DE2321000A1 - METHOD OF ISOLATION OF SPERM WITH A Y CHROMOSOME - Google Patents

Description

SCHERINGAG

* Patent Department

2321000 Berlin, April 17, 1973

METHOD OF INSULATION YOU SPERM WITH A ^ CHROMOSOME.

The invention "relates to a method and means of prediction of male offspring in mammals. she "also relates to a method for isolating sperm with a Y chromosome (Y sperm) from the sperm of mammals ·

In mammals the sex is distinguished by two Specified sperm types that have either an X or a Y chromosome. Fertilization with Y-sperm "leads to male." Offspring with X-sperm into female descendants. the The assumption that both forms of sperm must differ in size and weight has prompted a number of studies in which the separation of the X- and Y-sperm by sediments- tation, centrifugation, electrophoresis, changing the pH or flotation has been attempted. The desired separation was; however not achieved or only achieved in a small percentage.

The importance of the desired separation of the Y sperm from mammals is obvious to the person skilled in the art. She be therefore only explained here with a few examples. If a method for separation were available, it would have e.g. B. the animal breeders in the hand, by insemination of the animals with X or Y sperm, the sex of the descendants to be expected in the desired way. In the rearing of cattle,

409844/0868

Board of Directors: Hans-Jürgen Hamann ■ Karl Otto Mittelstenschcid Telex: 181777 »schb d · Telegrams: Scheringchemie Berlin Dr. Gerhard Raspe Dr. Horst Witzel _ ... "■" ..... " Deputy: Dr. Christian Brunn Dr. Heinz Hannse Postecheck account: Berlin-West Il 75 Chairman of the Supervisory Board: Dr. Eduard v. Schwarzenkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG CharloUenburg 93 HRB 283 and AG Kamen 5 HRB 71 SGHERINGAG Patent department

'"Jt- April 17, 1973

V 7321000

when the emphasis is on the meat product! qh. would be, with Y-. Sperm are inseminated. Since mutton generally after, the same Breeding time (e.g. "until weaning or weaning") heavier than Becoming sheep and meat production is in the foreground for sheep farmers, insemination would be used in sheep breeding mainly done with Y-sperm. · ·

Such a procedure is also of great importance for humans., The desire for a child of a certain gender is in many families a. one of the reasons for a high number of children. A method for separating X and Y sperm would make family planning much easier, and with it the population and significantly reduce the associated problems.

The present invention now provides a new method which remedies this wide need. The new method for isolating Y sperm from mammals is characterized by that the sperm, if necessary in a medium of lower density, e.g. B. a suitable solution or suspension, distributed, with a physiological compatible medium of greater density contacted, preferably underlayer, the combination stand for some time is left and, if necessary, the Y-sperm are then isolated from the respective contact medium.

409844/0868

Vontand: Harla-JOrgen Hamann · Karl Otto Mittelstenscheid Telex: 181777a schb d 'Telegrams: Scheringchemie Bertin Dc Gerhard Raspe ■ Dr. Horst Witzel. "..,.," ...., .. " Steflv .: Dr. Christian Brunn Dr. Heinz Hanns Postal check account: Berlin-West 11 75 Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppan Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottsnburg 93 HRB 283 and AG Kamen 5 HRB 71 SCHERINGAG Patent department

--F- April 17, 1973

According to the invention, Y sperm from the Sperm from all mammals can be isolated. Of special interest on the one hand is the isolation from the semen of mammals used in agriculture or breeding to other economic Find uses such as B. primates, horses, cattle, owls, sheep, goats, rabbits, etc., and on the other hand isolation from human semen.

The sperm from which the Y sperm are to be isolated can, can be used both as such and distributed in a suitable medium of lower density. This medium is not allowed weaken the pertility of the sperm to be separated, d. H. it must not damage or destroy the sperm. On the other hand, it must Medium may contain nutrients that may keep the sperm alive over a longer period of time. The medium must have "a suitable pH value, approximately in the range 7-.8, as well as an osmotic pressure at which the sperm are not compressed or burst. Such media are physiologically acceptable solutions, such as. B · Tyrode's solution, Ringer's solution, .Hank * s solution, physiological Saline solution, medium 199 »Eagle's medium, etc. in question, possibly with additives known to the person skilled in the art, such as salts, low molecular weight or higher molecular weight organic compounds, e.g. B. mono- or oligosaccharides, amino acids, peptides, proteins or Proteids, polymers, such as i.a. Polypyrrolidone, etc. to the

409844/0868

Board of Directors: Hans-Jdrgen Hamann - Karl Otto Mittelstenscheid Telex: 181 777 »echb d · Telegrams: Scheringchemie Berlin Dr. Gerhard Raspe - Dr. Horst jokes) Deputy: Dr. Christian Brunn - Dr. Heinz Hannse Postscheck account: Berlin-Weet 11 75 Chairman of the Supervisory Board: Dr. Eduard v. Schwarfzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Chariottenburg Θ3 HRB 283 and AG Kamen 5 HRB 71 SCHERING AG

desired conditions can be set «Should the isolated Y-sperm are first stored under appropriate conditions, e.g. Bo if they are for sperm banks or sperm depots are provided, sperm distributed in solutions is preferably assumed, in other cases, however, undiluted sperm can also be used Sperm will be run out.

To the media of higher density intended for contacting In general, the same preconditions are set as listed above for the lower density media, in which the sperm may be distributed. So there are all physiologically compatible solutions such. B. the above, in Consider that known by the pharmacologist or physiologist Additives can be adjusted to the desired conditions, in particular to the higher density. The additives include, for example Salts, low and high molecular weight organic compounds, among others. Mono- or oligosaccharides, amino acids, peptides, Proteins or Proteins, Polymers, such as. B. Polypyrrolidone, etc.

If some of the additives are not completely soluble in the starting solutions in order to achieve the desired density, suspensions, sols or gels can also be used as contacting media Find use. It is essential that the media provided for contacting have a higher density than that which may have been distributed

Board of Directors: Hans-Jürgen Hamann · Karl Olto Mitteütanschaid T.le ": 181 777" schb d · Telegram ": Scheringchtrni. Berlin Dr. Gerhard Raspe Dr. Hor * t Witzel Deputy: Dr. Christian Brunn Dr. Heinz Hanns. Poalacheek-Kbnto: Borlin-Weal 1175 Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen S HRB 71 '·

ORIGINAL INSPECTED

SCHERINGAG

Patent department

- * - April 17, 1973

Have sperm, as the density gradient has a decisive influence on the separation of the sperm is. An increase in the density gradient causes an increase in the separation. With one very low density gradient it is possible to separate the sperm to be underestimated in such a way that only motile from immotile sperm are separated without isolation from Y-sperm.

The density gradient required to separate the motile from the immotile sperm or to isolate Y sperm depends on the type and quality of the mammalian sperm to be used. In general, the density gradient required between the medium of lower density and medium of higher density is 1 to 50 $ With a density gradient of less than 3 $, a separation of motile and immotile sperm will predominantly take place.I ^{1 For} the separation of human sperm, the necessary density gradient for the separation of motile and immotile sperm is about 1 <fo to 5 fit, preferably 3 to 5 ° \ for the isolation of Y-sperm about 3 # "to 50 $, preferably 10 i ° to 25 <f ₀ .

The possibly distributed sperm can. With the contact medium higher Density can be brought into contact in any way. However, it must be ensured that no mixing of the possibly with sperm distributed in a medium of lower density

the contact medium of higher density occurs. For solutions or \ suspensions higher as a medium density is therefore the forthcoming

409844/096 8

Board of Directors: Hans-Jürgen Hamann · Karl Otto Mittelstentcheid 'Telex: 181777a achb d ■ Telegrams: Scheringchemie Berlin Dr. Gerhard Raspe Dr. Horst Witzel - ■■ _ Deputy: Dr. Christian Bruhn ■ Dr. Heinz Hannse Postscheck account: BerlintWest 11 75 Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen 5 HRB 71

SCHERINGAG

"Patent Department

■. - /> - April 17, 1973

-drawn contact form the underlaying of the * (-distributed) Sperm with the solution or suspension will be higher density. However, there is also the careful layering of the solution or Consider suspension of higher density with the (distributed) sperm in B. Will the sperm possibly distributed in a medium of lower density brought into contact with the fluid medium of higher density, so leads the one-time contact, z. B. the underlayer, "already leads to a clear, sufficient separation effect can now be increased by making multiple contacts. The multiple contact can be achieved on the one hand that not only with one medium, but with several, z. B. two or three media is contacted in each case of increasing density, so z. B. by subbing; of the distributed sperm with a solution of higher density and further sub-layering of this solution with a solution of even higher density. The multiple On the other hand, contacting can be achieved by the fact that the part of the already largely separated sperm, which in the higher density medium had entered, separated, recovered and re-contacted with a higher density medium · There is also the possibility of using the higher density medium, which contains the separated sperm, from the starting solution lower density and then with a medium higher density contact.

A09844 / 0868

Board of Directors: Hans-JUrgen Hamann ■ Kart Otto Mittelstenscheid Telex: 181777a schb d ■ Telegrams: Scheringchemie Berlin Dr Gerhard Raspe ■ Dr Horst Witzel _, '. ", _,.,",. ", .-

Deputy: Dr. Christian Brunn Dr. Heinz Hannse Postscheck account: Berlin-West! 175 Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen

Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 9a HRB 283 and AG Kamen 5 HRB 71 SCHERINGAG Patent department

The contacting of possibly distributed in a medium of lower density Sperm with the fluid medium of higher density can be in vessels of any desired shape, such as those skilled in the art for working with small amounts of fluid are generally used, for example: burettes, pipettes, separating tubes and columns with a suitable termination, etc. Since the shape of the vessels has no influence on this type of contact, many are available to the person skilled in the art Embodiments available that he can modify as desired. The time to complete the isolation (or separation) is from the mammalian species, the type of medium and the type of isolation or separation dependent. In the case of sub-stratification of the Sperm dispersed in a medium of lower density with a fluid medium will generally take times between 0.5 and 10 hours than suffice for implementation, preferably between 1 and 3 hours.

The practitioner who wants to isolate and inseminate the Y-sperm, the necessary work materials are expediently offered in the form of a set, which is preferably designed as a disposable pack will. The set then contains the following individual components, which have already been described above, in appropriate, preferred sterile., containers:

a) a vessel for working with small amounts of fluid,

409844/0868

D ™ G.rnard R ¹ aV ^JD / ° ^l Dr ^H HSi ^I rst ⁿ WiS5 ^l ° "° ^M '"" ^ltlen » ^{ch # i- Telel:} ' ^el " ⁷ ·« ^ch ° * ■ Tele _B rarnm ·: Scharingchtmie Berlin Deputy: Dr. Christian Bruhn ■ Dr. Heinz Hannse Postscheck account: Berlin-West 1176 Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen 5 HRB 71

ORIGINAL INSPECTED

SCHERINGAG

Patent department

• - ^ a - April 17, 1973

b) a meditation to absorb the sperm, as it -yorsteaend as Medium of lower density was designated,

c) a medium of higher density for contacting; since the higher density medium is generally composed of the lower density medium

. can be produced, the set "can be used as part c) also contain only the additive or additives to achieve the higher density,

d) an insemination syringe, as is generally known to the person skilled in the art is, and possibly.

e) a work instruction

The following examples serve to illustrate the invention. Before the separation, the sperm count and motility ^ "" Practical andrology, C. Schirren, Verlag Brothers Hartmann, Berlin 19717 determined. The isolation (or separation) that has taken place is determined by determining the X and Y sperm using the quinakridine technique / ~ Acta Mol. Med. germ. 28 (1972) 189-92_7. A possibly to be done The sperm are recovered by centrifugation.

409844 / 08-68-

OGeliard R _a J 6DrHorsw i "°" ° ^{Miltel8tonscheid Telei:} ' ^81777a « ^chb d" Telegrams: Scheringchemie Berlin Deputy: Dr. Christian Bruhn Dr. Heinz Hannse Postscheck account: Berlin-West 1175 Chairman of the Supervisory Board: Dr. Eduard v. Schwarzenkoppen Seat of the company: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen 6 HRB 71

SCHERNGAG

Patent department

April 17, 1973

Example 1. *

a) Fresh human sperm is mixed in a ratio of 1: 1 with Tyrode's solution and centrifuged to separate the sperm from the other sperm components. After determining the Spermienmotxlitat and number 50 10 χ sperm in 0.5 ml of Tyrode's solution "at pH 7.6 resuspended -. Simultaneously, a Tyrode's solution at pH 7.6 with bovine serum albumin (BSA) on a BSA concentration of 6 <f ₀ BSA adjusted. the sperm-containing Tyrode's solution is 0.9 ml of the BSA solution in a sealed Pasteur pipette underlaid · Mach one hour, the supernatant Tyrode solution and the BSA-Iiösung are separated. the sperm are recovered from the BSA solution, their The number and motility as well as the content of X and Y spennia are determined.

IM PM

70 25

0.5 ml ₆

(50 x 10 sperm in Tyrode solution)

0.9 ml R = 30th £ 6 BSA M = 90-95 (in Tyrode's solution) γ ₌ 63-67

409844 / 08-88

Board of Directors: Hans-JUrgen Hamann - KarlOtIo Mitlelslenscheid Dr. Gerhard Raspe ■ Dr. Horst jokes) Deputy: Dr. Christian Bruhn ■ Dr. Heinz Kannse Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen

Telex: 18t 777a schb d ■ Telegrams: Scheringchemie Berlin Postal check account: Berlin-West 1175

In the drawings, each mean

SCHERINGAG Patent department

- ST 17.04.1973

IM: initial motility PM: end motility

Recovery

M: sperm motility Y: io y sperm

b) 50 x 10. Sperm obtained according to Example 1a with 63 67 io Y-sperm are suspended in 0.5 ml Tyrode's solution and this underlay with 0.9 ml of a 10% BSA solution. After an hour, the supernatant Tyrode solution and the BSA solution are separated. The sperm are recovered from the BSA solution, their number and motility as well as the content of X and Y sperm are determined. The results are shown in drawing 2.

Drawing _2

IM = 90 PM = 40-60

0.5 ml r

(50 σ 10 sperm in Tyrode's solution)

0.9 ml R = 40

10 io BSA M = 95

(in Tyrode's solution) _γ _ η- ^

A09844 / 0868

Board of Directors: Hane-JUrgen Hamann · Karl Otto Miltelstenscheid Oc Gerhard Rasp «■ Dr. Horst Witzel Deputy: Dr. Christian Bruhn ■ Dr. Heinz Hannse Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen 5 HRB 7t

Telex: 181 777a schb d ■ Telegrams: Scheringchemie Berlin Postal check account: Berlin-West Jl 75

SCHEFiINGAG Patent department

- April 17, 1973

Example 2

Fresh human sperm is made in a 1: 1 ratio with Tyrode's solution mixed and centrifuged to separate the sperm. · After determining the sperm motility and number 50 χ 10 sperm are resuspended in 0.5 ml of Tyrode's solution and these underlayed with .0.8 ml of a 6 $ BSA solution. The $ 6 BSA solution is underlaid with 0.4 ml of a 15% BSA solution. After an hour and a half, the solutions are separated. The sperm are recovered from the BSA solutions, their number and motility as well as the content of X and Y sperm are determined. * The results can be found in drawing 3.

Drawing 3,

IN THE
FM

70 25

0.5 ml ₆

(50 x 10 sperm in Tyrode solution)

0.8 ml

6 <fo BSA

(in Tyrode's solution)

0.4 ml

15 1o BSA

(in Tyrode's solution)

R = 20 M = 90-95 Y = 60-65

R =

Y =

10 95 78-80

Example 3

a) Fresh human sperm is used in a ratio of 1: 1 Tyrode solution mixed and centrifuged to separate the sperm

Preface: Hane-jurgenHamann - Karl Otto MiUelalenscheid * Tele «: 1 Sl 777a achb d · Telegramma: Scheringchemi · Berlin Or. Gerhard Raspe ■ Dr. Horst Witzel Deputy: Dr. Christian Bruhn - Dr. Heinz Hanns. Poelscheok account: Berlin-West 11 75 Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen 5 HRB 71

SCHERINGAG

Patent department

--ET- April 17, 1973-

2321QOO

fused. After determining the sperm motility and number 50 χ 10 sperm cells are resuspended in 0.5 ml Tyrode's solution and these are underlaid with 0.9 ml of a 6% BSA solution. After one hour, the supernatant Tyrode solution and the BSA solution are separated. The BSA solution becomes the sperm recovered, their number and motility as well as the content of X- and Y-sperm determined. The results are shown in drawing 4.

Drawing:

IM = 70 FM = 25

0.5 ml ₆

(50 χ 10 sperm in Tyrode's solution)

0.9 ml

6 <fo BSÄ

(in Tyrode's solution)

R = 30

M = 90-95

Y = 63-67

b) 50 χ 10 sperm obtained according to example 3 a with 63 - 67 $ Y sperm are suspended in 0.5 ml of Tyrode's solution and this "layered with 0.9 ml of a 10 $ BSA solution - after a Hour the supernatant Tyrode solution and the BSA solution are separated. The sperm are recovered from the BSA solution, their number and motility as well as the content of X and Y sperm are determined. The results are shown in drawing 5 remove.

409844/0868

Board of Directors: Hans-JUrgen H »m» nn · K «rl OHo Mittelstenscheid Dr. Gerhard Raspe ■ Dr. Horst Witzel Deputy: Dr. Christian Bruhn ■ Dr. Heinz Hanns. Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Handelsr.gist.r: AG Charlottenburg 03 HRB 203 and AG Kamen δ HRB

Tele «: 181777a schb d ■ Telegrams: Scheringchemie Berlin Postal check account: Berlin-West 11 75

Drawing 5_

SCHERINGAG

Patent department

- 3 ^ - April 17, 1973

IM = 90

PM = 45-60

0.5 ml ₆

(50 x 10 sperm in Tyrode solution)

0.9 ml 10 io BSA

(in Tyrode's solution)

E = 45 M = 95 γ. = 74 -

c) 50 x 10 sperm obtained according to Example 3b with 74-78 y sperm are suspended in 0.5 ml Tyrode's solution and
this underlayed with 0.9 ml of a 20 $ BSA solution. After one hour, the supernatant Tyrode solution and the BSA solution are separated. "The sperm are recovered from the BSA solution, their number and motility as well as the content of X and Y sperm are determined. The results are shown in drawing 6.

IM = 95 PM = 92

0.5 ml (50 χ

Sperm in Tyrode's solution)

0.9 ml 20 io BSA (in ^ Tyrode solution)

R = M = Y =

409844/0868

Board of Directors: Hans-Jürgen Hamann ■ Karl Otto Mittelstenscheid Dr. Gerhard Rasp6 - Dr. Horst Witzel Deputy: Dr. Christian Brunn ■ Dr. Heinz Hanns. Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen 5 HRB

Telex: 181777a schb d ■ Telegrams: Scheringchemie Berlin Postal check account: Berlin-West 1175

SCHERINGAG

Patent department

- ■ April 17, 1973

Example 4 . -

a) Fresh human sperm is used in a ratio of 1: 1 Tyrode solution mixed and centrifuged to separate the sperm. After determining sperm motility and number 50 × 10 sperm are resuspended in 0.5 ml Tyrode's solution And this underlayed with 0.9 ml of a 3 $ BSA solution. After one hour, the supernatant Tyrode solution and the BSA solution separated. The sperm are recovered from the BSA solution, their number and motility as well as their content are determined X and y sperm determined. The results are shown in drawing 7. There will be predominantly a separation of the motile obtained from the immotile sperm.

IN THE

FM

65 25

0.5 ml ₆

(50 χ 10 sperm in Tyrode's solution)

0.9 ml

3 $> BSA

(in Tyrode's solution

R = 30-45 M = 85-90 Y = 57

409844/0868

Board of Directors: Hans-Jürgen Hamann ■ Karl Otto Mittelstenscheid Dr. Gerhard Rasp «· Or. Horst Witzel Deputy: Dr. Christian Bruhn ■ Dr. Heinz Hannse Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg S3 HRB 283 and AG Kamen 5 HRB

Telex: 1 81777a schb d ■ Telegrams: Scheringchemie Berlin Postscheck account: Berlin-West If 75

isr

SCHERINGAG Patent department

-O ¹ O ⁷ O ⁴ ' ¹⁹⁷³

Example 5 ^Λ -

Fresh human sperm is mixed with Tyrode's solution in a ratio of IiI and centrifuged to separate the sperm. After determining the sperm motility and number, 50 × 10 sperm are resuspended in 0.5 ml Tyrode's solution and these are underlaid with 0.5 ml of a 10% BSA solution. The 10 follow BSA solution is mixed with 0.3 ml of 15 $ BSA solution underlaid. The 15 $ BSA solution is underlaid with 0.3 ml of a 25 $ BSA solution. The upper layer is separated after an hour. After an hour and a half, the second layer is removed. After two hours, the remaining solutions are separated. The sperm are recovered from the BSA solutions, their number and motility as well as the content of X and Y sperm are determined. The results are shown in drawing 8.

Drawing £ 3

IM = 65
FM = 20

0.5 ml ₆

(50 x 10 sperm in Tyrode solution)

0.3 ml

10 1 » BSA

(in Tyrode's solution)

0.3 ml

15 1 » BSA

(in Tyrode's solution)

0.3 ml

25 Io BSA

(in Tyrode's solution)

Il
M.
Y

10 50 55

10 75

409844/0868

Board of Directors: Hans-Jürgen Hamann ■ Karl Otto Mittelstenscheid Dr. Gerhard Raspe Dr. Horst Witzol SIeIIv .: Dr. Christian Brunn Dr. Heinz Hanns " Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bcrqkaman Commercial register: AG Charlottenburg 93 HRB 283 U. AG Kamen 5 HRB 71

Y = 60 -

R = 10

M = 95

Y = 75

15 60 60

85 70

Telex: 181777a schb d - Telegrams: Scheringchemie Berlin Pottscheck account: Berlin-West It 75

SCHERINGAG Patent department

/ 6.

- April 17, 1973

2321Q-OO

Example 6 -. · »

a) Fresh rabbit sperm is mixed in a ratio of 1: 1 with Tyrode's solution mixed and centrifuged to separate the sperm from the other sperm components, after determining the spermatozoa motility and number are 50 x 10 sperm in 0.5 ml Tyrode-. solution resuspended at pH 7.6. - At the same time there is a Tyrode solution from pH 7.6 with bovine serum albumin (BSA) to one BSA concentration set at 6 $ BSA. The sperm-containing Tyrode solution is melted off with 0.9 ml of the BSA solution in a Pasteur pipette underlayed. After one hour, the supernatant Tyrode solution and the BSA solution are separated. The sperm are recovered from the BSA solution and their number and motility are determined. The results are the Refer to drawing 9.

^ Drawing J9

IM ΪΜ

75 75

0.5 ml
(50 x 10
Solution)

Sperm in Tyrode-

0.9 ml R '= 26

6 io BSA M = 95

(in Tyrode's solution)

409844 / 08.68

Board of Directors: Hans-Jurgen Hamann · Karl Otto Mittelstenscheid Dr. Gerhard Raape ■ Dr. Horst Witzel Deputy: Dr. Christian Brutin ■ Dr. Heinz Hannse Chairman »Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen

Tel «: 181777a schb d · Telegrams: Scheringchemi · Berlin Postal check account: Berlin-West It 75

SCHEFIINGAG Patent department

-τ / - April 17, 1973

b) 50 x 10 sperm obtained according to Example 6a are suspended in 0.5 ml of Tyrode's solution and this with 0.9 ml of a 10% BSA solution layered. After one hour, the supernatant Tyrode solution and the BSA solution are separated from the BSA solution, the sperm are recovered and their number and motility are determined The results are shown in drawing 10 refer to·

25 invoice 10_

IN THE
PM

95 80

0.5 ml ₆

(50 χ 10 sperm in Tyrode's solution)

0.9 ml 10 io BSA

(in Tyrode's solution)

R = 30-40 M = 75

Fertilization attempts show that predominantly male poets emerge, i.e. that a sufficient separation of the Y from the X-sperm is achieved

Example 7

Fresh rabbit sperm is mixed in a 1: 1 ratio with Tyrode's solution mixed and centrifuged to separate the sperm. 'After determining sperm motility and count 100 χ 10 sperm are resuspended in 0.5 ml of Tyrode's solution and

Board of Directors: Hans-Jürgen Hamann · Karl Otto Mittelstenscheid

Gerhard Raspo ■ Dr. Horst Witzel

Slellv .: Dr. Christian Bruhn ■ Dr. Heinz Hannse

Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppm

Company headquarters: Berlin and Bergkamen

Commercial register: AG Charlottenburg 93 HRB 283 and AG Kanwn S HRB

40984Λ / 0868

Telex: 181777a schb d · Tetegram ·: Scheringchamie Berlin Postal check account: Berlin-West 1175

SCHERINGAG Patent department

- IeT " April 17, 1973

this with 0.8 ml of a 6 μL BSA solution is added. the • 6 $ BSA solution is mixed with 0.4 ml of a 15 $ BSA solution layered. The upper layer is separated after an hour. After an hour and a half, the remaining solutions are separated. The sperm are recovered from the BSA solutions and theirs Number and motility determined. The results are shown in drawing 11.

2JL

IM = 75 PM = 70

0.5 ml - ₆

(100 x 10 sperm in Tyrode-

solution)

0.8 ml '. R = 10

6 <fo BSA. M = 90

(in Tyrode's solution)

0.4 ml R = 5

15 io BSA M = 98

(in Tyrode's solution)

409844/0868

Board of Directors: Hans-Jürgen Hamann * · Karl Otto Mittelstenscheid Dr. Gerhard Raspö ■ Dr. Horst Wltzel Deputy: Dr. Christian Bruhn ■ Dr. Heinz Hannse Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen 5 HRB 71

Telex: 18t 777a echo d ■ Telegrams: Scheringchemie Berlin Postal check account: Berlin-West U 75

SCHERINGAG Patent department

- 1? a - April 17, 1973 2321000.

Example 8 ^

Fresh human sperm is mixed with Tyrode's solution in a ratio of Is 1 mixed and centrifuged to separate the sperm from the other sperm components. After determining the sperm motility and number are 50 χ 10 sperm in 0.5 ml Tyrode's solution resuspended at pH 7.6. - At the same time there is an iDyrode solution adjusted with ovalbumin (OVA) to an OVA concentration of 10 # OVA and a pH of 7.3 - 7.4. The sperm neck. Dyrode solution is layered with 0.9 ml of the OVA solution in a melted Pasteur pipette. After one hour the supernatant Tyrode solution and the OVA solution are separated. The sperm are recovered from the OVA solution, their number and motility as well as the content of X and Y sperm are determined. The results are shown in drawing 12.

^ Drawing 12__

IM = 75-80 = 25-30

0.5 ml , ■

(50 x 10 sperm in Tyrode-

solution)

0.9 ml R.

19 36 OVA M (in Tyrode solution) _v

25 - 30 85 - 90 62 - 66

409844/0868

Board of Directors: Hans-JUrgen Hamann - KirlOlto Mittalttanacheid Dr. Gerhard Rasp «- Or. Horst Witzel Deputy: Dr. Christian Bruhn ■ Dr. Heinz Hanna. Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG ChartotUnburg 93 HRB 283 and AG Kamen 5 HRB 7t

Telex: 181777a achb d Telegram: Scheringchemie Berlin Postal check account: Berlin-West 1176

SCHERINGAG Patent department

Example 9 . ^

Fresh human sperm is made in a 1: 1 ratio with Tyrode's solution mixed and centrifuged to separate the sperm. After determining the sperm motility and number are 50 x 10 sperm are resuspended in 0.5 ml of Tyrode's solution and this underlayed with 0 * 8 ml of a 6 $ OVA solution · The 6 # OYA solution is underlaid with 0.4 ml of a 15 # OVA solution. After one hour the upper layer is separated off. After one and a half hours, the remaining solutions are separated. From the OVA solutions the sperm are recovered, their number and motility did as well as the content of X and Y sperm. The results can be found in drawing 13.

Drawing 13_

IM = 80

FM = 45

0.5 ml ₆

(50 x 10 sperm in! Eyrode-

Solution)

0.8 ml

$ 6, OVA

(in Tyrode's solution)

E = 15

M = 80-85

Y = 70-73

0.4 ml - E = 3 - 5

15 $> -BftftrtfVA M «45 - '50

(in Tyrode's solution) _y _ _6o _ _6?

409844/0868
Om * jn <A9rt g inputs on

input

Board of Directors: Hans-JOrgen Hamann · Kart Otto MitUlatanschcid Dr. Gerhard Rasp «■ Dr. Herat Witzel Stell ν .: Dr. Christian Bruhn - Or. Heinz Hannse Chairman of the Supervisory Board »: Dr. Eduard v. Schwirtzkoppan Company headquarters: Berlin and Bergkamen Handalaregistar: AG Charlottenburg 63 HRB 283 and AG Kamen S HRB

Tal ": t et 777" echb d · Tdtgramm ·: Scrteringchemie Berlin PUtltchTCk account: Berlin-West 1175

ORIGINAL IMSPECTED

Claims (1)

SCHERINGAG Patent department - ier- 17.4.1973 y 2321000 ' Claims * II Procedure for the isolation of Y sperm from the sperm of Mammals, characterized in that the sperm, optionally distributed in a suitable solution or suspension of lower density, larger with a physiologically compatible medium Density contacted, preferably underlayed, the combination is left to stand for some time and, if necessary, then the Y sperm are isolated from the respective medium. 2. The method according to claim 1, characterized in that human Sperm is used. 3. The method according to claim 1, that undistributed sperm is used will. 4. The method according to claim 3 »characterized in that as physiologically compatible medium of higher density a Tyrode solution mixed with bovine serum albumin is used. 5 · A method for separating motile and immotile sperm from the sperm of mammals, characterized in that the sperm is optionally distributed in a solution or suspension of lower density, with a physiologically compatible medium of higher density, the density gradient not being greater than. 5 i ° be \ 439844/0868 Board of Directors: Hans-Jttrgen Hamann · Karl Otto Mittelstenscheid Telex: 181777a schb d ■ Tetegrams: Scheringchemie Berlin Dr. Gerhard Raspo - Dr. Horst WiUeI _ ",", _ .. .... "" _ Represents: Dr. Christian Brunn Dr. Heinz Hannse Poetscheck account: Berlin-West 1175 Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen S HRB 71 SCHERINGAG Patent department . - April 17, 1973 is allowed to be contacted, preferably kept under scrutiny, and then the motile sperm are isolated from the higher density medium. 6. Method for the prediction of male descendants in mammals by fertilization with Y sperm, characterized in that Y sperm are used for fertilization obtained by the method according to claim 1. 7 · Means for predetermining the male sex of mammals, containing sperm produced by the method according to Claim 1 were obtained. ' 8. Composition according to claim 7, containing human sperm. 409844/0 8 68 Board of Directors: Hans-Jürgen Hamann - Karl Otto Mittelstenscheid Telex: 181777a schb d ■ Telegrams: Seheringchemie Berlin Dr. Gerhard Rasp * - Dr. Horst Witzel _, "",.,., Deputy: Dr. Christian Brunn Dr. Heinz Hannse Postscheck account: Berlin-West 11 75 Chairman of the Supervisory Board: Dr. Eduard v. Schwartzkoppen Company headquarters: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 283 and AG Kamen S HRB 7t SCHERINGAG Patent department - 2Ö - April 17, 1973 9. Set for applying the method according to claim 1, containing tend as individual components, a) a vessel for working with small amounts of fluid, b) a medium of lower density for the uptake of the sperm, c) a medium of higher density for contacting d) an insemanation syringe and, if necessary, e) a work instruction. 10. Set according to claim 9 »containing as an individual component a) a Pasteur pipette. 11. Set according to claim 9, containing as an individual component b) a lyrode solution. ■ 12. Set according to claim 9, containing as an individual component c) a Tyrode solution containing bovine serum albumin. 13. Set according to claim 9 »containing as a single stock c) the additive or additives to achieve the higher density. 14. Set according to claim 13 »containing bovine serum albumin as an additive . C ^ 409844/0868 Board of Directors: Hani-Jorflen Ham «nn · Karl OHo MiB ^ H« i «Ji« d Tel «: i 81 777« «chb d · Telegrams: Schwinger * ™ · Beriin Dr. Gerhard Raspe Dr. Hor »t Wilzel" "^ Deputy: Dr. Christian Bruhn ■ Dr. Heinz Hani ». Poattch.ck account: Berlin-Weit 1176 Chairman of the «Supervisory Board Dr. Eduard v. Schwartzkopp «n SiU of the company: Berlin and Bergkamen Commercial register: AG Charlottenburg 93 HRB 2S3 and AG Kamen 6 HRB 71