Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting

Intact, viable X and Y chromosome-bearing sperm populations of the rabbit were separated according to DNA content with a flow cytometer/cell sorter. Reanalysis for DNA of an aliquot from each sorted population showed purities of 86% for X-bearing sperm and 81% for Y-bearing sperm populations. Sorted sperm were surgically inseminated into the uterus of rabbits. From does inseminated with sorted X-bearing sperm, 94% of the offspring born were females. From does inseminated with sorted Y-bearing sperm from the same ejaculates, 81% of the offspring were males. The probability of the phenotypic sex ratios differing from 50:50 were p less than 0.0003 for X-sorted sperm and p less than 0.004 for Y-sorted sperm. Thus, the phenotypic sex ratio at birth was accurately predicted from the flow-cytometrically measured proportion of X- and Y-bearing sperm used for insemination.     

High pressure flow cytometric sorting damages sperm

Sexing sperm by high-speed flow cytometry subjects them to high pressure. The routine operating pressure of the MoFlo SX flow cytometer for sperm sorting for commercial production has been 50 pounds/square inch (psi), with a standard 70 microm standard nozzle tip. It was hypothesized that lowering the sorting pressure could reduce sperm damage. Therefore, a series of experiments using semen from six bulls, sorted with three MoFlo SX sorters, was conducted to determine optimal pressure. An additional experiment was done with stallion spermatozoa. In Experiment 1, sorting at 30 psi compared to 50 psi with the 70 microm nozzle tip increased sperm motility post-thaw at 30 min and 2h from 40.5 to 48.0% and 30.0 to 40.2%, respectively (P<0.05). In Experiment 2, 49, 43, 37, 31, and 25 psi resulted in 24.2, 32.8, 35.6, 37.5, and 39.8% progressively motile spermatozoa post-thaw (P<0.05). In Experiment 3, 3 pressures (50, 40, 30 psi)x2 sorting methods were further evaluated. At 50, 40, and 30 psi, respective mean sperm motilities at 30 min were 44.8, 48.6, and 49.6% (P<0.05), and percentage of live spermatozoa were 51.7, 55.7, and 57.8% (P<0.05). The improvement of post-sort sperm quality with lowered pressure was also evident in stallion spermatozoa. After sorting at 30, 40 and 50 psi were 40.6, 34.5 and 30.1% motile spermatozoa (P<0.1), and were 76.7, 72.5 and 67.8% (P<0.05) live spermatozoa (determined by SYBR-14/propidium iodide staining). In Experiment 4 sorter performance was evaluated with two pressures (40 and 50 psi)x2 staining concentrations of bovine spermatozoa (75 x 10(6) and 100 x 10(6)mL(-1)). Lowering pressure to 40 psi did not lower sort rate and purity when compared to 50 psi (P>0.05), and higher sperm concentration during staining increased sort rate (P<0.05). In conclusion, lowering pressure of the MoFlo SX flow cytometer for sperm sorting from 50 psi (standard pressure) to 40 psi clearly improved sperm quality without a significant decrease in sorter performance.     

Intra- and interboar variability in flow cytometric sperm sex sorting

To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intraboar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15 °C to 17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in three experiments. X- and Y-chromosome–bearing sperm were simultaneously separated according to the Beltsville sperm-sorting technology using a high-speed flow cytometer. In the first experiment, interboar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome–bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the receiver operating characteristics curve (0.88, P < 0.001). In the second experiment, a certain degree of intraboar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sortability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent 5 months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 hours of storage at 15 °C to 17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine artificial insemination programs.     

Flow cytometric sorting of non-human primate sperm nuclei

Pre-determination of the sex of offspring has implications for management and conservation of captive wildlife species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort spermatozoa of non-human primate species for use with assisted reproductive technologies. The objectives of this study were to: (i) determine the difference in DNA content between X- and Y-bearing spermatozoa (ii) sort sperm nuclei into X- and Y-enriched samples; and (iii) assess the accuracy of sorting. Spermatozoa were collected from two common marmosets (Callithrix jacchus), seven hamadryas baboons (Papio hamadryas) and two common chimpanzees (Pan troglodytes). Human spermatozoa from one male were used as a control. Sperm nuclei were stained (Hoechst 33342), incubated and analyzed using a high-speed cell sorter. Flow cytometric reanalysis of sorted samples (sort reanalysis, 10,000 events/sample) and fluorescence in situ hybridization (FISH; 500 sperm nuclei/sample) were used to evaluate accuracy of sorting. Based on fluorescence intensity of X- and Y-bearing sperm nuclei, the difference in DNA content between X and Y populations was 4.09 +/- 0.03, 4.20 +/- 0.03, 3.30 +/- 0.01, and 2.97 +/- 0.05%, for marmoset, baboon, chimpanzee and human, respectively. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94 +/- 0.9 and 93 +/- 0.8%, 94 +/- 0.7 and 94 +/- 0.5%, 91 +/- 0.9 and 97 +/- 0.6%, 94 +/- 0.6 and 94 +/- 0.9%, for marmoset, baboon, chimpanzee and human, respectively). These data indicate the potential for high-purity sorting of spermatozoa from non-human primates.     

Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X- and Y-sperm by sort reanalysis for DNA

Laboratory validation is essential in developing an effective method for separating X and Y sperm to preselect sex. Utilizing sexed sperm from a particular experiment to test fertility and achieve the subsequent phenotypic sex without knowing the likely outcome at conception is too costly for most applications. Further, research advances need to be built on an ongoing assessment with respect to the collection of data to continue progress towards achieving a successful outcome. The Beltsville Sperm Sexing Technology, which is based on the sorting of X- and Y-bearing sperm through the process of flow-cytometric sperm sorting, is also well suited for validation in the laboratory by "sort reanalysis" of the sperm X- and Y-bearing fractions for DNA content. Since the sexing technology is based on the use of Hoechst 33342, a permeant nuclear DNA stain for sorting X- and Y-bearing sperm, it also can be the marker for determining the proportions of X and Y populations by sort reanalysis. The process consists of using an aliquot of the sorted sperm and sonicating to obtain sperm nuclei. The uniformity of the nuclear staining is re-established through the addition of more Hoechst 33342. Separate analysis of each aliquot produces a histogram that is fitted to a double gaussian curve to determine proportions of X and Y populations. The relative breadths of the distributions of DNA of X- and Y-bearing sperm within a species affects interpretations of the histogram. Sort reanalysis is consistently repeatable with differences in X/Y DNA equal to or greater than 3.0%. This information on sex ratio of the sperm then provides the precise tool by which one can predict the outcome in terms of sex, from a particular sample of semen. Simple analysis of unsorted sperm to determine the proportions of X- and Y-bearing sperm based on DNA content is also an effective tool for validating sperm-sex ratio, whether it is in a sample assumed to be 50:50 or predicted to be something other than 50:50. This simple analysis provides for a check on the potential sex ratio of any sample of semen.     

Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 degrees C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 degrees C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples (P < 0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5 +/- 3.39%; 1.4 +/- 0.16 FPM versus 48.6 +/- 4.02%, 2.6 +/- 0.16 FPM; P < 0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0 +/- 19.50 spermatozoa) and distance (56.3 +/- 5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5 +/- 2.77%) and FPM rating (2.8 +/- 0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0 +/- 3.67 spermatozoa, 21.6 +/- 3.05 mm, 12.2 +/- 1.31% and 1.2 +/- 0.10 FPM, respectively; P < 0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0 +/- 1.2 mm; FT-GRADIENT: 57.2 +/- 0.76 mm; FT-CONTROL: 51.7 +/- 0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.     

Flow cytometric sorting of human sperm: MicroSort clinical trial update

This report provides a summary of MicroSort^® efficacy in separation of X- from Y-chromosome bearing human sperm (XSort^® and YSort^®, respectively), clinical outcomes, and the sex of the resultant babies when sorted sperm were used for intrauterine insemination (IUI), in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Clinical trial participants were married couples seeking reduced X-linked genetic disorder risk or family balancing. Sperm were stained with Hoechst 33342, sorted by flow cytometry, then used or cryopreserved for subsequent use. Fluorescence in situ hybridization (FISH) analysis determined the post-sort enrichment (purity) for X- and Y-bearing sperm. Birth and pediatric records were evaluated for incidence of congenital malformations. Between June 1994 and January 2007, patients underwent 3629 IUI cycles, 1642 IVF/ICSI cycles with fresh embryo transfer (ET) and 99 frozen embryo transfer (FET) cycles after MicroSort^®. Of 5871 total sorts, 74.9% were XSort^® and 25.1% were YSort^®. IVF/ICSI fertilization rate was 70.7% and 93.8% of 2PN embryos cleaved. The pregnancy rates for IUI, IVF/ICSI, and FET were 15.6, 32.0, and 33.3%, respectively, while miscarriage rates were 15.7, 14.3, and 33.3%, respectively. Post-sort purity averaged 87.9% (XSort^®) and 73.4% (YSort^®). A total of 1125 clinical pregnancies yielded 943 babies born and 167 ongoing pregnancies. For babies born, XSort^® resulted in 92.0% females and YSort^® yielded 81.5% males. Postnatal follow-up showed a 2.6% major congenital malformation rate, with no recurrent pattern or clustering of malformations. FISH results confirmed MicroSort^® enrichment of X- and Y-bearing sperm populations that closely corresponded with the sex of the resultant child. Fertilization, cleavage, spontaneous abortion, and pregnancy rates as well as incidence of major congenital malformations were comparable to those in literature reports utilizing unsorted sperm.     

Coulter counter-based evaluation of sperm volume to assess sperm viability of bull semen and application to X/Y sperm sorting

The Coulter Counter Hypo-Osmotic Swelling test (CC-HOS) was developed to provide insight into the membrane integrity (relative volume shift Vr) of sperm necessary for fertilization, and to identify the optimum buffer needed for the X/Y chromosome sorting process. Using the CC-HOS test on neat bovine semen, the mean relative volume shift Vr for July and August was 1.20 and 1.14, respectively, whereas mean Vr values ranged from 1.32 to 1.41 during September to November. There was an inverse relationship between Vr magnitude and environmental temperature; we inferred that this enhanced sperm viability during autumn relative to summer. A method was developed to measure the dynamics of volume change of sperm in the buffer (pH 6.5) used for the X/Y chromosome sorting process. When exposed to the buffer (4 mM K+, 153 mM Na+, 140 mM Cl(-)), sperm from Bull C had a mean modal volume of 22.8+/-0.2 fL during a 0-300 s time interval, which did not significantly vary from sperm volumes (21.88+/-0.66 fL for Bull A and 22.46+/-0.38 fL for Bull B) noted in isotonic Isoton II solution. However, when exposed to lower ionic concentrations (2 mM K+, 62 mM Na+, 47 mM Cl-), the mean volume of Bull C sperm increased to 29.2+/-1.5 fL and exhibited slower rates toward stabilized volumes relative to higher ionic concentration buffers. Utilization of volume swelling measurements for measuring the impact of ion concentrations in X/Y chromosome sorting process buffers illustrated the importance of its application for emerging sperm-based biotechnologies.     

Comparative motility of X and Y chromosome–bearing bovine sperm separated on the basis of DNA content by flow sorting

A combination of flow cytometric sperm sorting of X and Y chromosome–bearing sperm (X and Y sperm) and computer-assisted sperm analysis (CASA) for measuring sperm motility allows assessment of motion parameters in the two populations. Bull sperm were separated into X and Y populations by flow cytometry following staining with the DNA-binding dye Hoechst 33342. The motion parameters differed depending on sperm concentration. Decreasing sperm concentration resulted in higher velocities and straighter trajectories. The concentrations of control (stained-unsorted and unstained-unsorted) and flow-sorted sperm were therefore adjusted to similar numbers (5 × 10⁶ sperm per milliliter). Samples of sorted X and Y sperm and control sperm were transferred to prewarmed slides on a heated stage (37°C) and their motion video recorded for 2 min using a magnification of ×100 and a high-resolution camera. The sperm analysis was carried out on a Hobson Sperm Tracker (HST) using HST 7 software. The following motion parameters were measured: curvilinear, straight-line, and average path velocity; mean angular displacement (MAD); beat cross-frequency; amplitude of lateral head displacement; linearity (LIN); and straightness of path (STR). Sperm movement was unaffected by staining with Hoechst 33342, excitation by ultraviolet (UV) light, or the physical process of cell sorting. Significant differences were seen between X and Y sperm for MAD, LIN, and STR. No difference was observed for the other parameters. The results indicate that in a simple salts solution, Y bull sperm do not swim faster than X sperm but may be distinguished from X sperm on the basis of LIN and STR. Mol. Reprod. Dev. 50:323–327, 1998. Published 1998 Wiley-Liss, Inc.     

Assimilation efficiency of Vibrio bacterial protein biomass by the flagellate Pteridomonas : assessment using flow cytometric sorting

A flow cytometric sorting technique for direct determination of bacterial biomass assimilation by phagotrophic flagellates was developed and tested in laboratory culture experiments. Living Vibrio bacteria were quantitatively pulse-chase labelled with [(35)S]methionine tracer and fed to Pteridomonas flagellates. Flow sorting revealed that the isotopically labelled material is in either bacterial prey or flagellate predators and the egested bacterial debris contained negligible amounts of tracer. These experimental results confirm an earlier hypothesis that flagellates release metabolised bacterial proteins primarily in a dissolved form. The assimilation efficiency of the Vibrio protein biomass by Pteridomonas was low, only about 20%, independently of the amount of consumed bacterial biomass, confirming our earlier indirect estimates. Additionally, against expectations that cells decrease their metabolic activity whilst preparing for and engaged in division, we found that the precursor uptake rates by flow sorted bacterial cells at the S+G(2) cell cycle stages were constantly 1.5 times higher than those of cells at the G(1) stage.     

gamma-Glutamyltranspeptidase activity in intact leukocytes: flow cytometric analysis and sorting

A fluorescent method developed for visualizing gamma-glutamyltranspeptidase (GGT) in intact liver cells was adapted to leukocytes and used in a multiparameter flow cytometric study of blood and bone marrow cells from rats with subcutaneous implants of mammary carcinoma 5A. The severe granulocytosis caused by this non-metastatic tumor was preceded by a progressive rise in the percentage of leukocytes with high GGT fluorescence. Both granulocytes and small, immature cells of bone marrow showed increased GGT expression, whereas in blood this increase was attributable entirely to mature granulocytes. At 28 days (but not yet at 14 days) after carcinoma implantation, 20-30% of blood or bone marrow granulocytes constituted a distinct subpopulation in that their GGT fluorescence intensity range was much higher and did not overlap with the range for the rest of the population. The results indicate that fluorescent GGT assay of intact leukocytes provides a useful probe for flow cytometric analysis of population heterogeneity in leukoproliferative disorders.     

Swimming behavior of X and Y human sperm

Abstract. A laminar-flow fractionation method, developed primarily for removing dead sperm from human semen, was successfully modified to enrich X and Y sperm to 80% purity, and to characterize each enriched fraction for individual swimming behavior. Y-sperm fractions were rapidly detected by fluorescent cytogenetic staining. Subsequently, the degree of enrichment was quantitated with DNA extracted from each sperm fraction probed with a human male-specific recombinant DNA clone. In stationary fluid, X and Y sperm swam in circles with the same average speed. However, in a flowstream, X sperm shifted to a nearly straight path of movement in a significantly decreased angular velocity. This shift was four times more pronounced in X sperm than in Y sperm, especially after the initial transition from stationary fluid to flow. The velocity gradient across the flow axis was essential for separating X and Y sperm; uniform flow velocity did not separate them effectively.     

Swimming behavior of X and Y human sperm

A laminar-flow fractionation method, developed primarily for removing dead sperm from human semen, was successfully modified to enrich X and Y sperm to 80% purity, and to characterize each enriched fraction for individual swimming behavior. Y-sperm fractions were rapidly detected by fluorescent cytogenetic staining. Subsequently, the degree of enrichment was quantitated with DNA extracted from each sperm fraction probed with a human male-specific recombinant DNA clone. In stationary fluid, X and Y sperm swam in circles with the same average speed. However, in a flowstream, X sperm shifted to a nearly straight path of movement in a significantly decreased angular velocity. This shift was four times more pronounced in X sperm than in Y sperm, especially after the initial transition from stationary fluid to flow. The velocity gradient across the flow axis was essential for separating X and Y sperm; uniform flow velocity did not separate them effectively.     

Chromatin condensation in hamster sperm: A flow cytometric investigation

In this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine. © 1994 Wiley-Liss, Inc.     

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

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio-threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P <.05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P <.05). Treatment of sperm with DTT increased the activation rate (P < .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome-bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.     

Leukocytospermia and sperm preparation - a flow cytometric study

Background  

Leukocytes represent the predominant source of reactive oxygen species both in seminal plasma and in sperm suspensions and have been demonstrated to negatively influence sperm function and fertilization rate in assisted reproduction procedures. Peroxidase test is the standard method recommended by WHO to detect semen leukocytes but it may be inaccurate. The aims of this study were (i) to compare the efficiency of swim-up and density-gradient centrifugation techniques in removing seminal leukocytes, (ii) to examine the effect of leukocytes on sperm preparation, and (iii) to compare flow cytometry and peroxidase test in determining leukocyte concentration in semen using a multiparameter flow cytometric method.     

Improved flow cytometric sorting of X‐ and Y‐chromosome bearing sperm: Substantial increase in yield of sexed semen

The yield of flow cytometric sorted X‐ and Y‐chromosome‐bearing sperm in a given time period is an important factor in the strategies used for fertilization and the production of sex‐preselected offspring. This yield is dependent on the efficiency with which the modified flow cytometer/cell sorter analyzes the DNA of spermatozoa. The efficiency is directly related to the number of sperm with the correct orientation during DNA analysis. Currently, the efficiency of flow cytometric sperm sorting is low since orientation of the sperm head to laser excitation is rate limiting. To overcome this problem, a new nozzle was designed to enhance sperm orientation and tested under flow cytometric sorting conditions. The degree of orientation improvement was determined with different sample rates using viable sperm and dead sperm of several different species. There was at minimum, a two‐fold increase in the proportion of oriented sperm when comparing the new nozzle with the currently used modified flow cytometer/cell sorter employing a beveled needle. More than 60% of intact bull sperm and boar sperm were correctly oriented compared with 25% to 30% using the beveled needle system. A unique characteristic of the novel nozzle was that the proportion of oriented sperm was independent of sample rate and of sperm motility. The accuracy of DNA measurement together with high purity sorting was tested using the novel nozzle. The novel nozzle was unique in that accuracy of measurement and sorting performance were not diminished. Using the new nozzle, samples of 88% purity of sorted X‐sperm and Y‐sperm were obtained for viable bull and boar sperm. The yield of flow cytometric sorted X‐ and Y‐chromosome‐bearing sperm using the novel nozzle was, on average, twice that obtained by using the beveled needle system in conjunction with a standard equipment nozzle for orientation. Mol. Reprod. Dev. 52:50–56, 1999. Published 1999 Wiley‐Liss, Inc.     

Flow cytometric sorting of sperm: Influence on fertilization and embryo/fetal development in the rabbit

Viable, intact rabbit sperm, prepared, processed, and flow cytometrically sorted, were used in this study to determine the influence of flow sorting on fertilization and embryo development. In experiment I, flow-sorted or control (unstained and unsorted) sperm were surgically inseminated into the uterine horn of hormonally primed does (10 to 12 does per time point). At 42 hr postsurgical insemination, flushed embryos were assessed for development. Fetal development was determined at day 7, day 14, and day 21 post-surgical insemination. Embryos resulting from does surgically inseminated with control sperm at 42 hr post-insemination were observed to be at the early morula stage of development (>16 cell), whereas embryos from does inseminated with flow sorted sperm were at the 8- to 16-cell stage. No difference was observed between treatments at day 7, 14, or 21, however, there was a significant decrease in fetus number per doe inseminated with flow-sorted sperm over time. In experiment II, mature oocytes were flushed from the oviducts of superovulated does and coincubated in vitro (IVF) with flow-sorted or control rabbit sperm. Oocytes observed at 6 hr post-coincubation exhibited swollen sperm heads in the cytoplasm, demonstrating that fertilization had occurred (2 PN + T). There was a higher percentage of fertilized oocytes by 8 hr post-coincubation for both control (31%) and flow-sorted sperm (31%) when used for IVF. By 10 and 12 hr post-coincubation, little difference was observed in the number of fertilized oocytes between sperm treatments (52% and 66% for control vs. 57 and 54% for flow-sorted, respectively). These studies demonstrate that flow-sorted sperm are capable of fertilizing mature oocytes under in vitro conditions. In addition they show that flow sorting may not negatively influence fertilization events, but likely interferes during early embryonic and fetal development. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Factors governing the flow cytometric analysis and sorting of large biological particles

We have investigated the suitability of large flow cell tips for the flow cytometric analysis and sorting of large biological particles, including plant cells (pollen) and protoplasts. Using flow tips ranging in diameter from 79-204 micron, we have optimized conditions for the establishment of a stable hydrodynamic flow leading to accurate droplet production. We describe instrument modifications required for large particle sorting and demonstrate the use of these experimental conditions for the sorting to high purity of pollen and viable plant protoplasts possessing diameters as large as 95 micron. Our experiments have revealed a complex interaction among sorting efficiency, particle diameter, flow cell tip diameter and bimorphic crystal drive frequency. This interaction can be satisfactorily explained in terms of interference effects owing to phase differences between the particle-induced disturbance and the undulation driven by the bimorphic crystal.     

Flow sorting of X and Y chromosome-bearing spermatozoa into two populations

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation < 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.