history of commercializing sexed semen for cattle

www.theriojournal.com

Available online at www.sciencedirect.com

2008) 886–895
Theriogenology 69 (
History of commercializing sexed semen for cattle

D.L. Garner a,*, G.E. Seidel Jr.b

a GametoBiology Consulting, P.O. Box 1939, Graeagle, CA 96103-1939, USAb Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523-1683, USA

Received 19 December 2007; received in revised form 28 January 2008; accepted 28 January 2008

Abstract

Although the basic principles controlling the sex of mammalian offspring have been known for a relatively long time, recent

application of certain modern cellular methodologies has led to development of a flow cytometric system capable of differentiating

and separating living X- and Y-chromosome-bearing sperm in amounts suitable for AI and therefore, commercialization of this

sexing technology. After a very long history of unsuccessful attempts to differentiate between mammalian sperm that produce males

from those that produce females, a breakthrough came in 1981 when it was demonstrated that precise DNA content could be

measured. Although these initial measurements of DNA content killed the sperm in the process, they led to the ultimate

development of a sperm sorting system that was capable, not only of differentiating between live X- and Y-sperm, but of sorting

them into relatively pure X- and Y-sperm populations without obvious cellular damage. Initial efforts to predetermine the sex of

mammalian offspring in 1989 required surgical insemination, but later enhancements provided sex-sorted sperm in quantities

suitable for use with IVF. Subsequent advances in flow sorting provided minimal numbers of sperm sufficient for use in AI. It was

not until the flow cytometric sorting system was improved greatly and successful cryopreservation of sex-sorted bull sperm was

developed that efficacious approaches to commercialization of sexed semen could be implemented worldwide in cattle. A number

of companies now offer sex-sorted bovine sperm. Innovative approaches by a diverse group of scientists along with advances in

computer science, biophysics, cell biology, instrumentation, and applied reproductive physiology provided the basis for

commercializing sexed semen in cattle.

# 2008 Elsevier Inc. All rights reserved.

Keywords: Sexing; Sperm; Flow cytometry; Cattle; History

1. Historical background

The most sought after reproductive biotechnology of

all time, selection of sex at conception, has a long

history of great optimism, along with many disappoint-

ments. Much of this has been due to a lack of

understanding of the basic principles that govern sex

determination in mammals. This misunderstanding

* Corresponding author. Tel.: +1 530 836 0941;

fax: +1 530 836 0450.

E-mail address: [email protected] (D.L. Garner).

0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2008.01.006

goes back to at least the early Greeks when Democritus,

470-402 BC, suggested that the right testis produced

males, whereas the left testis produced females.

Why is there so much folklore about manipulating

sex ratios? Much of this is due to a lack of

understanding of probability laws. It is not all that

unusual to get 10 consecutive male births. The chances

of this happening are 1:1024 if the true sex ratio is

50:50. If there are 1 million cattle herds in North

America, getting 10 males in a row happens approxi-

mately 1000 times each year; so does 10 consecutive

females. Dairy farmers remember getting 10 of 12 bulls

in a row or 30 of 34, etc. and often ascribe it to changing

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http://dx.doi.org/10.1016/j.theriogenology.2008.01.006

D.L. Garner, G.E. Seidel Jr. / Theriogenology 69 (2008) 886–895 887

feed or some other coincidental event that occurred at

that particular time, even though the skewed sex ratio is

almost always due to chance alone.

There sometimes are distortions of sex ratios without

separating the male and female producing sperm. In

cattle, normal AI or embryo transfer (ET) results in 51%

males, whereas in vitro fertilization (IVF) results in

about 54% males [1]. Very old cows produce about 53%

male calves [2]. Management can play a role because

herds with very poor management had 49% males,

whereas herds with very good management averaged

53% males [2]. Others have suggested that the timing of

AI can alter the sex ratio [3]. However, alterations in the

sex ratio because of the timing of AI have not been

repeatable.

In the first half of the 20th century advances in the

biological sciences, especially genetics, resulted in

numerous discoveries, including identification of the

sex chromosomes. A conference entitled Sex Ratio at

Birth—Prospects for Control [4] was held at The

Pennsylvania State University to assess the current state

of the science as of 1971. Although some optimism was

generated, no substantive advances in sexing technol-

ogy were reported therein.

2. Theoretical differences

Mammalian males produce semen in which 50% of

the sperm carry the X-chromosome and 50% carry the Y-

chromosome. Many theoretical differences between the

male and female producing sperm have been suggested,

including physical differences such as size, weight and

density, swimming speed, electrical surface charges,

surface macromolecular proteins, differential effects of

pH, and differing effects of atmospheric pressure [4–13].

Although a few of these mean differences are real on a

population basis, the distributions overlap considerably,

analogous to sexing people by their height, but with the

additional problem that the differences are so small that it

is currently impossible to measure most of them

sufficiently accurate in individual sperm. Thus, sperm

are essentially identical in size, weight, electrical charge,

swimming speed, etc. The biggest problem that existed

prior to the 1980s was that there was no practical,

accurate in vitro method to determine success of attempts

to sex sperm. Therefore, there was no way to determine if

a particular method or variation actually sexed sperm

except to breed animals, and this made testing different

approaches very slow, expensive, and imprecise.

In 1981, an investor/entrepreneur, Mr. William

Goddard of Warwick Land Co., Providence, RI,

USA, approached faculty at Colorado State University

about investing in research on sexing sperm. The faculty

refused to accept funding for such research because of

not being aware of any promising approaches. Drs.

Rupert Amann and George Seidel, however, agreed to

organize a symposium on aspects of basic sperm

biology that might be relevant to sexing sperm as well as

updates from those working in this area; Warwick Land

Co. sponsored the symposium. It resulted in a book,

most of which is relevant a quarter century later [14].

However, one reviewer of the book observed that ‘‘It

appears that over the years a considerable amount of

time, effort and money have been expended producing

very few positive results.’’ Although more than 100

patents exist that claim successful sexing of sperm, most

procedures are no more efficacious than folk methods

were more than two millennia ago.

3. Breakthroughs

3.1. Sperm DNA content

In domestic cattle, the chromatin of each somatic cell

contains 60 chromosomes. Male gametes contain half

that number because the haploid X-chromosome-

bearing sperm that produce females carry 29 autosomes

plus the X-chromosome. The haploid Y-chromosome-

bearing sperm have the same 29 autosomes plus the

male determining Y-chromosome. According to Mor-

uzzi [15] the difference in total length of the bovine

chromosomes between those from bulls and cows is

approximately 4.2%.

The initial breakthrough occurred when one of the

most sophisticated weapons laboratories in the world,

Lawrence Livermore National Laboratory (LLNL), was

studying the health effects of radiation on humans using

mouse sperm as a model to indicate damage to the

germ-line DNA. Initial studies on the DNA stability of

mouse sperm and those from other mammals provided

results that were not interpretable, due to the flattened

shape of the sperm head [16,17]. This problem was

overcome by a flow cytometer that was developed by

Dr. Daniel Pinkel, which oriented the sperm so that

precise measurements of DNA content could be made

using the flattened side of the sperm head [18]. Otto

et al. [19] reported that one could also get precise DNA

content by measuring the sperm head first, termed

coaxial measurement, as utilized by the system that was

developed in Germany by Partec GmbH (Munster,

Germany). It was this system (Ortho’s Impulse

Cytophotometer, ICP-22; Ortho Diagnostic Systems,

Westwood, MA, USA [no longer in existence]) that was

used to provide the breakthrough in bull semen analyses

D.L. Garner, G.E. Seidel Jr. / Theriogenology 69 (2008) 886–895888

in 1981 [20]. Collaboration between Oklahoma State

University (OSU) and Lawrence Livermore National

Laboratory (LLNL) demonstrated the potential use of

flow cytometry to convincingly identify X- and Y-sperm

populations based on their DNA content differences. Dr.

Duane Garner, while a visiting scientist on sabbatical

leave at LLNL, submitted a research proposal entitled

‘‘Flow cytometric verification of the relative propor-

tions of X- and Y-chromosome-bearing sperm in bull

and boar semen’’ to the USDA Beltsville Agricultural

Research Center. This proposal was funded, resulting in

a cooperative research agreement between OSU,

USDA, and the US Department of Energy/LLNL. It

was this collaboration that led to the breakthrough first

reported by Gledhill et al. [20] at the conference

‘‘Prospects for Sexing Mammalian Sperm’’ which was

held in Denver, CO [14]. Garner et al. [21] showed that

this flow cytometric approach was capable of precisely

determining DNA content differences between X- and

Y-sperm from cattle, sheep, pigs, and rabbits. The

sperm, however, were killed in the process of making

them permeable to the membrane impermeant fluor-

escent dye, 40-6-diamindino-2-phenylindole (DAPI)

[21]. Sex selection in domestic animals became a

major objective once the ability to determine the

success of X- and Y-sperm separation was achieved

with flow cytometric analyses.

Flow cytometric examination of the DNA content of

killed, membrane denuded X- and Y-chromosome-

bearing sperm of cattle (Bos taurus and Bos indicus)

indicated a difference of about 3.7% [21]. According to

Garner et al. [21] the precision of this flow cytometric

system was sufficient to detect DNA content differences

between the X- and Y-chromosome-bearing sperm

among five breeds of cattle. Sperm from five Holstein

bulls averaged X–Y sperm DNA content differences of

about 4.98%, whereas those from Jersey, Angus, and

Hereford bulls averaged 4.24, 4.05, and 4.05%,

respectively. When sperm from Brahman bulls (B.

indicus) were examined, the average X–Y sperm

difference was 3.73% [21–23].

3.2. Sex sorting living sperm by DNA content

Thus far, we have concentrated on measuring DNA

content, not sorting sperm. Sperm sorting technology

was first developed at Lawrence Livermore National

Laboratory where Pinkel et al. [24] separated the X- and

O-sperm nuclei of the vole, Microtus oregoni, which

have 9% DNA content difference of its sex determining

chromosomes. This sperm sorting technology was then

implemented at the USDA Beltsville Agricultural

Research Center for application to domestic livestock

sperm. At that time, the staining system used to sort

sperm by DNA content removed the sperm membranes,

thus killing them [18,21,24]. It was not until Johnson

et al. [25–27] altered the staining process that removal

of cell membranes from sperm was no longer necessary

to achieve precise DNA staining They used the

membrane permeant bisbenzimidazole fluorescent

dye, Hoechst 33342, to stain the DNA in intact sperm.

Although the DNA content of living cells was first

determined using Hoechst 33342 to stain tissue culture

cells [28], the first reported use of this stain on living

mammalian sperm was by Morrell et al., who used such

living bull and rabbit sperm for insemination [29,30].

Although the purity of the sorted sperm populations

were not accurately determined, inseminations with

5 � 106 (bull) or 7.5 � 106 (rabbit) stained, sorted

sperm resulted in live offspring from both cows and

does.

In 1989, a major breakthrough in sperm sexing was

reported by Johnson et al. [31]. The USDA Beltsville

Research Center group reported production of live

offspring from sex-sorted, living rabbit sperm [31]. This

was the first verified report where the sex of offspring

had been predetermined at conception by sorting living

sperm into the respective X- and Y-chromosome-

bearing sperm populations. Sperm were stained with

Hoechst 33342, sorted according to their DNA content,

and then surgically inseminated into the oviducts of

rabbits [31]. Insemination of sex-sorted Y-chromo-

some-bearing sperm resulted in 81% males (17/21),

whereas insemination of X-chromosome-bearing sperm

resulted in 94% females (15/16).

Sperm DNA is stoichiometrically stained with

Hoechst 33342, and then the sperm are pumped in a

stream in front of a laser beam at specific wavelengths

[27,32–35]. The illuminated Hoechst 33342 stained

sperm emit a very bright blue fluorescence. This

fluorescence is rapidly measured using a photomulti-

plier tube (PMT) as the sperm flow single-file in front of

the PMT. A high speed computer is used to analyze the

relative fluorescence of the X- and Y-sperm populations

as they flow through the instrument in a fluidic stream.

A crystal vibrator is used to break the stream into

individual droplets, many of which contain a sperm.

The fluorescently stained sperm are sorted by DNA

content by placing opposite charges on droplets

containing X-sperm from those containing Y-sperm

(Fig. 1). The droplets fall past positive and negative

electrical fields, and since opposite charges attract, the

droplets separate into two streams for collection. A third

stream of uncharged droplets is discarded; these


Fig. 1. Dual-headed sperm flow cytometer/sperm sorter (Dako MoFlo1 SX) as currently used to commercially sort sexed sperm at Sexing

Technologies, Navasota, TX, USA. The system has been redesigned from the original MoFlo1 SX by K. Michael Evans so that the solid-state UV

laser beam could be split and directed into two sorting heads (nozzles).

droplets have sperm that could not accurately be sexed

(over half), no sperm, rarely two sperm, as well as dead

sperm. The gating out of dead sperm is a valuable fringe

benefit of this process. This sperm sorting technology is

known as the Beltsville Sperm Sexing Technology and

was patented by the USDA (US Patent #692958, 04/26/

1991), with Dr. Lawrence Johnson as the inventor.

4. Limited availability of sexed sperm

The commonly used insemination dose for cattle

usually is 20 � 106 or more cryopreserved sperm.

Initially sex-sorting X- and Y-sperm by flow cytometry

had a serious limitation because the instruments used

for sorting individual sperm by their DNA content were

too slow to produce adequate numbers for use in

artificial insemination. Initially a sperm sexing system

could only sort about 400,000 sperm/h. Thus, it would

take 25 h to sort one insemination dose of 10 � 106

sperm of each sex. Studies on the minimum number of

sperm per insemination dose required to achieve

acceptable pregnancy rates in cattle using non-return

to estrus estimates revealed that a 50% non-return rate

could be achieved with as few as 0.38 � 106 live sperm

post-thaw per insemination [36].

5. Initial IVF commercialization approach

One approach to overcome the limitation in sperm

numbers due to being able to sort only a few hundred

thousand sperm per hour was to use the sorted sperm for

IVF. Cran et al. [37] reported that Mastercalf Ltd. of the

United Kingdom produced male beef cattle embryos

using this approach. Sperm were sorted by modifying a

Becton Dickinson FACStar Plus flow cytometer/cell

sorter which could sort sperm at about 100 sperm/s at a

purity of 79% for X-sperm and 70% for Y-sperm. Twin

IVF embryos resulting from fertilization of oocytes with

these sex-sorted sperm were transferred, producing four

pregnancies. In a later experiment, embryos were

cryopreserved for future use. After thawing, two

embryos produced with sexed sperm were transferred

to each recipient cow. Of 106 cows that received twin

embryos, 35 calved resulting in 4 females (10%) and 37

males (90%) [38]. This IVF approach to using sex-

sorted sperm was impressive technically, but did not

achieve financial success.

6. Artificial insemination approach

In the early 1990s, the numbers of sperm that could

be sexed accurately and sorted per unit time was

discouragingly low, so artificial insemination was ruled

out for sexed sperm. Nevertheless, in 1994 a research

proposal was submitted by Dr. George Seidel of

Colorado State University to the National Association

of Animal Breeders (NAAB) to determine conception

rates in heifers with as few as 100,000 total unfrozen,

unsexed sperm. That proposal was rejected by the

NAAB Research Committee. However, Charles Allen


from Atlantic Breeders Cooperative, who was a member

of that Research Committee, contacted Colorado State

University and offered to fund the proposal using

research funds that Atlantic Breeders had available.

This rather bold move resulted in some enticing data

[39,40] and a subsequent ‘‘slippery slope’’ of events that

led to commercialization of sexed sperm for artificial

insemination.

In 1995, the data obtained the previous year with low

doses of unsexed, unfrozen semen led to a heroic

experiment in which bovine sperm were collected in the

early morning by Charles Allen at the Atlantic Breeders

Cooperative (now part of GENEX), Lancaster, PA, USA.

The freshly collected sperm samples were diluted 1:4 in a

HEPES-buffered extender and transported by car to

Beltsville, MD, USA (�160 km). The sperm were

prepared at that site for sexing by staining with Hoechst

33342 prior to sex-sorting from about 10 a.m. until

approximately 3 p.m. using flow cytometry. The sorted

sperm were then placed in an Equitainer1 (Hamilton

Research Inc., South Hamilton, MA, USA), so the

cooling to 5 8C would occur during shipment, and then

taken to the local airport where they were shipped by

commercial airlines to Denver, CO, USA (�2600 km).

The sexed sperm samples arrived in Colorado at

approximately 8 p.m.; partially due to the time zone

change from the east coast. The samples were then

transported by automobile to Fort Collins, CO

(�120 km), where they were loaded into straws for

insemination. The straws of sexed sperm were insemi-

nated into estrous-synchronized heifers at a local dairy at

approximately midnight. The sexed sperm were depos-

ited deep in the uterine horn ipsilateral to the ovary with

the largest follicle (as determined by ultrasonography).

Twenty-nine heifers were inseminated; 14 were verified

pregnant at 4 week of gestation, and 12 (41%) were

pregnant at 8 week. When inseminations were done

within 12 h of the sexing procedure conception rates

approached 50% [39]. Seidel et al. [40] found that

fertility decreased considerably, however, with sexed,

cooled, and transported sperm when inseminations took

place 17 h or more after sorting.

The conception rates from this initial trial were

encouraging enough that the USDA granted a license to

the Colorado State University Research Foundation

(CSURF), Fort Collins, CO, USA, to proceed with

commercialization of the Beltsville Sperm Sexing

Technology for sex sorting non-human mammalian

sperm. With issuance of this license a company, XY

Inc., was formed. This was a collaborative effort

between CSURF, Cytomation Inc. (a company that

manufactured flow cytometers) and private investors.

The USDA also granted an exclusive license for sex-

sorting human sperm to Genetics and IVF Institute

(GIVF), Fairfax, VA, USA. At GIVF, the Beltsville

Sperm Sexing Technology was adapted for use with

human sperm and commercialized as MicroSort1 [41].

Two options have been offered commercially: an

intrauterine insemination (IUI) approach or the use of

sexed sperm with IVF. When this system was used to

sort X-chromosome-bearing human sperm to increase

the probability of conceiving a girl (XSORT1), the

average purity was 88% X-sperm, with 92% of the

babies born using this sorting system being female (668

females from 726 births; http://www.microsort.net/

results.php). Using this system, sorting Y-chromo-

some-bearing human sperm proved to be slightly more

difficult with a YSORT1 purity of 73% resulting in

81% of the babies conceived being males (172 males of

211 births; http://www.microsort.net/results.php).

Overall clinical conception rates for sorted human

sperm using IUI are reported to be 32%. This approach

is especially appropriate for selecting female babies if

the probability of X-linked genetic disease is high for

males.

7. High speed flow cytometry

Cytomation Inc., a Fort Collins, CO, USA,

biotechnology company, had acquired the rights of

the high speed flow cytometer/sorter that had been

developed at Lawrence Livermore National Laboratory

by Van den Engh and Stokdijk [42]. This system was

commercialized at Cytomation as the MoFloTM

cytometer. The standard MoFlo1 was modified as a

sperm sorter by using a beveled injection needle similar

to what had been originally developed by Fulwyler [43]

and used by Pinkel et al. [18,24] to orient sperm. This

instrument, MoFlo SXTM can precisely measure sperm

DNA content from the flat surface of the sperm head. As

previously noted, this precision enabled measurement

of the small difference in DNA content between the X-

and Y-sperm. The original sperm sorter developed at the

USDA in Beltsville utilized the beveled injection needle

within the sorting nozzle of an Ortho flow cytometer/

cell sorter. This system was capable of sorting a few

hundred sexed sperm/s [32]. Later the sperm sorter

system was further enhanced by a novel modification of

the sorter nozzle whereby as many as 70% of the sperm

were oriented using the pressure of the hydrostatic

fluidic system [44,45]. This modification was upgraded

by Cytomation Inc. so that this new fluidic orientation

system resulted in analysis at rates exceeding

http://www.microsort.net/results.php




20,000 sperm/s and sorting up to 6000 or more sperm/s

each of X- and Y-sperm at 90+% accuracy.

In 2003, Cytomation Inc. was acquired by Dako, a

Danish biotechnology company. The name of the flow

cytometer manufacturer was first changed to DakoCy-

tomation A/S and then to DAKO A/S. The company

continues to produce the MoFlo SXTM sperm sorter, but

recently the flow cytometry instrumentation division

was purchased by Beckman Coulter (Fullerton, CA,

USA).

8. Cryopreservation of sex-sorted sperm

The availability of bovine sex-sorted sperm was

severely limited by the difficulty of collecting and

sorting adequate numbers of sexed sperm for use in AI,

and then having to ship the unfrozen sexed-sperm to a

site where the females to be inseminated were housed.

This is impractical, especially on a routine basis at the

distances described by Seidel et al. [39]. Efficacious

cryopreservation of sexed sperm circumvents this

problem. The first report of successful cryopreservation

of sex-sorted bovine sperm demonstrated that relatively

conventional cryopreservation methods using an egg-

yolk-Tris buffer medium worked well with sorted sperm

[46]. This advance enabled the sperm sorting systems to

be housed near where the sperm were collected, and the

females to be inseminated with sexed sperm could be

located almost anywhere. Successful cryopreservation

of sex-sorted sperm was followed by numerous field

trials to determine the potential of using cryopreserved,

sex-sorted bovine sperm commercially [47–50]. Many

of these advances in sexing sperm were reported at a

conference held in Maastricht, The Netherlands in 2000

[51].

9. Modifications of the sperm sexing system

An examination of the stability of sperm DNA

during and following the sex-sorting process indicated

that no significant damage occurred to the sperm

chromatin during the process [52,53]. Furthermore,

similar examinations of sperm viability, using SYBR-

14/propidium iodide staining, revealed that very little

damage occurred from staining the sperm with Hoechst

33342 or from exposure to the UV laser during sorting.

However, examination of sperm viability revealed that

some mechanical damage occurred during the sorting

process [23,52]. Lowering the fluidic pressure from 50

to 40 psi reduced the level of damage [53]. This change

increased the proportion of viable sex-sorted sperm that

could be recovered after sorting.

10. Normalcy of calves from sexed sperm

The incidence of chromosomal aberration suggested

that mammalian sperm chromatin might be damaged by

exposure to the Hoechst 33342 dye and UV laser

illumination during flow sorting [54]. However, no

obvious DNA damage was observed using the Sperm

Chromatin Structure Assay (SCSA) with sex-sorted

sperm [55]. Some concern remains over potential

genetic damage to the sperm DNA due to staining the

sperm with Hoechst 33342 and exposing them to

intense laser light during the sorting process. An

important point is that the wavelengths of laser light

used are not absorbed by DNA or proteins, thus

minimizing potential damage.

An examination of the data from calves was done

from field trials with sexed sperm, which included

calves from unsexed control semen in the same herds.

This large study found no increases in the abortion

rate, or differences in gestation length, neonatal death,

calving difficulty, birth weight, weaning weight, or

live births when sexed sperm were used for AI

compared to births when unsexed control sperm were

inseminated [56]. A recent report from commercial

applications shows that abortion rates in heifers

inseminated with sexed sperm (1.4%, n = 5495) did

not differ from that found for conventional semen

(1.9%, n = 4902) [57]. An important benefit, at least in

dairy cattle where mostly females are produced with

sexed sperm, is the naturally smaller birth weight of

the heifer calves. This lowers the incidence of

dystocia. The study by Tubman et al. [56] included

over 1000 calves resulting from sexed sperm, but

had insufficient numbers of calves to detect very

small differences in calf normalcy; it would take

tens of thousands of births to reveal small differences.

Furthermore, damage resulting in recessive

mutations would not be readily detected with this

approach. However, it appears that genetic damage to

sperm during sex-sorting is likely minimal or non-

existent.

11. Conception rates with sexed sperm in

commercial settings

Recommended commercial applications of sex-

sorted sperm have limited its use primarily to heifers

because of their inherent higher fertility and the limited

number of sexed sperm available. It is very difficult to

get unbiased field comparisons of sex-sorted sperm with

conventionally packaged unsexed sperm due to likely

biases in semen use.


DeJarnette et al. [57] reported conception rates of

44% (n = 16,587) across 121 Holstein herds where

heifers were inseminated with sperm sorted for 90% X-

sperm at Sexing Technologies (Navasota, TX, USA). In

this particular example, the conception rates of sexed

sperm (2.1 � 106 sperm/dose) were 85 � 3% of those

obtained at first service with conventional unsexed

semen (�20 � 106 sperm/dose). In 74% of these herds

the conception rates for sexed semen were at least 70%

of that achieved with conventional semen. Among 25

herds where more than 100 doses of sexed semen were

used (608 � 122 inseminations/herd), conception rates

averaged 48.2% and ranged from 33 to 72%. These

varying results among herds using sex-sorted semen

indicate that management level is an important factor to

consider when applying such techniques.

In another example, commercially sexed sperm were

used to inseminate both Jersey heifers and cows,

demonstrating that reasonable conception rates could be

achieved with sex-sorted sperm when used in well-

managed commercial dairy herds (Juan F. Moreno,

personal communication). Cattle were inseminated with

sperm from two bulls (A and B) that had been sex-sorted

for X-chromosome containing sperm at Sexing Tech-

nologies (Navasota, TX). The sex-sorted sperm were

packaged in 0.25-mL straws (2.1 � 106 sperm/straw),

cryopreserved and shipped to California for use at Jer-

Z-Boyz commercial dairy operations in Pixley, CA,

USA. Conception rates with sex-sorted sperm, deter-

mined by transrectal palpation 35–45 d post-insemina-

tion, were quite satisfactory, 471/825 (57%) for heifers

and 1265/3285 (39%) for cows. Although direct

comparisons may not be valid, conventionally pro-

cessed, unsorted, cryopreserved semen from many bulls

(�20 � 106 sperm/dose) from several bull studs

resulted in similar conception rates. As previously

reported [58], conception rates with sexed sperm were

lower in cows than in heifers. This difference between

heifers and cows also was noted when conventionally

processed semen was used.

Currently, it takes approximately 9 min for a single

sorter to produce one straw of sexed sperm, which is

approximately 7 straws/h at 2 � 106 sperm/dose. Var-

iations in sperm-sorting rate occur due to many factors

including initial semen quality factors such as sperm

concentration, viability, motility, and DNA staining

differences among bulls [59,60]. However, this sex pre-

selection system has produced over 2 � 106 commer-

cial doses of sexed, cryopreserved bovine sperm in the

USA during 2007. The MoFlo1SX sperm sorter has

undergone some recent modifications by K. Michael

Evans so that the solid-state laser is directed into two

separate sorting units, thereby doubling the output from

a single machine (Fig. 1).

12. Application to a number of species

In addition to cattle, flow cytometric sperm sexing

has been applied to sperm from a variety of mammalian

species including sheep [61–64], rabbits [31], swine

[65–69], horses [70,71], elk [72], cats [73], dolphins

[74], humans [41], non-human primates [75–77], and

dogs [78].

13. Commercial involvement

Four companies were initially involved in the

development of flow cytometric sorting to predetermine

the sex of offspring in cattle. These are American

Breeders Service (now ABS Global) which provided the

semen used at LLNL in 1981, Atlantic Breeders

Cooperative (now part of Genex), Mastercalf Ltd. (no

longer in existence), and XY Inc. (stock now owned by

Sexing Technologies). Other bull stud operations

assisting in the early field trials in the USA include

Advanced Dairy Genetics and Select Sires. Other

worldwide contributors are Cogent Ltd. (UK), Goyaike

Ltd. (Argentina), Hokkaido Genetics (Japan), and the

Livestock Improvement Association of Japan.

Recently, Monsanto (St. Louis, Mo, USA), devel-

oped a unique sperm sorting system that utilized 16

sorter nozzles instead of a single one. This system was

scheduled to become commercialized in 2006, but due

to apparent problems with lower conception rates than

were found in their early field trials, they abandoned this

effort. Genetic Resources International/Sexing Tech-

nologies of Navasota, TX, USA, has purchased the

related intellectual property and the sperm sex-sorting

equipment developed by Monsanto, and also has

acquired all stock of XY Inc.

14. Future

No method, other than flow sorting, has been shown

to repeatedly produce viable sexed sperm on a level

necessary for commercialization. However, it is

probable that simpler, better systems will be developed

for sexing sperm. Such developments should facilitate

not only a decrease in the cost of sexed sperm in cattle

but should speed application of sperm sexing to other

species. It is also likely that improvements in the current

system will increase the efficiency of the system and

improve the fertility of sexed sperm [14]. That one can

easily determine the effectiveness of a sexing procedure


with flow cytometry and the process termed sort

reanalysis [79] should facilitate development of better,

simpler methods of sexing sperm.

An important development toward gaining greater

access to superior sires is the sex-sorting of frozen-

thawed unsorted bull semen followed by successful

refreezing of the sorted sperm [80]. This advancement

preceded a sustained effort by de Graaf et al. [81] to

develop this with ram semen, whereby the birth of

offspring of a predetermined sex using frozen-thawed,

sex-sorted, and re-frozen ram sperm was achieved with

encouraging pregnancy rates.

In IVF experiments by Morton et al. [82], bovine

embryos from sex-sorted sperm had altered mRNA

expression patterns. This was not surprising, as it was

previously noted that embryos from sex-sorted sperm

developed somewhat slower than those from unsorted

sperm [83]. The use of sex-sorted bovine sperm in IVF

needs to be optimized because use of this approach to

sex pre-selection is likely to increase substantially.

Commercial sex pre-selection in cattle, using flow

cytometric sorting of sperm, was brought about by the

innovative approaches of a diverse group of scientists

and commercial interests, along with advances in

computer science, biophysics, cell biology, instrumen-

tation, and applied reproductive physiology. The

commercial success of flow cytometric separation of

X- and Y-sperm has stimulated renewed interests in

developing other, perhaps more efficient methods, to

pre-select the sex of mammalian offspring.

Acknowledgements

Many individuals contributed to the development

of commercialized sexed sperm, including many

cattle breeders and investors. Especially important

were farmers and ranchers who allowed us to use their

cattle for experiments. Individuals involved in devel-

oping the process include Charles Allen, Rupert

Amann, Zell Brink, David Cran, Patrick Doyle, Mike

Evans, Duane Garner, Roger Gerrits, Bart Gledhill,

Ronnie Green, John Hasler, Lisa Herickhoff, Michael

Holland, Larry Johnson, Kevin McSweeney, Gordon

Niswender, Dan Pinkel, Chris Polge, Wim Rens, John

Schenk, George Seidel, Tae-Kwang Suh, Marvin Van

Dilla, Glenn Welch, and many others.

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history of commercializing sexed semen for cattle

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