G.R. Welch

Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency.

Sex preselection that is based on flow-cytometric measurement of sperm DNA content to enable sorting of X- from Y-chromosome-bearing sperm has proven reproducible at various locations and with many species at greater than 90% purity. Offspring of the predetermined sex in both domestic animals and human beings have been born using this technology since its introduction in 1989. The method involves treating sperm with the fluorescent dye, Hoechst 33342, which binds to the DNA and then sorting them into X- and Y-bearing-sperm populations with a flow cytometer/cell sorter modified specifically for sperm. Sexed sperm are then used with differing semen delivery routes such as intra-uterine, intra-tubal, artificial insemination (deep-uterine and cervical), in vitro fertilization and embryo transfer, and intra-cytoplasmic sperm injection (ICSI). Offspring produced at all locations using the technology have been morphologically normal and reproductively capable in succeeding generations. With the advent of high-speed cell sorting technology and improved efficiency of sorting by a new sperm orienting nozzle, the efficiency of sexed sperm production is significantly enhanced. This paper describes development of the these technological improvements in the Beltsville Sexing Technology that has brought sexed sperm to a new level of application. Under typical conditions the high-speed sperm sorter with the orienting nozzle (HiSON) results in purities of 90% of X- and Y-bearing sperm at 6 million sperm per h for each population. Taken to its highest performance level, the HiSON has produced X-bearing-sperm populations at 85 to 90% purity in the production of up to 11 million X-bearing-sperm per h of sorting. In addition if one accepts a lower purity (75 to 80%) of X, nearly 20 million sperm can be sorted per h. The latter represents a 30 to 60-fold improvement over the 1989 sorting technology using rabbit sperm. It is anticipated that with instrument refinements the production capacity can be improved even further. The application of the current technology has led to much wider potential for practical usage through conventional and deep-uterine artificial insemination of many species, especially cattle. It also opens the possibility of utilizing sexed sperm for artificial insemination in swine once low-sperm-dose methods are perfected. Sexed sperm on demand has become a reality through the development of the HiSON system.

PRODUCTION OF PIGLETS PRESELECTED FOR SEX FOLLOWING IN VITRO FERTILIZATION WITH X AND Y CHROMOSOME-BEARING SPERMATOZOA SORTED BY FLOW CYTOMETRY

In vivo-matured porcine oocytes were fertilized in vitro with X and Y chromosome-bearing spermatozoa, and sorted for sex on the basis of DNA content by flow cytometry. Developmental competence of the sexed embryos was determined through established pregnancies after embryo transfer. Spermatozoa were stained with Hoechst 33342 and sorted using a flow cytometry cell sorter. Purity of sorting was 83% for Y spermatozoa and 92% for X spermatozoa. A total of 387 mature cumulus-oocyte-complexes (COC) was collected from 18 superovulated prepuberal gilts shortly before ovulation. In vitro fertilization with sorted spermatozoa was performed in 4 replicates. After 18 h of sperm- oocyte co-culture at 39 degrees C, the zygotes were placed into culture medium (NCSU-23) for another 24 h. The average cleavage rate was 56.2%. Ninety-two embryos produced from X-sorted sperm cells were transferred surgically into the uterus of 2 recipients. Two gilts farrowed and delivered 6 and 4 healthy female piglets, respectively. Additionally, 2 gilts were inseminated intratubally via surgical laparotomy with either X or Y sorted spermatozoa (2 x 10(5)) per oviduct. The 2 sows farrowed producing 15 piglets. Thirteen of the 15 piglets were of the predicted gender (85%).

Uterine horn insemination of heifers with very low numbers of nonfrozen and sexed spermatozoa

Abstract Experiments were conducted to determine 1) pregnancy rates of heifers inseminated with very low numbers of spermatozoa under ideal field conditions, and 2) pregnancy rates with low doses of sexed spermatozoa. In Experiment 1, semen from 3 Holstein bulls was extended to 1 × 105 or 2.5 × 105 sperm/0.1 ml; 2.5 × 106 total sperm/0.21 ml were used for the control. Semen was cooled to 5 °C, packaged into modified 0.25-ml French straws, and used 26 to 57 h after collection. Spermatozoa were inseminated 24 h after detection of estrus into the uterine horn of Holstein heifers ipsilateral to the ovary with the largest follicle, as determined by ultrasound 12 h after estrus was detected; side of ovulation was verified by detection of a corpus luteum (CL) by ultrasound 7 to 9 d post estrus. Pregnancy was determined 40 to 45 d post estrus. The side of ovulation was determined correctly in 262 of 286 heifers (92%), and pregnancy rates were nearly identical for ipsilateral and contralateral inseminations. Pregnancy rates were 48/118 (41%), 56/111 (50%), and 35/57 (61%) for 1 × 105, 2.5 × 105 and 2.5 × 106 sperm per insemination (P .05) among the heifers for the 3 AI technicians or the 3 bulls. In Experiment 2, freshly collected semen was transported from Lancaster, Pennsylvania to Beltsville, Maryland, and sorted into X- and Y-sperm populations based on DNA difference using a flow cytometer/cell sorter over a 6-h period. Sorting rates were about 100 sperm/sec of each sex at ~90% purity. Sorted spermatozoa were shipped ~2600 km by air, and in most cases cooled to 5 °C during shipping over 6 h in an Equitainer. Heifers were inseminated with 1 to 2 × 105 sorted X- or Y-spermatozoa in 0.1 ml within 9 to 29 h of sorting. The inseminate was either deposited into the uterine horn ipsilateral to the ovary with the largest follicle as determined by ultrasound at the time of insemination, or half of the inseminate was deposited into each uterine horn. None of 10 heifers became pregnant when inseminated with sexed spermatozoa shipped at ambient temperature. Of the 155 heifers inseminated with sexed spermatozoa cooled to 5 °C, 15 of 67 females (22.4%) inseminated 9 to 13 h post sorting calved, but only 2 of 78 (2.6%) inseminated at 17 to 29 h calved. Fourteen of the 17 calves bom (82%) were of the selected sex.

A novel nozzle for more efficient sperm orientation to improve sorting efficiency of X and Y chromosome-bearing sperm†

Efficient high-resolution detection of DNA for flow cytometric sorting of X and Y chromosome-bearing sperm is dependent on effectively orientating the sperm head to the laser beam in orthogonally configured flow systems. Normally, a beveled needle is required to enlarge the fraction of properly orientated sperm (flat side facing the laser beam). In this report, a modification to a standard jet-in-air nozzle for improved sperm orientation is presented. Inside the modified nozzle (novel nozzle), orientation forces are applied lower in the nozzle than in the current beveled injection needle system. The nozzle was tested with sperm heads from several species. This study shows that use of the nozzle to orientate cattle, swine, rabbit, mouse, and human sperm effectively improves the percentage of sperm that are properly oriented. The percentage of sperm heads oriented by use of the former system (beveled needle) ranges around 30% for most species. With the newly designed nozzle, that percentage ranges around 60%. At least a twofold increase in analysis is achieved. It was found that, unlike results with the beveled needle, the percentage of properly oriented sperm was independent of the sample rate. The introduced nozzle is a significant improvement over the beveled needle system for the analysis and sorting of sperm on the basis of DNA content. In addition to the improvement in sorted sperm production brought about by the novel nozzle when fitted to standard-speed cell sorters, it clearly also has significant potential for improving the efficiency of the Beltsville Sperm Sexing Technology for separating X and Y chromosome-bearing sperm when adapted to high-speed cell-sorting systems.

Effects of reactive oxygen species on sperm function

Reactive oxygen species (ROS) formation and membrane lipid peroxidation have been recognized as problems for sperm survival and fertility. The precise roles and detection of superoxide (SO), hydrogen peroxide (HP), and membrane lipid peroxidation have been problematic, because of the low specificity and sensitivity of the established chemiluminescence assay technologies. We developed flow cytometric assays to measure SO, HP, membrane lipid peroxidation, and inner mitochondrial transmembrane potential in boar sperm. These methods were sufficiently sensitive to permit detection of early changes in ROS formation in sperm cells that were still viable. Basal ROS formation and membrane lipid peroxidation in the absence of ROS generators were low in viable sperm of both fresh and frozen-thawed boar semen, affecting less than 4% of the sperm cells on average. However, this is not the case in other species, as human, bovine, and poultry sperm have large increases in sperm ROS formation, lipid peroxidation, loss of motility, and death in vitro. Closer study of the effects of ROS formation on the relationship between sperm motility and ATP content in boar sperm was conducted using menadione (mitochondrial SO generator) and HP treatment. Menadione or HP caused an immediate disruption of motility with delayed or no decrease in sperm ATP content, respectively. Overall, the inhibitory effects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to a ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.

Mitochondrial function and reactive oxygen species action in relation to boar motility

Flow cytometric assays of viable boar sperm were developed to measure reactive oxygen species (ROS) formation (oxidization of hydroethidine to ethidium), membrane lipid peroxidation (oxidation of lipophilic probe C(11)-BODIPY(581/591)), and mitochondrial inner transmembrane potential (DeltaPsi(m); aggregation of mitochondrial probe JC-1) during hypothermic liquid storage and freeze-thawing of boar semen and to investigate relationships among ROS, motility, DeltaPsi(m), and ATP production. Basal ROS formation and membrane lipid peroxidation were low in viable sperm of both fresh and frozen-thawed semen, affecting < or =4%. Sperm in fresh, liquid-stored and frozen-thawed semen appeared to be equally susceptible to the activity ROS generators xanthine/xanthine oxidase, FeSO(4)/ascorbate, and hydrogen peroxide (H(2)O(2)). Of the ROS generators tested, FeSO(4)/ascorbate was specific for membrane lipid peroxidation, whereas menadione, xanthine/xanthine oxidase, and H(2)O(2) were specific for oxidization of hydroethidine. Menadione (30microM) and H(2)O(2) (300microM) decreased (P<0.05) motility by 90% during 60min of incubation. Menadione decreased (P<0.05) the incidence of sperm with high DeltaPsi(m) by 95% during 60min of the incubation, although ATP content was not decreased (P>0.05) until 120min. In contrast, H(2)O(2) did not affect DeltaPsi(m) or ATP at any time. The formation of ROS was not associated with any change in viability (90%) for either menadione or H(2)O(2) through 120min. Overall, the inhibitory affects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to an ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.

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.

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.

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique.

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.

Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis

The only known and measurable difference between X‐ and Y‐chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations High resolution two‐dimensional (2‐D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2‐D electrophoresis. Up to 1,000 protein spots per gel could be detected and quantified. Comparison of the 2‐D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X‐ and Y‐chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation.