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In vitro deveropment of porcine oocytes fertilized in vitro with spermatozoa preincubated in two different media
IN VITRO DEVEROPMENT OF PORCINE OOCYTES FERTILIZED IN VITRO WITH SPERMATOZOA PREINCUBATED IN TWO DIFFERENT MEDIA Y.H.Choi, S.Saitoa and N.Oguri Laboratory of Horse Production Obihiro University of Agricukure and Veterinary Medicine Received for pubIication: August 25, 1992 Accepted: December 22, 1993 ABSTRACT This study was conducted to clarify the effects of sperm concentration and media during preincubation on fertikzation and development of porcine oocytes effect of porcine oviduct epit.hehaI ceII fertihzed in vitro 0. The aggregates (POECA) on in vitro development of IVF embryos was @so e+. Oocytes matured in vitro for 48 to 50 h were inr~m$ra~~~~ ~idr$maj spermatozoa preincubated at 2 sperm concentrations (1-2x 10 ml) for 3 h in either DuIbec&s phosphate buffered saline (PBS) of &-ackett and OIiphant medium (BO). For capacitation, spermatozoa were treated with heparin (1OOug / ml) for 15 min at 38.5 “C under 5% CO, in air. CIeavage and development to the bIastocyst stage were evahrated on Day 3 and Day 8 after cukure with or without POECA. The effect of sperm concentration on preincubation did not affect the fertihzation rate, but preincubation in PBS medium did result in a higher fertikzation rate (PcO.05) tban did the BO medium. The proportion of embryos undergoing cleavage and devekqrment to the blastocyst stage was significantIy higher (pcO.05) in the POECA co-cukure group than in the group without POECA co-cuhure. The present results indicate that PBS medium can be utilized as a simple preincubation medium for porcine spermatozoa and that the presence of POECA during in vitro cuhure improved the development of IVF oocytes to the blastocyst stage. Key words: sperm
concentration, preincubation medium, POECA, pig, oocytes INTRTODUCTION
In obtaining high fertilization rates in vitro in the pig, preincubation concentration of epididymaI and ejaculated spermatozoa has been e_xamined (1% 15). EpididymaI spermatozoa were preincubated at a low concentratron (0.8~10 cells / ml), and the penetration rate was low (11%). However, when the sperm Acknowledgements This work was supported by a contribution from the Japan Racing Association. The authors gratefuky acknowledge Dr. K. Sato and Dr. A. Saito for their valuable advice as weII as the veterinary doctors at the Meat Inspection Office in Kumamoto, Japan for the supply of porcine ovaries used in this $udy. present address: Feed Research Center, Nisshin FIour MiIIing Co.,Ltd. 1242-5, Nishinasuno, Tocbigi, 328-27 Japan. Theriogenology 44:287-294, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 1001 OS
0093-691 X/95/$1 0.00 SSDI 0093-691X(95)00178-B
289
Theriogenology
to the laboratory at 36 to 35 “C. About 1 h after slaughter, epididymal spermatozoa were extruded from the distal portion of the cauda epididymis by cc syringe. About 1 ml of epididymal negative air pressure with a 5spermatozoa was diluted with PBS or BO solution (3) supplemented with 4 mg / ml BSA, and then centrifuged 3 times for 10 min at 509xg and resuspended in the PBS and BO, respectively. Subsequently, epididymal spermatozoa were transferred to a 35mm petri dish for preincubation for 3 h. The concentration of epididymal spermatozoa in 2 ml preincubation media was / ml. For capacitation, hepaxin was calculated to be either l-2 or 4-5~10’ diluted with physiological saline for a concentration of 5 mg / ml. Then, 2 ~1 of diluted heparin was added to the 98 ul of the preincubated sperm suspention to yield final heparin concentration of 160 ug / ml. The heparin-treated epididymal spermatozoa were incubated in a tube at 38.5 “C under 5% CO, in air for 15 mm. The oocytes cultured for 48 to 50 h and were transferred to 50 ~1 of fertilization medium, BO solution containing 4 mg / ml BSA. A portion of the preincubated epididymal sperm suspention was introduced into the medium so that the final concentration at insemination was 5x 10” / ml. After 18 h in the fertilization medium, 326 oocytes were fixed, stained and examined for fertilization. In Vitro Culture The remaining oocytes were co-cultured with porcine oviduct epithelial cell aggregates (POECA) which were prepared from the oviductual tube of slaughtered gilts having corpus hemorrhagicum (1 to 2 d after ovulation). Oviducts w;~ pFW;dlac;to 15 ml of PBS ;ntizly tomrmove. co_mecbve tissues. epithehal cell were opened aggregates were scraped with a sterile glass slide and collected. Epithelial cell aggregates were washed twice with TCM 199 and 10% NBCS and were transferredtoa35mm petridishforculturinginthesamemedium for24h at 38.5 “C under 5% CO, in air. In vitro culture was conducted in 196 ~1 of modified KRB supplemented with 16% NBCS with about 56 to 109 POECA for 8 d at 38.5 “C under 5% CO, in air. Cleavage rates and development to the b&&cyst stage were examined microscopically at Day 3 and Day 8 of culture, respectively. The number of cells in the blastocysts was counted by the method of Ushijima et al. (19). The overall diameter of the blastocysts was determined with a screw ocular micrometer at xl00 magnification. Statistical Analysis statistical analyses of fertilization in 7 to 9 of the proportions replicates and cleavage rates, including development to the blastocyst stage in 5 to 7 replicates, were cakulated by a parametric generalized linear model following logit transformation. The number of nuclei and the diameter per blastocyst were compared by Student’s t- test. RESUL.TS There was no difference in fertilization rate between the 2 sperm concentrations used in preincubation when compared within the same medium (Table 1). But the PBS medium resulted in a higher fertilization ratemsdWa higher total percentage of fertilization fJkO.05) than the BO
290 pobPe~Y
Theriogenology
rangedfrom
13.2 to 20.596, with
no significant difference between
the treated groups.
The proportion of oocytes cleaving and developing to blastocyst stage was significantly higber (PcO.05) in the groups witb POECA co-culture tbau in those without POECA co-culture. There was no effect of different sperm Table 1.
In vitro fertilization of pig oocytes inseminated with epididymal spermatozoa preincubated at 2 different concentrations for 3 hours
Preincubation condition sperm concentration ( X10” /ml>
medium No.of oocytes examined
l-2
PBS Bo PBS I!0
4-5
Percentage of oocytes (mean + SEM> Fertilizeda
Polyspermic
Total
41.4k2.8 ;” 28.8k6.1 b 50.5k5.8 c d 29.Ok7.4 ’
20.5k5.6 18.9k7.6 24.1k6.0 13.2k9.2
61.9k6.9 ;; 47.6k9.0 b 74.5k4.3 d 42.2+8. 7
include the oocytes with swollen sperm head or male pronucleus. i-d Meansin the same column without a commonsuperscript differ significantly (P
In vitro development of oocytes co-cultured with or without POBCA after insemination with spermatozoa preincubated at 2 diferrent concentrations in 2 different media
Preincubation condition sperm concentration ( X10” /ml>
medium
l-2
PBS PBS Bo Bo
+ + -
PBS PBS Z
4-5
POECA No. of oocytes examined
Percentage of oocytes (mean + SEM) cleaved
developed to blastocyst
114
;;- ;=1;. $. c
1;.;;f-;
1;: 83
46: l* 28.5f
10: 7+2: 7 a 1.2k1.2 c
+
106 104
43.2+ 4.2a * 41.0+ 3: 5; D
+
127 143
;;- ;;
6: ga 6.4’
;* $I, c
1n.7fl. 7 -0: s+o: )j 11.0+2.4 0.5kO.5
:,c
a*b c a c
a-c Beans in the samecolumn without a commonsuperscript differ significantly (P
291
Theriogenology Table 3. Mean(-tSEH)nuclei numberand diameter of blastocysts developed in POECA co-culture under different preincubation conditions Preincubation condition sperm concentration ( x10” /ml) 1-2 4-5
medium
PBS Bo PBS Bo
No. of blstocysts examined
6 9 ;
Figure 1. Expandedblastocysts derived from IW- IVF porcine oocytes co-cultured with porcine ovidt1ct epithelial cell aggregates at 8 days of culture ( x200).
Blastocysts No. of nuclei 26.0 25.7 20.9 21.3
+6.3 *5. 9 t-4.0 +3. 5
Diameter ( ,um> 191.0 197.0 190.4 198.4
*lo. 9 -t 5.1 +- 6.9 +- 8.4
“igure 2. The nuclei of an expanded blastocyst (67 nuclei, x 100).
Theriogenology
292
concentrations or preincubation media on cleavage rate and deveiopmentai capacity (Table 2). The number of nuclei and the diameters of biastocysts are shown in Table 3, and did not differ under varying preiucubation conditions. Some of the blastocysts developed to the expanded blastocyst stage (Figure 1) and expanded blastocysthad 67 nuclei(Figure 2). DISCUSSION A high sperm concentration dming preincubation in a defined medium is important for penetrating oocytes (10, 15). Nagai et al. (15) reported that denuded ooqes were /su~y penetrated by @didymal qqmato~~;,wh$t had been premcubated m vitro at a high concen$rauon (4-16x10 ceils / ml did not affect the In our study, a sperm concentration of 1-5x10 fertilizationrates, but PBS medium as the preincubationmedium appeared to be superior to BO medium for attaikg a high fertilization rate. It was suggested that a high concentration of spermatozoa during preincubation may inuease the capacitation rate due to the increased amount of B-amino acids released by the concentrated boar spermatozoa (15). This study is the first to use PBS and BO as preincubationmedia in the fertilizationand development of porcine oocytes. Phosphate buffered saline is broadly used as the basic medium for washing spermatozoaand oocytes. Oocytes deveioped to biastocysts in vitro which bad heen co-cuhured with POECA. Previous studies on porcine morula stage embryos, fertilked aud matured in vitro, were obtained following culture in TCM199 supplemented with 26% estrous cow serum for 7 d (29). The results in our study support the beneficial role of the oviductai epitheiiai ceils in the development of porcine oocytes fertihzed and matured in vitro to the biastocyst stage. It has been postulated that rabbit oviduct epitheliai cells might secrete growth fktors or other embryotrophic substances which could enhance rabbit embryo development in vitro. Further, it has been reported that co-cuhure could provide a detoxification mechanism for damaging conditions (4). Gandolfi et al. (9) reported that sheep oviduct celis secrete proteins which bind to sheep embryos and can be transported.Thus, in some species such as the mbit, sheep and cow (8) certain proteins may be embryotrophic. The theory of enhancement in the development of early porcine embryos to blastocysts and protection of theembryosfromharmfuleIlvironm ents during in vitro culture by POECA might be supported by our experiment. The number of nuclei in the blastocysts was examined 8 d after the start of culture. The average number of nuclei per blastocyst was less than that reported by Davis and Day (7). This difference could be due to in vitro maturation, fertilization and culture. Some researchers have also reported that the ntm&er of nuclei in biastocysts derived in vitro was lower than for blastocysts derived in vivo (1,6). The outer diameter of biastocysts in our study was similar to the results of Wzmann et al. (16). However, hatching blastocysts were not observed in our study until 10 d of cuiture foilowing insenkation. It is possible that some factors essential for the initiation of the hatchiq process of biastocysts, may have been @aired during the long in vitro culture period. Other studies have reported hatching and hatched biastocysts developing in vitro from 2cell to early blastocyst embryos colkcted from oviducts aud cuitured in modified KRB supplemented with 10 %
Theriogenology lamb serum
293
and POEC (16,211.
In conclusion, our present results indicated that a sperm cow&ration of l- 5x lo8 cells/ ml during prekubation did not affect the fertihaation rate and that PBS can be used as the preincubation medium. Moreover, we found that oocytes matured and fert%aed in vitro under our experimental conditions were able to develop to the blastocyst stage after culture with POECA, which improved the development of IVM- IVF embryos to expanded blastocysts. REFERENCES 1. Arcbibong AE, Petters RM, Johnson BH. Development of porcine embryos from cell &a&s to blastocysts in culture medium supplemented %h po?$et%&tal fluid. Biol Reprod 198941.1076- 1083 2. Ball BA, Thomas PGA, Brim&o SP, Miller’ PG, EJlingk JE. Development of 1to 2cell equine embryos cocultured with oviductal epithelial cells. Theriogenology 1992;37:189 abstr. 3. Brachett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975;12:260-274. 4. Carrey EW,_Tobback C, Elliqton JE, Foote RH. Co-culture of rabbit 2- cell em&qq~215abbit eprthehal cells and other somatrc cells. Mol Rep Dev .
.
Cbeng *kTK, Moor RM, Polge C. In vitro fertilization of pig and sheep oocytes matured in vivo and in vitro. Tberiogenology 1986;25:146 abstr. 6. Davis DL. Culture and storage of pig embryos. J Reprod Fertil 1985;33 (Suppl):115- 124. 7. Davis DL, Day BN. Cleavage and bJastocyst formation by pig eggs in vitro. J Anim Sci 1978;46:104% 1053. 8. Eyestone WH, *First NL. Co- culture of early cattle. embryos to the blastocyst ~~15w7t$O ovrductal trssue or in condruoned medann. J Reprod Fertil 1989; 5.
9. Gandolfr F, Brevini TAL, Richardson L Brown CR, Moor RM. Characterization of proteins secreted by sheep o&h& epithelial cells and their function in embryonic development. Development 1989;106:303- 312. 10. Hamano S, Toyoda Y. In vitro fertihzation of pig eggs with ejaculated spermatozoa preincubated at higher sperm concentration. Jpn J Anim Reprod 1986;32:177-183. 11. fisher RL, Petters RM, Johnson BH. Effect of oviductual condition on the development of onecell porcine embryos in mouse or rat oviducts maintained in organ culture. J Exp Zool1989;249:235-239. 12. Krisher RL, Petters RM, Johnson BH, Bavister BD, Archilong AE. Development of porcine embryos from onecell stage to blastocyst in mouse oviducts maintained in organ culture. J Exp Zool1989;249:235-239. 13. Mattioli M, Bacci ML, Galeati G, Seren E. Development competence of pig oocytes matured and fertihzed in vitro. Theriogenology 1989;31:1201-1207. 14. Mermillod P, Boccart C, Dessy F. Use of rabbit or bovme oviduct epitheiial cell monolayer-s for supporting early development of bovine embryos. Theriogenology 1991;35:241 abstr of sperm. concentration during 15. Nagai T, Niwa aK,dekz kI.ffect preincubation in on fertihzatron III vrtro of pig follicular oocytes. J Reprod Fertil1984,70:271-275.
294
Theriogenology
16. Niemann H, Ihera MJ, Dziuk PJ. Developmentalcapacity, size and number of nucleiin pig embryos cuhured in vitro. Anim Reprod Sci 1983;5:311-321. 17. Pavlok A. Penetration of hamster and pig zona- free eggs by boar ejaculated spermatozoapreincubatedin vitro. IntJ FertiI1981;26:101-106. 18. PratherIS, Si MM, First NL. Culture of porcine embryos from the one- and two cell stage to ‘the bktstocyststage stage in sheep oviducts. Theriogenology 1991;35:1147-1151. 19. Ushijima M, Okuda T, Nakagawa A, Moji k, Ishida K, Murata H, Iguchi A, Etoh T. Relationshipbetween the cell number and qua&y of Day- 8 bovine blastocysts (in Japanese) Proc 3rd East Jpn Sot Anim Emb Trans 1988; Number9:37-38. 20. Vajta G, Machaty Z, Barandi Zs, !%ons A, Solti L. Transfer of in vitro ferti&ed and cuhivated swine embryos. Theriogenology 1991;35:289 abstr 21. White KL, Hehnke K, Richards LF, Southern LL, Thompson DL Jr, Wood TC. Early embryonic development in vitro by coculture with oviductaIepithehai &Is in pigs. BiolReprod 1989;41:425-430. 22. Yoshida M, Ishizaki Y, KawagishiH. Blastocyst formation by pig embryos resuhing from in vitro fertilization of oocytes matured in vitro. J Reprod FertiI1990;88:1-8.
concentration, preincubation medium, POECA, pig, oocytes INTRTODUCTION
In obtaining high fertilization rates in vitro in the pig, preincubation concentration of epididymaI and ejaculated spermatozoa has been e_xamined (1% 15). EpididymaI spermatozoa were preincubated at a low concentratron (0.8~10 cells / ml), and the penetration rate was low (11%). However, when the sperm Acknowledgements This work was supported by a contribution from the Japan Racing Association. The authors gratefuky acknowledge Dr. K. Sato and Dr. A. Saito for their valuable advice as weII as the veterinary doctors at the Meat Inspection Office in Kumamoto, Japan for the supply of porcine ovaries used in this $udy. present address: Feed Research Center, Nisshin FIour MiIIing Co.,Ltd. 1242-5, Nishinasuno, Tocbigi, 328-27 Japan. Theriogenology 44:287-294, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 1001 OS
0093-691 X/95/$1 0.00 SSDI 0093-691X(95)00178-B
289
Theriogenology
to the laboratory at 36 to 35 “C. About 1 h after slaughter, epididymal spermatozoa were extruded from the distal portion of the cauda epididymis by cc syringe. About 1 ml of epididymal negative air pressure with a 5spermatozoa was diluted with PBS or BO solution (3) supplemented with 4 mg / ml BSA, and then centrifuged 3 times for 10 min at 509xg and resuspended in the PBS and BO, respectively. Subsequently, epididymal spermatozoa were transferred to a 35mm petri dish for preincubation for 3 h. The concentration of epididymal spermatozoa in 2 ml preincubation media was / ml. For capacitation, hepaxin was calculated to be either l-2 or 4-5~10’ diluted with physiological saline for a concentration of 5 mg / ml. Then, 2 ~1 of diluted heparin was added to the 98 ul of the preincubated sperm suspention to yield final heparin concentration of 160 ug / ml. The heparin-treated epididymal spermatozoa were incubated in a tube at 38.5 “C under 5% CO, in air for 15 mm. The oocytes cultured for 48 to 50 h and were transferred to 50 ~1 of fertilization medium, BO solution containing 4 mg / ml BSA. A portion of the preincubated epididymal sperm suspention was introduced into the medium so that the final concentration at insemination was 5x 10” / ml. After 18 h in the fertilization medium, 326 oocytes were fixed, stained and examined for fertilization. In Vitro Culture The remaining oocytes were co-cultured with porcine oviduct epithelial cell aggregates (POECA) which were prepared from the oviductual tube of slaughtered gilts having corpus hemorrhagicum (1 to 2 d after ovulation). Oviducts w;~ pFW;dlac;to 15 ml of PBS ;ntizly tomrmove. co_mecbve tissues. epithehal cell were opened aggregates were scraped with a sterile glass slide and collected. Epithelial cell aggregates were washed twice with TCM 199 and 10% NBCS and were transferredtoa35mm petridishforculturinginthesamemedium for24h at 38.5 “C under 5% CO, in air. In vitro culture was conducted in 196 ~1 of modified KRB supplemented with 16% NBCS with about 56 to 109 POECA for 8 d at 38.5 “C under 5% CO, in air. Cleavage rates and development to the b&&cyst stage were examined microscopically at Day 3 and Day 8 of culture, respectively. The number of cells in the blastocysts was counted by the method of Ushijima et al. (19). The overall diameter of the blastocysts was determined with a screw ocular micrometer at xl00 magnification. Statistical Analysis statistical analyses of fertilization in 7 to 9 of the proportions replicates and cleavage rates, including development to the blastocyst stage in 5 to 7 replicates, were cakulated by a parametric generalized linear model following logit transformation. The number of nuclei and the diameter per blastocyst were compared by Student’s t- test. RESUL.TS There was no difference in fertilization rate between the 2 sperm concentrations used in preincubation when compared within the same medium (Table 1). But the PBS medium resulted in a higher fertilization ratemsdWa higher total percentage of fertilization fJkO.05) than the BO
290 pobPe~Y
Theriogenology
rangedfrom
13.2 to 20.596, with
no significant difference between
the treated groups.
The proportion of oocytes cleaving and developing to blastocyst stage was significantly higber (PcO.05) in the groups witb POECA co-culture tbau in those without POECA co-culture. There was no effect of different sperm Table 1.
In vitro fertilization of pig oocytes inseminated with epididymal spermatozoa preincubated at 2 different concentrations for 3 hours
Preincubation condition sperm concentration ( X10” /ml>
medium No.of oocytes examined
l-2
PBS Bo PBS I!0
4-5
Percentage of oocytes (mean + SEM> Fertilizeda
Polyspermic
Total
41.4k2.8 ;” 28.8k6.1 b 50.5k5.8 c d 29.Ok7.4 ’
20.5k5.6 18.9k7.6 24.1k6.0 13.2k9.2
61.9k6.9 ;; 47.6k9.0 b 74.5k4.3 d 42.2+8. 7
include the oocytes with swollen sperm head or male pronucleus. i-d Meansin the same column without a commonsuperscript differ significantly (P
In vitro development of oocytes co-cultured with or without POBCA after insemination with spermatozoa preincubated at 2 diferrent concentrations in 2 different media
Preincubation condition sperm concentration ( X10” /ml>
medium
l-2
PBS PBS Bo Bo
+ + -
PBS PBS Z
4-5
POECA No. of oocytes examined
Percentage of oocytes (mean + SEM) cleaved
developed to blastocyst
114
;;- ;=1;. $. c
1;.;;f-;
1;: 83
46: l* 28.5f
10: 7+2: 7 a 1.2k1.2 c
+
106 104
43.2+ 4.2a * 41.0+ 3: 5; D
+
127 143
;;- ;;
6: ga 6.4’
;* $I, c
1n.7fl. 7 -0: s+o: )j 11.0+2.4 0.5kO.5
:,c
a*b c a c
a-c Beans in the samecolumn without a commonsuperscript differ significantly (P
291
Theriogenology Table 3. Mean(-tSEH)nuclei numberand diameter of blastocysts developed in POECA co-culture under different preincubation conditions Preincubation condition sperm concentration ( x10” /ml) 1-2 4-5
medium
PBS Bo PBS Bo
No. of blstocysts examined
6 9 ;
Figure 1. Expandedblastocysts derived from IW- IVF porcine oocytes co-cultured with porcine ovidt1ct epithelial cell aggregates at 8 days of culture ( x200).
Blastocysts No. of nuclei 26.0 25.7 20.9 21.3
+6.3 *5. 9 t-4.0 +3. 5
Diameter ( ,um> 191.0 197.0 190.4 198.4
*lo. 9 -t 5.1 +- 6.9 +- 8.4
“igure 2. The nuclei of an expanded blastocyst (67 nuclei, x 100).
Theriogenology
292
concentrations or preincubation media on cleavage rate and deveiopmentai capacity (Table 2). The number of nuclei and the diameters of biastocysts are shown in Table 3, and did not differ under varying preiucubation conditions. Some of the blastocysts developed to the expanded blastocyst stage (Figure 1) and expanded blastocysthad 67 nuclei(Figure 2). DISCUSSION A high sperm concentration dming preincubation in a defined medium is important for penetrating oocytes (10, 15). Nagai et al. (15) reported that denuded ooqes were /su~y penetrated by @didymal qqmato~~;,wh$t had been premcubated m vitro at a high concen$rauon (4-16x10 ceils / ml did not affect the In our study, a sperm concentration of 1-5x10 fertilizationrates, but PBS medium as the preincubationmedium appeared to be superior to BO medium for attaikg a high fertilization rate. It was suggested that a high concentration of spermatozoa during preincubation may inuease the capacitation rate due to the increased amount of B-amino acids released by the concentrated boar spermatozoa (15). This study is the first to use PBS and BO as preincubationmedia in the fertilizationand development of porcine oocytes. Phosphate buffered saline is broadly used as the basic medium for washing spermatozoaand oocytes. Oocytes deveioped to biastocysts in vitro which bad heen co-cuhured with POECA. Previous studies on porcine morula stage embryos, fertilked aud matured in vitro, were obtained following culture in TCM199 supplemented with 26% estrous cow serum for 7 d (29). The results in our study support the beneficial role of the oviductai epitheiiai ceils in the development of porcine oocytes fertihzed and matured in vitro to the biastocyst stage. It has been postulated that rabbit oviduct epitheliai cells might secrete growth fktors or other embryotrophic substances which could enhance rabbit embryo development in vitro. Further, it has been reported that co-cuhure could provide a detoxification mechanism for damaging conditions (4). Gandolfi et al. (9) reported that sheep oviduct celis secrete proteins which bind to sheep embryos and can be transported.Thus, in some species such as the mbit, sheep and cow (8) certain proteins may be embryotrophic. The theory of enhancement in the development of early porcine embryos to blastocysts and protection of theembryosfromharmfuleIlvironm ents during in vitro culture by POECA might be supported by our experiment. The number of nuclei in the blastocysts was examined 8 d after the start of culture. The average number of nuclei per blastocyst was less than that reported by Davis and Day (7). This difference could be due to in vitro maturation, fertilization and culture. Some researchers have also reported that the ntm&er of nuclei in biastocysts derived in vitro was lower than for blastocysts derived in vivo (1,6). The outer diameter of biastocysts in our study was similar to the results of Wzmann et al. (16). However, hatching blastocysts were not observed in our study until 10 d of cuiture foilowing insenkation. It is possible that some factors essential for the initiation of the hatchiq process of biastocysts, may have been @aired during the long in vitro culture period. Other studies have reported hatching and hatched biastocysts developing in vitro from 2cell to early blastocyst embryos colkcted from oviducts aud cuitured in modified KRB supplemented with 10 %
Theriogenology lamb serum
293
and POEC (16,211.
In conclusion, our present results indicated that a sperm cow&ration of l- 5x lo8 cells/ ml during prekubation did not affect the fertihaation rate and that PBS can be used as the preincubation medium. Moreover, we found that oocytes matured and fert%aed in vitro under our experimental conditions were able to develop to the blastocyst stage after culture with POECA, which improved the development of IVM- IVF embryos to expanded blastocysts. REFERENCES 1. Arcbibong AE, Petters RM, Johnson BH. Development of porcine embryos from cell &a&s to blastocysts in culture medium supplemented %h po?$et%&tal fluid. Biol Reprod 198941.1076- 1083 2. Ball BA, Thomas PGA, Brim&o SP, Miller’ PG, EJlingk JE. Development of 1to 2cell equine embryos cocultured with oviductal epithelial cells. Theriogenology 1992;37:189 abstr. 3. Brachett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975;12:260-274. 4. Carrey EW,_Tobback C, Elliqton JE, Foote RH. Co-culture of rabbit 2- cell em&qq~215abbit eprthehal cells and other somatrc cells. Mol Rep Dev .
.
Cbeng *kTK, Moor RM, Polge C. In vitro fertilization of pig and sheep oocytes matured in vivo and in vitro. Tberiogenology 1986;25:146 abstr. 6. Davis DL. Culture and storage of pig embryos. J Reprod Fertil 1985;33 (Suppl):115- 124. 7. Davis DL, Day BN. Cleavage and bJastocyst formation by pig eggs in vitro. J Anim Sci 1978;46:104% 1053. 8. Eyestone WH, *First NL. Co- culture of early cattle. embryos to the blastocyst ~~15w7t$O ovrductal trssue or in condruoned medann. J Reprod Fertil 1989; 5.
9. Gandolfr F, Brevini TAL, Richardson L Brown CR, Moor RM. Characterization of proteins secreted by sheep o&h& epithelial cells and their function in embryonic development. Development 1989;106:303- 312. 10. Hamano S, Toyoda Y. In vitro fertihzation of pig eggs with ejaculated spermatozoa preincubated at higher sperm concentration. Jpn J Anim Reprod 1986;32:177-183. 11. fisher RL, Petters RM, Johnson BH. Effect of oviductual condition on the development of onecell porcine embryos in mouse or rat oviducts maintained in organ culture. J Exp Zool1989;249:235-239. 12. Krisher RL, Petters RM, Johnson BH, Bavister BD, Archilong AE. Development of porcine embryos from onecell stage to blastocyst in mouse oviducts maintained in organ culture. J Exp Zool1989;249:235-239. 13. Mattioli M, Bacci ML, Galeati G, Seren E. Development competence of pig oocytes matured and fertihzed in vitro. Theriogenology 1989;31:1201-1207. 14. Mermillod P, Boccart C, Dessy F. Use of rabbit or bovme oviduct epitheiial cell monolayer-s for supporting early development of bovine embryos. Theriogenology 1991;35:241 abstr of sperm. concentration during 15. Nagai T, Niwa aK,dekz kI.ffect preincubation in on fertihzatron III vrtro of pig follicular oocytes. J Reprod Fertil1984,70:271-275.
294
Theriogenology
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