Gender Preselection in Domestic Animals Using Flow Cytometrically Sorted Sperm Lawrence A. Johnson J Anim Sci 1992. 70:8-18.

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Gender Preselection in Domestic Animals Using Flow Cytometrically Sorted Sperm

Lawrence A. Johnson Germplasm & Gamete Physiology Laboratory, Beltsville, MD 20705

U.S. Department of Agriculture, ARS,

ABSTRACT: Preselecting the gender of offspring in both humans and animals has been of keen interest since the beginning of recorded history. Numerous attempts have been made to accomplish gender preselection in both humans and animals. However, virtually all attempts have ended in failure, primarily because they lacked a scientific basis upon which to determine a measurable difference. Some methods have been derived from folklore, in which case there was virtually no possibility for success. This paper deals briefly with the numerous physical separation methods that have been proposed and(or1 used to separate X- and Y-chromosome-bearing sperm, but the main

emphasis is given to the technique of flow cytometry for sperm validation and X- and Y-bearing sperm separation. Flow cytometric analysis of sperm DNA is very useful for evaluating the proportions of X- and Y-bearing sperm in a sample of semen. Similarly, flow cytometric sorting of Xand Y-bearing sperm has also proven itself to be the only laboratory method that skews the sex ratio of semen. This flow cytometric sorting method has been used successfully to produce progeny from rabbits and swine surgically inseminated from separate populations of X- and Ybearing sperm.

Key Words: Sex, DNA, Sperm, Flow Cytometry, X Chromosome, Y Chromosome

J. Anim. Sci. 1992. 7O(Suppl. 2l8-18

Introduction
Controlling the sex of offspring has been of interest to humans since the beginning of history. This interest has generally focused on some intervention prior to or after coitus or insemination or in vitro manipulation of sperm. The latter is of the keenest interest, because sex determination is based on the chromosome content of the fertilizing sperm. This paper will present some of the historical aspects of sex selection, briefly review the various methods that have been used, and then discuss in more detail the use of flow cytometric analysis and flow sorting for the purpose of preselecting the sex of domestic livestock progeny. Historical Aspects of Gender Preselection. Theories for controlling the sex of offspring have been

The author gratefully acknowledges the important contributions of Glenn Welch, Cindi Garbus-Gooch, James Flook, Mary Look, and Dan Pinkel toward the success of these studies and thanks Duane Garner and Phil Senger for their helpful comments on the manuscript.

prevalent since the time of the Greek philosophers. Some believed that females developed on the left side of the uterus and males on the right. Even as late as the 18th century there were those who thought that sperm from the right testes produced more males and that sperm from the left testes produced more females. Coincident to theories for gender control in humans, numerous approaches emerged and were applied to livestock. As was the case for humans, few were based on scientific fact. One of the first serious scientific studies to be conducted to control prenatal sex was reported by J. L. Lush (1925). The basis for Dr. Lushs research was the possible differential density of X- and Ybearing sperm in the rabbit. The progeny from the inseminations made with sperm separated by centrifugation failed to show altered sex ratios. Since then, innumerable reports have appeared describing a wide variety of methods to separate X- and Y-bearing sperm. The majority of these methods can be grouped under the broad heading physical separation methods. They are based on actual or perceived differences in the weight, density, size, motility, or surface charge of sperm.
8

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SEX SELECTION IN DOMESTIC ANIMALS

BASED ON DNA

For detailed reviews consult Kiddy and Hafs (19711, Amann and Seidel (19821, and Gledhill (1988). In many instances positive results were reported, but carefully controlled validation studies were lacking. In most cases, follow-up studies failed to confirm earlier results. In addition, there have been reports declaiming the use of H-Y antibody as a means of selecting only the X-bearing sperm (Zavos, 1985).The basic premise of the H-Y antigen method seems to be questionable: Hoppe and Koo (1984) were unable to show haploid expression of H-Y antigen by mouse sperm. Hoppe and Koo (1984) also reported that X- and Y-bearing sperm probably share the same surface antigen due to their origin in the same testicular milieu. The application of flow cytometric analysis of individual sperm for DNA content to determine the proportions of X- and Y-bearing sperm in a sample of semen that has been purported to skew the sex ratio has served a s a reliable check on procedures being developed (Pinkel et al., 1985; Johnson, 19881. In no case has there been evidence to support the claims made for various physical separation procedures or other non-DNA-based separation procedures (unpublished datal. Exploiting some aspect of X- and Y-bearing sperm differences is the ideal method on which sex preselection should be based. Only then is gender development of the fertilized egg known. This allows for the greatest economy in developing sexed embryos, because all manipulation occurs prior to fertilization. The first major scientific symposium concerning sex control in livestock was held in 1970 a t Penn State University. In the summation of that conference (Kiddy and Hafs, 1971) Dr. L. E. Casida observed the following: Were still testing many of the same ideas first considered a half a century or more ago. . .. What we really need is a new idea, a new approach. I suggest that the measurement of total DNA in the individual X- or Y-bearing sperm (Gledhill et al., 1976; Pinkel et al., 198219; Garner et al., 1983; Johnson and Pinkel, 1986) and subsequently the verified separation of X- and Y-chromosome-bearing sperm (Johnson et al., 198713, 1989; Johnson, 1991) represents a new approach to sexing semen. The development of modern flow cytometry/cell sorting technology is generally credited to Fulwyler (1965) and Kamentsky and Melamed (1967). Flow systems were commercialized in the 1970s and developed rapidly in conjunction with the computer revolution in the 1980s. Although the primary application has been in medical research and diagnosis with respect to blood cells, flow cytometry (FCMI can be a n effective tool for many types of cell suspensions. Obviously, sperm in suspension are readily adapted to flow analysis and sorting. The usefulness of flow cytometry is

illustrated by the ability to measure the relative DNA content of individual sperm a t a relatively rapid rate.

Flow Cytometry
Until the development of FCM, the only method available to evaluate the sex potential of a sample of semen was to determine the sex ratio among progeny. The limitation of fertility testing is obvious. High cost, time constraints, and, perhaps most important, delays in the evaluation of methodology all serve to diminish the incentive necess a r y for development of separation methods. The power of FCM lies in the fact that thousands of cells can be analyzed in a matter of seconds or minutes. Sex chromosomes were described in mammals just after the turn of the century (Guyer, 1910). In 1944, Avery and coworkers published experimental results that established DNA as the carrier of genetic information. Moruzzi (1979) identified numerous mammalian species with maximal differences in bulk chromatin between X- and Ybearing sperm that would be good candidates for sperm separation experiments. The basis for measurement was visual estimation of karyotypes. Gledhill et al. (1976) pioneered the use of flow cytometry for evaluating sperm DNA content to use as a n indicator of mutagenic events. Nuclear DNA content differs in X- and Y-chromosomebearing sperm of virtually all mammals, with some unique exceptions (e.g., the creeping vole, Microtus oregonil (Ohno, 1967; Pinkel et al., 1982a; Johnson and Clarke, 1989). The X-bearing sperm always carries more total DNA than the Y-bearing sperm because the X chromosome is larger than the Y chromosome. Although the DNA content of the autosomes does not differ between the X- and Ybearing sperm, the heterologous DNA in the sex chromosomes do differ. This difference in DNA mass between the X and Y chromosome of sperm is still the only established and measurable differential parameter. By accurately measuring the DNA content of a s few as 1,000 sperm, one can determine the ratio of Y to X sperm Ke., sperm sex ratio). This measurement can be done on virtually any sample of semen that has at least a 3% difference in DNA mass between X- and Y-bearing sperm (Johnson and Pinkel, 1986; Johnson et al., 1987a).

Flow Cytometric Instrumentation for Sperm

High-resolution flow cytometric DNA analysis of sperm nuclei is more difficult than such analysis of other types of cells because of the inordinate

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10

JOHNSON

compactness of chromatin in the morphologically flat, paddle-shaped sperm head characteristic in some mammals. The dense packing of the chromatin causes a high index of refraction. The difference in refractive index between the sperm nuclei and the surrounding medium, coupled with the flat shape of the sperm head, results in preferential emission of light in one plane of the cell. Because of these properties, the orientation of the sperm head with respect to the excitation laser beam and the optical detectors that collect the emitted

.
I

Figure 1. Illustration of desired orientation of the sperm relative to the beveled exit end of the sample insertion tube and intersection with the laser beam. The sperm take up and are aligned in the same plane as the ribbon-shaped stream caused by the beveling of the sample insertion tube. The laser beam is incident on the flat surface of the sperm. The edge of the sperm that emits the brighter fluorescence is directed to the 90-degree detector. The forward fluorescence detector receives the emitting fluorescence from the flat face of the sperm head, opposite to the other face, which is excited by the laser beam. Note: Size relationships of sperm, needle, and fluids are not shown in actual proportions.

fluorescence is critical for resolution. Therefore, to adequately resolve the differences in DNA between X- and Y-chromosome-bearing sperm, one must use a flow cytometer (Pinkel et al., 1982131 in which the fluorescent signal collected is independent of the orientation of the nuclei. Pinkel et al. (1982b)demonstrated that flow cytometric analysis for DNA to differentiate X-and Y-bearing sperm is possible using either an epi-illumination flow cytometer or a specially built orienting flow cytometer. Garner et al. (1983) then used the epiillumination flow cytometer to demonstrate the potential of routine sperm evaluation for domestic animals. This study showed that X- and Y-bearing sperm from the various farm animals could be differentiated so as to determine the ratio of Y to X in a sample of semen. Cockerel sperm were useful in establishing the validity of the DNA analysis because they are homogametic rather than heterogametic; therefore, one would expect the DNA content to be equal in all avian sperm (Garner et al., 1983; Johnson, 19861. Orientation of sperm is critical to the potential for sorting sperm. Thus, the modification of a n orthogonal type flow cytometer was considered to be essential. This led to the modification of a commercial flow cytometer (Epics V, Coulter Corporation, Hialeah, FL; Johnson and Pinkel, 1986). To control the orientation of the sperm head in the flow stream one must first control the shape of the fluid stream in which the sperm head is suspended (Figure 1). This is done by beveling the exit orifice of the sample insertion tube from 20 to 30 degrees. This modification to a commercially produced flow cytometer/cell sorter with orthogonal optical geometry along with the addition of a forward fluorescent detector is essential for measuring sperm DNA (Johnson and Pinkel, 1986). This system, which has been in operation in my laboratory for over 9 yr, produces nearly uniform orientation of sperm nuclei, collects the fluorescent signal from both the flat side and the edges of the nuclei (Johnson et al., 1987a1, and is capable of cell sorting (Johnson et al., 1987b; Johnson and Clarke, 1988). The fluorescent signal emitted from the sperm edge @@degree angle from laser emission) is brighter than that emitted from the flat side (Figure 1 forward detector). The edge emission is used to characterize the orientation of the sperm heads as they pass the laser beam. The emitting light is collected by the %)-degree detector. Misoriented sperm give off less light and therefore can be electronically gated out of the analysis. In general, about 65 to 95% of the sperm heads are properly oriented. This percentage is species- and sample-dependent. Of the paddle-shaped sperm that we have analyzed, rabbit sperm has proven the easiest to orientate, for reasons still unknown.

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SEX SELECTION IN DOMESTIC ANIMALS BASED ON DNA

11

The complete schematic of the flow system is shown in Figure 2.

Flow Cytometric Analysis

The ability to confirm the purity of X or Y sample enrichment can be monitored directly on an individual sperm basis, allowing a distinct advantage to sperm separation research. Formerly, one was forced to use the sex ratio of progeny to determine the efficiency of separation. Determining sex ratio among progeny can now be delayed until one is virtually certain of the success of the procedure, reducing cost and allowing one to plan the critical fertility experiments with much higher precision. Sperm are stained with a DNA-specific dye (Hoechst 33342, Calbiochem, L a Jolla, CAI. These fluorescently stained sperm heads are introduced under pressure into the flow cytometer in liquid suspension. The sperm enter the sample insertion tube and are oriented as they exit the beveled end of the tube into sheath fluid. This procedure maintains the integrity of the sample stream (laminar flow) as the two fluids (sample and sheath) pass out through the 76-micron orifice of the flow cell nozzle. The stream containing the sperm then intersects a laser beam generated by a n Innova 90-5 Argon-ion laser (Coherent, Palo Alto, CAI. The lasing occurs in the ultraviolet (351, 364 nm) wavelengths at up to 200 mW of power. The fluorescently stained nuclei are individually excited by the laser beam, giving off fluorescent signals proportional to the amount of DNA that has been bound by the dye. These signals are received by the photomultiplier tubes, amplified, converted from analog to digital format, and outputed as a frequency distribution. Flow cytometric analysis of mammalian sperm DNA content in a given sample of semen results in a biomodal distribution representing X- and Ychromosome-bearing sperm populations. Data collected from a single sample can be transferred to another computer and fitted to a pair of Gaussian distributions whose means, relative areas, and coefficients of variation are adjusted to give the best least squares fit to the data (Johnson et al., 1987a). The percentage of separation of the two populations is calculated by the difference = 100 [x - y/.5 (x + yll, where x and y are the respective channel means for the two peaks. Figure 3 illustrates typical histograms for several species from which semen has been analyzed to determine sperm sex ratio. Although the 3 to 4% DNA difference between X- and Y-bearing sperm is small (Table 11, FCM has the resolving power to repeatably distinguish generally nontoxic to living cells. This is not

Sperm Suspension-)

chi

DEFLECTION PLATES

1 BOo Detector IOo Detector1

y\
COLLECTION
TUBES'

Fluorescence Per Sperm

Figure 2. This schematic drawing illustrates the flow cytometric analysis and sorting system as modified for measurement of DNA in sperm. The sample of suspended sperm is drawn into the flow cell through a stainless steel insertion tube, where it is injected into sheath fluid from the beveled end of the sample insertion tube; both streams exit the orifice of the flow cell nozzle. The sperm flow in single file and intersect the laser beam, which excites the fluorescent dye. The light given off is collected by the two optical detectors. Analysis of the sample is now complete. If sorting is done to the sample, circuits are activated so that a charging pulse ( + or -) is given to the respective X- or Y-bearing sperm, which are encased in liquid droplets resulting from high-speed vibration of the flow cell. As the droplets fall, they pass through an electrostatic field that pulls the charged droplets containing sperm into separate tubes. The tubes containing a small amount of Test-Yolk semen extender receive the sorted sperm. The viable sperm are then used for surgical insemination or in vitro fertilization protocols. An aliquot of the sorted sperm is taken for reanalysis as described in the text.

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12

JOHNSON

X- from Y-bearing sperm (Johnson et al., 1987b; Johnson and Clarke, 19881. This can only be done with efficiency and consistency if one is using the beveled tip and forward detector as modifications to the standard orthogonal flow cytometer. Sperm Preparation and Staining. Because sperm can be held in suspension, preparations from

virtually any species, including birds, can be analyzed by flow cytometry (Figures 2 and 4). Sperm may be washed to remove seminal plasma, although it is not essential. The sperm are then subjected to sonication for about 10 s (Figure 41 to break off the tails (Johnson et al., 1987a). The sperm are then stained with Hoechst 33342,a vital

c31:

BULL

500 1

BOAR

400
400

Y
300

X
300

%Y
200

x-Y C . V .

I
loo
0

49

3.9% 1.1%

XY x-Y
200 C.V.

=
100

49

3.5%
1.2%

I
0 100

II
200

300

lool
0 1
0

I
X
300

J \ 200

Erc

CHINCHILLA
500

STALLION
Y

5001

aOo

aoo
300

200

XY x-Y CV
'

48 4.2%

1.6%

100

I \

RELATIVE

DNA

CONTENT

Figure 3. Histograms resulting from the flow cytometric analysis of sperm heads (nuclei] from several species. Note the spacing of the X- and Y-chromosome-bearing peaks depending on the species and the percentage of difference in DNA content between the X- and Y-bearing sperm. Turkey exhibits a single peak, because the male turkey is homogametic. Therefore, one would expect all sperm to carry the same DNA complement.
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SEX SELECTION IN DOMESTIC ANIMALS BASED ON DNA

13

fluorochrome that preferentially binds to the adenine-thymine regions of the DNA helix (Muller and Gautier, 1975). The mechanism of attachment is binding rather than intercalation. Thus, the dye is important relative to simple flow cytometric DNA analysis of sperm nuclei (Johnson et al., 1987a1; however, when one is interested in maintaining viability for insemination of intact sperm (Figure 41,the use of Hoechst 33342 is essential (Johnson et al., 1989; Johnson, 19911. Flow cytometric analysis to determine sperm sex ratio of semen is currently routine procedure for most mammals, excluding humans (Johnson, 1988). Human sperm are characterized by a more angular or bullet-shaped head, which makes orientation difficult. Also, the DNA difference between X- and Y-bearing sperm is less than 3.0% (Johnson and Welch, 19911, which necessitates increased precision in the analysis. Samples of semen that have been prepared by one of numerous physical separation procedures have been received from various sources. The sources have been both commercial and academic. The samples have been prepared as sperm nuclei and flow cytometrically analyzed for sperm sex ratio. Analysis of over 300 samples prepared by various separation methods has resulted in 50:50 proportions in all samples (Johnson, 1988; Upreti et al., 1988). This technique is a n essential tool to

Table 1. Percentage of Difference in DNA content between X- and Y-chromosome-bearing sperm as determined by flow cytometric analysis
%

Species Turkey Human Rabbit Swine Cattle Dog Horse Sheep Dorcus gazelle Muntjac Chinchilla Microtus oregoni Microtus oregoni *Creeping vole, habitating in Oregon. bCreeping vole, habitating in Washington

Difference
0 2.9 3.0 3.6 3.8 3.9 4.1 4.2 4.3 6.3
7.5
9.2

12.5b

predict the potential outcome of using the tested sperm for fertilization and for laboratory evaluation of various methods. There has been absolutely no indication to date that any method, except one based on flow cytometry and DNA, has any validity for producing sexed sperm. It has also

PREPARATION OF SPERM FOR FLOW SORTING AND REANALYSIS SORTING VIABLE SPERM REANALYSIS OF SORTED SPERM FOR DNA
2X

I O X l o 6 sperm
Dilute

Hoechst 33342 (5 ug/106 sperm) Incubate 1 hr 35C Sort into Test-Yolk sur gicaI insem inat io n

n n

i o 5 sperm

Sonicate (for 15 sec) Add 2.5 ug ofHoechst 33342

Flow cytometric DNA analysis For proportionsof X and Y sperm

X and Y sperm

a B

Figure 4. This chart illustrates the means by which viable sperm are prepared for flow sorting based on DNA and for reanalysis of the sorted sperm for DNA content. Diluents that have been used with good success are BTS, pH 7.2; Tris, pH 6.9; HEPES, pH 7.4; and PBS, pH 7.2. The sonication apparatus is a Branson Model 200 operated at an output of 4 at a duty cycle setting of 55% using a microprobe.
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14

JOHNSON

been shown to be a good predictor of the ultimate sex of offspring conceived by sorted, viable sperm (Johnson et al., 1989; Johnson, 1991).

Flow Cytometric Sorting of Sperm

Applications for the modified flow cytometer/ cell sorter developed for sperm DNA analysis (Johnson and Pinkel, 1986) work well for sorting both nucleated sperm cells and viable, intact sperm. Flow sorting involves vibrating the flow chamber, causing the descending stream carrying the sperm nuclei away from the laser to break into small, uniform droplets. At the flow rates commonly used for sperm, about 1 in 16 droplets actually contains a sperm (7.0%). After a sperm heads DNA mass is identified by its respective pulse height as clearly belonging in the left ( Y )or right (XI peak of the frequency distribution, timing and charging circuits are activated, and droplets carrying sperm are electrically charged positive or negative according to the DNA content of the sperm within the droplet (Figure 2). The charged droplet (as well as uncharged droplets containing no sperm) then passes through a n electrostatic field of about 2,000 V. The droplets carrying Xbearing sperm (given a positive charge) are deflected toward the negative pole of the highvoltage plate, and the droplets carrying the Y bearing sperm (given a negative charge) are deflected toward the positive pole of the highvoltage plate. The deflected droplets carrying X and Y sperm, respectively, then fall into collection tubes (Figure 2). The droplets containing no sperm or sperm with a n undesired pulse height (outside or between the sort windows that have been set on each peak; Figures 2 and 5) are left uncharged and thus fall into the discard tube. The sperm that are deflected are electronically selected from a region of the X- and Y-bearing sperm peaks (sorting windows; Figure 5). The purities of the collected X and Y sperm populations are determined by cytometric reanalysis of respective samples. Resultant histograms are computer-fitted to double Gaussian peaks as described earlier. Sorting Sperm Nuclei. Because sperm nuclei (without tails) a r e the easiest to orient with respect to the laser beam, they sort with highest purity. We have sorted sperm nuclei from bulls, boars, rams, chinchillas, creeping voles (Johnson et al., 1987b; Johnson and Clarke, 19881,rabbits (Johnson et al., 19891, stallions, humans, and dogs (unpublished data). Purities ranged from 80% to > 95%. Farm animal species gave purities > 90%. Purity determinations were made through cytometric reanalysis of sorted samples. Purities of sort from several species are shown in Table 2, and histograms from reanalyzed sperm heads a r e shown in

150

2co

253

150

200

250

5
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z
- ,

150

200

250

150

200

250

500

6ooi
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150

200

250

150

250

250

RELATIVE DPIA CONTENT

Figure 5. Representative DNA bimodal distributions of bull, boar, and ram sperm nuclei showing sort windows as hatched areas; the left (A, C, E) panels illustrate the unsorted samples, with sort windows placed on the resulting peaks. The right panels (B, D, E] are an overlay of the reanalysis of each sorted X- and Ybearing-sperm nuclei population. The peaks correspond to the sort windows depicted in the left panels (A, C, E) and represent sperm that were deflected into separate tubes, based on the amount of DNA present in the sperm head. See text for a more detailed explanation of this process. Values for proportions of X or Y were calculated from computer-fitting the histograms to double Gaussian peaks (fromJohnson and Clarke, 1988; copyright 1988, Wiley-Liss. Reprinted by permission of Wiley-Liss, a division of John Wiley and Sons, Inc.).
@

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SEX SELECTION IN DOMESTIC ANIMALS BASED ON DNA

15

Table 2 . Comparative flow cytometric reanalysis for DNA of sorted sperm nuclei and sorted, intact sperm from three different species
Mean X-Y DNA difference, Soecies Boar Bull Rabbit Boar Bull Rabbit
3.7
3.9 3.0
O h

O/O

of Y sperm in Y-sort f SEM Sperm nuclei

010

of X sperm in X-sort i SEM

n
4 9 3

91.2 f 2.6 92.8 f .9 84.3 f 5.7

92.7 f 1.6 90.4 f 1.4 88.0 f 2.2

Intact sperm
_ .

76.2 f 4.3 80.5 f 1.9 80.8 f 1.9

90.0 f .7 88.9 f 1.9 .8 85.9 f

5 6 10

Figure 5. The viability of the sorted sperm nuclei was determined by microinjecting sperm heads into hamster eggs (Johnson and Clarke, 1988) and culturing the eggs through to the pronuclear stage. Ram sperm were also microinjected into sheep eggs and subsequently cultured to the 16-cell stage (Clarke et al., 1988). Sorting Intact, Viable Sperm. In our initial studies it was thought that only sperm without tails would sort with purity, because tail position is uncontrollable. However, a certain percentage of the intact sperm do orient to the laser in the proper manner. Further, it was found that beveling the insertion tube enhanced sorting efficiency for intact sperm as well as for nuclei. Ultimately, it was found that hydrodynamic orientation varied according to species and flow rate (Johnson and Welch, 1991). Preparation and staining of intact sperm differs from preparation of sperm nuclei. The main difference is in the lack of sonication and the added step of incubation needed to effect rapid stain penetration (Figure 4). Although our initial success of sorting intact sperm came in 1986 (unpublished datal, it was only after a significant amount of experimentation that we were able to develop the proper conditions for maintaining sperm viability after sorting over a n extended period (Johnson et al., 1989; Johnson, 1991). The sorting principle is that described earlier under the heading Flow Cytometric Sorting of Sperm. Figure 8 illustrates the original separation of Xand Y-bearing sperm from nuclei through sorting of intact, viable sperm and the subsequent reanalysis of the sorted X and Y sperm populations. There has been one other report of sorting viable sperm (Morrell et al., 1988). In that study, a n unmodified flow cytometer was used to sort Hoechst 33342-treated sperm. However, without modified instrumentation, reanalysis proportions of X- or Y-bearing sperm could not be performed. Fertility results from the sorted sperm did not produce significant shifts in the sex ratios of

offspring (Morrell et al., 19881. Nuclei Versus Intact, Viable Sperm Sorting. Table 2 lists a comparison of both sperm nuclei and sorted, intact sperm for bulls, boars, and rabbits. Aliquots reanalyzed and calculated for DNA content show proportions of X- and Y-bearing sperm. The results illustrate the fact that sperm nuclei (tailless) can be sorted at a higher purity than can the intact sperm. Speed of Sorting. Flow rates for sorting nuclei for most species generally range from 700 to 1,000 sperm per second. This results in a deflection sort rate of 100 sperm per second in each direction. Effectively, this totals 400,000 sperm heads per hour in each collection tube. Intact sperm flow a t a higher rate of 2,500 per second, resulting in 90 to 110 sperm per second deflected. This results in X and Y sort rates of 400,000 intact sperm per hour. Sorted sperm tend to average 75% motility, decreasing viable sperm to 300,000 per hour. Variables affecting sorting rates include uniformity of staining, individual ejaculate characteristics, and species. Recent results using improved hydrodynamic orientation of viable sperm and a significantly upgraded data acquisition system indicate that sorting speed can be increased to 1 x loe sperm per hour (unpublished datal.

Fertility Using Flow-Sorted Sperm

Flow-sorted X- and Y-chromosomebearing sperm from the rabbit and the boar have been surgically inseminated into does and sows, respectively (Johnson et al., 1989; Johnson, 1991). Bull sperm has been sorted and used for in vitro fertilization experiments (unpublished data). Ram sperm has been sorted and used for surgical insemination into the oviduct of ewes (Johnson and Rexroad, unpublished data). Swine. Sperm were collected from mature boars, sorted into X- and Y-bearing populations, and

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JOHNSON

inseminated into the isthmus of the oviduct of gilts whose estrous cycles were synchronized by feeding Regumate@ followed by injections with pregnant mares serum gonadotropin and human chorionic gonadotropin to induce ovulation. Inseminations were made by mid-ventral laparotomy 2 to 4 h after expected ovulation (Johnson, 19911. A summary of the results in terms of deviation from the theoretical 50:50 sex ratio obtained from progeny produced from 19 litters is shown in Figure 7. There was a significant deviation in the sex ratio of progeny produced from X and Y sperm

INTACT SPERM
LY
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. . . .

. . . . .

. . . . .

LLI

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3

X-SORT 06X

populations compared with the theoretical 50:50 ratio. Rabbits. Sperm from mature bucks were sorted into X- and Y-bearing populations and inseminated into the tip of the uterine horn of New Zealand White does. Does had been primed with human chorionic gonadotropin and were scheduled to ovulate approximately 4 h after surgery. A summary of the actual and predicted sex of offspring is shown in Figure 7. There was a significant deviation in the sex ratio of progeny produced from the inseminations with sorted sperm. Offspring born from sorted X sperm were 94% female and from sorted Y sperm were 81% male. Preselection for Sex. Aliquots of sorted sperm reanalyzed for DNA content were used as predictors of the sex of offspring. Higher purities of sorted rabbit sperm (86 and 81% X and Y, respectively) than of boar sperm (80 and 77% X and Y, respectively) were obtained. As illustrated in Figure 7, the rabbit sperm reanalysis served as a better predictor of offspring, attributable to greater consistency in the original rabbit sperm samples. Rabbit sperm also have a higher orientation percentage than do boar sperm. Sheep. Fifteen ewes were laporatomized and sorted sperm were inseminated into the oviducts. From this preliminary experiment two ewes lambed. Both offspring were predicted to be males; however, one female and one male were born (Johnson and Rexroad, unpublished datal. In Vitro Fertilization. Preliminary results using sorted bull sperm for in vitro fertilization of cow and pig eggs indicate that the process is promising because fertilization and cleavage have been obtained (unpublished data). Optimum conditions for sperm number, extender composition, and dilution to suit the specific needs of in vitro fertilization and sorted sperm are not yet completed.

. .
1

200

250

RELATIVE DNA CONTENT

Figure 6. Typical histograms from the analysis and sorting of rabbit sperm, which is representative of sperm from farm animals. (A] Typical for sperm nuclei, the calculated difference in DNA between the X and Y peaks is 3.0%. (B) Intact, viable rabbit sperm that were sorted. ( C )A histogram representing the reanalysis of Ybearing sperm that had been sorted. (D) A histogram representing the reanalysis of X-bearing sperm that had been sorted. The vertical dotted lines illustrate the actual relationship, in terms of relative DNA content, of the various analyses. All the analyses were conducted using the same instrument conditions (from Johnson et al., 1989; reprinted by permission).

Future Prospects for Sexed Semen

Sperm separated with flow cytometric cell sorting have several limitations for artificial insemination, primarily the low number of sperm sorted in any given time period. Maintaining viability through 40 to 50 h of sorting for cattle would be difficult. Swine require a much larger insemination dose, which increases the sorting time substantially. However, with the current development of in vitro fertilization with sorted sperm, high sperm number requirements could be reduced. However, the added expense of embryo transfer would then become a factor. There is no doubt that these techniques have potential for providing

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SEX SELECTION IN DOMESTIC ANIMALS BASED ON DNA

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RABBITS
Percent males
81
81

SWINE
Percent males

80

80

80

Actual Births

II

60
50
40

60 50
40

20

20

Figure 7. The bar graphs shown illustrate the similarity of predicted percentages of male progeny based on reanalysis of aliquots from the sorted samples of X- and Y-bearing sperm for DNA compared to the actual sex of the offspring born for rabbits and swine [Johnson et al., 1989; Johnson, 1991). The values are based on 13 litters of rabbits -and 19 litters of swine. The values in the b&s represent the percentage for each group.

sexed sperm for specialized markets. Other factors that must be considered are the necessity for cryopreservation of sorted sperm and the possible impact of increased embryonic mortality from sorted sperm inseminations (Johnson et al., 1989; Johnson, 1991). Perhaps the most logical and cost-effective means of producing sexed semen would be to use a sex-specific surface membrane marker that would lend itself to antibody development. Such a marker does not exist at the present time. Efforts to identify such a marker are in progress (Fenner et al., 1991). The promise of such a procedure is hard to minimize in that antibody-tagged sperm could be readily separated by affinity chromatographic procedures or by the more recent separation technology using micromagnetic beads or fluids. However, it also should be stated that there is no evidence to date that would suggest that there is a sex-specific surface markerk) on which to develop such a sexing method. Given the progress made in the field of gender preselection over the past 10 yr, it is very possible that 10 yr hence we will see a practical sexing procedure for livestock semen. If the past 10 yr have taught us anything, it is that the approach to

this problem must have a sound scientific basis that can be monitored throughout or it is likely doomed to failure. The advances in gene technology in recent years offer numerous possibilities for researching X- and Y-bearing sperm separation/ sex determination at the gene level.

Literature Cited
Amann, R. P., and G. E. Seidel (Ed.). 1982. Prospects for Sexing Mammalian Sperm. Colorado University Press, Boulder. Avery, 0. T., C. M. MacLeod, and M. McCarty. 1944. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a deoxyribonucleic acid fraction isolated from pneumococcus Type 111. J. Exp. Med. 79:137. Clarke, R. N., C. E. Rexroad, Jr., A. M. Powell, and L. A. Johnson. 1988. Microinjection of ram spermatozoa into homologous and heterologous oocytes. Biol. Reprod. 38(Suppl. 1):
75.

Fenner, G.P., L. A. Johnson, W. R. Hruschka, and D. J. Bolt. 1992. Two-dimensional electrophoresis and densistometric analysis of solubilized bovine sperm plasma membrane proteins detected by silver staining and radioiodination. Arch. Androl. (In press). Fulwyler, M. J. 1965.Electronic separation of biological cells by volume. Science 150:910.

Downloaded from jas.fass.org by on November 29, 2009.

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JOHNSON
Johnson, L. A., J. P. Flook, M. V. Look, and D. Pinkel. 1987b. Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. Gamete Res. 18:l. Johnson, L. A,, and D. Pinkel. 1986.Modification of a laser-based flow cytometer for high resolution DNA analysis of mammalian spermatozoa. Cytometry 7:268. Johnson, L. A,, and G. R. Welch. 1991.Flow sorting of X and Y sperm based on DNA: Influence of viability and head shape on hydrodynamic orientation. Cytometry Suppl. 5336. Kamentsky, L. A., and R. R. Melamed. 1967.Spectrophotometric cell sorter. Science 156:1364. Kiddy, C. A., and H. D. Hafs [Ed.). 1971. Sex Ratio at BirthProspects for Control. J. Anim. Sci. Symp. Suppl., Champaign, IL. Lush, J. L. 1925. The possibility of sex control by artificial insemination with centrifuged spermatozoa. J. Agric. Res.
30:893.

Garner, D. L., B. L. Gledhill, D. Pinkel, S. Lake, D. Stephenson, M. A. Van Dilla, and L. A. Johnson. 1983. Quantification of the X and Y chromosome-bearing spermatozoa of domestic animals by flow cytometry. Biol. Reprod. 28312. Gledhill, B. L. 1988.Selection and separation of X- and Y-chromosome bearing mammalian sperm. Gamete Res. 20377. Gledhill, B. L., S. Lake, L. L. Steinmetz, J. W. Gray, J. R. Crawford, P. N. Dean, and M. A. Van Dilla. 1976. Flow microfluorometric analysis of sperm DNA content: Effect of cell shape on the fluorescence distribution. J. Cell. Physiol.
87:367.

Guyer, M. F. 1910. Accessory chromosomes in man. Biol. Bull. (Woods Hole) 19:210. Hoppe, P. C., and G. C. Koo. 1984. Reacting mouse sperm with monoclonal H-Y antibodies does not influence sex ratio of eggs fertilized in vitro. J. Reprod. Immunol. 6:l. Johnson, L. A. 1986. Flow cytometry: Analysis and sorting of X and Y chromosome-bearing spermatozoa. In: P. C. Augustine, H. D. Danforth, and M. R. Bakst [Ed.) Beltsville Symposia in Agricultural Research X. Biotechnology for Solving Agricultural Problems. pp 121-134. Martinus Nijhoff, Dordrecht, The Netherlands. Johnson, L. A. 1988. Flor cytometric determination of sperm sex ratio in semen purportedly enriched for X or Y bearing sperm. Theriogenology 29:265. Johnson, L. A. 1991. Sex preselection in swine: Altered sex ratios in offspring following surgical insemination of flow sorted X- and Y-bearing sperm. Reprod. Domest. Anim. 26309. Johnson, L. A,, and R. N. Clarke. 1988.Flow sorting of X and Y chromosome-bearing mammalian sperm: Activation and pronuclear development of sorted bull, boar and ram sperm microinjected into hamster oocytes. Gamete Res. 21:
335.

Morrell, J. M., K. D. Keeler, D. E. Noakes, N. M. Mackenzie, and D. W. Dresser. 1988. Sexing of sperm by flow cytometry. Vet. Rec. 122322. Moruzzi, J. F. 1979. Selecting a mammalian species for the separation of X- and Y-chromosome-bearingspermatozoa. J. Reprod. Fertil. 57319. Muller, W., and F. Gautier. 1975.Interactions of heteroaromatic compounds with nucleic acids. Eur. J. Biochem. 542.58. Ohno, S. 1967. Sex Chromosomes and Sex-Linked Genes. p 141. Springer-Verlag, New York. Pinkel, D., D. L. Garner, B. L. Gledhill, S. Lake, D. Stephenson, and L. A. Johnson. 1985.Flow cytometric determination of the proportions of X and Y chromosome-bearing sperm in samples of purportedly separated bull sperm. J. Anim. Sci.
60:1303.

Johnson, L. A., and R. N. Clarke. 1989. Sperm DNA and sex chromosome differences between two geographical populations of the creeping vole, Microtus oregoni. Mol. Reprod. Dev. 27:159. Johnson, L. A., J. P. Flook, and H. W. Hawk. 1989. Sex preselection in rabbits: Live births from X and Y sperm separated by DNA and cell sorting. Biol. Reprod. 41:199. Johnson, L. A., J. P. Flook, and M. V. Look. 1987a.Flow analysis of X and Y chromosome-bearing sperm for DNA using a n improved preparation method and staining with Hoechst 33342. Gamete Res. 17:203.

Pinkel, D., B. L. Gledhill, S. Lakes, D. Stephenson, and M. A. Van Dilla. 1982a.Sex preselection in mammals? Separation of sperm bearing Y and 0 chromosomes in the vole Microtus oregoni. Science 218:904. Pinkel, D., B. L. Gledhill, M. A. Van Dilla, D. Stephenson, and G. Watchmaker. 198213. High resolution DNA measurements of mammalian sperm. Cytometry 3:1. Upreti, G. C., P. C. Riches, and L. A. Johnson. 1988. Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient. Gamete Res. 2033. Zavos, P. 1985. Sperm separation attempts via the use of albumin gradients in rabbits. Theriogenology 23:875.

Downloaded from jas.fass.org by on November 29, 2009.

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