- Email: [email protected]
Ovulation, fertilization and pronucleus development in superovulated gilts
Theriogenology 41:447-452,
1994
OVULATION, FERTILIZATION AND PRONUCLEUS DEVELOPMENT IN SUPEROVULATED GILTS J. Laurincik, P. Hyttell, D. Rath2 and J. Pivko Research Institute of Animal Production, Department of Reproduction and Embryology, Nitra, Slovak Republic 1 The Royal Veterinary and Agricultural University, Department of Anatomy and Physiology, Frederiksberg C, Denmark 2 Institut l?ir Tierzucht und Tierverhalten, Mariensee, (FAL), Neustadt, Germany Received for publication: Accepted:
January December
27, 1993 7,
1993
ABSTRACT Estrus was synchronized in 45 gilts by ingestion of Zinc-Methallibur in the feed for 15 d On Day 16 each gilts was treated with PMSG ( 1200 IU i.m.) followed in 72 h by hCG ( 500 IU i.m.). Gilts were inseminated 24 and 36 h after the onset of estrus followed by slaughter of groups (n = 4 or 5) at 40 h, 44 h, 48 h, 52 h, 56 h, 60 h and 64 h after hCG injection. Ovaries were evaluated macroscopically and oocytesfembryos were recovered by flushing the oviducts. The ovulation rate increased from 38% to 87% from 40 to 45 h and remained constant thereafter. At 40 h, 36% of oocytes were penetrated by a single spermatozoon. The rate of fertilization increased from 36% (40 h) to 59% (44 h), to 65% (48 h), to 73% (52 h), to 76% (56 h), 80% (60 h) and to 64% (64 h). At 40 h all fertilized ova contained a decondensed sperm head. Afler another 4 to 8 h early pronuclei were common, and 52 h after hCG treatment opposed pronuclei were predominant. The first cleavages were recorded 64 h after hCG injection. Key words: Ovulation, fertilization, pronucleus development, gilts INTRODUCTION At present the microinjection technique is the most favourable method to introduce foreign DNA constructs into the pronuclei of zygotes or nuclei of 2-cell embryos in small laboratory and farm animals (2, 10, 17, 25). This intervention requires large numbers of zygotes synchronized at the pronucleus stage of development. This is not easy in the pig because of the difficulty in determining the onset and duration of the ovulation period, factors crucial for the recovery of zygotes at well-defined developmental stages. It has been reported that proestrous gilts ovulate approximately 40 h afler administration of hCG (9) and that ovulation is completed over 1 to 3 h period (1,3,22). Such hCG treatment, in combination with exogenous gonadotropins Acknowledgement : The work was supported by the Danish Agricultural and Veterinary Research Council.
Copyright
Q 1994 Butterworth-Heinemann
448
Theriogenology
is commonly used in superovulation regimes for in vivo production of zygotes. Using this strategy, normal regulation of follicle recruitment and selection for ovulation may be bypassed. However, one must consider that, at least in cows, folliculogenesis and ovulation induced by exogenous gonadotropins may lead to deviant follicle and oocyte maturation (15). The objective of the present study was to monitor the duration of ovulation, the timing of fertilization and pronucleus development in synchronized gilts superovulated with PMSGIhCG treatments. MATERIALS AND METHODS Es&us was synchronized in 45 cycling gilts at the age of about 6 months by ingestion of Zinc-Methallibur ( Evertas P, WBVL, Pohori-Chotoun, &echo-Slovakia ) in the feed ( 0.125 g in 2 kg of the complete feed mixture ) for 15 days. On Day 16 each gilt was treated with PMSG (Amex, Leo, Denmark; 1200 IU i.m. ) followed in 72 h by hCG ( Praedyn, Leciva, Prague, Czecho-Slovakia; 500 IU i.m. ) Semen was collected by the hand-glove method, evaluated for motility and diluted to approximately three billion spermcells per insemination dose. Semen was used on the day of collection and the gilts were inseminated artificially 24 h and 36 h after onset of estrus. Groups of gilts were slaughtered 40 h (n = 5), 44 h (n = 5), 48 h (n = 4), 52 h (n = 4), 56 h (n = 4), 60 h (n = 4) or 64 h (n = 4) after hCG injection. The ovaries were examined and the numbers of non-ovulated (> 7 mm) (24) and ovulated follicles were recorded. The oviducts were flushed twice with 20 ml TCM 199e (Sevac, Prague, Czecho-Slovakia), supplemented with 2.92 mM Ca-lactate, 2 mM Na-pyruvate, 33.9 mM Na-bicarbonate, 4.34 mM Hepes (Serva, Heidelberg, Germany) (21), 10 &ttl Gentamicin and 20% heat-inactivated estrous cow serum (ECS). Oocytes and embryos present in the flushing medium were evaluated under a stereomicroscope (x 125) for morphology of the cumulus investment, the presence of polar bodies, and cleavage. Subsequently, they were f&d in acetic alcohol (I:3 v/v) and stained with aceto-orcein (1%) 24 h later (28). All stained preparations were evaluated under a phase-contrast microscope (x 400) for fertilization, pronucleus development, and position of paternal and maternal pronuclei (13). Oocytes or zygotes were classified to be degenerated when no chromosomal structures or pronuclei were recognized after staining. Statistical comparisons were performed by Student’s t-test, while proportions were analyzed by the Chi-square test. In the case of a significant result a mutual comparison of groups was done by means of the U-test. RESULTS Ovulation rate and recovery rate of ovulated ova are given in Table 1. Forty h after hCG administration only 38% of the follicles had ovulated while 4 h later 65% had done so. At 48 h 87% of the follicles had ovulated, and no further increase in the ovulation rate was recorded up to 64 h. The rates of fertilization and degeneration are given in Table 2. At 40 h after hCG application 10 out of 22 recovered ova displayed expanded cumulus investments. At all later time intervals all ova were denuded. Among the 10 ova, 8 (36% from all recovered) had undergone normal
449
Theriogenology
fertilization. However, a gradual increase of the fertilization rate up to 80% at 60 h was noted. Data of the further developmental steps are given in Table 2; they differ significantly (pcO.01) from the 40 h values. No correlation between time interval and rate of polyspermy was observed. Table 1. Ovulation rate and recovery rate of early embryonic stages at different times after hCG injection. Recovery Time Gilts Follicles rate ovulated after hCG non-ovulated n n % h n n % % Sa 40 3ga 22 5 66 62a 40 78’ 59 65’ 46 5 31 35b 44 82’ 60 87’ 49 4 9 13c 48 83’ 72 91C 60 4 7 9c 52 90’ 76 86c 69 4 12 14c 56 89’ 68 8gc 61 4 9 12c 60 86c 61 91C 53 4 6 9c 64 Values with different superscripts in the same column are significantly different. a,b PCO.05 ac P
Rates of fertilization, polyspermy and degeneration of ova collected at different times after hCG injection.
Tie Ova Unfertiliied after recovered h n n O/O 12 55a 40 22 14 30b 44 46 8c 48 49 4 8 14c 52 60 12 18’ 56 69 8 13c 60 61 3 6’ 64 53 Values with diierent superscripts in a,b ~~0.05 a,c ~co.01.
Normal
Fertilized Polyspermv
Degenerate
36a
n 2
9
n 0
;;
2;:
;
1; b
;
44 53 49 32 the same
4 73c 4 7 2 76’ 2 3 a 1 80’ 3 5 a 64’ 8 16 b 10 column are significantly different.
n 8
O/O
%
%
Oa 4a 15b 6a 3a 2a 14b
The chronological events of pronucleus formation and development are given in Table 3. At 40 h after hCG injection the maternal chromatin in all fertilized ova was in Ana- or Telophase II. The paternal chromatin in these ova was identified as sperm heads, which were slightly swollen. At 44 h sperm head enlargement was observed in 70% of the fertilized ova, while the remaining 30% had 2 spherical, synchronously developing pronuclei. The size of each was about l/3 of the fklly developed pronuclei. The pronuclei were located at each pole of the zygote, and the sperm tail was associated with the paternal pronucleus. At the 48 h interval 53% of the ova had developing pronuclei as described above, while 28% displayed opposed pronuclei in the central
Theriogenology
450
region of the ooplasm. The maternal pronucleus was located towards the second polar body, being slightly smaller than the other. At this stage the paternal pronucleus had lost its association with the sperm tail. At 52 h 52% of the ova displayed apposed pronuclei, and at 56 h and 60 h 7576% had reached this stage of development. At 64 h 48% of the embryos developed to the 2-cell stage, while 37% displayed apposed pronuclei, which on some occasions had achieved identical size. Throughout the investigated period a certain proportion of the fertilized ova showed early stages of fertilization, i.e. sperm head decondensation. Table 3.
Pronucleus development in fertilized ova recovered at different times after hCG injection.
Time Ova Sperm head after recovered in ooplasm h
n
40 44 48 52 56 60 64
8 27 32 44 53 49 53
Develooment of nronuclei Apposed Early pronuclei pronuclei
n
%
n
8 19 6 2 1 5 3
100 70 19 5 2 10 9
0 8 17 19 12 7 2
% 0 30 53 43 23 14 6
n 0 0 9 23 40 37 12
% 0 0 2s 52 75 76 37
Cleavage
n
%
0 0 0 0 0 0 15
0 0 0 0 0 0 48
DISCUSSION The pattern and duration of ovulation in pigs can only be estimated retrospectively. Earlier studies have shown that gilts ovulate 39 to 40 h after the onset of spontaneous oestrus (12) and 40 (9) to 42 h (23) after hCG administration. Hunter (13) observed that 60 to 100% of the gilts had completed ovulation 44 to 46 h after hCG application. Similar data were observed by Pope et al. (24), where 68 to 95% of the gilts had completed ovulation about 4 h after the onset of estrus. Gur tidings are in accordance with these data. The maximum rate of ovulation was reached at 44 to 48 h after hCG, but in contrast to the observation of other authors (1,3,22) the duration of the ovulation period (4 to 8 h) was slightly prolonged based on the ovulation rate between 40 h and 52 h after hCG application. Although a significant increase in the ovulation rate happened between 40 h and 44 h it seems that the superovulatory treatment hampers the course of normal ovulation. Fertilization is believed to occur almost immediately after entry of the mature oocytes into the oviduct, containing capacitated spermatozoa (7,14,16,26). Our data support this hypothesis because both ovulation and fertilization rates increased almost simultaneously. As published earlier in pigs (1 l), cattle (6,16) and sheep (7) the oocyte sheds the accompanying cumulus cells during or soon after ovulation, allowing the spermatozoa to contact the surface of the zona pelhmida directly. Accordingly, in the present study less than one half of
451
Theriogenology
ova recovered 40 h after hCG treatment were surrounded by cumulus cell layers. However, in a few cumulus invested oocytes these cumulus cells did not serve as a barrier against fertilization. Under in vivo conditions, fertilization of pig oocytes is reported to be monospermic in most cases (95%) (13,14). The rate of polyspermy in our experiment ranged from 3 to 16%. The entry of more than one spermatozoon may be associated with an abnormal hormonal environment at the time before fertilization (8,13) or may be caused by a delayed activation of the cortical granule release. Polyspermy in experiments for in vitro fertilization of in vivo matured oocytes was caused by a delayed migration of cortical granules (5). In those experiments superovulatory treatment was used to synchronize donor animals as it was in our experiments. The hormonal induction of ovulation may be the reason for asynchronous maturation and development of the cell compartments. Upon gamete fusion the ovum is activated and a series of nuclear changes in both the maternal chromosomes and the fertilizing spermatozoon take place. The transformation of the sperm head from a slightly swollen structure into an early pronucleus with intact membrane covering is reported to require 1S-2 h, and appearance of the pronuclei can be observed about 6 h after mating (13). The pronucleus development found in the present experiment resembled this pattern although the time sequence was apparently longer. More than 12 to 16 h were required before the majority of the sperm heads had developed into male pronuclei. It is hypophesized that this delay also was induced by the hormonal pretreatment. In comparison to spontaneous ovulation maternal and paternal pronuclei developed well synchronized. These data correspond with earlier observations in cattle (16), hamsters (27) and man (18). No enlarged sperm heads were observed within each interval after insemination. Although a delayed penetration can not be excluded, the missing enlargement is interpretated as an insufficient mechanism of cytoplasmic substances such as male pronucleus growth factor (MPGF), which is responsible for male pronucleus formation (4,14). It is concluded that the ovulation wave in superovulated gilts has a duration of about 8 h. Both ovulation and fertilization rates increased continuously and reached the maximum 44 h after hCG. Synchronous pronucleus development characterized by decondensed sperm heads, early pronuclei, and opposed pronuclei were predominantely present at 44 h, 48 h and 52 h, respectively. REFERENCES Betteridge KJ, Raeside JI. Observation of the ovary by peritoneal cannulation in pigs. Res Vet Sci 1962; 3:390398 Brem G, Brenig B, Goodman HM, Selden RC, Graf F, Kruff B, Springman K, Hondele J, Meyer J, Winnaher EL, Kr&rsslich H. Production of transgenic mice, rabbits and pigs by microinjection into pronuclei. Zuchthyg 1985; 20:251-256. Burger JF. Sex physiology of pigs. Onderstepoort J vet Res 1952; 25 (Suppl 1): 120-131 (1952). Calvin HI, Grosshans K, Blake EJ. Estimation and manipulation of glutathione levels in prepubertal mouse ovaries and ova: Relevance to sperm nucleus transformation in the fertilized egg. Gamete Res 1986; 14:265275.
452
Theriogenology
Cran DG, Cheng WTK. The cortical reaction in pig oocytes during in vivo and in vitro fertilization. Gamete Res 1986; &241-251. 6. Crozet N. Ultrastructural aspects of in vivo fertilization in the cow. Gamete Res 1984; 10:241-251. 7. Crozet N, Dumont M. The site of the acrosome reaction during in vivo penetration of the sheep oocyte. Gamete Res 1984; 10:97-105. 8. Day BN, Polge C. Effects of progesterone on fertilization and egg transport in the pig. J Reprod Fertil 1968; 17:227-230. 9. Dziuk PJ, Baker RD. Induction and control of ovulation in swine. 1962; Anim Reprod Sci 1:697-699 10. Hammer RE, Pursel VG, Rexroad CE, Wall RI, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 1985; 3 15:680-683. 11. Hunter RI-IF.Fertilization in the pig: Sequences of nuclear and cytoplasmic events. J Reprod Fertil 1972; 29: 395-406. 12. Hunter RI-IF.Fertilization of pig eggs in vivo and in vitro. J Reprod Fertil 1990; 40 (SuppI): l-226. 13. Hunter RHF. Ovulation in the pig: Timing of the response to injection of human chorionic gonadotropin. Res Vet Sci 1972; 13:356-361. 14. Hunter RI-IF.Polyspermic fertiliition in pigs during the luteal phase of the estrus cycle. J Exp 2001 1967; 165:451-466. 15. Hyttel P, Callesen H, Greve T, Schmidt M. Oocyte maturation and sperm transport in superovulated cattle. Theriogenology 1991; 35:91-108. 16. Hyttel P, Greve T, Callesen H. Uhrastructure of in-vivo fertilization in superovulated cattle. J Reprod Fertil 1988; 82:1-13. 17. Kremer D, Minhas B, Capehart J. Gene transfer into pronuclei of cattle and sheep zygotes. In: Bandury Report Costantini F, Jaenisch R (eds.).New York, Cold Spring Harbor Press, 1985; 221-227. 18. Lassalle B, Testart J. Sequential transformations of human sperm nucleus in human egg. J Reprod Fertil 1991; 91:393-402. 19. Laurincik J, Picha J, Pichova D, Oberfranc M. Timing of laparoscopic aspiration of preovulatory oocytes in heifers. Theriogenology 1991; 35:415-43. 20. Laurincik J, Pivko J, Grafenau P, OberBanc M. Recovery of oocytes by aspiration from preovulatory follicles in heifers. Vet Med (P&a) 1988; 33:727-74. 21. Pavlok A_Tomer H, Motlik J, Fulka J, Kauffold P, Duschinski U. Fertilization of bovine oocytes in vitro: Effect of different sources of gametes on fertilization rate and frequency of fertilization anomalies, Anim Reprod Sci 1988; 16:207-213. 22. Pitkjanen IG. The time of ovulation in sows. Svinovodstvo 1958; 12:38-40. 23. Polge C. Fertilization in the pig and horse. J Reprod Fertil 1978; 54:461-470. 24. Pope WF, Wilde MH, Xie S. Effect of electrocautery of nonovulated day 1 follicles on subsequent morphological variation among day 11 porcine embryos. Biol Reprod 1988; 39:882-887. 25. Purse1VG, Miller KF, Pinkert CA, Palmiter RD, Brinster RL. Development of l-cell and 2-cell pig ‘ovaafter microinjection of genes. J Anim Sci 1987; 65 (Suppl 1):402 abstr. 26 Thibault C. Analyse comparee de la fecondation et des anomalies chez la brebis, la vache et la lapine. Anual Biotech Anim Biochem Biophys 1967; 7:5-23. 27 Wright SJ, Longo FJ. Sperm nuclear enlargement in fertilized hamster eggs is related to meiotic maturation of the maternal chromatin. J exp Zool 1988; 247:155-165. 28. Xu KP, Greve T. In vitro fertilization of bovine follicular oocytes: a detailed analysis of early events. J Reprod Fertil 1988; 82:127-134. 5.
1994
OVULATION, FERTILIZATION AND PRONUCLEUS DEVELOPMENT IN SUPEROVULATED GILTS J. Laurincik, P. Hyttell, D. Rath2 and J. Pivko Research Institute of Animal Production, Department of Reproduction and Embryology, Nitra, Slovak Republic 1 The Royal Veterinary and Agricultural University, Department of Anatomy and Physiology, Frederiksberg C, Denmark 2 Institut l?ir Tierzucht und Tierverhalten, Mariensee, (FAL), Neustadt, Germany Received for publication: Accepted:
January December
27, 1993 7,
1993
ABSTRACT Estrus was synchronized in 45 gilts by ingestion of Zinc-Methallibur in the feed for 15 d On Day 16 each gilts was treated with PMSG ( 1200 IU i.m.) followed in 72 h by hCG ( 500 IU i.m.). Gilts were inseminated 24 and 36 h after the onset of estrus followed by slaughter of groups (n = 4 or 5) at 40 h, 44 h, 48 h, 52 h, 56 h, 60 h and 64 h after hCG injection. Ovaries were evaluated macroscopically and oocytesfembryos were recovered by flushing the oviducts. The ovulation rate increased from 38% to 87% from 40 to 45 h and remained constant thereafter. At 40 h, 36% of oocytes were penetrated by a single spermatozoon. The rate of fertilization increased from 36% (40 h) to 59% (44 h), to 65% (48 h), to 73% (52 h), to 76% (56 h), 80% (60 h) and to 64% (64 h). At 40 h all fertilized ova contained a decondensed sperm head. Afler another 4 to 8 h early pronuclei were common, and 52 h after hCG treatment opposed pronuclei were predominant. The first cleavages were recorded 64 h after hCG injection. Key words: Ovulation, fertilization, pronucleus development, gilts INTRODUCTION At present the microinjection technique is the most favourable method to introduce foreign DNA constructs into the pronuclei of zygotes or nuclei of 2-cell embryos in small laboratory and farm animals (2, 10, 17, 25). This intervention requires large numbers of zygotes synchronized at the pronucleus stage of development. This is not easy in the pig because of the difficulty in determining the onset and duration of the ovulation period, factors crucial for the recovery of zygotes at well-defined developmental stages. It has been reported that proestrous gilts ovulate approximately 40 h afler administration of hCG (9) and that ovulation is completed over 1 to 3 h period (1,3,22). Such hCG treatment, in combination with exogenous gonadotropins Acknowledgement : The work was supported by the Danish Agricultural and Veterinary Research Council.
Copyright
Q 1994 Butterworth-Heinemann
448
Theriogenology
is commonly used in superovulation regimes for in vivo production of zygotes. Using this strategy, normal regulation of follicle recruitment and selection for ovulation may be bypassed. However, one must consider that, at least in cows, folliculogenesis and ovulation induced by exogenous gonadotropins may lead to deviant follicle and oocyte maturation (15). The objective of the present study was to monitor the duration of ovulation, the timing of fertilization and pronucleus development in synchronized gilts superovulated with PMSGIhCG treatments. MATERIALS AND METHODS Es&us was synchronized in 45 cycling gilts at the age of about 6 months by ingestion of Zinc-Methallibur ( Evertas P, WBVL, Pohori-Chotoun, &echo-Slovakia ) in the feed ( 0.125 g in 2 kg of the complete feed mixture ) for 15 days. On Day 16 each gilt was treated with PMSG (Amex, Leo, Denmark; 1200 IU i.m. ) followed in 72 h by hCG ( Praedyn, Leciva, Prague, Czecho-Slovakia; 500 IU i.m. ) Semen was collected by the hand-glove method, evaluated for motility and diluted to approximately three billion spermcells per insemination dose. Semen was used on the day of collection and the gilts were inseminated artificially 24 h and 36 h after onset of estrus. Groups of gilts were slaughtered 40 h (n = 5), 44 h (n = 5), 48 h (n = 4), 52 h (n = 4), 56 h (n = 4), 60 h (n = 4) or 64 h (n = 4) after hCG injection. The ovaries were examined and the numbers of non-ovulated (> 7 mm) (24) and ovulated follicles were recorded. The oviducts were flushed twice with 20 ml TCM 199e (Sevac, Prague, Czecho-Slovakia), supplemented with 2.92 mM Ca-lactate, 2 mM Na-pyruvate, 33.9 mM Na-bicarbonate, 4.34 mM Hepes (Serva, Heidelberg, Germany) (21), 10 &ttl Gentamicin and 20% heat-inactivated estrous cow serum (ECS). Oocytes and embryos present in the flushing medium were evaluated under a stereomicroscope (x 125) for morphology of the cumulus investment, the presence of polar bodies, and cleavage. Subsequently, they were f&d in acetic alcohol (I:3 v/v) and stained with aceto-orcein (1%) 24 h later (28). All stained preparations were evaluated under a phase-contrast microscope (x 400) for fertilization, pronucleus development, and position of paternal and maternal pronuclei (13). Oocytes or zygotes were classified to be degenerated when no chromosomal structures or pronuclei were recognized after staining. Statistical comparisons were performed by Student’s t-test, while proportions were analyzed by the Chi-square test. In the case of a significant result a mutual comparison of groups was done by means of the U-test. RESULTS Ovulation rate and recovery rate of ovulated ova are given in Table 1. Forty h after hCG administration only 38% of the follicles had ovulated while 4 h later 65% had done so. At 48 h 87% of the follicles had ovulated, and no further increase in the ovulation rate was recorded up to 64 h. The rates of fertilization and degeneration are given in Table 2. At 40 h after hCG application 10 out of 22 recovered ova displayed expanded cumulus investments. At all later time intervals all ova were denuded. Among the 10 ova, 8 (36% from all recovered) had undergone normal
449
Theriogenology
fertilization. However, a gradual increase of the fertilization rate up to 80% at 60 h was noted. Data of the further developmental steps are given in Table 2; they differ significantly (pcO.01) from the 40 h values. No correlation between time interval and rate of polyspermy was observed. Table 1. Ovulation rate and recovery rate of early embryonic stages at different times after hCG injection. Recovery Time Gilts Follicles rate ovulated after hCG non-ovulated n n % h n n % % Sa 40 3ga 22 5 66 62a 40 78’ 59 65’ 46 5 31 35b 44 82’ 60 87’ 49 4 9 13c 48 83’ 72 91C 60 4 7 9c 52 90’ 76 86c 69 4 12 14c 56 89’ 68 8gc 61 4 9 12c 60 86c 61 91C 53 4 6 9c 64 Values with different superscripts in the same column are significantly different. a,b PCO.05 ac P
Rates of fertilization, polyspermy and degeneration of ova collected at different times after hCG injection.
Tie Ova Unfertiliied after recovered h n n O/O 12 55a 40 22 14 30b 44 46 8c 48 49 4 8 14c 52 60 12 18’ 56 69 8 13c 60 61 3 6’ 64 53 Values with diierent superscripts in a,b ~~0.05 a,c ~co.01.
Normal
Fertilized Polyspermv
Degenerate
36a
n 2
9
n 0
;;
2;:
;
1; b
;
44 53 49 32 the same
4 73c 4 7 2 76’ 2 3 a 1 80’ 3 5 a 64’ 8 16 b 10 column are significantly different.
n 8
O/O
%
%
Oa 4a 15b 6a 3a 2a 14b
The chronological events of pronucleus formation and development are given in Table 3. At 40 h after hCG injection the maternal chromatin in all fertilized ova was in Ana- or Telophase II. The paternal chromatin in these ova was identified as sperm heads, which were slightly swollen. At 44 h sperm head enlargement was observed in 70% of the fertilized ova, while the remaining 30% had 2 spherical, synchronously developing pronuclei. The size of each was about l/3 of the fklly developed pronuclei. The pronuclei were located at each pole of the zygote, and the sperm tail was associated with the paternal pronucleus. At the 48 h interval 53% of the ova had developing pronuclei as described above, while 28% displayed opposed pronuclei in the central
Theriogenology
450
region of the ooplasm. The maternal pronucleus was located towards the second polar body, being slightly smaller than the other. At this stage the paternal pronucleus had lost its association with the sperm tail. At 52 h 52% of the ova displayed apposed pronuclei, and at 56 h and 60 h 7576% had reached this stage of development. At 64 h 48% of the embryos developed to the 2-cell stage, while 37% displayed apposed pronuclei, which on some occasions had achieved identical size. Throughout the investigated period a certain proportion of the fertilized ova showed early stages of fertilization, i.e. sperm head decondensation. Table 3.
Pronucleus development in fertilized ova recovered at different times after hCG injection.
Time Ova Sperm head after recovered in ooplasm h
n
40 44 48 52 56 60 64
8 27 32 44 53 49 53
Develooment of nronuclei Apposed Early pronuclei pronuclei
n
%
n
8 19 6 2 1 5 3
100 70 19 5 2 10 9
0 8 17 19 12 7 2
% 0 30 53 43 23 14 6
n 0 0 9 23 40 37 12
% 0 0 2s 52 75 76 37
Cleavage
n
%
0 0 0 0 0 0 15
0 0 0 0 0 0 48
DISCUSSION The pattern and duration of ovulation in pigs can only be estimated retrospectively. Earlier studies have shown that gilts ovulate 39 to 40 h after the onset of spontaneous oestrus (12) and 40 (9) to 42 h (23) after hCG administration. Hunter (13) observed that 60 to 100% of the gilts had completed ovulation 44 to 46 h after hCG application. Similar data were observed by Pope et al. (24), where 68 to 95% of the gilts had completed ovulation about 4 h after the onset of estrus. Gur tidings are in accordance with these data. The maximum rate of ovulation was reached at 44 to 48 h after hCG, but in contrast to the observation of other authors (1,3,22) the duration of the ovulation period (4 to 8 h) was slightly prolonged based on the ovulation rate between 40 h and 52 h after hCG application. Although a significant increase in the ovulation rate happened between 40 h and 44 h it seems that the superovulatory treatment hampers the course of normal ovulation. Fertilization is believed to occur almost immediately after entry of the mature oocytes into the oviduct, containing capacitated spermatozoa (7,14,16,26). Our data support this hypothesis because both ovulation and fertilization rates increased almost simultaneously. As published earlier in pigs (1 l), cattle (6,16) and sheep (7) the oocyte sheds the accompanying cumulus cells during or soon after ovulation, allowing the spermatozoa to contact the surface of the zona pelhmida directly. Accordingly, in the present study less than one half of
451
Theriogenology
ova recovered 40 h after hCG treatment were surrounded by cumulus cell layers. However, in a few cumulus invested oocytes these cumulus cells did not serve as a barrier against fertilization. Under in vivo conditions, fertilization of pig oocytes is reported to be monospermic in most cases (95%) (13,14). The rate of polyspermy in our experiment ranged from 3 to 16%. The entry of more than one spermatozoon may be associated with an abnormal hormonal environment at the time before fertilization (8,13) or may be caused by a delayed activation of the cortical granule release. Polyspermy in experiments for in vitro fertilization of in vivo matured oocytes was caused by a delayed migration of cortical granules (5). In those experiments superovulatory treatment was used to synchronize donor animals as it was in our experiments. The hormonal induction of ovulation may be the reason for asynchronous maturation and development of the cell compartments. Upon gamete fusion the ovum is activated and a series of nuclear changes in both the maternal chromosomes and the fertilizing spermatozoon take place. The transformation of the sperm head from a slightly swollen structure into an early pronucleus with intact membrane covering is reported to require 1S-2 h, and appearance of the pronuclei can be observed about 6 h after mating (13). The pronucleus development found in the present experiment resembled this pattern although the time sequence was apparently longer. More than 12 to 16 h were required before the majority of the sperm heads had developed into male pronuclei. It is hypophesized that this delay also was induced by the hormonal pretreatment. In comparison to spontaneous ovulation maternal and paternal pronuclei developed well synchronized. These data correspond with earlier observations in cattle (16), hamsters (27) and man (18). No enlarged sperm heads were observed within each interval after insemination. Although a delayed penetration can not be excluded, the missing enlargement is interpretated as an insufficient mechanism of cytoplasmic substances such as male pronucleus growth factor (MPGF), which is responsible for male pronucleus formation (4,14). It is concluded that the ovulation wave in superovulated gilts has a duration of about 8 h. Both ovulation and fertilization rates increased continuously and reached the maximum 44 h after hCG. Synchronous pronucleus development characterized by decondensed sperm heads, early pronuclei, and opposed pronuclei were predominantely present at 44 h, 48 h and 52 h, respectively. REFERENCES Betteridge KJ, Raeside JI. Observation of the ovary by peritoneal cannulation in pigs. Res Vet Sci 1962; 3:390398 Brem G, Brenig B, Goodman HM, Selden RC, Graf F, Kruff B, Springman K, Hondele J, Meyer J, Winnaher EL, Kr&rsslich H. Production of transgenic mice, rabbits and pigs by microinjection into pronuclei. Zuchthyg 1985; 20:251-256. Burger JF. Sex physiology of pigs. Onderstepoort J vet Res 1952; 25 (Suppl 1): 120-131 (1952). Calvin HI, Grosshans K, Blake EJ. Estimation and manipulation of glutathione levels in prepubertal mouse ovaries and ova: Relevance to sperm nucleus transformation in the fertilized egg. Gamete Res 1986; 14:265275.
452
Theriogenology
Cran DG, Cheng WTK. The cortical reaction in pig oocytes during in vivo and in vitro fertilization. Gamete Res 1986; &241-251. 6. Crozet N. Ultrastructural aspects of in vivo fertilization in the cow. Gamete Res 1984; 10:241-251. 7. Crozet N, Dumont M. The site of the acrosome reaction during in vivo penetration of the sheep oocyte. Gamete Res 1984; 10:97-105. 8. Day BN, Polge C. Effects of progesterone on fertilization and egg transport in the pig. J Reprod Fertil 1968; 17:227-230. 9. Dziuk PJ, Baker RD. Induction and control of ovulation in swine. 1962; Anim Reprod Sci 1:697-699 10. Hammer RE, Pursel VG, Rexroad CE, Wall RI, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 1985; 3 15:680-683. 11. Hunter RI-IF.Fertilization in the pig: Sequences of nuclear and cytoplasmic events. J Reprod Fertil 1972; 29: 395-406. 12. Hunter RI-IF.Fertilization of pig eggs in vivo and in vitro. J Reprod Fertil 1990; 40 (SuppI): l-226. 13. Hunter RHF. Ovulation in the pig: Timing of the response to injection of human chorionic gonadotropin. Res Vet Sci 1972; 13:356-361. 14. Hunter RI-IF.Polyspermic fertiliition in pigs during the luteal phase of the estrus cycle. J Exp 2001 1967; 165:451-466. 15. Hyttel P, Callesen H, Greve T, Schmidt M. Oocyte maturation and sperm transport in superovulated cattle. Theriogenology 1991; 35:91-108. 16. Hyttel P, Greve T, Callesen H. Uhrastructure of in-vivo fertilization in superovulated cattle. J Reprod Fertil 1988; 82:1-13. 17. Kremer D, Minhas B, Capehart J. Gene transfer into pronuclei of cattle and sheep zygotes. In: Bandury Report Costantini F, Jaenisch R (eds.).New York, Cold Spring Harbor Press, 1985; 221-227. 18. Lassalle B, Testart J. Sequential transformations of human sperm nucleus in human egg. J Reprod Fertil 1991; 91:393-402. 19. Laurincik J, Picha J, Pichova D, Oberfranc M. Timing of laparoscopic aspiration of preovulatory oocytes in heifers. Theriogenology 1991; 35:415-43. 20. Laurincik J, Pivko J, Grafenau P, OberBanc M. Recovery of oocytes by aspiration from preovulatory follicles in heifers. Vet Med (P&a) 1988; 33:727-74. 21. Pavlok A_Tomer H, Motlik J, Fulka J, Kauffold P, Duschinski U. Fertilization of bovine oocytes in vitro: Effect of different sources of gametes on fertilization rate and frequency of fertilization anomalies, Anim Reprod Sci 1988; 16:207-213. 22. Pitkjanen IG. The time of ovulation in sows. Svinovodstvo 1958; 12:38-40. 23. Polge C. Fertilization in the pig and horse. J Reprod Fertil 1978; 54:461-470. 24. Pope WF, Wilde MH, Xie S. Effect of electrocautery of nonovulated day 1 follicles on subsequent morphological variation among day 11 porcine embryos. Biol Reprod 1988; 39:882-887. 25. Purse1VG, Miller KF, Pinkert CA, Palmiter RD, Brinster RL. Development of l-cell and 2-cell pig ‘ovaafter microinjection of genes. J Anim Sci 1987; 65 (Suppl 1):402 abstr. 26 Thibault C. Analyse comparee de la fecondation et des anomalies chez la brebis, la vache et la lapine. Anual Biotech Anim Biochem Biophys 1967; 7:5-23. 27 Wright SJ, Longo FJ. Sperm nuclear enlargement in fertilized hamster eggs is related to meiotic maturation of the maternal chromatin. J exp Zool 1988; 247:155-165. 28. Xu KP, Greve T. In vitro fertilization of bovine follicular oocytes: a detailed analysis of early events. J Reprod Fertil 1988; 82:127-134. 5.