of somatic cell nuclear transfer embryos reconstructed from oocytes matured in vitro with follicle shells in miniature pig

The developmental competence of domestic pig oocytes that were transferred to somatic cell nuclei of miniature pig was examined. A co-culture system of oocytes with follicle shells was used for the maturation of domestic pig oocytes in vitro. Co-cultured oocytes progressed to the metaphase II stage of meiosis more quickly and more synchronously than non co-cultured oocytes. Oocytes were enucleated and fused with fibroblast cells of Potbelly miniature pig at 48 h of maturation. The blastocyst formation rate of nuclear transfer (NT) embryos using cocultured oocytes (24%) was significantly higher (p < 0.05) than that of non-co-cultured oocytes (13%). Cleaved embryos at 48 h after nuclear transfer using co-cultured oocytes were transferred to the oviducts of 14 G?ttingen miniature pigs and four Meishan pigs. Estrus of all G?ttingens returned at around 20-31 days of pregnancy. Two of the four Meishans became pregnant. Three and two cloned piglets were born after modest number of embryo transfer (15 and 29 embryos transferred), respectively. These results indicated that oocytes co-cultured with follicle shells have a high developmental competence after nuclear transfer and result in full-term development after embryo transfer.     

New developments reproductive technologies in deer

In vitro embryo production is the platform for advanced reproductive technologies, such as cloning. The in vitro embryo production system developed for farmed red deer (Cervus elaphus) evolved along similar lines to that pioneered by other domestic species researchers. However, applying existing in vitro embryo production methods from these other species resulted in limited success and has necessitated developing a species-specific methodology for red deer based on the their physiology. Analysis of oviduct fluid led to the development of a semi-defined fertilization and culture media system, Deer Synthetic Oviduct Fluid (DSOF), which resulted in successful culture of red deer embryos to the blastocyst stage. Transvaginal ultrasound-guided ovarian examination and ovum pickup has enabled the study of seasonality constraint and propagation from selected female genetics, respectively. During the 4-month breeding season (April-July), 15% of cleaved oocytes developed to blastocysts, whereas no blastocysts developed from oocytes collected after July. The process of developing an in vitro embryo production system for farmed red deer may serve as a beneficial model for the propagation of endangered cervine species.     

Testing the effects of genetic crossing distance on embryo survival within a metapopulation of brown trout (Salmo trutta)

Predicting progeny performance from parental genetic divergence can potentially enhance the efficiency of supportive breeding programmes and facilitate risk assessment. Yet, experimental testing of the effects of breeding distance on offspring performance remains rare, especially in wild populations of vertebrates. Recent studies have demonstrated that embryos of salmonid fish are sensitive indicators of additive genetic variance for viability traits. We therefore used gametes of wild brown trout (Salmo trutta) from five genetically distinct populations of a river catchment in Switzerland, and used a full factorial design to produce over 2,000 embryos in 100 different crosses with varying genetic distances (F _ST range 0.005–0.035). Customized egg capsules allowed recording the survival of individual embryos until hatching under natural field conditions. Our breeding design enabled us to evaluate the role of the environment, of genetic and non-genetic parental contributions, and of interactions between these factors, on embryo viability. We found that embryo survival was strongly affected by maternal environmental (i.e. non-genetic) effects and by the microenvironment, i.e. by the location within the gravel. However, embryo survival was not predicted by population divergence, parental allelic dissimilarity, or heterozygosity, neither in the field nor under laboratory conditions. Our findings suggest that the genetic effects of inter-population hybridization within a genetically differentiated meta-population can be minor in comparison to environmental effects.     

Future prospectives on animal biotechnology

Abstract

Farm animal reproduction is entering the era of embryo engineering ‐ a part of the new biotechnology revolution that has been sweeping the nation during the early 1980s. This comes at a time when the $70 billion livestock industry is hard‐pressed for survival. Not since the commercial development of artificial insemination (AI) techniques in the 1950s has any new technical research development caused such a stir in the livestock community. The genetic impact of artificial insemination (AI) in the cattle industry these last 40 years cannot be questioned. Nearly three‐fourths of the dairy cattle in the United States are now being artificially inseminated. Also, commercial processing of bull semen has been and still is a major agribusiness success story, grossing millions of dollars annually. With the development of embryo transfer (ET) technology in the mid‐1970s, animal reproduction again entered a new age of technical advancement. It appears that AI and embryo methodology are just the beginning of a new age in animal reproduction technology. Recent developments in molecular biology and genetic engineering now offer a new dimension in research and development for future application to seed stock farm animals. New molecular technologies will most certainly change the traditional approach to animal breeding, thus allowing the livestock producer to select breeding stock on genotype rather than phenotype. In the future, researchers will be able to study whole animal biology to a depth never before dreamed using molecular biology.     

广西巴马小型猪克隆胚的构建及胚胎移植

通过胚胎移植验证构建的广西巴马小型猪克隆胚是否可以发育到期.利用刺入式手术胚胎移植法,将0.5～1.5日龄巴马小型猪克隆胚移植到2头巴马小型猪和2头陆川猪的输卵管壶腹部.其中2头巴马小型猪和1头陆川猪返情,另外一头陆川猪于2007年10月13日产下1头克隆雄性巴马小型猪.说明巴马小型猪克隆胚能够在受体猪体内发育到期并产仔.     

Factors affecting the survival of sheep embryos after transfer within a MOET program

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rate of genetic improvement in sheep. However, better realization of this potential requires maximum survival rates of transferred embryos of high genetic merit after transfer into recipient ewes. These studies were therefore conducted to investigate the effect of both embryonic and recipient ewe factors on the survival rate of transferred embryos. Survival rate was similar after transfer of morula or blastocyst stage embryos, and these were higher (P<0.05) than for very early morulae and early morulae. Advanced embryos (Day 5 blastocyst) had an advantage (P<0.05) in survival rate over retarded embryos (Day 6 morula). Grades 1 and 2 embryos survived significantly (P<0.05) better than Grades 3 or 4 embryos. There was no difference in embryo survival rate following transfer to recipients with different numbers of corpora lutea. In general, age or parity of recipient ewes did not affect embryo survival rate, although a higher (P<0.05) embryo survival rate was observed for yearling recipients. Buserelin (GnRH agonist) treatment of recipient ewes 5 or 6 days after transfer of embryos (Day 12 of the cycle) did not improve embryo survival rate. These results confirm that both embryonic and recipient factors can play an important role in the success of a MOET program in sheep.     

Birth of the Japanese Monkey (Macaca fuscata) infant following in-vitro fertilization and embryo transfer

The first successful birth by in-vitro fertilization (IVF) and embryo transfer (ET) in the Japanese monkey was described. IVF was carried out by using oocytes collected after ovarian stimulation and sperms collected by rectal electro-ejaculation. The embryos were incubated for 36–66 hours and then transferred to the fallopian tube of the recipient via the fimbria under laparoscopic observations. Four recipients received their own embryos and six recipients received donor embryos. Two recipients of six that received donor embryos became pregnant after receiving one 3-cell and one 2-cell embryos, and one 4-cell and one 2-cell embryos, respectively. On healthy terminated male infant was delivered 166 days after ET, but the other aborted on day 128. This successful birth indicates the usefulness of our IVF/ET method for systematic indoor artificial breeding and preservation of endangered primates species.     

Altered apoptosis/autophagy and epigenetic modifications cause the impaired postimplantation octaploid embryonic development in mice

Polyploids are pervasive in plants and have large impacts on crop breeding, but natural polyploids are rare in animals. Mouse diploid embryos can be induced to become tetraploid by blastomere fusion at the 2-cell stage and tetraploid embryos can develop to the blastocyst stage in vitro. However, there is little information regarding mouse octaploid embryonic development and precise mechanisms contributing to octaploid embryonic developmental limitations are unknown. To investigate the genetic and epigenetic mechanisms underlying octaploid embryonic development, we generated mouse octaploid embryos and evaluated the in vitro/in vivo developmental potential. Here we show that octaploid embryos can develop to the blastocyst stage in vitro, but all fetus impaired immediately after implantation. Our results indicate that cell lineage specification of octaploid embryo was disorganized. Furthermore, these octaploid embryos showed increased apoptosis as well as alterations in epigenetic modifications when compared with diploid embryos. Thus, our cumulative data provide cues for why mouse octaploid embryonic development is limited and its failed postimplantation development.     

Cryopreservation of canine embryos

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.     

Intergeneric embryo transfer between water buffalo and domestic cattle

The feasibility of reciprocal embryo transfer between water buffalo (Bubalus bubalis) and domestic cattle (Bos taurus) was studied. Eight mature water buffalo females were superovulated and bred naturally to a male water buffalo. Sixteen water buffalo embryos were recovered nonsurgically using routine bovine embryo transfer procedures. No pregnancies resulted after transfer of thirteen water buffalo embryos to synchronized Holstein heifers. Physiological age of the water buffalo embryos was ahead of chronological age when compared to bovine embryo development.Two cattle embryos were transferred nonsurgically to two synchronized water buffalo recipients. One recipient was diagnosed pregnant; however, she subsequently aborted between 2.5 and 3.0 months of gestation.The significance of successful intergeneric embryo transfer between cattle and water buffaloes would be the provision of a model for the preservation of endangered genera and species for which common recipients are readily available.The reproductive anatomy as well as the reproductive physiology of the estrous cycle of the donor and the recipient species must be reasonably similar. Minor endocrinological incompatibilities might be circumvented by transfer of alien embryos to mated recipients. Problems of immunological incompatibilities might be minimized by modifying the immune response of the recipient and/or the antigenicity of the embryo by microsurgical manipulation.     

Oocyte activation after intracytoplasmic injection with sperm frozen without cryoprotectants results in live offspring from inbred and hybrid mouse strains

Re-establishment of mouse strains used for mutagenesis and transgenesis has been hindered by difficulties in freezing sperm. The use of intracytoplasmic sperm injection (ICSI) enables the production of embryos for the restoration of mouse lines using sperm with reduced quality. By using ICSI, simplified sperm-freezing methods such as snap freezing can be explored. We examined the capacity of embryos from the inbred C57Bl/6J and 129Sv/ImJ mouse strains, commonly used for transgenic and N-ethyl-N-nitrosourea mutagenesis purposes to develop to blastocysts in vitro and to term following ICSI with sperm frozen without cryoprotectant. The results were compared to F1 (C57BlxCBA) hybrid embryos. Following freezing, sperm were immotile but could fertilize oocytes at similar rates to fresh sperm. However, embryo development in vitro to the blastocyst stage was reduced in all three strains. No pups were born from C57Bl/6J or 129Sv/ImJ embryos obtained from frozen sperm following transfer to foster females, and only a limited number of F1 embryos developed to term. Activation of oocytes injected with frozen sperm with 1.7 mM Sr2+ (SrCl2) did result in the birth of pups in all three strains. We conclude that the inability of sperm frozen without cryoprotectants to effectively activate oocytes may affect embryo development to term and can be overcome by strontium activation. This may become an effective strategy for sperm preservation and the restoration of most popular strains used for genetic modifications.     

Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus)

A novel, minimally invasive, transabdominal embryo collection method (transabdominal method) was developed as an alternative to a standard abdominal incision for embryo collection in the common marmoset. The abdominal incision method was used for 304 flushes using 36 female animals, whereas the transabdominal method was used for 488 flushes using 48 females; successful embryo collection rates were 48.0% and 48.4% (P > 0.05), respectively. These techniques were successfully duplicated at another institute (German Primate Center, DPZ). At that institution, successful embryo collection rates were 88.9% and 77.8% for the abdominal incision and transabdominal methods, respectively (P > 0.05), whereas the average numbers of preimplantation embryos obtained per flush were (mean ± SD) 1.91 ± 0.35 and 1.71 ± 0.14 (P > 0.05). The transabdominal method reduced animal stress, did not require incisional wound healing, and enabled successive embryo recoveries to be done much sooner. More embryos in early developmental stages (zygotes/morulae) were recovered using the transabdominal method (76.1%) than the abdominal incision method (52.6%, P < 0.01). In contrast, recovery of arrested or abnormal embryos was not significantly different between these two methods (9.8% and 8.3%). To verify developmental ability of embryos recovered by the transabdominal method, transfer of 28 normal embryos to 14 surrogate mothers yielded a nidation rate of 57%. Five females sustained term pregnancies and eight neonates were born. This novel transabdominal method will facilitate progress in marmoset developmental biology and embryology.     

Surgical transfer of in vivo produced farmed European polecat (Mustela putorius) embryos

Surgical embryo transfer of farmed European polecat (Mustela putorius) was investigated as part of an ex situ preservation project. The long-term objective of the project is to develop effective technology for ex situ conservation of the European mink (Mustela lutreola), which is a highly endangered aboriginal European species. Twenty European polecat females, which served as a model species for the European mink, were humanely killed 4-9 days after first mating and embryos were recovered from oviducts and uteri. Donor-recipient pairs (n = 16) were generated by mating the donors (n = 20) once a day for 2 consecutive days with fertile males and by mating the corresponding recipients (n = 16) on the same days with vasectomized males. An embryo recovery rate of 70% (200 recovered embryos/284 corpora lutea) was achieved from 20 donors. Morulae and blastocysts were recovered between Days 5 and 9 after first mating and were regarded as the best developmental stages for uterine embryo transfer. A total of 172 embryos were transferred surgically under general anaesthesia into the ovarian third of the left uterine horn of 16 recipients with a thin glass capillary. Eleven recipients (69%) produced 72 pups equivalent to an average success rate of 42% (72 pups/172 transferred embryos). The average litter size was 4.5 (range 0-9). These results with this model species, farmed European polecat, demonstrate the potential of embryo transfer as an effective method for the preservation of the endangered European mink (M. lutreola). These species are closely related and have a similar reproductive physiology. However, success of applying embryo transfer in conserving European mink is still dependent on further studies both into its reproductive physiology and developing of improved flushing techniques for anaesthetized donors and the successful transfer of frozen-thawed embryos.