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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Animal Sciences and Industry, Kansas State University, Manhattan, 66506
Abstract
Polyspermic fertilization and embryo quality are important issues for the in vitro production of pig embryos. We hypothesized that oocyte donor (prepubertal gilt vs. sow) affects polyspermy and blastocyst development in vitro and that the sexual maturity of the oocyte donor affects the response to sperm concentration in the fertilization medium. In Exp. 1, oocytes of sows and gilts were mounted and stained 12 h after insemination to provide fertilization data. In Exp. 2, putative embryos were developed in vitro to 144 h postinsemination before mounting. In both experiments, cumulus–oocyte complexes (COC) were collected from ovaries of prepubertal gilts and adult sows. Sperm were added after maturation of COC for 40 to 44 h. Sperm from two boars at 0.5 to 5.0 x 106 sperm/mL was used for insemination. More (P < 0.01) monospermic fertilizations were observed in oocytes derived from gilts than for oocytes from sows. There were fewer (P < 0.02) penetrated sperm per fertilized oocyte in oocytes from gilts compared with sows. There were effects of semen donor (boar) on the percentage of monospermic (P < 0.01) and polyspermic (P < 0.002) fertilizations, and on the number of penetrated sperm/fertilized oocyte (P < 0.02). In Exp. 2, cleavage and blastocyst formation was evaluated at 2 and 6 d postinsemination, respectively. More (P < 0.001) blastocysts developed from sow-derived oocytes than from gilt-derived oocytes. More (P < 0.05) total cells per blastocyst were observed in embryos from sow-derived oocytes than from gilt-derived oocytes. Semen donor affected the percentage of oocytes cleaving (P < 0.02), and a boar x sperm concentration interaction affected (P < 0.05) the incidence of blastocyt formation. Results indicate that sexual maturity of the donor is not responsible for the high incidence of polyspermy in porcine in vitro fertilization. However, blastocyst development is improved by the use of oocytes from sows rather than from prepubertal gilts.
Key Words: Embryo Culture • In Vitro Fertilization • Pigs • Prepubertal Females • Sexual Maturity
Introduction
Steady progress has been made in the production of pig embryos in vitro (Abeydeera, 2001
, 2002). Porcine oocytes can be fertilized at a high rate. However, the in vitro-produced (IVP) embryos differ from in vivo embryos in that the incidence of polyspermy is much higher and the blastocysts produced in vitro are generally inferior in cell number and in their ability to produce pregnancies.
Nearly all studies involving IVP embryos in the pig utilize prepubertal gilts as oocyte donors. However, studies of bovine and ovine IVP embryos indicate limited success when prepubertal females provide oocytes. Oocytes derived from prepubertal calves can be fertilized at a rate similar to that obtained with cows (Duby et al., 1995; Looney et al., 1995; Revel et al., 1995). Similarly, fertilization rates of oocytes obtained from prepubertal and cyclic sheep are not different (O’Brien et al., 1997). Nevertheless, developmental competence of embryos produced from prepubertal donors is low. Abnormal cortical granule patterns (Duby et al., 1995) and calcium oscillations, along with delayed sperm aster formation have been demonstrated (Damiani et al., 1996) for calf oocytes. These results suggest calf oocytes undergo incomplete cytoplasmic maturation in vitro.
In light of these considerations, the present study evaluated the hypothesis that results of porcine in vitro fertilization (IVF) and in vitro production of embryos would be improved by using sow-derived vs. prepubertal gilt-derived oocytes. Because high sperm concentrations in the IVF medium increase the incidence of polyspermic penetration (Abeydeera and Day, 1997), we also hypothesized that oocytes from prepubertal and mature donors might respond differently to sperm concentration in the fertilization medium. Therefore, oocytes from sows and prepubertal gilts were exposed to four sperm concentrations and semen from two boars was utilized to evaluate differences in their ability to undergo normal fertilization and development.
Materials and Methods
Experimental Design
Two experiments were conducted, one to obtain fertilization data (Exp. 1) and the other to evaluate development of IVP embryos (Exp. 2). Each experiment consisted of three replicates. In each experiment, more than 1,400 cumulus–oocyte complexes (COC) were cultured. Fertilization was accomplished in 100-µL drops, and each drop contained 30 to 35 COC. Percentage data were calculated from each drop. In each experiment, there were 48 fertilization drops and equal numbers of drops were assigned across treatments.
Treatment main effects were oocyte donor (gilt or sow), semen donor (Boar 162 or 18-7), and sperm concentration (0.5, 1.0, 2.5, and 5.0 x 106 sperm/mL) applied in a 2 x 2 x 4 factorial treatment structure. Boars used for semen collection were No. 162 (line 327, Pig Improvement Co. USA, Franklin, KY), and No. 18-7 (lines C22 x 326, Pig Improvement Co. USA). Boar 162 was approximately 2 yr old and supplied semen with progressive motility, ranging from 65 to 70% immediately after collection. Boar 18-7 was approximately 8 mo of age and initial progressive motility ranged from 75 to 80%. Further analyses of sperm characteristics were not performed. These experiments were approved by the Institutional Animal Care and Use Committee.
Culture Media
Unless otherwise noted, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). All cultures were conducted in a humidified atmosphere containing 5% CO2 in air and at 38.7°C. Oocyte maturation medium was tissue culture medium 199 (Gibco, Grand Island, NY) supplemented with 0.1% polyvinylalcohol, 3.05 mM glucose, 0.91 mM sodium pyruvate, 75 µg/mL of potassium penicillin, 50 µg/mL of streptomycin sulfate, 0.5 µg/mL of ovine LH (catalog No. L-5269), 0.5 µg/mL of porcine FSH (catalog No. F-2293), and 10 ng/mL of murine EGF (catalog No. E-4127; Abeydeera et al., 1998; 2000).
The medium used for IVF was a modified Tris-buffered medium containing 113.1 mM NaCl, 3.0 mM KCl, 7.5 mM CaCl2•2H2O, 20.0 mM Tris (crystallized free-base; Fisher Scientific, Fair Lawn, NJ), 11.0 mM glucose, 5.0 mM sodium pyruvate, 1 mM caffeine, and 0.1% BSA (catalog No. A-7888; Abeydeera and Day, 1997). Embryo development was accomplished in North Carolina State University 23 medium containing 0.4% BSA (catalog No. A-8022; Petters and Reed, 1991).
Recovery and Maturation of Oocytes
Ovaries were obtained from prepubertal gilts and multiparous sows at two local slaughterhouses. Gilt slaughter was by electrical stunning followed by exsanguination and scalding. Captive bolt stunning was used at the facility supplying sow ovaries, and carcasses were skinned with the aid of a mechanical hide puller. The interval from stunning to ovary collection for both sows and gilts was less than 5 min.
Ovaries were transported to the laboratory in 0.85% saline supplemented with 5 mL/L of antibiotic–antimycotic solution (catalog No. A-7292) containing 2,500 IU/L of penicillin, 2.5 mg/L of streptomycin, and 125 µg/L of amphoterecin B. For gilts, only ovaries with no corpora lutea and with no follicles larger than 6 mm in diameter were used. For both age groups, COC from 3- to 6-mm follicles were aspirated into 50-mL conical tubes by an 18-gauge needle attached to a vacuum pump (22 mL of H2O/min). Interval from ovary collection to oocyte aspiration was 3 to 6 h. Oocytes with evenly distributed cytoplasm and at least three layers of compact cumulus cells were selected and washed three times in maturation medium. Then, 60 to 70 COC were placed into each well of a Nunc 4-well multidish (Nunc, Roskilde, Denmark) containing 500 µL of maturation medium. The medium in each multidish was covered with paraffin oil (Ovoil-100, IVF Science, Gothenburg, Sweden) and had been equilibrated with 5.0% CO2 in air at 38.7°C. After 20 to 22 h of incubation, COC were washed in maturation medium and transferred to fresh maturation medium without porcineFSH or ovineLH, but with murineEGF, for an additional 20 to 22 h.
In Vitro Fertilization
At the end of the maturation period, cumulus cells were removed by pipetting the oocytes in each well into a 15-mL disposable centrifuge tube (Fischer Scientific) containing 0.1% hyaluronidase and vortexing for 30 s at a setting of 3 on a VWR vortexer (Scientific Industries, Bohemia, NY). The oocytes from each maturation well were washed three times in IVF medium and divided into two groups of 30 to 35 and placed into the wells of a Nunc 4-well multidish containing 50 µL of fertilization medium and covered with paraffin oil. Before addition of sperm, dishes were kept in the incubator for 20 to 40 min during sperm preparation.
The sperm-rich fraction was collected from two boars by digital pressure with a gloved hand. Semen (30 mL) was supplemented with 2.25 mg of potassium penicillin G and 1.5 mg of streptomycin sulfate added directly to the semen and incubated for 18 h at 20°C. Following incubation, semen was centrifuged (80 x g) for 3 min and the top 5 mL was removed for use. This aliquot was washed two times by centrifugation at 500 x g for 3 min and resuspending in Dulbecco’s PBS (Gibco) supplemented with 0.1% BSA (catalog No. A-8022). After washing, the sperm pellet was resuspended in IVF medium. After appropriate dilutions, 50-µL of the sperm suspension was added to the 50 µL drops containing the oocytes to achieve final sperm concentrations of 0.5, 1.0, 2.5, and 5.0 x 106 sperm/mL. Oocytes were co-incubated with the sperm for 5 to 6 h.
After exposure to sperm, oocytes were transferred to development medium (500 µL) in Nunc plate wells covered with paraffin oil. For fertilization data (Exp. 1), putative embryos were mounted 12 h after insemination to assess sperm penetration and the incidence of monospermic and polyspermic fertilization. The percentage of cleaved and blastocyst formation was evaluated 48 and 144 h postinsemination, respectively, by phase-contrast microscopy at 50 and 100x (Exp. 2). At 144 h, blastocysts were mounted on microscope slides under coverslips supported with paraffin:vaseline (1:1, vol/vol) at the corners. Slides were placed into 25% acetic acid in ethanol overnight to remove cellular lipids. Slides were stained with 1.0% orcein in 45% acetic acid for visualization of genetic material and evaluated using phase contrast microscopy (160 and 400x). The presence of a female pronucleus and swollen sperm head or male pronucleus with its corresponding sperm tail indicated fertilization. Zygotes were classified as cleaved if they had undergone one or more cell divisions by 48 h postinsemination. At 144 h postinsemination, embryos with cavities were tentatively identified as blastocysts, and if they contained 20 or more cells, were recorded as blastocysts for data analysis.
Statistical Analyses
Statistical models included main effects of donor type (sow or gilt), boar, sperm concentration, and their interactions. The percentage of monospermic and polyspermic oocytes, immature oocytes (Exp. 1), cleaved embryos, and blastocysts (Exp. 2) were calculated for each well and data were evaluated using the GLM procedure and LSMEANS statement of SAS (SAS Inst., Inc., Cary, NC).
Results
Experiment 1
Approximately 10% of both gilt- and sow-derived oocytes did not mature to Metaphase II (Figure 1). The percentage of oocytes fertilized did not differ (P > 0.10) between sow and gilt oocytes. However, the percentage fertilized by only one sperm was greater (P < 0.01) for gilt than sow oocytes, and the number of fertilizing sperm per fertilized oocyte (swollen head or male pronucleus) was less (P < 0.02) for gilt (1.3 ± 0.05) than for sow (1.4 ± 0.05) oocytes. No female age x boar or female age x sperm concentration interactions (P > 0.35) were detected for fertilization rates.
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). Increasing concentrations of sperm from Boar 162, but not Boar 18-7, resulted in more fertilized oocytes. Furthermore, when added at the same sperm concentration, the sperm from Boar 18-7 fertilized more (P < 0.02) oocytes compared to sperm from Boar 162, except at the highest sperm concentration. The percentage of monospermic fertilizations were greater (P < 0.001) for Boar 18-7 than for Boar 162 (47.0 ± 2.3 vs. 33.8 ± 2.3). Polyspermic fertilizations were also greater (P < 0.01) for Boar 18-7 (24.8 ± 2.5) than for Boar 162 (12.9 ± 2.5). Increasing sperm concentration from 0.5 to 5 million/mL increased (P = 0.02) the percentage of oocytes fertilized with one sperm from 32.3 ± 3.3 to 47.9 ± 3.3. The percentage of oocytes fertilized with more than one sperm increased (P < 0.01) from 8.8 ± 3.5 to 28.1 ± 3.5 over the same sperm concentration range. There was a tendency for a boar x sperm concentration interaction to affect the percentage of polyspermic (P < 0.10) fertilizations. Increasing sperm concentration for Boar 162, but not 18-7, markedly increased polyspermic fertilizations.
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Experiment 2
Cleavage and blastocyst development was evaluated at 48 h and 144 h postinsemination, respectively. There was a tendency (P = 0.10) for more sow than gilt oocytes to cleave and more (P < 0.001) sow than gilt oocytes became blastocysts (Figure 3). Blastocysts developing from sow oocytes contained more (P < 0.05) total cells than did blastocysts from gilt oocytes.
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). Increasing sperm concentrations appeared to increase the oocytes cleaving for Boar 162 but not for Boar 18-7. Regardless of sperm concentration, fewer (P < 0.02) oocytes cleaved following addition of sperm from Boar 162 (51.6 ± 3.1%) compared with sperm from Boar 18-7 (63.0 ± 3.1%). Numerically, the greatest cleavage rate (70.2 ± 6.2), for gilt and sow oocytes combined, occurred following insemination with 0.5 x 106 sperm/mL from Boar 18-7.
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). For Boar 18-7, the most blastocysts were produced by addition of 0.5 x 106 sperm/mL and for Boar 162, a sperm concentration of 2.5 x 106 sperm/mL produced the most blastocysts. For Boar 18-7, the addition of more than 0.5 x 106 sperm/mL decreased (P < 0.05) the rate of blastocyst production. For all sperm concentrations, blastocysts produced by fertilizing with sperm from Boar 162 had more (P = 0.05) cells than blastocysts developing after fertilization with sperm from Boar 18-7 (37.7 ± 1.7 vs. 31.2 ± 2.4). As sperm concentration increased, there was a decrease (P < 0.01) in the number of cells per blastocyst (Figure 5).
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The present study examined the hypothesis that the incidence of polyspermic fertilization and blastocyst development are affected by the sexual maturity of the oocyte donor. Gilt- and sow-derived oocytes had similar rates of nonmaturing oocytes, but more gilt oocytes were fertilized by only one sperm. Marchal et al. (2001) reported that sow oocytes had a higher maturation rate and a lower sperm penetration rate than gilt oocytes but had similar rates of polyspermic fertilization if porcine follicular fluid was included in the oocyte maturation medium. Marchal et al. (2001) observed that gilt oocytes underwent polyspermic fertilization almost twice as frequently as sow oocytes if follicular fluid was not used. In the present studies, follicular fluid was not used and no differences were found in the incidence of polyspermy for sow- and gilt-derived oocytes. In fact more gilt- than sow-derived oocytes were fertilized by only one sperm. Therefore the hypothesis that sexual maturity of the female donor explains the high incidence of polyspermic fertilization in porcine IVF is not supported.
A secondary hypothesis tested was that the oocytes of prepubertal gilts are more sensitive to the number of sperm used for IVF. This hypothesis was not supported because no interactions for age of oocyte donor x sperm concentration were observed. The boar x sperm concentration interactions observed provide a rigorous test of gamete behavior, as affected by oocyte donor, over a wide range of sperm concentrations and fertilization rates. Oocytes from both sows and gilts responded with greater fertilization rates to increasing sperm concentrations for semen from Boar 162. However, fertilization rate appears to have been maximal for the lowest sperm concentration tested for Boar 18-7. Boar differences are known to affect pig IVF results (Xu et al., 1996 a,b), and the two boars used in the present report were selected because preliminary results indicated that their sperm produced different fertilization rates in vitro. The results reported here are consistent with those preliminary observations.
Monospermic and polyspermic fertilizations increased with increasing sperm concentration for both sow and gilt oocytes. There were no interactions of oocyte source and boar or sperm concentration, and the response to sperm concentration and boar seemed to be similar. It may be that when sperm numbers are reduced, the incidence of polyspermic penetration is reduced due to decreased chances of interaction with multiple sperm before the physiological blocks to polyspermy are effective. The present results support this interpretation for both sows and gilts.
Somewhat more sow- than gilt-derived oocytes cleaved and embryos derived from sow oocytes developed to blastocysts at higher rates than embryos derived from gilt oocytes. Blastocysts from sow-derived oocytes contained more total cells than blastocysts from gilt-derived oocytes Marchal et al. (2001) also observed a higher incidence of blastocyst formation for sow oocytes fertilized in vitro.
The effect of donor age is consistent with reports of the developmental superiority of bovine embryos produced from adult cows compared with prepubertal calf oocytes (Duby et al., 1995; Looney et al., 1995; Damiani et al., 1996). Delayed sperm aster formation, insufficient Ca++ release, and abnormal cortical granule migration were identified as reasons for the lack of developmental competence of calf oocytes (Duby et al., 1995; Damiani et al., 1996). Cortical granule exocytosis has been evaluated as normal in oocytes from prepubertal gilts (Wang et al., 1997, 1998). Identifying cytostructural and biochemical differences in prepubertal gilt and sow oocytes before and during maturation in vitro may be fruitful areas for investigation.
The present studies may also be relevant to reports comparing oocytes and embryos from pubertal vs. postpubertal gilts. Koenig and Stormshak (1993) found that gilts ovulate a higher percentage of immature oocytes and tend to ovulate more oocytes displaying chromosomal nondysjunction at the pubertal vs. third postpubertal estrus. Menino et al. (1989) observed that three- to eight-cell embryos collected from pubertal gilts were less likely to develop to blastocyts in vitro than similar stage embryos collected from the same gilts at their third estrus. Similarly, Pinkert et al. (1989), Hajdu et al. (1994), and Peters et al. (2001) found that embryos collected from prepubertal gilts, which were induced to ovulate with exogenous hormones, were less likely to form blastocysts in vitro than embryos collected from postpubertal gilts. It may be that the relatively poorer development by prepubertal gilt oocytes in the present report is related to poorer in vitro development by gilt oocytes ovulated at spontaneous or induced puberty and developed in vitro. Furthermore, evidence of deficiencies of oocytes released at the pubertal estrus comes from some studies showing a lower embryo survival in gilts mated or inseminated at puberty vs. a postpubertal estrus (Archibong et al., 1987; 1992). However, Knott et al. (1984) did not observe this effect, and Rhodes et al. (1991) found no effect of pubertal status at insemination on total gestational conceptus losses.
For oocytes inseminated with sperm from Boar 18-7, the sperm concentration in the fertilization medium was inversely related to the percentage of oocytes that formed blastocysts, but this relationship was not exhibited for sperm from Boar 162. Higher sperm concentrations also resulted in fewer cells per blastocyst. The general tendency was for lower sperm concentrations to produce lower rates of polyspermic fertilization, more blastocysts, and blastocysts with more cells. This relationship may be consistent with the report of Han et al. (1999a), who observed that polyspermic porcine zygotes developed into blastocysts with fewer inner cell mass cells compared with blastocysts from monospermic oocytes. However, in the report of Han et al. (1999a), the total cell number per blastocyst was similar for the two types of fertilization.
Blastocysts developing from polyspermic zygotes can produce pregnancies and live offspring (Han et al., 1999b). The present results indicate that although blastocysts can develop from polyspermic oocytes, sperm concentrations that result in high polyspermy rates also produce fewer blastocysts with fewer total cells than sperm concentrations that minimize polyspermy. Whether these results are directly attributable to polyspermy or result from other conditions created by higher sperm numbers in the fertilization medium cannot be determined from the present experiments.
In summary, the present results indicate that the use of prepubertal gilts as oocyte donors is not the cause of elevated rates of polyspermic fertilization in porcine IVF. However, sow oocytes are more likely to produce blastocysts in vitro and their blastocysts contain more cells. Whether the blastocysts produced from sow oocytes are better for producing pregnancies and offspring should be evaluated.
Implications
For in vitro-produced pig embryos to be used for agricultural or biotechnological applications, the incidence of polyspermy must be reduced and the quality of blastocysts, including cell number, improved. The present results do not implicate sexual maturity of the oocyte donor as a cause of polyspermy in porcine in vitro fertilization. However, oocytes harvested from sows produced more blastocysts and blastocysts with more cells. Sperm concentrations that increased polyspermy resulted in fewer blastocysts and fewer cells per blastocyst. Therefore, sow oocytes may offer advantages for porcine in vitro-produced embryos, and sperm concentration should be optimized for each sperm donor.
Footnotes
1 Contribution No. 03-366-J from the Kansas Agric. Exp. Stn. 
2 Present address: ViaGen, Inc., Athens, GA 30602.
3 Correspondence: 256 Weber Hall (phone: 785-532-1224; fax: 785-532-7059; e-mail: ddavis{at}oznet.ksu.edu).
Received for publication May 12, 2003. Accepted for publication August 27, 2003.
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0.05). Increasing sperm concentration increased both monospermic (P < 0.01) and polyspermic (P = 0.02) fertilizations for both boars.
