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ISSN : 1738-2432(Print)
ISSN : 2288-0151(Online)
Reproductive & developmental Biology Vol.36 No.1 pp.1-6

Analysis of Sexed Sperm by Flow Cytometry in Hanwoo (Korean Native Cattle)

Han-Jun Yoo1, Kyung-Jin Lee2, Yong-Seung Lee2, Pil-Sang Yoon1, Joung-Jun Park1, Hyeong-Cheol Kim3, Choon-Keun Park2,†
2College of Animal Life Sciences, Kangwon National University
1Developmental Biotechnology Laboratory, Myung-poom Hanwoo Consulting, 3Hanwoo Experiment Station, National Livestock Research Institute
Received: 19 January 2012, Accepted: 26 March 2012


This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm wereseparated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without nearUV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability (84±1.15 %, p<0.05) comparedto other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability (79±3 %,p<0.05) than those of the X and Y sperm (44.7±1.67 and 41.7±1.2 %) separated by differences of DNA content. On theother hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities(24.3±1.2 and 25.7±0.9 %, p<0.05) compared to that of the sorted Y sperm (13.7±1.2 %) in the hypoosmotic swellingtest. The proportion acrosome reaction in the sorted X sperm was higher (55.0±1.7 and 45.0±1.5 %) than those of thesorted Y-sperm (32.3±0.9 %, p<0.05). However, the sperm morphologies of the sorted groups were not significantlydifferences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm.Further study is needed to determine the optimal procedures to enhance male and female embryos and sortingaccuracy.

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Sex-sorting of mammalian spermatozoa has applications for genetic improvement of farm animals, in humans for the control of sex-linked disease, and in wildlife as a captive management strategy and for the repopulation of endangered species. Also, higher degree of female selection will have an impact on the genetic development of the population as females will contribute with up to 15 % on genetic selection, which was based so far on sires (Weigel and Barlass, 2003). This technology was the most highly developed for bovine semen and was introduced into commercial application. Recently, several American AI centers have contracted for the technology and offer sexed semen from their bulls worldwide. 

 The current technology to sort X and Y chromosome bearing sperm population requires individual identification and selection of spermatozoa in a modified high-speed flow cytometer (Johnson and Welch, 1999). The success of the technology will depend mainly on the fertilizing capacity of the sorted spermatozoa, as this is the most affecting and economically relevant factor (Rath and Johnson, 2008). Sex selection by flow cytometry involves the staining of spermatozoa with Hoechst 33342, penetrate the living sperm membrane and bind to the DNA of the highly condensed chromatin of the sperm nucleus (Johnson et al., 1987), in combination with the impact of an ultraviolet laser beam, Johnson et al. (1989) postulated that fluorochrome dyes reduce embryonic viability by mid-gestation. Co-incubation with Hoechst dye as well as the sorting process itself diminished the percentage of live spermatozoa. The damaged ability to fertilize and carry the embryo to term may be the result of the combined effects of the dye and UV laser or of either individually. Higher laser intensity is more damaging than the lower laser intensity as shown for rabbit (Johson et al., 1996) and bovine (Schenk and Seidel, 2007) spermatozoa. The majority of the studies with sexed sperm evaluated a specific feature such as their motility and integrity of the DNA and acrosome (Suh et al., 2005; Mocé et al., 2006) or higher pregnancy loss for sexed sperm compared to non-sexed sperm (Bodmer et al., 2005; Underwood et al., 2010).

 Therefore, the objective of the present study was to investigate sperm ability to produce embryos after sexsorting by flow cytometry in Hanwoo (Korean native cattle).


Preparation of Sheath Fluid Buffer

 Sheath fluid is an isotonic buffer saline solution that is pumped through the flow chamber, causing single cells to flow through the middle of the stream. Preparation of sheath fluid buffer, known as HEPES sheath flow, used in stallions (Buss, 2005) was performed from each of the regents shown in Table 1 in a mixture with distilled water. Briefly, metal ions such as CaCl2 · H2O, MgCl2․6H2O (Sigma-Aldrich, St. Louis, MO, USA), which are involved in chelation, are dissolved in 800 ml distilled water. After they are completely dissolved, other powder regents are added, and then the remaining liquid components are dissolved to incomplete compounds of sheath flow. After all of the powder is dissolved, the sheath flow buffer is adjusted to 1 L with distilled water and to pH 7.2. The filtration was performed through 0.2 μm pore size filters. Generally, this solution was prepared as a five-fold stock solution and was stored at 4℃ before use. The shelf-life of the solution was considered to be only one week because instability of the stream during flow cytometric sperm sorting is observed after longer storage times. Additionally, phosphate buffered saline (PBS) and tris-buffered, Triladyl(Minitüb, Tiefenbach, Germany) were used as flow buffers in Hanwoo sperm sexing procedures (data not shown).

Table 1. Composition of HEPES sheath fluid buffer for sex-sorting of bull sperm using flow cytometry

Cryopreservation and Thawing of Sperm

 For all experiments herein, Korean native cattle (Hanwoo) semen was collected using an artificial vagina and a teaser at the Hoengseong Livestock Cooperative Farm. Immediately after collection, motility and concentration of each bull sperm sample was assured using a phase-contrast microscope and a hemocytometer, respectively. Collected semen (volume: 5~15 ml; density: 2~10×108 ml; live ratio >75%) was diluted 1:1 (v/v) with Triladyl containing 20 % egg yolk, and the semen tube was placed in a 500 ml, 35℃ water jacket before being cooled to 4℃ over 6 h in a refrigerator. The second dilution to a concentration of 10×106 sperm per ml was then performed with an aliquot of semen diluted with the same freezing extender as was used in the first dilution. At this point, sperm were loaded into 0.5 ml straws, frozen and submerged in liquid nitrogen for storage. For use, semen straws were thawed for 45 s in a 37℃ water bath and dried. Semen was placed on a continuous density gradient tube to remove non-viable sperm and cryodiluent

Sperm Pretreatment

 Percoll, a sterile colloidal silica suspension and isotonic salt gradient solution, was diluted to a 65 % fraction with 10x HEPES buffer. The 65 % Percoll was prepared by placing 1 ml in a 1.5 ml micro tube. The semen (0.5 ml) was placed over the gradient and centrifuged at 1,700 rpm for 15 min. The sperm pellets were assessed for concentration and resuspended in 1 ml of HEPES sheath flow buffer. The sperm was then subjected to one more washing step in order to improve the resolution of flow cytometry for sexing.

Hoechst 33342 Staining

 Sperm preparation and staining were based on the method described by Johnson et al. (1989) in order to maintain viability through sorting and fertilization. Briefly, pretreated aliquots of fresh or frozen- thawed semen were stained with 40 μM Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) and were incubated for 30 min at 38℃ to foster fluorochrome penetration. Finally, extra fluoro-dye, which was not bound to the adenine-thymine regions of the sperm DNA, was eliminated via centrifugation. Also, an unstained control sample was prepared as a reference comparison of intensity of Hoechst 33342 on flow cytometry during fluorescence compensation. Generally, samples were prepared for separation in several loading tubes because stained sperm aggregate during sperm sorting after 18~25 min.

Sex-Sorting of Sperm

 Intact sperm were sorted using a flow cytometer/cell sorter (BD FACs Aria Ⅱ; BD, USA). Intact, viable Hanwoo sperm were flow-cytometrically separated into X and Y populations on the basis of relative DNA contents with near UV laser beam (Fig. 1). Also, sperm were performed the pre-sorting without near UV laser compare stained sperm. Twenty to forty percent of living intact sperm were oriented with this process, and a sorting rate of approximately 50 to 200 sperm/sec was achieved for each population. The sperm cells were measured and sorted using a 100 μm nozzle and a HEPES sheath fluid pressure of 20 psi. Sperm were sorted in single-cell mode into each 5 ml round tube that had been treated with 0.4 % BSA solution to prevent the sperm from sticking to the wall of the tube, resulting in a final concentration of at least 1~5 × 106 ml per tube. The tubes were then stored at room temperature (25℃; RT) until analysis. After a total of 1~5 × 106 sperm cells were collected, the sperm were concentrated via centrifugation at 1,500 rpm for 7 min. The resultant pellet was resuspended with HEPES buffer and 2 % egg yolk and was processed for immediate use.

Fig. 1. Visualized histogram of X- and Y-chromosomes after Hoechst 33342 staining and a dot plot of sperm cell population. Hanwoo sperm were flow-cytometrically separated into X and Y populations based on differences in DNA content. Twenty to forty percent of living intact sperm were oriented with this process, and sorting rates of approximately 50 to 200 sperm/sec were achieved for each population.

Sperm Analysis

 The LIVE/DEAD Sperm Viability Kit (Molecular Probes, Eugene, OR, USA) was used in the fluorescence-based assay to analyze the viability of sperm. Membrane-permanent SYBR 14 nucleic acid stain labels live sperm with green fluorescent tags, and membrane-impermanent propidium iodide labels the nucleic acids of membrane-compromised sperm with red fluorescent tags. Diluted semen samples in PBS were prepared in lighttight tubes. A final SYBR-14 concentration of 40 nM was achieved through addition of 2 μl of diluted SYBR-14 dye to each sample of diluted semen. After incubation for 5~10 min at 37℃, 2 μl of propidium iodide was added to the sample of diluted semen to produce a final propidium iodide concentration of 4.8 μM. The samples were incubated for 5~10 min 37℃ and then analyzed either under a fluorescence microscope equipped with equivalent filters or with flow cytometry.

 For the evaluation of sperm morphology, semen samples in PBS were centrifuged at 1,500 rpm for 5 min. The supernatant was removed, and the sperm were spread evenly over a glass slide. The smear was allowed to air-dry for 5 to 10 min and was stained with rose bengal (Sigma-Aldrich, St. Louis, MO, USA). The morphologic characteristics of 300 sperm were analyzed under a light microscope.

 The status of the sperm acrosome was assessed using the Coomassie Brilliant blue G-250 dye method (Moller et al., 1990) with slight modifications. Briefly, the dye solution was prepared by suspending 15 mg of Coomassie Brilliant blue G-250 dye in 10 ml of 3.5% perchloric acid and then filtering it through filter paper. Multitest slides with fixed sperm were incubated with the dye for 2 min and then washed in distilled water. The slides were covered with a coverslip using mounting solution (PBS containing 10 % glycerol). All slides were randomized and scored blindly based on observed sperm. Sperm with an intact acrosome exhibited blue stain over the sperm head. Such staining was not detectable on the sperm that had undergone the acrosome reaction (AR). To induce the acrosome reaction (AR), the sperm were subjected to capacitation, which took place in the medium containing heparin via incubation at 37℃ for 1 h under 5 % CO₂ in air. The calculation of the acrosome reaction used the difference ratio between the AR status before and after induction of the acrosome reaction.

 The hypo-osmotic swelling test (HOST) is based on the semi-permeability of the intact cell membrane, which causes sperm to swell under hypo-osmotic conditions, when an influx of water results in an expansion of cell volume (Drevius and Eriksson, 1966). The test method was introduced by Jeyendran et al. (1984). Briefly, HOST solution (150 mosm/l) was prepared by dissolving 0.735 g sodium citrate and 1.351 g fructose in 100 ml of distilled water. The solution was stored at 4℃ until use. Before the solution was used in the experiment, it was warmed in a 1 ml closed eppendorf tube at 37℃ for about 5 min. Then, 1 ml of HOST solution was mixed with 0.1 ml of diluted semen and incubated at 37℃ for 30 min. A drop of diluted semen was placed onto a dry glass slide and covered with a cover slip. A total of 300 sperm were counted in different fields at 400× using a phase-contrast microscope, and swelling sperm were identified based on changes in the coiled shape of the tail.

Statistical Analysis

 Statistical analysis was performed with analysis of variance (ANOVA) using SAS (version 9.1, SAS Institute Inc., Cary, NC, USA). Differences among sperm analysis mean values from the sperm sorting method results were processed using Duncan's multiple range tests. Significance was defined at a level of p<0.05.


 Pre-sorted sperm had significantly higher viability (84±1.15 %, p<0.05) compared to those of the other sorted groups in frozen- thawed semen. Also, significantly lower viability sorted sperm were analyzed sorted Y sperm (62.7±1.2 %) by differences of DNA content in exceptional control group (p<0.05). The fresh semen showed significantly higher viability in pre-sorted sperm (79±3 %, p<0.05) more than separated into X and Y sperm (44.7±1.67 and 41.7±1.2 %) by differences of DNA content in exceptional control group (Fig. 2). A comparison between fresh sperm and frozen-thawed sperm showed that frozen-thawed sorted sperm tended to have higher viability than fresh-sorted sperm.

Fig. 2. Viability of fresh and frozen-thawed sperm sorted using the near UV laser beam at a low flow rate (p<0.05). Pre-sort mean sperm sorted without near UV laser beam. Hoechst mean sperm sorted according to differences in DNA content.

 The results of sperm analysis are shown in Table 2. In the hypoosmotic swelling test (HOST), the pre-sorted and sorted X sperm according to differences in DNA content had significantly higher viabilities (24.3±1.2 and 25.7±0.9 %, p<0.05) than did the sorted Y sperm (13.7±1.2 %). The same results were observed in the acrosome reaction experiment. In short, the pre-sorted and sorted X sperm were significantly more viable (55.0±1.7 and 45.0±1.5 %) than was the sorted Y sperm (32.3±0.9 %, p<0.05). However, there were no significant morphological differences between the two sorting methods.

Table 2. Sperm analysis using the hypoosmotic swelling test, abnormal morphology and acrosome reaction rate in frozen-thawed sperm sorted at a low flow rate


 Previous experiments have only focused on increasing the viability and motility of sorted sperm. This problem was solved by changing the nozzle type from the 100 micron closed loop type (assembled type with oring) to the normal type (requires assembly with oring). However, other limitations, such as fertility and concentration of sorted sperm, which compromise the sperm’s ability to produce offspring, have been reported. To minimize these limitations, the Percoll gradient and BSA were tested during sperm preparation. Moreover, an additional experiment (data not shown) was conducted to examine the differences between three different collection media: HEPES buffer containing 2 % egg yolk or 10 % seminal plasma, and 0.4 % BSA HEPES buffer containing 2 % egg yolk in order to achieve the highest viability and storability before sperm analysis. Test was performed where egg yolk, BSA and seminal plasma from boar semen were added to the collection tube before or after sperm sorting. The Percoll gradient and egg yolk effected viability, and the egg yolk and seminal plasma increased motility after sperm sorting. The research described here was conducted in parallel to another study, which also used additives, such as TEST-yolk (boar: Johnson, 1995) or Androhep EnduraguardTM (Minitüb, Germany; ram: Hollinshead et al., 2003) or XY Talp and egg yolk (bull: Schenk et al., 1999) or glucose-skim milk extender (stallion:Buchanan et al., 2000), all of which provide protection from the combined effects of dilution by sheath fluid and physical damage by projection into the collection tube (Maxwell and Johnson, 1999). Also, numerous studies (Mousset-Siméon et al., 2004; Samardzija et al., 2006) have examined the benefits of various gradient separation methods such as PureSperm  or BoviPureTM (Nidacon, Göteborg, Sweden) instead of Percoll  (Sigma-Aldrich, St Louis, MO, USA) to remove non-viable sperm, in addition to the use of cryodiluent prior to IVF. Another physical characteristic affected by the process of sexing was integrity of the membrane. This change may have been due to mechanical stress (Garner, 2006); in that regard, decreased pressure during the process of sexing increased the survival of sexed sperm, and consequently rates of fertilization (Suh et al., 2005) and pregnancy (Schenk et al., 2009).

 The acrosome reaction is the final step in the multi event process of capacitation. Thus, semen samples with high percentages of acrosome reacted spermatozoa would most probably have originated from bulls that produced semen that was generally more capacitated, and such semen would potentially perform differently in similar IVF conditions (Blondin et al., 2009). Thundathil et al. (1999) demonstrated that the proportion of capacitated spermatozoa in frozen non-sexed semen is positively correlated to in vivo field fertility. Another study, again using frozen non-sexed semen, demonstrated that different bulls will result in different capacities to produce embryos in an IVF system (Palma et al., 2008).

 In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.


 The authors are grateful to researcher Joung-Hee Park, Biotechnology Analysis Lab, Central Laboratory Kangwon National University, Korea and gratefully acknowledges the Hoengseong Livestock Cooperative and Institute of Animal Resources of Kangwon National University for providing the semen samples and analysis.


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