The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than spermatozoa from Holstein, Hereford, and Angus bulls; spermatozoa from Brahman bulls had smaller X-Y differences (P less than 0.004). It is suggested from the evidence obtained in these studies that flow cytometry can be used to assess the proportion of X- and Y-spermatozoa in semen of domestic animals and is thereby applicable to verification of the effectiveness of enrichment techniques for X- or Y-spermatozoa.

A high-speed flow system for quantitative determination of fluoresence of cells containing fluorochrome has been developed. Feulgen-DNA distributions in populations of tissue culture cells and human leukocytes havebeen measured at a rate of 10(4) to 10(5) cells per minute and compare well with results of other independent methods.

The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +7 degrees.Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAF'I) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm.They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative DNA content of these two populations in sperm from normal mice and those with the Cattanach [7 to XI translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

A flow microfluorometer was used to measure metaphase chromosomes in suspension at rates up to 100,000 per min. Chromosomes from cells of the Chinese hamster M3-1 cell line were isolated, stained for DNA with the fluorescent dye ethidium bromide, and analyzed for DNA content. Nine distinct peaks were resolved that correspond well with independent chromosomal DNA measurements made with a high-resolution scanning cytophotometer. Chromosomes were sorted from each peak by an electronic cell sorter. Visual examination of each fraction indicated the purity of the sorted chromosomes. This novel technology allows separation of purified populations of individual chromosomes suitable for biochemical and biological characterizations. Metaphase chromosomes can be described by several properties, including length, area, DNA content, and sedimentation velocity (1, 2). Of these, DNA content is the most fundamental chromosomal descriptor and the only one insensitive to shape and degree of compaction. Heretofore, chromosomal DNA content has been measured cytophotometrically in conventional slide preparations (2-4); the method is complex and time-consuming, and cannot be used to prepare fractions of purified chromosomes. Bulk techniques such as centrifugation can sort the chromosomes, but only into gross fractions of low purity (5). The advent of flow microfluorometry and flow sorters allows application of precise DNA measurement, rapid processing, and individual sorting to metaphase chromosomes. We report here the first such application. Individual chromosomes of a clonal derivative (650A) from the M3-1 Chinese hamster cell line were isolated, stained for DNA with ethidium bromide, and analyzed by the flow system. A distribution of DNA for the chromosomes in suspension was obtained that showed nine distinct peaks. The chromosomes corresponding to each peak were separated for visual scoring by an electronic cell sorter. The relative DNA content (peak mean), chromosomal frequency (peak area), and chromosome type (peak content as determined visually) were compared with measurements of similar parameters made with a high-resolution scanning cytophotometer on chromosomes from metaphase spreads of cells from the same culture. The agreement was excellent. Chromosome spreads of cells from the same culture were also prepared for karyotyping and analysis by CYDAC (computer-oriented image analysis microscope system) according to procedures described elsewhere (2).Chromosome Staining. For flow system analysis, the chromosomes, still suspended in the Pipes solution, were stained for DNA by adding an equal volume of 0.004% ethidium bromide in Pipes buffer. The chromosomes were uniformly stained within 10 min; they remained in the stain solution throughout flow system analysis. Chromosomal fluorescence was stable from 0.1 hr to 2 days after staining.Other DNA dyes were used also. Propidium iodide, substituted for ethidium bromide, gave almost identical results. Chromosomes prepared according to the procedure of Burki et al. (5) and sta...

Theory predicts that small angle light scattering by spherical particles of 5 to 20 p. diam is nearly proportional to volume and insensitive to particle refractive index. A flow system photometer with helium-neon laser light source measures the scattering between 0.5 and 2.0 0 from individual particles at 10 4 to 10 5 /min. Volume distributions of mammalian cells and plastic microspheres agree with other independent determinations.A large desorption cryostat with a heat leak of about 150 mW is described. With the help of an electronic temperature regulator it is capable of maintaining any temperature between 4.2 and 19.0 K to an accuracy of better than 0.5%, using only 200 g of activated charcoal.