In-vitro production of cattle embryos: problems with pregnancies and parturition

Transcription

1 Human Reproduction Vol. 15, (Suppl. 5) pp , 2000 In-vitro production of cattle embryos: problems with pregnancies and parturition John F.Hasler Em Tran, Inc., 197 Bossier Road, Elizabethtown, PA 17022, USA Using complex media containing serum and somatic cells, pregnancy rates following transfer of single, day 7 advanced-stage blastocysts approached 60%, while pregnancy rates of morulae or day 8 blastocysts were substantially lower. Pregnancies resulting from in-vitroderived embryos were characterized by the following features: sex ratio skewed in favour of males; increased spontaneous abortion rate throughout gestation; reduced intensity of labour in recipients; and increases in birth weights, dystocias, calf mortality, and fetal abnormalities. In an attempt to improve the normality of pregnancies, a field trial was conducted within the commercial in-vitro programme at Em Tran. Following fertilization in vitro, equal numbers of zygotes were put into a Menezo's B2-buffalo rat liver cell (B2-BRL) coculture system with (S+) or without (S-) 10% serum for the first 72 h of in-vitro culture. On day 4, all embryos were moved to fresh medium and cultured to day 7 in B2-BRL with serum. The efficiency of blastocyst production from oocytes (19.6 versus 17.8%) and the pregnancy rate at 60 days (47.8 versus 47.1%) did not differ between S+ and S-. Likewise, there was no difference between S+ and S- in the percentage of male calves (53.9 versus 54.3%), abortions (13.1 versus 11.9%), percentage of live calves (78.8 versus 81.4%), or congenital abnormalities (4.3 versus 3.3%). Key words: bovine/calves/embryo transfer/m vitro/ parturition Introduction The production of calves from in-vitro procedures became established commercially on an international basis during the 1990s. Several commercial embryo transfer programmes have reported on the production of pregnancies from infertile, genetically superior donor cows (Looney et al, 1994; Hasler et al, 1995; Bousquet et al, 1999), whereas reproductively healthy donors have been utilized in other programmes (Sauve, 1998; van Wagtendonkde Leeuw et al, 1998). Commercial embryo transfer programmes have also been based on production of in-vitro-derived embryos from slaughterhouseprocured ovaries (Lu and Polge, 1992; Sinclair et al, 1995; Kuwayama et al., 1996; Lazzari and Galli, 1996). The pregnancy rates resulting from transfers of in-vitro-derived embryos have averaged -50% at 60 days of gestation, slightly lower than the pregnancy rates from in-vivo embryos. A number of reports, however, have detailed problems with pregnancies resulting from the transfer of in-vitro-derived bovine embryos (Hasler et al., 1995; Walker et al, 1996; Farin and Farin, 1997; Kruip and den Daas, 1997; van Wagtendonk-de Leeuw et al, 1998, 1999). These problems have included heavier birth weights, extended gestation periods, higher rates of abortion and increased rates of perinatal mortality. In sheep, the birth weight of lambs was heavier when they resulted from IVF-derived embryos cultured in medium containing serum versus media containing bovine serum albumin (BSA) (Walker et al., 1992; Thompson et al, 1995). European Society of Human Reproduction and Embryology 47

2 J.F.Hasler The first section of the present study constitutes a retrospective analysis of the results over a 4 year period of oocyte retrieval from donor cows, pregnancy rates following transfer of embryos produced from in-vitro maturation (IVM), fertilization (IVF) and culture (IVC), and the outcome of the resulting pregnancies. The second section of the study examined the possible effects on pregnancies of the inclusion of serum during the first 3 days of bovine in-vitro embryo culture within the context of an on-going commercial embryo transfer programme. Materials and methods Oocyte recovery and in-vitro maturation For IVC experiments, bovine ovaries were transported to Em Tran from a slaughterhouse in a plastic bag, with no additional fluid added, within an insulated box at a temperature of C. Upon arrival in the laboratory, the ovaries were rinsed with 28 C tap water and placed for 5 min into 28 C tap water containing 1% Nolvasan solution (Aveco Co., Inc., Fort Dodge, IA, USA) and 1% 7X cleaning solution (ICN Biochemicals, Inc., Costa Mesa, CA, USA), after which the ovaries were rinsed copiously with tap water and maintained at ambient temperature (23-27 C) prior to aspiration. A 19-gauge -inch needle attached to a Pioneer Pro-Pump (Pioneer Medical, Inc., Madison, CT, USA) was used to aspirate the contents of all follicles between 2 and 10 mm in diameter into a 50 ml centrifuge tube. For production of genetically valuable embryos for transfer into recipients, oocytes were collected by transvaginal ultrasound-guided aspiration from client-owned cows, primarily Holsteins, with an Aloka 500 ultrasound monitor (Corometrics Medical Systems, Inc., Wallingford, CT, USA) and a 5 MHz sector scanning transducer housed together in a vaginal probe with a stainless steel guide containing a 17-gauge single lumen needle. Aspiration pressure was provided by a foot pedal-operated Pioneer Pro-Pump set at -50 mmhg. The follicular aspirate was rinsed with PBS through an Em-Con filter (Immuno Systems, Spring Valley, WI, USA). For IVC experiments, all oocytes of slaughterhouse origin with three or more compact layers of 48 cumulus cells were utilized for maturation and were not graded on the basis of cytoplasmic appearance, whereas all oocytes collected from donor cows with one or more layers of cumulus were utilized. All culture procedures, including IVM, IVF and IVC, were conducted in 4-well plates with no oil overlay (Nunclon, Roskilde, Denmark). Unless stated otherwise, all chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA). Following aspiration of slaughterhouse ovaries or oocyte retrieval on donor cows, oocytes were rinsed five times in a modified Tyrode's Medium (TALP; Bio Whittaker, Inc., Walkersville, MD, USA). Oocytes were placed into maturation medium within 5-9 h after the ovaries were collected at the slaughterhouse or within 30 min after oocyte retrieval. Groups of experimental oocytes or all the oocytes from individual donors were matured for h in 0.5 ml TCM-199 with Earle's salts, supplemented with 2.2 g/1 sodium bicarbonate (Gibco BRL, Grand Island, NY, USA), 10% heat-inactivated fetal calf serum (FCS; Hyclone Laboratories, Logan UT, USA), 4 ug FSH and 6 jxg LH (Sioux Biochemicals, Sioux Center, IA, USA). The oocytes were incubated at 39 C in a humidified atmosphere of 5% CO2 in air. In-vitro fertilization At the end of IVM, oocytes were rinsed twice in TALP and placed in 0.5 ml of fertilization medium, which consisted of modified Tyrode-lactatepyruvate medium (Fert-TALP; Bavister and Yanagimachi, 1977). Frozen semen was thawed in a 35 C water bath and layered on a discontinuous gradient of Percoll (Sigma) in a 15 ml centrifuge tube. The Percoll gradient was composed of 2 ml of 90% Percoll overlaid with 2 ml of 24% Percoll, both of which were prepared with Hepes Buffer Sperm TALP (SP-TALP; Parrish et al., 1988). After 30 min of centrifugation at 700 g, the sperm pellet was recovered, resuspended in SP-TALP and the sperm concentration determined with a haemocytometer. Depending on the bull, spermatozoa were added to fertilization wells at a concentration ranging from 0.1 to 0.5Xl0 6 /ml. At the time spermatozoa were added to the fertilization wells, 20 (xmol/1 penicillamine, 10 imol/l hypotaurine,

3 1 imol/l epinephrine and 2 ig/ml heparin were also added. After 18 h in IVF, oocytes were removed, rinsed twice in TALP, vortexed for 2 min to remove cumulus cells and then placed into in-vitro culture. In-vitro culture Experiment 1 During a 4 year period, following vortexing, presumed zygotes from donor cows were placed in 0.5 ml of either Menezo's B2 medium (Laboratoire C.C.D., Paris, France) containing 10% FCS or TCM-199 with 10 g/1 BSA (A-4503, Sigma) and 10% FCS (Hyclone) on a monolayer of buffalo rat liver cells (BRL; American Type Culture Collection, Rockville, MD, USA). The BRL were plated at a concentration of , or cells at 24, 48 or 72 h before use. On the fourth day of IVC, the embryos were transferred to fresh co-culture wells and cultures were continued to day 8 or 9. Experiment 2 Following IVF, presumptive zygotes derived from slaughterhouse-derived ovaries were cultured in B2-BRL co-culture either with or without 10% serum during days 1-4 (72 h) of IVC. On day 4, all embryos were transferred to fresh co-culture wells containing 10% serum. Six replicates were conducted. Experiment 3 Presumptive zygotes from the donor cows were allocated randomly to B2-BRL co-culture either with or without 10% serum. On day 4 of IVC, all embryos were transferred to B2-BRL co-culture including 10% serum. During the time period over which Experiment 3 was conducted (15 months), all embryos from an additional group of Holstein cows, at the request of the owner of the cows, were cultured exclusively on TCM-199-BRL with serum present throughout IVC. In both cases, incubation was conducted at a temperature of 39 C in a humidified atmosphere of 4% CO 2 in air. Most of the resulting embryos were transferred into recipients as described below. In-vitro production of cattle embryos Evaluation of embryos In-vitro-derived embryos in all three experiments were evaluated for stage (morula, M; early blastocyst, EB; mid-blastocyst, MB; expanded blastocyst, XB; hatched blastocyst, HB) and grade (excellentgood, 1; fair, 2) as described previously (Hasler et al., 1995). In-vivo-derived embryos in Experiment 1 were evaluated for stage in the same manner as in-vitro embryos, whereas grade was defined as: excellent, 1; good, 2; and fair, 3; as described previously (Hasler et al., 1987). Transfer of embryos In Experiments 1 and 3, blastocysts and late morulae (referred to hereafter as 'embryos'), were removed from culture medium on day 7 or 8 and rinsed in TALP prior to loading in a 0.25 ml straw. Single embryos were transferred non-surgically to the uterine horn ipsilateral to the corpus luteum of Holstein heifers that had been observed in oestrus between 2 days before (+2) and the same day as the IVF of the embryos (0 synchrony). Pregnancy was determined by rectal palpation at days of gestation. The sex of many of the pregnancies produced in this study was determined by ultrasound examination at Em Tran, Inc. before the recipients were transported to the farms of the donor owners. Also, whenever possible, the sex of the resulting calves was recorded. In addition, prior to transfer, biopsies were removed from some embryos via micromanipulation and sex was determined by polymerase chain reaction (Bredbacka et al, 1995). Data analysis Differences between and within treatments in embryo production, sex ratio, pregnancy rate, abortions and the number of calves dead at birth were determined by % 2 analysis. Results Experiment 1 Experiment 1 represents a retrospective analysis during a 4 year period in which -300 different donor females, primarily Holstein cows, underwent oocyte retrieval from one to as many as 167 times on a once or twice per week basis. The number and type of oocytes recovered from 500 consecutive oocyte retrieval sessions were determined during two periods of weeks -1 year apart. The mean number of oocytes collected per donor during 49

4 J.F.HasIer Table I. Mean number of different types of oocytes recovered from 1000 ultrasound-guided aspirations of 125 Holstein cows Oocyte type Cumulus Mean ± SD/donor % total Range I II III IV Total >4 layers 1-3 layers Nude Expanded 1.7 dt jt dt dt ± 4.5 I II III Oocyte Type % Cleaved a % Blastocysts % Hatched Figure 1. Potential for fertilization and embryonic development of different types of immature bovine oocytes aspirated from slaughterhouse-derived ovaries. Number of oocytes: Type I, 571; II, 228; III, 289; IV, 151. Percentages are based on the number of oocytes placed in in-vitro maturation. Type I, >4 layers of cumulus; Type II, 1-3 layers; Type III, nude; Type IV, expanded cumulus. [Adapted from Hasler, J.F. (1994) Commercial applications of in vitro fertilization in cattle. Compendium, 16, ] the two periods was not different and the data were combined and presented in Table I. In the present study, only Type I, II and IV oocytes (i.e. having cumulus cells attached) were considered usable and, as shown in Table I, an average total of 4.5 of these three types were recovered. The developmental potential of different types of oocytes was determined also by utilizing slaughterhouse-derived oocytes that were cultured in TCM-199-BRL co-culture following IVM and IVF. The development of these oocytes into blastocysts after 7 days in culture is shown in Figure 1. It is clear that Type I oocytes, which are those invested with four or more compact layers of cumulus cells, have the greatest potential for developing into blastocysts. Although 40% of Type II oocytes started cleaving after IVF, only 8% developed into blastocysts. More striking was the finding that 38% of nude oocytes (Type III) also started cleaving but < 1 % of them developed into blastocysts. During the first 6 months of the commercial IVF programme, most blastocysts were transferred to recipients on the first day they appeared in IVC (i.e. day 7, 8 or 9). In addition, some morulae were transferred on day 7. As the relationship between pregnancy rate and embryo age and quality became more apparent, most blastocysts of lower quality were discarded on days 8 and 9. As shown in Table II, transfer of grade 1 embryos, regardless of stage, resulted in higher pregnancy rates than did grade 2 embryos on both days 7 and 8. Within stage on day 7, however, only transfers of grade 1 EB and MB produced significantly higher pregnancy rates than corresponding stages classified grade 2. On day 8, only grade 1 XB resulted in a 50

5 In-vitro production of cattle embryos Table II. The effect of stage, age and morphological grade of bovine IVF-derived embryos upon pregnancy rates following transfer into Holstein heifers Embryo stage No. transfers (% pregnant) Day 7 Day 8 Morula Blastocyst Early Mid Expanded Hatched Total Grade (45) 1114 (53) a 1305 (56) a 1069 (63) 91 (57) 3914 (56) a Grade 2 83 (31) 259 (36) b 296 (46) b 54 (57) 692 (41) c Grade 1 1(0) 31 (39) 96 (49) 188 (47) a 45 (45) 361 (48) a Grade 2 2(0) 30 (27) 45 (31) 24 (33) d 101 (30) b ad Values in rows within days differ significantly (a versus b: P < 0.01, a versus c: P < 0.001, a versus d, b versus c: P < 0.05). Table III. Pregnancy rates following transfer of fresh and frozen bovine in-vivo- and IVF-derived embryos into Holstein heifers at one location Embryo IVF (fresh) IVF (fresh) IVF (fresh) IVF (frozen) IVF (frozen) In vivo (fresh) In vivo (frozen) Embryo age (days) No. transfers Percentage pregnant (relative to embryo grade) Grade 1 Grade 2 Grade 3 All grades 56 a 48 b b 20 c 76 d 64 e 41 a 30 b C - a " e Values within a column with different superscripts differ significantly (P < 0.05). significantly higher pregnancy rate than grade 2. Transfer of grade 1 day 7 embryos at the EB, MB or XB stages resulted in higher pregnancy rates (P < 0.05) than transfer of corresponding day 8 embryos. The pregnancy rates for all IVF and in-vivo embryos transferred over a 4 year period at Em Tran, Inc. were compared (Table III). The highest pregnancy rate was achieved with grade 1, fresh day 7 in-vivo embryos. Significantly lower pregnancy rates were achieved, in order, by in-vivo frozen day 7, fresh day 7 IVF, fresh day 8 IVF and frozen day 7 IVF and, lastly, by frozen day 8 IVF embryos. The sex ratios of calves resulting from embryos cultured in TCM-199 or in B2 media were not different. Males comprised 54.3% of all the a 43 b b 20 c 67 d 64 d in-vitro-derived calves and fetuses in this study, which differed significantly (P < 0.001) from 50%. Abnormalities noted with pregnancies and calves in this study included the following: large calves, increased gestation period, decreased intensity of labour, increased abortions throughout gestation, congenital malformations, increased perinatal mortality and an increased rate of hydroallantois. Precise quantitative data were not obtained from some clients. The overall loss at birth of IVF calves was 14.9% (205/1376), although there was no difference in the incidence of calf loss at birth between calves produced in the TCM-199 and B2 co-culture systems. An incidence of - 1% hydroallantois was diagnosed among pregnant Holstein recipients carrying IVF-derived fetuses; all of these recipients died prior to term. Although birth 51

6 J.F.Hasler Table IV. Production of bovine blastocysts from slaughterhouse-derived oocytes cultured in Menezo's (BRL) co-culture with or without serum for the first 72 h of culture B2-buffalo rat liver cell B2-BRL co-culture No. oocytes No. cleaved (%) No. blastocysts (%) Day 7 Day 8 Serum 516 No serum (days 1-4) (58.9) 340 (67.4) Percentages are based on the number of oocytes placed in in-vitro maturation. weights were not recorded, there were numerous reported cases of a higher than normal incidence of large calves. A defining characteristic of the IVF-derived pregnancies, reported by numerous owners of recipients, was poorly defined labour at term. As a result, because recipients were not observed to be in labour, calvings often went unassisted, resulting in increased perinatal losses. When calves were delivered by scheduled Caesarean section, the survival rate was nearly 98%. Experiment 2 Similar numbers of zygotes cultured in B2-BRL with or without serum during the first 72 h of coculture developed into blastocysts at days 7 and 8 (Table IV). By day 4 of culture, there was some sloughing of BRL cells from the bottom of the culture wells. However, this was not accompanied by any observable influence on the percentage of oocyte cleavage or the rate of embryo development. Experiment 3 There was no significant difference among the three culture systems in the efficiency of embryo production from the oocytes that were placed into IVM, which ranged from 17.8 to 19.6% (Table V). Also, there were no significant differences in 60 day pregnancy rates in recipient heifers resulting from the transfer of embryos produced in the three different culture systems. The sex of embryos, as determined by three different methods (as described in Materials and methods), was not different for embryos produced among the three different culture systems (Table V). The percentage of males for each separate treatment and for all treatments combined (54.2%) did not differ 155 (30.0) 162(32.1) 183 (35.4) 186 (36.9) significantly from a sex ratio of 50:50. There were also no differences in the distribution of embryo stages produced in B2-BRL co-culture with or without serum (Table VI). However, B2-BRL cocultures, either with or without serum, produced a lower percentage of mid blastocysts and a higher percentage of expanded and hatched blastocysts than did the TCM-199-BRL co-culture system. There were no differences in the percentage of spontaneous abortions, live births or dystocias among the three different co-culture systems (Table VII). There was no way to differentiate accurately between Caesarean deliveries that were scheduled prior to the onset of labour versus those that were performed as the result of calving problems in recipients. Nevertheless, there was no difference in the survival rate of calves following Caesarean delivery among the three co-culture systems. There was, however, a significantly lower survival rate (P < 0.05) following natural delivery of all calves combined for the three co-culture systems (187/ 228, 82.0%) compared to the total of all calves that survived Caesarean delivery (55/59, 93.2%). There were congenital abnormalities in calves and cases of hydroallantois observed in recipients that received embryos from either of the two B2 coculture systems but neither problem was observed in the pregnancies resulting from embryos cultured in TCM-199. Similar to Experiment 1, a frequently reported characteristic of the pregnancies resulting from all three culture systems was that labour was not clearly pronounced in the recipients. Discussion With a few exceptions, the -300 donors used in this study were infertile cows with an average age of >8 years. Oocytes were collected successfully 52

7 In-vitro production of cattle embryos Table V. Embryo production and pregnancy rate following transfer of embryos derived from oocytes retrieved and cultured in Menezo's B2-buffalo rat liver cell (BRL) co-culture with or without serum for the first 72 h of culture or in TCM-199-BRL co-culture with serum B2-BRL Serum TCM-199-BRL No. oocytes No. embryos % embryos/oocytes No. transfers 11 No. pregnant (%) No. males/calves (%) b (47.8) 186/345 (53.9) No Serum (47.1) 152/280 (54.3) a Does not include embryos that were biopsied or frozen. b Includes biopsied embryos, pregnancies detected by ultrasound, and calves. Morulae Early blastocysts Mid blastocysts Expanded blastocysts Hatched blastocysts Total 84 (13.2) 186 (27.2) 191 (29.9) 155 (24.3) 22 (3.4) a 638 Serum (50.4) 36/65 (55.4) Table VI. Distribution of different stages of embryos derived from oocytes retrieved and cultured in Menezo's B2-buffalo rat liver cell (BRL) co-culture with or without serum for the first 72 h of culture or in TCM-199-BRL co-culture with serum Embryo stage No. embryos (% total) B2-BRL Serum No serum ab Means in rows without common superscripts differ significantly (P < 0.05). TCM-199-BRL Serum 52 (9.7) 27 (8.6) b 149 (27.8) 95 (30.4) 183 (34.2) a 139 (44.4) b 137 (25.6) a 51 (16.3) b 14 (2.7) a 1 (0.3) b from all donors and at least one pregnancy was The overall pregnancy rate of 54% for grade produced from over 95% of the donors. Neverthe- 1, day 7 IVF embryos in the present study is less, the average number of oocytes recovered via significantly lower than the 76% rate for in-vivo oocyte retrieval from donor cows in the commercial embryos of comparable grade and age transferred programme at Em Tran was lower than that reported contemporaneously into the same pool of recipient in other studies involving fertile cattle (Kruip et al., heifers. In addition, we previously reported a 71% 1994; Gibbons et al., 1995; Bols et al., 1997). As pregnancy rate following transfer of more than shown previously (Hasler et al., 1995), there was 7000 in-vivo embryos into Holstein heifer recipia great deal of variation not only in the number of ents (Hasler et al., 1987). Other commercial oocytes collected from different donors, but in embryo transfer programmes have also reported the efficacy of in-vitro embryo development, the pregnancy rates for IVF embryos that are lower viability following transfer and the rate of abor- than those normally achieved with in-vivo embryos tions. The economic demand for pregnancies from (Looney et al., 1994; van Wagtendonk-de Leeuw some donors resulted in oocyte retrievals being et al., 1998; Bousquet et al., 1999). It is clear that conducted as many as 167 times from one donor in-vitro embryo production systems are not yet with a resulting 176 embryos sired by 23 different capable of yielding cattle embryos equal in viability bulls (Hasler, 1998). to embryos of in-vivo origin. These differences 53

8 J.F.Hasler Table VII. Pregnancy and calving characteristics following transfer of embryos derived from oocytes retrieved and cultured in Menezo's B2-buffalo rat liver cell (BRL) co-culture with or without serum for the first 72 h of culture or in TCM-199-BRL co-culture with serum Total no. pregnancies a No. abortions (%) No. calvings No. live calves (%) No. dead calves (%) No. Caesareans No. live calves (%) No. dead calves (%) B2-BRL Serum (13.1) (78.8) 24 (21.2) (88.5) 3 (11.5) No. congenital malformations (%) b 4/139 (2.9) No. hydroallantois (%) c 2/273 (0.7) includes intact and biopsied embryos. b % of calvings and Caesareans combined. c % of total pregnancies. have been discussed in a number of excellent reviews (Bavister, 1995; Thompson, 1996; Gardner and Lane, 1999). It has been reported that male cattle embryos produced in vitro grow more rapidly than female embryos in IVC (Avery etal, 1991; Xu etal, 1992; Carvalho et al, 1996). The overall percentage of males in the whole of the present study (54%) was significantly higher than 50% and may reflect this reported difference in growth rate, since several studies showed that the overall sex ratio of invitro-derived cattle embryos did not deviate from 1:1 (King et al, 1991; Avery et al, 1992). Skewing of the sex ratio in favour of males following transfer of in-vitro-derived bovine embryos has also been reported variously as 55.5% (van Wagtendonk-de Leeuw et al., 1998), 60% (Reichenbach et al, 1992), 62% (Guyader-Joly et al, 1993; Massip et al, 1995), and 57.6% (Agca et al, 1998). The 54% males observed in Experiment 3 did not differ in frequency from 50% males, probably due to the small sample size. However, the 54.3% males recorded in Experiment 1 (n = 2039) did differ from 50% and also from the 51.1% males reported for 1752 calves produced from in-vivo-derived embryos (King et al, 1985) and the 50.5% males out of Holstein calves that were a product of artificial insemination (Foote, 1977). 54 No serum (11.9) (81.4) 13 (18.6) (95.2) 1 (4.8) 2/94 (2.1) 1/201 (0.5) TCM-199-BRL Serum 65 7 (10.7) (91.1) 4 (8.9) (100) The embryos cultured in B2-BRL in the present study developed more rapidly than those cultured in TCM-199-BRL, which is in agreement with previous comparisons between the two culture systems (Hasler et al, 1995; Farin et al, 1997; Krisher, et al, 1998). However, there was no difference in the pregnancy rates resulting from the transfer of embryos produced in the two culture systems nor were there any detectable differences in the normality of the pregnancies. The problems with pregnancies and calves described in the present study are part of what has come to be known as the 'large offspring syndrome' (Young et al, 1998) in sheep and cattle. Largerthan-normal offspring is only one of a number of features associated with the syndrome. The heavier birth weights and higher-than-normal rates of abortion and perinatal losses in the present study have been reported in other studies in IVF-derived pregnancies in cattle (Van Soom et al, 1994; Kruip and den Daas, 1997; van Wagtendonk-de Leeuw et al, 1998). The embryos in the initial part of the present study were all produced in either B2 or TCM-199 co-cultured with BRL cells and serum. Because serum has been implicated as a cause of heavier birth weights of in-vitro-produced lambs (Walker et al, 1992; Thompson et al, 1995), the co-culture of bovine embryos without serum was investigated in the present study. Farin and James

9 (1996) previously showed that bovine embryos could be successfully cultured in TCM-199 with cumulus cell co-culture without serum for the first 72 h. They reported that serum restriction did not affect the proportion of embryos reaching compact morula or blastocyst stages but that the morphological quality was on average lower than for embryos exposed to serum throughout culture. Kruip and den Daas (1997) analysed information from 30 data sets on transfers of bovine IVF embryos obtained worldwide and reported that abortion rates were higher, gestation lengths and birth weights were increased, and there were higher incidences of dystocia relative to pregnancies produced by artificial insemination or embryo transfer of in-vivo-derived embryos. Specifically, the problems with pregnancies and calvings described previously (Hasler, 1998) and in the present study closely parallel those described by van Wagtendonk-de Leeuw et al. (1998) for bovine pregnancies produced by IVF embryos cultured in TCM-199 in a BRL monolayer system. Using synthetic oviduct fluid (SOF) in a non-co-culture system, Thompson et al. (1998) reported that bovine blastocyst development was accelerated when serum or charcoal-treated serum was added on day 5 compared to media containing only BSA throughout culture. Transfer of embryos following culture resulted in similar pregnancy and embryo survival rates for the three treatments and no birth weight differences were detected. Two other recent studies also failed to demonstrate any difference in birth weights or calving ease following transfer of embryos cultured in SOF with or without serum (Jacobsen et al, 1999; Tricoire, et al, 1999). However, in both of these studies, the small numbers of pregnancies produced (5-16) per treatment may have been a factor in the failure to demonstrate a significant difference between treatments. In the present study, serum restriction for the first 72 h of culture in B2-BRL affected neither the percentage of embryos becoming blastocysts or the distribution of different stages of blastocysts ranging from early to hatched. Also, of equal importance in a commercial embryo transfer programme, serum restriction did not influence the pregnancy rate of embryos that were transferred into recipients. Unfortunately, however, serum In-vitro production of cattle embryos restriction during the first 72 h of in-vitro culture was not accompanied by a decrease in the incidence of abortions, dystocias or congenital problems. Sinclair and colleagues (1999) recently compared pregnancies in sheep resulting from the transfer of in-vivo-derived embryos versus those cultured in TCM-199 with granulosa cell co-culture or in SOF with or without human serum. Fetuses were significantly heavier and growth coefficients for fetal liver and heart were greater from cocultured and serum-supplemented embryos compared to control embryos. The allometric coefficients for fetal liver and heart derived from embryos cultured in SOF without serum were not different from controls, however. In addition to this research, Holland Genetics has recently reported that the average birth weight of calves produced from SOF without serum was lighter than that of calves produced from TCM-199 co-culture with serum (van Wagtendonk et al, 2000). These findings strongly suggest that culture of in-vitro-derived bovine and ovine embryos in a semi-defined medium without serum or co-culture supplementation may result in an improvement in the normality of pregnancies. As a result of Experiment 2, the use of serum-free co-culture for the first 72 h is considered to pose a minimal risk to the efficacy of a commercial embryo production programme that utilizes oocytes collected via oocyte retrieval from valuable donor cows. Commercial embryos destined for transfer into recipients at Em Tran are currently being cultured without serum in sequential media developed by Gardner and Lane that are known as G1.2/G2.2 (Barnes et al, 1995; Gardner et al, 1998). Although embryo cell counts and pregnancy rates of G1.2/G2.2 embryos are quite acceptable (Hasler et al, 2000), it has not yet been established whether the use of these media has resulted in an improvement in pregnancy normality. Commercial IVF procedures at Em Tran were offered to the cattle industry starting in January The first donors to enter the IVF programme were exclusively aged, infertile cows. Demand for IVF grew rapidly due primarily to the high rate of success in producing embryos and pregnancies from infertile donors. Subsequently, a number of reproductively normal heifers and females that 55

10 J.F.Hasler were 1-4 months pregnant were entered into the programme. However, after ~2 years, the demand for IVF decreased markedly when word of the various problems with IVF-derived pregnancies circulated throughout the cattle industry. Currently, demand for IVF is at a very modest level and involves almost exclusively infertile donors. Wider acceptance of IVF by the cattle industry is dependent on an improvement in the normality of pregnancies, even at the possible expense of the efficiency of embryo production or pregnancy rate. There is probably a legitimate niche application for IVF to be used on infertile cattle and on genetically superior donors during early pregnancy and also on virgin heifers from whom offspring sired by multiple bulls are desired. It is clear that due to current low efficiencies and high expense, IVF in cattle is not economically competitive with traditional embryo transfer procedures for most donors. In summary, the present study has demonstrated that immature oocytes can be recovered from previously infertile cattle with the use of specialized ultrasound-guided aspiration equipment. The oocytes can be matured and fertilized and then cultured in vitro in either B2-BRL or TCM-199- BRL, producing blastocysts which reach the expanded stage by day 7. In-vitro-produced embryos transferred to recipients resulted in pregnancy rates comparable to those described by other investigators. A number of problems with the invitro-derived pregnancies and offspring, similar to those described by other authors, were noted. Restriction of serum during the first 72 h of culture did not have a detectable effect on embryo development or viability nor did it improve the normality of pregnancies resulting from transferred embryos. References Agca, Y., Monson, R.L., Northey, D.L. et al. (1998) Transfer of fresh and cryopreserved IVP bovine embryos: Normal calving, birth weight and gestation lengths. Theriogenology, 50, Avery, B., Madison, V. and Greve, T. (1991) Sex and development in bovine in vitro fertilized embryos. Theriogenology, 35, Avery, B., Jorgenson, C.B., Madison, V. and Greve, T. (1992) Morphological development and sex of bovine in wyro-fertilized embryos. Mol. Reprod. Dev., 32, Barnes, F.L., Crombie, A., Gardner, D.K. et al. (1995) Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum. Reprod., 10, Bavister, B.D. (1995) Culture of preimplantation embryos: Facts and artifacts. Hum. Reprod. Update, 1, Bavister, B.D. and Yanagimachi, R. (1977) The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol. Reprod., 16, Bols, P.E.J., Lein, A., Ysebaert, M.T. etal. (1997) Effects of long term treatment with bovine somatotropin on oocyte and blastocyst yield after OPU/IVF. Theriogenology, 47, 315 (Abstr.) Bousquet, D., Twagiramungu, H., Morin, N. et al. (1999) In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach. Theriogenology, 51, Bredbacka, P., Kankaanpaa, A. and Peippo, J. (1995) PCR-sexing of bovine embryos: a simplified protocol. Theriogenology, 44, Carvalho, R.V., Del Campo, M.R., Palasz, A.T. et al. (1996) Survival rates and sex ratio of bovine IVF embryos frozen at different developmental stages on day 7. Theriogenology, 45, 489^98. Farin, P.W. and Farin, C.E. (1997) Perspectives on large calves following transfer of embryos produced in vitro. IETS Embryo Transfer Newslett., 15, Farin, C.E. and James, B.E. (1996) Effect of early serum restriction on development of bovine embryos produced in vitro. Biol. Reprod., 54 (Suppl. 1), 91 (Abstr.). Farin, C.E., Hasler, J.F., Martus, N.S. and Stokes, J.E. (1997) A comparison of Menezo's B2 and tissue culture medium-199 for in vitro production of bovine blastocysts. Theriogenology, 48, Foote, R.H. (1977) Sex ratios in dairy cattle under various conditions. Theriogenology, 8, Gardner, D. and Lane, M. (1999) Embryo culture systems. In Trounson, A. and Gardner, D.K. (eds), Handbook of In vitro Fertilization. CRC Press, Boca Raton, FL, pp Gardner, D., Schoolcraft, W.B., Wagley, L. et al. (1998) A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization. Hum. Reprod., 13, Gibbons, J.R., Krisher, R.L., Carlin, S.K. et al. (1995) In vitro embryo production after microinjection and ovarian dynamics following transvaginal follicular oocyte aspiration. Theriogenology, 43, Guyader-Joly, C, Charbonnier, G., Durand, M. et al. (1993) Ability of in vitro produced bovine embryos to develop to term. Proc. 9th Meeting of the AETE, Lyon, p. 208 (Abstr.). 56

11 Hasler, J.F. (1994) Commercial applications of in vitro fertilization in cattle. Compendium, 16, Hasler, J.F. (1998) The current status of oocyte recovery, in vitro embryo production, and embryo transfer in domestic animals, with an emphasis on the bovine. J. Anim. Sci., 76 (Suppl. 3), Hasler, J.F. (2000) In vitro culture of bovine embryos in Menezo's B2 medium with or without coculture and serum: the normalcy of pregnancies and calves resulting from transferred embryos. Anim. Reprod. Sci., 60-61, Hasler, J.F., McCauley, A.D., Lathrop, W.F. and Foote, R.H. (1987) Effect of donor-embryo-recipient interactions on pregnancy rate in a large-scale bovine embryo transfer program. Theriogenology, 27, Hasler, J.F., Henderson, W.B., Hurtgen, P.J. et al. (1995) Production, freezing and transfer of bovine IVF embryos and subsequent calving results. Theriogenology, 43, Hasler, J.F, Lane, M, Musser, J. et al. (2000) Culture of bovine embryos in the sequential media G1.2/G2.2. Theriogenology, 53, 295 (Abstr.). Jacobsen, H., Holm, P., Schmidt, M. etal. (1999) In vitro embiyo production versus artificial insemination: delivery type, birth weight, and blood chemistry of the newborn calf. Theriogenology, 51, 226 (Abstr.). King, K.K., Seidel, G.E., Jr and Elsden, R.P. (1985) Bovine embryo transfer pregnancies. I. Abortion rates and characteristics of calves. J. Anim. Sci., 61, King, W.A., Yadav, K.P., Lu, K.P. et al. (1991) The sex ratios of bovine embryos produced in vivo and in vitro. Theriogenology, 36, Krisher, R.L., Gibbons, J.R. and Gwazdauskas, F.C. (1998) Effectiveness of Menezo's B2 medium with buffalo rat liver cells for development of in vitro matured/m vitro fertilized bovine oocytes. J. Assist. Reprod. Genet., 15, Kruip, Th.A.M., Boni, R., Wurth, Y.A. et al. (1994) Potential use of ovum pick-up for embryo production and breeding in cattle. Theriogenology, 42, Kruip, Th.A.M. and den Daas, J.H.G. (1997) In vitro produced and cloned embryos: effects on pregnancy, parturition and offspring. Theriogenology, 47, Kuwayama, M., Hamano, S., Kolkeda, A. and Matsukawa, K. (1996) Large scale in vitro production of bovine embryos. Proc. 13th Int. Cong, on Animal Reproduction, 71 (Abstr.). Lazzari, G. and Galli, C. (1996) In vitro embryo production and its application to cattle breeding. Proc. 12th Scientific Meeting of the AETA, Lyon, pp Looney, C.R., Lindsey, B.R., Gonseth, C.L. and Johnson, D.L. (1994) Commercial aspects of oocyte retrieval and in vitro fertilization (IVF) for embryo production in problem cows. Theriogenology, 41, Lu, K.H. and Polge, C. (1992) A summary of two In-vitro production of cattle embryos years' results in large scale in vitro bovine embryo production. Proc. 12th Int. Cong, on Animal Reproduction, The Hague, The Netherlands, 3, Massip, A., Mermillod, P., Van Longendonckt, A. et al. (1995) Survival and viability of fresh and frozenthawed in vitro bovine blastocysts. Reprod. Nutr. Dev., 35, Parrish, J.J., Susko-Parrish, J., Winer, M.A. and First, N.L. (1988) Capacitation of bovine sperm by heparin. Biol. Reprod., 38, Reichenbach, H.D., Liebrich, J., Berg, U. and Brem, G. (1992) Pregnancy rates and births after unilateral or bilateral transfer of bovine embryos produced in vitro. J. Reprod. Fertil., 95, Sauve, R. (1998) Ultrasound guided follicular aspiration and in vitro fertilization. In Program of the 8th annual meeting of the Brazilian Embryo Transfer Association, Porto Alegro, Brazil, 26, Sinclair, K.D., Broadbent, P.J. and Dolman, D.F. (1995) In vitro produced embryos as a means of achieving pregnancy and improving productivity in beef cows. Anim. Sci., 60, Sinclair, K.D., McEvoy, T.G., Maxfield, E.K. et al. (1999) Aberrant fetal growth and development after in vitro culture of sheep zygotes. J. Reprod. Fertil., 116, Thompson, J.G. (1996) Defining the requirements for bovine embryo culture. Theriogenology, 45, 27^-0. Thompson, J.G., Gardner, D.K., Pugh, P.A. etal. (1995) Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol. Reprod., 53, Thompson, J.G., Allen, N.W., McGowan, L.T. et al. (1998) Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer. Theriogenology, 49, Tricoire, H., Touze, J-L. and Mermillod, P. (1999) Effect of fetal calf serum on the quality of in vitro produced cattle embryos. Theriogenology, 51, 257 (Abstr.). Van Soom, A., Mijten, P., Van Vlaenderen, I. et al. (1994) Birth of double-muscled Belgian blue calves after transfer of in vitro produced embryos into dairy cattle. Theriogenology, 41, van Wagtendonk-de Leeuw, A.M., Aerts, B.J.G. and den Daas, J.H.G. (1998) Abnormal offspring following in vitro production of bovine preimplantation embryos: a field study. Theriogenology, 49, van Wagtendonk-de Leeuw, A.M., Mullaart, E., de Roos, A.P.W. et al. (2000) Effects of different reproduction techniques: Al, ET, or IVP, on health and welfare of bovine offspring. Theriogenology, 53, Walker, S.K., Heard, T.M. and Seamark, R.F. (1992) In vitro culture of sheep embryos without co-culture: successes and perspectives. Theriogenology, 37,

12 J.F.Hasler Walker, S.K., Hartwich, K.M. and Seamark, R.F. (1996) The production of unusually large offspring following embryo manipulation: concepts and challenges. Theriogenology, 45, Xu, K.P., Yadav, B.R., Rorie, R.W. et al. (1992) Development and viability of bovine embryos derived from oocytes matured and fertilized in vitro and co-cultured with bovine oviductal epithelial cells. J. Reprod. Fertil, 94, 33^3. Young, L.E., Sinclair, K.D. and Wilmut, I. (1998) Large offspring syndrome in cattle and sheep. Rev. Reprod., 3,