ELSEVIER
`
`FLOW CYTOMETRIC
`HIGH-SPEED
`SEX PRESELECTION:
`X AND Y SPERM FOR MAXIMUM
`EFFICIENCY
`
`SORTING OF
`
`L.A. Johnson and G.R. Welch
`
`Research Service
`and Gamete Physiology Laboratory, Agricultural
`Germplasm
`U.S. Department of Agriculture,
`Beltsville, MD 20705 USA
`
`Received
`
`September 23, 1999
`for publication:
`Accepted: October 13, 1999
`
`ABSTRACT
`
`to
`
`sex
`
`that is based on flow-cytometric measurement of sperm DNA content
`Sex preselection
`from Y-chromosome-bearing
`sperm has proven reproducible
`at various
`enable sorting of X-
`locations and with many species at greater than 90% purity. Offspring of the predetermined
`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 cytometerkell
`sorter modified specifically
`for sperm. Sexed sperm are then used
`with differing
`semen delivery
`routes such as i&a-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
`in
`performance
`level, the HiSON has produced X-bearing-sperm
`populations
`at 85 to 90% purity
`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.
`by Elswier
`Science
`inc.
`PublIshed
`Key words: sexed sperm, offspring,
`
`flow cytometry, Hoechst 33342, X- and Y-sperm
`
`thanks to Wim Rens for his technical expertise
`Special
`Acknowledgments
`the original orienting
`nozzle.
`
`in the development
`
`of
`
`Theriogenology
`Publiihed
`
`52:1323-1341,
`by Elsmier
`Science
`
`IQQQ
`Inc.
`
`XkW$-sw
`OOQ3-691
`PII SOOQ3-691
`
`front matter
`X(QQ)OO22W
`
`

`

`1324
`
`Theriogenology
`
`INTRODUCTION
`
`Sex preselection has been a long sought goal of the livestock producer. The efficiency of
`livestock improvement can be greatly enhanced by the utilization of recent advancements in
`agricultural biotechnology. Interest in this area stems from biblical times and, with recent
`advances in the technology, it has become a reality. Predetermining the sex of offspring can be
`done with precision and repeatability using flow cytometric sperm sorting, which is based on
`measuring the relative difference in DNA content in mammalian sperm and sorting the sperm
`using a modified flow cytometer/cell sorter (17). The application of the sexing technology to
`mammals has been proven in several species by the production of offspring (swine, cattle, sheep,
`rabbit, horse, human) from sexed sperm. The method utilizes the sex-specific difference in
`sperm DNA for the separation of X- and Y-chromosome-bearing spermatozoa using flow-
`cytometric sperm sorting (15). Skewed sex ratios have been demonstrated in numerous
`laboratory and field experiments. Among them are rabbits (15) swine (16, 39), cattle (4, 5,44,
`45), and sheep (6). Offspring of the desired sex have also been born after using the sorting
`technology in sheep (14,3). The method also has been adapted to human sperm (21) and in
`clinical trials has proven success&l at producing children of the desired sex (8,9). All of these
`results have been produced using the original standard-speed flow sorting of sperm, which
`produces about 350,000 each of viable X- and of Y-bearing sperm per h (15). On the average,
`the skewing of the sex ratio in the offspring produced has been about 90% with a range of 75 to
`100% of the desired sex. Various reviews of the sexing technology document the progress over
`the past 10 years in particular (14, 19,25, 26).
`
`Two aspects of the technology have been substantially altered and have resulted in
`significant enhancements to the sperm-sexing technology noted above. Sperm sorting for x/Y
`separation is dependent on the sperm’s orientation (17) to the laser beam so as to reduce
`variability sufficiently to distinguish the small difference in DNA content of the sperm. Due to
`the compactness of the sperm chromatin, differential fluorescence is exhibited from the edge of
`the cell compared to the more transparent flat side of the sperm head. This leads to variable
`DNA fluorescence that masks the small (3 to 4%) X/Y DNA differences of many mammals. A
`significant enhancement in orientation is gained by the use of a new orienting nozzle system (41,
`42) which we have fitted to a high-speed sperm sorter (27). Improvement in orientation has
`increased from 25% oriented to 70% oriented. This innovation replaces the beveled needle of the
`original system (17).
`
`The objective of this paper is to chronicle the development of a successful method to
`preselect the sex of offspring through the use of sperm sorting and to illustrate the improvements
`that have been made in the original technology. Throughout this paper, in discussin high-speed
`sorting technology equipped with an orienting nozzle, we are referring to a MoFlo@ high-speed
`cell sorter modified for sperm sorting (17) and equipped with an orienting nozzle (4 1).
`Therefore, throughout the paper, the high-speed sperm sorter with orienting nozzle will be
`referred to as HiSON.
`
`a Cytomation, Inc., Fort Collins, CO, USA
`
`

`

`Theriogenology
`
`DNA AS A MARKER
`
`Basis For Using DNA Content of Sperm as a Sex-specific Marker Measured by Flow Cytometry
`
`In 19 10, Guyer (13) reported the presence of sex chromosomes. One of the first significant
`attempts to preselect sex was conducted in 1925 by Lush (31) without reference to DNA. That
`study showed no skewing of the sex ratio in rabbits and pigs based on centrifugation. Several
`groups carried out research on sex preselection and much of the work
`in the area was reviewed
`a symposium held at Penn State University
`in 1970 (29).
`
`in
`
`Once flow cytometry came on the scene in the late 1960’s (28), interest was stimulated in
`measuring DNA
`in individual cells for many purposes, especially for cancer diagnosis. Another
`aspect of that early work was that DNA could be a sensitive indicator of mutagenic events
`associated with various weapons systems being developed or used at that time. Most of the early
`work on measuring DNA of sperm was done in the United States at the National Laboratories at
`Los Alamos, NM and at Livermore, CA since that is where the flow-cytometer
`instrumentation
`was being pioneered. Gledhill et al. (12) reported the use of flow cytometry for measuring DNA
`in sperm to determine changes that might occur with genetic damage. Asymmetric shape was
`shown to cause differential fluorescence. Coupled with random orientation, the differential
`fluorescence masked the known difference in DNA content between X- and Y-bearing sperm SO
`that the difference could not be measured. It was found that flow-cytometer
`fluid streams could
`be changed from cylindrical to flat (10) and when applied to sperm (7) the asymmetrical-shaped
`sperm could be analyzed with a flow- cytometer/analyzer for DNA content.
`
`In 1979, Moruzzi (35) drew attention to the use of DNA as a potential marker for sex
`preselection with the publication of data that showed a variety differences between X and Y
`sperm for numerous species in DNA content measured by differences in chromosome length. An
`average 6.6% difference in DNA among several species was reported. A high-resolution,
`orthogonally configured, experimental laser-based flow cytometer that could orient sperm was
`built (37) and used to show a difference in DNA content in fixed mouse sperm nuclei of 3.2%.
`The ability to measure a small difference in DNA was confirmed in nuclei of livestock sperm
`ranging from 3.6 to 4.0% using a simpler analytical but non-sorting flow cytometer (11) based on
`mercury-lamp excitation. Another symposium (2) served to bring the sex-preselection research
`up to date and demonstrated not only a broad interest in the area but also updated the potential
`technologies available to separate X and Y sperm. The knowledge gained over a period of 71
`years ( 19 lo- 198 1) contributed to the developmental successes of the most recent 18 years in
`which DNA was successfully shown to be the only effective marker for separating viable X and
`Y sperm (15).
`
`

`

`1326
`
`Thet-iogenology
`
`SORTING OF SPERM NUCLEI
`
`Development of Sperm Sorting Technology With Standard-Speed Cell Sorters 1983 to 1988
`
`Commercial cell-sorting instrumentation arrived in the 1970’s and its performance improved
`dramatically in 198Obwith the development of more advanced data-acquisition equipment. We
`acquired a cell sorter in 1982 and modified it to sort sperm nuclei (17). This system formed the
`basis for the current technology as various protocols were developed that led to sorting sperm
`nuclei of several mammals (18, 26; Figure 1). The sperm used in these studies had been
`sonicated to remove the tails since tails negatively affected the orientation of the sperm to the
`laser beam. The utilization of Hoechst 33342’ a bisbenzimide fluorescent vital dye sensitive to
`ultraviolet light was found to give improved separation over previous dyes (20). The dye is non-
`intercalating and binds to the minor groove of the DNA helix. In 1988, Johnson and Clarke (23)
`showed that sperm nuclei sorted using Hoechst 33342 bound to the DNA were capable of
`fertilization, indicating that the replicating DNA was functional in the presence of the bound dye
`and withstood flow-cytometric sorting conditions. Sort-reanalysis was developed to provide
`continuous validation of the proportion of X- and Y-bearing sperm in a particular semen sample
`(26) or sorted-sperm population in the laboratory (48).
`
`Stallion I! x-Y=3.7%
`
`Y
`
`x
`
`Y
`Chinchilla
`x-Y=7.5%
`
`DNA Content
`
`IllI
`
`Figure 1.
`
`Flow cytometric histograms produced from ejaculated semen from 8 common species
`illustrating the inherent difference (X-Y) in relative DNA content between X- and Y-
`chromosome-bearing sperm. The difference in DNA from the 2.8% for human sperm
`to 7.5% for Chinchilla IanPier illustrates the difference in DNA associated with
`chromosome size in domesticated animals and man.
`
`b Epics V, Coulter Corporation, Miami, FL, USA
`
`’ Calbiochem-Behring Corporation, La Jolla, CA, USA
`
`

`

`Theriogenology
`
`1327
`
`VIABLE SPERM SORTING
`
`Development of Viable Sperm Sorting with Standard Speed Sperm Sorter 1989 to 1991
`
`Hoechst 33342 was found to be highly permeable to the living sperm membrane and that through
`incubation of the dye/sperm suspension at (32 to 39°C) staining uniformity was enhanced. This
`fmding led to the production of offspring from populations of viable X-bearing sperm and populations
`of Y-bearing sperm (15). A key factor in the development was the use of a concentrated buffer
`solution in which to catch the sperm being sorted. As the fluid builds up in the collection tube, sperm
`swim to the bottom and become accustomed to the more concentrated egg-yolk environment
`maintaining their viability. A total of -350,000 sperm could be sorted by this original method in an
`hour. Rabbits were born with litters showing sex ratios of 94% females and 8 1% males (15), and
`litters of pigs were born with 74% females and 68% males (16) all by intra-uterine or intratubal
`insemination, respectively (Figure 4). Rabbits and pigs were all morphologically normal and were
`reproductively capable in adulthood showing no negative effects of the sorting process and this was
`repeated in succeeding generations.
`
`Expansion of Viable Sperm Sorting to Other Livestock and Humans 1992 to 1997
`
`Viable sperm sorting (standard speed) was expanded to collaborations with other groups. A
`sorting facility established by Animal Biotechnology Cambridge Ltd., UK was used to sort bull sperm
`for use in conjunction with in vitro fertilization (IVF). Six calves of the correct sex were born from
`embryos produced from sexed sperm (4). A field demonstration of the capability of the technology
`(5) was the birth in 1995 of 41 calves, 90% being male, after production and transfer of embryos
`produced from sorted sperm (Figure 4). Utilization of sexed sperm in the most economical way
`suggests that freezing sorted sperm would enhance the utility of sexed sperm to the industry. We
`applied standard freezing technology to bull sperm and found with slight adjustments for
`concentrating the sperm that we could achieve such a goal. Sorted bull sperm was frozen and thawed
`and average sperm motility was 30%, and acrosomal integrity, 40%. One and 2 million sperm per
`0.5-ml straws were used (19). The first lamb produced by intra-cytoplasmic sperm injection of sexed
`sperm was reported in 1996(3).
`
`We applied the method to human beings by sorting human sperm (21). Fluorescence in situ
`hybridization (FISH) was needed to verify the purities of the sorted X- and Y-bearing human sperm
`due to the small X/Y difference in DNA (2.8%) and inconsistency of sort reanalysis where small DNA
`differences exist. Clinical trials were initiated and the first child from sorted human X-bearing sperm
`was born in 1995 to a family that carried the X-linked disease, hydrocephalus. Subsequently, the
`clinical appl$ation of the technology in man has resulted in more than 50 births (8,9) under the name
`MicroSort@ for human sperm.
`
`Low-dose insemination for cattle using a deep-uterine-insemination technique with sexed sperm
`
`was demonstrated through collaboration with Seidel and coworkers in 1997 (44). The technique was
`used with sexed sperm and provided a useful semen-delivery avenue needed to bring sexed semen to
`
`d Genetics and IVF Institute, Fairfax, VA, USA
`
`

`

`1328
`
`Theriogenology
`
`producers. Semen was brought to Beltsville from Atlantic Breeders Cooperative, Lancaster, sorted
`into X and Y populations and shipFed by air to Colorado where it was inseminated using the deep-
`uterine technique. A total of 2x 10 sperm per dose was sufficient to get pregnancies and produce 17
`calves (Figure 4). The average percentage of females from sorted X sperm was 82%.
`
`With improved techniques for pig IVF, sexed sperm were used to produce 2 litters of pigs
`totaling 10 female piglets (Figure 4). This was the first successful use of sexed sperm to produce
`sexed offspring from sexed embryos in the pig (39).
`
`Conversion of Standard Speed Sorting to High-Speed Sorting With Orienting Nozzle 1996 to 1999
`
`Achieving gains in sperm throughput as well as efficiency of sorting was necessary for greater
`applicability of flow-sorted sexed sperm for livestock production using AI. Improving the efficiency
`of sperm orientation was a key factor since it would increase the number of sperm available for
`sorting. This effort led to the development of the orienting nozzle described below (41; Figure 2).
`The second major factor was the commercial development of the high-speed sorter that we were able
`to acquire and modify. In and of itself, the high-speed sorter with the standard beveled needle (Figure
`2) would sort up to 2 million sperm per hour. However, when we adapted the orienting nozzle to the
`high-speed sorter (HiSON) the capability increased to 6 million sperm of each sex per hour.
`
`CONVERSION TO HIGH SPEED SORTING
`
`Preparation of Sperm for High Speed Sorting (HiSON) Based on DNA Content
`
`Ejaculated semen was collected into a warmed container and transported, maintaining
`temperature, to the laboratory. Sperm concentration and percentage of motile sperm were determined
`by standard methods. Proportion of stain per number of sperm is important to maintaining staining
`uniformity. The semen extender used for maintaining sperm viability and the stain was Beltsville TS
`(16) for the boar and HEPES-BSA (42) for bull sperm. Most semen extenders appropriate to the
`species will work for this purpose; however, egg yolk and milk and high levels of BSA as constituents
`should be avoided since they inhibit uniformity of staining. The staining stock solutions were:
`Hoechst 33342, (5 mg/mL in water), and food coloring, FD&C #40; 25 mg/mL (22,27). The food
`coloring penetrates the membrane of the dead sperm and quenches the intensity of fluorescence of the
`dead sperm, thus eliminating dead sperm from the viable sperm population. Propidium iodide can
`serve the same purpose but has the drawback of being an intercalating dye (24).
`
`Ejaculated sperm (150 x 1 06) were aliquoted into a fmal volume 1 .O ml of extender that was
`equilibrated to semen temperature (- 35 “C). The optir@ amount of stain for most species was 40 pg
`(8 FL of 5 mg/rnL stock) Hoechst 33342 per 150 x 10 sperm. Sperm number prepared per mL or per
`sample tube can be varied as long as the proportion of Hoechst 33342 per sperm is maintained. The
`sperm-Hoechst suspension was incubated 60 min at 3 5 “C (15) and then moved from the heating block
`to a styrofoam container maintained at room temperature. To stain dead sperm, 1 pl of FD&C 40 was
`mixed gently and allowed to sit for 5 min. Samples were filtered through a 30-u nylon mesh prior to
`sorting. Sperm are then sorted. In preparing for a long sort (3 h or more), it has been found
`advantageous to stain new samples of sperm for the second 3 h period.
`
`

`

`Theriogenology
`
`1329
`
`a) Beveled
`needle
`
`5.5mmw
`
`Orientation
`
`nozzle’s interior
`
`for sperm (17). Shown areOthe
`Figure 2. Schematic diagram of basic cell sorter (47) modified
`additional photomultiplier
`(PMT) and optical detector in the forward position
`(0 ). The
`impact of using the Beltsville elliptical nozzle
`instead of the beveled needle for orienting
`sperm to the laser beam in a typical modified cell sorter configured
`for sorting sperm is
`shown in the two histograms. Because the hydrodynamic
`forces that orient the stream of
`fluid are of shorter duration and fkrther away (5.5 mm) from the laser beam when using a
`beveled needle, sperm lose orientation again before they reach the laser beam. This is
`compared
`to the hydrodynamic
`forces using an orienting nozzle which
`is subjected to a
`longer duration of orientation
`forces and is a shorter distance (1 mm) from laser beam of
`sperm passing through
`the orienting nozzle. This results in 2 to 3 times the number of
`oriented sperm (70% versus 25%) that are available to be sorted because they fall within
`the gate of the 90”PMT.
`Increased sorting speed and effkiency
`in utilizing
`sperm applied
`to the high-speed sperm sorter is the result of using the orienting nozzle (b) versus the
`beveled needle (a). Configuration
`(b) also requires a sample injection needle, but it can be
`cylindrical and unmodified. Another modification used with high-speed sorting is the
`addition of an elliptical beam shaping optic (49) (not illustrated). This beam shaping optic
`is fitted in the laser path just before the intersection of the laser beam and the sample
`stream. (Adapted
`from 4 1,27).
`
`

`

`1330
`
`Theriogenology
`
`Set Up of HiSON for Sorting X- and Y-Chromosome-Bearing Sperm
`
`The high speed sorter equipped with an orienting nozzle (27; Figure 2) is optimally adjusted
`for sperm sorting using beadse and nuclei of sperm from the species to be sorted. System
`parameters can be adjusted according to the experiment orft the investigator’s determination.
`Our common parameters are: sheath pressure (3.62 kg/cm ); nozzle with 60-p orifice; sort
`deflection=1 drop; 100 mW laser power (35 1,364 nm); average flow rates from 10,000 to
`15,00O/sec. To achieve higher throughpy we have used flow rates of 30,000 per sec. The lasers
`are argon-ion lasers that are water cooled operated with ultraviolet optics. A gate is set on
`oriented sperm (Figure 2) using the 90” fluorescence histogram. The 0” fluorescence histogram
`displays the DNA content of oriented sperm only, and sort windows are established according to
`the speed and enrichment of the specific population desired (Figure 3).
`
`& Sperm eliminated from sort
`
`q Unresolved X and Y sperm
`
`t
`
`180
`
`200
`
`I /
`
`\
`
`24 -0
`2 20
`DNA Content
`
`260
`
`280
`
`Figure 3.
`
`Illustration of a typical histogram of flow-sorting set-up for sperm. Sort windows
`selecting for X- and Y-chromosome-bearing sperm are based upon the relative DNA
`content. The outer boundaries (arrows) remain constant for a given species. The
`interior boundaries when set as shown, to eliminate the area (A) in which X and Y
`sperm are not resolved (B), can result in X and Y purities near 95%. Sort speeds can
`be increased at the expense of reducing the purity of X and Y sperm by reducing the
`number of channels within (A), which always encompasses area (B).
`
`e Fluoresbrite BB 4.5~ beads, Polysciences Inc., Warrington, PA, USA
`
`f Models 90-5 and 307; Cf>‘erent Inc., Palo Alto, CA, USA
`
`

`

`Theriogenology
`
`1331
`
`Collection of Sorted X- and Y-Chromosome Bearing Sperm
`
`Maintaining sperm viability in high-dilution conditions of cell sorting is dependent on
`providing a nutrient environment within the collection tube. The procedures described are
`adapted from the original 1989 (15) method to adjust for high-speed sorting conditions. Tubes
`used for collecting sorted sperm can be of any size to fit the type of sort being done. A 0.6~mL or
`1 S-mL microfuge or 15mL conical tube have worked effectively, the former for short sorts, the
`latter for sorts of longer duration. To reduce sperm loss through adhesion, tubes should be
`precoated by filling with a 1% BSA solution for 1 h prior to use to neutralize any charge on the
`tube surface. A volume of 0.05 to 0.5 mL of TEST-yolk (2 or 20%) should be added to the
`coated tubes, depending on size of tube. Generally a minimum of 50 ul of TEST-yolk added to
`the smaller tubes and 0.5 mL in the larger tubes is satisfactory to provide a concentrated haven
`for the sorted sperm. Two percent TEST-yolk is effective to collect sorted sperm for IVF (39,
`30), while up to 20% works for sorted sperm to be used for insemination. Sorted sperm were
`centrifuged from 350 x g to 700 x g for 10 to 15 min, depending on size of tube. The supernatant
`was aspirated and the pellet rediluted according to the particular usage. TEST-yolk extender has
`been very effective for most species. However, other extenders using egg yolk to give a
`concentrated environment can also be used. Validation of sorted sperm populations were
`conducted using “sort reanalysis” as described (48).
`
`Evaluation of X and Y sorted suerm membranes. Sperm quality should be monitored once
`
`sperm have been sorted using standard assessment techniques. Sperm motility and live/dead
`sperm evaluations are helpful to test for the ability of the sperm to fertilize ova. Membrane
`changes do take place during the process. Chlortetracycline has been used to delineate the
`membrane effects of sorting in vitro (32). The primary conclusion to be drawn from numerous
`studies is that a higher proportion of membranes are acrosome reacted or pre-capacitated in
`sperm that have been sorted (33). This is one reason sorted sperm do not need to be capacitated
`before they are used for IVF. Addition of seminal plasma to the staining buffer as well as the
`collection medium appears to be beneficial. However, these findings have been shown in vitro
`and await in vivo testing (33).
`
`Swimming velocitv of X and Y suerm. For more than 25 years there has been controversy
`surrounding the question of whether or not Y sperm swim faster than X sperm due to their
`slightly lower weight. Although the conclusions were drawn relative to human sperm, it is likely
`that sperm of other species would respond similarly should the hypothesis be true. We
`conducted a study to test the hypothesis using computer-assisted motion analysis (CASA). There
`was no difference with respect to the swimming velocity between Y sperm and X sperm of the
`bovine (36) as measured by CASA.
`
`Performance Parameters Under Routine Sorting Conditions With HiSON
`
`Assuming a flow rate of 14,00O/sec with sperm that were 80% motile at the outset, one
`could expect the following output: About 1400 to 1700 sperm/set each of X and of Y would be
`sorted with a resulting purity of X or Y at approximately 90%. Losses to the sort are the dead
`sperm, and the misoriented sperm. Assuming an orientation rate of 70% and a sort-window
`
`

`

`1332
`
`elimination of 30% (mixed population of X and Y sperm) there is a final yield of about 20 to
`30% of the total sperm with which one started (10 to 15% for each population of X- or Y-bearing
`sperm). Many factors affect flow integrity including doublets, large aggregates, staining
`uniformity, resolution, sperm viability, orientation, and coiled tails (43). It was also established
`that, as sperm pass through the flow cell of the sperm sorter, they go either head first or tail first
`with equal frequency (43). No data have been produced to determine if there is an advantage of
`head or tail first in the integrity of the sort. Placement of sort windows is also a factor in the
`outcome. Various parameters can be adjusted for given sorts that would affect flow rate, sort
`rate, production of sorted sperm per h as well as fertilization. Table 1 shows parameters that will
`produce 6 million sperm per h in each direction (6 million X and 6 million Y sperm) for a 1 -h
`sort total of 12 million sperm. Sorting parameters are shown that were used to obtain 6 million
`sperm per h at 90% purity. These are average results that on any given day can be influenced by
`the factors mentioned above to increase the production by 20% or reduce the production rate by
`20%.
`
`Table 1. Mean values for boar sperm sorts using a HiSON and operating at 3.16 kg/cm2 over
`2.5 h at conditions described in the text illustrating a routine setup and sorting
`experiment (N=4)
`Flow rate
`
`Spermlsec % Oriented Sperm/set Sperm/h
`
`Sort rate
`
`No. of sperm
`sorted
`
`Sort reanalysisa
`
`% x
`
`% Y
`
`14,000
`89
`91
`15 x lo6
`6x lo6
`1,700
`65.1
`a Sort reanalysis was performed to determine purity using a 300~pl volume aliquoted from each
`sorted sample (48).
`
`Table 2 illustrates the potential production using higher flow rates for 11 million X sperm/h
`at 87% purity using the HiSON. 1~ reasing the flow rate to 30,000 sperm/set and utilizing a
`higher sheath pressure (4.22 kg/cm ) and droplet formation of 100 kHz while increasing the size
`of the orientation gate for X-bearing-sperm can yield X sorts of over 10 million per h with 85 to
`90% purity.
`
`Table 2. Mean values for boar-sperm sorts using HiSON and operating at 4.22 kg/cm2 over
`1 .O h at 100 kHz droplet formation(N=3)
`X Sort rate
`% x sort
`
`Y Sort rate
`
`%Y sort
`reanalysis
`
`Flow rate/set
`
`per set
`
`per h
`
`reanalysis per set
`
`per h
`
`3,150 11.3 x lo6
`30,000
`77
`7.2 x lo6
`2,000
`87
`d Sort reanalysis to determine purity was performed using a 300~ul volume aliquoted from the
`sorted sample (48).
`
`

`

`Theriogenology
`
`1333
`
`Adjustment of parameters such as this are possible to achieve a specialized outcome, i.e.
`only one population. The sorting of X sperm alone is the easiest due to the fact that it carries the
`most DNA and, therefore, is the brightest in response to the exciter-laser beam, which places it
`on the upper end of the histogram. Consequently, the sorting of X sperm at higher speeds results
`in a purer sample than can be obtained with Y sperm. This is due to the fact that slightly
`misanalyzed X sperm will
`fall into the Y-sort window.
`
`Data to illustrate the near upper limits of the HiSON system in terms of viable intact sperm
`production rate with the current optics and configuration are shown in Table 3. Through the use
`of sort reanalysis the proportionate purity of X sperm was determined at various rates. Three
`of viable sperm from each of three boars were done (N=9). The increased production rate
`sorts
`of X sperm was accomplished by including more of the sperm in the X sort window
`(Figure 3)
`which includes more of area B in the sorted X population, and thus results in a reduction of the
`purity of X sperm.
`In comparison to Table 2, it should be noted that a lower Khz droplet
`formation i3used in table 3 (75 Khz) versus 100 Khz as well as a lower pressure (3.92 versus
`4.22 kg/cm ). More optimum sorting conditions were obtained in the lower pressures and the
`lower Khz settings.
`
`Table 3. Comparison of various collection rates of flow sorted sperm and the effect on purity
`of the sorted X population of sperm
`Number of sperm sorted per h with respective % purity of X sperm
`
`Boar
`
`9x106
`
`10.8 x lo6
`
`12.6 x lo6
`
`14.4 x lo6
`
`16.2 x lo6
`
`18 x lo6
`
`1
`
`2
`
`3
`
`86
`
`83
`
`83
`
`86
`
`83
`
`90
`
`82
`
`81
`
`81
`
`80
`
`78
`
`81
`
`77
`
`79
`
`78
`
`76
`
`73
`
`71
`
`73
`78
`80
`81
`86
`84
`Mean
`a Sort reanalysis to determine purity was performed using a 300~~1 volume aliquoted from the
`sorted sample (48). Flow rates averaged 24,000 sperm/set at 75 Khz and 3.92 kg/cm2.
`
`the data from sorts at the highest pressure that we have used for sorting
`Table 43hows
`(4.22 kg/cm ). There was a 10% increase in DAR in the sorted sperm illustrating a mild effect of
`HiSON sorting conditions on the integrity of the acrosomal membranes of boar sperm.
`
`Differences
`
`in Sperm DNA Among Species Affects the Sorting of Sperm
`
`The most critical differences in sorting procedures relative to species are the differences in
`DNA between X and Y sperm within a species. For example the Chinchilla langier has a 7.5%
`difference (Figure 1) making it relatively easy to get a pure sort (98 to 100%) since the bimodal
`split (Figure 1) is nearly complete. The common species of domestic animals are different in X-
`Y DNA in varying degrees: rabbit (3.0%), pig (3.6%), bull (3.8%), horse (3.7%), and sheep
`
`

`

`1334
`
`Theriogenology
`
`the sort windows.
`in setting
`the more care is required
`the DNA difference,
`(4.2%). The narrower
`Most domestic animals mentioned
`have the paddle shaped sperm with a clear difference between
`edge and flat side.
`
`Table 4.
`
`of fresh control semen with sorted sperm from the boar to determine
`Comparison
`effect of high speed sort conditions on acrosomal
`integrity.a
`
`the
`
`Control
`
`Sorted
`
`NAR
`
`96.9
`
`87.9
`
`Acrosomal
`
`Integrity
`
`(%)
`
`DAR
`
`2.3
`
`10.5
`
`MAR
`
`0.4
`
`0.7
`
`LAC
`
`0.3
`
`1.1
`
`a Boar sperm were stained and sorted under 4.22 kg/cm2 pressure; control boar sperm were not
`stained and not sorted. Sperm were fixed in glutaraldehyde
`and evaluated
`for acrosomal
`integrity. A small volume was sorted (50 ul) onto 2% Test-Yolk buffer (5 ~1) and immediately
`fixed with equal volume of 2% glutaraldehyde
`(38). Equivalent numbers of control sperm were
`pipetted and similarly
`fixed. N=22 observations and 5 boars. Normal Apical Ridge
`(NAR);
`Damaged Apical Ridge (DAR); Missing Apical Ridge (MAR) and Loose Acrosomal Cap (LAC).
`
`than domestic animal sperm and are
`Man, on the other hand, has sperm that are smaller
`shaped less like a paddle but more angular or lobular. However, differential
`fluorescence
`is also
`characteristic
`of human sperm allowing
`their selection according
`to their orientation
`to the laser
`beam. The sperm of man has a DNA difference of 2.8% (21), requiring
`exact tuning of the
`sperm sorter and refining sperm sorting procedures. Also
`it is particularly
`difficult
`to g