Enrichment of high-quality spermatozoa in bovine semen: relative effectiveness of three filtration matrixes.

To be practical, any method for improving bull semen must yield a large quantity of motile spermatozoa. Some separation methods based on physical properties, e.g. filtration, chromatography, centrifugation, washing and pooling, have been reported as satisfactory, but generally are not repeatable. Nevertheless, filtration methods appear to allow the attainment of an acceptable number of spermatozoa, thus allowing such a technique to be introduced in the production of standard bovine semen doses for artificial insemination. The aim of this work was to evaluate systematically the relative effects of three filtration matrixes (silica oxide, glass beads or Sephadexätrade mark) on the improvement of whole ejaculate quality. Analysis of the type of matrix and the volume and height of the filtration column was performed. The only characteristic of the columns that appears to influence ejaculate quality after filtering is the matrix volume. While all matrixes produced improvement of semen quality, SephadexTM was better than the other matrixes tested. An explanation for the mechanism of column filtration is proposed.

Accelerator mass spectrometry in biomedical dosimetry: relationship between low-level exposure and covalent binding of heterocyclic amine carcinogens to DNA.

Accelerator mass spectrometry (AMS) is used to determine the amount of carcinogen covalently bound to mouse liver DNA (DNA adduct) following very low-level exposure to a 14C-labeled carcinogen. AMS is a highly sensitive method for counting long-lived but rare cosmogenic isotopes. While AMS is a tool of importance in the earth sciences, it has not been applied in biomedical research. The ability of AMS to assay rare isotope concentrations (10Be, 14C, 26Al, 41Ca, and 129I) in microgram amounts suggests that extension to the biomedical sciences is a natural and potentially powerful application of the technology. In this study, the relationship between exposure to low levels of 2-amino-3,8-dimethyl[2-14C]imidazo[4,5-f]quinoxaline and formation of DNA adducts is examined to establish the dynamic range of the technique and the potential sensitivity for biological measurements, as well as to evaluate the relationship between DNA adducts and low-dose carcinogen exposure. Instrument reproducibility in this study is 2%; sensitivity is 1 adduct per 10(11) nucleotides. Formation of adducts is linearly dependent on dose down to an exposure of 500 ng per kg of body weight. With the present measurements, we demonstrate at least 1 order of magnitude improvement over the best adduct detection sensitivity reported to date and 3-5 orders of magnitude improvement over other methods used for adduct measurement. An additional improvement of 2 orders of magnitude in sensitivity is suggested by preliminary experiments to develop bacterial hosts depleted in radiocarbon. Expanded applications involving human subjects, including clinical applications, are now expected because of the great detection sensitivity and small sample size requirements of AMS.

Quantification and classification of human sperm morphology by computer-assisted image analysis.

A quantitative, semi-automated method for classifying human sperm based on objective measurements of head shapes and sizes has been developed. Air-dried smears of semen from eight healthy men were stained with the Feulgen reaction and 283 sperm were selected as prototypic examples of the 10 morphology classes used in our classification system. Sperm heads were imaged through a microscope (NA = 1.3), sampled at 0.125-micron intervals, and measured on an image analysis system. Measurements included stain content, length, width, perimeter, area, and arithmetically derived combinations. Additionally, each sperm image was optically sectioned at right angles to its major axis to give a measure of lengthwise heterogeneity of shape. Linear stepwise discriminant analysis was used to identify the more powerful parameters and to create a model employing eight parameters. The jackknifed classification procedure distinguished normal from abnormal sperm with 95% accuracy and correctly assigned 86% of the sperm to one of 10 shape classes. Most of the misclassification errors occurred among closely related classes. The results demonstrate the ability of automated image analysis to classify individual sperm into clinically familiar shape categories.

Selection and separation of X- and Y- chromosome-bearing mammalian sperm.

Preselection of the gender of offspring is a subject that has held man's attention since the beginning of recorded history. Most scientific hypotheses for producing the desired sex of offspring address separation of X- and Y-bearing sperm, and most have had limited, if any success. Eight of these hypotheses and their experimental verifications are discussed here. Three hypotheses are based on physical characteristics of sperm, one on supposed differences in size and shape, another on differences in density, and a third on differences in surface charge. There has been no experimental verification of differences based on size and shape, and the results from attempts to verify separation of X- and Y-bearing sperm based on density have been mixed. Electrophoresis may provide a method for separating X- and Y-bearing sperm, but it is currently unproven and would be of little practical utility, since sperm motility is lost. A fourth hypothesis employs H-Y antigen to select preimplantation embryos. This method reliably produces female offspring, but does not permit the selection of male offspring and does not work on sperm. There are two applications of the theory that X- and Y-bearing sperm should be separable by flow fractionation. Flow fractionation using thermal convection, counter-streaming sedimentation, and galvanization is highly promoted by its originator but has not gained wide acceptance due to lack of independent confirmation. Flow fractionation by laminar flow is said to provide up to 80% enrichment of both X- and Y-bearing sperm; however, this method also has not been confirmed by other workers or tested in breeding trials. The sixth theory discussed is that of separation through Sephadex gel filtration. This method may provide enrichment of X-bearing sperm, but, again, other experimenters have not been able to adequately confirm the enrichment. The best-known approach to sperm separation is that employing albumin centrifugation, yet even with this method, not all researchers have been able to confirm a final fraction rich in Y sperm, and trials in animals have given contradictory results. The most reliable method for separating X- and Y-bearing sperm is use of flow cytometric and flow sorting techniques. These techniques routinely separate fractions with a purity greater than 80% and can be above 90%. Unfortunately, these methods do not always work for human samples. Furthermore, as with electrophoretic approaches, the methods identify and separate only chemically fixed sperm and provide limited biological applications.(ABSTRACT TRUNCATED AT 400 WORDS)

How large should my study be so that I can detect an altered sex ratio?

The world literature on manipulations purported to alter the gender of offspring is a quagmire full of reports presenting inadequate data and contradictory conclusions. Many are due to inappropriate statistical design or analysis. The authors present simple, nontechnical reference tables for determination of the minimum sample size necessary to detect a change in sex ratio (1) from a theoretical value, and (2) between two experimental populations. Simple methods for analyzing the results of gender manipulation experiments are described. Technical details are included in a separate section.

Sex chromosome ratios determined by karyotypic analysis in albumin-isolated human sperm.

Human sperm that had been processed for Y-enrichment (male sex preselection) according to a currently favored albumin density gradient procedure were analyzed karyotypically for the proportion of X and Y chromosomes with the use of the human sperm/hamster egg system. This method allows direct inspection of haploid chromosome complements from human sperm. In 290 albumin-isolated sperm from six men, there were 57.2% X- and 42.8% Y-bearing chromosome complements; 201 unprocessed concurrent control sperm from five of the men had 50.2% X and 49.8% Y complements. The observed shift in sex chromosome ratio in processed samples, a decrease in Y-bearing sperm, was not statistically different from that of unprocessed controls (P = 0.13) but was significantly different when compared with the theoretic X/Y ratio of 50/50 (P = 0.016). A total of 3187 historical control karyotypes were also reviewed, with an overall sex chromosome ratio (X/Y) of 49.8/50.2. The control groups did not differ significantly from the expected 50/50. The Y-enrichment of processed sperm was not confirmed.

Flow cytometric determination of the proportions of X- and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm.

A rapid assay for determining the proportions of X- and Y-chromosome-bearing sperm in semen samples would benefit research aimed at sex ratio control through sperm separation. It also would be of value for quality control should a separation technique be developed. Flow cytometric methods capable of measuring sperm DNA content precisely enough to resolve and quantify the X and Y populations in many mammalian species have been developed. They are effective for fresh and cryopreserved sperm of most domestic animals. Results are reported of flow cytometric analyses of bull sperm samples from seven commercial and academic sources after processing with procedures purported to separate the X and Y populations. In no case was enrichment of either sperm population observed. Breeding trials carried out by the sources of two of the sets of samples showed these procedures were ineffective in altering the sex ratio.

Estimation of the frequency of malformed sperm by slit scan flow cytometry.

We have investigated the utility of Slit Scan Flow Cytometry (SSFCM) for measuring the frequencies of malformed sperm heads in control and mutagen treated B6C3F1/CRL mice. In SSFCM, fluorescence profiles of sperm heads stained with the DNA-specific fluorescent dye acriflavine were recorded for sperm flowing lengthwise through a 2.5-microns-thick laser beam. Malformed sperm were detected as having fluorescence profiles that differed substantially from an average fluorescence profile for sperm from untreated mice. Specifically, a sum of squared difference (SSD) value was calculated for the fluorescence profile of each sperm according to the equation (Formula: see text) where c(i) and t(i) are the ith values for the fluorescence profiles from control and test sperm, respectively. Profiles whose SSD exceeded a threshold value of 20 were considered to be from malformed sperm. We measured fluorescence profiles for 500 sperm per mouse from five control mice, five mice injected intraperitoneally daily for 5 days with a total of 375 mg/kg of body weight methyl methane sulfonate (MMS), and for 30 mice injected intraperitoneally daily for 5 days with total doses of procarbazine ranging from 125 mg/kg to 1,250 mg/kg. Sperm were collected from the caudae epididymides 35 days after the last injection. Frequencies of malformed sperm in these samples were also estimated by visual analysis. All samples were analyzed in double blind fashion. The visual and SSFCM malformed sperm frequencies for the samples from control, MMS-treated, and procarbazine-treated mice were correlated (r = 0.83). A dose effect was seen with both the visual and SSFCM estimates for the sperm from the procarbazine-treated mice.

Effect of LH on steroidogenesis by hamster follicles isolated at defined stages of development.

Multiple follicular growth was stimulated in groups of immature female hamsters by the administration of PMSG. The ovaries were removed 72-78 or 96-102 h later. The animals at 96-102 h were subdivided according to whether the concentration of plasma progesterone was within the range of the concentrations found at the previous period (0.45-13.18 nmol/l; Group I) or above (Group II). Thecal and granulosa cells from batches of isolated follicles (591-740 micron) were cultured together in the presence or absence of LH and the products of steroidogenesis were measured in the culture fluid. The results showed that there was a significant increase in the production of progesterone and 17 alpha-hydroxyprogesterone in follicles from Groups I and II at 96-102 h. In the presence of LH there was a significant increase in the yield of progesterone and 17 alpha-hydroxyprogesterone over the control values at all time periods. There were, however, reductions in the yields of 17 alpha-hydroxyprogesterone and androstenedione in the presence of LH at 96-102 h compared with 72-78 h and in Group II compared with Group I, although the concentrations of 17 alpha-hydroxyprogesterone were approximately 40-fold those of androstenedione. In addition, the production of oestradiol decreased significantly in follicles from both groups at 96-102 h and was reduced further in the presence of LH. It is concluded that the major rate-limiting step of steroidogenesis in hamster follicles undergoing the oestrogen-progestagen shift involves the side-chain cleavage of 17 alpha-hydroxyprogesterone.

Half-life of FSH and LH in the ferret.

Anoestrous and oestrous ferrets were injected with luteinizing hormone-releasing hormone (LRH) or a long-acting analogue and subsequently hypophysectomized. Spayed ferrets were hypophysectomized without prior treatment with gonadotrophin releasing factor, and serial blood samples collected from all animals in order to follow the rate of decline in plasma gonadotrophin concentration. The half-life of LH in the spayed female (around 2 h) was much longer than that of the hormone released from the hypophysis of anoestrous females by LRH (25 min) or by the analogue (19 min). The half-life of FSH released by LRH or analogue in anoestrous females was approximately 65 min, while that discharged by the analogue in oestrous females was about 4 h. The fall in plasma FSH concentration in spayed females after hypophysectomy was too slow to allow calculation of a half-life.

Cytometry of deoxyribonucleic acid content and morphology of mammalian sperm.

Because spermatogenesis is exquisitely sensitive to external influences, sperm can serve as a biological dosimeter. Advances in interpreting induced sperm abnormalities require a better understanding of sperm characteristics. This report reviews the application of several methods for automated, quantitative detection of shape changes, methods that are faster and more sensitive than conventional subjective techniques. Variability of sperm deoxyribonucleic acid content as a bioassay of genetic damage is explored, and limitations of the bioassay are discussed. New flow cytometric techniques that could lead to sexing mammalian sperm are examined.

Radiation-induced DNA content variability in mouse sperm.

Mouse sperm collected from the cauda epididymidis 35 days after acute testicular X-ray exposure and fluorescently stained for DNA show dose-dependent increases in the coefficient of variation (CV) of flow cytometrically obtained fluorescence distributions. By comparing dose-response curves obtained with three protocols which overcome the optical and cytochemical difficulties of sperm measurement in different ways we conclude the response is due to X-ray-induced DNA content variability. In the range between 0 and 600 rad the dose dependence of the square of CV of the DNA content variability, delta CV2D, is described by delta CV2D = Bx + Cx2, with 0 less than or equal to B less than or equal to 0.23 X 10(-2) and C = (0.44 +/- 0.06) X 10(-4). The dose x is measured in rad and delta CVD is expressed in percent. Computer modeling of the shapes of the fluorescence distributions show that at 600 rad 30 to 40% of the sperm have abnormal DNA content. Some have errors as large as two whole chromosomes, but it is not clear whether they are due to whole chromosome nondisjunction or a finer fragmentation of the genome. Exposures to benzo(a)pyrene and mitomycin C cause no detectable DNA content variability. We conclude mouse sperm DNA content measurements are not sensitive to small amounts of aneuploidy and as such will only be useful in detecting agents that produce substantial DNA content variability. Another animal with a smaller number of chromosomes might be more favorable. These sperm measurement techniques may find additional application in other areas of reproductive biology, such as the determination of the relative numbers of X and Y chromosome-bearing sperm in semen that may be artificially enriched in one population.

Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry.

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.

Sex preselection in mammals? Separation of sperm bearing Y and "O" chromosomes in the vole Microtus oregoni.

The two sex determining sperm populations of the vole Microtus oregoni were separated according to DNA content by use of flow sorting instrumentation. Although the sperm were not viable, they should be useful for addressing the question of haploid expression of genes linked to sex chromosomes and for efficiently searching for biochemical markers that differentiate the two populations.

Quantitative analysis of radiation-induced changes in sperm morphology.

When developing spermatogenic cells are exposed to radiation, chemical carcinogens or mutagens, the transformation in the morphology of the mature sperm can be used to determine the severity of the exposure. In this study five groups of mice with three mice per group received testicular doses of X irradiation at dosage levels ranging from 0 rad to 120 rad. A random sample of 100 mature sperm per mouse was analyzed five weeks later for the quantitative morphologic transformation as a function of dosage level. The cells were stained with gallocyanin chrome alum (GCA) so that only the DNA in the sperm head was visible. The ACUity quantitative microscopy system at Lawrence Livermore National Laboratory was used to scan the sperm at a sampling density of 16 points per linear micrometer and with 256 brightness levels per point. The contour of each cell was extracted using conventional thresholding techniques on the high-contrast images. For each contour a variety of shape features was then computed to characterize the morphology of that cell. Using the control group and the distribution of their shape features to establish the variability of a normal sperm population, the 95% limits on normal morphology were established. Using only four shape features, a doubling dose of approximately 39 rad was determined. That is, at 39 rad exposure the percentage of abnormal cells was twice that occurring in the control population. This compared to a doubling dose of approximately 70 rad obtained from a concurrent visual procedure.

High resolution DNA content measurements of mammalian sperm.

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 (DAPI) 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 X] 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.