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Gender Preselection in Cattle with Intracytoplasmically Injected, Flow Cytometrically Sorted Sperm Heads¹

^a Maebashi Institute of Animal Science, Livestock Improvement Association of Japan, Inc., Maebashi, Gunma, 371–0121, Japan

	ABSTRACT

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We investigated the development to the blastocyst and subsequent live-offspring stages of in vitro-matured bovine oocytes intracytoplasmically injected with flow cytometrically sorted bull sperm heads. Bull sperm heads, prepared by ultrasound sonication, were distinguished and sorted on the basis of their relative DNA contents using a flow cytometer/cell sorter modified for sorting sperm. By fluorescence in situ hybridization, the proportion of sperm confirmed as having Y specific DNA in the fraction sorted for the Y sperm was 82%. Injection with single sorted sperm heads of in vitro-matured oocytes (cultured for 24 h) resulted in 46.6% cleavage and 6.9% blastocyst development rates. Embryo transfer of 48 blastocysts (Days 7–8) to recipients (one per recipient) resulted in 20.8% pregnancy and 20.8% normal live offspring production rates. The birth of 8 male and 2 female calves represents an 80% sex preselection accuracy rate.

	INTRODUCTION

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Sex control of offspring has been investigated at both the sperm and embryonic levels. Sex confirmation of embryos is currently performed by polymerase chain reaction (PCR) and DNA analysis of embryonic cells recovered by bisection or biopsy. Sex preselection using X or Y sperm in methods such as artificial insemination (AI), in vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI) has been recognized to be more efficient than embryonic sex confirmation.

The most successful method developed of separating X and Y chromosome-bearing sperm is a flow cytometric sorting method using a cell sorter modified [1] for sorting sperm heads [2] and for living sperm [3]. Flow cytometrically sorted X and Y sperm have been successfully used to produce offspring in rabbits [3] and swine [4] using surgical insemination, in cattle [5, 6] and swine [7] using IVF, and in sheep [8] using ICSI. By ICSI, a sex-preselected lamb was born. More recently, in cattle [9] and sheep [10], pregnancies and live births were achieved by nonsurgical AI (depositing sperm deep in the uterine horn) with small populations (10⁵) of sorted X or Y sperm.

Goto et al. [11] demonstrated the birth of bovine offspring using ICSI. Mouse pups were produced by ICSI of eggs using sperm heads. Although there were no differences between the developmental rates of mouse eggs to blastocysts produced using whole sperm and sperm heads only, the number of offspring produced using sperm heads was lower than that using whole sperm [12]. Goto et al. [13] also reported that in cattle, developmental rates of oocytes to blastocysts with ICSI using sperm heads were lower than those with whole sperm.

Johnson et al. [2] demonstrated that bull sperm nuclei (heads) allowed a higher proportion to be sorted for a given purity. For the number of sperm required for a calf production, ICSI with single sperm heads is an efficient method, and optimal storage conditions of sorted sperm heads for ICSI should be established. In farm animals, the production rates of offspring derived from ICSI oocytes using flow-sorted sperm is lower than that from IVF [6, 8]. In sheep, Catt et al. [8] produced one male offspring from 28 recipients of embryos produced by ICSI with sorted Y sperm. In cattle, there are no reports to date of calf production using blastocysts derived from oocytes injected with sorted X or Y sperm heads.

The present study was an attempt to demonstrate the production of normal offspring using ICSI of flow cytometrically-sorted bull sperm heads.

	MATERIALS AND METHODS

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Flow Cytometer Modifications

The EPICS 753 flow cytometer (Coulter Co., Hialeah, FL) was modified by the procedures described by Johnson and Pinkel [1]. The forward light scatter detector was replaced with an extra fluorescence detector, and the end of the insertion needle (which influences the orientation of the sperm head in the sheath fluid) was ground into a beveled shape.

Sperm Head Preparation

Frozen semen (from two Japanese Black bulls) in 0.5-ml plastic straws (bull A: 32 straws with 3 ejaculates, bull B: 40 straws with 12 ejaculates) was thawed at 38°C and washed three times with PBS-PVP (145.4 mM NaCl, 8.1 mM Na₂HPO₄, 1.9 mM NaH₂PO₄2H₂O, 0.05% polyvinyl pylorydon (K-90; PVP) (Nacalai Tesque Inc., Kyoto, Japan) by centrifugation at 350 x g for 5 min. The washed sperm suspension (5 million sperm/ml) was sonicated for 20 sec using half the power output (100 W) of an ultrasound sonicator (Ohtake Works Co., Tokyo, Japan). By this procedure, 90–95% of the sperm had their tails removed. The sperm heads (5 million/ml) were subsequently washed by centrifugation with PBS-PVP and stained by incubation in PBS-PVP containing 9 µM (final concentration) bisbenzimide 33342 (Sigma Chemical Co., St. Louis, MO) at 37°C for 1 h [3].

Flow Sorting and Confirmation of the X and Y Sperm Heads

Stained sperm heads were excited by ultraviolet light from a 90–5 Innova (Coherent Inc., Palo Alto, CA) argon laser operating at 150–200-mW power. Sperm heads appropriately oriented in the sheath fluid (PBS-PVP) were detected by the side fluorescence detector of the flow cytometer/cell sorter. By measuring the fluorescence intensity (relative DNA content) with the forward fluorescence detector, the oriented sperm heads were sorted into X and Y populations [2]. Flow rates ranged from 500 to 700 sperm heads per second, and 100 000–150 000 sperm heads (150 000 X and 100 000 Y sperm heads per hour) were sorted from a sample (5 million sperm heads) into empty microfuge tubes (1.5 ml). Upon sorting, the respective sperm head samples were assessed for X or Y by fluorescence in situ hybridization (FISH) using a Y-specific DNA probe (B.C.1.2) [14]. DNA probes were prepared by means of amplifying and labeling by PCR. The oligonucleotide primers used in PCR were synthesized by a DNA synthesizer by means of information for DNA sequences of B.C.1.2. [14]. The amplification was carried out by a thermal cycler. Briefly, sorted sperm heads (about 10 000) were made to adhere to a glass slide by means of centrifugation (350 x g, 10 min) and fixed with methanol-acetic acid solution (3:1). In situ hybridization was carried out using a digoxigenin-11-dUTP (Dig; Boehringer Mannheim Co., Mannheim, Germany)-labeled DNA (B.C.1.2) probe, for 1–4 h at 37°C. After being washed with single-strength SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.2) at 73°C and 4-strength SSC at 37°C, sperm heads were stained with an anti-Dig-fluorescein Fab fragment (Boehringer) and counterstained with propidium iodide (Sigma). The fluorescent Y-specific DNA signal was detected by fluorescence microscopy (BX2; Olympus Co., Tokyo, Japan). Three hundred to 500 sperm heads were examined in the FISH slide. At examination, unsorted sperm heads were also counted and identified for evaluation of sperm head labeling. The sperm heads sorted for Y were suspended (200 000 sperm heads/ml) in PBS-PVP and stored at -30°C.

In Vitro Oocyte Maturation

Slaughterhouse-derived bovine oocytes (Japanese Black) were cultured in 25 mM HEPES-buffered TCM 199 medium (Gibco, Grand Island, NY) supplemented with 5% calf serum (Gibco) and 10 mM hypotaurine (Sigma) in an atmosphere of 5% CO₂, 95% air at 38.5°C for 22–26 h. Cumulus cells were removed from the matured oocytes by incubation in PBS containing 500 IU/ml hyaluronidase (Sigma) for 5 min, followed by gentle mechanical pipetting.

ICSI with X and Y Sorted Sperm Heads

ICSI was performed according to the procedure described by Goto et al. [11] and Li et al. [15]. In vitro-matured oocytes displaying the first polar body were placed in groups of ten in a 10-µl drop of PBS containing 3 mg/ml BSA, and a single sorted sperm head was injected into the ooplasm using an injection pipette (internal diameter 5–10 µm). Oocytes were activated by a 5-min incubation in PBS containing 7% ethanol once before and twice after ICSI (at 30 min and 60 min).

In Vitro Culture and Embryo Transfer

Sperm head-injected oocytes were cultured in CR1-aa medium (114.7 mM NaCl, 3.1 mM KCl, 26.2 mM NaHCO₃, 5 mM hemicalcium lactate, 0.4 mM sodium pyruvate, 20 ml/L basal medium Eagle (BME) amino acids solution [Sigma], 10 ml/L MEM nonessential amino acids solution [Gibco], 10 ml/L L-glutamic acid [Gibco], 3 mg/ml BSA) supplemented with 5% calf serum (Gibco) in an atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C. Embryos that developed to the expanded blastocyst stage at 7–8 days after ICSI were nonsurgically transferred (one embryo per recipient) to the uteri of 48 recipient cows (Holstein) on Days 7–9 of the estrous cycle (without artificial synchronization of estrus). Pregnancy was diagnosed by rectal palpation 60–90 days later.

	RESULTS

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The proportion of sperm confirmed by FISH as having Y chromosome-specific DNA in the fraction sorted for the Y chromosome was 82% (Table 1). When in vitro-matured oocytes cultured for 22, 24, and 26 h were used for ICSI, the percentages that developed to the blastocyst stage were 4.8%, 6.9%, and 3.5%, respectively (Table 2). Among the 48 cows that received a single embryo (derived from ICSI using Y sperm heads), 10 were confirmed to be pregnant at 60–90 days post-embryo transfer, and 10 normal live calves were delivered (8 male and 2 female). Gestation lengths were 286, 279, 279, 285, 288, 293, 300, 287, 278, and 279 days, and the birth weights were 30, 35, 33, 35, 25, 45, 40, 30, 35, and 40 kg, respectively.

	DISCUSSION

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Our modified flow cytometer/cell sorter enabled us to achieve 82% accuracy in separation of Y sperm heads and an overall 80% sex preselection accuracy. We examined the accuracy of separation of the sorted bull X and Y sperm heads both by FISH using a Y-specific DNA probe and by the sex of the calves. FISH is thought to be a reliable method for confirmation of X and Y sperm because it has the potential to get accurate results for individual sperm from a small sample. The accuracy of X and Y sperm separation reported here is comparable to that obtained by Johnson [16], Welch et al. [17], and Cran et al. [5], perhaps because our cell sorter was modified in the same way as the cell sorters in the studies cited [5, 16, 17].

More than 80% of the oocytes cultured for 20–22 h developed to the metaphase II stage in our experiments. These metaphase II oocytes were fertilized with unsorted and capacitated bull spermatozoa and developed to transferable blastocysts in vitro (30%). It has been demonstrated that bovine oocytes matured in vitro for 24–26 h and injected intracytoplasmically with sperm are able to develop to the blastocyst stage at a higher rate than oocytes cultured for 20–22 h [15]. Our results showed that 6.9% of oocytes that were matured in vitro for 24 h and then received injections of sorted bull sperm heads developed to the blastocyst stage. Oocytes used for ICSI require a longer in vitro-maturation period than those used in IVF [15]. The effects of maturation time on blastocyst development after ICSI observed in this study were similar to those reported previously [15]. In these experiments, in vitro-matured bovine oocytes (24 h) injected with sorted Y sperm heads developed into blastocysts and live offspring. Recently, sex-predetermined live offspring were obtained by AI (depositing sperm deep in the uterus horn) using sorted bull sperm [9]. In ordinary bovine AI, at least 10 million sperm are necessary for successful pregnancies. It would be difficult to separate 10 million X or Y motile sperm by the flow sorting method in a single day. Seidel et al. [9] inseminated cows with sorted bull sperm (0.1–0.2 million sperm/0.1 ml) by depositing the sperm deep into the uterine horn and achieved a lower pregnancy rate (50% at 8 wk after insemination) than with ordinary AI. Cran et al. [10] inseminated sheep with a low dose (10⁵) of sorted ram sperm by the laparoscopic intrauterine AI procedure and obtained a lower offspring production rate than that obtained by the ordinary AI procedure. For ordinary bovine IVF, many motile sperm are required for capacitation. Although the ICSI method requires technical skill and special equipment, theoretically it requires only a single sperm to fertilize an oocyte.

Although Cran et al. [6] reported fertilization rates of 79% and 80% using oocytes that were fertilized in vitro with sorted X and Y motile sperm, respectively, there are few reports about the development to blastocysts from oocytes fertilized in vitro with sorted sperm. Rath et al. [7] obtained porcine cleaved embryos (56.2%) by in vitro fertilization with sorted X sperm. In our study, 46.6% of in vitro-matured oocytes were fertilized by sorted Y sperm head injection, and 6.9% of the oocytes developed to blastocysts. Although the fertilization rate of oocytes with ICSI is lower than that with IVF, these results are comparable to those in previous reports using ICSI with sorted sperm [8, 18] and unsorted sperm heads [13]. Catt et al. [8] produced cleaved embryos (46%) at 6 days after ICSI with sorted ram sperm. Goto et al. [13] produced bovine blastocysts (0.4%) using ICSI with unsorted bull sperm heads. There may be some methodological differences between previous studies and our study, such as in the oocyte maturation periods, sperm injection technique, and/or in vitro embryo culture systems. By using intracytoplasmic sperm head injection, Kuretake et al. [12] produced offspring in the mouse. They showed that mouse oocytes injected with sperm heads developed to blastocysts but not to live offspring at a rate similar to that achieved with ICSI and whole sperm. The live offspring production rate after embryo transfer was much lower for blastocysts produced with ICSI of sperm heads than for those produced by ICSI with whole sperm. Furthermore, the sperm centriole, which is essential for fertilization and development to offspring in other species was not essential in the mouse. It is not certain whether bull sperm tails (especially the centrioles) are essential for fertilization and development of bovine oocytes. A study to assess the presence or absence of the centrioles in the sonicated bull sperm heads should be performed. For ICSI, we used bull sperm heads that had been stored at -30°C without cryoprotectant. Kuretake et al. [12] reported the effects on fertilization and blastocyst development both of the amount of time between sperm tail splitting and injection and of the addition of glycerol and BSA to the freezing buffer used in the cryopreservation of the sperm heads. Bull sperm heads may be affected similarly to those of the mouse, but this remains to be confirmed.

We transferred one blastocyst per recipient in the present study; however, it has been reported that a higher pregnancy rate can be achieved using two embryos per recipient. Catt et al. [8] demonstrated that a male lamb was born from 28 recipients implanted with between 5 and 15 embryos derived from oocytes injected with sorted Y ram sperm. Cran et al. [6] reported a 33% pregnancy and offspring rate in recipients implanted with two embryos derived from IVF with flow-sorted Y bull sperm. Rath et al. [7] produced 10 offspring from 92 transferred embryos (10.8%) derived from IVF with sorted X sperm in swine. The offspring production rates of ICSI and IVF with sorted sperm are indeed lower than those of ordinary ICSI and IVF. The calf production rate in this study is low but comparable to previous studies using ICSI and IVF with sorted sperm. McNutt and Johnson [19] found that sorted intact rabbit sperm might not have negatively influenced fertilizing ability, but interfered with early embryonic development after fertilization. The reason for the low rate remains to be determined. It is possible that the flow sorting procedure itself affects the fertilizing ability of sperm at both the sperm and embryonic levels. For statistical analysis purposes, we require additional experiments, but here we report the birth of 10 calves from 48 recipients using sorted Y sperm heads. Experiments are in progress to establish reproducibility and more statistically significant results.

	ACKNOWLEDGMENTS

 
The authors thank Dr. J. Masaki and Dr. L.A. Johnson for invaluable suggestions. We wish to thank S. Sakamoto, H. Yokosawa, Y. Okada, and the staff of the Federation of Akagi Dairy Co-operative Associations for their skilled technical assistance, and Dr. P.J. Kotaras for his kind assistance with English usage.

	FOOTNOTES

 
¹ This work was supported by a grant from the Agriculture and Livestock Industries Corporation and Ministry of Agriculture, Forestry and Fisheries.

² Correspondence: Yoshiaki Minato, Maebashi Institute of Animal Science, Livestock Improvement Association of Japan, Inc., 316 Kanamarumachi, Maebashi, Gunma, 371–0121, Japan. FAX: 81 27 269 9526; res-liaj{at}tohgoku.or.up

³ Current address: Research Farm, Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano 399–4598, Japan.

Accepted: December 14, 1998.

Received: August 6, 1998.

	REFERENCES

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