Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
  • A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes

Definitions

• This invention relates to a method of prese- lecting the sex of offspring by sorting sperm into X and Y chromosome-bearing sperm based on differences in DNA content.
• Gender of animal offspring is important to livestock producers. Because the dairy farmer has little use for most bull calves, the use of sexed semen to pro ⁇ cute only females would make mild production more effi ⁇ cient. Swine farmers would produce pork more efficiently if they were able to market only female swine, because females grow faster than males.
• the ability to specify male or female offspring should shorten the time required for genetic improvements, since desirable traits are often associated with one or the other parent.
• Planning the sex of cattle offspring is already practiced on a limited basis. This procedure consists of removing embryos from the cow, identifying their potential gender, and re- implanting only those of the desired gender.
• an ability to separate sperm into male-producing and female- producing groups before they are used for artificial insemination could enhance the overall value of offspring produced by embryo transfer.
• Every living being has a set of paired chromo ⁇ somes, which carry all the genetic material necessary to maintain life and also to propagate new life.
• All but one pair of chromosomes are called autosomes and carry genes for all the characteristics of the body, such as skin, hair and eye color, mature size, and body characteristics. The remaining pair are called sex chromosomes. They carry the genetic material that specifies gender.
• One sex chromosome is called X, the other Y.
• a sperm from the male or an egg from the female contains one of each pair of autosomes; in addition, in mammals the egg always contains an X chromosome, while the sperm always carries either an X or Y chromosome.
• the offspring When a sperm and egg unite and the sperm carries the Y chromosome, the offspring is male (XY); however, if the sperm carries an X chromosome when it unites with the egg, the resulting offspring is female (XX).
• XY deoxy- ribonucleic acid
• XX deoxy- ribonucleic acid
• the X chromosome is larger and contains slightly more DNA than does the Y chromosome.
• the difference in total DNA between X-bear- ing sperm and Y-bearing sperm is 3.4% in boar, 3.8% in bull, and 4.2% in ram sperm.
• the amount of DNA in a sperm cell is stable. Therefore, the DNA content of individual sperm can be monitored and used to differ ⁇ entiate X- and Y-bearing sperm.
• a flow cytometer measures the amount of fluo ⁇ rescent light given off when the sperm, previously treated with a fluorescent dye, pass through a laser beam. The dye binds to the DNA.
• the fluorescent light is collected by an optical lens assembly; the signal is transported to a photomultiple tube, amplied, and ana ⁇ lyzed by computer.
• the X chromosome contains more DNA than the Y chromosome
• the female sperm (X) takes up more dye and gives off more fluorescent light than the male sperm (Y) .
• the sperm must pass single file through the laser beam, which measures the DNA content of indi ⁇ vidual sperm.
• a suspension of single cells stained with a fluorochrome is made to flow in a narrow stream intersecting an excitation source (laser beam).
• an excitation source laser beam
• optical detectors collect the emitted light, convert the light to electrical signals, and the electrical signals are analyzed by a multichannel analyzer.
• the data are displayed as multi-or single-parameter histograms, using number of cells and fluorescence per cell as the coordinates.
• fluorescence measurement is most accurate when the sperm fluorescence is excited and measured on an axis perpen ⁇ dicular to the plane of the sperm head [Pinkel et al (2), supra] .
• hydrody- namics are used to orient tailless sperm so that DNA content can be measured precisely on 60 to 80% of the sperm passing in front of the laser beam.
• the modified Epics V system used in this study can measure the DNA content of tailless sperm from most species at the rate of 50 to 150 sperm per second [Johnson and Pinkel, supra] .
• Intact sperm (with tails), whether viable or nonviable, cannot be oriented as effectively as tailless sperm nuclei [Johnson (2), supra] .
• a 90-degree detector can be used to select the population of properly oriented intact sperm to be measured by the 0 degree detector. Since no hydrodynamic orientation is attempted, the sample flow rate can be much higher, which compensates somewhat for the fact that only 15 to 20% of intact sperm pass through the laser beam with proper orientation.
• the overall flow rate was approximately 2500 intact sperm per second.
• the intact X-and Y-bearing sperm fractions were sorted simul ⁇ taneously from the population of input sperm at a rate of 80-90 sperm of each type per second.
• a nontoxic DNA stain must be selected.
• a pre- ferred stain is Hoeschst bisbenzimide H 33342 fluoro ⁇ chrome (Calbiochem-Behring Co., La Jolla, CA) .
• this fluorochrome is the only DNA binding dye that is nontoxic to sperm.
• Concentration of the fluoro ⁇ chrome must be minimal to avoid toxicity, and yet be sufficient to stain sperm uniformly and to detect the small differences in the DNA of X and Y sperm with ini- mal variation.
• a suitable concentration was found to be 5 g/ml, but this may be varied from 4 to 5 ⁇ / ug/ l .
• the sperm must be incubated with stain at suf ⁇ ficient temperature and time for staining to take place, but under mild enough conditions to preserve viability. Incubation for 1 hour at 35°C would also be effective. Incubation time has to be adjusted according to tempera ⁇ ture; that is, 1.5 hr. for 30°C; 1 hr for 39°C.
• Sheath fluid used in sorting cells must be electrically conductive and isotonic. A concentration of 10 mM phosphate buffered saline provided the necessary electrical properties, and 0.1% bovine serum albumin was added to enhance sperm viability by providing protein support for metabolism and viscosity for the sperm. The sheath fluid must be free of sugars and excess salts. Dilution of sperm as occurs in sorting tends to reduce viability of the cells. To overcome this problem sperm were collected in test egg yolk extender [Graham et al, J. Dairy Sci. 55: 372 (1972)] modified by adjusting the pH and adding a surfactant. Details of the composi- tion of the extender are shown in Example 1. The surfac ⁇ tant is believed to enhance capacitation of the sperm prior to fertilization.
• sperm After being sorted, sperm were surgically inseminated into the uteri of rabbits.
• Inseminations were also made to determine the comparability of predicted sex of offspring to phenotypic sex. As the data in Table II indicate, the predictabil ⁇ ity of the phenotypic sex based on DNA analysis of the separated intact sperm was very high. Reanalysis of the sorted Y population used for insemination indicated that 81% of the sperm were Y-bearing. The sex ratio of off ⁇ spring from these inseminations was identical to that predicted. These values were significantly different from theoretical 50:50 sex rates (P ⁇ .003).
• the pheno ⁇ typic sex ratio of offspring born of does inseminated with either sorted X-bearing or sorted Y-bearing sperm was different (P ⁇ .0002 for X and P ⁇ .001 for Y) from the theoretical (50:50) sex ratio expected from untreated semen.
• Embryonic mortality was significant in the does inseminated with sorted intact sperm. With a reasonably high fertilization rate (Table I), one would expect a kindling rate of near 80% and litter size of about six from does of this age and breed. However, the kindling rate across the three treatment groups averaged 28%, with an average litter size of 3.9. The cause of the apparent high rate of embryonic death is thought to be due to the fluorochrome binding to the DNA and/or to the effect of -li ⁇
• DNA can be used as a differentiating marker between X- and Y-bearing sperm, that DNA can be used to accurately predict the sex of offspring from separated X- and Y-bearing sperm popula ⁇ tions, and that flow sorting is an effective means for separating viable X- and Y-bearing sperm populations suitable for production of offspring.
• the samples were incubated for 1 hr at 35°C.
• Intact sperm were sorted on a modified EPICS V flow cytometer/cell sorter.
• the stained intact sperm were excited in the ultraviolet (UV; 361 and 364 n ) lines of a 5-watt 90-5 Innova Argon-ion laser operating at 2000mW. Data were collected as 256- channel histograms.
• PBS phosphate- buffered saline
• the composition of the extender was N-tris(hy- droxymethyl)-methyl-2-amino ethane sulfonic acid, 2.16 g; tris hydroxymethyl aminomethane, 0.51 g; dextrose, 0.1 g; streptomycin sulfate, 0.13 g; penicillin G, 0.08 g; egg yolk, 12.5 ml; Equex STM (Nova Chemical Sales, Scituato, MA), 0.5%; and distilled water, 50 ml. This mixture was centrifuged, and only the supernatant was used. The sorted sperm were concentrated by incubating at room temperature for 1 hr, after which the more dilute frac ⁇ tion was removed and the remainder was used for insemina ⁇ tion 1 to 4 hr later.
• Example 2 Example 2
• Mature New Zealand White does were injected with 150 international units of human chorionic gonado- tropin (HOG) to induce ovulation, which was expected to occur 10 hr later.
• HOG human chorionic gonado- tropin
• the does were surgically prepared by injection with Ketamine hydrochloride containing acepromazine and anes ⁇ thetized under halothane and oxygen.
• the uterus was exposed by midline incision, and 100 ⁇ X of sorted or unsorted sperm was placed into the lumen of the anterior tip of each uterine horn through a 21-guage needle. Standard management practices were used in caring for the rabbits.
• These does were sacrificed 40 hr post-insemina ⁇ tion; uteri were flushed and recovered eggs evaluated. All fertilized eggs recovered were classified as morula. The results of these experiments are shown in Table I.
• Example 3 shows the results of inseminations made into the tip of the uterine horn: the number of does that kindled and the phenotypic sex of the offspring compared to the predicted sex.
• Predicted sex of off ⁇ spring was based on reanalysis of sorted intact sperm to determine relative DNA content. For reanalysis, the sorted sperm was sonicated for 10 sec and centrifuged at 15,000 g, the supernatant was discarded, and the pellet was resuspended in 9 JU bisbenzimide H 33342. Phenotypic sex of the offspring was determined soon after birth and confirmed at later ages up to 10 weeks. Recombined X and Y is the sorted X and Y sperm populations recombined immediately before insemination.
• Example 4 Using the methods of Examples 1, 2, and 3, viable swine sperm was sorted into viable X and Y chro ⁇ mosome-bearing populations. Two litters (18 pigs) from surgically inseminated boar semen produced 88% females from X-sorted sperm and 67% males from Y-sorted sperm. It is understood that the foregoing detailed description is given mainly by way of illustration and that modification and variation may be made therein with ⁇ out departure from the spirit and scope of the invention.

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• 1990
  • 1990-04-30 JP JP2507796A patent/JP2552582B2/ja not_active Expired - Lifetime
  • 1990-04-30 AU AU56642/90A patent/AU623016B2/en not_active Expired
  • 1990-04-30 WO PCT/US1990/002324 patent/WO1990013303A1/en not_active Ceased
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  • 1990-04-30 DK DK90907724.0T patent/DK0471758T3/da active
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• 1991
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