Exhibit 1010
`Select Sires, et al. v. ABS Global
`
`Exhibit 1010
`Select Sires, et al. v. ABS Global
`
`

`

`J. Dairy Sci. 100:10314–10331
`https://doi.org/10.3168/jds.2017-13138
`© American Dairy Science Association®, 2017.
`A 100-Year Review: Reproductive technologies in dairy science1
`S. G. Moore*2 and J. F. Hasler†‡
`*Division of Animal Sciences, University of Missouri, Columbia 65211
`†Vetoquinol USA, Fort Worth, TX
`‡427 Obenchain Rd., Laporte, CO 80535
`
`ABSTRACT
`
`Reproductive technology revolutionized dairy pro-
`duction during the past century. Artificial insemina-
`tion was first successfully applied to cattle in the early
`1900s. The next major developments involved semen
`extenders, invention of the electroejaculator, progeny
`testing, addition of antibiotics to semen during the
`1930s and 1940s, and the major discovery of sperm
`cryopreservation with glycerol in 1949. The 1950s and
`1960s were particularly productive with the develop-
`ment of protocols for the superovulation of cattle with
`both pregnant mare serum gonadotrophin/equine
`chorionic gonadotrophin and FSH, the first success-
`ful bovine embryo transfer, the discovery of sperm
`capacitation, the birth of rabbits after in vitro fertiliza-
`tion, and the development of insulated liquid nitrogen
`tanks. Improved semen extenders and the replacement
`of glass ampules with plastic semen straws followed.
`Some of the most noteworthy developments in the
`1970s included the initial successes with in vitro cul-
`ture of embryos, calves born after chromosomal sexing
`as embryos, embryo splitting resulting in the birth of
`twins, and development of computer-assisted semen
`analysis. The 1980s brought flow cytometric separation
`of X- and Y-bearing sperm, in vitro fertilization leading
`to the birth of live calves, clones produced by nuclear
`transfer from embryonic cells, and ovum pick-up via
`ultrasound-guided follicular aspiration. The 20th cen-
`tury ended with the birth of calves produced from AI
`with sexed semen, sheep and cattle clones produced by
`nuclear transfer from adult somatic cell nuclei, and the
`birth of transgenic cloned calves. The 21st century has
`seen the introduction of perhaps the most powerful bio-
`technology since the development of artificial insemina-
`tion and cryopreservation. Quick, inexpensive genomic
`analysis via the use of single nucleotide polymorphism
`genotyping chips is revolutionizing the cattle breeding
`industry. Now, with the introduction of genome editing
`
`Received May 9, 2017.
`Accepted July 11, 2017.
`1 This review is part of a special issue of the Journal of Dairy Science
`commissioned to celebrate 100 years of publishing (1917–2017).
`2 Corresponding author: moorestep@missouri.edu
`
`technology, the changes are becoming almost too rapid
`to fully digest.
`Key words: artificial insemination, multiple ovulation
`and embryo transfer, in vitro embryo production, sexed
`semen
`
`INTRODUCTION
`
`
`
`Artificial insemination was the first reproductive
`technology applied to cattle, initially in Russia and
`Denmark during the early 1900s (Ivanoff, 1922; Perry,
`1945). The primary driving force behind AI was its
`potential to increase the rate of genetic gain in livestock
`populations by widespread use of sires with elite genetic
`merit. Cooperative AI centers began in Denmark in
`1936 and were replicated internationally (Perry, 1945).
`Not since the invention of the milking machine has a
`technology had such an effect. For farmers, transition
`away from natural service breeding required changes in
`herd reproductive management. In addition to genetic
`gains, AI breeding avoided the need to have bulls on
`each farm and contributed to improved safety for farm
`employees. Today, use of AI has grown to the extent
`that internationally approximately 130 million cattle
`are submitted for AI annually (Vishwanath, 2003).
`Birth of the first calves from the use of frozen–thawed
`semen (Polge and Rowson, 1952) and embryos (Wilmut
`and Rowson, 1973) represented important milestones
`during the past century. Both developments were criti-
`cal to the feasibility and growth of large-scale AI and
`embryo transfer (ET) operations globally because it
`became no longer essential to use only unfrozen (fresh)
`semen and embryos. Today, the vast majority of in-
`seminations and transfers are performed with frozen–
`thawed semen and embryos, respectively (Vishwanath,
`2003; Hasler, 2014).
`Techniques for multiple ovulation and ET for cattle
`were developed in the 1940s and 1950s (Casida et al.,
`1943; Rowson, 1951; Willett et al., 1951; Dziuk et al.,
`1958); however, large-scale ET operations were not es-
`tablished in North America until the 1970s, in Europe
`until the 1980s, and in South America until the 1990s
`(Hasler, 2014). In vitro developments in oocyte matura-
`tion and sperm capacitation, fertilization, and embryo
`10314
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`100-YEAR REVIEW: REPRODUCTIVE TECHNOLOGIES IN DAIRY SCIENCE
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`10315
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`Table 1. Key events in our understanding of reproductive biology before the early 1900s1
`
`Event
`
`de Graaf describes testis structure.
`de Graaf describes follicle structure.
`Van Leuwenhoek and Ham describe spermatozoa.
`Spallanzani reports AI of a bitch followed by birth of pups.
`Spallanzani reports that sperm survived chilling.
`Von Baer describes oocytes.
`Kölliker demonstrates that the testes produce sperm.
`Barry observes the fusion of sperm and oocyte.
`Leydig describes Leydig cells.
`Sertoli describes Sertoli cells.
`Waldeyer describes the ovary and oocyte formation.
`Von Ebner describes spermatogenesis.
`Dreisch demonstrates artificial embryo twinning in the sea urchin (an invertebrate).
`Heape performs the first mammalian embryo transfer.
`Spemann demonstrates artificial embryo twinning in the salamander (a vertebrate).
`1Data from Marshall (1910); Genetic Science Learning Center (2014).
`
`Year
`
`1668
`1672
`1678
`1784
`1803
`1827
`1841
`1843
`1851
`1865
`1870
`1871
`1885
`1890
`1902
`
`culture during the 1970s and 1980s led to the birth of
`the first completely in vitro embryo produced calves in
`1987 (Lu et al., 1987). The ovum pick-up (OPU) meth-
`od for repeated oocyte recovery from live donor females
`was developed during the late 1980s by Pieterse et al.
`(1988). Protocols for in vitro embryo production (IVP)
`were further developed in the 1990s as an alternative to
`multiple ovulation and ET by combining OPU, in vitro
`fertilization (IVF), and ET (Looney et al., 1994). The
`practice of IVP has grown rapidly since the 2000s, with
`large-scale commercial operations established primarily
`in South America (Hasler, 2014).
`Cloning techniques for production of identical sheep
`began in the 1970s, first by embryo splitting (Willad-
`sen, 1979) and subsequently replaced by nuclear trans-
`fer (Willadsen, 1986; Prather et al., 1987). A far more
`powerful technology, however, involved what is referred
`to as somatic cell nuclear transfer (SCNT), allowing
`the cloning of an animal whose genetics and morphol-
`ogy were already known. Dolly the sheep was the first
`example of success with SCNT (Wilmut et al., 1997).
`The technique was also applied to the production of
`transgenic cattle (Cibelli et al., 1998) and has so far
`found its greatest use in production of transgenic and
`gene-edited animals for research or pharmaceutical use.
`Examples include development of cattle with mastitis
`resistance (Liu et al., 2014) and polled traits (Carlson
`et al., 2016). Nuclear transfer can be combined with
`genomic selection to further accelerate genetic gain by
`reducing the generation interval (Kasinathan et al.,
`2015).
`Production of offspring of predetermined sex has
`been long sought after by livestock producers. Sort-
`ing of X- and Y-chromosome-bearing sperm by flow
`cytometry has been possible since the 1980s (Garner
`et al., 1983), but the initial procedures killed sperm.
`It was not until 1989 that the first offspring (rabbits)
`
`from sexed semen were born (Johnson et al., 1989), and
`it was 1993 before the first calf from sex-sorted semen
`was born (Cran et al., 1993). In recent years, the use
`of sexed semen has grown internationally to the extent
`that bovine semen is currently being sex sorted in ap-
`proximately 15 countries.
`It is a credit to the many scientists, farmers, vet-
`erinarians, and breeding organizations that have
`translated the basic science to on-farm and laboratory
`technologies. In this review, we highlight the scientific
`advances that contributed to the development of re-
`productive technologies in dairy science. To put the
`advances of the past century in perspective, key events
`in reproductive biology preceding the early 1900s are
`summarized in Table 1. The review begins with the
`development of AI and continues chronologically with
`each advancement (see Appendix Table A1).
`
`DEVELOPMENTS IN AI
`
`Before the development of AI, cows were bred by nat-
`ural service and bulls were often shared among farms.
`It was in Denmark in 1936 that the first large-scale
`bovine AI organization was established before similar
`organizations were established in the United States and
`worldwide (Perry, 1945). Some producers and breeders
`initially opposed the use of AI because early procedures
`for collection, handling, and insemination were cumber-
`some (Polge, 2007; Wilmot, 2007). The benefits of AI
`over natural service, however, soon became obvious.
`Artificial insemination was critical to increasing the
`reproductive potential of sires with elite genetic merit.
`The reduction in disease transmission between animals
`and the opportunity to evaluate sperm production and
`characteristics provided additional importnant benefits
`(Ivanoff, 1922; Perry, 1945). Most important, AI en-
`abled precise genetic evaluation of bulls via hundreds
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`Journal of Dairy Science Vol. 100 No. 12, 2017
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`10316
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`MOORE AND HASLER
`
`of thousands of daughters. Its growth during the past
`80 yr to approximately 130 million inseminations in-
`ternationally per year (Vishwanath, 2003) was driven
`by some key advancements, primarily in the collection,
`extension, and preservation of sperm.
`
`AI Techniques
`
`Techniques for AI were developed initially for mares
`and were translated for cattle by teams in Russia and
`Denmark (Ivanoff, 1922; Perry, 1945). The earliest
`method for AI of livestock was to deposit semen in
`the vagina, as would be the case with natural service.
`The speculum method was developed next; this in-
`volved viewing the cervical os with a light source and
`shallowly inseminating the semen into the cervix. The
`concept of low sperm dose insemination was introduced
`with the development of the speculum method. Kozlova
`(1935) reported similar fertility rates after insemination
`of 0.2 mL of semen into the cervix compared with 4
`mL of semen into the vagina (cited in Salisbury and
`VanDemark, 1951). Even though the vaginal deposi-
`tion and speculum methods were both relatively easy
`to perform, neither were very efficient (Olds, 1978).
`Large numbers of sperm were required, low numbers
`of pregnancies were achieved, and the large effort to
`maintain good hygiene with either method was not
`conducive to widespread adoption of AI. The success
`of AI was transformed by the development of the recto-
`vaginal method by Danish veterinarians around 1937,
`which remains the method of AI still practiced today.
`With this method, a gloved hand in the rectum holds
`the cervix and guides the insemination gun through
`the cervix. Its superiority compared with the specu-
`lum method was exemplified by data collated by Olds
`(1978). Across 9 studies, average nonreturn rates were
`48.9% for the speculum method compared with 60.1%
`for the rectovaginal method. The other major benefit of
`the rectovaginal technique compared with the vaginal
`deposition method was that much fewer sperm were
`required. Adoption of the rectovaginal method was also
`greatly assisted by the development of the stainless
`steel insemination gun, shoulder-length plastic gloves,
`replacement of glass ampules with plastic semen straws
`(Cassou, IMV Technologies, L’Aigle, France), and dis-
`posable plastic sheaths. Together these developments
`made the procedure of AI easier to perform, improved
`animal hygiene and biosecurity, and culminated in
`greater fertility.
`
`Semen Collection
`
`Early efforts at semen collection were cumbersome,
`with large losses of sperm and a high risk of disease
`
`Journal of Dairy Science Vol. 100 No. 12, 2017
`
`transfer between animals. A bull mounted a cow and
`ejaculated in the vagina. Semen was removed from the
`vagina in a sponge or bag that was already in situ with
`a spoon or a syringe (Ivanoff, 1922; Perry, 1945). Al-
`ternatively, semen was ejaculated from bulls after tran-
`srectal massage of the ampullae and accessory glands;
`however, contamination of the sample with urine and
`low concentrations of sperm were common problems
`(Case, 1925; Miller and Evans, 1934). The artificial va-
`gina (AV) was a major advancement in the process of
`collecting clean semen samples. The AV was first devel-
`oped for the collection of semen from dogs by Amantea
`in 1914 and was modified for use with bulls by research-
`ers in Russia in the 1930s. Bulls were trained to mount
`teaser animals and to ejaculate into the AV. The AV
`has been a great resource for researchers also because
`it is now possible to collect clean samples that are not
`contaminated by vaginal secretions. Electroejaculators
`were invented in the 1930s (Gunn, 1936), and their
`use with bulls was first reported in 1954 (Dziuk et al.,
`1954). The technique is primarily used with bulls that
`are not suitable for semen collection by teaser mount-
`ing and AV.
`As demand for AI sires began to overtake supply dur-
`ing the 1940s, methods to maximize semen collection
`received increased focus. It became clear that exposure
`of bulls to several positive stimuli was important. In-
`ducing sexual excitement in bulls by allowing longer
`periods of time with the mount animal and some false
`mounting before ejaculation were demonstrated to
`increase the concentration and motility of sperm (Col-
`lins et al., 1951). It also became common practice for
`semen to be collected 2 to 3 times per day at 3- to 4-d
`intervals (Bratton and Foote, 1954; Hafs et al., 1959).
`This change had the major effect of increasing sperm
`production. For example, the number of motile sperm
`collected per week was 60% greater with no loss in fer-
`tility when semen collection was increased from once to
`twice per 8-d period (Bratton and Foote, 1954). At this
`rate, 30 billion sperm could be collected per week from
`Holstein bulls (enough to inseminate 3,000 cows with
`10 million sperm each).
`
`Semen Evaluation
`
`One of the indirect benefits for producers from us-
`ing AI versus natural service has been the knowledge
`that only high-quality semen is used to inseminate their
`livestock. Initial assessments of each ejaculate involved
`measurement of the volume and the sperm concen-
`tration to estimate the number of sperm collected
`(Salisbury et al., 1943) and a visual assessment of the
`proportion of sperm displaying progressive motility in
`a diluted sample at a magnification of 400× (Elliott,
`
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`100-YEAR REVIEW: REPRODUCTIVE TECHNOLOGIES IN DAIRY SCIENCE
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`10317
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`1978). These assessments provide important informa-
`tion on the suitability of the ejaculate for processing,
`the reproductive performance of the sire, and the num-
`ber of doses that can be produced and are required for
`accurate dilution and packaging of semen. Criteria for
`assessing sperm morphology were developed by Wil-
`liams (1920), who also reported associations between
`abnormal sperm and fertility. Hence, sperm morphol-
`ogy is routinely assessed by commercial semen process-
`ing labs; ejaculates typically are required to have ≥65%
`morphologically normal sperm to be further processed.
`Cell sorting became feasible in the 1960s with the
`development of flow cytometry (Fulwyler, 1965; Dit-
`trich and Goehde, 1968). The ability to sort, count,
`and assess sperm cells gave semen processing centers
`a major impetus to automate aspects of their quality
`control procedures and was central to the establish-
`ment of sex-sorted sperm (to be discussed later). Sperm
`staining and flow cytometry have been combined to
`assess various aspects of sperm quality (Garner et al.,
`1986). Today this methodology is used to assess sperm
`membrane integrity, acrosome status, sperm energetics,
`and sperm DNA integrity.
`The accuracy and reliability of sperm motility as-
`sessments were greatly advanced by the development
`of computer-assisted semen analysis (Liu and Warme,
`1977). These systems were developed to provide objec-
`tive measurement of sperm velocity and to determine
`the proportion of the sperm population with total and
`progressive motility. More recently, some units are ca-
`pable of assessing sperm morphology. Even though it is
`possible to assess several sperm quality characteristics,
`fertility remains the definitive test.
`
`Semen Processing and Preservation
`
`The ability to preserve sperm viability until insemi-
`nation was a major advance in the establishment of
`organized AI because of the difficult logistics created
`by the fragmented nature of farms in rural areas. The
`basic principle of preserving sperm viability is to slow
`down or inhibit its metabolic activity by cooling to low
`temperatures. Spallanzani demonstrated in an 1803 re-
`port that sperm could survive chilling. The next major
`demonstrations of the potential to preserve sperm via-
`bility were performed by Walton (1926) with pregnancy
`in rabbits in Edinburgh, Scotland, after insemination
`with semen collected 48 h previously in Cambridge,
`England, and by Edwards et al. (1938) with pregnancy
`in cows in the Netherlands after insemination with se-
`men collected 57 h previously in England. The afore-
`mentioned studies demonstrated that storage of raw,
`undiluted semen at 5°C with ice was sufficient to reduce
`
`the metabolism of the sperm and extend their survival,
`typically for 2 to 4 d.
`It became obvious to researchers early on that a
`single ejaculate contained sufficient sperm for thou-
`sands of inseminations, yet only 10 to 20 inseminations
`were possible with undiluted semen (Ivanoff, 1922).
`Initially semen was diluted with a medium containing
`glucose, phosphate buffer, and sodium sulfate. A major
`breakthrough reported in 1939 was the success of egg
`yolk phosphate buffer as a semen extender (Phillips,
`1939). It was later demonstrated that boiled milk could
`replace egg yolk as a suitable semen extension medium
`with comparable fertility (Thacker and Almquist,
`1953). Semen extenders were distinct from previous
`media because of their ability to extend the motility
`and fertility of sperm (Foote, 1978). Phillips (1939)
`demonstrated that high levels of sperm motility were
`maintained for more than 150 h after the addition of an
`equal-volume mixture of egg yolk and phosphate buffer
`solution to semen stored at 5 to 10°C; pregnancy was
`achieved even from extended semen stored up to 180
`h. Subsequent replacement of phosphate buffer with ci-
`trate buffer achieved the same preservation ability but
`with the added benefit of dispersing fat globules from
`the egg yolk (Salisbury et al., 1941). This characteristic
`greatly improved the clarity of the extended semen and
`the ability to examine sperm microscopically (Salisbury
`et al., 1941). Fertility was greatest with 2-d-old semen
`compared with 1-d-old semen because of a decrease
`in the proportional number of abnormal sperm from
`the first to the second day (Salisbury and Flerchinger,
`1967). In practice, semen was discarded 2 d after col-
`lection because fertility declined when older semen was
`used (Salisbury and Flerchinger, 1967).
`During the 1940s, awareness of microorganisms in
`semen ejaculates and their implications for fertility
`increased (Gunsalus et al., 1941; Prince et al., 1949)
`as a consequence of the greater incidence of bacte-
`rial growth in semen extended with nutrient-rich egg
`yolk. The negative effect of some microorganisms on
`fertility was clearly demonstrated when the addition of
`antibiotics (penicillin and streptomycin) to egg yolk-
`extended semen reduced bacterial growth (Almquist et
`al., 1949) and increased 60- to 90-d nonreturn rates by
`11% (Foote and Bratton, 1950). Extension of semen
`with antibiotics has been standard practice ever since.
`This in conjunction with improved sanitary procedures
`and disease screening made a substantial contribution
`to the eradication of venereal diseases in particular.
`It seems appropriate here to reiterate that the afore-
`mentioned studies were performed at a time when it
`was still not possible to maintain the viability of bovine
`sperm after the freeze–thaw process. In the absence of a
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`10318
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`MOORE AND HASLER
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`cryoprotectant, the two mechanisms through which the
`freeze–thaw process damages sperm are the formation
`of internal ice crystals that affect sperm structure and
`an increase in osmotic pressure, and the interaction be-
`tween the two (Pickett and Berndtson, 1978). Working
`with unfrozen (fresh) semen with a useful life span lim-
`ited to 2 d required some innovative thinking to maxi-
`mize the number of inseminations before the semen had
`to be discarded. In some regions, semen processing labs
`dispatched packaged semen daily to distant AI techni-
`cians by airplane (Underwood, 2012). Such programs
`became irrelevant with perhaps the most important
`breakthrough in the development of the AI industry.
`In 1949, the cryoprotective properties of glycerol on
`sperm after the freeze–thaw process were reported
`(Polge et al., 1949). Sperm of fowl frozen with glycerol
`at −79°C (the temperature of solid carbon dioxide)
`resumed normal motility after thawing (Polge et al.,
`1949). This was the first report that living cells could
`survive the freeze–thaw process. The same success was
`subsequently demonstrated with bovine sperm (Polge
`and Rowson, 1952). The suitability of glycerol is largely
`attributed to its ability to buffer electrolytes in sperm
`(Lovelock, 1953). The arrival of the first calves from the
`AI of frozen–thawed semen in Reading and Cambridge
`in England (Polge and Rowson, 1952) heralded a new
`era that revolutionized the AI industry. With the abil-
`ity to successfully cryopreserve sperm, extenders suit-
`able for frozen semen were developed. Studies reported
`in 1965 demonstrated that the motility of sperm was
`18 percentage units greater in egg yolk-extended semen
`buffered with Tris compared with no Tris (Davis et al.,
`1963a,b). Egg yolk extenders using Tris and glycerol
`achieved extensive use in semen processing labs because
`of good results whether the semen was stored at 5°C,
`stored at ambient temperature, or frozen (Vishwanath
`and Shannon, 2000).
`The main risk to sperm viability during the freeze–
`thaw process is the period when sperm are exposed to
`temperatures ranging from −15°C to −60°C (Mazur,
`1984). Hence, methods for processing semen during
`freezing and thawing were developed that maximized
`postthaw sperm motility and fertility. First, the ejacu-
`late is partially extended at 37°C to buffer the sperm
`and to provide antibiotic and thermal protection;
`then it is cooled to 5°C. Slow cooling over a period
`of a few hours was demonstrated to improve postthaw
`sperm motility and fertility (Graham et al., 1957; Mar-
`tin, 1965), probably because it permits the sperm to
`adjust to low temperatures before cryopreservation.
`The final extension is then performed to achieve the
`desired sperm concentration per straw, typically 10 to
`20 million sperm per frozen dose. In the original work
`of Polge and Rowson (1952), extended semen with
`
`Journal of Dairy Science Vol. 100 No. 12, 2017
`
`glycerol was frozen to −79°C with solid carbon dioxide
`and alcohol. Unfortunately, solid carbon dioxide was
`cumbersome, and sperm were damaged by the forma-
`tion of crystals. Storage of semen in liquid nitrogen
`was also difficult until glass vacuum containers were
`replaced by insulated liquid nitrogen tanks developed
`in 1954 by American Breeders Service (Deforest, WI)
`and Linde Air Products (Murray Hill, NJ). The freez-
`ing process was later modified so that semen was fro-
`zen with liquid nitrogen vapor to −196°C (Forgason
`et al., 1961) or with programmable freezers (Almquist
`and Wiggin, 1973). Liquid nitrogen was subsequently
`adopted as the preferred coolant for freezing semen
`because postthaw sperm viability was greater and it
`facilitated longer term, lower maintenance storage
`of frozen semen compared with solid carbon dioxide
`(Fowler et al., 1961). Similar to when freezing semen,
`the primary risk to sperm viability after thawing is
`when sperm are exposed to the temperature window
`of −15°C to −60°C (Mazur, 1984). Sperm survival is
`dependent on the effects of freezing and thawing, and
`sperm cooled rapidly should be thawed rapidly (Mazur,
`1984). A thawing temperature of 35°C applied for 45 s
`is broadly practiced and increases sperm motility but
`not nonreturn rates when compared with semen thawed
`at other temperatures (Pickett and Berndtson, 1978;
`Vishwanath and Shannon, 2000).
`Frozen semen brought about many advantages, in-
`cluding indefinite storage of sperm, ease of transport,
`a greater choice of sires, and, most influentially, the
`international trade of semen of sires with elite genetic
`merit. However, the freeze–thaw process does result in
`damage to about 50% of the sperm, and consequently
`fertility performance of frozen–thawed semen has con-
`sistently been inferior to that of liquid semen (Shannon
`and Vishwanath, 1995). Despite the limited viability of
`liquid semen, fresh-semen AI programs are available in
`New Zealand and Ireland, for example, and have the
`advantage of maximizing the number of inseminations
`per ejaculate because fewer sperm can be packaged
`without a reduction in fertility. Typical doses for fresh
`semen usage are 1 to 5 million sperm per straw com-
`pared with 10 to 20 million sperm per straw for frozen
`semen (Shannon and Vishwanath, 1995). The major
`developments on extenders for liquid semen began with
`development of the Illini variable temperature diluent
`by VanDemark and Sharma (1957). The distinguishing
`feature was that extended semen was saturated with
`CO2, which had the effect of immobilizing and preserv-
`ing sperm for about 3 d, but the results were variable.
`The Cornell University extender developed by Foote
`et al. (1960) was based on the same principle as the
`Illini variable temperature diluent but was described
`as “self-carbonating”—that is, CO2 was released into
`
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`100-YEAR REVIEW: REPRODUCTIVE TECHNOLOGIES IN DAIRY SCIENCE
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`10319
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`the extended semen by the action of citric acid on
`sodium bicarbonate (Vishwanath and Shannon, 2000).
`Nonreturn rates of 76% by 60 to 90 d were reported
`with liquid semen extended with the Cornell University
`extender and stored for up to 4 d at 5°C (Foote et al.,
`1960). Continuing advancements in technologies meant
`that it became possible to store liquid semen at ambi-
`ent temperatures (18–24°C) for up to 4 d without loss
`of fertility following the development of the Caprogen
`extender in 1965 (Shannon, 1965; Shannon and Curson,
`1984). Caprogen works on the basis that saturation of
`extended semen with N2 gas reduces O2 concentration
`in the semen (Shannon, 1965). These authors also
`demonstrated that the viability of sperm stored in the
`liquid state was greater when stored at the ambient
`New Zealand temperature compared with storage at
`5°C (Shannon and Curson, 1984).
`
`Sexed Semen
`
`In a review of the history of sexed semen use in
`cattle, Garner and Seidel (2008) provided the following
`statement: “The most sought after reproductive bio-
`technology of all time, selection of sex at conception,
`has a long history of great optimism, along with many
`disappointments.” The recorded historical attention to
`determination of sex goes back at least to the early
`Greeks, when Democritus (470–402 BC) suggested that
`the right testis produced males whereas the left testis
`produced females. Many unsuccessful studies and a
`large number of inoperative registered patents for the
`sex separation of sperm cells, based on a variety of
`different principles, have been produced in the past 50
`yr. Currently, only the separation of stained X- and
`Y-chromosome-bearing sperm by flow cytometry cell
`sorting has proven successful.
`Initial breakthroughs involving laminar flow cytom-
`etry at the Lawrence Livermore National Laboratory in
`Livermore, California, led to success determining the
`DNA content differences between X- and Y-sperm from
`cattle, sheep, pigs, and rabbits (Garner et al., 1983).
`Two critical advances facilitated this process: (1) the
`use of the DNA-specific live cell stain Hoechst 33342
`(membrane-permeable fluorophore) that allowed sort-
`ing of living sperm and (2) modifications to cell flow
`sorting analysis (dual orthogonal fluorescence detec-
`tion) that created sufficient signal-to-noise ratio to al-
`low sperm differentiation based on the small (3.8%) dif-
`ference in chromosomal DNA content between X- and
`Y-chromosome-bearing bovine sperm. Ultimately, a live
`stain (food coloring) was included in the process, which
`allowed sperm to be sorted simultaneously for both sex
`
`and viability. Critical to the development of sexed se-
`men technology was the announcement by Johnson et
`al. (1989) at the USDA laboratories in Beltsville, Mary-
`land, of the live birth of rabbits, with 94% female pups,
`from sexed sperm. The work led to a US patent cover-
`ing the technical details of flow sorting sperm for sex
`(Johnson, 1992). The original sexing patent by Johnson
`has long expired, but more than 200 patents related to
`all aspects of the production, freezing, and use of sexed
`bovine sperm have been registered by XY Inc. (now XY
`LLC) and Sexing Technologies (Navasota, TX).
`Live calves resulting from IVF of oocytes with sexed
`bull semen were first reported by Cran et al. (1993).
`Cogent, located in Chester, United Kingdom, was the
`first company to commercialize sexed semen for AI.
`Currently, Sexing Technologies/XY LLC has licensed
`approximately 40 semen processing facilities to produce
`sexed semen in 15 countries. It has been estimated that
`semen from approximately 1,800 sires was sorted glob-
`ally in 2013; the majority of the sires were Holstein-
`Friesian and Jersey. In the United States, between 4.5
`and 5 million straws of sexed semen were processed in
`2016, of which more than 90% were from dairy sires.
`There are 2 limiting factors involving the current tech-
`nology used to sex semen. First, the processing of sexed
`semen involves flow cytometers that are both expensive
`to purchase and expensive to operate. Depending on
`factors such as individual bull variation and the de-
`sired accuracy of sex selectivity, approximately 7 to
`12 straws/h can be processed. Consequently, multiple
`flow cytometers are used simultaneously to speed up
`the processing of each ejaculate. This is despite the
`fact that in most cases only 2 million sexed sperm are
`packaged per straw compared with the 10 to 20 million
`unsexed sperm in conventional straws. The accuracy of
`selection of either X- or Y-chromosome-bearing sperm
`is 90%, and the accuracy is closely related to the speed
`of sorting. Second, the process of sexing sperm results
`in varying levels of damage to the cells. This is re-
`flected in a decrease in conception rates and embryo
`production following AI in both dairy heifers and cows
`compared with unsexed frozen semen (DeJarnette et
`al., 2009; Kaimio et al., 2013; Mikkola and Taponen,
`2017). In terms of offspring performance, there is evi-
`dence for (DeJarnette et al., 2009; Healy et al., 2013;
`Siqueira et al., 2017) and against (Tubman et al., 2004;
`DeJarnette et al., 2009) negative health consequences
`in calves generated from sex-sorted semen, and 1 study
`to date (Siqueira et al., 2017) has reported less milk
`production from cows generated from in vitro-produced
`sexed embryos compared with cows generated from
`timed AI, although potential mechanisms have not
`been determined.
`
`Journal of Dairy Science Vol. 100 No. 12, 2017
`
`Exhibit 1010
`Select Sires, et al. v. ABS Global
`
`

`

`10320
`MOORE AND HASLER
`DEVELOPMENTS IN EMBRYO TECHNOLOGIES
`
`Superovulation and ET are seen