In vitro Fertilization and Development of Pig Oocytes Inseminated with Boar Sperm by Different Sperm Washing Media after Thawing of the Frozen Straws

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing 500 μl mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to 24°C over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were resuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain 1×10 sperm/ml and cooled to 5°C over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with 2×10 sperm concentration. Oocytes were coincubated for 6 h in 500 μl mTBM fertilization. At 6 h after IVF, oocytes were transferred into 500 μl NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 2 : 164-167)


INTRODUCTION
Since Mattioli et al. (1989) succeeded in getting piglets from IVM/IVF oocytes in pigs, in vitro production of pig embryos have been developed gradually.However, efficient production of pig embryos through IVM/IVF techniques has been hampered by the high incidence of polyspermy and a low incidence of male pronuclear formation.
Several procedures using frozen-thawed boar semen have been reported for in vitro fertilization of in vitro or in vivo matured porcine oocytes.However, the results have not been consistent sufficiently because of day-to-day variations between ejaculates, even under identical laboratory conditions.Potential explanations may be due to boar effects, to the purity of the sperm rich fraction, and to different semen treatment protocols for IVF.Little is known about the physiology of sperm capacitation.Furthermore, adequate in vitro procedures to accomplish optimal spermoocyte interactions such as coordinated binding of spermatozoa to the zona pellucida are still not available.
This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the same boar sperm by different sperm washing media after thawing of the 5 ml frozen straws.

Oocyte collection and in vitro maturation
Ovaries were collected from prepubertal gilts at a local slaughter house and transported to the laboratory in 0.9% NaCl solution containing 75 µg/ml potassium penicillin G, 50 µg/ml streptomycin sulfate and 0.1% BSA at 30-35°C.Cumulus oocyte complexes (COCs) were aspirated from antral follicles (3 to 6 mm in diameter) using a 20 gauge needle fixed to a 10 ml disposable syringe.COCs were washed three times in mTLP-PVA and were washed two times with a maturation medium.30-40 COCs were transferred to 500 µl of the same medium that had been covered with mineral oil in a 4-well multidish (Nunc, Roskilde, Denmark) and equilibrated at 38.5°C, 5% CO 2 in air.The medium used for oocyte maturation was tissue culture medium (TCM) 199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 µg/ml insulin, 2 µg/ml vitamin B 12 , 25 mM HEPES, 10 µg/ml bovine apotransferrin, 150 µM cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 µg/ml sodium penicillin G, 50 µg/ml streptomycin sulfate and 10% pFF.After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5°C, 5% CO 2 in air.

Frozen semen processing
Semen was collected from one Duroc boar twice a week.The sperm-rich fraction (30 to 60 ml) of ejaculate was collected into an insulated vacuum bottle.Semen was slowly cooled to room temperature (20 to 23°C) by 2 h after collection.Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min at 800 g and the supernatant solution was poured off.One volume of concentrated sperm was resuspended with 1 volume of lactose, egg yolk and N-acetyl-D-glucosamine (LEN) diluent (the first diluent to provide 1.0×10 9 sperm/ml) at room temperature (Yi et al., 2002).
Semen was cooled in a refrigerator to 5°C over a 2 h period and 1 volume a LEN+4% glycerol diluent (the second diluent) was added to 1 volume of cooled semen.Straws (Minitub Gmbh, Landshut, Germany) were immediately filled with 5 ml of semen and steel or glass balls were used to seal the ends of the straws.The air bubble was adjusted to the center of each straw and the straws were horizontally placed on an aluminum rack and set into a liquid nitrogen tank containing liquid nitrogen (LN).The straws were situated 5 cm above the LN, and kept at that level for 20 min before the straws were transferred into LN storage.

In vitro fertilization and culture of oocytes
After the completion of culture of oocytes for IVM, cumulus cells were removed with 0.1% hyaluronidase in mTLP-PVA and washed two times with mTBM.Thereafter, 30-40 oocytes were transferred into each well of a 4-well multidish containing 500 µl mTBM fertilization medium that had been covered with mineral oil and equilibrated at 38.5°C, 5% CO 2 in air.The dishes were kept in a CO 2 incubator until spermatozoa were added for insemination.
For IVF, one frozen 5 ml straw was thawed at 52°C in 40 sec and was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature.Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM solution, respectively and nonpreincubated) with 2×10 7 /ml sperm concentration.At 6 h after IVF, oocytes were transferred into 500 µl NCSU-23 culture medium containing 0.4% BSA for further culture.

Examination of oocytes
At 12 and 48 h after insemination, oocytes were fixed for 48 h in 25% acetic acid (v:v) in ethanol at room temperature, and stained with 1% (w:v) orcein in 45% (v:v) acetic acid to examine sperm penetration, polyspermic oocyte, male pronucleus and cleaved oocytes under a phasecontrast microscope at ×400 magnification.Blastocysts on day 6 were stained with Hoechst 33342 and were counted nucleus number under fluorescent microscope (Olympus, Japan).
Unless otherwise mentioned, all chemicals used in this study were purchased from Sigma Chemical Co.(St.Louis, MD, USA).

Statistical analysis
Analyses of variance (ANOVA) were carried out using the SAS package (SAS, 1996) in a completely randomized design.Duncan's multiple range test was used to compare mean values of individual treatment, when the F-value was significant (p<0.05).

RESULTS
The effects of washing media after thawing of 5 ml straws on fertilization parameters were shown in Table 1.The present study showed that sperm motility reduced dramatically during culture in each treatment (about 42.9-50.0%at the start of culture to 15.0-29.3% at 6 h of culture).Oocytes inseminated with the mTLP-PVA and mTBM washing showed 51.7 and 53.9% sperm penetration, 2.5 and 4.1% polyspermy, and 43.3 and 43.9 male pronuclear formation, respectively.Sperm penetration and male pronuclear formation were higher in the BTS, mTLP-PVA and mTBM washing treatments than the unwashed treatment (p<0.05).
As shown in Table 2, the percentage of cleaved oocytes was higher in the BTS, mTLP-PVA and mTBM washing treatments than the unwashed treatment, and the percentage of blastocysts from cleaved oocytes were higher in the mTLP-PVA washing treatment than the unwashed, BTS, and mTBM washing treatments.

DISCUSSION
In this study, the motility of frozen boar sperm after thawing was 42.9-50.0%in each treatment, but the motility reduced dramatically during culture.We found out that frozen-thawed boar sperm, even with a low motility, contributed to the penetration rates of oocytes.Watson (1995) reported that the cryopreserved sperm somehow leaded to acceleration of capacitation-like process.Martinez et al. (1996) indicated that, under the in vitro conditions studied, boar sperm underwent capacitation and a true acrosome reaction during co-incubation with oocytes even when not washed or preincubated.Polyspermic penetration has been the persistent problem in IVM-IVF systems (Funahashi and Day, 1997).Polyspermic penetration of oocytes in vitro was not due to delayed or incomplete cortical granule exocytosis (Wang et al., 1998) but more likely to a delayed zona reaction and/or simultaneous sperm penetration (Wang et al., 1999).Funahashi and Nagai (2001) reported that adenosine increased the normal penetration of oocytes by frozenthawed boar sperm, and that the number of partially acrosome-reacted sperm at insemination might be one of the primary causes of polyspermy in porcine IVF system.In this study, we found out extremely low incidence of polyspermic penetration.These results were consistent with the above reports of Martinez et al. (1996), Wang et al. (1998Wang et al. ( , 1999) ) and Funahashi and Nagai (2001).
In general, semen pellet and straw were thawed and washed three times in mBO medium (Wang et al., 1995), TALP medium (Rath and Niemann, 1997), Dulbecco's PBS (Abeydeera and Day, 1997;Abeydeera et al., 1998), mTCM199 medium (Kikuchi et al., 1999;Yamauchi and Nagai, 1999) and BTS (Gandhi et al., 2001;Marchal et al., 2002).This study was the first to compare the in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws.The sperm penetration, male pronuclear formation, and percentage of cleaved oocytes were higher in the BTS, mTLP-PVA and TBM washing treatments than the unwashed treatment.These results indicated that the sperm washing after thawing of 5 ml frozen-straw was an important factor for higher sperm penetration and male pronuclear formation.The removed egg yolk particles in the LEN diluent during washing may be one of factors to improve the sperm penetration and male pronuclear formation.
The percentage of cleaved oocytes was higher in the BTS, mTLP-PVA and mTBM washing treatments than the unwashed treatment.The percentage of blastocysts from cleaved oocytes was higher in the mTLP-PVA washing treatment than the unwashed, BTS and mTBM washing treatments.The above results demonstrated that the sperm washing media contributed to sperm penetration rate, monospermy, cleavage rate and blastocyst formation of oocytes.
In conclusion, we found out that the washing of frozenthawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium was very important to improve the blastocyst formation of oocytes.

Table 1 .
Effect of washing medium after thawing of 5 ml straw on fertilization parameters of pig oocytes in mTBM % of sperm motility 1

Table 2 .
Effect of washing medium after thawing of 5 ml straw on development ability of pig oocytes after maturation and fertilization in vitro