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In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars
ELSEVIER
IN VITRO FERTILIZATION OF PORCINE OOCYTES WITH FRESH AND FROZEN-THAWED EJACULATED OR FROZEN-THAWED EPIDIDYMAL SEMEN OBTAINED FROM IDENTICAL BOARS D. Rath and H. Niemann lnstitut fur Tierzucht und Tierverhalten, Mariensee, (FAL), 31535 Neustadt, Germany
Received for publication: Accepted:
March 5, 1996 September 26, 1996
ABSTRACT The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocytecomplexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozenthawed epididymal semen samples showed motility rates (72.2&6%) similar to those of spermatozoa in fresh semen (76.4ti.5°h), while sperm motility decreased in frozenthawed ejaculated semen (40.2&9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-tell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozenthawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen. 0 1997 by Elsevier Science Inc. Keywords: porcine semen, IVF, epididymal spermatozoa, ejaculate, deep freezing INTRODUCTION Several procedures using fresh (13,19,31) and frozen-thawed (14) boar semen have been reported for in vitro fertilization (IVF) of in vitro (11, 33) or in vivo matured
Acknowledgments We thank B.Sieg, A. Frenzel and K.G. Hadeler for their able technical assistance. Theriogenology 47:785-793, 0 1997 by Elsevier Science
1997 Inc.
0093~691XI97/$17.00 PII SOO93-691X(97)00034-4
Theriogenology
786
(1,19) porcine COC. However, the results have not been consistent sufficiently. Even under identical laboratory conditions, day-today variations between ejaculates occur which hamper comparative analysis of experiments. 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 sperm-oocyte interactions such as coordinated binding of spermatozoa to the zona pellucida are still not available (23). To the best of our knowledge, there is no report in which the fertilizing capacity of fresh or frozen-thawed ejaculate or of frozen-thawed epididymal semen obtained from identical boars has been investigated. The objective of this study thus was to compare the in vitro fertilizing capacity of such spermatozoa in order to standardize the semen preparation protocol for IVF. MATERIALS AND METHODS In vivo matured COC were fertilized in vitro using fresh and frozen-thawed ejaculated semen or frozen-thawed epididymal semen from identical boars. Fertilization rates were tested at 8 and 18 h after fertilization. Prepuberal German Landrace gilts, with an average age of 5.5 mo and a body weight of 86.4 + 6.2 kg, were superovulated with intramuscular injection of 1500 IU PMSG (Intergonan, Vemie, Kempen, Germany) followed by 500 IU hCG (Ekluton, Vemie) 72 h later. Gilts were slaughtered 38 h thereafter to collect the oocytes. Follicles of 5 to 10 mm in diameter were punctured, and the oocytes were prepared for IVF (19). Briefly, matured COC were washed twice after aspiration in fertilization medium (TALP supplemented with 3OhBSA and 25 mMol caffeine; Sigma Deisenhofen, Germany). For IVF, 5 oocytes per microdrop (40 ul), covered with silicone oil (DC 200; Serva, Heidelberg, Germany), together with 10 ul of semen were co-cultured in a humidified atmosphere at 39°C and 5% COZ.After 8 h of co-culture, randomly selected oocytes from all microdrops (n = 211) were fixed with acetic acid, stained after 24 h with 1% (w/v) aceto-orcein and evaluated under a light microscope at x 200 to 400 magnification. The remaining unfixed oocytes (n = 485) were transferred into 2 ml of culture medium (NCSU 23; 18) 18 h after fertilization, and were cultured for another 30 h, after which they were stained with aceto-orcein. Sperm-rich fractions of ejaculates were collected from 3 boars of known fertility. Motility was estimated under a phase-contrast microscope at x 200 magnification, and 5 ml of the semen were immediately diluted at a 1:2 ratio with Androhep extender (Minitub, Tiefenbach, Germany). The semen was centrifuged twice (550 x g) and the remaining sperm pellet was resuspended with 20 ml capacitation medium (TCM 199 supplemented with 3% BSA; Sigma). The sperm concentration was adjusted to 2x1@ sperm/ml, and semen was incubated in a humidified atmosphere at 39’C and 5% C& for -2 h. After the incubation period, aliquots of the semen samples were diluted in fertilization medium (TALP supplemented with 3% BSA and 25 mMol caffeine; Sigma) for a final concentration of 4000 spermatozoa per fertilizable oocyte within 10 ul of fertilization medium.
Theriogenology
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Sperm-rich fractions of the ejaculates were collected from the same boars and were immediately diluted at a 1:2 ratio with Androhep extender (Minitub). After cooling to +1X (2 h), semen samples were centriiged for 10 min at 800 x g, and the pellets were resuspended with cooling extender (lactose-monohydrate 115.8 g/L, egg yolk 20%, Hz0 for a total of 1000 ml; 30) for a concentration of 3x1@ sperm/ml. Within 2 h the semen was cooled to +5”C and the final sperm concentration was adjusted to 2x108 sperm/ml using a freezing extender (cooling extender with 6% glycerol and 1.5% orvuses-paste) for a final glycerol concentration of 2%. Immediately after the dilution, semen was stored in 0.25ml plastic straws (Minitub) containing 5x10’ sperm cells and frozen in nitrogen vapor 4 cm above the liquid nitrogen. After 20 min, the straws were plunged into liquid nitrogen and stored until use. Prior to IVF the straws were thawed in warm water (38°C for 20 set). After estimation of the motility under a phase contrast microscope (x 200 magnification), the semen samples were centrifuged at 800 x g for 3 min, and the sperm pallet was resuspended with fertilization medium TALP. In vitro fertilization, in vitro culture and staining were performed as described above for freshly capacitated semen samples. The final sperm concentration was adjusted to 750 spermatozoa per fertilizable oocyte. Immediately after slaughter, the epididymides of the 3 boars were removed and spermatozoa were flushed with Androhep extender from the ductus deferens in caudal direction into the cauda epididymis. The collected semen was centrifuged for 10 min at 800 x g and prepared for freezing, thawing and lVF, as described above. Data were compared by ANOVA using SIGMA STAT (Jandel Cooperation, San Rafael, CA). All data are given as mean f SEM. Differences were considered to be significant at PcO.05. RESULTS After final dilution for h/F, frozen-thawed epididymal semen samples showed sperm motility rates (72.2&.6%) similar to those of fresh semen (76.4i4.5%); however, motility in frozen-thawed ejaculated semen was decreased (40.2&4%; PC 0.05). Significant differences in motility were observed among boars for ejaculated semen but not for epididymal semen (Table 1). Eight hours after h/F, higher rates of pronucleus formation (Table 2) were found with frozen-thawed epididymal semen than with frozenthawed ejaculated semen. The better fertilizing capacity of frozen-thawed epididymal semen was confirmed by the cleavage rates (Tables 3 and 4) obtained 48 h after IVF. Significantly more embryos developed to the 2- to 4cell stage than those fertilized with fresh or frozen-thawed ejaculated semen, whereas more embryos remained at the metaphase 2 stage or were activated (1 pronucleus) when fresh semen was used for IVF. Cleavage rates varied among boars (PcO.05) when ejaculated semen was used, but no variation was seen after lVF with frozen-thawed epididymal spermatozoa.
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Theriogenology
Table 1. Sperm motility (%) in fresh and frozen-thawed epididymal semen in 3 boars Boar 1 x (Oh)
ejaculated
semen and in
Boar 3
Boar 2
iSEM
x (%)
kSEM
‘ii (%)
iSEM
Unprocessed semen
84.2ad
2.0
74.2ae
5.9
77.7ae
5.2
Fresh semen prepared for IVF
74.2bd
4.9
74.2ad
3.8
80.8ae
4.9
Frozen-thawed ejaculated semen
5O.Od
11.0
28.2be
7.5
42.5bd
9.9
Frozen-thawed epididymal semen
76.7bd
2.6
69.2ad
7.4
70.8ad
6.7
a* Values with different superscripts within a column differ significantly between treatments (PC 0.05). d-e Values with different superscripts within a row differ significantly between boars (PC 0.05).
Table 2. Percentage of pronucleus development of in vivo matured porcine oocytes 8 hours after in vitro fertilization with 3 different types of semen IVF with Fresh semen
x (%)
&EM
Frozen-thawed ejaculated semen x (W)
&EM
Frozen-thawed epididymal semen X (%)
&EM
No. of oocvtes
73
Germinal vesicle
5.5
2.9
4.4
2.9
7.2
2.3
28.9b
6.8
36.7b
8.2
9.5=
2.4
Degenerated
18.2
4.6
12.8
5.4
10.5
4.2
Penetrated a
14.1
5.2
11.5
6.9
9.1
2.3
1 pronucleus
24.4
5.6
20.1
5.4
18.4
3.1
2 pronuclei
8.9
4.0
2.2b
2.2
23.oC
4.7
> 2 pronuclei
Ob
12.3bc
7.5
22.3c
5.3
Metephase II
44
94
ANOVA: Values with different superscripts within a row differ significantly (P< 0.05). a Expanded sperm head and corresponding tail in the cytoplasm, no visible pronuclei.
Theriogenology
789
Table 3. Developmental rates of in-vivo matured porcine oocytes 48 hours after in vitro fertilization with 3 different types of semen IVF with Fresh semen
‘ii
rw
Frozen-thawed ejaculated semen
*SEM
x (%) 127
X (%) 212
iSEM
13.3
3.8
25.6
4.2
22.5
3.1
11.0
4.8
12.4
2.9
2.4
1.0
Metaphase II
38.2a
13.4
23.lb
5.7
4.4b
1.8
1 pronucleus
15.9a
6.9
4.2b
1.7
0.8b
0.8
2 pronuclei
5.7
2.5
11.8a
3.5
4.2b
1.4
Penetration/ expanded sperm head
1.3
0.9
2.3a
1.3
ob
4.6
2.3
6.0
1.7
No. of oocytes
146
Degenerated oocyte Germinal vesicle
> 2 pronuclei
0
&EM
Frozen-thawed epididymal semen
2 cell
2.4a
1.7
5.3a
2.0
21 .ob
2.8
14 cell
i2.2a
4.0
i0.7a
2.9
38.7b
3.2
Values with different superscripts differ significantly (PI. 0.05).
between treatment groups
Table 4. Cleavage rates of in vivo matured porcine oocytes 48 hours after in vitro fertilization with spermatozoa from 3 different boars and after 3 different semen treatments IVF with Fresh semen
x
(Oh)
Frozen-thawed ejaculated semen
Frozen-thawed epididymal semen
kSEM
T? (%)
*SEM
x (%)
kSEM
Boar 1
10.8ac
2.4
27.iac
5.3
654ad
5.6
Boar 2
6O.obd
4.1
22.iac
6.7
59.4ad
6.5
Boar 3
2.4ac
1.4
2.8bc
1.0
64.oad
6.2
a-b Values with different superscripts in a column differ significantly between boars (PC 0.05). c-d Values with different superscripts in a row differ significantly between treatments (PC 0.05).
790
DISCUSSION The present study is the first to compare the in vitro fertilizing capacity of fresh and frozen-thawed ejaculated semen and frozen-thawed epididymal semen obtained from identical boars under uniform lVF conditions. In previous work sperm rich fractions that were used for lVF had undergone different capacitation procedures (1,4,13,15,16,19,25,31,32). Frozen-thawed ejaculated semen (20,26,27,28) and fresh (5,7,14) or frozen-thawed (8,9,14) epididymal spermatozoa were used for IVF after diierent pre-treatments. Sperm fertilizing capacities reported in these studies were not comparable because the conditions for IVF and the genetic material were different. Epididymal semen was superior for lVF because the standard freezing protocol (30) using 25Ct-ul plastic straws led to high motility rates after thawing. In a previous study (29) we had shown that small (25O-pl) plastic straws were superior to the 5-ml macrotubes, which were developed for Al with frozen-thawed boar semen (30) because crystallization occurs more homogeneously at the outer and inner parts of the small straw. In our current study, short equilibration periods at 15°C and 4°C resulted in still higher motility rates and subsequently improved h/F and cleavage rates. Another explanation for the improved NF rates in our study may be that epididymal spermatozoa never had contact with seminal plasma. As one of its main physiological functions seminal plasma protects sperm cells from gaining final fertilizing capacity when migrating through the female genital tract (17,24). Thus, the spermatozoa come into contact with decapacitation factors present in the seminal plasma. The biochemical structure of decapacitation factors is mostly unknown. Peptides with low molecular weight (12,22), or glycoproteins (6,21) steroids (10) or cholesterol (3) have all been thought to be implicated in this process. Recently, it has been reported that porcine sperm cells carry proteins of low molecular weight on their surface which are essential for gamete recognition (23). One of these sperm adhesins, called AWN, while added to the sperm surface in the epididymis seems to be activated by components of the seminal plasma. Deactivation of AWN has been shown to occur during sperm migration through the female genital tract and AWN returns to its basic activity when reaching the oocyte (24). Therefore, decapacitation must be avoided under in vitro conditions since the timing of fertilization has to be established by the IVF system itself. In general, epididymal spermatozoa need to be capacitated as a prerequisite for inducing the acrosome reaction. This process includes changes in sperm membrane fluidity and permeability (24). Membrane stability seems to be reduced by the freezing procedure, which allows for the acrosome reaction to occur in the presence of COC without further treatment. The idea of adding seminal plasma to the epididymal semen was considered; however, due to our technical limitations (slaughter of the boars), it was not possible to include this step into the experimental design. The adjustment of sperm concentration is essential for avoiding polyspermic fertilization. In a previous study (19) we determined the minimal number of spermatozoa necessary to fertilize an oocyte without decreasing the fertilization rate is 4ooo freshly capacitated sperm cells. When we compared different concentrations of
Theriogenology
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frozen-thawed spermatozoa, we found that 750 sperm/oocyte resulted in lower polyspermy rates (30.5%) than 1000 (37.4%) 2000 (53.8%) or 4000 (69.4%; P
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10. Langiais J, Zoolinger M, Plante L, Chapdelaine A, Bleau, G Roberts, KD. Localization of cholestryl sulfate in human spermatozoa in support of a hypothesis of the mechanism of capacitation. Proc Natl Acad Sci (USA) 1981; 78: 7288 7270. 11. Mattioli M, Bacci ML, Galeati G, Seren, E. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology 1989; 31: 1201 - 1207. 12. MC Rorie RA, Williams WL. Biochemistry of mammalian fertilization. Ann Rev Biochem 1974; 43: 777 - 803. 13. Nagai T, Niwa K, lritani A. Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes. J Reprod Fertil 1984; 70: 271 - 275. 14. Nagai T, Takahashi T, Masuda H, Shioya Y, Kuwayama M, Fukushima M, lwasaki S, Hanada A. In vitro fertilizatiocl of pig oocytes by frozen boar spermatozoa. J Reprod Fertill988; 84: 585 - 591. 15. Nagai T, Moor RM. Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. Mot Reprod Dev 1990; 28: 377 - 382. 18. Naito K, Fukuda Y, Toyoda Y. Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro. Gamete Res 1988; 21: 289 - 295. 17. Oliphant G, Reynolts AB, Thomas TS. Sperm surface components involved in the control of the acrosome reaction. Amer J Anat 1985; 174: 289-283. 18. Petters RM, Reed ML Addition of taurine and hypotaurine to culture medium improves development of one- and two-cell pig embryos in vitro. Theriogenology 1991; 35: 253 abstr. 19. Rath D. Experiments to improve in vitro fertilization techniques for in vivo-matured porcine oocytes. Theriogenology 1992; 37: 885 - 898. 20. Rath D, Niemann H, Sieg B, Frenzel A. In vitro Befruchtung von Schweineoocyten mit unterschiedlich aufbereitetem Ebersperma. Reprod Dom Anim 1995; 30 (Suppl3): 78 abstr. 21. Reyes A, Oliphant G, Brackett BG. Partial purification and identification of a reversible capacitation factor from rabbit seminal plasma. Fertil Steril 1975; 28: 148 - 157. 22. Robertson RT, Bhalla VK, Williams BL. Purification in the peptide nature of decapacitation factor. Biochem Biophys Res Commun 1971; 45: 1331 - 1338. 23. Topfer-Petersen E. Molecular mechanisms of spem+oocyte interactions in the pig. Reprod Dom Anim 1998; 31 (Suppl4): 93 - 100. 24. Topfer-Petersen E, Calvete JJ, Dostalova ZZ, Reinert M, Waberski D, Sanz L, Hubner T. One year in the life of the spermadhesin family. Fertilitat 1995; 11: 233241. 25. Toyoda Y. Fertilization of pig eggs in vitro with special reference to capacitation of boar spermatozoa with ionophore. Jap J Anim Reprod 1985; 31: 40 - 44. 26. Wang WH, Niwa K, Okuda K. In vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa. J Reprod Fertil 1991; 93: 491 498.
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27. Wang WH, Abeydeera LR, Fraser LR, Niwa K. Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen-thawed ejaculated spermatozoa incubated in a protein-free chemically defined medium. J Reprod Fertil 1995; 104: 305313. 28. Wang WH, Abeydeera LR, Okuda K, Niwa K. Penetration of porcine oocytes during maturation in vitro by cryopreserved, ejaculated spermatozoa. Biol Reprod 1994; 50: 510 - 515. 29. Weitze KF, Fazano F, Rath D. Deep freezing of boar semen in macro- and minitubes. In Johnson LA, Larsson K (eds), Deep Freezing of Boar Semen. Uppsala, Sweden: University press 1985; 268 - 275. 30. Westendorf P, Richter L. Treu H. Zur Tiefgefrierung von Ebersperma. Labor- und Besamungsergebnisse mit dem Hiilsenberger Pailettenverfahren. Dtsch Tier&%1 Wochenschr 1975; 82: 261 - 267. 31. Yoshida M. In vitro fertilization of pig oocytes matured in vivo. Jap J Vet Sci 1987; 49: 711 - 718. 32. Yoshida M. Improved viability of two-cell stage pig embryos resulting from in vitro fertilization of oocytes matured in vivo. Jap J Anim Reprod 1989; 35: 34 - 37. 33. Yoshida M, lshitaki Y, Kawagishi H. Blastocyst formation by pig embryos resulting from in vitro fertilization of oocytes matured in vitro. J Reprod Fertil 1990; 88: 1-8.
IN VITRO FERTILIZATION OF PORCINE OOCYTES WITH FRESH AND FROZEN-THAWED EJACULATED OR FROZEN-THAWED EPIDIDYMAL SEMEN OBTAINED FROM IDENTICAL BOARS D. Rath and H. Niemann lnstitut fur Tierzucht und Tierverhalten, Mariensee, (FAL), 31535 Neustadt, Germany
Received for publication: Accepted:
March 5, 1996 September 26, 1996
ABSTRACT The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocytecomplexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozenthawed epididymal semen samples showed motility rates (72.2&6%) similar to those of spermatozoa in fresh semen (76.4ti.5°h), while sperm motility decreased in frozenthawed ejaculated semen (40.2&9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-tell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozenthawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen. 0 1997 by Elsevier Science Inc. Keywords: porcine semen, IVF, epididymal spermatozoa, ejaculate, deep freezing INTRODUCTION Several procedures using fresh (13,19,31) and frozen-thawed (14) boar semen have been reported for in vitro fertilization (IVF) of in vitro (11, 33) or in vivo matured
Acknowledgments We thank B.Sieg, A. Frenzel and K.G. Hadeler for their able technical assistance. Theriogenology 47:785-793, 0 1997 by Elsevier Science
1997 Inc.
0093~691XI97/$17.00 PII SOO93-691X(97)00034-4
Theriogenology
786
(1,19) porcine COC. However, the results have not been consistent sufficiently. Even under identical laboratory conditions, day-today variations between ejaculates occur which hamper comparative analysis of experiments. 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 sperm-oocyte interactions such as coordinated binding of spermatozoa to the zona pellucida are still not available (23). To the best of our knowledge, there is no report in which the fertilizing capacity of fresh or frozen-thawed ejaculate or of frozen-thawed epididymal semen obtained from identical boars has been investigated. The objective of this study thus was to compare the in vitro fertilizing capacity of such spermatozoa in order to standardize the semen preparation protocol for IVF. MATERIALS AND METHODS In vivo matured COC were fertilized in vitro using fresh and frozen-thawed ejaculated semen or frozen-thawed epididymal semen from identical boars. Fertilization rates were tested at 8 and 18 h after fertilization. Prepuberal German Landrace gilts, with an average age of 5.5 mo and a body weight of 86.4 + 6.2 kg, were superovulated with intramuscular injection of 1500 IU PMSG (Intergonan, Vemie, Kempen, Germany) followed by 500 IU hCG (Ekluton, Vemie) 72 h later. Gilts were slaughtered 38 h thereafter to collect the oocytes. Follicles of 5 to 10 mm in diameter were punctured, and the oocytes were prepared for IVF (19). Briefly, matured COC were washed twice after aspiration in fertilization medium (TALP supplemented with 3OhBSA and 25 mMol caffeine; Sigma Deisenhofen, Germany). For IVF, 5 oocytes per microdrop (40 ul), covered with silicone oil (DC 200; Serva, Heidelberg, Germany), together with 10 ul of semen were co-cultured in a humidified atmosphere at 39°C and 5% COZ.After 8 h of co-culture, randomly selected oocytes from all microdrops (n = 211) were fixed with acetic acid, stained after 24 h with 1% (w/v) aceto-orcein and evaluated under a light microscope at x 200 to 400 magnification. The remaining unfixed oocytes (n = 485) were transferred into 2 ml of culture medium (NCSU 23; 18) 18 h after fertilization, and were cultured for another 30 h, after which they were stained with aceto-orcein. Sperm-rich fractions of ejaculates were collected from 3 boars of known fertility. Motility was estimated under a phase-contrast microscope at x 200 magnification, and 5 ml of the semen were immediately diluted at a 1:2 ratio with Androhep extender (Minitub, Tiefenbach, Germany). The semen was centrifuged twice (550 x g) and the remaining sperm pellet was resuspended with 20 ml capacitation medium (TCM 199 supplemented with 3% BSA; Sigma). The sperm concentration was adjusted to 2x1@ sperm/ml, and semen was incubated in a humidified atmosphere at 39’C and 5% C& for -2 h. After the incubation period, aliquots of the semen samples were diluted in fertilization medium (TALP supplemented with 3% BSA and 25 mMol caffeine; Sigma) for a final concentration of 4000 spermatozoa per fertilizable oocyte within 10 ul of fertilization medium.
Theriogenology
787
Sperm-rich fractions of the ejaculates were collected from the same boars and were immediately diluted at a 1:2 ratio with Androhep extender (Minitub). After cooling to +1X (2 h), semen samples were centriiged for 10 min at 800 x g, and the pellets were resuspended with cooling extender (lactose-monohydrate 115.8 g/L, egg yolk 20%, Hz0 for a total of 1000 ml; 30) for a concentration of 3x1@ sperm/ml. Within 2 h the semen was cooled to +5”C and the final sperm concentration was adjusted to 2x108 sperm/ml using a freezing extender (cooling extender with 6% glycerol and 1.5% orvuses-paste) for a final glycerol concentration of 2%. Immediately after the dilution, semen was stored in 0.25ml plastic straws (Minitub) containing 5x10’ sperm cells and frozen in nitrogen vapor 4 cm above the liquid nitrogen. After 20 min, the straws were plunged into liquid nitrogen and stored until use. Prior to IVF the straws were thawed in warm water (38°C for 20 set). After estimation of the motility under a phase contrast microscope (x 200 magnification), the semen samples were centrifuged at 800 x g for 3 min, and the sperm pallet was resuspended with fertilization medium TALP. In vitro fertilization, in vitro culture and staining were performed as described above for freshly capacitated semen samples. The final sperm concentration was adjusted to 750 spermatozoa per fertilizable oocyte. Immediately after slaughter, the epididymides of the 3 boars were removed and spermatozoa were flushed with Androhep extender from the ductus deferens in caudal direction into the cauda epididymis. The collected semen was centrifuged for 10 min at 800 x g and prepared for freezing, thawing and lVF, as described above. Data were compared by ANOVA using SIGMA STAT (Jandel Cooperation, San Rafael, CA). All data are given as mean f SEM. Differences were considered to be significant at PcO.05. RESULTS After final dilution for h/F, frozen-thawed epididymal semen samples showed sperm motility rates (72.2&.6%) similar to those of fresh semen (76.4i4.5%); however, motility in frozen-thawed ejaculated semen was decreased (40.2&4%; PC 0.05). Significant differences in motility were observed among boars for ejaculated semen but not for epididymal semen (Table 1). Eight hours after h/F, higher rates of pronucleus formation (Table 2) were found with frozen-thawed epididymal semen than with frozenthawed ejaculated semen. The better fertilizing capacity of frozen-thawed epididymal semen was confirmed by the cleavage rates (Tables 3 and 4) obtained 48 h after IVF. Significantly more embryos developed to the 2- to 4cell stage than those fertilized with fresh or frozen-thawed ejaculated semen, whereas more embryos remained at the metaphase 2 stage or were activated (1 pronucleus) when fresh semen was used for IVF. Cleavage rates varied among boars (PcO.05) when ejaculated semen was used, but no variation was seen after lVF with frozen-thawed epididymal spermatozoa.
788
Theriogenology
Table 1. Sperm motility (%) in fresh and frozen-thawed epididymal semen in 3 boars Boar 1 x (Oh)
ejaculated
semen and in
Boar 3
Boar 2
iSEM
x (%)
kSEM
‘ii (%)
iSEM
Unprocessed semen
84.2ad
2.0
74.2ae
5.9
77.7ae
5.2
Fresh semen prepared for IVF
74.2bd
4.9
74.2ad
3.8
80.8ae
4.9
Frozen-thawed ejaculated semen
5O.Od
11.0
28.2be
7.5
42.5bd
9.9
Frozen-thawed epididymal semen
76.7bd
2.6
69.2ad
7.4
70.8ad
6.7
a* Values with different superscripts within a column differ significantly between treatments (PC 0.05). d-e Values with different superscripts within a row differ significantly between boars (PC 0.05).
Table 2. Percentage of pronucleus development of in vivo matured porcine oocytes 8 hours after in vitro fertilization with 3 different types of semen IVF with Fresh semen
x (%)
&EM
Frozen-thawed ejaculated semen x (W)
&EM
Frozen-thawed epididymal semen X (%)
&EM
No. of oocvtes
73
Germinal vesicle
5.5
2.9
4.4
2.9
7.2
2.3
28.9b
6.8
36.7b
8.2
9.5=
2.4
Degenerated
18.2
4.6
12.8
5.4
10.5
4.2
Penetrated a
14.1
5.2
11.5
6.9
9.1
2.3
1 pronucleus
24.4
5.6
20.1
5.4
18.4
3.1
2 pronuclei
8.9
4.0
2.2b
2.2
23.oC
4.7
> 2 pronuclei
Ob
12.3bc
7.5
22.3c
5.3
Metephase II
44
94
ANOVA: Values with different superscripts within a row differ significantly (P< 0.05). a Expanded sperm head and corresponding tail in the cytoplasm, no visible pronuclei.
Theriogenology
789
Table 3. Developmental rates of in-vivo matured porcine oocytes 48 hours after in vitro fertilization with 3 different types of semen IVF with Fresh semen
‘ii
rw
Frozen-thawed ejaculated semen
*SEM
x (%) 127
X (%) 212
iSEM
13.3
3.8
25.6
4.2
22.5
3.1
11.0
4.8
12.4
2.9
2.4
1.0
Metaphase II
38.2a
13.4
23.lb
5.7
4.4b
1.8
1 pronucleus
15.9a
6.9
4.2b
1.7
0.8b
0.8
2 pronuclei
5.7
2.5
11.8a
3.5
4.2b
1.4
Penetration/ expanded sperm head
1.3
0.9
2.3a
1.3
ob
4.6
2.3
6.0
1.7
No. of oocytes
146
Degenerated oocyte Germinal vesicle
> 2 pronuclei
0
&EM
Frozen-thawed epididymal semen
2 cell
2.4a
1.7
5.3a
2.0
21 .ob
2.8
14 cell
i2.2a
4.0
i0.7a
2.9
38.7b
3.2
Values with different superscripts differ significantly (PI. 0.05).
between treatment groups
Table 4. Cleavage rates of in vivo matured porcine oocytes 48 hours after in vitro fertilization with spermatozoa from 3 different boars and after 3 different semen treatments IVF with Fresh semen
x
(Oh)
Frozen-thawed ejaculated semen
Frozen-thawed epididymal semen
kSEM
T? (%)
*SEM
x (%)
kSEM
Boar 1
10.8ac
2.4
27.iac
5.3
654ad
5.6
Boar 2
6O.obd
4.1
22.iac
6.7
59.4ad
6.5
Boar 3
2.4ac
1.4
2.8bc
1.0
64.oad
6.2
a-b Values with different superscripts in a column differ significantly between boars (PC 0.05). c-d Values with different superscripts in a row differ significantly between treatments (PC 0.05).
790
DISCUSSION The present study is the first to compare the in vitro fertilizing capacity of fresh and frozen-thawed ejaculated semen and frozen-thawed epididymal semen obtained from identical boars under uniform lVF conditions. In previous work sperm rich fractions that were used for lVF had undergone different capacitation procedures (1,4,13,15,16,19,25,31,32). Frozen-thawed ejaculated semen (20,26,27,28) and fresh (5,7,14) or frozen-thawed (8,9,14) epididymal spermatozoa were used for IVF after diierent pre-treatments. Sperm fertilizing capacities reported in these studies were not comparable because the conditions for IVF and the genetic material were different. Epididymal semen was superior for lVF because the standard freezing protocol (30) using 25Ct-ul plastic straws led to high motility rates after thawing. In a previous study (29) we had shown that small (25O-pl) plastic straws were superior to the 5-ml macrotubes, which were developed for Al with frozen-thawed boar semen (30) because crystallization occurs more homogeneously at the outer and inner parts of the small straw. In our current study, short equilibration periods at 15°C and 4°C resulted in still higher motility rates and subsequently improved h/F and cleavage rates. Another explanation for the improved NF rates in our study may be that epididymal spermatozoa never had contact with seminal plasma. As one of its main physiological functions seminal plasma protects sperm cells from gaining final fertilizing capacity when migrating through the female genital tract (17,24). Thus, the spermatozoa come into contact with decapacitation factors present in the seminal plasma. The biochemical structure of decapacitation factors is mostly unknown. Peptides with low molecular weight (12,22), or glycoproteins (6,21) steroids (10) or cholesterol (3) have all been thought to be implicated in this process. Recently, it has been reported that porcine sperm cells carry proteins of low molecular weight on their surface which are essential for gamete recognition (23). One of these sperm adhesins, called AWN, while added to the sperm surface in the epididymis seems to be activated by components of the seminal plasma. Deactivation of AWN has been shown to occur during sperm migration through the female genital tract and AWN returns to its basic activity when reaching the oocyte (24). Therefore, decapacitation must be avoided under in vitro conditions since the timing of fertilization has to be established by the IVF system itself. In general, epididymal spermatozoa need to be capacitated as a prerequisite for inducing the acrosome reaction. This process includes changes in sperm membrane fluidity and permeability (24). Membrane stability seems to be reduced by the freezing procedure, which allows for the acrosome reaction to occur in the presence of COC without further treatment. The idea of adding seminal plasma to the epididymal semen was considered; however, due to our technical limitations (slaughter of the boars), it was not possible to include this step into the experimental design. The adjustment of sperm concentration is essential for avoiding polyspermic fertilization. In a previous study (19) we determined the minimal number of spermatozoa necessary to fertilize an oocyte without decreasing the fertilization rate is 4ooo freshly capacitated sperm cells. When we compared different concentrations of
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791
frozen-thawed spermatozoa, we found that 750 sperm/oocyte resulted in lower polyspermy rates (30.5%) than 1000 (37.4%) 2000 (53.8%) or 4000 (69.4%; P
792
Theriogenology
10. Langiais J, Zoolinger M, Plante L, Chapdelaine A, Bleau, G Roberts, KD. Localization of cholestryl sulfate in human spermatozoa in support of a hypothesis of the mechanism of capacitation. Proc Natl Acad Sci (USA) 1981; 78: 7288 7270. 11. Mattioli M, Bacci ML, Galeati G, Seren, E. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology 1989; 31: 1201 - 1207. 12. MC Rorie RA, Williams WL. Biochemistry of mammalian fertilization. Ann Rev Biochem 1974; 43: 777 - 803. 13. Nagai T, Niwa K, lritani A. Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes. J Reprod Fertil 1984; 70: 271 - 275. 14. Nagai T, Takahashi T, Masuda H, Shioya Y, Kuwayama M, Fukushima M, lwasaki S, Hanada A. In vitro fertilizatiocl of pig oocytes by frozen boar spermatozoa. J Reprod Fertill988; 84: 585 - 591. 15. Nagai T, Moor RM. Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. Mot Reprod Dev 1990; 28: 377 - 382. 18. Naito K, Fukuda Y, Toyoda Y. Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro. Gamete Res 1988; 21: 289 - 295. 17. Oliphant G, Reynolts AB, Thomas TS. Sperm surface components involved in the control of the acrosome reaction. Amer J Anat 1985; 174: 289-283. 18. Petters RM, Reed ML Addition of taurine and hypotaurine to culture medium improves development of one- and two-cell pig embryos in vitro. Theriogenology 1991; 35: 253 abstr. 19. Rath D. Experiments to improve in vitro fertilization techniques for in vivo-matured porcine oocytes. Theriogenology 1992; 37: 885 - 898. 20. Rath D, Niemann H, Sieg B, Frenzel A. In vitro Befruchtung von Schweineoocyten mit unterschiedlich aufbereitetem Ebersperma. Reprod Dom Anim 1995; 30 (Suppl3): 78 abstr. 21. Reyes A, Oliphant G, Brackett BG. Partial purification and identification of a reversible capacitation factor from rabbit seminal plasma. Fertil Steril 1975; 28: 148 - 157. 22. Robertson RT, Bhalla VK, Williams BL. Purification in the peptide nature of decapacitation factor. Biochem Biophys Res Commun 1971; 45: 1331 - 1338. 23. Topfer-Petersen E. Molecular mechanisms of spem+oocyte interactions in the pig. Reprod Dom Anim 1998; 31 (Suppl4): 93 - 100. 24. Topfer-Petersen E, Calvete JJ, Dostalova ZZ, Reinert M, Waberski D, Sanz L, Hubner T. One year in the life of the spermadhesin family. Fertilitat 1995; 11: 233241. 25. Toyoda Y. Fertilization of pig eggs in vitro with special reference to capacitation of boar spermatozoa with ionophore. Jap J Anim Reprod 1985; 31: 40 - 44. 26. Wang WH, Niwa K, Okuda K. In vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa. J Reprod Fertil 1991; 93: 491 498.
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27. Wang WH, Abeydeera LR, Fraser LR, Niwa K. Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen-thawed ejaculated spermatozoa incubated in a protein-free chemically defined medium. J Reprod Fertil 1995; 104: 305313. 28. Wang WH, Abeydeera LR, Okuda K, Niwa K. Penetration of porcine oocytes during maturation in vitro by cryopreserved, ejaculated spermatozoa. Biol Reprod 1994; 50: 510 - 515. 29. Weitze KF, Fazano F, Rath D. Deep freezing of boar semen in macro- and minitubes. In Johnson LA, Larsson K (eds), Deep Freezing of Boar Semen. Uppsala, Sweden: University press 1985; 268 - 275. 30. Westendorf P, Richter L. Treu H. Zur Tiefgefrierung von Ebersperma. Labor- und Besamungsergebnisse mit dem Hiilsenberger Pailettenverfahren. Dtsch Tier&%1 Wochenschr 1975; 82: 261 - 267. 31. Yoshida M. In vitro fertilization of pig oocytes matured in vivo. Jap J Vet Sci 1987; 49: 711 - 718. 32. Yoshida M. Improved viability of two-cell stage pig embryos resulting from in vitro fertilization of oocytes matured in vivo. Jap J Anim Reprod 1989; 35: 34 - 37. 33. Yoshida M, lshitaki Y, Kawagishi H. Blastocyst formation by pig embryos resulting from in vitro fertilization of oocytes matured in vitro. J Reprod Fertil 1990; 88: 1-8.