In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep

ELSEVIER

IN VITRO AND IN VIVO DEVELOPMENTAL CAPAClTY OF OOCYTES FROM PREPUBERTAL AND ADULT SHEEP J. K. O’Brien,* S. L. Catt, K. A. Ireland, W. M. C. Maxwell and G. Evans Department of Animal Science, University of Sydney, NSW 2006, Australia Received for publication: Accepted:

October 14, 1996 December 5, 1996

ABSTRACT Development to the blastocyst stage and survival following embryo transfer were assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The rates of maturation, fertilization and cleavage in vitro did not differ significantly between oocytes from prepubertal and adult sheep. The proportion of cleaved zygotes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal than for those from adult sheep (15.4% and 34.1% respectively). There were no differences in the pregnancy rate and number of lambs born following transfer of blastocyst stage embryos derived from prepubertal and adult sheep to adult recipients. These data show that embryos derived from prepubertal lamb oocytes have reduced developmental potential in vitro but, of those which do reach the blastocyst stage, they have equal capacity to develop to term as embryos derived from adult sheep. 0 J.K. O’Brien et al 1997 Key words: sheep, prepubertal, in vitro culture, embryo transfer INTRODUCTION Assisted breeding technologies such as nuclear transfer (23, 48), in vitro maturation, fertilization and culture (17, 27, 40), and intracytoplasmic spermatozoa injection (22,45) are making possible the production of large numbers of embryos from live or slaughtered animals. The use of oocytes derived from prepubertal animals in conjunction with embryo transfer and the aforementioned procedures could increase the rate of genetic gain in livestock breeding programs through a reduction of the generation interval (6, 28, 29. 38, 49). The production of viable embryos following in vitro maturation and in vitro or in vivo fertilization has been reported for prepubertal cattle (1, 3, 18, 35), sheep (2, 11, 12, 19, 31) and goats (26). Several researchers have shown that fewer embryos develop in vitro from oocytes derived from prepubertal animals than from adult animals (cattle: 20,21,32,37; sheep: 30,31; pigs: 34). Acknowledgments This research was supported by grants from The University of Sydney Research Grants Scheme and The Australian Research Council. The authors thank Dr C. R. Earl for his helpful advice, Dr. K. A. J. Sedgers for his obstetrical advice and assistance, and MS A. M. Hansen, MS K. Heasman, Mr A. Souter and Mr A. Scherer for their technical assistance. *Correspondence and reprint requests. Theriogenology 0 J.K. O’Brien

47:1433-1443, et al. 1997

1997

0093-691X/97/$17.00 PII SOO93-691X(97)00134-9

1434

Theriogenology

In vivo development following the transfer of in vitro produced embryos from prepubertal animals to adult recipients has been investigated, and the establishment of pregnancies or production of live offspring has been demonstrated for cattle (1, 18) and sheep (2,13). The former studies have utilized prepubertal animals which had undergone various hormone treatments to stimulate follicle and oocyte growth prior to collection of oocytes. Comparative studies involving the transfer of embryos derived from unstimulated prepubertal animals have only been reported for cattle (37), and pregnancies were lost after Day 21. The aim of the present study was to examine the developmental capacity of oocytes from unstimulated prepubertal and normally cyclic adult sheep after 1) in vitro maturation, fertilization and culture to the blastocyst stage and 2) transfer of these blastocyst-stage embryos to adult recipients. MATERIALS AND METHODS In Vitro Maturation, Fertilization and Culture Methods of in vitro embryo production in the present study have been described previously (31). Ovaries were collected from 60 prepubertal crossbred (predominantly Merino, 4 to 6 mo), and 84 adult purebred Merino sheep (3 to 5 yr) at an abattoir on 4 separate days. They were transported at 37°C to the laboratory and washed 3 times in a PBS solution containing 250 IU/ml penicillin (Sigma, St Louis, MO, USA), 95 IU/ml streptomycin (Sigma) and 50 pg/ml neomycin (Sigma; PBS+). Oocytes were aspirated from follicles 2 to 4 mm in diameter using a 23g needle and 2ml syringe and washed 3 times in a HEPES-buffered Medium 199 (25 mM HEPES; Sigma; H199) containing 10% (v/v) fetal bovine serum (FBS; Multise?, Cytosystems P/L, Castle Hill, NSW, Australia; H199+). Those oocytes with a homogenous ooplasm and surrounded by several layers of cumulus cells were matured in multiwell dishes (Nunclon; Inter Med. Roskilde, Denmark), with each well containing 30 to 40 cumulus-oocyte complexes and 500 ~1 of maturation medium under paraffin oil (Ajax Chemicals, Auburn, NSW, Australia), for 22 h at 39°C in a humidified atmosphere of 5% CO2 in air. The maturation medium was Medium 199 (with Earle’s salts, L-glutamine, 2200 mg/l sodium bicarbonate, 25 mM HEPES, Gibco BRL, Grand Island, NY) supplemented with 0.3 mM pyruvate, 0.3 mM glutamine, 10% FBS (v/v), lO@ml FSH (Folltropin-V; Vetmpharm, Essendon, Victoria, Australia) and 10 pg/rnl LH (Lutropin-V; Vetrepharm). The average time from slaughter of animals to the beginning of culture was 3 h and 55 min f 7 min. Following in vitro maturation, oocytes were washed twice in H199+ followed by gentle pipetting to dissociate surrounding cumulus cells to leave 1 or 2 layers of corona cells intact. Groups of 30 to 40 oocytes were transferred to multiwe dishes (Nunclon), each well containing 500 pl of fertilization medium under a layer of paraffin oil. The fertilization medium comprised bicarbonate-buffered synthetic oviduct fluid medium (SOF, 40) supplemented with 2% sheep serum (v/v; collected and processed from a ewe on Day 3 of the estrous cycle). Oocytes were inseminated with frozen-thawed semen prepared by centrifugation on a Percoll (Sigma) gradient. The final concentration of spermatozoa used for inseminations was 0.3 x 106 and 0.5 x 106 motile sperm/ml for prepubertal and adult oocytes, respectively. After 18 h of culture in fertilization medium under the same conditions described for oocyte maturation, presumptive zygotes were washed twice in H199+ to remove spermatozoa and cell debris. They were then transferred to 500 l.tl of SOF supplemented with 0.3% BSA (v/v; Sigma A-7030, Lot # 24H0174) and MEM-

Theriogenology

1435

nonessential and essential amino acids (Gibco-BRL) under a layer of paraffin oil, and cultured for 6 or 7 d at 39°C under low oxygen tension (5% CO2,5% 02.90% N2). Oocyte and Embryo Assessment The number of oocytes undergoing maturation and fertilization was determined by removing approximately 10 oocytes from each group on Day 1 (day of insemination being Day 0). Oocytes were fixed in ethanol:acetic acid (3:l) for 24 h and stained with 0.5% orcein and examined under phase contrast microscopy (x 400 magnification). The criteria for maturation were a metaphase II nucleus and an abstricted polar body, while fertilization was confirmed by the presence of 2 pronuclei and 2 polar bodies. The number of oocytes cleaving was assessed visually (light microscopy, x 100 magnification) on Day 2 post insemination, and the number of oocytes developing to the blastocyst stage was assessed on Days 5, 6 and 7 post insemination. Criteria for blastocyst formation was the presence of a blastocoele cavity. Five developmental stages were assigned: early blastocyst (blastocoele cavity beginning to form), blastocyst (blastocoele cavity formed), expanded blastocyst (enlarged zona pellucida), hatching blastocyst and hatched blastocyst. Blastocysts were therefore classified in terms of their 1) growth rate during in vitro culture (day of culture the blastocyst stage was observed) and 2) developmental stage at transfer. Embryo Transfer Estrus was synchronized in 50 mature Merino ewes (4 to 5 yr old, 38.1 f 0.60 kg live weight) by treatment with progestagen-impregnated intravaginal sponges (Chrono-gest 30; Intervet. Castlehill, NSW, Australia) inserted for 12 d. Ewes received PMSG (400 IU im, Folligon; Intervet) at the time of sponge removal. Embryo transfer was performed by mini-laparotomy 6 d following the onset of estrus on 2 separate days (Replicates 1 and 2). Recipient ewes were treated with local anaesthetic (5ml, Lignocaine; Ilium, Troy Laboratories, Smithfield, NSW, Australia), and the ovaries were visualized by manipulation with a probe to check for the presence of corpora lutea under standard laparoscopic procedures (14). Four ewes had not ovulated while 1 ewe had an adhered reproductive tract, and were excluded. The uterus was located by laparoscopy and the tip of either the right or left uterine horn was exteriorised through a 2cm mid-ventral incision. The uterus was punctured with a sterile paper clip close to the utero-tubal junction. Two blastocysts from the same treatment group (prepubertal or adult) which had developed to the blastocyst stage on the same day (Day 5, 6 or 7). and at the same developmental stage (early blastocyst, blastocyst, expanded blastocyst, hatching blastocyst and hatched blastocyst) following 6 or 7 d of in vitro culture, were transferred to each recipient. Blastocysts were deposited using a 10~1 capillary pipette (Unopette; Becton Dickinson, Rutherford, NJ) attached to a lml syringe. The uterine horn was returned into the abdominal cavity and the incision was sutured. Recipient ewes received antibiotics (5ml Penstrep; Iiium, Troy Laboratories, Smithfield, NSW, Australia). Pregnancy Diagnosis Pregnancy status in recipient ewes was determined by ultrasonography, either 67 (Replicate 1) or 45 (Replicate 2) days following transfer.

1436 Synchronization of Partuition and Lamb Management Due to the relatively small size of the recipient ewes compared with the large oocyte donor (abattoir) ewes, parturition was induced to minimize dystociaassociated mortalities. This was performed by an intramuscular injection of 15 mg estradiol benzoate (Intervet) on Days 146 and 142 of pregnancy (Replicates 1 and 2, respectively). Ewes were penned and kept under observation from the time of induction treatment. Obstetrical assistance was given when necessary, and all lambs were identified. Birthweight was recorded within 1 h of lambing. Statistical Analysis Statistical differences in the proportion of oocytes undergoing maturation and fertilization and cleaved oocytes developing to the blastocyst stage were determined by the Chi-square test. Proportional data for pregnancy status, twinning, sex ratio and the number of lambs born were also analyzed by the Chi-square test. Lamb birthweight means were compared by Student’s t - tests. A value of P c 0.05 was considered significant. RESULTS In Vitro Maturation, Fertilization and Development in Culture The proportions of oocytes undergoing fertilization and development to the blastocyst stage are presented in Table 1. The proportion of oocytes undergoing maturation and fertilization, and the proportion of zygotes that had cleaved 48 h after insemination did not differ between prepubertal and adult sheep. However, there was an increased incidence of polyspermic fertilization in prepubertal oocytes (30.1%) compared with that in adult oocytes (17.6%; P < 0.05). In addition, the proportion of oocytes developing to the blastocyst stage was significantly greater for oocytes obtained from adult (34.1%) than from prepubertal sheep (15.4%; P < 0.001). The development of embryos to any of the blastocyst stages by Day 5 of culture only occurred in embryos derived from adult sheep. Development to blastocyst stages occurred predominantly by Day 6 of culture for both prepubertal and adult derived embryos (Figure 1). Embryos derived from prepubertal and adult animals were morphologically indistinguishable at each developmental stage. Embryo Transfer Data for the number of recipient ewes pregnant, as determined by ultrasound, and the number of lambs born are presented in Table 2. The number of lambs born as a proportion of the number of embryos transferred was not different for oocytes derived from prepubertal(7/20; 35.0%) and adult sheep (2W70; 40.0%).

166

250

Prepubertal sheep

Adult sheep

250 (1OO)a

166 (1OO)a

No. of oocytes matured(%)

175 (7o.o)a

119 (71.7)a

No. of oocytes fertilized (%) (%I

44 (17.6)b

50 (3O.l)a

~Y@S

No. of polyspermic

205 (82.0)a

136 (81.9)a

No. of oocytes cleaved (%)

70/205 (34.1)d

21/136 (15.4)~

No. of oocytes forming blastocysts/ no. cleaved (%)

Values with a different superscript within the same column are significantly different. abPcO.05; cdP
No. of oocytes cultured

Oocyte source

Table 1. Maturation, fertilization and early embryo development of oocytes from prepubertal and adult sheep.

Theriogenology

0

&pubertal sheep

n Adult sheep

*

-m Day 5

Day 6

Day 7

Day of culture Figure 1. Day of culture and development to the blastocyst stage of prepubertal and adult sheep oocytes matured and cultured in vitro to the blastocyst stage. * Significantly different within day of culture (PcO.05). There was also no difference in the in vivo embryo survival rate between embryos transferred after 6 (15/33; 45.5%) and 7 (20/57; 35.1%) days of culture. Although not statistically significant, the number of lambs born as a proportion of the number of embryos transferred was greatest when embryos were transferred at the expanded blastocyst stage (lW40; 45.0%) compared with that of the early blastocyst (l/4; 25.0%), blastocyst (7/22; 31.8%) and hatching or hatched blastocyst (9/24; 37.5%) stages (prepubertal and adult data pooled). Table 2. In vivo survival of transferred in vitro-produced embryos derived from prepubertal and adult sheep. Source of oocytes

No. of recipients

No. of recipients diagnosed pregnant by ultrasound (%)

No. lambs born/ no. of embryos transferred (8)

Prepubertal sheep

10

6/10 (60.0)

7/20 (35.0)

Adult

35

23l35 (65.7)

28/70 (40.0)

sheep

1439

Theriogenology

Parturition and Lambing Performance A week prior to lambing, 1 ewe died, and post mortem examination indicated a twisted segment of bowel was the cause of death. The lamb from the ewe was recovered, but data for its birthweight was not included in the statistical analyses. Following estradiol benzoate treatment, parturition had occurred in all ewes within 26.6 + 10 h and 34.6 + 8 h (Replicates 1 and 2, respectively). The incidence of twin pregnancies that resulted from the transfer of prepubertal and adult derived embryos was not significantly different (l/10 vs 5/35, respectively). The mean birthweight of lambs born from singleton pregnancies did not differ between those derived from prepubertal and adult animals (4.22 + 0.30 kg vs 4.09 f 0.19 kg, respectively), and was not affected by the day the blastocyst stage was first observed in culture, by the blastocyst developmental stage or by the age of blastocyst stage embryo at transfer (data not presented). The mean birthweight of lambs born from twin pregnancies was significantly lower than those derived from singleton pregnancies (2.92 f 0.25 kg vs 4.12 f. 0.16 kg; PcO.001). as would be expected. Of the 28 recipient ewes that lambed, 4 died of postparturient complications due to pregnancy toxemia, 1 died of a peritonitis following caesarean section, and 1 had a prolapsed uterus and was euthanazed. Of the 34 lambs born, 21 required obstetrical assistance and 3 caesareans were performed. Eight lambs were stillborn and 4 died during the following week. The total sex ratio of lambs was shifted towards females (female: 23/35, 65.7%; male: 12/35, 34.3%), but was not different from the expected ratio of 50%. DISCUSSION The results from the present study show an increased incidence of polyspermic fertilization and a reduced in vitro developmental capacity of embryos derived from oocytes of prepubertal compared with that of adult sheep confirming previous findings (31). While this has been observed in other species (cattle: 20.32.37; pigs: 34), several studies have shown similar rates of in vitro development to the blastocyst stage of embryos derived from in vitro maturation and fertilization (cattle: 3; sheep: 12). Oocytes in those studies were obtained from animals previously treated with gonadotrophins, and this in vivo hormone exposure may have improved the ability of oocytes to undergo normal maturation, fertilization and subsequent development in vitro. Previous studies have demonstrated the beneficial effect of premating hormone therapy on the fertility of peripubertal sheep (5). Estrogen- and progesterone-treated lambs had a lower incidence of embryonic mortality following mating at their third estrus than lambs receiving no treatment. However, a study involving a similar hormone pretreatment of prepubertal cattle found an increase in the number of harvestable oocytes but no improvement in the in vitro development and in vivo survival following embryo transfer (37). Those authors suggest that inadequate gonadotrophin exposure was responsible for the reduced developmental capacity of oocytes due to the very young age of the animals (3 mo). In the sheep, primary oocytes enclosed in primordial follicles begin to grow at birth, with 2 to 5 primordial follicles having a diameter of less than 0.06 mm entering the growth phase each day (44). Once follicles enter the growth phase they are dependent on gonadotrophins (25) and take 6 mo to reach a preovulatory size (8). Hence, when oocytes of prepubertaI animals are removed from antral follicles for maturation and fertilization in vitro, subsequent abnormal and compromised development may result from inadequate exposure to hormones and other follicular components they would otherwise encounter.

Theriogenology

An earlier study (24) reporting poor in vivo survival of transferred in vivo matured and fertilized oocytes from prepubertal compared with those from adult sheep attributed the lower reproductive performance to poor oocyte quality. Several recent studies have shown disturbances during maturation of oocytes obtained from prepubertal animals. Differences in glutamine metabolism and ultrastructure (31). protein deficiencies (20) and sensitivity of Ca*+ release mechanisms (9, 10) have also been observed between in vitro matured oocytes obtained from prepubertal and adult animals. In the present study, the rates of in vitro maturation, in vitro fertilization and cleavage were similar for prepubertal and adult oocytes, but a higher incidence of polyspermic fertilization was observed with prepubertal oocytes. However, inseminating in vitro matured oocytes from prepubertal sheep with a low concentration of spermatozoa (0.3 x 106 sperm/ml vs 0.5 x 106 sperm/ml) only slightly reduced the incidence of polyspermic fertilization observed in a previous study (30.1 vs 34.68, respectively; 31). The development of embryos to any of the blastocyst stages by Day 5 of culture only occurred in embryos derived from adult sheep. Despite this feature and the reduced proportion of prepubertal derived oocytes developing to the blastocyst stage by Day 7 of culture, embryos from both the prepubertal and adult derived animals were morphologically indistinguishable at each developmental stage. The in vivo viability of in vitro produced embryos as assessed by birth of lambs following embryo transfer was similar for blastocysts derived from prepubertal and adult sheep. In a previous study, cell numbers of blastocysts derived from prepubertal and adult sheep oocytes were similar at all morphological stages (31). These fmdings are supported by the present study if the cell number is used as an indication of viability, since comparable rates of in vivo developmental capacity of blastocyst stage embryos from prepubertal and adult animals was observed. However, the incidence of early embryonic mortality was not recorded, and differences between the 2 groups may have been present. In vitro culture of embryos has been associated with abnormal development, manifested as retarded morphological development at the blastocyst stage such as a reduction in embryo cell numbers (4.33.36.46). The transfer of embryos cultured in vitro has also resulted in reduced pregnancy rates (15,41) and increased birthweight, gestation length and incidence of dystocia and lamb mortalities (16, 42, 46, 47). Furthermore, significantly greater growth rates and testis weight of male lambs derived from in vitro produced embryos compared with that of lambs derived from natural matings have been reported (7). The birthweight and growth weight disparities appear to be associated with the inclusion of sera in the culture medium, since normal birthweights in a previous study (43) and normal birthweights in the present study were observed following the transfer of embryos cultured in media containing BSA and amino acids in place of sera. The large mortality rate observed in the present study can be attributed to the small physical size of the recipient ewes, to their relatively poor body condition score due to inadequate pasture growth during the last trimester of gestation, and to the subsequent high incidence of dystocia. The present study has demonstrated that oocytes from prepubertal and adult sheep have a different capacity for in vitro development. However, once development to the blastocyst stage has been achieved, in vivo development appears to be similar for oocytes derived from both prepubertal and adult sheep. Further investigation into the process of cytoplasmic maturation in prepubertal oocytes is

1441

Theriogenology

needed in order to improve the rate of fertilization and subsequent development to the rblaStOCySt * Stage. REFERENCES 1. Armstrong DT, Holm P, Irvine B, Petersen BA, Stubbings RB, McLean D, Stevens G, Seamark RF. Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration. Theriogenology 1992; 38667-678. 2. Armstrong DT, Irvine B, Earl CR. In vitro fertilization (IVF) of follicular oocytes from juvenile lambs and their developmental competence in vitro and in vivo. Biol Reprod 1994; 52(Suppl); 189 abstr. 3. Armstrong DT, Irvine B, Earl CR, McLean D, Seamark RF. Gonadotrophin stimulation regimens for follicular aspiration and in vitro embryo production from calf oocytes. Theriogenology 1994; 42:1227-1236. 4. Batt PA, Gardner DK, Cameron AWN. Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro. Reprod Fertil Dev 1991; 3:601-607. 5. Beck NFG, Davies MCG. The effect of stage of breeding season or pre-mating oestrogen and progestagen therapy on fertility in ewe lambs. Anim Prod 1994; 59~429-434. 6. Brash LD. Advanced breeding techniques for wool sheep improvement. Wool Tech Sheep Breed 1994; 42:327-337. 7. Brown BW. Radziewic T. In vitro production of sheep embryos and growth rates of resultant lambs. Proc 13th Inter Congr Anim Reprod 1996; P22-16 abstr. 8. Cahill LP, Mauleon P. Influences of season, cycle and breed on follicular growth rates in sheep. J Reprod Fertil 1980; 58:321-328. 9. Damiani P, Fissore RA, Cibelli JB, Rob1 JM, Duby RT. Evaluation of cytoplasmic maturation of calf oocytes. Theriogenology 1995; 43:191 abstr. 10. Duby RT, Damiani P, Looney CR, Fissom RA, Rob1 JM. Prepubertal calves as oocyte donors: promises and problems. Theriogenology 1996; 45: 121- 130. 11. Earl CR, Irvine BJ. Armstrong DT. Development of techniques for the production of viable embryos from six- to seven-week-old lambs. Proc Aust Anim Prod 1994; 20~428 abstr. 12. Earl CR, Irvine BJ, Kelly JM, Rowe JP, Armstrong DT. Ovarian stimulation protocols for oocyte collection and in vitro embryo production from 8-to 9week-old lambs. Theriogenology 1995; 43:203 abstr. 13. Earl CR, Rowe JP, Kelly JM, DeBarro TM. Pregnancy rates for fresh and frozen ovine IVP embryos from juvenile and adult donors. Proc 13th Inter Congr Anim Reprod 1996; P18-17 abstr. 14. Evans G and Maxwell WMC. Salamon’s Artificial Insemination of Sheep and Goats.Sydney: Butterworths, 1987; 154-166. 15. Gandolfi F, Moor RM. Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells. J Reprod Fertil 1987; 81:23-28. 16. Hohn P, Walker SK, Petersen BA, Ashman RJ, Seamark RF. In vitro vs in vivo culture of ovine IVM-IVF ova: effect on lambing. Theriogenology 1994; 41:217 abstr. 17. Hunter RHF, Lawson RAS, Rowson LEA. Maturation, transplantation and fertilization of ovarian oocytes in cattle. J Reprod Fertill972; 29:203-213. 18. Kajihara Y, Blakewood EG, Myers MW, Kometani N, Goto K, Godke RA. In vitro maturation and fertilization of follicular oocytes obtained from calves. Theriogenology 1991; 35:220 abstr.

1442

Theriogenology

19. Ledda S, Bogliolo L, Calvia P. Leoni G, Naitana S. Developmental competence of follicular oocytes from juvenile lambs matured in vitro in different conditions. J Reprod Fertil1996; 17(Suppl):28 abstr. 20. LCvesque JT, Sirard MA. Proteins in oocytes from calves and adult cows before maturation: relationship with their developmental capacity. Reprod Nutr Dev 1994; 34:133-139. 21. Looney CR, Damiani P, Lindsey BR, Long CR, Gonseth CL, Johnson DL, Duby RT. Use of prepubertal heifers as oocyte donors for IVF:effect of age and gonadotrophin treatment. Theriogenology 1995; 43:269 abstr. 22. Markert CL. Fertilization of mammalian eggs by sperm injection. J Exp Zoo1 1983; 228: 195-201. 23. McGmth J, Solter D. Nuclear and cytoplasmic transfer in mammalian embryos. In Gwatkin RBL (ed), Developmental Biology. Vo14: Manipulation of Mammalian Development. New York-London: Plenum Press, 1985; 37-55. 24. McMillan WH, McDonald MF. Survival of fertilized ova from ewe lambs and adult ewes in the uteri of ewe lambs. Anim Reprod Sci 1985; 8:235-240. 25. McNeilly AS, Jonassen JA, Fraser HM. Suppression of follicular development after chronic LHRH immunoneutralization in the ewe. J Reprod Fertill986; 76:481-490. 26. Mogas T, Izquierdo MD, Palomo MJ, Paramio MT. Effect of hormones, serum source and culture system on the IVM and IVF of prepubertal goat oocytes and subsequent embryo development. Theriogenology 1995; 43:284 abstr. 27. Moor RM, Trouson AO. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil1977; 49:101-109. 28. Nicholas FW. The genetic implications of multiple ovulation and embryo transfer in small dairy herds. Proc 30th Annual Meeting EAAP Harrogate 1979; CG1.11. 29. Nicholas FW. Genetic improvement through reproductive technology. Anim Reprod Sci 1996; 42:205-214. 30. O’Brien JK, Dwarte D, Ryan JP, Maxwell WMC, Evans G. (1995). Developmental capacity, energy metabolism and ultrastructure of oocytes from prepubertal and adult sheep. Proc Aust Sot Reprod Biol 1995; 27:30 abstr. 31. O’Brien JK, Dwarte D, Ryan JP, Maxwell WMC, Evans G. Developmental capacity, energy metabolism and ultrastructure of oocytes from prepubertal and adult sheep. Reprod Fertil Dev 1996; 8:1029-1037. 32. Pahna GA, Clement-Sengewald A, Kreft H. In vitro production of cattle embryos from calf oocytes. Theriogenology 1993; 39:278 abstr. 33. Papaioannou VE, Ebert KM. Development of fertilized embryos transferred to oviducts of immature mice. J Reprod Fertill986; 76:603-608. 34. Pinkert CA, Kooyman DL, Baumgartner A, Keisler DH. In-vitro development of zygotes from superovulated prepubertal and mature gilts. J Reprod Fertil1989; 87:63-66. 35. Presicce GA, Jiang S, Simkin M, Yang X. Oocyte quality and embryo development in prepubertal calves. Biol Reprod 1995; 52(Suppl):127 abstr. 36. Quinn P, Warnes GM, Walker SK, Seamark RF. Culture of preimplantation sheep and goat embryos. In: Lindsay DR and Pearce DT (eds.), Reproduction in sheep. Canberra: Australian Academy of Science and Wool Corporation, 1984; 289290. 37. Revel F, Mermillod P, Peynot N, Renard JP, Heyman Y. Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows. J Reprod Fertil 1995; 103:115-120. 38. Smith C. Use of embryo transfer in genetic improvement of sheep. Anim Prod 1986; 42:81-88.

1443

39. Staigmiller RB, Moor RM. Effect of follicle cells on the maturation and

40. 41. 42.

43. 44. 45. 46. 47. 48. 49.

developmental competence of ovine oocytes matured outside the follicle. Gamete Res 1984; 9:221-229. Tervit HR, Whittingham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972; 30:493-497. Tervit HR, Rowson LEA. Birth of lambs after culture of sheep ova in vitro for up to 6 days. J Reprod Fertil 1974; 38:177-179. Thompson JG, Simpson AC, Pugh PA, McGowan LT. James RW, Berg DK, Payne SR, Tervit HR. Effect of glucose level in culture medium on survival of in vitro cultured sheep embryos following transfer to recipient ewes. Proc NZ Sot Anim Prod 1992; 52:255-256. Thompson JG, Gardner DK, Pugh PA, McMillan WH, Tervit HR. Lamb birth weight following transfer is affected by the culture system used for preelongation development of embryos. Biol Reprod 1995; 1385-1391. Turnball KE, Braden AWH, Mattner PE. The pattern of follicular growth and atresia in the ovine ovary. Aust J Biol Sci 1977; 30:229-241. Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod 1976; 15:467-470. Walker SK, Heard TM, Seamark RF. In vitro culture of sheep embryos without co-culture: successes and perspectives. Theriogenology 1992; 37: 11 l- 126. Walker SK, Hartwich KM, Seamark RF. The production of unusually large offspring following embryo manipulation: concepts and challenges. Theriogenology 1996; 45:111-120. Willadsen SM. Nuclear transplantation in sheep embryos. Nature (Lond). 1986; 320:63-65. Wray NR, Goddard ME. MOET breeding schemes for wool sheep 1. Design alternatives. Anim Prod 1994; 59:71- 86.