Germplasm & Gamete Physiology Laboratory, Agricultural Research Service
U.S. Department of Agriculture, Beltsville, Maryland 20705 USA
Considerable progress has been made in the improvement and simplification of preservation procedures routinely used for sperm, oocytes and embryos, especially in cattle. Conventional programmable freezing and vitrification of germplasm and embryos have given veterinarians, scientists and producers alternatives in their herd reproduction practices. While continuously being improved upon for use in cattle, there are few methods available, tested or proven effective with germplasm and embryos of other common domestic farm animals. This paper will discusses concepts and breadth of germplasm preservation without elaborating on detailed technical aspects. It will then discuss novel development of germplasm preservation in a common farm species other than cattle, illustrating how modern science can be applied to solve some of the most difficult biological questions. Finally, it will discuss conceptually how germplasm preservation may be applied and implemented in propagating desirable genetics on a global basis.
in situ (¹n sº“t›, s¶“-) adv. --in si·tu adj. In the original position. [Latin in sit¿ : in, in + sit¿, ablative of situs, place.]
ex (µks) prep. 1. Not including; without [Latin. See eghs .] eghs. Important derivatives are: ex-, exotic, external, extra-, strange, extreme. eghs. Out. 1. Variant eks. a. EX, EX-from Latin ex, ex-, out of, away from
preservation (prµz”…r-v³“sh…n) n. from preserve (pr¹-zûrv“) –tr. 1. To maintain in safety from injury, peril, or harm; protect. 2. To keep in perfect or unaltered condition; maintain unchanged. 3. To keep or maintain intact. 4. To prepare for future use. 5. To prevent from decaying or spoiling.
• ex situ = out of original position
• preservation = maintain intact, unchanged, protected; prepared for future use
germplasm: sperm, egg or oocyte (gametes); germ cells that carry haploid chromosomes
embryo: fertilized egg or oocyte possessing diploid chromosomes from the sire and dam
inferred definition ex-situ germplasm preservation: the maintenance and indefinite protection of sperm, eggs and embryos outside the reproductive tract.
Preservation potential Today, large numbers of animals are transported by air freight from countries or regions in which nucleus herds are located to countries or regions in which new breeding units are being established. Transportation costs are extremely high, risk of disease transmission is a threat, and health testing and import/export bureaucracy and requirements are difficult and involved. These costs and associated constraints could be considerably reduced by shipping gametes or embryos rather than live animals. The use of embryos in addition to sperm represents a major increase in the efficiency of transmitting improved genetic potential. The ability to preserve maternal genetic information indefinitely would enable the transmission of improved genetic potential in a form other than the live animal. Desirable breeds possessing beneficial production characteristics or valuable disease resistance traits are not in any one country or region, yet they are desired globally to provide optimal production and disease resistance. Disease transmission and other health concerns limit live animal transport and subsequent propagation globally. Implementation of methodologies for long-term embryo preservation and transfer would provide a foundation for effective utilization of the world’s best genetic resources on a global basis while modernizing production and enhancing genetic improvement programs.
The development of a repeatable method for the long-term preservation of gametes or embryos would provide numerous applications, including: transport of paternal and/or maternal germplasm, rapid regeneration or expansion of new and existing lines, the ability to increase selection pressure in nucleus herds, extraction or rescue of healthy stock from diseased herds, improve or eliminate quarantine conditions and provide a method for the international export/import of potential breeding stock. While maintaining genetic resources through embryo banking, successful embryo preservation would enhance the further development of other animal production technologies such as sperm sexing, artificial insemination, in vitro fertilization and non-surgical embryo transfer. With the increasing use of animals in human biomedical research, embryos from invaluable genetic or transgenic lines used as organ donors for xenotransplantation could be preserved for future use. Collectively, these technologies could be instrumental in the continuous production of animals of high genetic merit capable of having a significant impact on the improvement of livestock and human medicine.
Genetic Repository Determining what kind of genetic resources are necessary for genetic repopulation and maintenance of genetic diversity are dependent on a number of factors which include: 1) species involved; 2) population status of the specie; 3) gamete availability; 4) laboratory expertise; 5) available biotechnologies for the specie; 6) germplasm research and repository systems; 7) contingency plans for germplasm maintenance, deposition and strategic use; 8) strategic planning for germplasm use in repopulation. For example, in cattle, most of the technology for cryopreserving bull sperm and cow embryos are highly commercialized and satisfactory for use in germplasm conservation. However, the swine industry is much different. Do to physiological and cellular differences between pigs and most other mammals, their germplasm and embryos are more difficult to handle in vitro as well as for long term preservation. Laboratory expertise is crucial for developing new or adapting current technologies to work with each and every species, and no two species are much alike. The more rare an animal becomes, the more difficult it becomes to obtain the germplasm or embryos
needed, as well as adapting existing technologies to their unique embryos. A strong and well funded research program with state-of-the-art equipment and well trained personnel are required.
Gamete Preservation For over 40years, bull studs around the world have been peddling frozen bull semen for use in artificial insemination (AI). In the US, over 90% of the dairy cattle are bred by AI. Within the last few years, boar studs have been established where most semen is extended and maintained at 18oC for up to 7 days. Over 30% of pigs bred in the US today are by bred AI, and its use has been increasing dramatically over just the last 2 years. The producers telephone or email in orders then rely on over-the-ground delivery of semen to be used with AI. The network is quite vast, and fully commercialized businesses. However, most other farm animal species do not have this type of commercialized support, and thus, for these species, most of the reproductive biotechnologies remain at the research level.
In cattle and swine, the use of sperm for germplasm preservation has many advantages: 1) ease of collection; 2) reliable protocols for semen maintenance and preservation; 3) renewable resource over time; 4) reliable insemination protocols; 5) high pregnancy rates with AI; 6) minimal disease transmission with AI. However, there are some disadvantages to using preserved semen coupled with AI: 1) frozen sperm must be monitored and maintained on the stud or the farm; 2) timing of AI to the estrus cycle of the female becomes crucial for timing of insemination; 3) training is necessary for handling preserved semen and performing AI; 4) germplasm itself contains only a haploid component of chromosomes, i.e. there is no maternal DNA present; 5) reliable protocols are available for cattle and swine, while protocols for other species are experimental or confined to research laboratories; 6) protocols for rare or endangered animals require a model system as genetic resources from them are in low to no availability.
Eggs, or as we in science refer to them as oocytes, are the least understood and least researched area of gamete preservation. Because of their size (such a large individual cell), their volume to surface area ratio and high water content make them extremely sensitive to cooling and/or cryopreservation. Although successful methodology is available in laboratories, that is where it is confined at the present day. Oocyte preservation would be useful for maintaining maternal genetics. However, there is a much simpler way to preserve maternal genetics... embryo preservation.
Embryo Preservation At the time of fertilization, the joining of a single sperm cell from the male with a mature oocyte or egg from the female results in the formation of an embryo. The embryo contains haploid chromosomes from both the sire and dam, that join together to make a diploid eucaryote. Thus, by utilizing embryo transfer, both maternal and paternal genetics can be conserved over time and germplasm can be propagated from a diploid chromosome source. During early embryonic development, embryos reside in the uterine horns (in vivo), where they develop rapidly and change morphologically (appearance, structure) over time prior to implantation and the establishment of pregnancy. While in the uterus, the morula and blastocyst stage embryos can be surgically and/or transferred back into surrogate embryo recipients. After transfer, these embryos can develop into normal, live offspring. Non-surgical embryo transfer in cattle is commercialized globally and completed on farm. Surgical embryo collection and transfer methods are routine procedures in many pig reproduction laboratories. Other species are confined to the laboratory, and reliable procedures are not fully developed. The important message is, the use of embryos in addition to sperm represents a major increase in the efficiency of transmitting improved genetic potential.
Preservation vs. Cryopreservation Whether the source of germplasm propagation be gametes or embryos, they must be preserved for short or long term for prospective use over time. There are a few options available. Short term storage includes: 1) culture; 2) hypothermic storage; long term storage = cryopreservation. However, these techniques are typically used with freshly recovered gametes and embryos, or previously cultured embryos. The problem is this: fresh or cultured gametes and embryos are perishable entities, and can only survive in vitro (outside the reproductive tract) for only a short period of time in culture.
Depending on the species, sperm can remain viable post ejaculation for up to 24-48 h at room temperature, although viability is reduced over time. If extended in a nutritious medium, sperm can prolong their fertilizing capacity and viability up to 7 days. However, extended semen does survive over these lengths of time, but their viability is continuously decreasing. Refrigerating extended bull sperm can help prolong its fertilizing capacity, however, boar semen may not be cooled below 15oC, as boar sperm possesses a high lipid content that is extremely sensitive to cooling, and death will occur. Bovine sperm can easily be frozen and maintained indefinitely. It is the basis for the AI industry and is responsible for the practical commercialization of the cattle AI. To a limited extent, boar semen can be cryopreserved. However, 2x109 sperm are needed to AI a female. Boar sperm are extremely sensitive to cooling below 15oC, and although protected with present day extenders, many sperm perish after freezing, and too few are often available for use in AI. Much research needs to be continued in boar semen preservation to make it as easy and reliable as it is in cattle.
Oocytes can be maintained in culture for a short period of time. Depending on the species, freshly ovulated mature oocytes can be cultured up to 10-15 h before they lose there developmental competence. Immature oocytes collected from ovarian follicles can be cultured up to 24-48 h and matured in vitro, depending on the species. However, oocyte culture is terminal if the oocytes are not fertilized within their maturation window. Oocyte cryopreservation is very dismal, as in most species maybe 10-20% survive any form of cold storage, and very few retain normal developmental competence.
Embryos can be cultured up to seven days, however, the longer the culture period in vitro, the less likelihood of individual embryo development in vivo after transfer. Whether it be sperm, eggs or embryos, the problem is directly related to mother nature. We as scientists have not created the ideal culture medium which will support in vitro embryo development equivalent to the in vivo environment mother nature has provided in the reproductive tract. So, how do we get around this and still be able to handle embryos in vitro? We could figure out mother nature’s recipe for success, of which science has yet to crack. Or rather, if there was methodology available for the indefinite storage of embryos, then the factor of culturing embryos in vitro would be minimized, and they could be transferred shortly after recovery from long term preservation.
Patience and persistence needed...
This part of the manuscript is an illustration of how preservation methodology was established for a species whose gametes and embryos are extremely sensitive to cooling. Explained is the following: 1) why the gamete and embryonic cells from this species are sensitive to chilling injury; 2) what research was committed to determine what parts of the embryos are permanently disrupted during cooling or cryopreservation; 3) what inhibitory methods were employed to deter chilling injury; 4) application of the new methodology for the production of live offspring after transfer of cryopreserved embryos; 5) Practical situations where this new technology will be an invaluable tool for animal production.
Methods exist to adequately, but not optimally, preserve germplasm and embryos from genetically superior animals of most of our livestock species except the pig. While methods exist to preserve boar sperm, little success until recently has been realized in preserving pig oocytes and embryos. As stated previously, the use of embryos in addition to sperm represents a major increase in the efficiency of transmitting improved genetic potential.
In 1989, the first live offspring were reported after transfer of frozen pig embryos. For many years, isolated reports in the scientific literature stated production of live offspring after transfer of frozen embryos. Unfortunately, many of these reports were never or could never be repeated in controlled scientific studies. Why are cow embryos so easy or routine to cryopreserve, yet this technology has never worked for pig embryos? Evolution is one contributing factor to consider. During evolutionary development of mammals, the pig must have developed a little differently than other domestic farm animals, or even humans. One pronounced evolutionary difference was in early embryonic development. Unlike cattle and human embryos, pig embryos suffer from severe sensitivity to hypothermic (below normal body temperature) conditions, which limit their ability to withstand many conventional methods of cryopreservation. This sensitivity has been attributed to a high lipid content of pig embryos. Cattle and human embryos have less lipid. Simply, pig embryos cooled below 15oC do not implant after surgical transfer to recipients. This sensitivity of pig embryos to chilling is not apparent in embryos at the morula to blastocyst stage from other domestic and laboratory species, such as cattle, sheep, rodents and rabbits. Thus, conventional methods of cryopreservation (such as programmable rate freezing) that rely on slow, controlled cooling during early ice formation are not suitable nor viable for whole pig embryo cryopreservation.
Our laboratory has been involved in developing vitrification procedures for cryopreservation of pig embryos. Vitrification is the rapid cooling of liquid medium in the absence of ice crystal formation. The solution forms an amorphous glass as a result of rapid cooling by direct submersion of the embryo in a plastic straw into liquid nitrogen. The glass retains the normal molecular/ionic distributions of a liquid but remains in an extremely viscous, supercooled form. The glass is devoid of all ice crystals, and embryos are not subjected to the physical damage that is associated with ice crystal formation during freezing. Vitrification is simple, inexpensive, and has been developed as a viable alternative to conventional freezing of domestic and laboratory animal embryos. Cattle embryos have been successfully cryopreserved with vitrification and under field conditions without any reduction in pregnancy rate.
For pig embryos, we hypothesized that vitrification might reduce the possibility of both intracellular and extracellular ice formation while rapid cooling of embryos could bypass or out-race detrimental chilling-induced cellular changes that take place during slow cooling. Vitrification worked as we obtained 30-40% survival and subsequent development of vitrified hatched blastocysts in vitro. Good, yes, but not good enough. Finding any survival and development after vitrification was great, but at the going rate, you would need about 100 embryos to even come close to a normal litter size after cryopreservation and embryo transfer. The question became: What is happening in the other 60-70% of the embryos being destroyed by cryopreservation?
We observed major cellular disruption following vitrification. Cryopreservation of any kind can be extremely disruptive to the cellular organization of embryos. Ice crystal formation can lyse plasma membranes, storage in liquid nitrogen can denature critical intracellular functions and organelles, and the central cytoarchitecture (cell skeleton or cytoskeleton) of a cell can be destroyed. The cytoskeleton is complex network of protein constituents, microfilaments and microtubules, distributed throughout the a cell. They give 3-dimensionality and mechanical strength to the surface of a cell, including the membrane, and provides a system of fibers which impart polarity while regulating cell shape, cell movement, and the plane of cell division. Documenting cellular damage during or after cryopreservation provides useful information for understanding cellular sensitivities to cryopreservation and can lead to improved protocols for embryo cryopreservation. Recently, research has characterized critical cellular players in the hypothermic sensitivity of pig embryos.
Utilizing advanced flourescent, laser scanning confocal microscopy, we observed that vitrified embryos which were damaged or destroyed after cryopreservation had disrupted outer membranes and cytoskeleton. During cryopreservation, solutes used to protect cellular structures wreak havoc on the entire embryonic cytoskeleton, cause its disruptions, then leave the embryo partially or wholly destroyed. At this point, we knew the following: 1) pig embryos at the appropriate stage could survive cryopreservation; 2) 60-70% of cryopreserved embryos did not survive after recovery from cryopreservation; 3) the plasma membrane and its linked cytoskeleton were disrupted in many pig embryos following cryopreservation.
We hypothesized that cytoskeletal disruption from cryopreservation might be prevented if we could stabilize the cytoskeleton prior to cryopreservation. Hatched blastocyst stage pig embryos were vitrified under the influence of cytoskeletal stabilization with a microfilament inhibitor. Embryonic survival and subsequent development in vitro improved 2.5-fold (80-90%). We then wanted to check the in vivo developmental competence of cytoskeletal stabilized and vitrified pig embryos. In our first trials, 2 of 5 recipients of stabilized/vitrified embryos farrowed 10 live and normal offspring, being the first report of live offspring produced after transfer of pig embryos cryopreserved by vitrification. Of these mature offspring, 2 boars and 2 gilts successfully proved their reproductive capacity, as the boars each inseminated 3 gilts, and the 2 gilt offspring were mated and farrowed normal litters. In our second trial, utilizing trans-oviductal uterine catheterization (TUC) for surgical embryo transfer, 4 of 7 recipients farrowed 26 live offspring. Stabilized vitrification is a viable method for the long term preservation of pig embryos, a first for maternal genetics in swine.
This validated technology will provide a foundation for effective utilization and transport of the world’s best genetic resources on a global basis while modernizing pork production and enhancing genetic improvement programs. Although an important breakthrough, much work still needs to be continued in order to gain the ability to cryopreserve earlier stage pig embryos that are essential for the pathogen-free propagation of desirable genetics. Thus, more intensive research needs to be conducted to characterize cellular and molecular disruptions during and after cryopreservation, so that we can further improve methodology for preserving earlier stage, developmentally competent swine embryos.
As stated earlier, until recently, the swine industry has had no successful method for preserving pig embryos. The ability to preserve maternal genetic information indefinitely would enable the transmission of improved genetic potential in a form other than the live animal, a first for maternal genetics in swine. Desirable breeds possessing beneficial production characteristics or valuable disease resistance traits are not in any one country or region, yet need to be globally available to ensure optimal production and disease resistance. Disease transmission and other health concerns limit live animal transport and subsequent propagation globally. Implementation of methodologies for long-term embryo preservation and transfer in swine would provide a foundation for effective utilization of the world’s best genetic resources on a global basis while modernizing pork production and enhancing genetic improvement programs.
Practical Applications The development of a repeatable method for the long-term preservation of pig embryos would provide numerous applications, including: transport of maternal germplasm, rapid regeneration or expansion of new and existing lines, the ability to increase selection pressure in nucleus herds, extraction or rescue of healthy stock from diseased herds, improve or eliminate quarantine conditions and provide a method for the international export/import of potential breeding stock. While maintaining genetic resources through embryo banking, successful embryo preservation would enhance the further development of other animal production technologies such as sperm sexing, artificial insemination, in vitro fertilization and non-surgical embryo transfer. With the increasing use of swine in human biomedical research, embryos from invaluable genetic or transgenic lines of pigs used as organ donors for xenotransplantation could be preserved for future use. Collectively, these technologies could be instrumental in the continuous production of animals of high genetic merit capable of having a significant impact on the improvement of the world swine population and human medicine.
For example, genetic seedstock companies have nucleus herds throughout the world, or want desirable genetics outside their region or country. However, the desired genetics must come from a non-disease validated herd, or a herd that is positive for any such disease, or from a region or country that is positive for such diseases. The bottom line is, the pigs must come from possible infected sources, which is a terrible risk to take for genetics advancement in their lines. International policy dictates that the animals cannot come into the country with the possibility of bringing in potential devastating disease risks with them. How do you get around this dilemma? Embryos. Embryos can be recovered from the reproductive tracts of donor females from the herd maintaining the desirable genetics. After recovery, embryos are washed to remove any possible pathogens that may be on the surface of the embryos that were acquired from the mother while residing in vivo. This is an example for genetic rescue from a diseased herd. That is great, but, as said before, embryos are perishable, and must be preserved if remaining in vitro for any extended amount of time. If we could preserve embryos indefinitely, embryos from the desired genetics herd could be collected, washed, preserved, and then shipped to the location where the genetic repopulation is to occur. The existing lines could now be regenerated in a disease-free environment, and eventually integrated into breeding programs.
In another example, a researcher or genetic supplier has over 20 years in development of specific nucleus herd or invaluable lines of pigs. All these pigs are produced and propagated in one huge confinement facility. The 20 year old wiring in the barn fails, and there is a fire; or some disease or sickness comes over the entire herd. Bottom line... the entire herd is devastated or wiped out. The only pigs of this type in the entire world were in this barn, and now everything is gone. All those years of work, toil and trouble... gone! Not a pig left! What do you do? If you had embryos stored indefinitely, by regeneration through embryo transfer, you could feasiblely have your line of pigs back up as a reproducing herd within a couple of years.
In a final example, a company has produced an invaluable line of pigs developed for human organ transplantation. If a devastating incidence would occur to this herd, the whole company could perish. They could preserve embryos for an insurance policy on their unique line, insuring indefinite recovery possibility for their line anytime in the future, for any purpose.
The cattle industry has had this technology available since the early 1980s, and it is primarily used for the propagation of genetically or economically superior animals. Embryo preservation and transfer revolutionized cattle breeding on a global basis. These technologies are in their relative infancy for the swine industry, and the possibilities are limitless.
PROPAGATION OF GLOBAL GENETICS
Considerations, Cooperation and Vision
A future scenario Desirable genetics for a specific disease resistant line of pigs has been located in a country. The herd happens to be partially positive with an infective pathogen, inhibiting the transport of breeder pigs out of the herd. A research team is sent in to isolate, cryopreserve and recover 500 pathogen-free embryos from this line. The aseptic embryos are transported out of the country to be transferred into recipients and propagated in a pathogen-free environment. After ample propagation while maintaining genetic diversity, founder-line pigs from this line will be integrated into hybrid production systems.
A future solution A future solution will be developed following common scientific method.
Objective: Can we develop a pathogen-free system for the global propagation of swine genetics?
Rationale: The development of a repeatable method for the long-term preservation of pathogen-free pig embryos would provide numerous practical applications, including: transport of maternal germplasm, rapid regeneration or expansion of new and existing lines, the ability to increase selection pressure in nucleus herds, extraction of healthy stock from diseased herds, improve or eliminate quarantine conditions and provide a method for the international export/import of potential breeding stock. While maintaining genetic resources through embryo banking, successful embryo cryopreservation would enhance the further development of other animal production technologies such as sperm sexing, artificial insemination, in vitro fertilization and non-surgical embryo transfer. With the increasing use of swine in human biomedical research, embryos from valuable genetic or transgenic lines of pigs used as organ donors in xenotransplantation could be preserved for future use. Collectively, these technologies could be instrumental in the continuous production of animals of high genetic merit capable of having a significant impact on the improvement of the world swine population and human medicine.
1) Establishment of a global network of swine genetics, based on gene mapping for production, reproduction and disease resistance traits. This will allow researchers, industry and producers access to information on desirable global genetics. 2) Implementation of disease control guidelines, to minimize any possibility for accidental or unwanted disease contamination; including facility, animal, embryo and human contamination. These guidelines will establish methodology for the access to, and embryo collection procedures from a possible infected herd, embryo handling procedures as defined by international transport guidelines set forth and governed by the International Embryo Transfer Society. 3) Locate herds containing lines of desirable genetics regionally, nationally or internationally. 4) Propagation of embryos from line(s) of animals of desired genetics by established laboratory methodologies. 5) Aseptic collection and preservation of embryos. 6) Long-term (indefinite) storage of embryos in a Germplasm Repository. Regional Repositories will be set up internationally in association with proposed bio-secure zones. 7) Establishment of global pathogen-free bio-secure zones for the propagation of rescued genetics. Internationally procure zones with unique bio-secure features, such as islands, where contaminated traffic can be fully controlled. Cryopreserved embryos will be aseptically transferred into pathogen-free recipient animals. Farrowing and development of F-1 founder lines will occur in the bio-secure zones. Propagation will continue to fulfill minimal requirements of founder line animals needed, as well as founder F-1 and F-2 line embryos for long-term preservation. 8) Release of germplasm and/or live animals for integration into hybrid production systems. 9) Continual location, propagation and inventory maintenance for desired lines of animals in a genetic conservation program.
1) Preservation and conservation of valuable genetic resources in reasonable and economical manner.
2) Development of pathogen-free system for the rescue of valuable swine genetics.
3) Establishment of global bio-secure zones for propagation of valuable lines of pigs.
In conclusion, it is quite evident from the cattle industry what an impact germplasm or embryo preservation can have on the global production herds. Although the swine industry is far behind cattle, research has uncovered developmental differences between species and allowed solutions to be found. If we can do it with pigs, we can do it with other domestic farm animal species too. All it takes is asking the right questions and having good experimental method, while being patient observant and persistent. Good luck.