Changes to porcine blastocyst vitrification methods and improved litter size after transfer

The objective was to improve the protocol that was used to obtain the first reported piglets from transferred vitrified and warmed zona-intact blastocysts. Blastocysts were collected from superovulated sows and gilts, centrifuged to polarize lipid, vitrified, warmed and cultured for 24h or transferred immediately. Removing the zona pellucida after warming increased the number of cells in the surviving blastocysts (zona-free 60.8+/-4.3, zona-intact 39.1+/-2.8; P<0.05). Thinning the zona pellucida produced similar results to zona removal. Changing the basal medium of the vitrification and warming solutions from modified PBS to phosphate buffered NCSU-23 increased the number of cells (44.7+/-2.2 versus 56.0+/-3.9, respectively; P<0.05). Reducing the plunge temperature of the liquid nitrogen from -196 degrees C to less than -204 degrees C improved the embryo survival rate (61.9% versus 82.9%, respectively; P<0.05). These modifications were incorporated into the vitrification protocol that was used to vitrify and warm 105 blastocysts (that were subsequently transferred into four recipients). Three recipients became pregnant, farrowing three litters (average litter size, 5.3; 18.8% embryo survival in farrowing sows). Changing the warming protocol to using sucrose rather than ethylene glycol resulted in a trend towards improved embryo survival (73.5% versus 91.2%) but this was not statistically significant. Incorporating this modification, 203 blastocysts were vitrified, warmed and transferred into seven recipients. Five became pregnant and 36 fetuses were recovered (average litter size 7.2; 24.8% embryo survival in pregnant sows) at Day 40 of pregnancy. In conclusion, changes made to the vitrification protocol improved pregnancy rate and in vivo embryo survival compared to an earlier study using the original protocol.     

Effects of asynchrony on rabbit blastocyst development

The development of synchronously and asynchronously transferred rabbit morulae (recovered at Day 3 p.c.) and blastocysts (recovered at Day 4 p.c.) was investigated before the anticipated time of implantation. The results obtained with various techniques (evaluation of gross morphology, measurement of diameter, thymidine incorporation, light and electron microscopy) led basically to the same conclusions. Embryos being asynchronously transferred to the uterus of recipient rabbits survived, at least in terms of certain cellular functions like cell proliferation, for more than 2 days. Development, however, was clearly retarded and ultrastructural examination revealed substantial cell damage. Some blastocysts showed, even after 3 days, normal growth and cell proliferation indicating considerable differences between individuals in the ability to compensate for suboptimal developmental conditions before implantation. In general, this ability was greater in the transferred Day-3 morulae than in the Day-4 blastocysts. Embryonic growth and the ability to dissolve the zona pellucida, to synthesize crystalloid bodies and to differentiate extraembryonic endoderm indicated the maintenance of some developmental functions under asynchronous conditions. Blastocyst development was influenced by the progestational stage of the recipient. At 1 day after transfer into asynchronous older uteri, blastocyst diameters were larger and cell proliferation was increased compared with all other groups, suggesting an attempt of the blastocyst to adjust to the more advanced maternal milieu. Development in asynchronous younger uteri was delayed. No comparable differences in development were found in cultured embryos for which the media had been supplemented with flushings from the same progestational uterine stages as used for transfer. Thymidine incorporation in cultured embryos did not differ between the various supplements (P greater than 0.05) and was generally lower than in chronologically aged asynchronously transferred embryos (P less than 0.05 for Day-3 and P less than 0.001 or P greater than 0.05 for Day-4 embryos).     

Viability of bovine demi- and quarter-embryos after transfer

The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6 13 ) for heifers and 33.3% (4 12 ) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8 13 ) for heifers and 8.3% (1 12 ) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.     

Micromanipulation of sheep morulae to produce monozygotic twins

Donor ewes were treated with an intravaginal sponge containing 30 mg fluorogestone acetate (FGA) which was removed 12 days later. On the mornings of days 10, 11, and 12, each animal received 30 mg horse anterior pituitary (HAP) extract. Donors were mated twice daily during oestrus. Egg recovery was attemped seven days after progestagen with-drawal, when late morula/early blastocysts could be expected. The zona pellucida was opened with the aid of a micromanipulator, and the embryo was removed by gentle positive pressure from an evacuating pipette. After removal from the zona pellucida, the embryo was bisected using a fine glass needle; each demi-embryo was immediately placed either into the original zona pellucida or into one from an evacuated oocyte. The original opening was closed and both halves were transferred bilaterally into previously synchronized recipients. From 17 embryos split and transferred to 17 ewes, nine became pregnant and produced seven sets of monozygotic twins and two singletons.     

Decreased embryonic survival of in-vitro fertilized oocytes in rats is due to retardation of preimplantation development

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2 and allocated to 3 groups. On the evening of Day 0, rats in Groups I and II were allowed to mate. Embryos were collected on Day 4 (Group I, control morulae) or Day 5 (Group II, control blastocysts) and were transferred into the oviduct or uterine horn of Day-4 pregnant recipient rats. On the transfer side of the recipients, the bursa had been peeled from around the ovary to prevent endogenous oocytes from entering the oviduct. For Group III, unmated donors were killed 65-67 h after PMSG injection. Ovulated oocytes recovered from the oviducts were fertilized in vitro and transferred 16-18 h later. Embryos developing from in-vitro fertilized (IVF) oocytes were recovered on Day 5, separated into morulae (Group IIIm) and blastocysts (Group IIIb) and transferred into Day-4 pregnant recipients similar to control embryos. Some embryos from each group were used to determine the mean number of cells/embryo. Embryo recipients were killed on Day 20. After transfer, the development of IVF oocytes was retarded compared to control embryos. IVF morulae contained significantly fewer cells/embryo than did control morulae but were able to implant and grow to fetuses, in proportions similar to controls, if transferred into the oviduct of the recipients. These results suggest that the developmental potential of rat oocytes fertilized in vitro is limited due to asynchrony between the embryo and the uterine environment at the time of implantation, rather than possible defects incurred by the oocyte during the fertilization procedure.     

Developmental capacity of bovine oocytes collected from ovaries of individual heifers and fertilized in vitro

Bovine follicular oocytes from individual heifers (n=49) were separately matured, fertilized with frozen-thawed spermatozoa and cultured with cumulus cells. Although there were great variations in the number (mean+/-SD=19.1+/-11.9) of oocytes collected from individual heifers and the percentages of the oocytes cleaved 48 hours after insemination (mean+/-SD=69.5+/-18.4) and developed to the morula stage 7 days after insemination (mean+/-SD=10.9+/-10.9), there were significant correlations between the numbers of oocytes collected and cleaved (the correlation coefficient: r= 0.9336) or developed to morula stage (r=0.6633), indicating that oocytes from different heifers have the same developmental ability after in vitro fertilization. Ten morulae and 12 blastocysts which were obtained 7 and 8 days after insemination were transferred, one by one, to each uterine horn of 11 recipients. At Day 60 of pregnancy, 8 (80%) fetuses were identified in 4 (80%) of 5 recipients into which 10 embryos were transferred at Day -1 of synchrony. However, only 3 (25%) fetuses were identified in 2 (40%) of 6 recipients into which 12 embryos were transferred at Day 0 or +1 of synchrony.     

Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes

Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins. The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.     

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida

In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepes-buffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida.     

Survival of sheep demi-embryos in vivo and in vitro

Sheep embryos (morulae and blastocysts) were bisected either by microscalpel or by microneedle after dissolving the zona pellucida with acidified Tyrode's solution. Fourteen and 11 cryopreserved demi-embryos failed to develop when transferred to recipients or placed in culture, respectively. When fresh demi-embryos were cultured in Dulbecco's phosphate buffered saline (DPBS) plus fetal calf serum (FCS) or Whitten's medium, the survival rate was 26% compared to 68% for whole embryos (P<0.01), and there was a suggestion that the presence of a zona pellucida was beneficial to survival. When two demi-embryos each within a zona pellucida were transferred into each of 10 ewes, six of them lambed to produce a total of eight lambs, including two sets of identical twins. Of 10 ewes receiving two demi-embryos without zonae pellucidae, three lambed to produce a total of four lambs, including one set of identical twins. Of 10 ewes that each received two whole embryos, 10 lambed to produce a total of 16 lambs. There was a suggestion that the zona pellucida might enhance the survival of demi-morulae but not demi-blastocysts.     

Transfer of cow blastocysts after sexing

We obtained 174 Charolais cow blastocysts at day 13 (D(13)) after superovulation and slaughter of 24 donor heifers. Using de Wecker's scissors, we sampled 1 10 to 1 3 of the total blastocyst volume. After colcemid treatment, hypotonic shock, fixation and Giemsa staining, the sex of 149 blastocysts (60 %) was determined. The main difficulty in sexing was the absence of metaphase or the presence of incomplete metaphases on the preparations. During manipulation, the portion of the blastocyst to be transferred was stored at 37 degrees C in a medium of 20 % fetal calf serum containing 80 % B(2) under an atmosphere of 90 % nitrogen, 5 % O(2) and 5 % CO(2) for 6 to 7 hours. After sex determination, the blastocysts were put into an artificial insemination straw and placed with a catheter at the base of the uterine horns. Twenty-eight blastocysts were transferred into both horns of 14 recipient Charolais heifers; 4 heifers (28 %) had an extended cycle and 3 (21 %) were pregnant. When 2, 3 or 4 blastocysts were transferred into the ipsilateral horn with the corpus luteum, 20 to 25 % of the recipients had an extended cycle and 37, 40 and 75 %, respectively, were pregnant. The number of surviving embryos per recipient never exceeded 2.     

Effect of centchroman on tubal transport and preimplantation embryonic development in rats

A single oral administration of centchroman (1.25 mg/kg) to adult female rats within 24 h of mating induced slight acceleration in the rate of transport of embryos through the oviducts. The compound did not seem to produce any deleterious effect on preimplantation embryonic development since well organized and apparently normal embryos were collected from the genital tract up to Day 12 of pregnancy. The recovery rate of embryos from centchroman-treated rats was, however, significantly reduced after Day 4 of pregnancy. There was some stimulation in the rate of cleavage of embryos and morula to blastocyst transformation, but retardation in the shedding of the zona pellucida. The rate of blastocyst formation was not altered when 6-8-cell embryos collected from the oviducts of control rats were transferred to the uteri of control or centchroman-treated females. A delay in zona shedding was observed in the centchroman-treated recipients.     

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post co?tum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.     

Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares

In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) development of four- to eight-cell embryos to blastocysts compared to medium alone (11/15 vs 0/6) during 5 days in vitro. Embryos co-cultured with OEC were smaller (P < 0.05) and more delayed in development than Day-7 uterine blastocysts. There was no difference in the Day-30 survival rate of co-cultured blastocysts (3/8) or Day-7 uterine blastocysts (5/8) after transfer to recipient mares. These results indicate that co-culture with OEC can support development of four- to eight-cell equine embryos in vitro and that co-cultured embryos can continue normal development after transfer to recipient mares.     

Births of calves derived from embryos produced by intracytoplasmic sperm injection without exogenous oocyte activation

Tail-cut bovine spermatozoa were microinjected into ooplasmic lipid polarised, in vitro matured bovine oocytes using a piezomicropipette-driving system. No exogenous oocyte activation treatment was used. Of the sperm-injected oocytes, 86.3% were activated, 71.8% cleaved and 22.7% developed to the blastocyst stage. The average cell count of the blastocysts was 122.5 +/- 15 and a majority (81.8%) of the blastocysts were cytologically normal (diploid). When transferred to recipient cows, 5 of 8 blastocysts developed to fetuses and 4 of 7 recipients became pregnant. Normal offspring were born.     

Production of monozygotic twins following the transfer of bisected embryos in the goats

Embryos at the morula, blastocyst and hatched blastocyst stage were obtained from superovulated and naturally ovulated Japanese native goats. They were bisected into halves with a glass needle, and transferred immediately or after culture (for morula) to recipients. None of five does which received bisected morula became pregnant. Three of nine goats became pregnant after transfer of bisected hatched blastocysts, six of eleven recipients became pregnant. Four of them produced monozygotic twins and the remaining two produced singles. The present study demonstrated that the hatched blastocyst is suitable for bisection in the goat.     

Invitro fertilization of cow ova

Embryos were recovered from superovulated heifers and transferred non-surgically through the cervix of heifers via an ureteral catheter. The ureteral catheter was passed into the anterior portion of the uterine horn ipsilateral to the corpus luteum through a series of concentric stainless steel tubes. One morula or blastocyst was transferred to each of 95 heifers.Eighteen heifers (19%) were pregnant 55 to 60 days later. The pregnancy rate was not significantly influenced by treatment of the vagina with antibiotics before transfer; whether a morula or blastocyst was transferred; or whether ovulation occurred from the left or right ovary. There were no significant differences among the three technicians. Fifty of the 77 recipients that were not pregnant at 60 days of gestation were observed in estrus after 18–23.5 or 37–44.5 days, representing normal or double the normal estrous cycle lengths. However, 11 showed a shortened return to estrus (9–26 days), and 16 had a delayed return to estrus (25–33 or 48–55 days). We conclude that this technique results in premature luteolysis in some recipients and probably in more early embryonic death than normal.     

Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report

A method for producing identical twin calves is described in which Day 7 frozen-thawed bovine embryos in 12.5% sucrose solution were bisected using a fine microsurgical blade. The resulting bisected embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/-1 d), two bisected embryos per recipient. The pregnancy rate when both halves remained in the same zona pellucida was 50% (5 10 ); the pregnancy rate was 1 5 for morulae and 4 5 for blastocysts. The pregnancy rate for unfrozen morulae bisected in PBS and transferred without zona pellucida was 27% (4 15 ). The in vitro survival rate of embryos bisected in 12.5% sucrose when both halves remained in the original zona pellucida was 82% (18 22 ), which was higher than when embryos were bisected in PBS (53%, 9 17 ).     

Effect of human leukemia inhibitory factor on in vitro development of parthenogenetic bovine morulae

The present study was conducted to investigate the effect of human leukemia inhibitory factor (hLIF) addition to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS) on the development of in vitro matured and parthenogenetically activated bovine oocytes. The oocytes matured for 30 h were exposured to ethanol (7%, 7 min) and cytochalasin B (5 mug/ml, 5 to 6 h). The treated oocytes were cultured for 5 d in SOFM supplemented with HS, and Day-5 morulae were cultured for 2 d in SOFM supplemented with HS and with or without hLIF (5000 U/ml) to investigate the subsequent in vitro development to the blastocyst stage. Of the 1531 oocytes that were parthenogenetically activated, 592 (37.5%) cleaved to the 2- to 8-cell stage and 174 (13.8%) developed to the morula stage. The addition of hLIF at the morula stage resulted in a significantly (P<0.01) higher rate of development to the blastocyst stage in the medium with hLIF (55.9%) than without hLIF (28.9%). The mean cell number per blastocyst developed in the medium with hLIF was also significantly (P<0.01) higher than that developed in the medium without hLIF. To evaluate the viability, 6 parthenogenetically developed blastocysts were transferred to 3 recipient heifers (2 embryos per heifer), while in 2 other recipient heifers estrus was prolonged after transfer. The plasma progesterone levels of the 2 recipient heifers at the 28th day after transfer were 8.1 ng/ml and 9.0 ng/ml, but pregnancy was not observed by ultrasonic scanning. The present results indicate that the addition of hLIF to in vitro-produced, Day-5 parthenogenetic bovine morulae significantly improves the subsequent development to the blastocysts stage; however, the present method still does not promote for development of parthenogenetic fetuses in cattle.     

Successful piglet production after transfer of blastocysts produced by a modified in vitro system

Porcine in vitro production (IVP) systems, including in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes and their subsequent in vitro culture (IVC), have been modified by many researchers, but are still at a low level because of a low developmental rate of embryos to the blastocyst stage and their poor qualities. Our objectives were to establish reliable IVP procedures for porcine blastocysts and to examine the ability of the blastocysts to develop to term after transfer to recipients. Porcine cumulus-oocyte complexes were matured in vitro under 5% O(2) or 20% O(2), fertilized in vitro under 5% O(2), and subsequently cultured under 5% O(2) in 1) IVC medium supplemented with glucose (IVC-Glu) from Day 0 (the day of IVF) to Day 6; 2) IVC-Glu from Days 0 to 2, then IVC medium supplemented with pyruvate and lactate (IVC-PyrLac) from Days 2 to 6; 3) IVC-PyrLac from Days 0 to 2, then IVC-Glu from Days 2 to 6; and 4) IVC-PyrLac from Days 0 to 6. There were no significant differences in blastocyst formation rates on Day 6 between the 5% O(2) and 20% O(2) conditions (19.9% and 14.0%, respectively). However, the quality of blastocysts, as evaluated by the total cell number, was better after IVM under 5% O(2) than under 20% O(2) (mean cell number, 43.5 and 37.8, respectively). When IVP embryos were cultured in IVC-PyrLac from Days 0 to 2 and subsequently in IVC-Glu from Days 2 to 6, the rate of blastocyst formation (25.3%) and cell number (48.7) were higher than the rates (5.8% to 18.1%) and numbers (35.4 to 37.1) with the IVC-Glu then IVC-Glu, the IVC-Glu then IVC-PyrLac, and the IVC-PyrLac then IVC-PyrLac regimens, respectively. We then prepared conditioned medium (CM) from culture of porcine oviductal epithelial cells for 2 days in IVC-PyrLac and evaluated its effect on development to the blastocyst stage. Cultivation in CM for the first 2 days, followed by IVC-Glu for a further 4 days, had a significantly greater effect in increasing the number of cells in the blastocyst (58.3) than did in IVC-PyrLac (48.4). Finally, we evaluated the ability of blastocysts, generated by IVM under 5% O(2) and IVC in CM, to develop to term. When Day 5 expanding blastocysts (mean cell number, 49.7) were transferred to an estrus-synchronized recipient (50 blastocysts per recipient), the recipient remained pregnant and farrowed eight normal piglets. Furthermore, when Day 6 expanded blastocysts (mean cell number, 80.2) were transferred to two estrus-synchronized recipients, both gilts remained pregnant and farrowed a total of 11 piglets. These results suggest that an excellent piglet production system can be established by using this modified IVP system, which produces high-quality porcine blastocysts. This system has advantages for the generation of cloned and transgenic pigs.     

Pregnancies, calves and calf viability after transfer of in vitro produced bovine embryos

Pregnancy, parturition and calf survival following the transfer of embryos produced in vitro were monitored. A total of 44 blastocysts was transferred in pairs to 1 uterine horn ipsilateral to the corpus luteum (CL) of 22 synchronized heifers. At Day 42 of development 14 recipients (64%) were pregnant; the calving rate was also 64%. The twinning rate was 9/14 at Day 42 and 7/14 at birth, for an overall fetal mortality rate of 9%. The average gestation length was 281 and 275 d for single and twin pregnancies, respectively. Blood samples from recipients were collected for determination of bovine pregnancy associated glycoprotein (bPAG) from 2 wk after transfer and throughout the pregnancy. During the first trimester of pregnancy, the bPAG concentration was significantly higher in twin than in single bearing heifers, and the perinatal increase in bPAG was correlated positively with the total weight of the fetus(es). The percentage of male calves was 43%. The birth weight of twin individuals was 25 +/- 1 kg, which was 78% of the birthweight of the singletons (32 +/- 2 kg). One singleton calf was oversized, weighing 58 kg (80% more than the median weight of the other singletons). Stillbirths occurred in 21% of the twins, butin none of the singletons. Calf mortality during the first 14 d was higher for twins (4/11) than for singletons (1/7) due to infections and cerebellar hypoplasia. Karyotyping the calves detected no cytogenetically recognizable abnormalities. All calves were negative for BVD virus and IBR antibodies. The results of this study showed that although the incidence of fetal loss was low, there was an unacceptable high perinatal mortality of the calves. Thus it is likely that the blood supply through the placenta of animals pregnant with twins was impaired or it is possible that these fetuses and calves had increased stress susceptibility caused by the in vitro conditions. Furthermore, the birth of 1 oversized calf, 2 calves with cerebellar hypoplasia and 5 calves succumbing to infections seems to indicate that a proportion of in vitro produced calves may suffer from factors inherent in the in vitro production system.