Theriogenology 81 (2014) 675–682
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Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence Hyun-Tae Jo a, Jae-Il Bang a, Seong-Su Kim a, Byung-Hyun Choi a, Jong-In Jin a, Heyng-Lyool Kim c, In-Suk Jung d, Tae-Kwang Suh d, Nasser Ghanem a, e, Zhongde Wang f, Il-Keun Kong a, b, * a
Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea b Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea c Dairy Cattle Improvement Center, National Agricultural Cooperation Federation, Goyang-si, Gyeonggi-do, Republic of Korea d Korea Sexing Biotech Inc., Daegu, Gyeongsangbuk-do, Republic of Korea e Department of Animal Production, Faculty of Agriculture Cairo University, Giza, Egypt f Department of Animal, Dairy, and Veterinary Sciences, School of Veterinary Medicine, Center for Integrated BioSystems, Utah State University, Logan, UT, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2013 Received in revised form 6 November 2013 Accepted 7 November 2013
The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry–mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and
Keywords: Bovine Embryo development rate Intracytoplasmic sperm injection In vitro fertilization Karyotyping Sex-sorted sperm
Hyun-Tae Jo and Jae-Il Bang contributed equally to this study. * Corresponding author. Tel.: þ82 55 772 1942; fax: þ82 55 772 1949. E-mail address: [email protected] (I.-K. Kong). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.11.010
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usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Intracytoplasmic sperm injection (ICSI) is a useful technique because it can overcome infertility in humans, cattle, and other domestic animals. It is also a good alternative in cases in which in vitro fertilization (IVF) does not produce a high number of embryos, and the number of viable sorted spermatozoa is still insufficient for artificial insemination (AI) [1–3]. Intracytoplasmic sperm injection is also useful in the production of embryos with a desired sex and specific genotype in certain farm animals. Although Uehara and Yanagimachi [4] were the first to inject mammalian sperm directly into mature hamster oocytes, the first healthy bovine calves were born in 1990 using ICSI with spermatozoa obtained from a dead bull [5]. To improve the efficiency of ICSI, several different oocyte activation methods and sperm treatments have been tested in human [6,7], mouse [8], sheep [9], horse [10], pig [11], cat [12], monkey [13], and bovine [14]. For example, bovine oocytes are activated chemically [14–18] and physically [19], whereby sperm are pretreated before ICSI to increase the fertilization rate [20–22]. All of these treatments have substantially improved the fertilizing capacity of sperm, and the subsequent development of embryos after ICSI [20–22]. The male-to-female ratio of embryos is approximately 50:50 under normal reproductive conditions. In the dairy cattle industry, female is the desired sex and most of the male calves are euthanized with little financial return to farmers, resulting in a global loss of billions of dollars and significantly in the reduction of profit margin of the dairy industry. One of the best ways to predetermine offspring sex is to use ICSI with sex-sorted sperm where the Y chromosome–bearing sperm (Y-sperm) and X chromosome–bearing sperm (X-sperm) are separated. Ericsson, et al. [23] introduced a relatively simple tool for sex-sorting sperm using albumin gradient method. Corson, et al. [24] reported the successful conception of an XX karyotype after Sephadex filtration of human sperm. The birth of healthy female children was also reported with sorted sperm via IVF or ICSI followed by intrauterine embryo transfer [25]. Sperm separation can also be accomplished by an immunological approach, which depends on differences in surface proteins on X- and Y-chromosome sperm [26]. Flow cytometry, on the basis of the difference of DNA contents between X- and Y-sperm, was first used by Johnson, et al. [27] to sort X- and Y-sperm, and live births of rabbits with predetermined sex were achieved through AI. Offspring were also produced from sorted sperm in other domestic animals and humans either by IVF [25,28,29] or by AI [10]. Lu, et al. [30] reported that the percentage of oocytes fertilized with sorted sperm by flow cytometry was similar to that of oocytes fertilized with unsorted sperm. However, the embryo developmental rate after IVF was reduced when oocytes were fertilized with sorted sperm
compared with unsorted sperm [30,31], illustrating that flow cytometry procedure might affect sperm integrity, morphology, and ultrastructural features [32–35]. Similar to the results from IVF using sorted sperm, the advancement of technologies for ICSI using sorted sperm has resulted in the birth of the first male offspring in sheep [9] and normal calves in cattle [36] despite the previous reports that sorted sperm were unable to support IVF blastocyst formation at the rate comparable with that of unsorted counterparts in cattle [30,37]. The IVF blastocyst development rate using sorted sperm has increased from less than 20% in the late 1990s to greater than 30% in recent reports by utilizing different fertilization and culture systems in bovine [30,38,39]. Nevertheless, the developmental potential of bovine IVF embryos produced from sorted sperm are still inferior to the one produced from unsorted sperm. Therefore, this study was carried out to investigate whether ICSI can be an alternative method to IVF for efficient production of bovine embryo using sorted sperm. 2. Materials and methods Unless indicated, all reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Care and use of animals was conducted in accordance with guidelines prescribed by Gyeongsang National University (approval no. GNU-130902-A0059). 2.1. In vitro maturation Oocyte recovery and in vitro maturation were performed as described by Jeong, et al. [40]. Briefly, oocytes were aspirated from 2- to 8-mm antral follicles obtained from the ovaries of Korean native cows with an 18-ga needle attached to a vacuum pump. Good quality oocytes having at least three layers of intact cumulus cells with uniform cytoplasm were used for IVM. Cumulusoocyte complexes (COCs) were briefly washed 2 to 3 times in Tyrode’s lactate-HEPES (TL-HEPES) before washing in maturation medium (TCM199 supplemented with 10% [vol/vol] fetal bovine serum, 1 mg/mL estradiol17b, 10 mg/mL follicle-stimulating hormone, 0.6 mM cysteine, and 0.2 mM Na-pyruvate) and cultured in IVM medium at 38.5 C in a humidified atmosphere of 5% CO2 in air for 22 to 24 hours. 2.2. Sperm sorting and freezing of bull spermatozoa Flow sorting and cryopreservation were performed as described by Schenk, et al. [41] and Suh and Schenk [42]. Briefly, aliquots of spermatozoa from four candidate bulls (Korean National Agriculture Cooperative Federation [NACF; http://rd.dcic.co.kr]) (Table 1) were diluted in modified Tyrode’s albumin-lactate-pyruvate buffer [41], stained with 125 mM Hoechst 33342 at 200 106
H.-T. Jo et al. / Theriogenology 81 (2014) 675–682 Table 1 Developmental competence of IVF blastocysts derived from four different bull’s sperm. Bull
No. of oocytes
Stage of embryo development, n (%)
A B C D
265 267 283 273
161 190 128 225
Cleavage (60.8)a,b (71.2)a,b (45.2)b (82.4)a
Blastocyst 23 66 31 79
(8.7)b (24.7)a (11)b (28.9)a
This experiment was performed five times. Values with different superscripts in the same column are significantly different (P < 0.05).
spermatozoa per mL for 45 minutes at 34 C, and then further diluted to 100 106 spermatozoa/mL with Tyrode’s albumin lactate pyruvate buffer containing 4% (wt/vol) egg yolk and 0.002% (wt/vol) food coloring dye (FD&C #40; Warner Jenkinson, St. Louis, MO, USA). Stained spermatozoa were sorted for the X chromosome–bearing population by the MoFlo XDP system, equipped with a 70-mm internal diameter nozzle and laser source of 150 mW. Sorted spermatozoa were collected in a 50-mL centrifuge tube containing TRIS catch fluid supplemented with 20% (wt/vol) egg yolk [41]. The unsorted control (i.e., the sample was not passed through the sorter) was prepared from the same ejaculate as the sorted sperm. Sperm samples in plastic tube racks were cooled to 5 C in a cold room for 90 minutes. Thereafter, samples were diluted with an equal volume of 12% (vol/vol) glycerol containing TRIS extender (SortEnsure; XY Inc., Navasota, TX, USA) and centrifuged at 850g for 20 minutes using a swinging-bucket centrifuge precooled to 5 C. After the supernatant was aspirated, the resulting sperm pellet was diluted and the final sperm concentration was adjusted to 20 106 spermatozoa per mL. Sorted and unsorted samples were packaged into 0.5mL straws containing 2 106 and 20 106 total spermatozoa, respectively, and frozen in static liquid nitrogen vapor using a routine procedure on racks [41].
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2.4. Intracytoplasmic sperm injection For ICSI, 2 mL of the sorted and unsorted sperm suspension was transferred to 10 mL of tissue culture medium containing 10% (vol/vol) polyvinylpyrrolidone (PVP) under paraffin oil to prevent spermatozoa from sticking to the inner surface of the micropipette and to reduce their motility. The ICSI was performed using a Narishige micromanipulator (Narishige, Tokyo, Japan) and an IX71 model inverted microscope (Olympus, Tokyo, Japan) at 200 magnification in 20 mL droplets of tissue culture medium under mineral oil in a tissue culture dish (NUNC Thermo Scientific, Shanghai, China) maintained at 37 C. Oocytes were held at the 6- or 12-o’clock position of the first polar body by the holding pipette. During injection, the pipette containing the spermatozoa was inserted into the ooplasm at the 3-o’clock position of the first polar body. A small volume of ooplasm was aspirated into the injection pipette to rupture the cytoplasmic membrane. Thereafter, the aspirated ooplasm and spermatozoa were expelled into the ooplasm with a minimum volume of medium. Oocytes were chemically activated with 2 mM inomycin for 5 minutes and 5 mM N-6 dimethylaminopurine at 38.5 C in a humidified atmosphere of 5% CO2 in air for 4 hours. 2.5. In vitro culture After ICSI and IVF, presumptive zygotes were placed into a 4-well dish containing 700 mL of a modified CR1-aa medium [45] supplemented with 44 mg/mL sodium pyruvate, 14.6 mg/mL glutamine, 100 IU/mL penicillin and 100 mg/ mL streptomycin sulfate, 3 mg/mL bovine serum albumin and 310 mg/mL glutathione (IVC-I) and incubated at 38.5 C in a humidified atmosphere of 5% CO2 in air for 3 days. Cleaved embryos were then cultured for an additional 4 days in the same medium, except that bovine serum albumin was replaced with 10% (vol/vol) fetal bovine serum (IVC-II) for 4 days. 2.6. Karyotyping
2.3. Sperm preparation and in vitro fertilization Sorted and unsorted frozen semen from a proven Holstein bull (D semen; Table 1) provided by the NACF (http://rd.dcic.co.kr) were used in this study. After 22 to 24 hours of maturation, sorted and unsorted semen were thawed in a water bath set at 38.5 C for 1 minute and then washed with Dulbecco’s PBS (D-PBS) by centrifugation at 750g for 5 minutes. The pellet was diluted in 500 mL of Tyrode’s lactate solution (TL-Fert) [43,44] supplemented with heparin (20 mg/mL) and incubated at 38.5 C in a humidified atmosphere of 5% CO2 in air for 15 minutes. The capacitated motile spermatozoa were then diluted in IVF medium (Tyrode’s lactate solution supplemented with 6 mg/mL BSA, 22 mg/mL sodium pyruvate, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin) to 1 to 2 106 spermatozoa per mL. The matured COCs, which included capacitated sperm, were fertilized in 700 mL of IVF at 38.5 C in a humidified atmosphere of 5% CO2 in air for 22 to 24 hours.
Karyotyping was performed as previously described by Ulloa Ulloa, et al. [46]. In brief, Day-7 blastocysts were placed into IVC-II medium containing 10 mg/mL colcemid (Biological Industries Ltd., Kibbutz Beit Haemek, Israel) and incubated for 4 to 6 hours in a humidified atmosphere of 5% CO2 in air before karyotyping. The embryos were then washed with 400 mL of 0.9% (wt/vol) sodium citrate for 20 minutes, followed by fixation with fixative solution A (methanol:acetic acid:distilled water ¼ 5:1:4) and fixative solution B (methanol:acetic acid ¼ 3:1). They were then washed with 0.9% (wt/vol) sodium citrate for 1 to 2 minutes, and were individually transferred onto a clean glass slide and a droplet of acetic acid was used to separate the blastomeres of each embryo. Finally, the embryo chromosomes were spread out and fixed with several drops of fixative solution B. The slides were air dried, stained with 4% (wt/vol) Giemsa solution for 5 minutes, and rinsed with distilled water. The karyotype of each embryo was evaluated using phase contrast microscopy (Carl Zeiss Axioskop,
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Table 2 Pronuclear formation rate of bovine embryos derived from usIVF, ssIVF, usICSI, and ssICSI. Group
No. of oocytes
2 PNa (%)
usIVF ssIVF usICSI ssICSI
45 39 78 72
32 9 57 54
(71.1)A (23.1)B (73.1)A (75)A
Othersb (%) 13 30 21 18
(28.9)B (76.9)A (26.9)B (25.0)B
A,B
Values with different superscripts in the same column are significantly different (P < 0.05). Abbreviations: ICSI, intracytoplasmic sperm injection; PN, pronucleus; ss, sorted sperm; us, unsorted sperm. a 2 male and female PN. b Metaphase II, 1 PN per sperm head, and 3 PN (polyspermy).
Germany) and MacProbe software version 4.1 (Perceptive Scientific Imaging, League City, TX, USA). 2.7. Pronuclear and apoptotic cell analysis by 4,6 diamino-2phenylindole and terminal deoxynucleotidyl transferase dUTP nick end labeling staining To visualize pronuclear formation in sorted and unsorted sperm after 16 hours of IVF, inseminated oocytes were fixed in 4% (wt/vol) paraformaldehyde in PBS containing 0.1% (wt/vol) PVP (PBS-PVP) and then washed three times in PBS-PVP [47]. The inseminated oocytes were stained with 2 mg/mL of 4,6 diamino-2-phenylindole (DAPI) for 30 minutes at room temperature in the dark. The oocytes were washed twice in PBS-PVP and mounted onto glass slides under coverslips. Pronuclear formation was observed under an IX71 model epifluorescence microscope (Olympus) equipped with a mercury lamp. To stain apoptotic cells, blastocysts were washed 2 to 3 times in PBS-PVP and fixed in a 4-well dish containing 700 mL of 4% (wt/vol) paraformaldehyde in PBS-PVP for 1 hour at room temperature. Apoptotic cell analysis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed according to the instructions provided by the manufacturer in the In Situ Cell Death Detection Kit (Roche Diagnostics Corp., Indianapolis, IN, USA). Briefly, the fixed embryos were washed twice with PBS-PVP before permeabilization (0.5% [vol/vol] Triton X100, 0.1% [wt/vol] sodium citrate) for 30 minutes at room
temperature. After permeabilization, the embryos were washed twice in PBS-PVP and then incubated in fluorescence-conjugated dUTP and terminal deoxynucleotide transferase for 1 hour at 37 C in the dark. The TUNEL stained embryos were then washed in PBS-PVP and counterstained with Hoechst 33342 (10 mg/mL) in PBS-PVP for 10 minutes at room temperature in the dark to label nuclei. The blastocysts were washed twice in PBS-PVP to remove excess Hoechst 33342 and mounted onto glass slides under coverslips. The number of cells per blastocyst was counted under an IX71 model epifluorescence microscope. The TUNEL positive (i.e., apoptotic) cells were stained bright red, and the total cell number was determined according to blue fluorescence. Embryos subjected to the TUNEL assay were randomly selected from three different in vitro culture experiments. 2.8. Statistical analysis Sperm ability (unsorted and sorted sperm), pronuclear formation, and embryo development rate (cleavage and blastocyst) were expressed as percentages (%). Embryo quality (total and apoptotic cell numbers) was expressed as mean standard deviation. All experiments were performed at least three times. Significant differences were determined by Chi-square and Kruskal-Wallis tests using SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined as P < 0.05. 3. Results For selecting of semen from a bull with high fertility and for IVF embryo production, semen from four different breeding bulls obtained from NACF were used. Cleavage rate of IVF embryos from the sperm of bull D (before sexsorting) was higher than that of bull C (82.4% vs. 45.2%; P < 0.05), but not significantly different among bulls D, A and B (82.4% vs. 60.8% vs. 71.2%). However, blastocyst developmental competence was higher in bulls B and D compared with bulls A and C (24.7% and 28.9% vs. 8.7% and 11%; P < 0.05), respectively (Table 1). In addition, we also examined embryo developmental competence by IVF with sex-sorted sperm from four bulls. The cleavage rate was
Fig. 1. Images of pronuclear formation in 4,6 diamino-2-phenylindole–stained bovine embryos derived from sex-sorted and unsorted sperm by IVF and intracytoplasmic sperm injection (ICSI). *, Metaphase II (chromatin); arrowhead, pronucleus; and arrow, polar body. PB, Polar body; PN, Pronucleus.
H.-T. Jo et al. / Theriogenology 81 (2014) 675–682 Table 3 In vitro development competence of bovine embryos derived from sexsorted and unsorted sperm IVF and ICSI. Group
No. of oocytes
Stage of embryo development, n (%)
usIVF ssIVF usICSI ssICSI
222 335 195 235
159 146 181 186
Cleavage (71.6)b (43.6)c (92.8)a (79.1)b
Percentage of female sex
Blastocysts 67 9 56 58
(30.2)a (2.7)b (28.7)a (24.7)a
58.8b 100a 57.8b 100a
This experiment was performed at least over four independent times. Values with different superscripts in the same column are significantly different (P < 0.05). Abbreviations: ICSI, intracytoplasmic sperm injection; ss, sorted sperm; us, unsorted sperm.
lower in bull B than in bull C and D (37.2% vs. 53.8% and 54.2%; P < 0.05), but no significant difference was observed among bulls A, C and D (49.0% vs. 53.8% and 54.2%, Supplementary Table 1). However, the blastocyst developmental competence using sex-sorted sperm was not different in all the experimental groups (A: 4.1% vs. B: 5.8% vs. C: 6.1% vs. D: 6.3%; P > 0.05; Supplementary Table 1). Therefore, the sperm from bull D was selected for subsequent experiments. To compare embryo developmental competence using IVF and ICSI with that using unsorted and sorted sperm, we first investigated the effect of sex-sorting of sperm on PN formation rates in IVF and ICSI embryos by DAPI staining (Table 2 and Fig. 1). Pronuclear formation (judged by 2PN) rates were 71.1% in the usIVF, 23.1% in the ssIVF, 73.1% in the usICSI, and 75% in the ssICSI groups (Table 2 and Fig. 1). Nonfertilization and abnormal pronuclear formation pattern, such as metaphase II, one PN per sperm and greater than or equal to three PNs (polyspermy), were observed 28.9% in the usIVF, 76.9% in the ssIVF, 26.9% in the usICSI, and 25% in the ssICSI groups (Table 2 and Fig. 1).
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Cleavage rates of the usICSI group were higher than those of the usIVF, ssIVF and ssICSI groups (92.8% vs. 71.6%, 43.6%, and 79.1%, respectively; P < 0.05), but the rate of the ssIVF group was significantly lower than that of other groups (Table 3). Blastocyst developmental competence in the ssIVF group was lower than that in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7%, and 24.7%, respectively; P < 0.05). To confirm sex ratio of the embryos, we conducted karyotyping assay of blastocysts derived from all the groups. As shown in Table 3 and Figure 2, all of the embryos in the ssIVF and ssICSI groups were females, whereas there were 58.8% and 57.8% female embryos in the usIVF and usICSI groups, respectively (Table 3 and Fig. 2). Finally, we investigated the quality of blastocysts by counting the total and apoptotic cell numbers. We found that total cell number in the usIVF, ssIVF, usICSI, and ssICSI groups was not significantly different (127.1 29.9, 117.8 19.8, 122.8 25.7, and 123.5 34.2, respectively). Moreover, apoptotic cell number was not significantly different among four groups (2.9 1.2, 2.0 2.1, 3.2 2.7, and 3.6 2.7, respectively) (Table 4 and Fig. 3). Therefore, the lower blastocyst formation rate in the ssIVF group seems not to compromise blastocyst quality. 4. Discussion In the present study, we used the sex-sorted sperm for ICSI and IVF to produce female bovine embryos. To the best of our knowledge, this is the first study to investigate the effect of sex-sorted sperm by flow cytometry on the developmental competence and quality of bovine embryos derived from IVF and ICSI. It is well known that animal-toanimal variation can contribute to IVF efficiency and embryo developmental competence when sex-sorted bovine sperm are used [48], therefore we analyzed the fertility of
Fig. 2. Chromosome analysis of embryos derived from unsorted and sorted sperm by intracytoplasmic sperm injection (ICSI) and IVF. (A) Karyotype of a normal diploid female embryo (2n ¼ 60,XX), (B) Karyotype of a normal diploid male embryo (2n ¼ 60,XY).
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Table 4 Comparison of total and apoptotic cell numbers in usIVF, ssIVF, usICSI, and ssICSI blastocysts. Group
No. of blastocysts
No. of total cells (mean standard deviation)
usIVF ssIVF usICSI ssICSI
12 11 12 12
127.1 117.8 122.8 123.5
29.9 19.8 25.7 34.2
No. of apoptotic cells (mean standard deviation) 2.9 2.0 3.2 3.6
1.2 2.1 2.7 2.7
Abbreviations: ICSI, intracytoplasmic sperm injection; ss, sorted sperm; us, unsorted sperm.
unsorted and sex-sorted sperm collected from four different bulls by IVF, and selected the one with higher developmental competence for IVF and ICSI. Our results demonstrated a significant reduction in embryo developmental competence when sex-sorted sperm was used for IVF compared with unsorted sperm, a finding in agreement with other studies. For example, Bermejo-Alvarez, et al. [49] reported a lower embryo production rate by IVF with sorted sperm. Our results also confirmed that IVF embryos produced with sex-sorted sperm significantly reduced the cleavage rate compared with unsorted sperm, because the process of sex-sorting might damage sperm and subsequently reduce its fertility [30,50]. In contrary, embryos derived from ICSI with sorted and unsorted sperm have very similar blastocyst developmental competence in vitro (Table 3). Moreover, blastocyst formation rate of the ssICSI group is about five-fold higher
than that in the ssIVF group (Table 3). It was established that the fertility of sperm in ICSI is not coincident with their performance in IVF [22]. Thus, IVF efficiency is not a reliable indicator for sperm fertility when blastocyst formation is used as the endpoint. Our data demonstrated that ICSI is a much better option than IVF when sex-sorted sperm is used for fertilization and that sperm sorting does not affect the ability of sperm to fertilize the oocyte via ICSI. Because the sex-sorted sperm is less mobile, ICSI also provides a guaranteed way for sperm–egg fusion. The total number of normal and apoptotic cells has widely been used as an indicator for embryo quality. For example, a high number of total cells and fewer apoptotic cells usually result in a higher rate of implantation and calving [25]. In this study, we found that there was no difference in either total or apoptotic cell numbers between blastocysts derived from sorted and unsorted sperm used for IVF and ICSI. These results demonstrated that embryo production efficiency and quality were not affected by sorted sperm from a single bull [51]. It is not surprising that neither sperm processing (sorted vs. unsorted) nor fertilization procedure (IVF vs. ICSI) influenced blastocyst production efficiency and quality, because culture conditions determine embryo quality [52]. In the present study, karyotyping analysis was used to confirm the sex of embryos. We found that the percentage of female embryos was approximately the same for IVF (58.8%) and ICSI (57.8%) using unsorted sperm, whereas sorted sperm resulted in the production of female embryos
Fig. 3. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of blastocysts derived from unsorted and sorted sperm by intracytoplasmic sperm injection (ICSI) and IVF. (A) usIVF, (B) ssIVF, (C) usICSI, and (D) ssICSI. White arrow, apoptotic nuclear (red spot). ss, sex-sorted; us, unsorted.
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only (100%). Within both X- and Y-sorted sperm groups, nine of 10 transferable embryos were of the predicted sex [51]. In another two studies, the proportion of embryos of predicted sex was 96% [48,53]. Bermejo-Alvarez, et al. [49] reported 88% and 89% of embryos to be female and male, respectively, after IVF with X- and Y-sorted sperm. 4.1. Conclusions In this study, we demonstrated that sex-sorted bovine sperm could be used to successfully produce bovine embryos of a predetermined sex by the ICSI technique. Moreover, the developmental competence of preselected female embryos derived from ICSI with sex-sorted sperm is much greater than those derived from IVF with sex-sorted sperm. Acknowledgments This work was partly supported by a grant from the Next-Generation BioGreen 21 Program (gs1) (No. PJ00958702), Rural Development Administration, IPET (gs2) (Grant No. 110020-5) and a scholarship from the BK21 program, Republic of Korea. Hyun-Tae Jo, Seong-Su Kim and Byung-Hyun Choi were supported by BK21 fellowships from the Graduate School of Gyeongsang National University, Republic of Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.theriogenology.2013.11.010. References [1] Rath D, Johnson LA, Dobrinsky JR, Welch GR, Niemann H. Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Theriogenology 1997;47:795–8. [2] Abeydeera LR, Johnson LA, Welch GR, Wang WH, Boquest AC, Cantley TC, et al. Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry. Theriogenology 1998;50:981–8. [3] Rath D, Long CR, Dobrinsky JR, Welch GR, Schreier LL, Johnson LA. In vitro production of sexed embryos for gender preselection: highspeed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer. J Anim Sci 1999;77:3346–52. [4] Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod 1976;15:467–70. [5] Goto K, Kinoshita A, Takuma Y, Ogawa K. Fertilization of bovine oocytes by the injection of immobilized, killed spermatozoa. Vet Rec 1990;127:517–20. [6] Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17–8. [7] Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;8:1061–6. [8] Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995;52:709–20. [9] Catt SL, Catt JW, Gomez MC, Maxwell WM, Evans G. Birth of a male lamb derived from an in vitro matured oocyte fertilized by intracytoplasmic injection of a single presumptive male sperm. Vet Rec 1996;139:494–5. [10] Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, et al. Production of live foals from sperm-injected oocytes harvested from pregnant mares. J Reprod Fertil Suppl 2000;56:503–12.
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H.-T. Jo et al. / Theriogenology 81 (2014) 675–682 Supplementary Table 1 Developmental competence of blastocysts derived from sex-sorted sperm IVF. Bull
No. of oocytes
A B C D
145 121 212 246
Stage of embryo development, n (%) Cleavage 71 45 114 130
(49.0)a,b (37.2)a (53.8)b (54.2)b
Blastocyst 6 7 13 15
(4.1) (5.8) (6.1) (6.3)
Values with different superscripts in the same column are significantly different (P < 0.05). This experiment was performed three times.
682.e1
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Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence Hyun-Tae Jo a, Jae-Il Bang a, Seong-Su Kim a, Byung-Hyun Choi a, Jong-In Jin a, Heyng-Lyool Kim c, In-Suk Jung d, Tae-Kwang Suh d, Nasser Ghanem a, e, Zhongde Wang f, Il-Keun Kong a, b, * a
Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea b Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea c Dairy Cattle Improvement Center, National Agricultural Cooperation Federation, Goyang-si, Gyeonggi-do, Republic of Korea d Korea Sexing Biotech Inc., Daegu, Gyeongsangbuk-do, Republic of Korea e Department of Animal Production, Faculty of Agriculture Cairo University, Giza, Egypt f Department of Animal, Dairy, and Veterinary Sciences, School of Veterinary Medicine, Center for Integrated BioSystems, Utah State University, Logan, UT, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2013 Received in revised form 6 November 2013 Accepted 7 November 2013
The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry–mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and
Keywords: Bovine Embryo development rate Intracytoplasmic sperm injection In vitro fertilization Karyotyping Sex-sorted sperm
Hyun-Tae Jo and Jae-Il Bang contributed equally to this study. * Corresponding author. Tel.: þ82 55 772 1942; fax: þ82 55 772 1949. E-mail address: [email protected] (I.-K. Kong). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.11.010
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usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Intracytoplasmic sperm injection (ICSI) is a useful technique because it can overcome infertility in humans, cattle, and other domestic animals. It is also a good alternative in cases in which in vitro fertilization (IVF) does not produce a high number of embryos, and the number of viable sorted spermatozoa is still insufficient for artificial insemination (AI) [1–3]. Intracytoplasmic sperm injection is also useful in the production of embryos with a desired sex and specific genotype in certain farm animals. Although Uehara and Yanagimachi [4] were the first to inject mammalian sperm directly into mature hamster oocytes, the first healthy bovine calves were born in 1990 using ICSI with spermatozoa obtained from a dead bull [5]. To improve the efficiency of ICSI, several different oocyte activation methods and sperm treatments have been tested in human [6,7], mouse [8], sheep [9], horse [10], pig [11], cat [12], monkey [13], and bovine [14]. For example, bovine oocytes are activated chemically [14–18] and physically [19], whereby sperm are pretreated before ICSI to increase the fertilization rate [20–22]. All of these treatments have substantially improved the fertilizing capacity of sperm, and the subsequent development of embryos after ICSI [20–22]. The male-to-female ratio of embryos is approximately 50:50 under normal reproductive conditions. In the dairy cattle industry, female is the desired sex and most of the male calves are euthanized with little financial return to farmers, resulting in a global loss of billions of dollars and significantly in the reduction of profit margin of the dairy industry. One of the best ways to predetermine offspring sex is to use ICSI with sex-sorted sperm where the Y chromosome–bearing sperm (Y-sperm) and X chromosome–bearing sperm (X-sperm) are separated. Ericsson, et al. [23] introduced a relatively simple tool for sex-sorting sperm using albumin gradient method. Corson, et al. [24] reported the successful conception of an XX karyotype after Sephadex filtration of human sperm. The birth of healthy female children was also reported with sorted sperm via IVF or ICSI followed by intrauterine embryo transfer [25]. Sperm separation can also be accomplished by an immunological approach, which depends on differences in surface proteins on X- and Y-chromosome sperm [26]. Flow cytometry, on the basis of the difference of DNA contents between X- and Y-sperm, was first used by Johnson, et al. [27] to sort X- and Y-sperm, and live births of rabbits with predetermined sex were achieved through AI. Offspring were also produced from sorted sperm in other domestic animals and humans either by IVF [25,28,29] or by AI [10]. Lu, et al. [30] reported that the percentage of oocytes fertilized with sorted sperm by flow cytometry was similar to that of oocytes fertilized with unsorted sperm. However, the embryo developmental rate after IVF was reduced when oocytes were fertilized with sorted sperm
compared with unsorted sperm [30,31], illustrating that flow cytometry procedure might affect sperm integrity, morphology, and ultrastructural features [32–35]. Similar to the results from IVF using sorted sperm, the advancement of technologies for ICSI using sorted sperm has resulted in the birth of the first male offspring in sheep [9] and normal calves in cattle [36] despite the previous reports that sorted sperm were unable to support IVF blastocyst formation at the rate comparable with that of unsorted counterparts in cattle [30,37]. The IVF blastocyst development rate using sorted sperm has increased from less than 20% in the late 1990s to greater than 30% in recent reports by utilizing different fertilization and culture systems in bovine [30,38,39]. Nevertheless, the developmental potential of bovine IVF embryos produced from sorted sperm are still inferior to the one produced from unsorted sperm. Therefore, this study was carried out to investigate whether ICSI can be an alternative method to IVF for efficient production of bovine embryo using sorted sperm. 2. Materials and methods Unless indicated, all reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Care and use of animals was conducted in accordance with guidelines prescribed by Gyeongsang National University (approval no. GNU-130902-A0059). 2.1. In vitro maturation Oocyte recovery and in vitro maturation were performed as described by Jeong, et al. [40]. Briefly, oocytes were aspirated from 2- to 8-mm antral follicles obtained from the ovaries of Korean native cows with an 18-ga needle attached to a vacuum pump. Good quality oocytes having at least three layers of intact cumulus cells with uniform cytoplasm were used for IVM. Cumulusoocyte complexes (COCs) were briefly washed 2 to 3 times in Tyrode’s lactate-HEPES (TL-HEPES) before washing in maturation medium (TCM199 supplemented with 10% [vol/vol] fetal bovine serum, 1 mg/mL estradiol17b, 10 mg/mL follicle-stimulating hormone, 0.6 mM cysteine, and 0.2 mM Na-pyruvate) and cultured in IVM medium at 38.5 C in a humidified atmosphere of 5% CO2 in air for 22 to 24 hours. 2.2. Sperm sorting and freezing of bull spermatozoa Flow sorting and cryopreservation were performed as described by Schenk, et al. [41] and Suh and Schenk [42]. Briefly, aliquots of spermatozoa from four candidate bulls (Korean National Agriculture Cooperative Federation [NACF; http://rd.dcic.co.kr]) (Table 1) were diluted in modified Tyrode’s albumin-lactate-pyruvate buffer [41], stained with 125 mM Hoechst 33342 at 200 106
H.-T. Jo et al. / Theriogenology 81 (2014) 675–682 Table 1 Developmental competence of IVF blastocysts derived from four different bull’s sperm. Bull
No. of oocytes
Stage of embryo development, n (%)
A B C D
265 267 283 273
161 190 128 225
Cleavage (60.8)a,b (71.2)a,b (45.2)b (82.4)a
Blastocyst 23 66 31 79
(8.7)b (24.7)a (11)b (28.9)a
This experiment was performed five times. Values with different superscripts in the same column are significantly different (P < 0.05).
spermatozoa per mL for 45 minutes at 34 C, and then further diluted to 100 106 spermatozoa/mL with Tyrode’s albumin lactate pyruvate buffer containing 4% (wt/vol) egg yolk and 0.002% (wt/vol) food coloring dye (FD&C #40; Warner Jenkinson, St. Louis, MO, USA). Stained spermatozoa were sorted for the X chromosome–bearing population by the MoFlo XDP system, equipped with a 70-mm internal diameter nozzle and laser source of 150 mW. Sorted spermatozoa were collected in a 50-mL centrifuge tube containing TRIS catch fluid supplemented with 20% (wt/vol) egg yolk [41]. The unsorted control (i.e., the sample was not passed through the sorter) was prepared from the same ejaculate as the sorted sperm. Sperm samples in plastic tube racks were cooled to 5 C in a cold room for 90 minutes. Thereafter, samples were diluted with an equal volume of 12% (vol/vol) glycerol containing TRIS extender (SortEnsure; XY Inc., Navasota, TX, USA) and centrifuged at 850g for 20 minutes using a swinging-bucket centrifuge precooled to 5 C. After the supernatant was aspirated, the resulting sperm pellet was diluted and the final sperm concentration was adjusted to 20 106 spermatozoa per mL. Sorted and unsorted samples were packaged into 0.5mL straws containing 2 106 and 20 106 total spermatozoa, respectively, and frozen in static liquid nitrogen vapor using a routine procedure on racks [41].
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2.4. Intracytoplasmic sperm injection For ICSI, 2 mL of the sorted and unsorted sperm suspension was transferred to 10 mL of tissue culture medium containing 10% (vol/vol) polyvinylpyrrolidone (PVP) under paraffin oil to prevent spermatozoa from sticking to the inner surface of the micropipette and to reduce their motility. The ICSI was performed using a Narishige micromanipulator (Narishige, Tokyo, Japan) and an IX71 model inverted microscope (Olympus, Tokyo, Japan) at 200 magnification in 20 mL droplets of tissue culture medium under mineral oil in a tissue culture dish (NUNC Thermo Scientific, Shanghai, China) maintained at 37 C. Oocytes were held at the 6- or 12-o’clock position of the first polar body by the holding pipette. During injection, the pipette containing the spermatozoa was inserted into the ooplasm at the 3-o’clock position of the first polar body. A small volume of ooplasm was aspirated into the injection pipette to rupture the cytoplasmic membrane. Thereafter, the aspirated ooplasm and spermatozoa were expelled into the ooplasm with a minimum volume of medium. Oocytes were chemically activated with 2 mM inomycin for 5 minutes and 5 mM N-6 dimethylaminopurine at 38.5 C in a humidified atmosphere of 5% CO2 in air for 4 hours. 2.5. In vitro culture After ICSI and IVF, presumptive zygotes were placed into a 4-well dish containing 700 mL of a modified CR1-aa medium [45] supplemented with 44 mg/mL sodium pyruvate, 14.6 mg/mL glutamine, 100 IU/mL penicillin and 100 mg/ mL streptomycin sulfate, 3 mg/mL bovine serum albumin and 310 mg/mL glutathione (IVC-I) and incubated at 38.5 C in a humidified atmosphere of 5% CO2 in air for 3 days. Cleaved embryos were then cultured for an additional 4 days in the same medium, except that bovine serum albumin was replaced with 10% (vol/vol) fetal bovine serum (IVC-II) for 4 days. 2.6. Karyotyping
2.3. Sperm preparation and in vitro fertilization Sorted and unsorted frozen semen from a proven Holstein bull (D semen; Table 1) provided by the NACF (http://rd.dcic.co.kr) were used in this study. After 22 to 24 hours of maturation, sorted and unsorted semen were thawed in a water bath set at 38.5 C for 1 minute and then washed with Dulbecco’s PBS (D-PBS) by centrifugation at 750g for 5 minutes. The pellet was diluted in 500 mL of Tyrode’s lactate solution (TL-Fert) [43,44] supplemented with heparin (20 mg/mL) and incubated at 38.5 C in a humidified atmosphere of 5% CO2 in air for 15 minutes. The capacitated motile spermatozoa were then diluted in IVF medium (Tyrode’s lactate solution supplemented with 6 mg/mL BSA, 22 mg/mL sodium pyruvate, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin) to 1 to 2 106 spermatozoa per mL. The matured COCs, which included capacitated sperm, were fertilized in 700 mL of IVF at 38.5 C in a humidified atmosphere of 5% CO2 in air for 22 to 24 hours.
Karyotyping was performed as previously described by Ulloa Ulloa, et al. [46]. In brief, Day-7 blastocysts were placed into IVC-II medium containing 10 mg/mL colcemid (Biological Industries Ltd., Kibbutz Beit Haemek, Israel) and incubated for 4 to 6 hours in a humidified atmosphere of 5% CO2 in air before karyotyping. The embryos were then washed with 400 mL of 0.9% (wt/vol) sodium citrate for 20 minutes, followed by fixation with fixative solution A (methanol:acetic acid:distilled water ¼ 5:1:4) and fixative solution B (methanol:acetic acid ¼ 3:1). They were then washed with 0.9% (wt/vol) sodium citrate for 1 to 2 minutes, and were individually transferred onto a clean glass slide and a droplet of acetic acid was used to separate the blastomeres of each embryo. Finally, the embryo chromosomes were spread out and fixed with several drops of fixative solution B. The slides were air dried, stained with 4% (wt/vol) Giemsa solution for 5 minutes, and rinsed with distilled water. The karyotype of each embryo was evaluated using phase contrast microscopy (Carl Zeiss Axioskop,
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Table 2 Pronuclear formation rate of bovine embryos derived from usIVF, ssIVF, usICSI, and ssICSI. Group
No. of oocytes
2 PNa (%)
usIVF ssIVF usICSI ssICSI
45 39 78 72
32 9 57 54
(71.1)A (23.1)B (73.1)A (75)A
Othersb (%) 13 30 21 18
(28.9)B (76.9)A (26.9)B (25.0)B
A,B
Values with different superscripts in the same column are significantly different (P < 0.05). Abbreviations: ICSI, intracytoplasmic sperm injection; PN, pronucleus; ss, sorted sperm; us, unsorted sperm. a 2 male and female PN. b Metaphase II, 1 PN per sperm head, and 3 PN (polyspermy).
Germany) and MacProbe software version 4.1 (Perceptive Scientific Imaging, League City, TX, USA). 2.7. Pronuclear and apoptotic cell analysis by 4,6 diamino-2phenylindole and terminal deoxynucleotidyl transferase dUTP nick end labeling staining To visualize pronuclear formation in sorted and unsorted sperm after 16 hours of IVF, inseminated oocytes were fixed in 4% (wt/vol) paraformaldehyde in PBS containing 0.1% (wt/vol) PVP (PBS-PVP) and then washed three times in PBS-PVP [47]. The inseminated oocytes were stained with 2 mg/mL of 4,6 diamino-2-phenylindole (DAPI) for 30 minutes at room temperature in the dark. The oocytes were washed twice in PBS-PVP and mounted onto glass slides under coverslips. Pronuclear formation was observed under an IX71 model epifluorescence microscope (Olympus) equipped with a mercury lamp. To stain apoptotic cells, blastocysts were washed 2 to 3 times in PBS-PVP and fixed in a 4-well dish containing 700 mL of 4% (wt/vol) paraformaldehyde in PBS-PVP for 1 hour at room temperature. Apoptotic cell analysis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed according to the instructions provided by the manufacturer in the In Situ Cell Death Detection Kit (Roche Diagnostics Corp., Indianapolis, IN, USA). Briefly, the fixed embryos were washed twice with PBS-PVP before permeabilization (0.5% [vol/vol] Triton X100, 0.1% [wt/vol] sodium citrate) for 30 minutes at room
temperature. After permeabilization, the embryos were washed twice in PBS-PVP and then incubated in fluorescence-conjugated dUTP and terminal deoxynucleotide transferase for 1 hour at 37 C in the dark. The TUNEL stained embryos were then washed in PBS-PVP and counterstained with Hoechst 33342 (10 mg/mL) in PBS-PVP for 10 minutes at room temperature in the dark to label nuclei. The blastocysts were washed twice in PBS-PVP to remove excess Hoechst 33342 and mounted onto glass slides under coverslips. The number of cells per blastocyst was counted under an IX71 model epifluorescence microscope. The TUNEL positive (i.e., apoptotic) cells were stained bright red, and the total cell number was determined according to blue fluorescence. Embryos subjected to the TUNEL assay were randomly selected from three different in vitro culture experiments. 2.8. Statistical analysis Sperm ability (unsorted and sorted sperm), pronuclear formation, and embryo development rate (cleavage and blastocyst) were expressed as percentages (%). Embryo quality (total and apoptotic cell numbers) was expressed as mean standard deviation. All experiments were performed at least three times. Significant differences were determined by Chi-square and Kruskal-Wallis tests using SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined as P < 0.05. 3. Results For selecting of semen from a bull with high fertility and for IVF embryo production, semen from four different breeding bulls obtained from NACF were used. Cleavage rate of IVF embryos from the sperm of bull D (before sexsorting) was higher than that of bull C (82.4% vs. 45.2%; P < 0.05), but not significantly different among bulls D, A and B (82.4% vs. 60.8% vs. 71.2%). However, blastocyst developmental competence was higher in bulls B and D compared with bulls A and C (24.7% and 28.9% vs. 8.7% and 11%; P < 0.05), respectively (Table 1). In addition, we also examined embryo developmental competence by IVF with sex-sorted sperm from four bulls. The cleavage rate was
Fig. 1. Images of pronuclear formation in 4,6 diamino-2-phenylindole–stained bovine embryos derived from sex-sorted and unsorted sperm by IVF and intracytoplasmic sperm injection (ICSI). *, Metaphase II (chromatin); arrowhead, pronucleus; and arrow, polar body. PB, Polar body; PN, Pronucleus.
H.-T. Jo et al. / Theriogenology 81 (2014) 675–682 Table 3 In vitro development competence of bovine embryos derived from sexsorted and unsorted sperm IVF and ICSI. Group
No. of oocytes
Stage of embryo development, n (%)
usIVF ssIVF usICSI ssICSI
222 335 195 235
159 146 181 186
Cleavage (71.6)b (43.6)c (92.8)a (79.1)b
Percentage of female sex
Blastocysts 67 9 56 58
(30.2)a (2.7)b (28.7)a (24.7)a
58.8b 100a 57.8b 100a
This experiment was performed at least over four independent times. Values with different superscripts in the same column are significantly different (P < 0.05). Abbreviations: ICSI, intracytoplasmic sperm injection; ss, sorted sperm; us, unsorted sperm.
lower in bull B than in bull C and D (37.2% vs. 53.8% and 54.2%; P < 0.05), but no significant difference was observed among bulls A, C and D (49.0% vs. 53.8% and 54.2%, Supplementary Table 1). However, the blastocyst developmental competence using sex-sorted sperm was not different in all the experimental groups (A: 4.1% vs. B: 5.8% vs. C: 6.1% vs. D: 6.3%; P > 0.05; Supplementary Table 1). Therefore, the sperm from bull D was selected for subsequent experiments. To compare embryo developmental competence using IVF and ICSI with that using unsorted and sorted sperm, we first investigated the effect of sex-sorting of sperm on PN formation rates in IVF and ICSI embryos by DAPI staining (Table 2 and Fig. 1). Pronuclear formation (judged by 2PN) rates were 71.1% in the usIVF, 23.1% in the ssIVF, 73.1% in the usICSI, and 75% in the ssICSI groups (Table 2 and Fig. 1). Nonfertilization and abnormal pronuclear formation pattern, such as metaphase II, one PN per sperm and greater than or equal to three PNs (polyspermy), were observed 28.9% in the usIVF, 76.9% in the ssIVF, 26.9% in the usICSI, and 25% in the ssICSI groups (Table 2 and Fig. 1).
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Cleavage rates of the usICSI group were higher than those of the usIVF, ssIVF and ssICSI groups (92.8% vs. 71.6%, 43.6%, and 79.1%, respectively; P < 0.05), but the rate of the ssIVF group was significantly lower than that of other groups (Table 3). Blastocyst developmental competence in the ssIVF group was lower than that in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7%, and 24.7%, respectively; P < 0.05). To confirm sex ratio of the embryos, we conducted karyotyping assay of blastocysts derived from all the groups. As shown in Table 3 and Figure 2, all of the embryos in the ssIVF and ssICSI groups were females, whereas there were 58.8% and 57.8% female embryos in the usIVF and usICSI groups, respectively (Table 3 and Fig. 2). Finally, we investigated the quality of blastocysts by counting the total and apoptotic cell numbers. We found that total cell number in the usIVF, ssIVF, usICSI, and ssICSI groups was not significantly different (127.1 29.9, 117.8 19.8, 122.8 25.7, and 123.5 34.2, respectively). Moreover, apoptotic cell number was not significantly different among four groups (2.9 1.2, 2.0 2.1, 3.2 2.7, and 3.6 2.7, respectively) (Table 4 and Fig. 3). Therefore, the lower blastocyst formation rate in the ssIVF group seems not to compromise blastocyst quality. 4. Discussion In the present study, we used the sex-sorted sperm for ICSI and IVF to produce female bovine embryos. To the best of our knowledge, this is the first study to investigate the effect of sex-sorted sperm by flow cytometry on the developmental competence and quality of bovine embryos derived from IVF and ICSI. It is well known that animal-toanimal variation can contribute to IVF efficiency and embryo developmental competence when sex-sorted bovine sperm are used [48], therefore we analyzed the fertility of
Fig. 2. Chromosome analysis of embryos derived from unsorted and sorted sperm by intracytoplasmic sperm injection (ICSI) and IVF. (A) Karyotype of a normal diploid female embryo (2n ¼ 60,XX), (B) Karyotype of a normal diploid male embryo (2n ¼ 60,XY).
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Table 4 Comparison of total and apoptotic cell numbers in usIVF, ssIVF, usICSI, and ssICSI blastocysts. Group
No. of blastocysts
No. of total cells (mean standard deviation)
usIVF ssIVF usICSI ssICSI
12 11 12 12
127.1 117.8 122.8 123.5
29.9 19.8 25.7 34.2
No. of apoptotic cells (mean standard deviation) 2.9 2.0 3.2 3.6
1.2 2.1 2.7 2.7
Abbreviations: ICSI, intracytoplasmic sperm injection; ss, sorted sperm; us, unsorted sperm.
unsorted and sex-sorted sperm collected from four different bulls by IVF, and selected the one with higher developmental competence for IVF and ICSI. Our results demonstrated a significant reduction in embryo developmental competence when sex-sorted sperm was used for IVF compared with unsorted sperm, a finding in agreement with other studies. For example, Bermejo-Alvarez, et al. [49] reported a lower embryo production rate by IVF with sorted sperm. Our results also confirmed that IVF embryos produced with sex-sorted sperm significantly reduced the cleavage rate compared with unsorted sperm, because the process of sex-sorting might damage sperm and subsequently reduce its fertility [30,50]. In contrary, embryos derived from ICSI with sorted and unsorted sperm have very similar blastocyst developmental competence in vitro (Table 3). Moreover, blastocyst formation rate of the ssICSI group is about five-fold higher
than that in the ssIVF group (Table 3). It was established that the fertility of sperm in ICSI is not coincident with their performance in IVF [22]. Thus, IVF efficiency is not a reliable indicator for sperm fertility when blastocyst formation is used as the endpoint. Our data demonstrated that ICSI is a much better option than IVF when sex-sorted sperm is used for fertilization and that sperm sorting does not affect the ability of sperm to fertilize the oocyte via ICSI. Because the sex-sorted sperm is less mobile, ICSI also provides a guaranteed way for sperm–egg fusion. The total number of normal and apoptotic cells has widely been used as an indicator for embryo quality. For example, a high number of total cells and fewer apoptotic cells usually result in a higher rate of implantation and calving [25]. In this study, we found that there was no difference in either total or apoptotic cell numbers between blastocysts derived from sorted and unsorted sperm used for IVF and ICSI. These results demonstrated that embryo production efficiency and quality were not affected by sorted sperm from a single bull [51]. It is not surprising that neither sperm processing (sorted vs. unsorted) nor fertilization procedure (IVF vs. ICSI) influenced blastocyst production efficiency and quality, because culture conditions determine embryo quality [52]. In the present study, karyotyping analysis was used to confirm the sex of embryos. We found that the percentage of female embryos was approximately the same for IVF (58.8%) and ICSI (57.8%) using unsorted sperm, whereas sorted sperm resulted in the production of female embryos
Fig. 3. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of blastocysts derived from unsorted and sorted sperm by intracytoplasmic sperm injection (ICSI) and IVF. (A) usIVF, (B) ssIVF, (C) usICSI, and (D) ssICSI. White arrow, apoptotic nuclear (red spot). ss, sex-sorted; us, unsorted.
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only (100%). Within both X- and Y-sorted sperm groups, nine of 10 transferable embryos were of the predicted sex [51]. In another two studies, the proportion of embryos of predicted sex was 96% [48,53]. Bermejo-Alvarez, et al. [49] reported 88% and 89% of embryos to be female and male, respectively, after IVF with X- and Y-sorted sperm. 4.1. Conclusions In this study, we demonstrated that sex-sorted bovine sperm could be used to successfully produce bovine embryos of a predetermined sex by the ICSI technique. Moreover, the developmental competence of preselected female embryos derived from ICSI with sex-sorted sperm is much greater than those derived from IVF with sex-sorted sperm. Acknowledgments This work was partly supported by a grant from the Next-Generation BioGreen 21 Program (gs1) (No. PJ00958702), Rural Development Administration, IPET (gs2) (Grant No. 110020-5) and a scholarship from the BK21 program, Republic of Korea. Hyun-Tae Jo, Seong-Su Kim and Byung-Hyun Choi were supported by BK21 fellowships from the Graduate School of Gyeongsang National University, Republic of Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.theriogenology.2013.11.010. References [1] Rath D, Johnson LA, Dobrinsky JR, Welch GR, Niemann H. Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Theriogenology 1997;47:795–8. [2] Abeydeera LR, Johnson LA, Welch GR, Wang WH, Boquest AC, Cantley TC, et al. Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry. Theriogenology 1998;50:981–8. [3] Rath D, Long CR, Dobrinsky JR, Welch GR, Schreier LL, Johnson LA. In vitro production of sexed embryos for gender preselection: highspeed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer. J Anim Sci 1999;77:3346–52. [4] Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod 1976;15:467–70. [5] Goto K, Kinoshita A, Takuma Y, Ogawa K. Fertilization of bovine oocytes by the injection of immobilized, killed spermatozoa. Vet Rec 1990;127:517–20. [6] Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17–8. [7] Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;8:1061–6. [8] Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995;52:709–20. [9] Catt SL, Catt JW, Gomez MC, Maxwell WM, Evans G. Birth of a male lamb derived from an in vitro matured oocyte fertilized by intracytoplasmic injection of a single presumptive male sperm. Vet Rec 1996;139:494–5. [10] Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, et al. Production of live foals from sperm-injected oocytes harvested from pregnant mares. J Reprod Fertil Suppl 2000;56:503–12.
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H.-T. Jo et al. / Theriogenology 81 (2014) 675–682 Supplementary Table 1 Developmental competence of blastocysts derived from sex-sorted sperm IVF. Bull
No. of oocytes
A B C D
145 121 212 246
Stage of embryo development, n (%) Cleavage 71 45 114 130
(49.0)a,b (37.2)a (53.8)b (54.2)b
Blastocyst 6 7 13 15
(4.1) (5.8) (6.1) (6.3)
Values with different superscripts in the same column are significantly different (P < 0.05). This experiment was performed three times.
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