Theriogenology 65 (2006) 943–957
www.journals.elsevierhealth.com/periodicals/the
Flow cytometric sexing of mammalian sperm
Duane L. Garner *
Department of Animal Biotechnology, University of Nevada, Reno, P.O. Box 1939,
Graeagle, CA 96103-1939, USA
Abstract
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 2 106/
dose remains at 70–80% of unsexed sperm at normal doses of 10 to 20 106 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.
# 2005 Elsevier Inc. All rights reserved.
Keywords: Predetermination of sex; X- and Y-sperm; DNA content; Sperm sexing; Flow cytometer; Sperm
sorter
* Tel.: +1 530 836 0941; fax: +1 530 836 0450.
E-mail address: [email protected].
0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2005.09.009
944
D.L. Garner / Theriogenology 65 (2006) 943–957
1. Introduction
Mature male gametes are small, haploid cells that can be accurately analyzed for DNA
content because this attribute is stable in healthy sperm. High resolution measurement of
the DNA content of sperm was first achieved by flow cytometric analysis of
demembranated spermatids or sperm nuclei [1,2]. The precision of this DNA measurement
is such that the difference in DNA content between mammalian X- and Y-chromosome
bearing sperm of a variety of mammals has been determined [2,3]. This preparation
process, however, severely damaged sperm due to the aggressive removal of the tail and the
membranes surrounding the nuclei prior to staining with the membrane impermeant dye,
40 -6-diamidino-2-phenylindole (DAPI) [2]. It was not until the membrane permeant
bisbenzimidazole DNA-binding dye, Hoechst 33342, was employed that accurate
measurement of DNA content was achieved in living sperm [4]. Precise measurement of
the DNA content difference between X- and Y-chromosome bearing sperm of mammals
has provided an effective means of separating viable gametes carrying either the X- or Ychromosome with an accuracy of 85–95% [5–8]. Flow cytometric sex-sorting of sperm
according to their DNA content is patented [9] and has been sub-licensed for non-human
mammals to XY Inc., through Colorado State University. Sperm sexing has been the topic
of several recent reviews [7,8,10,12–14].
The objectives of this review are [1] to reexamine some of the parameters that will need
optimization before flow cytometric sexing of mammalian sperm that can be expanded for
commercial application to a greater variety of species and [2] to update the current status of
sperm sexing in species in which it has been applied.
2. Sperm DNA content differences
The initial documentation of a DNA content difference between the mammalian Xchromosome-bearing sperm from sperm carrying the Y-chromosome was established
for human spermatids with a Phywe or ICP 22 flow cytometer [1]. This finding led to
development of a rapid, high resolution method that minimized variability associated
with the orientation of the sperm head by hydrodynamically forcing each sperm into a
particular orientation as it passed through the flow cytometer for measurement. This
orienting instrument allowed accurate measurement of the fluorescence emitted from
the flat surface of the sperm nucleus for some portion of the cells as they flowed through
the instrument [2,15,16]. Application of this innovative measurement of X- and Ychromosome bearing sperm in semen samples of domestic livestock, including bulls,
boars, rams and rabbits revealed differences among these domestic species in the
magnitude of the DNA content difference between the sex-determining gametes [3].
Although the DNA content of all cells for each mammalian species is highly conserved
some DNA content differences do occur even among breeds of cattle [3]. It is not
surprising that a difference occurs in DNA content of the X- and Y-sperm nuclei from
the European breeds (Bos taurus) and those animals originating in Asia (Bos indicus)
because of the differing sizes of the Y-chromosomes of these two species (Fig. 1).
Subtle X–Y differences, however, have been observed in sperm nuclei within a species
D.L. Garner / Theriogenology 65 (2006) 943–957
945
Fig. 1. Subtle X–Y sperm DNA content differences among sperm nuclei from four European breeds of cattle (Bos
taurus) and the Brahman cattle originating in Asia (Bos indicus) that had been stained with 40 -6-diamimidino-2phenylindole (DAPI). Sperm nuclei from Brahman bulls had smaller X–Y differences than the European breeds
and sperm nuclei from Jersey bulls had greater differences in the DNA content of the X- and Y-chromosome
bearing sperm nuclei than those from Holstein or Hereford bulls. Dairy breeds are shown as (&) while beef breeds
are illustrated as ( ) and Brahman as (&). X–Y differences of the DNA content of the sperm nuclei are shown
within the column for each species. No significant differences were noted within breeds, but means of the square
root transformed data showed that sperm from Jersey bulls had larger X–Y DNA differences than gametes from
Angus, Hereford and Holstein whereas sperm from Brahman bulls had smaller X–Y differences than the European
breeds. Adapted from Garner et al. [3].
in that sperm from Jersey bulls exhibited a greater X–Y difference than those from
Holstein or Hereford bulls [3].
Recently flow cytometric sexing of sperm from wildlife species has documented DNA
content differences between the X- and Y-chromosome bearing sperm of the common
chimpanzee: 3.3%, hamadryas baboon: 4.2%, African elephant: 4.0% and giraffe: 4.4%
[17]. DNA content measurements have provided the precision necessary to identify the Xand Y-chromosome bearing sperm populations in semen from at least 23 mammalian
species [7,8,11,12,17] (Fig. 2).
Fig. 2. X–Y sperm DNA content differences among mammalian sperm from 23 species as measured by flow
cytometry ( ) and the seven species from with offspring have been produced form sex-sorted sperm (&). X–Y
differences are shown above the display for each species (Compiled from [3,7,8,11,13,17]).
946
D.L. Garner / Theriogenology 65 (2006) 943–957
3. Sperm head size and shape differences
An important attribute that differs among the sperm of mammals is the shape of the
head. Most domesticated mammals have a flattened, oval shaped heads with the exception
of rodents and monotremes [18]. A comparison of the dimensions of the sperm heads of
some mammals is shown (Table 1). The effectiveness of utilizing DNA content differences
between the X- and Y-chromosome carrying sperm depends not only on relative DNA
differences, but also on the ability to precisely orient these gametes at the time of
measurement in the flow cytometer/cell sorter. Mammalian sperm with flattened, oval
heads tend to be more readily oriented in a sperm sorter using hydrodynamics than those
gametes possessing more rounded or angular shaped heads (Table 1). This attribute can be
expressed using actual species differences in DNA content as reflected by the presence of
the X- or Y-chromosome and the area making up more flattened side or profile of the sperm
head. A comparison of this particular attribute is shown for a few mammalian species
(Table 1). This approximation suggests that the easiest sperm to separate in flow cytometer/
cell sorter would be from the bull because the area of the head profile is 34.5 mm2 with an
X–Y difference in DNA content of 3.8% resulting in the highest sorting index of 131. It is
not surprising that the most notable success has been with bull sperm. Sperm sorting
indices for the boar, ram, rabbit, cat, dog, horse and man are in Table 1. This simple
approximation suggests that the attributes of human sperm make them nearly four times
more difficult to sort than sperm from bulls or boars. In addition, species differences exist
in the rates by which living sperm can be stained with Hoechst 33342. These uptake
differences are small, but the highest resolution of DNA content can only be obtained by
optimizing the sperm staining procedure for each species or even individual males within a
species [19]. However, the process is even more complex than that because the ability to
successfully sort sperm also must take into account the relative susceptibilities of gametes
to laser exposure, high dilution, elevated pressure and resistance to the several changes in
media composition that occur during the sexing process. This makes sex-sorting of sperm
not only different for each mammalian species, but differences in sorting efficiency also
exist for the sperm of individual males within a species.
4. Sex-sorting sperm
The ability to differentiate between X- and Y-chromosome bearing sperm has not only
provided a means to determine the efficacy of any sperm enrichment approach, but is the basis
for the sperm sexing system that is now used commercially to predetermine the sex of offspring
[7,8,11,12]. The efficiency of the sorting system was enhanced by identification of the dead or
damaged sperm so that only living intact sperm were actually sorted [21]. Damaged or dead
sperm were first identified in Hoechst 33342-stained population by their uptake of a membrane
impermeant dye, propidium iodide (PI) [21]. Compromised gametes were identified so that
they could be gated out and disposed off as waste along with those sperm not measured
properly. Substitution of the food dye, FD&C #40 (Warner Jenkinson, St. Louis, USA) for the
PI, a DNA dye that intercalates between the base pairs, provided greater safety because it
simply quenches the Hoechst 33342 fluorescence and is not mutagenic [22,23].
Table 1
Dimensions and profiles of sperm heads and flow cytometric sorting indices for some domestic mammals and mana
Bull
Boar
Ram
Rabbit
Cat
Dog
Horse
Man
Length (mm)
Head sagittal section
9.1
9.0
8.1
7.7
7.7
7.0
6.5
4.6
Width (mm)
Head profile
4.7
5.0
4.0
4.5
3.2
3.5
3.4
3.2
Area (mm2)
X–Y difference (%)
Sorting indexb
34.5
3.8
131
37.5
3.6
115
26.6
4.2
112
28.0
3.0
84
19.0
4.2
80
20.9
3.9
82
15.2
3.9
59
10.8
2.8
31
a
b
D.L. Garner / Theriogenology 65 (2006) 943–957
Dimension
Compiled from Mann [67] Mann and Lutwak-Mann [68], Johnson [7], Welch and Johnson [6], Garner [11], Garner and Seidel [13] and Seidel and Garner [14].
An approximation of the ability to flow cytometric sort sperm consisting of the head profile area (mm2) X–Y Sperm DNA difference (%).
947
948
D.L. Garner / Theriogenology 65 (2006) 943–957
Sex-sorting of cattle sperm was brought to the point that of commercialization by
development of a more sophisticated orienting nozzle for the sperm sorter by Rens et al.
[20] and successful cryopreservation of the sorted gametes [23]. The sorter nozzle was
further enhanced by development of a ceramic version (Cytonozzle1) of the SX MoFlo1
system (DakoCytomation, Fort Collins, USA) that is used commercially. As to the
efficiency of the system, 10 insemination doses of sexed bovine sperm at 2 106 live
sperm/dose for each sex can be produced per hour of actual sorting under optimal
conditions. Production rates of sexed sperm, however, are considerably less in practice
[12]. Unfortunately, the efficiency of flow sorting Hoechst 33342 stained bovine sperm is
marginal because only one-third of the sperm passing through the system can be sorted
with the threshold set to achieve 90% accuracy [12]. Another 20% of the sorted sperm are
lost during the sperm concentration and packaging processes [12]. Furthermore, sorting
efficiency can be much less with poor quality ejaculates. Nonetheless, flow sorting of Xand Y-chromosome bearing sperm has resulted in normally-appearing calves from
numerous bulls and for offspring of seven species [8,12,24] (Fig. 3).
5. Damage to sperm during sorting
Successful sperm sexing must take into account the relative susceptibilities of gametes
to staining, laser exposure, high dilution, elevated pressure and resistance to the several
changes in media composition that occur during the sexing process [25]. In addition, to
developing more optimal media for use in sorting sperm for each species, it may be
necessary to modify the flow cytometer/cell sorter nozzle so that sorting can be more
efficient for sperm for each species. The fluorescence signals emitted when Hoechst 33342stained sperm are illuminated with the 351 and 364 nm lines of an argon laser, thereby
providing an accurate measure of the DNA content of X- and Y-chromosome bearing
sperm for sorting [7,11,12,26,27]. Examination of the integrity of the DNA (Sperm
Chromatin Structure Assay, SCSA, 28) of bovine sperm during various steps in the sexsorting process indicated that mechanical injury, not only exposure to Hoechst 33342 and/
or 150 mW of laser illumination, was a factor (Fig. 4) [11]. Comparison of DNA integrity
increases over control (unsorted controls, with no exposure to an Argon laser or the DNAbinding dye, Hoechst 33342 stain) of thawed, cryopreserved bovine sperm showed that
when sperm were sorted with no stain and no laser illumination DNA integrity was reduced
by only 1.8% when compared to unsorted, unstained sperm. For sperm sorted with
illumination, but without stain DNA damage was increased by 2.0%, while sperm sorted
with stain but no illumination increased the DNA damage by another 1.5% while sperm
sorted with both stain, and laser illumination increased the potential DNA damage by 1.5%.
(Fig. 4) [29]. DNA integrity was measured by the SCSA for two ejaculates from six bulls. It
is expressed as a DNA fragmentation index (%DFI) [28].
Sperm viability tests with SYBR-14 and PI [30,31] showed the mechanical stresses of
sorting and centrifugation increased the dead or damaged sperm by 18.6%, while sorting
without staining added another 6.8% and staining with Hoechst 33342 without laser
exposure added 3.6% (Fig. 5) [12,32]. The combined use of Hoechst 33342 staining and
laser exposure only increased the proportion of dead or damaged sperm by an additional
D.L. Garner / Theriogenology 65 (2006) 943–957
949
Fig. 3. Illustration of an assembled Cytonozzle1 (a); a disassembled nozzle showing the flow chamber, tapered
injection needle and the ceramic tip [surrounded by dotted lines] (b); a profile of the ceramic tip (c); sagittal section
of the tip showing the narrowest elliptical orienting configuration [shaded cross sections of the tip interior are
shown on the immediate left of the tip] (d); and a sagittal section after rotation of the tip 908 the illustrate the widest
portion of the elliptical internal bore of the tip [shaded cross sections of the tip interior are shown on the immediate
left of the tip] (e).
0.3% (Fig. 5) [12,32]. Lowering the fluidic pressure during the sorting process from 50 to
near 40 psi increased the survivability of sorted gametes while maintaining resolution of
the X- and Y-chromosome bearing sperm [33,34]. The lower pressure may minimize
damage, but may not be sufficient to orient gametes from species producing sperm with
rounded or angular heads. Optimizing sorting conditions may be necessary for each species
to achieve acceptable sexing efficiency of mammalian species producing sperm with
higher susceptibilities to processing and those with more rounded or angular heads.
Concerns on the safety of sperm sexing relative to potential genetic risks have been
expressed [35]. Staining with Hoechst 33342 and UV-laser irradiating sperm during flow
950
D.L. Garner / Theriogenology 65 (2006) 943–957
Fig. 4. Comparison of the stepwise increases in DNA damage occurring during the sexing process as compared to
the previous treatment and in addition to that observed in control samples (unsorted controls, with no exposure to
an Argon laser illumination at 354–361 nm light or the DNA-binding dye, Hoechst 33342 stain) of thawed,
cryopreserved bovine sperm samples processed as sorted with no stain and no laser illumination showing
mechanical injury [1], sorted with illumination but no stain [2], sorted with stain but no illumination [3], or sorted
with both stain and laser illumination [4]. DNA integrity was measured by the Sperm Chromatin Structure Assay
(SCSA) and is expressed as a DNA fragmentation index (%DFI) [28]. Adapted from Garner [11].
sorting tended to increase the incidence of chromosome aberrations [36]. In this study,
after Hoechst-stained and UV-laser irradiated sperm nuclei were microinjected into
hamster eggs, the number of DNA breaks and triradial and quadriradial exchanges
between chromosomes that developed, increased slightly. In a recent study, no
genotoxic effects of sexing sperm with Hoechst 33342 were found in cells of resulting
pigs [37].
Fig. 5. Mechanical stresses induced during sorting and processing of sexed sperm resulted in the largest increase
in the percentage points of damaged sperm. Increases in the proportions of living, SYBR-14 stained sperm and
sperm that took up propidium iodide (PI) as an indication of dead or membrane damaged cells over that found at
each previous step starting from that found in control samples (unsorted controls, with no exposure to an Argon
laser illumination at 354–362 nm light or the Hoechst 33342 stain had a mean live-dead proportions of 62.1%
SYBR-14 positive with 36.1% PI positive) of thawed, cryopreserved bovine sperm samples with sorting. Sperm
damage increased by 18.6% when sperm were sorted with no stain and no laser illumination [1], another 6.8%
when sorted with illumination but no Hoechst 33342 stain [2], and by 3.6% when sorted with Hoechst 33342 stain
but no illumination [3], and only by 0.3% when sorted with both Hoechst 33342 stain and laser illumination [4].
Adapted from Seidel and Garner [12].
D.L. Garner / Theriogenology 65 (2006) 943–957
951
Although we have information on cellular toxicity, sperm staining efficiency, the
embryonic development of embryos resulting from Hoechst-stained sperm and the
normalcy of offspring, we know little concerning either the fate of the Hoechst stain within
the female reproductive tract or the overall long-term effect of the use of this fluorophore
for sexing mammalian sperm. It seems reasonable to examine other membrane permeant
DNA-specific dyes that are excited at longer excitation wavelengths because these tend to
be less mutagenic, especially those that bind to the small groove of the DNA rather than
intercalate between base pairs. Reasonable fertility levels, however, have been attained in a
large number of field trials with cattle sperm [5,12,26,38,39] and with Hoechst-stained
boar sperm [26,40,41]. Although the numbers are small relative from an epidemiological
standpoint, the normalcy of the progeny resulting from the use of sexed sperm stained with
Hoechst 33342 and laser illumination has remained similar to the normalcy levels for
progeny from unstained, unsexed sperm [12,24,42]. When identical doses of sexed and
unsexed sperm were compared to normal dose controls, pregnancy rates of the low dose
controls were close to the normal dose controls, indicating that the damage due to sexing
sperm was also expressed as lower pregnancy rates [5,39].
The survival of sperm after sex-sorting is extremely important in that a considerable
investment has been made in these gametes. When higher proportions of sperm after
sorting retain their fertility after storage, either cryopreserved and thawed or as fluid semen,
fewer sperm need to be sorted for each dose. Thus, cryopreservation and storage of sorted
sperm needs careful re-examination. Development of enhanced cryopreservation methods
such as the Multi-Thermal Gradient freezing system should enable more of the sorted
sperm to survive cryopreservation and thawing [43]. Although sorting results in
elimination of the dead and damaged sperm, those cells surviving the sexing process tend
to degenerate faster than unsorted control gametes from bulls and boars [44,45], but
apparently not from stallions [46] and rams [19]. Any gain in the efficiency either of sorting
sperm or increases in cryopreservation or storage, reduces the time required to sort each
dose of sexed sperm [47].
Implementation of sperm sexing technology for domestic species:
Cattle: The principal force that drives the implementation of sperm sexing technology
by the domestic livestock industries is economics [14,48]. Although many seek to utilize
sexing technology, very few potential users have the capital necessary to implement and
maintain such a program. Seidel [14] suggested that the yearly operating costs for the
first year of a sex-sorting facility would exceed US$ 2 million. Such costs are beyond the
range of most companies or cooperatives that currently process and market semen.
Sperm sexing technology was first implemented nearly 20 years after the principle of
selecting sperm by their DNA content was first established. At this time more than
20,000 calves have been born from sperm sorted for sex-chromosome differences based
on DNA content. Most of these calves were produced in the United States, United
Kingdom, Argentina and Mexico, with lesser numbers in several other countries.
Currently, sexed bovine sperm can be purchased from companies in the United
Kingdom, Argentina, and some regions of the United States while licensing/
commercialization of this technology for cattle is in various phases of development
in several other countries. Successful cryopreservation of sexed sperm was a major
952
D.L. Garner / Theriogenology 65 (2006) 943–957
factor that facilitated commercialization of bovine sperm sexing [5]. Although, sexsorted bovine sperm tend to degenerate faster than unsorted control gametes,
cryopreservation overcomes this problem allowing sexed bovine sperm to be stored
indefinitely so that they can be transported and used almost anywhere in the world.
Successful use of sexed sperm, however, requires strict management practices even
when limited to use in heifers [14]. Insemination of lactating dairy cows with low doses
of sexed semen has not produced acceptable pregnancy rates [14] from published
studies. Precise packaging of sperm/insemination dose is essential to minimize
inadvertent production of straws containing fewer sperm than the 2 106 currently used
in most insemination doses of cryopreserved, sexed bovine sperm. Minimizing losses
during sorting, centrifugation and packaging are essential because nearly 60% of the
total sperm in the sample are excluded or lost during various processing steps [13]. The
need for improvement in the sorting system is obvious.
Swine: Predetermination of sex using flow cytometry/cell sorting was established first
using rabbit sperm [27], but swine was the first large animal species where the feasibility
of using this technology was demonstrated [49]. Currently, sperm sexing technology has
not been implemented in swine production operations, but the increased use of AI in pigs
should alter this, particularly with genetic companies seeking to produce large numbers
of seedstock gilts in multiplier herds. The increased use of AI in swine, especially as
recently seen in North America, could augment this development. Not only does the
broader use of sexed sperm in swine require more efficient AI, but the inseminations
must be with sperm numbers far below what is currently used in AI. An important
development along this line is the porcine Firflex catheter (Magapor, Spain) whereby
low numbers of sexed sperm can be deposited deep within the tract near the tip of the
uterine horn [45,50–52]. Major efforts to refine the AI system so that sex-sorted boar
sperm could be successfully inseminated using low sperm numbers is ongoing in Spain
[50,51], Germany [45,53,54], Australia [8,54] and in North America [52,55]. The
adoption of sperm sexing for production of founder animals with superior genetic
qualities should increase the availability of animals with superior genetic traits to swine
producers.
Sheep: Sex was predetermined in sheep first by ICSI of sex-sorted ram sperm [56]. As
with pigs, the initial inseminations were surgical producing several lambs by depositing
low doses of freshly sorted ram sperm directly into the uteri of estrous-synchronized
ewes [57]. Recently, Hollinshead et al. [58] produced successful pregnancies in ewes
inseminated with 4 106 sex-sorted, cryopreserved–thawed ram sperm. Although
pregnancy rates in ewes were low, live lambs resulted from insemination with either
freshly-sorted or thawed, cryopreserved sex-sorted ram sperm. Improvements in
diluents and increasing the numbers of sperm per dose along with more precise control
of the time of insemination relative to ovulation might improve pregnancy rates [58–60].
Horses: Development of the hysteroscopic method for inseminating of mares with low
numbers of sperm has made predetermination of sex in this species a reality [61,62].
However, the cost of equipment and the inability to use semen from many stallions will
likely limit this application. Perhaps, development of a flexible catheter, similar to the
Firflex1 (Magapor, Spain) that has been used in swine, would be a more practical
approach to placing low numbers of sexed sperm in the upper regions of the uterine
D.L. Garner / Theriogenology 65 (2006) 943–957
953
horns. An effort should be made to gain a greater understanding of the susceptibility of
stallion sperm to the in vitro manipulation required for sperm sexing. Most equine sperm
needs to be separated from its seminal plasma prior to processing for sperm sexing. This
adds additional stress to the sample even prior to the sexing procedure.
6. Application to endangered and exotic species
Recent efforts to extend sperm sexing to more endangered and exotic species are
encouraging [17,63,64]. The potential value of being able to control the sex of offspring of
endangered and exotic animals is obvious. The use of sperm sexing as a management tool
could minimize inbreeding within captive populations through carefully planned breeding
programs.
Elk: Reasonable pregnancy rates have been obtained with electro-ejaculated sperm from
a bull elk that had been sex-sorted, cryopreserved using methods similar to those for
cattle, and inseminated into cow elk [65]. The 3.6% difference in DNA content between
X- and Y-chromosome-bearing sperm of the American Bison [13] suggests that sorting
bison sperm to predetermine sex of offspring would be feasible.
Cats: Much of the interest in developing assisted reproductive technologies in the
domestic cat has arisen because this animal is a good model for the large exotic felids
that are endangered or becoming rapidly so. Ejaculated cat sperm have been sex-sorted
and used in IVF to produce day 7 blastocysts (Garner, Sondergard, Pope, Harris and
Dresser, unpublished [13]).
Marine mammals: Application of sperm sexing technology to the various marine
mammals in captivity such as the Killer Whale would assist management of these and
other exotic marine mammals. A 4.1% difference in DNA content between the X- and Ychromosome-bearing sperm has been established for the Atlantic Bottle Nosed Dolphin
and Pacific White-sided Dolphin (Table 1) [13].
7. Sexing thawed, cryopreserved sperm
The birth of lambs of predetermined sex produced by IVF of oocytes with frozen–
thawed, sex-sorted and re-frozen–thawed ram sperm greatly expands the possibility of
using currently cryopreserved sperm in sexing programs [19]. Success with sorting
previously cryopreserved bull sperm has been noted also [66]. Although the efficiency of
this process may be limited, such a development could expand the use of genetically
superior sires in some sperm sexing operations.
8. Need for further flow sorter development
Development of a dedicated sperm sorter is needed because the technology currently in
use could be greatly simplified and made more efficient. The flexibility of general flow
954
D.L. Garner / Theriogenology 65 (2006) 943–957
cytometers, including software, is not necessary for sorting sperm. The excitation power
produced by the expensive, large water-cooled Argon lasers is probably not needed with
the development of new powerful solid-state lasers. The amount of sheath fluid that ends up
in the sperm collection vessel could be reduced if the interior chamber of the nozzle was
redesigned so that less fluid passes around the sample stream during the sorting process.
Such a nozzle could reduce sperm losses during the sorting because it would minimize
dilution of sperm during the sorting process. Likewise, a collection vessel that facilitates
fluid removal during the sorting similar to that used to collect embryos could remove some
of the unneeded fluid prior to centrifugation to re-concentrating the sorted sperm. A sperm
sorter nozzle could be developed with a straight rather than curved sample injection system
along with making it disposable to minimize cleaning and sterilizing between semen
samples. It should be relatively simple to design and manufacture a dedicated sperm sorter
with disposable components.
Acknowledgement
The author thanks George Seidel Jr. for his constructive suggestions on the manuscript.
References
[1] Otto FJ, Hacker U, Zante J, Schumann J, Göhde W, Meistrich ML. Flow cytometry of human sperm.
Histochemistry 1979;62:249–54.
[2] Pinkel D, Lake S, Gledhill BL, Van Dilla MA, Stephenson D, Watchmaker G. High resolution DNA content
measurements of mammalian sperm. Cytometry 1982;3:1–9.
[3] Garner DL, Gledhill BL, Pinkel D, Lake S, Stephenson D, Van Dilla MA, et al. Quantification of the X- and
Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry. Biol Reprod 1983;28:312–21.
[4] Johnson LA, Flook JP, Look MV. Flow cytometry of X- and Y-chromosome-bearing sperm for DNA using an
improved preparation method and staining with Hoechst 33342. Gamete Res 1987;17:203–12.
[5] Seidel Jr GE, Schenk JL, Herickhoff LA, Doyle SP, Brink Z, Green RD. Insemination of heifers with sexed
sperm. Theriogenology 1999;52:1407–20.
[6] Welch GR, Johnson LA. Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X- and
Y-sperm by sort reanalysis for DNA. Theriogenology 1999;52:1343–52.
[7] Johnson LA. Sexing mammalian sperm for production of offspring: the state of the art. Anim Reprod Sci
2000;60–61:93–107.
[8] Maxwell WMC, Evans G, Hollinshead FK, Bathgate R, deGraaf SP, Eriksson BM, et al. Integration of sperm
sexing technology into the art toolbox. Anim Reprod Sci 2004;82–83:79–85.
[9] Johnson LA. Method to preselect the sex of offspring. United States Patent #5 1991 135:759.
[10] Johnson LA, Seidel GE Jr. (Eds.). Current status of sexing mammalian sperm. Theriogenology 1999;
52:1267–1484.
[11] Garner DL. Sex-sorting mammalian sperm: concept to application in animals. J Androl 2001;22:519–26.
[12] Seidel Jr GE, Garner DL. Sexing mammalian sperm by flow cytometry. Reproduction 2002;124:733–43.
[13] Garner DL, Seidel Jr GE.. Past, present and future perspectives on sexing sperm. Can J Anim Sci
2003;83:375–84.
[14] Seidel Jr GE. Economics of selecting for sex: the most important genetic trait. Theriogenology 2003;59:585–
98.
[15] Fulwyler MJ. Hydrodynamic orientation of cells. J Histochem Cytochem 1977;25:781–3.
[16] Dean PN, Pinkel D, Mendelsohn ML. Hydrodynamic orientation of sperm heads for flow cytometry. Biophys
J 1978;23:7–13.
D.L. Garner / Theriogenology 65 (2006) 943–957
955
[17] O’Brien JK, Crichton EG, Evans KM, Evans G, Schenk JL, Stojanov T, et al. Sex ratio modification using
sperm sorting and assisted reproductive technology—a population management strategy. In: Proceedings of
the 2nd International Symposium on Assisted Reproductive Technology for the Conservation and Genetic
Management of Wildlife; 2002. p. 224–31.
[18] Austin CR. Fertilization. Englewood Cliffs, NJ: Prentice-Hall, 1965.
[19] Hollinshead FK, O’Brien JK, Gillan L, Meyers M, Maxwell WMC, Evans G. Birth of lambs of a
predetermined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–
thawed ram spermatozoa. Reproduction 2004;127:557–68.
[20] Rens W, Welch GR, Houck DW, van Oven CH, Johnson LA. Slit-scan flow cytometry for consistent high
resolution DNA analysis of X- and Y-chromosome bearing sperm. Cytometry 1996;25:191–9.
[21] Johnson LA, Welch GR, Garner DL. Improved flow sorting resolution of X- and Y-bearing viable sperm
using dual staining and dead cell gating. Cytometry 1994;17(Suppl. 7):83.
[22] Johnson LA, Welch GR. Sex preselection: high-speed flow cytometric sorting of X- and Y-sperm for
maximum efficiency. Theriogenology 1999;52:1134–323.
[23] Schenk JL, Suh TK, Cran DG, Seidel Jr GE. Cryopreservation of flow-sorted bovine sperm. Theriogenology
1999;52:1375–91.
[24] Tubman LM, Brink Z, Suh TK, Seidel Jr GE. Characteristics of calves produced with sperm sexed by flowcytometry/cell sorting. J Anim Sci 2004;82:1029–36.
[25] Maxwell WMC, Long CR, Johnson LA, Dobrinsky JR, Welch GR. The relationship between membrane
status and fertility of boar spermatozoa after flow cytometric sorting in the presence or absence of seminal
plasma. Reprod Fertil Dev 1998;10:433–40.
[26] Johnson LA. Gender preselection in domestic animals using flow cytometrically sorted sperm. J Anim Sci
1992;70(Suppl. 2):8–18.
[27] Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: live births from X- and Y-sperm separated by
DNA and cell sorting. Biol Reprod 1989;41:199–203.
TM
[28] Evenson DP, Larson K, Jost LK. The sperm chromatin structure assay (SCSA ): clinical use for detecting
sperm DNA fragmentation related to male infertility and comparisons with other techniques. Androl Lab
Corner J Androl 2002;23:25–43.
[29] Garner D, Schenk J, Seidel Jr G. Chromatin stability in sex-sorted sperm. In: Proceedings of the VIIth
International Congress of Andrology Montreal. Andrology in the 21st Century, Canada: Short Communication; 2001. p. 3–7.
[30] Garner DL, Johnson LA, Yue ST, Roth BL, Haugland RP. Dual DNA staining assessment of bovine sperm
viability using SYBR-14 and Propidium Iodide. J Androl 1994;15:620–9.
[31] Garner DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide.
Biol Reprod 1995;53:276–84.
[32] Garner DL, Suh TK. Effect of Hoechst 33342 staining and laser illumination on viability of sex-sorted bovine
sperm. Theriogenology 2002;57:746 [Abstracts].
[33] Suh TK, Schenk JL. Pressure during flow sorting of bull sperm affects post-thaw motility characteristics.
Theriogenology 2003;59:516 [Abstracts].
[34] Campos-Chillon LF, de la Torre JF. Effect of concentration of sexed bovine sperm sorted at 40 and 50 psi on
developmental capacity of in vitro produced embryos. Theriogenology 2003;59:506 [Abstracts].
[35] Meistrich ML. Possible genetic risks of sperm sorting. Cytometry 1996;S113(Suppl. 8):134 [Abstracts].
[36] Libbus GL, Perreault SD, Johnson LA, Pinkel D. Incidence of chromosome aberrations in mammalian
sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting. Mutat Res 1987;182:
265–74.
[37] Parilla I, Vasquez JM, Cuello C, Gil MA, Roca J, Di Berardino D, et al. Hoechst 33342 stain and UVexposure
do not induce genotoxic effect in flow-sorted boar spermatozoa. Reproduction 2004;128:615–21.
[38] Morrell JM, Dresser DW. Offspring from inseminations with mammalian sperm stained with Hoechst 33342,
either with or without flow cytometry. Mutat Res 1989;224:177–218.
[39] Seidel Jr GE, Schenk JL. Field trials with sexed bovine semen. In: Proceedings of the 19th Technical
Conference on Artificial Insemination & Reproduction; 2002. p. 64–9.
[40] Garner DL, Dobrinsky JR, Welch GR, Johnson LA. Porcine sperm viability, oocyte fertilization and embryo
development after staining sperm with SYBR-14. Theriogenology 1996;45:1103–13.
956
D.L. Garner / Theriogenology 65 (2006) 943–957
[41] Guthrie HD, Johnson LA, Garrett WM, Welch GR, Dobrinsky JR. Flow cytometric sperm sorting effect of
varying laser power on embryo development in swine. Mol Reprod Dev 1992;61:87–92.
[42] Doyle SP, Seidel Jr GE, Schenk JL, Herickhoff LA, Cran D, Green RD. Artificial insemination of lactating
Angus cows with sexed semen. In: Proceedings of the Western Section, vol. 50 American Society of Animal
Science; 1999. p. 203–5.
[43] Arav A, Zero Y, Shturman H, Gacitua H. Successful pregnancies in cows following double freezing of large
volume of semen. Reprod Nutr Sci Dev 2002;42:583–6.
[44] Rath D, Sieg B, Leigh J, Kline P, Besseling M, Krüger C, et al. Current perspectives of sperm sorting in
domestic farm animals. In: Proceedings of the 19th Meeting Association of Europeenne de Transfert
Embryonnaire; 2003. p. 125–8.
[45] Rath D, Ruiz S, Sieg B. Birth of female piglets following intrauterine insemination of a sow using flow
cytometrically sexed boar semen. Vet Rec 2003;152:400–1.
[46] Morris L, Hollinshead FK, Evans G, Maxwell WMC. The longevity and acrosome status of flow sorted
stallion. Spermatozoa 2003;59:512 [Abstracts].
[47] Centurion F, Vasquez JM, Calvete JJ, Roca J, Sanz L, Parilla I, et al. Influence of porcine spermadhesins on
the susceptibility of boar spermatozoa to high dilution. Biol Reprod 2003;69:640–6.
[48] Amann RP. Issues affecting commercialization of sexed sperm. Theriogenology 1999;52:1441–57.
[49] Johnson LA. Sex selection in swine Altering sex rations in offspring following surgical insemination with
flow sorted X- and Y-bearing sperm. Reprod Domest Anim 1991;26:195–204.
[50] Martinez EA, Gil MA, Rieke A, McCauley TC, Cantley TC, Vazquez JM, et al. Successful non-surgical deep
intrauterine insemination in gilts and sows during metestrus. In: Proceedings of the Sixth International
Conference on Pig Reproduction; 2001. p. 133.
[51] Martinez EA, Vazquez JM, Roca J, Lucas X, Gill MA, Parrilla I, et al. Successful non-surgical intrauterine
insemination with small numbers of spermatozoa in sows. Reproduction 2001;122:289–96.
[52] Johnson LA, Rath D, Vasquez JM, Maxwell W.M.C.. Dobrinsky JR. Pre-selection of sex in swine for
production of offspring: an update on the process and application. Theriogenology 2005;63:615–24.
[53] Probst S, Rath D. Production of piglets using intracytoplasmic sperm injection (ICSI) with flow cytometrically sorted boar semen and artificially activated oocytes. Theriogenology 2003;59:961–73.
[54] Bathgate R, Eriksson G, Maxwell WMC, Evans G. Low dose deep intrauterine insemination of sows with
fresh and frozen–thawed spermatozoa. Theriogenology 2005;63:553–5.
[55] Abeydeera LR, Johnson LA, Welch GR, Wang WH, Boquest AC, Cantley TC, et al. Birth of piglets
preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X- and Ychromosome bearing sperm sorted by high speed flow cytometry. Theriogenology 1998;50:981–8.
[56] Catt SL, Catt JW, Gomez MC, Maxwell WMC, Evans G. Birth of a male lamb derived from an in vitro
matured oocyte fertilized by intracytoplasmic injection of a single presumptive male sperm. Vet Rec
1996;139:494–5.
[57] Cran DG, McKelvey WAC, King ME, Dolman DF, McEvoy TG, Broadbent PJ, et al. Production of lambs by
low dose intrauterine insemination with flow cytometrically sorted and unsorted sperm. Theriogenology
1997;47:267 [Abstracts].
[58] Hollinshead FK, O’Brien JK, He L, Maxwell WMC, Evans G. Pregnancies after insemination of ewes with
sorted, cryopreserved ram spermatozoa. Proc Soc Reprod Biol 2001;32:20 [Abstracts].
[59] de Graaf SP, Gillan L, Evans G, Maxwell WMC, O’Brien JK. Effect of flow cytometric sorting and freezing
diluent preparation on in vitro quality of ram spermatozoa. In: Proceedings of the 15th International
Congress on Animal Reproduction, 8–12 August, Volume 2; 2004 [Abstracts].
[60] de Graaf SP, Gillan L, Evans G, Maxwell WMC, O’Brien JK. The effect of sheath fluid on the quality of sexsorted ram spermatozoa. Reprod Fertil Dev 2004;16:284–5.
[61] Lindsey AC, Morris LHA, Allen WR, Schenk JL, Squires EL, Bruemmer JE. Hysteroscopic insemination of
mares with low numbers of nonsorted or flow sorted spermatozoa. Equine Vet J 2002;34:128–32.
[62] Lindsey AC, Schenk JL, Graham JK, Bruemmer JE, Squires EL. Hysteroscopic insemination of low numbers
of flow sorted fresh and frozen/thawed stallion spermatozoa. Equine Vet J 2002;34:121–7.
[63] O’Brien JK, Hollinshead FK, Evans G, Maxwell WMC. In vitro developmental capacity of in vitro produced
sexed embryos derived from sex-sorted and re-cryopreserved frozen–thawed ram sperm. Reprod Fertil Dev
2004;16:286. 332 [Abstracts].
D.L. Garner / Theriogenology 65 (2006) 943–957
957
[64] O’Brien JK, Hollinshead FK, Evans KM, Evans G, Maxwell WMC. Flow cytometric sorting of frozen–
thawed spermatozoa in sheep and non-human primates. Reprod Fertil Dev 2003;15:367–75.
[65] Schenk JL, DeGrofft DL. Insemination of cow elk with sexed frozen semen. Theriogenology 2003;59:514
[Abstracts].
[66] Hollinshead FK, O’Brien JK, Maxwell WMC, Evans G. Assessment of in vitro sperm characteristics after
flow cytometric sorting of frozen–thawed bull spermatozoa. Theriogenology 2004;62:958–68.
[67] Mann T. The biochemistry of semen and of the male reproductive tract. London: Methuen, 1964.
[68] Mann T, Lutwak-Mann C. Male reproductive function and semen. Heidelberg, New York: Berlin, SpringerVerlag, 1981.