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Xenogenous fertilization of laboratory and domestic animals in the oviduct of the pseudopregnant rabbit
THERIOGENOLOGY
XENOGENOUS FERTILIZATION OF LABORATORY AND DOMESTIC ANIMALS IN THE OVIDUCT OF THE PSEUDOPREGNANT RABBIT* P. J. Hirst, F. J. DeMayo, and W. Richard Dukelow Endocrine Research Unit, Michigan State University East Lansing, Michigan 48824
ABSTRACT Xenogenous fertilization was accomplished using bovine, porcine, and hamster follicular oocytes. The xenogenous fertilization rates for bovine and porcine follicular oocytes in the oviduct of the pseudopregnant rabbit were 13.4% and 2.0%, respectively. Temperatures of ovary, during transport to the laboratory, of 00 or 370 C had no effect on xenogenous fertilization rates of bovine oocytes. In vitro culture in 50 ~g/ml FSH did not alter the xenogenous fertilization rates of bovine oocytes. Fertilization was observed with oocytes recovered 40 to 75 hr after insemination. Two cell embryos were recovered 70 to 75 hr after insemination. Ligation of the rabbit oviduct, number of ova deposited and sperm concentration did not affect the xenogenous fertilization rates of hamster ova. Cleavage of xenogenously fertilized hamster oocytes occurred between 28 and 29 hours after insemination. INTRODUCTION The fertilization of ova in the oviduct of a foreign species, I xenogenous fertilization, has been attempted with hamster (i), squirrel monkey (i), bovine (2,3,4) and human ova (5). Xenogenous fertilization of bovine oocytes has been attempted in the oviducts of the estrous ewe (2), estrous rabbit (2), prepuberal gilt (3) and pseudopregnant rabbit (4) but have only been successful in the estrous ewe and prepuberal gilt. Sreenan (2) matured oocytes in vitro from non-gonadotropin primed cattle for 27-32 hr. Of these oocytes, 82 were transferred to the oviduct of estrous rabbits previously inseminated with bull sperm. No fertilization occurred. However when 375 bovine oocytes matured in vitro were transferred to the oviduct of previously inseminated estrous ewes, 198 (52.8%) were recovered and 17 (8.6%) were fertilized. Bedirian et al. (3), deposited 70 oocytes recovered from gonadotropin stimulated prepuberal heifers in the oviduct of prepuberal gilts previously inseminated with bovine semen. Of these, 27 (38.5%) were recovered and six
*Supported, in part, by grants from the National Institutes of Health and the March of Dimes Birth Defects Foundation.
ACKNO~fLEDGEMENTS The authors express their appreciation to Dr. T. Asakawa for his assistance and expertise regarding karyotypic analysis of the fertilized embryos.
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THERIOGENOLOGY
(22.2%) were fertilized. Trounson et al. (4) cultured follicular oocytes from gonadotropin stimulated cattle for 24 hr. Of these, 491 oocytes were transferred to the oviducts of pseudopregnant rabbits previously inseminated with bovine semen. At intervals of 12 hr to 7 days, a total of 337 oocytes (68.6%) were recovered, of which 25 (7.4%) were cleaved. However, this cleavage rate did not differ from oocytes recovered from non-inseminated pseudopregnant rabbits and these authors concluded that fertilization had not occurred. Since the pseudopregnant rabbit oviduct provides an excellent environment for embryo culture (6) and because of our recent studies on xenogenous fertilization of hamster and squirrel monkey oocytes in the pseudopregnant rabbit oviduct (with fertilization rates of 60% and 36%, respectively) (i), we attempted the xenogenous fertilization of unstimulated bovine and porcine follicular oocytes. Additionally the effects of varying numbers of ova deposited, sperm concentration, temperature of ovaries during transport to the laboratory, and recovery time were investigated using domestic animals and hamsters. MATERIALS AND METHODS Ovaries from mixed breeds of sows and cows were obtained at slaughter and transported at either 0 ° or 37°C in 0.15 M NaC1 solution. A second pool of bovine ovaries was available from ovariectomies as part of another experiment. Oocytes were aspirated from all visible antral follicles using a 25 g needle and a 3 ml syringe and transported to a watch glass. The syringe contained 0.2 - 0.3 ml of 37°C collection medium (Table I). TABLE I.
MEDIUM* USED FOR OVUM HANDLINE AND SPERM DILUTIONS ~g" Quantity
Source
Medium 199 (a) GG-Free Fetal Bovine Serum(b) Pyruvic Acid(d) Gentamicin (antibiotics) Penicillin-Streptomycin
80% Gibco(C)Laboratories 20% Gibco(C)Laboratories 115.2 g/ml Sigma Chemical Co. 0.i mg/ml Schering Corp. i00 units/ml & N.A.B., Miami i00 ~g/ml Hyaluronidase (e) i mg/ml Sigma Chemical Co. Heparin Sodium (f) i unit/ml Upjohn Co. a) with 25 mM Hepes Buffer, Earle's Salts, and L-glutamine b) Mycoplasma tested and virus screened, heat inactivated at 56°C for 30 minutes c) Grant Island Biological Company, Grand Island, N.Y. d) Sodium Salt, Crystalline Type II e~ from bovine testes, lyophilized powder type 1-5 f) from beef lung g) Media used for sperm dilutions lacked hyaluronidase and heparin. *Medium was filtered through a 0.45 ~m millipore filter into sterile vacutainers and stored at 4oc until ready to be used.
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THERIOGENOLOGY
Semen samples were either fresh or frozen depending on the availability of males on the day of insemination. The artificial vagina collection method for both bulls and boars was used. Semen was washed twice in medium and centrifuged at 1058 xg for five minutes. The sperm were held at 37°C until used. Bovine frozen semen was obtained in straws or ampules from Michigan Animal Breeders Corporation (MABC, East Lansing, MI). Porcine semen that had been frozen by International Boar Semen, United Suppliers, Eldora, Iowa using the technique of Pursel and Johnson (7) was also used. Ampules or straws of bovine frozen semen were thawed at 37°C. The spermatozoa were then diluted with 2-3 ml of medium. Procine frozen semen was placed in a test tube that was held at 37°C. After the pellet was completely thawed 2-5 ml of medium were added. Sperm motility was recorded at time of thawing and dilution. Adult, female New Zealand White rabbits were induced to ovulate with (i.v.) lO0 IU of hCG (A.P.L., Ayerst, Montreal) 24 hr prior to surgery. Rabbits were anesthetized with sodium pentobarbital and the plane of anesthesia was maintained with ether inhalation. Through a midventral incision the reproductive tract was exposed. A visual observation of the ovaries for the presence of a fresh corpus luteum was made. Oocytes were transferred using a micropipette to the fimbriated end of the oviduct and at a depth of approximately 2 cm into the ampulla. The ova were transferred in five ~i aliquots with 22 to 41 ova placed in each oviduct. Spermatozoa, of the appropriate species, were then deposited into each oviduct using a 0.25 ml glass syringe with a 20 gauge, i;~" needle with a 5 cm length of polyethylene tubing (P.E. 90, Clay-Adams, N.Y.). The flexible tubing was introduced into the oviductal fimbria to a depth of 2 cm and 0.05 ml of diluted sperm suspension was deposited. After insemination each oviduct was usually ligated at the tubal-uterine junction using 00 chromic gut suture to prevent egg loss. The incision was closed and prophylactic penicillin given. Later the rabbits were killed by cervical dislocation. The oviducts were dissected and flushed from the tubal-uterine junction with a 25 gauge needle and 2 ml of medium. Oocytes were located with a dissecting microscope and transferred to an 8 chamber culture slide (Lab-tek, Napier, Illinois) with 0.25 ml of medium. Ova were then observed under a phase contrast microscope for evidence of fertilization. The criteria for fertilization used were the appearance of two polar bodies and two pronuclei, or normal cleavage. The ova were stained using Giemsa stain (8). The procedure for xenogenous fertilization of hamster oocytes has been described by DeMayo et al. (i). The effects of number of ova deposited on the fertilization rate was determined with ova numbers in the following categories: a) <20; b) 20-29; c) 30-39; and d) >40. The effects of sperm concentration on xenogenous hamster fertilization was evaluated with sperm concentration categories of 106 , 107 , 108 , and 109 sperm/ml. Additionally the effect of recovery at 28, 29, 30, or 32 hr after insemination on the xenogenous fertilization rate of hamster ova was studied.
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THERIOGENOLOGY
RESULTS The number and percent of oocytes recovered and fertilized are shown in Table II. Fertilization rates of 13.4% and 2.0% for bovine and porcine, respectively, were achieved. TABLE II.
Oocytes Bovine Porcine
The recovery and xenogenous fertilization rates of bovine and porcine follicular oocytes in the oviduct of the pseudopregnant rabbit. Recovered/transferred 261/582 148/410
(%)
(44.8) (36.1)
Fertilized/recovered 35/261 3/148
(%)
(13.4) (2.0)
Table III shows the effects of two different temperatures of ovary transport, 0 ° and 37oc, on the recovery and fertilization rates of bovine oocytes. Transporting the ovaries at 0°C resulted in better recovery rates from the rabbit oviducts (X 2 = 7.54; P<0.01) than did 37°C. But there was no difference in fertilization rate. TABLE III.
Temperature 0°C 37°C
Temperature effects on ovary transport on recovery and xenogenous fertilization rates of bovine oocytes. Oocytes recovered/transferred 88/228 88/259
(%)
(38.6) (34.0)
Oocytes fertilized/recovered 10/88 16/88
(%)
(11.4) (18.2)
Temperature effects on the transport of porcine ovaries are shown in Table IV. TABLE IV.
Temperature 0°C 37°C
Temperature effects on ovary transport on recovery and xenogenous fertilization rates of porcine oocytes. Oocytes recovered/transferred 83/241 65/169
(34.4) (38.5)
(%)
Oocytes fertilized/recovered 1/83 2/65
(%)
(1.2) (3.1)
There was no significant influence of the handling of the ovaries on the subsequent recovery and fertilization rates. Oocytes were recovered at varying times after insemination and the results are shown in Table V. Maximal recovery rates for bovine oocytes were 50-55 hr after insemination, whereas the highest fertilization rates were obtained at intervals of 70-75 hr post-insemination.
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Two-cell stage embryos were obtained 50-70 hr after insemination. Fertilized ova recovered before 50 hr were only at the two polar body: two pronuclei stage. In most cases sperm tails could be seen after staining with Giemsa. TABLE V.
Recovery and xenogenous fertilization rates of bovine oocytes recovered at varying times after insemination
Recovery time (hr. post-insemination) 40 50 65 70
-
Oocytes recovered/ transferred (%)
45 55 70 75
5/44 (11.4) 103/207 (49.8) 61/154 (39.6) 7/82 (8.5)
Oocytes fertilized/ recovered (%) 1/5 (20.0) 10/103 ( 9 . 7 ) 13/61 (21.3) 2/7 (28.6)
Table Vl illustrates the effect of in vitro maturation of bovine oocytes with and without FSH in the medium. Incubation was carried out for 20 hours in a 37°C, 5% CO 2 in air environment. There was no significant difference between the treated and control groups. Fertilization rates of 2/23 (8.7%) for FSH supplemented medium and 1/34 (2.9%) for the control medium were achieved. The recovery from the oviduct was 54 hr after insemination. Of the two fertilized ova that were incubated in medium supplemented with FSH, one was recovered as a two-cell embryo. The other was at the 2 polar body stage (after staining a sperm tail was seen in the cytoplasm of this ovum). The control treatment yielded one fertilized ovum at the two polar body stage. TABLE Vl.
Effect of culture in vitro for 20 hours in medium supplemented with 50 ~g/ml FSH, on recovery and xenogenous fertilization rates of bovine oocytes.
Oocytes recovered/ transferred (%) FSH Control
23/30 34/37
Oocytes fertilized/ recovered (%)
(76.7) (91.9)
2/23 1/34
(8.7) (2.9)
Stage of Fertilization 2 cell, 2 pb 2 pb
Another experiment was carried out using hamster oocytes to determine the value of tubal ligation. The results of this trial are shown in Table VII. Oocytes were recovered 32 hr after insemination. Fourty-one ova were deposited in each oviduct of the rabbit. One oviduct was ligated and the other was not. The data show that at a recovery time of 32 hr or less after insemination, there is no advantage with tubal ligation. The criterion for fertilization in this trial was development to the two-cell stage.
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THERIOGENOLOGY
TABLE VII.
Effects of ligation at the uterotubal junction, on recovery and xenogenous fertilization rates of hamster embryos. (recovery 32 hours after insemination) Oocytes recovered/ transferred (%)
Nonligated oviduct Ligated oviduct
24/41 22/41
Fertilized oocytes/ ova recovered (%)
(58.5) (53.7)
4/24 3/22
(16.7) (13.6)
The number of hamster ova placed in the pseudopregnant rabbit oviduct was not a significant factor affecting the xenogenous fertilization or cleavage rate (Table VIII). However the recovery of ova was significantly increased (X222.44; P<.05) when greater than forty eggs were placed in the rabbit oviduct. TABLE VIII.
Oocytes
Effects of number of oocytes deposited in the rabbit oviduct on the recovery, xenogenous fertilization and cleavage rates of hamster oocytes. Oocytes recovered/ transferred (%)
<20 20-24 30-34 >40 TOTAL
6/32 24/93 83/275 66/133
(18.8) (25.8) (30.2) (49.6)
179/533
(33.6)
Oocytes fertilized/ recovered (%) 3/6 10/24 59/83 41/66
2 cell embryos/ recovered (%)
(50.0) (41.7) (71.1) (62.1)
1/6 3/24 22/83 10/66
113/179 (63.1)
(16.7) (12.5) (26.5) (15.2)
36/179 (20.1)
The concentration of hamster spermatoza, deposited in the rabbit oviduct, had no effect on the xenogenous fertilization rate or cleavage rate (Table IX) but a trend towards increased fertilization occurred with the two higher sperm concentrations. TABLE IX.
The effects of sperm concentration on the recovery, xenogenous fertilization and cleavage rate of hamster oocytes.
Sperm (cells/m_l) Concentration 106 107 108 109 TOTAL
72
Oocytes recovered/ transferred (%) 59/121 33/97 37/240 50/75
(48.8) (34.0) (15.4) (66.7)
179/533 (33.6)
Oocytes fertilized/ recovered (%)
2 cell embryos/ recovered (%)
33/59 18/33 26/37 36/50
(55.9) (54.5) (70.3) (72.0)
7/59 5/33 11/37 13/50
(11.9) (15.2) (29.7) (26.0)
113/179
(63.1)
36/179 (20.1)
JANUARY 1981 VOL. 15 NO. 1
THERIOGENOLOGY
The recovery of hamster ova at 28, 29, 30, and 32 hours (Table X) after the addition of sperm showed no significant increase in the fertilization rates, however, the percentage of cleaved ova was significantly greater after 29 hr (P<.05). TABLE X.
Recovery, ienogenous fertilization and cleavage rates of hamster oocytes recovered at varying times after insemination.
Recovery time (hr post-insemination) 28 29 30 32
Oocytes recovered/ transferred (%) 52/132 44/130 67/152 16/119
Oocytes fertilized/ recovered (%)
(39.4) (33.9) (44.1) (13.5)
25/52 31/44 45/67 12/16
(48.1) (70.5) (67.2) (75.0)
Cleaved/ recovered 5/52 11/44 16/67 4/16
(%)
(9.6) (25.0) (23.9) (25.0)
DISCUSSION The oviductal environment of the pseudopregnant rabbit is suitable for the xenogenous fertilization of porcine and bovine oocytes even though fertilization rates do vary between species. Earlier studies, cited in the introduction, failed to achieve fertilization in rabbit oviducts but this probably reflects the use of estrous rabbits and the concomitant rapid rate of ovum transport through the oviduct. A second variable between previous and present studies is the site of deposition of the sperm suspension, which was vaginal in the earlier studies and oviductal in the present experiments. The temperature of ovarian transport (0 ° vs 37°C) did not affect the xenogenous fertilization rate of either porcine or bovine oocytes although a higher recovery rate from the rabbit oviduct was noted with bovine oocytes transported at 0°C. Fertilized bovine oocytes were recovered as early as 40 hr and as late as 75 hr after insemination. These were the temporal limits of these studies. Two cell embryos were observed at a recovery t%me of 70 hr which is similar to the time scale observed for normal in vivo or in vitro maturation and fertilization. Neither hamster nor cattle xenogenously fertilized embryos cleaved beyond the two cell stage. Previous studies (i) on xenogenous fertilization have produced eight cell squirrel monkey embryos but hamster embryos never progressed beyond the two cell stage, a phenomenon similar to that observed with in vitro fertilization in this species. The domestic animals used in these studies were not pretreated with gonadotropins. The oocytes aspirated from the follicle were generally immature with no polar body extrusion until after later maturation. In lieu of gonadotropin priming, in vitro maturation of the oocyte with FSH (and marginal LH contamination) was attempted but the fertilization rate was not affected. These preliminary studies suggest strongly the importance of future studies on in vivo maturation within the donor animal or by alternate means with in vitro culture.
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THERIOGENOLOGY
Because of the ease of xenogenous fertilization of hamster ova, this animal provides a useful model for developing techniques which can subsequently be applied to domestic species. The studies relating to the need for ligation of the tubal-uterine junction emphasize this approach. Ligation did not alter recovery or fertilization rate of the oocytes recovered from the rabbit oviduct within 32 hr. It is possible that differences might have been observed with later recovery times. By a similar test system it was determined that the number of oocytes placed in the oviduct did not alter fertilization rate but a higher percentage of recovery was noted when more than 40 oocytes were placed in each oviduct. Cleavage of xenogenously fertilized hamster oocytes occurred about 29 hrs after insemination, a value comparable with natural, in vivo fertilization. These results demonstrate that xenogenous fertilization of porcine and bovine oocytes can occur in the oviduct of the psuedopregnant rabbit. There are several important applications of such studies. If the embryos produced by this procedure are normal (which must ultimately be demonstrated by the successful transfer to a recipient mother and live birth following pregnancy) then this represents a source of embryos for transfer or for in vitro evaluation of the effects of chemical or environmental insult on embryonic development. This would include drugs having mutagenic or teratogenic effects. A second application might relate to the use of the procedure for assessing fertilization rates of specific sires. Current methods of fertility assessment are either time-consuming (nonreturn rates) or utilize sperm penetration of the zona-free ova of a foreign species (normally hamster). If xenogenous fertilization rates of bovine oocytes, fertilized by bovine sperm, are correlated with non-return fertility rates, then the technique has application for rapid screening of the fertility of bulls for potential use in artificial insemination. REFERENCES i.
DeMayo, F.J., Mizoguchi, H., and Dukelow, W.R. Fertilization of Squirrel Monkey and Hamster Ova in the Rabbit Oviduct (Xenogenous Fertilization). Science 2 0 8 : 1 4 6 8 (1980).
2.
Sreenan, J. In Vitro Maturation and Attempted Fertilization of Cattle Follicular Oocytes. J. Agric. Sci. (Camb.) 7 5 : 3 9 3 (1970).
3.
Bedirian, K.N., Shea, B.F., and Baker, R.D. Fertilization of Bovine Follicular Oocytes in Bovine and Porcine Oviducts. Can. J. Anim. Sci. 5 5 : 2 5 1 (1975).
4.
Trounson, A.O., Willadsen, S.M., and Rowson, L.E.A. Fertilization and Development Capability of Bovine Follicluar Oocytes Matured In Vitro and In Vivo and Transferred to the Oviducts of Rabbits and Cows. J. Reprod. Fert. 5 1 : 3 2 1 (1977).
5.
Edwards, R.G., Donahue, R.P., Baramki, T.A., and Jones, H.W. Preliminary Attempts to Fertilize Human Oocytes Matured In Vitro. Am. J. Obst. & Gynec. 9 6 : 1 9 2 (1966).
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6.
Adams, C.E., Rowson, L.E.A., Hunter, G.L., and Bishop, G.P. Long Distance Transport of Sheep Ova. Proc. 4th Intern'l Congr. Animal Reprod. and AI. The Hague 2 : 3 8 1 (1961).
7.
Pursel, V.G. and Johnson, L.A. Freezing of Boar Spermatozoa: Fertilizing Capacity with Concentrated Semen and a New Thawing Procedure. J. Anim. Sci. 4 0 : 9 9 (1975).
8.
Mizoguchi, H. and Dukelow, W.R. Gradual Fixation Method for Chromosomal Studies of Squirrel Monkey Oocytes After Gonadotropin Treatment. J. Med. Prim. (In Press) (1981).
JANUARY 1981 VOL. 15 NO. 1
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XENOGENOUS FERTILIZATION OF LABORATORY AND DOMESTIC ANIMALS IN THE OVIDUCT OF THE PSEUDOPREGNANT RABBIT* P. J. Hirst, F. J. DeMayo, and W. Richard Dukelow Endocrine Research Unit, Michigan State University East Lansing, Michigan 48824
ABSTRACT Xenogenous fertilization was accomplished using bovine, porcine, and hamster follicular oocytes. The xenogenous fertilization rates for bovine and porcine follicular oocytes in the oviduct of the pseudopregnant rabbit were 13.4% and 2.0%, respectively. Temperatures of ovary, during transport to the laboratory, of 00 or 370 C had no effect on xenogenous fertilization rates of bovine oocytes. In vitro culture in 50 ~g/ml FSH did not alter the xenogenous fertilization rates of bovine oocytes. Fertilization was observed with oocytes recovered 40 to 75 hr after insemination. Two cell embryos were recovered 70 to 75 hr after insemination. Ligation of the rabbit oviduct, number of ova deposited and sperm concentration did not affect the xenogenous fertilization rates of hamster ova. Cleavage of xenogenously fertilized hamster oocytes occurred between 28 and 29 hours after insemination. INTRODUCTION The fertilization of ova in the oviduct of a foreign species, I xenogenous fertilization, has been attempted with hamster (i), squirrel monkey (i), bovine (2,3,4) and human ova (5). Xenogenous fertilization of bovine oocytes has been attempted in the oviducts of the estrous ewe (2), estrous rabbit (2), prepuberal gilt (3) and pseudopregnant rabbit (4) but have only been successful in the estrous ewe and prepuberal gilt. Sreenan (2) matured oocytes in vitro from non-gonadotropin primed cattle for 27-32 hr. Of these oocytes, 82 were transferred to the oviduct of estrous rabbits previously inseminated with bull sperm. No fertilization occurred. However when 375 bovine oocytes matured in vitro were transferred to the oviduct of previously inseminated estrous ewes, 198 (52.8%) were recovered and 17 (8.6%) were fertilized. Bedirian et al. (3), deposited 70 oocytes recovered from gonadotropin stimulated prepuberal heifers in the oviduct of prepuberal gilts previously inseminated with bovine semen. Of these, 27 (38.5%) were recovered and six
*Supported, in part, by grants from the National Institutes of Health and the March of Dimes Birth Defects Foundation.
ACKNO~fLEDGEMENTS The authors express their appreciation to Dr. T. Asakawa for his assistance and expertise regarding karyotypic analysis of the fertilized embryos.
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THERIOGENOLOGY
(22.2%) were fertilized. Trounson et al. (4) cultured follicular oocytes from gonadotropin stimulated cattle for 24 hr. Of these, 491 oocytes were transferred to the oviducts of pseudopregnant rabbits previously inseminated with bovine semen. At intervals of 12 hr to 7 days, a total of 337 oocytes (68.6%) were recovered, of which 25 (7.4%) were cleaved. However, this cleavage rate did not differ from oocytes recovered from non-inseminated pseudopregnant rabbits and these authors concluded that fertilization had not occurred. Since the pseudopregnant rabbit oviduct provides an excellent environment for embryo culture (6) and because of our recent studies on xenogenous fertilization of hamster and squirrel monkey oocytes in the pseudopregnant rabbit oviduct (with fertilization rates of 60% and 36%, respectively) (i), we attempted the xenogenous fertilization of unstimulated bovine and porcine follicular oocytes. Additionally the effects of varying numbers of ova deposited, sperm concentration, temperature of ovaries during transport to the laboratory, and recovery time were investigated using domestic animals and hamsters. MATERIALS AND METHODS Ovaries from mixed breeds of sows and cows were obtained at slaughter and transported at either 0 ° or 37°C in 0.15 M NaC1 solution. A second pool of bovine ovaries was available from ovariectomies as part of another experiment. Oocytes were aspirated from all visible antral follicles using a 25 g needle and a 3 ml syringe and transported to a watch glass. The syringe contained 0.2 - 0.3 ml of 37°C collection medium (Table I). TABLE I.
MEDIUM* USED FOR OVUM HANDLINE AND SPERM DILUTIONS ~g" Quantity
Source
Medium 199 (a) GG-Free Fetal Bovine Serum(b) Pyruvic Acid(d) Gentamicin (antibiotics) Penicillin-Streptomycin
80% Gibco(C)Laboratories 20% Gibco(C)Laboratories 115.2 g/ml Sigma Chemical Co. 0.i mg/ml Schering Corp. i00 units/ml & N.A.B., Miami i00 ~g/ml Hyaluronidase (e) i mg/ml Sigma Chemical Co. Heparin Sodium (f) i unit/ml Upjohn Co. a) with 25 mM Hepes Buffer, Earle's Salts, and L-glutamine b) Mycoplasma tested and virus screened, heat inactivated at 56°C for 30 minutes c) Grant Island Biological Company, Grand Island, N.Y. d) Sodium Salt, Crystalline Type II e~ from bovine testes, lyophilized powder type 1-5 f) from beef lung g) Media used for sperm dilutions lacked hyaluronidase and heparin. *Medium was filtered through a 0.45 ~m millipore filter into sterile vacutainers and stored at 4oc until ready to be used.
68
JANUARY 1981 VOL. 15 NO, 1
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Semen samples were either fresh or frozen depending on the availability of males on the day of insemination. The artificial vagina collection method for both bulls and boars was used. Semen was washed twice in medium and centrifuged at 1058 xg for five minutes. The sperm were held at 37°C until used. Bovine frozen semen was obtained in straws or ampules from Michigan Animal Breeders Corporation (MABC, East Lansing, MI). Porcine semen that had been frozen by International Boar Semen, United Suppliers, Eldora, Iowa using the technique of Pursel and Johnson (7) was also used. Ampules or straws of bovine frozen semen were thawed at 37°C. The spermatozoa were then diluted with 2-3 ml of medium. Procine frozen semen was placed in a test tube that was held at 37°C. After the pellet was completely thawed 2-5 ml of medium were added. Sperm motility was recorded at time of thawing and dilution. Adult, female New Zealand White rabbits were induced to ovulate with (i.v.) lO0 IU of hCG (A.P.L., Ayerst, Montreal) 24 hr prior to surgery. Rabbits were anesthetized with sodium pentobarbital and the plane of anesthesia was maintained with ether inhalation. Through a midventral incision the reproductive tract was exposed. A visual observation of the ovaries for the presence of a fresh corpus luteum was made. Oocytes were transferred using a micropipette to the fimbriated end of the oviduct and at a depth of approximately 2 cm into the ampulla. The ova were transferred in five ~i aliquots with 22 to 41 ova placed in each oviduct. Spermatozoa, of the appropriate species, were then deposited into each oviduct using a 0.25 ml glass syringe with a 20 gauge, i;~" needle with a 5 cm length of polyethylene tubing (P.E. 90, Clay-Adams, N.Y.). The flexible tubing was introduced into the oviductal fimbria to a depth of 2 cm and 0.05 ml of diluted sperm suspension was deposited. After insemination each oviduct was usually ligated at the tubal-uterine junction using 00 chromic gut suture to prevent egg loss. The incision was closed and prophylactic penicillin given. Later the rabbits were killed by cervical dislocation. The oviducts were dissected and flushed from the tubal-uterine junction with a 25 gauge needle and 2 ml of medium. Oocytes were located with a dissecting microscope and transferred to an 8 chamber culture slide (Lab-tek, Napier, Illinois) with 0.25 ml of medium. Ova were then observed under a phase contrast microscope for evidence of fertilization. The criteria for fertilization used were the appearance of two polar bodies and two pronuclei, or normal cleavage. The ova were stained using Giemsa stain (8). The procedure for xenogenous fertilization of hamster oocytes has been described by DeMayo et al. (i). The effects of number of ova deposited on the fertilization rate was determined with ova numbers in the following categories: a) <20; b) 20-29; c) 30-39; and d) >40. The effects of sperm concentration on xenogenous hamster fertilization was evaluated with sperm concentration categories of 106 , 107 , 108 , and 109 sperm/ml. Additionally the effect of recovery at 28, 29, 30, or 32 hr after insemination on the xenogenous fertilization rate of hamster ova was studied.
JANUARY 1981 VOL. 15 NO. 1
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THERIOGENOLOGY
RESULTS The number and percent of oocytes recovered and fertilized are shown in Table II. Fertilization rates of 13.4% and 2.0% for bovine and porcine, respectively, were achieved. TABLE II.
Oocytes Bovine Porcine
The recovery and xenogenous fertilization rates of bovine and porcine follicular oocytes in the oviduct of the pseudopregnant rabbit. Recovered/transferred 261/582 148/410
(%)
(44.8) (36.1)
Fertilized/recovered 35/261 3/148
(%)
(13.4) (2.0)
Table III shows the effects of two different temperatures of ovary transport, 0 ° and 37oc, on the recovery and fertilization rates of bovine oocytes. Transporting the ovaries at 0°C resulted in better recovery rates from the rabbit oviducts (X 2 = 7.54; P<0.01) than did 37°C. But there was no difference in fertilization rate. TABLE III.
Temperature 0°C 37°C
Temperature effects on ovary transport on recovery and xenogenous fertilization rates of bovine oocytes. Oocytes recovered/transferred 88/228 88/259
(%)
(38.6) (34.0)
Oocytes fertilized/recovered 10/88 16/88
(%)
(11.4) (18.2)
Temperature effects on the transport of porcine ovaries are shown in Table IV. TABLE IV.
Temperature 0°C 37°C
Temperature effects on ovary transport on recovery and xenogenous fertilization rates of porcine oocytes. Oocytes recovered/transferred 83/241 65/169
(34.4) (38.5)
(%)
Oocytes fertilized/recovered 1/83 2/65
(%)
(1.2) (3.1)
There was no significant influence of the handling of the ovaries on the subsequent recovery and fertilization rates. Oocytes were recovered at varying times after insemination and the results are shown in Table V. Maximal recovery rates for bovine oocytes were 50-55 hr after insemination, whereas the highest fertilization rates were obtained at intervals of 70-75 hr post-insemination.
70
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Two-cell stage embryos were obtained 50-70 hr after insemination. Fertilized ova recovered before 50 hr were only at the two polar body: two pronuclei stage. In most cases sperm tails could be seen after staining with Giemsa. TABLE V.
Recovery and xenogenous fertilization rates of bovine oocytes recovered at varying times after insemination
Recovery time (hr. post-insemination) 40 50 65 70
-
Oocytes recovered/ transferred (%)
45 55 70 75
5/44 (11.4) 103/207 (49.8) 61/154 (39.6) 7/82 (8.5)
Oocytes fertilized/ recovered (%) 1/5 (20.0) 10/103 ( 9 . 7 ) 13/61 (21.3) 2/7 (28.6)
Table Vl illustrates the effect of in vitro maturation of bovine oocytes with and without FSH in the medium. Incubation was carried out for 20 hours in a 37°C, 5% CO 2 in air environment. There was no significant difference between the treated and control groups. Fertilization rates of 2/23 (8.7%) for FSH supplemented medium and 1/34 (2.9%) for the control medium were achieved. The recovery from the oviduct was 54 hr after insemination. Of the two fertilized ova that were incubated in medium supplemented with FSH, one was recovered as a two-cell embryo. The other was at the 2 polar body stage (after staining a sperm tail was seen in the cytoplasm of this ovum). The control treatment yielded one fertilized ovum at the two polar body stage. TABLE Vl.
Effect of culture in vitro for 20 hours in medium supplemented with 50 ~g/ml FSH, on recovery and xenogenous fertilization rates of bovine oocytes.
Oocytes recovered/ transferred (%) FSH Control
23/30 34/37
Oocytes fertilized/ recovered (%)
(76.7) (91.9)
2/23 1/34
(8.7) (2.9)
Stage of Fertilization 2 cell, 2 pb 2 pb
Another experiment was carried out using hamster oocytes to determine the value of tubal ligation. The results of this trial are shown in Table VII. Oocytes were recovered 32 hr after insemination. Fourty-one ova were deposited in each oviduct of the rabbit. One oviduct was ligated and the other was not. The data show that at a recovery time of 32 hr or less after insemination, there is no advantage with tubal ligation. The criterion for fertilization in this trial was development to the two-cell stage.
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TABLE VII.
Effects of ligation at the uterotubal junction, on recovery and xenogenous fertilization rates of hamster embryos. (recovery 32 hours after insemination) Oocytes recovered/ transferred (%)
Nonligated oviduct Ligated oviduct
24/41 22/41
Fertilized oocytes/ ova recovered (%)
(58.5) (53.7)
4/24 3/22
(16.7) (13.6)
The number of hamster ova placed in the pseudopregnant rabbit oviduct was not a significant factor affecting the xenogenous fertilization or cleavage rate (Table VIII). However the recovery of ova was significantly increased (X222.44; P<.05) when greater than forty eggs were placed in the rabbit oviduct. TABLE VIII.
Oocytes
Effects of number of oocytes deposited in the rabbit oviduct on the recovery, xenogenous fertilization and cleavage rates of hamster oocytes. Oocytes recovered/ transferred (%)
<20 20-24 30-34 >40 TOTAL
6/32 24/93 83/275 66/133
(18.8) (25.8) (30.2) (49.6)
179/533
(33.6)
Oocytes fertilized/ recovered (%) 3/6 10/24 59/83 41/66
2 cell embryos/ recovered (%)
(50.0) (41.7) (71.1) (62.1)
1/6 3/24 22/83 10/66
113/179 (63.1)
(16.7) (12.5) (26.5) (15.2)
36/179 (20.1)
The concentration of hamster spermatoza, deposited in the rabbit oviduct, had no effect on the xenogenous fertilization rate or cleavage rate (Table IX) but a trend towards increased fertilization occurred with the two higher sperm concentrations. TABLE IX.
The effects of sperm concentration on the recovery, xenogenous fertilization and cleavage rate of hamster oocytes.
Sperm (cells/m_l) Concentration 106 107 108 109 TOTAL
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Oocytes recovered/ transferred (%) 59/121 33/97 37/240 50/75
(48.8) (34.0) (15.4) (66.7)
179/533 (33.6)
Oocytes fertilized/ recovered (%)
2 cell embryos/ recovered (%)
33/59 18/33 26/37 36/50
(55.9) (54.5) (70.3) (72.0)
7/59 5/33 11/37 13/50
(11.9) (15.2) (29.7) (26.0)
113/179
(63.1)
36/179 (20.1)
JANUARY 1981 VOL. 15 NO. 1
THERIOGENOLOGY
The recovery of hamster ova at 28, 29, 30, and 32 hours (Table X) after the addition of sperm showed no significant increase in the fertilization rates, however, the percentage of cleaved ova was significantly greater after 29 hr (P<.05). TABLE X.
Recovery, ienogenous fertilization and cleavage rates of hamster oocytes recovered at varying times after insemination.
Recovery time (hr post-insemination) 28 29 30 32
Oocytes recovered/ transferred (%) 52/132 44/130 67/152 16/119
Oocytes fertilized/ recovered (%)
(39.4) (33.9) (44.1) (13.5)
25/52 31/44 45/67 12/16
(48.1) (70.5) (67.2) (75.0)
Cleaved/ recovered 5/52 11/44 16/67 4/16
(%)
(9.6) (25.0) (23.9) (25.0)
DISCUSSION The oviductal environment of the pseudopregnant rabbit is suitable for the xenogenous fertilization of porcine and bovine oocytes even though fertilization rates do vary between species. Earlier studies, cited in the introduction, failed to achieve fertilization in rabbit oviducts but this probably reflects the use of estrous rabbits and the concomitant rapid rate of ovum transport through the oviduct. A second variable between previous and present studies is the site of deposition of the sperm suspension, which was vaginal in the earlier studies and oviductal in the present experiments. The temperature of ovarian transport (0 ° vs 37°C) did not affect the xenogenous fertilization rate of either porcine or bovine oocytes although a higher recovery rate from the rabbit oviduct was noted with bovine oocytes transported at 0°C. Fertilized bovine oocytes were recovered as early as 40 hr and as late as 75 hr after insemination. These were the temporal limits of these studies. Two cell embryos were observed at a recovery t%me of 70 hr which is similar to the time scale observed for normal in vivo or in vitro maturation and fertilization. Neither hamster nor cattle xenogenously fertilized embryos cleaved beyond the two cell stage. Previous studies (i) on xenogenous fertilization have produced eight cell squirrel monkey embryos but hamster embryos never progressed beyond the two cell stage, a phenomenon similar to that observed with in vitro fertilization in this species. The domestic animals used in these studies were not pretreated with gonadotropins. The oocytes aspirated from the follicle were generally immature with no polar body extrusion until after later maturation. In lieu of gonadotropin priming, in vitro maturation of the oocyte with FSH (and marginal LH contamination) was attempted but the fertilization rate was not affected. These preliminary studies suggest strongly the importance of future studies on in vivo maturation within the donor animal or by alternate means with in vitro culture.
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Because of the ease of xenogenous fertilization of hamster ova, this animal provides a useful model for developing techniques which can subsequently be applied to domestic species. The studies relating to the need for ligation of the tubal-uterine junction emphasize this approach. Ligation did not alter recovery or fertilization rate of the oocytes recovered from the rabbit oviduct within 32 hr. It is possible that differences might have been observed with later recovery times. By a similar test system it was determined that the number of oocytes placed in the oviduct did not alter fertilization rate but a higher percentage of recovery was noted when more than 40 oocytes were placed in each oviduct. Cleavage of xenogenously fertilized hamster oocytes occurred about 29 hrs after insemination, a value comparable with natural, in vivo fertilization. These results demonstrate that xenogenous fertilization of porcine and bovine oocytes can occur in the oviduct of the psuedopregnant rabbit. There are several important applications of such studies. If the embryos produced by this procedure are normal (which must ultimately be demonstrated by the successful transfer to a recipient mother and live birth following pregnancy) then this represents a source of embryos for transfer or for in vitro evaluation of the effects of chemical or environmental insult on embryonic development. This would include drugs having mutagenic or teratogenic effects. A second application might relate to the use of the procedure for assessing fertilization rates of specific sires. Current methods of fertility assessment are either time-consuming (nonreturn rates) or utilize sperm penetration of the zona-free ova of a foreign species (normally hamster). If xenogenous fertilization rates of bovine oocytes, fertilized by bovine sperm, are correlated with non-return fertility rates, then the technique has application for rapid screening of the fertility of bulls for potential use in artificial insemination. REFERENCES i.
DeMayo, F.J., Mizoguchi, H., and Dukelow, W.R. Fertilization of Squirrel Monkey and Hamster Ova in the Rabbit Oviduct (Xenogenous Fertilization). Science 2 0 8 : 1 4 6 8 (1980).
2.
Sreenan, J. In Vitro Maturation and Attempted Fertilization of Cattle Follicular Oocytes. J. Agric. Sci. (Camb.) 7 5 : 3 9 3 (1970).
3.
Bedirian, K.N., Shea, B.F., and Baker, R.D. Fertilization of Bovine Follicular Oocytes in Bovine and Porcine Oviducts. Can. J. Anim. Sci. 5 5 : 2 5 1 (1975).
4.
Trounson, A.O., Willadsen, S.M., and Rowson, L.E.A. Fertilization and Development Capability of Bovine Follicluar Oocytes Matured In Vitro and In Vivo and Transferred to the Oviducts of Rabbits and Cows. J. Reprod. Fert. 5 1 : 3 2 1 (1977).
5.
Edwards, R.G., Donahue, R.P., Baramki, T.A., and Jones, H.W. Preliminary Attempts to Fertilize Human Oocytes Matured In Vitro. Am. J. Obst. & Gynec. 9 6 : 1 9 2 (1966).
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6.
Adams, C.E., Rowson, L.E.A., Hunter, G.L., and Bishop, G.P. Long Distance Transport of Sheep Ova. Proc. 4th Intern'l Congr. Animal Reprod. and AI. The Hague 2 : 3 8 1 (1961).
7.
Pursel, V.G. and Johnson, L.A. Freezing of Boar Spermatozoa: Fertilizing Capacity with Concentrated Semen and a New Thawing Procedure. J. Anim. Sci. 4 0 : 9 9 (1975).
8.
Mizoguchi, H. and Dukelow, W.R. Gradual Fixation Method for Chromosomal Studies of Squirrel Monkey Oocytes After Gonadotropin Treatment. J. Med. Prim. (In Press) (1981).
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