Small Ruminant Research 29 1998.

8388

A protocol for in vitro maturation and fertilization of sheep oocytes

S. Wang
a

a, )

, Y. Liu

a,b

, G.R. Holyoak a , R.C. Evans a , T.D. Bunch

Department of Animal, Dairy and Veterinary Sciences, Utah State Uniersity, Logan, UT 84322-5600, USA b Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100094, China Accepted 21 June 1997

Abstract Two in vitro maturation IVM. and fertilization IVF. protocols were evaluated with the objective of improving upon a protocol that will optimize IVF-assisted reproduction in sheep. There was no attempt to compare possible confounding effects within protocol. The addition of follicle-stimulating hormone FSH. and luteinizing hormone LH. to the maturation IVM. medium significantly enhanced ovine oocyte maturation as compared to human chorionic gonadotropin hCG. alone. Oocytes fertilized in vitro with spermatozoa treated with calcium ionophore A23187 and caffeine in Brackett and Oliphant medium Protocol 1. significantly improved oocyte fertilization and embryo development in vitro than with spermatozoa separated by swim-up in synthetic oviduct fluid containing estrus ewe serum Protocol 2.. Embryos derived from oocytes matured in medium containing FSH and LH and fertilized with Protocol 1 were transferred to synchronized recipients and resulted in the birth of lambs. This protocol may be useful for assisted reproduction in sheep. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: In vitro maturation; Fertilization; Sheep oocytes; Follicle-stimulating hormone; Human chorionic gonadotropin

1. Introduction A considerable effort in reproductive biology has been focused on the development of procedures for the in vitro maturation IVM. and in vitro fertilization IVF. of oocytes for basic and applied research, especially as they apply to livestock reproduction Bavister, 1995.. Pregnancies have resulted from the transfer of IVMrIVF produced embryos in several domesticated animals we.g., cattle Brackett et al., 1982; Xu et al., 1987., swine Cheng et al., 1986., goats Hanada, 1985; Younis et al., 1991; de Smedt
)

Corresponding author.

et al., 1992; Keskintepe et al., 1994., horses Palmer et al., 1991., and sheep Crozet et al., 1987; Czlonkowska et al., 1991.x. Efficiency and consistency are factors influencing the selection of an in vitro embryo production protocol for application in sheep reproduction. Addition of follicle-stimulating hormone FSH. and luteinizing hormone LH. to maturation medium has improved embryo production in vitro Bavister et al., 1992; Keefer et al., 1993; Keskintepe et al., 1994.. Human chorionic gonadotropin hCG. is often used as a substitute for LH Ward et al., 1991.. Whether hCG is as good as or better than FSH and LH for in vitro maturation of sheep oocytes needs to be investigated.

00921-4488r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 9 2 1 - 4 4 8 8 9 7 . 0 0 0 9 8 - 9

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S. Wang et al. r Small Ruminant Research 29 (1998) 8388

Estrus ewe serum EES. has been used in fertilization media during the in vitro production of ovine embryos Walker et al., 1994, 1996.. Estrus ewe serum is an undefined biological product and variation in bioactivity results in inconsistencies in fertilization rates. To abate variation in fertilization rate, calcium ionophore A23187 and caffeine have been used for in vitro capacitation of bull Zhang et al., 1992. and goat Hanada, 1985. spermatozoa. Calcium ionophore A23187 has not been evaluated in sheep in vitro fertilization systems. In this study, we evaluated two IVM and IVF protocols to determine which has the greater efficacy to enhance assisted reproduction in sheep. We compared IVM protocols that use either FSH and LH or hCG alone. We also compared two IVF protocols, whereby oocytes were fertilized in vitro either with spermatozoa treated with calcium ionophore A23187 and caffeine in Brackett and Oliphant medium or with spermatozoa separated by swim-up in synthetic oviduct fluid containing estrus ewe serum. Sheep embryos produced in what we considered the best protocols were transferred to synchronized recipients to determine whether the IVMrIVF system would result in the development of normal lambs.

2. Materials and methods 2.1. Experiment 1: In itro maturation of oocytes This experiment compared two protocols for in vitro maturation of ovine oocytes. Sheep ovaries were collected from a local abattoir located 72 km from our laboratory and maintained at 308C in phosphate buffered saline containing 100 IU potassium penicillin-G and 100 m g streptomycin sulfate per ml. Oocytes were recovered by slicing the ovaries in Hepes TALP Parrish et al., 1988.. Cumulus cell compacted oocytes were randomly distributed into experimental treatments TRT.. The time from the collection of ovaries to when the oocytes were placed into maturation medium was approximately 5 h. For TRT 1, oocytes n s 134. were matured in tissue culture medium-199 TCM-199; Cat. No. 12340-022, Gibco BRL, Life Technologies, Grand Island, NY, USA. containing 10% volrvol. fetal bovine serum FBS; Cat. No. A 1111-L, HyClone Laboratories,

Logan, UT, USA., 0.5 m grml of FSH Cat. No. F 8001, Sigma Chemical, St. Louis, MO, USA., 5.0 m grml of LH Cat. No. L 9773, Sigma Chemical., 1.0 m grml of estradiol Cat. No. E 2258, Sigma Chemical., 100 IU of potassium penicillin-G, and 100 m grml of streptomycin sulfate. For TRT 2, oocytes n s 147. were matured in the same medium as in TRT 1 except that FSH and LH were replaced with 4.0 m grml of hCG Cat. No. C 1063, Sigma Chemical.. The concentration of hCG used in TRT 2 was based upon the dose used for in vitro maturation of goat oocytes Ling et al., 1992.. Oocytes were cultured in plastic 4-well petri dishes NunclonR , Nunc, Naperville, IL, USA. under a humidified 5% CO 2 atmosphere at 398C for 27 h. Approximately 30 to 50 ova were placed in each well containing 400 m l of IVM medium. After 27 h of culture, oocytes were fixed in a solution of acetic acid:ethanol 1:3. and stained with 1% wt.rvol. Lacmoid. Stained oocytes were examined for nuclear developmental status at 600 = with phase contrast microscopy based on the following classification: 1. matured oocytes as shown by the formation of a metaphase spindle with expulsion of the first polar body; 2. unmatured oocytes in which no metaphase II plate was observed; and 3. degenerated oocytes in which the nucleic structure was not recognizable. The Chi-square test was used to determine whether the differences between the two treatments were significant. 2.2. Experiment 2: In itro fertilization of oocytes This experiment compared two IVF protocols using a randomized complete block design with two treatment protocols in each block. Each block consisted of oocytes from the same collection of ovaries from a local abattoir 5 replications.. One protocol contained calcium ionophore A23187 and caffeine in the IVF medium and the other estrus ewe serum EES.. Estrus ewe serum was separated from blood collected from two St. Croix sheep in estrus day 0. and combined. The serum was stored at y808C until it was used. Sheep oocytes were recovered by slicing ovaries and matured in vitro in TCM-199 containing 10% volrvol. FBS, 0.5 m grml of FSH, 5.0 m grml of LH, 1.0 m grml of estradiol, 100 IU of potassium penicillin-G, and 100 m grml of streptomycin sul-

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fate, in a humidified 5% CO 2 atmosphere at 398C for 27 h. Oocytes were then assigned to one of two IVF protocols. For Protocol 1, freshly collected ram semen was washed twice with Brackett and Oliphant medium BO; Brackett and Oliphant, 1975. containing 10 mM caffeine and 0.3% wt.rvol. bovine serum albumin BSA; Cat. No. A 6003, Sigma Chemical. and the spermatozoa lightly packed by centrifuging at 800 rpm 250 g. for 5 min Beckman, CPR Centrifuge.. The sperm pellet was resuspended with BO medium containing 1 mM caffeine and 0.6% BSA and the concentration was adjusted to approximately 30 = 10 6 spermatozoa per ml. The spermatozoa were then capacitated with 0.1 m M calcium ionophore A23187 Cat. No. C 7522, Sigma Chemical. in BO medium containing 1 mM caffeine and 0.6% BSA for 1 min as described by Zhang et al. 1992.. The treated spermatozoa were then added into the IVF medium BO medium containing 0.6% BSA. at the concentration of approximately 1.5 million spermrml. The oocytes n s 406. were added and cultured for 6 h. Plastic 4-well petri dishes NunclonR , Nunc. were used to culture oocytes. Approximately 30 to 50 ova were placed in each well containing 400 m l of IVF medium. For Protocol 2, freshly collected ram semen was washed twice with synthetic oviduct fluid SOF; Carolan et al., 1995. containing 0.3% wt.rvol. BSA Cat. No. A 6003, Sigma Chemical. and spermatozoa lightly packed by centrifuging at 800 rpm 250 g. for 5 min Beckman, CPR Centrifuge.. Washed sperm pellet was resuspended with SOF containing 5% volrvol. EES. A total of 100 m l sperm suspension was placed in the bottom of 1.5 ml of SOF containing 5% volrvol. EES for swimming up. The concentration of separated spermatozoa was adjusted to approximately 1.5 millionrml using SOF containing 5% volrvol. EES. Oocytes n s 353. were added to this medium containing the sperm and incubated for 17 h. Plastic 4-well petri dishes NunclonR , Nunc. were used to culture oocytes. Approximately 30 to 50 ova were placed in each well containing 400 m l of IVF medium. Although oocyte numbers allocated to treatments in Protocols 1 and 2 were randomly assigned, there were less oocytes in Protocol 2 primarily due to losses that occured in handling.

After in vitro fertilization, the ova from Protocol 1 and 2 were washed three times with TCM-199 containing 10% FBS and cultured in vitro IVC. in TCM-199 containing 10% FBS. Plastic 4-well petri dishes NunclonR , Nunc. were used to culture oocytes. Approximately 30 to 50 ova were placed in each well containing 400 m l of IVC medium. During all of the IVM, IVF, and IVC steps, oocytes were maintained in a humidified 5% CO 2 atmosphere at 398C. Cleavage rate was determined at 45 h after fertilization. Embryonic development was evaluated on day 9 of culture. Two-way analysis of variance ANOVA. was used to determine the significance between the effects of treatments on embryo development. The IVMrIVF produced embryos of what we judged to be the best protocol in Experiments 1 and 2 were transferred into recipients synchronized with progestogen 40 mg of fluorogestone pessaries, Intervet, Boxmeer, Netherlands.. Oocytes n s 24. were collected from one donor ewe during the breeding season and matured in vitro in TCM-199 containing 10% FBS, 0.5 m grml of FSH, 5.0 m grml of LH, and 1.0 m grml of estradiol as described for TRT 1 of Experiment 1. The oocytes were then fertilized in vitro with ram sperm capacitated with 0.1 m M calcium ionophore A23187 and caffeine as described in Protocol 1 for Experiment 2. The IVMrIVF derived ova were cultured in vitro as described previously. Eleven of day 5 embryos IVF s day 0. were surgically transferred into seven recipients.

3. Results and discussion As shown in Table 1, the rate of oocyte maturation was greater P - 0.05. in TRT 1 70.1%. which contained FSH and LH than in TRT 2 50.3%. which contained hCG. The occurrence of degenerated oocytes was less P - 0.05. in TRT 1 3.0%. than in TRT 2 13.6%.. Supplementation of maturation medium with reproductive hormones e.g., FSH and LH. has been shown in other studies to improve in vitro embryo production Bavister et al., 1992; Keefer et al., 1993.. The results from Experiment 1 indicate that the IVM protocol that supplemented FSH and LH in the medium was more effective in promoting oocyte maturation than the protocol with hCG.

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S. Wang et al. r Small Ruminant Research 29 (1998) 8388

Table 1 Comparison of two in vitro maturation protocols that contained either FSH and LH TRT 1. or hCG TRT 2. for the maturation of ovine oocytes n s 134 and 147, respectively. Treatment Matured oocytes N TRT 1: FSH and LH TRT 2: hCG
a,b

Unmatured oocytes N 36 53 % 26.9 36.1

Degenerated oocytes N 4 20 % 3.0 a 13.6 b

% 70.2 a 50.3 b

94 74

Means within columns without common superscripts differ P - 0.05..

The IVF Protocol in which spermatozoa were treated with calcium ionophore A23187 and caffeine in BO medium resulted in better fertilization and embryo development than the protocol which used EES in SOF medium. As shown in Table 2, the cleavage rate was greater P - 0.01. in Protocol 1 BO q ionophore A23187 and caffeine, 60.2%., than that in Protocol 2 EES in SOF medium, 40.0%.. The production of morulae and blastocysts was greater P - 0.05. in Protocol 1 36.5%. than in Protocol 1 22.9%. Fig. 1.. Because there were several variables differing between the two IVF protocols, such as media, the handling of spermatozoa, and the number of hours of oocytersperm coincubation, it would be inappropriate to ascribe these differences to any specific component in the medium. Possibly, however, the better IVF results from Protocol 1 may be related to the involvement of calcium ionophore A23187 and caffeine in the IVF medium. Spermatozoa will undergo an acrosomal reaction when exposed to ionophore A23187, which increases intracellular calcium Yovich et al., 1994; Luconi et al., 1996.. Caffeine in the fertilization medium facili-

tates Caqq uptake by the spermatozoa, thus, contributing to the acrosomal reaction Nagai et al., 1994.. Although EES has been reported to have a beneficial effect during in vitro production of ovine embryos Huneau et al., 1994., its biological activity varies depending on the source and may even be detrimental to sperm capacitation. Calcium ionophore A23187 and caffeine, unlike EES, are chemically defined components in IVF medium. The use of chemically defined components in medium for in vitro embryo production facilitates the application of in vitro techniques, provides for more consistent results and aids in the evaluation of other factors in the in vitro embryo production system Bavister et al., 1992; Carolan et al., 1995.. Table 3 shows the transfer results of IVMrIVF derived embryos. Twenty-four oocytes were collected from one donor ewe, matured in vitro in the medium containing FSH and LH, and then fertilized with spermatozoa capacitated with calcium ionophore A23187 and caffeine. Eleven morula-stage embryos were transferred to synchronized recipients and re-

Table 2 Comparison of embryonic development of ovine ova by the use of two protocols of in vitro fertilization nb s 406 and 353. Protocola Protocol 1 Protocol 2 Cleaved 249 142 % 60.2 40.0 d
e

M q Bc 155 83

% 36.5e 22.9 d

a Protocol 1: In vitro matured ovine oocytes were fertilized in vitro with ram spermatozoa treated with calcium ionophore and caffeine in Brackett and Oliphant BO. medium. Protocol 2: In vitro matured ovine oocytes were fertilized in vitro with ram spermatozoa separated by swim-up in synthetic oviduct fluid SOF. containing 5% volrvol. estrus ewe serum EES.. b Data obtained by use of a randomized complete block design with two treatment protocols in each replication. Each replication consisted of oocytes from the same collection of ovaries. c M q B indicates that oocytes developed into morulae or blastocysts. d,e Means within columns without common superscripts differ P - 0.05..

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Fig. 1. The sheep oocytes collected for in vitro embryo production top left., developed into 8-cell embryos top right., blastocysts bottom left. and hatching blastocysts bottom right. after IVM and IVF.

sulted in the birth of two live lambs, thus, showing that viable embryos can be produced in vitro by the use of the protocols described above. The birth of

only two lambs from 11 embryos, however, indicates that there is still room to improve on this technology if it is to have practical application.

Table 3 Lambs born from transferred embryos that were matured in vitro in medium containing FSH and LH TRT 1 in Experiment 1., and fertilized with spermatozoa treated with calcium ionophore A23187 and caffeine Protocol 1 in Experiment 2. Oocytes collected 2 to 8 cell embryos 16 cell embryos Morulaea Lambs from ET. ET s embry transfer a These embryos were transferred. 24 1 2 11 2

4. Conclusions Based on the comparison of two protocols of IVM and IVF and the birth of live IVF-ET lambs, the use of IVM medium containing FSH plus LH for in vitro maturation and the protocol involving the treatment of spermatozoa with calcium ionophore A23187 and caffeine in BO medium for in vitro fertilization may be a useful protocol for IVF assisted reproduction in sheep.

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S. Wang et al. r Small Ruminant Research 29 (1998) 8388 stimulating hormone and luteinizing hormone during bovine in vitro maturation and development following in vitro fertilization and nuclear transfer. Mol. Reprod. Dev. 36, 469474. Keskintepe, L., Darwish, G.M., Kinemer, A.T., Brackett, B.G., 1994. Term development of caprine embryos derived from immature oocytes in vitro. Theriogenology 42, 527535. Ling, L., Jufen, Q., Yong, Z., 1992. In vitro fertilization of goat ovarian oocytes. Theriogenology 37, 247, Abstr... Luconi, M., Krausz, C., Forti, G., Baldi, E., 1996. Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol. Reprod. 55, 207216. Nagai, T., Takenaka, A., Mori, T., Hirayama, M., 1994. Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture. Mol. Reprod. Dev. 37, 452 456. Palmer, E., Bezard, J., Magistrini, M., Duchamp, G., 1991. In vitro fertilization in the horse: A retrospective study. J. Reprod. Fert. 44, 375384. Parrish, J.J., Susko-Parrish, J., Winer, M.A., First, N.L., 1988. Capacitation of bovine sperm by heparin. Biol. Reprod. 38, 11711180. de Smedt, V., Crozet, N., Ahmed-Ali, M., Martino, A., Cognie, Y., 1992. In vitro maturation and fertilization of goat oocytes. Theriogenology 37, 10491060. Walker, S.K., Hill, J.L., Bee, C.A., Warnes, D.M., 1994. Improving the rate of production of sheep embryos using in vitro maturation and fertilization. Theriogenology 41, 331, Abstr... Walker, S.K., Hill, J.L., Kleemann, D.O., Nancarrow, C.D., 1996. Development of ovine embryos in synthetic oviductal fluid containing amino acids at oviductal fluid concentrations. Biol. Reprod. 55, 493497. Ward, D.N., Bousfield, G.R., Moore, K.H., 1991. Gonadotropins. In: Perry, T.C. Ed.., Reproduction in Domestic Animals, 4th edn. Academic Press, New York, NY, pp. 2580. Xu, K.P., Greve, T., Callasen, H., Hytel, P., 1987. Pregnancy resulting from cattle oocytes matured and fertilized in vitro. J. Reprod. Fert. 81, 501504. Younis, A.I., Zuelke, K.A., Harper, K.M., Oliverira, M.A.L., Brackett, B.G., 1991. In vitro fertilization of goat oocytes. Biol. Reprod. 44, 11771182. Yovich, J.M., Edirisinghe, W.R., Yovich, J.L., 1994. Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: A prospective, double-blind, randomized controlled study. Fert. Steril. 61, 902910. Zhang, L., Denniston, R.S., Godke, R.A., 1992. A simple method for in vitro maturation, in vitro fertilization and co-culture of bovine oocytes. J. Tiss. Cult. Meth. 14, 107112.

Acknowledgements This research was supported by grants from the Utah Department of Agriculture agreement no. 902720 and USDA-APHIS-VS agreement number 949-1-49-0233-C and by the Utah Agricultural Experiment Station, Utah State University, Logan. Approved as journal paper no. 4892. The authors thank Utah Beef and Lamb Inc., Ogden, UT, for their donation of the sheep ovaries.

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