Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

Sperm sexing technology-the transition to commercial application. An introduction to the symposium "update on sexing mammalian sperm"

Over the past decade, procedures for sexing mammalian sperm by flow cytometry/cell sorting have been refined sufficiently for large-scale commercial application in cattle; millions of doses of sexed sperm are sold each year for artificial insemination. Furthermore, more than a hundred babies are born annually from use of sexed human sperm via artificial insemination or in vitro fertilization, and to a lesser extent by injection of single sperm into oocytes. Semen sexing technology is also being applied for various objectives in a number of other species. Accuracy of sexing is around 90% in most species. However, this technology is still rather expensive, and fertility of sexed sperm is lower than unsexed controls in most instances. Speakers in this symposium, “Update on sexing mammalian sperm,” held in San Diego, California on 3 January 2009, presented the latest research on sexing sperm, including documentation of success rates. The written versions were peer-reviewed and follow in this issue of Theriogenology.     

Sexing mammalian sperm--intertwining of commerce, technology, and biology

Sperm from many mammalian species can be sexed by flow cytometry/cell sorting at about 90% accuracy without damaging them unduly. However, because sperm are evaluated one at a time, in series, the number of sexed sperm produced per unit time is limited. Furthermore, the equipment required currently is expensive, in the order of 300,000 US dollars per machine. Despite these limitations, commercialization of this technology has begun with bovine semen, in part by inseminating cattle with relatively low number of sperms. No other approach to sexing sperm in any practical way is likely to be available within the next few years. The constraints for commercial application of sexed sperm in cattle can be somewhat lowered fertility, the high costs of equipment and skilled personnel, and costs of intellectual property such as licensing fees and royalty payments. Most economic analyses indicate that farmers can afford to pay 10-20 US dollars more per dose of sexed sperm than unsexed sperm if costs must be recouped by selling milk or meat. When the product is breeding stock or for certain niche applications, a higher price for sexed semen may be justified. Sexed sperm will be used more broadly in cattle only when improved production and/or efficiency can compensate for the extra costs of purchasing sexed sperm.     

Preselection of sex of offspring in swine for production: current status of the process and its application

It is estimated that as many as 30,000 offspring, mostly cattle, have been produced in the past 5 years using AI or some other means of transport with spermatozoa sexed by flow cytometric sperm sorting and DNA as the marker of differentiation. It is well documented that the only marker in sperm that can be effectively used for the separation of X- and Y-chromosome bearing spermatozoa is DNA. The method, as it is currently used worldwide, is commonly known as the Beltsville Sperm Sexing Technology. The method is based on the separation of sperm using flow cytometric sorting to sort fluorescently (Hoechst 33342) labeled sperm based on their relative content of DNA within each population of X- and Y-spermatozoa. Currently, sperm can be produced routinely at a rate of 15 million X- and an equal number of Y-sperm per hour. The technology is being applied in livestock, laboratory animals, and zoo animals; and in humans with a success rate of 90-95% in shifting the sex ratio of offspring. Delivery of sexed sperm to the site of fertilization varies with species. Conventional AI, intrauterine insemination, intra-tubal insemination, IVF with embryo transfer and deep intrauterine insemination are effectively used to obtain pregnancies dependent on species. Although sperm of all species can be sorted with high purity, achieving pregnancies with the low numbers of sperm needed for commercial application remains particularly elusive in swine. Deep intrauterine insemination with 50-100 million sexed boar sperm per AI has given encouragement to the view that insemination with one-fiftieth of the standard insemination number will be sufficient to achieve pregnancies with sexed sperm when specialized catheters are used. Catheter design, volume of inseminate, number of sexed sperm are areas where further development is needed before routine inseminations with sexed sperm can be conducted in swine. Cryopreservation of sex-sorted sperm has been routinely applied in cattle. Although piglets have been born from frozen sex-sorted boar sperm, freezing and processing protocols in combination with sex-sorted sperm are not yet optimal for routine use. This review will discuss the most recent results and advances in sex-sorting swine sperm with emphasis on what developments must take place for the sexing technology to be applied in commercial practice.     

History of commercializing sexed semen for cattle

Although the basic principles controlling the sex of mammalian offspring have been known for a relatively long time, recent application of certain modern cellular methodologies has led to development of a flow cytometric system capable of differentiating and separating living X- and Y-chromosome-bearing sperm in amounts suitable for AI and therefore, commercialization of this sexing technology. After a very long history of unsuccessful attempts to differentiate between mammalian sperm that produce males from those that produce females, a breakthrough came in 1981 when it was demonstrated that precise DNA content could be measured. Although these initial measurements of DNA content killed the sperm in the process, they led to the ultimate development of a sperm sorting system that was capable, not only of differentiating between live X- and Y-sperm, but of sorting them into relatively pure X- and Y-sperm populations without obvious cellular damage. Initial efforts to predetermine the sex of mammalian offspring in 1989 required surgical insemination, but later enhancements provided sex-sorted sperm in quantities suitable for use with IVF. Subsequent advances in flow sorting provided minimal numbers of sperm sufficient for use in AI. It was not until the flow cytometric sorting system was improved greatly and successful cryopreservation of sex-sorted bull sperm was developed that efficacious approaches to commercialization of sexed semen could be implemented worldwide in cattle. A number of companies now offer sex-sorted bovine sperm. Innovative approaches by a diverse group of scientists along with advances in computer science, biophysics, cell biology, instrumentation, and applied reproductive physiology provided the basis for commercializing sexed semen in cattle.     

Livestock embryo sexing: A review of current methods, with emphasis on Y-specific DNA probes

Control of the sex ratio of domestic species is potentially of great commercial importance to agriculture. While sexing of spermatozoa would be the most advantageous approach, studies to date suggest that this technology is unlikely to be available in the near future. As an alternative, four methods of sexing embryos have been developed. The use of X-linked enzymes and a serological assay involving H-Y antigen are noninvasive methods which have the advantage of allowing all embryos to be sexed, but these methods are not always accurate. Cytogenetic analysis and the use of Y-specific DNA probes are invasive methods which are limited by the accessibility of embryonic material for biopsy, but they are highly accurate. Each method is reviewed, with an emphasis on the use of Y-linked probes, and each is seen to have both advantages and limitations; the difficulty is in achieving a method that provides both an accurate sexing procedure and an acceptable pregnancy rate after embryo transfer. While no single method currently available fulfills all the criteria for a commercial method of embryo sexing, the potential for the development of an ideal method does exist.     

Application of sperm sorting and associated reproductive technology for wildlife management and conservation

Efforts toward the conservation and captive breeding of wildlife can be enhanced by sperm sorting and associated reproductive technologies such as sperm cryopreservation and artificial insemination (AI). Sex ratio management is of particular significance to species which naturally exist in female-dominated social groups. A bias of the sex ratio towards females of these species will greatly assist in maintaining socially cohesive groups and minimizing male-male aggression. Another application of this technology potentially exists for endangered species, as the preferential production of females can enable propagation of those species at a faster rate. The particular assisted reproductive technology (ART) used in conjunction with sperm sorting for the production of offspring is largely determined by the quality and quantity of spermatozoa following sorting and preservation processes. Regardless of the ART selected, breeding decisions involving sex-sorted spermatozoa should be made in conjunction with appropriate genetic management. Zoological-based research on reproductive physiology and assisted reproduction, including sperm sorting, is being conducted on numerous terrestrial and marine mammals. The wildlife species for which the technology has undergone the most advance is the bottlenose dolphin. AI using sex-sorted fresh or frozen-thawed spermatozoa has become a valuable tool for the genetic and reproductive management of captive bottlenose dolphins with six pre-sexed calves, all of the predetermined sex born to date.     

Economics of selecting for sex: the most important genetic trait

Over 20,000 calves have resulted from artificial insemination (AI) of cattle with sexed, frozen/thawed sperm in the course of experimentation in several countries, and from commercial sales in the United Kingdom. This technology likely will become commercially available in many countries within a few years. Accuracy of the process is about 90% for either sex, and resulting calves appear to be no different from non-sexed controls in birthweight, mortality, rate of gain, and incidence of abnormalities. The main determinants of the extent of use of sexed sperm will be pregnancy rate and cost. Fertility of low doses (1.5 x 10(6)-2 x 10(6)) of sexed, frozen sperm for AI of heifers usually has been in the range of 70-80% of unsexed sperm at normal doses (10 x 10(6)-20 x 10(6) sperm) in well managed herds; it has been lower in poorly managed herds, and likely will be lower with lactating dairy cows. It is expected that fertility of sexed sperm will increase significantly due to very recent improvements in the hydrodynamics of the sexing process and potential improvements in cryopreservation procedures. It is unclear how sexed sperm will be priced; the cost of sexed sperm for cattle will likely be more than double the cost of unsexed sperm in most markets. The economic benefit of using sexed sperm also will depend on the baseline fertility of the herd since at lower fertility, it takes more doses of semen per calf produced. It is noted that for a small percentage of elite cattle, the economics of using sexed sperm do not depend primarily on increased production or efficiency of producing meat or milk, but rather on factors such as scarcity, tradition, cattle show winnings, and biosecurity during herd expansion. Until sorting efficiencies improve and costs decline, sales likely will be limited primarily to these niche markets. With near normal fertility and a premium for sexing in the range of US$ 10 per insemination dose, sexed sperm likely would become economically and environmentally beneficial for many, if not most populations of cattle being bred by AI.     

Sex ratio determination in bovine semen: a new approach by quantitative real time PCR

Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction. Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies. In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV=2.58%) and reproducibility (CV=2.57%) was shown. The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P>0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11+/-0.56% for ejaculates and 50.17+/-0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.     

What affects fertility of sexed bull semen more, low sperm dosage or the sorting process?

Until now it has been unclear to what extent the reduced fertility with sexed semen in the dairy industry is caused by too few sperm per AI dose, or by the effect of flow cytometric sorting, which is the established procedure for sexing semen. Therefore, we evaluated the effects of low sperm numbers per dose with and without sorting on non-return rates after 56 days (NRR56); in addition, we evaluated the effects of bulls, in order to further optimize use of sexed semen.Based on results of using sexed semen from seven Holstein bulls, an overall numerical decline of 13.6% in NRR56 was observed (P < 0.05). About two-thirds of this decline (8.6%) was due to the low dose (P < 0.05), and a third (5.0%) due to the process of sorting (P < 0.05). The effect of low dosage and sorting differed among bulls. We observed a sex ratio of 91.6% females for sexed semen from the first 131 calves born.Currently the best way to increase fertility of sexed semen is by closely monitoring fertility so that the highest fertility bulls are used, and by improving farm animal management. However, to make substantial progress, more in depth studies are needed on the sexing technology, especially on aspects such as sorting procedures and sperm dosage.     

Cryopreservation of flow-sorted bovine spermatozoa

Experiments were designed to maximize sperm viability after sorting by flow cytometry and cryopreservation. Experiments concerned staining sperm with Hoechst 33342 dye, subsequent dilution, interrogation with laser light, and postsort concentration of sperm. Concentrating sorted sperm by centrifugation to 10 to 20 x 10(6) sperm/ml reduced adverse effects of dilution. Exposing sperm to 150 mW of laser light resulted in lower percentages of progressively motile sperm after thawing than did 100 mW. Sorted sperm extended in a TRIS-based medium had higher postthaw sperm motility after incubation for 1 or 2 h than sperm extended in egg-yolk citrate (EYC) or TEST media, and equilibrating sperm at 5 degrees C for 3 or 6 h prior to freezing was superior to an equilibration time of 18 h. For sorting sperm 4 to 7 h postcollection, it was best to hold semen at 22 degrees C neat instead of at 400 x 10(6)/ml in a TALP buffer with Hoechst 33342. Current procedures for sexing sperm using flow cytometry result in slightly lower postthaw motility and acrosomal integrity compared to control sperm. However, this damage is minor compared to that caused by routine cryopreservation. Fertilizing capacity of flow-sorted sperm is quite acceptable as predicted by simple laboratory assays, and sexed bovine sperm for commercial AI may be available within 2 years.     

哺乳动物精子分离方法的研究进展

分离X、Y精子,用于人工授精或体外受精,是目前实现哺乳动物性别控制的最有效手段。本文对哺乳动物精子分离及分离精子纯度评估方法的研究历史及现状作一回顾和总结。     

Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.     

Factors affecting commercial application of embryo technologies in New Zealand: a modelling approach

New reproductive technologies include sexed sperm and embryo-based technologies. The technology of sperm sexing, for various reasons, is not available in New Zealand and its use has not been modelled. Embryo technologies are however already in use on a limited scale and various scenarios for their use in both the dairy and beef industries in New Zealand have been modelled. This review briefly discusses the various technologies available and some of their potential strengths and weaknesses. In the dairy industry, modelling has been used to simulate the production of breeding bulls for large breeding companies and the production of replacement heifers in dairy herds. For the beef industry, similar modelling has been carried out to determine the opportunities for more efficient beef production.All the models confirmed that at current levels of performance, embryo-based reproductive technologies are usually not profitable in New Zealand except in niche market situations where the returns from the resulting offspring are significantly greater than can be obtained from natural mating or artificial insemination (AI) reproduction systems. This is confirmed by the low uptake of these technologies in this country to date. Even if performance lifts to levels similar to AI, profitability is likely to occur only if the costs of pregnancies to embryo-based reproductive technologies can occur at prices less than two to four times greater than AI or natural mating. This break-even requirement depends on the returns that can be achieved and the advantages that can be captured by the technology over and above those available from AI or natural mating. Two new uses for reproductive technologies in dairy cattle could be the proliferation of novel or rare genotypes from gene discovery programs and improving the female reproductive rate for optimal marker assisted selection. In both these uses the technology is not at present competing with AI or natural mating. The challenge exists therefore for the biological scientists to satisfy these requirements, coupled with the ethical and human factors involved in the introduction of any new technology.Potential end users of the technologies have been surveyed. They are quite positive about the technologies provided they can use them profitably and are keen to obtain more information about them.     

Functional integrity of sex-sorted, frozen-thawed boar sperm and its potential for artificial insemination

Despite being developed in its present form 20 years ago, sex-sorting of mammalian sperm is still a work in progress. While relatively successful in cattle and sheep, the unique challenges of incorporating sex-sorted sperm into pig production have not yet been overcome. Generally speaking, boar sperm survive freeze-thawing less well and are required in larger numbers for insemination, while in vitro embryo production of pig embryos is less successful compared to other domestic species [Niemann H, Rath D. Progress in reproductive biotechnology in swine. Theriogenology 2001;56:1291-1304]. Due to the large number of sperm required for artificial insemination in pigs, a technique of storing sperm after sorting must be developed while adequate numbers of sperm are allocated into X- or Y-chromosome-bearing enriched pools. Cryopreservation is perhaps the ideal method of storage between sorting and insemination, as it allows unlimited time to build up a sperm bank, whereas liquid-storage requires the use of sperm within days of sorting. The limited number of studies investigating the survivability of sex-sorted, frozen-thawed boar sperm have produced promising in vitro results but poor in vivo outcomes. Before fertility can be improved, the causes of any damage to sperm function during the sex-sorting and freeze-thawing procedures must be more fully understood. Once defined, the source of damage may be minimised and this would lead to increased success rates after in vivo application of sex-sorted, frozen-thawed boar sperm.     

Overview of sexing sperm

Hundreds of thousands of off springs have been born as a result of AI with sexed sperm. Although this technology has been used for many species, the overwhelming majority of pregnancies have been in cattle, nearly all as a result of sperm that were sexed and subsequently frozen. The technology for sexing sperm has not changed greatly in the past 7 years, but refinements have speeded up the process and reduced damage to sperm. The process of commercialization of sexed sperm has accelerated recently. However, this technology is characterized by high costs, complexity of implementation and lower pregnancy rates than with control sperm. Nevertheless, sexed, frozen bovine sperm are being produced commercially in many countries, although from a limited number of bulls. The main application of sexed sperm to date has been to breed dairy heifers to produce female calves. Because of the slow speed of sexing sperm, fewer sperm are used per insemination dose of sexed than conventional sperm, and pregnancy rates with this product are often only slightly decreased. Successful use of sexed sperm requires excellent management of cattle, careful handling of sperm and use of skilled inseminators. As costs decline, sexed sperm will be used increasingly for cattle breeding, horse breeding and niche applications in other species.     

Advances in flow cytometry for sperm sexing

This review presents the key technological developments that have been implemented in the 20 years since the first reports of successful measurement, sorting, insemination and live births using flow cytometry as a proven physical sperm separation technique. Since the first reports of sexed sperm, flow technology efforts have been largely focused on improving sample throughput by increasing the rate at which sperm are introduced to the sorter, and on improving measurement resolution, which has increased the proportion of cells that can be reliably measured and sorted. Today, routine high-purity sorting of X- or Y-chromosome-bearing sperm can be achieved at rates up to 8000 s⁻¹ for an input rate of 40,000 X- and Y- sperm s⁻¹. With current protocols, straws of sex-sorted sperm intended for use in artificial insemination contain approximately 2 × 10⁶ sperm. The sort rate of 8000 sperm s⁻¹ mentioned above corresponds to a production capacity of approximately 14 straws of each sex per hour per instrument.     

Production of Plant Made Pharmaceuticals: From Plant Host to Functional Protein

In the last two decades plants have emerged as valuable alternatives to mammalian cells for the production of pharmaceuticals and their potential as expression systems was shown by the commercial availability and acceptance of several plant made therapeuticals in clinical trials. Plants have many advantages over yeast, insect and bacterial expression systems such as the potential to properly fold the expressed proteins and the synthesis of more human-like N-glycans on the proteins. However, several constraints, such as expression yields, downstream processing and structural authenticity, currently limit the widespread use of plant expression systems. In this review, the focus is on the current limitations of plant systems for the production of pharmaceuticals and the possibilities to overcome these obstacles. A comparison is made with insect cell and yeast expression systems. Furthermore, the importance of glycosylation, in particular N-glycosylation for the biological function(s) of therapeutics in the human body will be discussed in detail and an overview of the state of art in the humanization of the N-glycosylation pathway in plants is provided.     

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.