Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses

The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30-90 of gestation. Fetal and placental characteristics were studied from Days 30-90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30-90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35-60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.     

In vitro and in vivo survival of bisected sheep embryos derived from frozen-thawed unsorted, and frozen-thawed sex-sorted and refrozen-thawed ram spermatozoa

Ovine IVP embryos were derived from frozen-thawed unsorted and frozen-thawed sex-sorted spermatozoa that had been refrozen and thawed. The embryos were bisected and cultured in vitro, or transferred to recipient ewes to determine their survival in vitro and in vivo. Oocyte progression to the blastocyst stage was similar for unsorted (97/232, 41.8%) and sex-sorted spermatozoa (113/286, 39.5%; P > 0.05). Embryo survival in vitro post-bisection was similar for demi-embryos derived from unsorted and sex-sorted sperm, and embryos bisected at the blastocyst and expanded blastocyst stage (P > 0.05). A higher proportion of recipient ewes were pregnant at Day 63 after transfer of two intact embryos derived from unsorted (17/21, 80.9%) than two demi-embryos derived from unsorted (5/15, 33.3%) or sex-sorted spermatozoa (7/17, 41.2%). The number of fetuses per original embryo at Day 63 did not differ among groups (unsorted intact: 23/42, 54.8%; unsorted demi: 7/15, 46.7%; sex-sorted demi: 10/17, 58.8%) and twin pregnancies were observed in all groups. Embryo survival to term was high, and was not significantly different among intact (unsorted: 22/42, 52.4%) and demi-embryos (unsorted: 4/15, 26.7%; sex-sorted spermatozoa: 7/17, 41.2%; P > 0.05). Dizygotic twins (n = 6 sets) were born after the transfer of two intact embryos derived from unsorted spermatozoa, but only singleton lambs resulted from the transfer of demi-embryos. In conclusion, bisected IVP embryos successfully developed into morphologically normal lambs. However, embryo survival to term was neither increased nor decreased by embryo bisection.     

Pregnancy and fetal characteristics after transfer of vitrified in vivo and cloned bovine embryos

This study was conducted to examine pregnancy progression and fetal characteristics following transfer of vitrified bovine nuclear transfer versus in vivo-derived embryos. Nuclear transfer (NT) was conducted using cumulus cells collected from an elite Holstein-Friesian dairy cow. Expanding and hatching blastocysts on Day 7 were vitrified using liquid nitrogen surface vitrification. Day 7 in vivo embryos, produced using standard superovulation procedures applied to Holstein-Friesian heifers (n=6), were vitrified in the same way. Following warming, embryos were transferred to synchronized recipients (NT: n=65 recipients; Vivo: n=20 recipients). Pregnancies were monitored by ultrasound scanning on Days 25, 45 and 75 and a sample of animals were slaughtered at each time point to recover the fetus/placenta for further analyses. Significantly more animals remained pregnant after transfer of in vivo-derived embryos than NT embryos at all time points: Day 25 (95.0 versus 67.7%, P<0.05), Day 45 (92.8 versus 49.1%, P<0.01) and Day 75 (70.0 versus 20.8%, P<0.0). There was no significant difference (P=0.10) in the weight of the conceptus on Day 25 from NT transfers (1.14+/-0.23 g, n=8) versus in vivo transfers (0.75+/-0.19 g, n=8). On Day 45, there was no significant difference in the weight of either fetus (P=0.393) or membranes (P=0.167) between NT embryos (fetus: 2.76+/-0.40, n=12; membranes: 59.0+/-10.0, n=11) or in vivo-derived embryos (fetus: 2.60+/-0.15, n=6; membranes: 41.8+/-5.2, n=4). However, on Day 75 the weight of the fetus and several of the major organs were heavier from NT embryos. These data suggest that morphological abnormalities involving the fetus and the placenta of cloned pregnancies are manifested after Day 45.     

Splitting of porcine embryos at the four-cell or morula stage

Porcine embryos obtained from estrus-induced prepuberal gilts were split at the 4-cell and morula stage. The in vitro development of the demi-embryos was compared with that of intact control embryos. The intact control embryos developed according to expectation. The in vitro survival of the demi-embryos was inferior to that of the respective controls. The splitting of 4-cell embryos was easier to accomplish and more efficient than the splitting of morulae. The in vitro development of the 4-cell embryos was also slightly better, although this difference was not significant. In vitro development of demi-embryos originating from morulae split at room temperature was slightly although nonsignificantly inferior to that of demi-embryos from morulae split on a warming stage at 37 degrees C. Between 14 and 18 demi-embryos were transferred to synchronous recipient gilts after 24 to 48 h of culture. Of 3 gilts receiving split 4-cell embryos, 1 gilt aborted 2 normal fetuses on Day 90 of pregnancy and 1 carried a single piglet to term. Of the 4 gilts receiving split morulae, 1 gilt had 4 normal and 3 degenerated fetuses upon slaughter on Day 35 of pregnancy.     

Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography.In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of Day 21 samples, 41% of Day 25, 95% of Day 35, 96% of Day 42, 99% of Day 49 and in 100% of samples of Days 56 and 63. Concentrations of PSPB increased (P < 0.05) from Days 21 to 42 and from Days 56 to 63, with a plateau between Days 42 to 56. Demi embryo pregnancies had higher (P < 0.05) plasma PSPB concentrations on Days 35 and 42 than whole embryo pregnancies. Progesterone supplementation had a positive effect (P < 0.01) on PSPB concentrations from Days 35 to 63. Concentrations of PSPB were similar in non-supplemented whole and demi embryo pregnancies from Days 7 to Day 63. In contrast, in supplemented recipients, demi embryo pregnancies had higher (P < 0.05) PSPB concentrations on Days 25 to 42 than whole embryo pregnancies. No significant correlation was found between P4 and PSPB concentrations or between the concentrations of these hormones and embryonic measurements on Day 42. In conclusion, demi embryos experienced a compensatory growth until Day 42 of pregnancy, attaining a similar size to that of whole embryos and originating conceptuses producing similar plasma PSPB concentrations to those of whole embryo derived conceptuses. Embryonic growth and conceptus secretion of PSPB were positively stimulated by early pregnancy exogenous P4 treatment.     

In vitro development to blastocysts of early porcine embryos produced in vivo or in vitro

The objective of this study was to compare the development of porcine embryos from the 2- and 4-cell stages to the blastocyst stage after in vivo or in vitro fertilization and in vivo or in vitro culture. Early-stage embryos were collected either from superovulated gilts 36 h after the second mating or after in vitro fertilization (IVF) of in vivo-matured oocytes, both followed by in vitro culture to the blastocyst stage. Blastocysts collected from superovulated donors served as controls. In the first experiment, a total of 821 2- and 4-cell embryos derived from in vivo-fertilized oocytes was cultured either in medium NCSU 23, modified Whittens' medium or modified KRB for 5 d. Significantly (P < 0.05 and P < 0.001) more embryos overcame the 4-cell block and developed to the blastocyst stage in medium NCSU 23 than in the 2 other culture media. Hatching was only observed in medium NCSU 23. In the second experiment, embryos derived from in vivo-matured oocytes fertilized in vitro were cultured in medium NCSU 23. Of 1869 mature oocytes 781 (41.8%) cleaved within 48 h after in vitro fertilization. A total of 715 embryos was cultured to the morula and blastocyst stages, and 410 (57.3%) overcame the developmental block stage, with 358 embryos (50.1%) developing to the morula and blastocyst stages. None of the embryos hatched, and the number of nuclei was significantly (P < 0.05) lower compared with that of in vivo-fertilized embryos (18.9 +/- 9.8 vs 31.2 +/- 5.8). In the third experiment, 156 blastocysts derived from in vitro fertilization and 276 blastocysts derived from in vivo fertilization and in vitro culture were transferred into synchronized recipients, while 164 blastocysts were transferred immediately after collection into 6 recipients, resulting in a pregnancy rate of 83.3%, with 35 piglets (on average 7.0) born. From the in vitro-cultured embryos, 58.3% (7/12) of the recipients remained pregnant at Day 35 after transfer, but only 33.3% maintained pregnancy to term, and 14 piglets (on average 3.5) were born. In contrast, the transfer of embryos derived from in vitro-fertilized oocytes did not result in pregnancies. It is concluded that 1) NCSU 23 is superior to modified Whittens' medium and modified KRB and 2) blastocysts derived from in vitro fertilization have reduced viability as indicated by the lower number of nuclei and failure to induce pregnancy upon transfer into recipients.     

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.     

Interaction between season and culture with insulin-like growth factor-1 on survival of in vitro produced embryos following transfer to lactating dairy cows

Culture of bovine embryos in the presence of insulin-like growth factor-1 (IGF-1) can increase pregnancy rates following transfer to heat-stressed, lactating dairy cows. The objective of the present experiment was to determine whether the effect of IGF-1 on post-transfer embryo survival was a general effect or one specific to heat stress. Lactating recipients (n=311) were synchronized for timed-embryo transfer at four locations. Embryos were produced in vitro and cultured with or without 100 ng/mL IGF-1. At Day 7 after anticipated ovulation (Day 0), a single embryo was randomly transferred to each recipient. Pregnancy was diagnosed at Day 21 by elevated plasma progesterone concentrations, at Days 27-32 by ultrasonography, and at Days 41-49 by transrectal palpation. Transfers were categorized into two seasons, hot or cool (based on the month of transfer). There was a tendency (P<0.09) for an interaction between embryo treatment and season for pregnancy rate at Day 21; this interaction was significant at Days 30 and 45 (P<0.02). Recipients receiving IGF-1 treated embryos had higher pregnancy rates in the hot season but not in the cool season. There was a similar interaction between embryo treatment and season for overall calving rate (P<0.05). There was also an interaction between season and treatment affecting pregnancy loss between Days 21 and 30; recipients that received IGF-1 treated embryos had less pregnancy loss during this time period in the hot season but not in the cool season. The overall proportion of male calves born was 77.5%. In conclusion, treatment of embryos with IGF-1 improved pregnancy and calving rates following the transfer of in vitro produced embryos into lactating recipients, but only under heat-stress conditions.     

Pregnancy and interferon tau regulate major histocompatibility complex class I and beta2-microglobulin expression in the ovine uterus

Major histocompatibility complex (MHC) class I molecules, consisting of an alpha chain and beta2-microglobulin (beta2MG), play an important role in immune rejection responses by discriminating self and nonself and are increased by type I interferons during antiviral responses. Interferon tau (IFNtau), the pregnancy-recognition signal in ruminants, is a type I interferon produced by the ovine conceptus between Days 11 and 21 of gestation. In study 1, expression of MHC class I alpha chain and beta2MG mRNA and protein was detected primarily in endometrial luminal epithelium (LE) and glandular epithelium (GE) on Days 10 and 12 of the estrous cycle and pregnancy. On Days 14-20 of pregnancy, MHC class I and beta2MG expression increased only in endometrial stroma and GE and, concurrently, was absent in LE and superficial ductal GE (sGE). Although neither MHC class I nor beta2MG proteins were detected in Day 20 trophectoderm, beta2MG mRNA was detected in conceptus trophectoderm. In study 2, cyclic ewes were ovariectomized on Day 5, treated daily with progesterone to Day 16, received intrauterine infusions between Days 11 and 16 of either control serum proteins or recombinant ovine IFNtau, and were hysterectomized on Day 17. The IFNtau increased MHC class I and beta2MG expression only in endometrial stroma and GE. During pregnancy, MHC class I and beta2MG gene expression is inhibited in endometrial LE and sGE but, paradoxically, is stimulated by IFNtau in the stroma and GE. The silencing of MHC class I alpha chain and beta2MG genes in the endometrial LE and sGE during pregnancy recognition and establishment may be a critical mechanism preventing immune rejection of the conceptus allograft.     

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.     

Morphologic and histologic comparisons between in vivo and nuclear transfer derived porcine embryos

Nuclear transfer (NT) is an inefficient but invaluable tool of the biotechnology industry. This study looked at abnormalities associated with peri-implantation NT porcine embryos. Four experimental groups were examined: nonpregnant animals, in vivo pregnant animals, NT recipients, and manipulation control embryos (MC). Embryos (Day 10, 12, or 14) were evaluated for embryonic disc diameter, gross morphology, nucleoli density, and mitotic figure index. Day 12 (P < or = 0.03) and Day 14 (P < or = 0.01) NT embryos had increased numbers of nucleoli, and Day 14 NT embryos had an increased (P < or = 0.03) mitotic index compared to in vivo and MC embryos. In vivo produced Day 14 embryos had increased (P < or = 0.01) disk diameters when compared to other embryos except for MC Day 14, which also showed increases (P < or = 0.01) in disk diameter except when compared to in vivo produced Day 12 and Day 14 embryos. In vivo produced Day 12 had greater (P < or = 0.03) disk diameters when compared to NT and MC embryos except for MC Day 14, and in vivo produced Day 14 embryos, which had a significantly increased (P < or = 0.01) disk diameter. In vivo produced Day 14 embryos were morphologically more advanced (P < or = 0.01) than Day 14 NT and MC counterparts. NT embryos develop at a slower rate than their in vivo produced counterparts. The increase in nucleoli and mitotic index of NT embryos suggest the cell cycle may be affected or the NT embryos are employing other means to compensate for slow development. The techniques used during NT also appear to compromise embryo development.     

Expression of the interferon tau inducible ubiquitin cross-reactive protein in the ovine uterus.

Ubiquitin cross-reactive protein (UCRP) is a 17-kDa protein that shows cross-reactivity with ubiquitin antisera and retains the carboxyl-terminal Leu-Arg-Gly-Gly amino acid sequence of ubiquitin that ligates to, and directs degradation of, cytosolic proteins. It has been reported that bovine endometrial UCRP is synthesized and secreted in response to conceptus-derived interferon-tau (IFNtau). In the present studies, UCRP mRNA and protein were detected in ovine endometrium. Ovine UCRP mRNA was detectable on Day 13, peaked at Day 15, and remained high through Day 19 of pregnancy. The UCRP mRNA was localized to the luminal epithelium (LE), stromal cells (ST) immediately beneath the LE, and shallow glandular epithelium (GE) on Day 13, but it extended to the deep GE, deep ST, and myometrium of uterine tissues by Day 15 of pregnancy. Western blotting revealed induction of UCRP in the endometrial extracts from pregnant, but not cyclic, ewes. Ovine UCRP was also detected in uterine flushings from Days 15 and 17 of pregnancy and immunoprecipitated from Day 17 pregnant endometrial explant-conditioned medium. Treatment of immortalized ovine LE cells with recombinant ovine (ro) IFNtau induced cytosolic expression of UCRP, and intrauterine injection of roIFNtau into ovariectomized cyclic ewes induced endometrial expression of UCRP mRNA. These results are the first to describe temporal and spatial alterations in the cellular localization of UCRP in the ruminant uterus. Collectively, UCRP is synthesized and secreted by the ovine endometrium in response to IFNtau during early pregnancy. Because UCRP is present in the uterus and uterine flushings, it may regulate endometrial proteins associated with establishment and maintenance of early pregnancy in ruminants.     

Survival of biopsied and sexed bovine demi-embryos

The viability of sex-diagnosed bovine demi-embryos was investigated after transfer. Day-7 morulae and blastocysts were subjected to splitting and biopsy in PBS + 4mg/ml polyvinylpyrrolidone + 200mM sucrose using a microblade. The biopsy (approximately 2 to 8 blastomeres) was transferred to a tube, and its presence in the tube was verified by examination under a stereomicroscope. After proteinase K treatment, repetetive male-specific DNA was amplified by the polymerase chain reaction (PCR). No autosomal control primers were used in the PCR. Instead, the absence of a characteristic Y-specific product together with the amplification of non-specific products was considered an indication of a female sample. The biopsied demi-embryos were transferred either singly or in pairs to synchronous heifer or cow recipients 6 to 10 h after flushing. Sex diagnosis was carried out within 6 to 7 h. Of 19 original embryos, 7 were diagnosed as males and 5 as females. The DNA of the biopsies of the remaining 7 embryos did not result in any amplification products. Since 5 of these samples were seen in the tubes prior to PCR, the corresponding embryos were considered "potential females." The sex of the last 2 samples could not be determined. Nine of 10 embryos were correctly sexed as revealed by calving data. Of the 38 transferred demi-embryos, 16 had developed to live fetuses as detected by ultrasonography on Day 65 of pregnancy. Eleven live calves and three stillborn calves were delivered. After bisection, biopsy and single transfer, 6 live calves were born from 7 original embryos (86%). After transfer of both halves into the same recipient, only 5 live calves from 12 original embryos were produced (42%). None of the 4 manipulated Grade-2 embryos survived to term, nor did any of the 4 manipulated blastocysts. Of the 14 original Grade-1 morulae manipulated and transferred, 15 were live fetuses at Day 65, and 11 live calves were born.     

Superovulation, collection and transfer of embryos and demi-embryos from Boran(Bos indicus ) cows and heifers

Twenty-three Boran(Bos indicus ) cows and heifers were superovulated with pregnant mare serum gonadotropin (PMSG); a total of four embryos and 4.1 +/- 0.3 (mean +/- SEM) ova per ova-producing donor resulted. Follicle stimulating hormone (FSH-P) was then used to superovulate 49 Boran cows for a total of 106 superovulations, of which 63 (59.4%) produced an average of 3.7 +/- 0.4 (mean +/- SEM) embryos. The embryo production was not influenced by either the season or the number of times(one to five) the cows were superovulated. A higher pregnancy rate was obtained when the selection of Boran recipients was based on their plasma-progesterone values (overall 52.5%, single embryos 63.3%, twin demi-embryos 45.8%) than when they were selected by palpation per rectum only (overall 43.8%, single embryos 50%, twin demi-embryos 36.4%). The twinning rate of twin demiembryos was 62.5%, whereas only single calves were born after transfer of two embryos per recipient. No pregnancies were produced following transfer of twin demi-embryos without zonae pellucidae. Transferring single demi-embryos gave a low pregnancy rate (13.3%). Twelve donor Boran cows (21 superovulations) bred with their fathers resulted in a high rate of early embryonic death; additionally, only 20.9% (overall) of the recipients became pregnant. Estrus synchronization of Boran cows with a progesterone releasing intravaginal device (PRID) for a short period (7 d) combined with one injection of prostaglandin (Day 6) produced a larger number of good quality recipients (70.5%) than using double prostaglandin injections (60%).     

Effects of interval between fusion and activation, cytochalasin B treatment, and number of transferred embryos, on cloning efficiency in goats

To improve the efficiency of somatic cell nuclear transfer (SCNT) in goats, we evaluated the effects of the interval between fusion and activation (1 to 5 h), cytochalasin B (CB) treatment after electrofusion, and the number of transferred embryos on the in vivo and in vitro development of cloned caprine embryos. The majority of the reconstructed embryos had condensed chromosomes and metaphase-like chromosomes at 2 and 3 h after fusion; cleavage and blastocyst rates from those two groups were higher (P < 0.05) than those of embryos activated 1, 4, or 5 h after fusion. Treatment with CB between fusion and activation improved in vitro and in vivo development of nuclear transfer (NT) goat embryos by reducing the fragmentation rate (P < 0.05). Although there were no significant differences in NT efficiency, pregnancy rate and kids born per recipient were increased by transfer of 20 or 30 embryos per recipient compared with 10 embryos. We concluded that CB treatment for 2 to 3 h between fusion and activation was an efficient method for generating cloned goats by somatic cell NT. In addition, increasing the number of embryos transferred to each recipient resulted in more live offspring from fewer recipients.     

Progestin implants can rescue demi-embryo pregnancies in goats: a case study

Survival after transfer of demi-embryos (i.e., half-embryos produced by embryo splitting) to recipients usually is lower than survival after transfer of intact embryos. Reduced survival after demi-embryo transfer could be due to loss of viability after splitting, failure of a viable demi-embryo to prevent corpus luteum (CL) regression in the recipient female, or a combination of factors. From a retrospective analysis of pregnancy and embryo survival rates after demi-embryo transfer in sheep and goats, we report the rescue of caprine demi-embryo pregnancies in which CL regression occurred at the end of diestrus despite the presence of a viable conceptus in the uterus with progestin implants. Day 5 or 6 morulae and blastocysts were flushed from superovulated ewes and does and split into demi-embryos of approximately equal halves. Demi-embryos were either transferred fresh to synchronized recipients of the homologous species or frozen in liquid nitrogen. Approximately half of the recipient does and ewes were treated with norgestomet implants on Day 10 of the embryo transfer cycle and again 2 wk later. Serum collected on Day 25 from recipients with implants was assayed for progesterone to determine if a CL of pregnancy had been maintained. Pregnancy was diagnosed by ultrasonography on Day 35 of gestation. Corpus luteum regression occurred despite the presence of a viable conceptus in the uterus in 6 of 55 progestin-treated caprine demi-embryo recipients and in 0 of 66 ovine demi-embryo recipients. Five of the caprine pregnancies were maintained to term with norgestomet implants and produced 5 live kids. The sixth fetus, which was carried by a progestin implant-treated 8-mo-old doeling, died at approximately 50 d of gestation. These results suggest that, at least in goats, some demi-embryos may provide inadequate signaling for maternal recognition of pregnancy, and such pregnancies can be rescued with progestin treatment to the doe.     

In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes

The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embryos derived from adult ewes and from unstimulated (16- to 24-week-old) and hormone-stimulated prepubertal (3- to 5-week-old) ewes were assessed. Cleavage was lower for hormone-stimulated (617/1025; 60.2%) than unstimulated prepubertal (117/169; 69.2%) and adult ewe oocytes (184/267; 68.9%; P < 0.05). Blastocyst formation by Day 7 (from zygotes) was similar for unstimulated (45/117; 38.5%), hormone-stimulated prepubertal (229/617; 37.1%) and adult ewes (101/184; 54.9%). Blastocysts derived from hormone-stimulated prepubertal ewes developed mainly on day 7, compared with Day 6 for adult and unstimulated prepubertal ewes. Pregnancy rates (day 60) and embryonic loss (between Days 20 and 60) did not differ after transfer to adult recipient ewes of adult, unstimulated and hormone-stimulated prepubertal-derived fresh or frozen-thawed embryos. The number of lambs born as a proportion of embryos transferred did not differ for fresh and frozen embryos derived from adult ewes (3/16; 18.8% and 1/12; 8.3%, respectively) and unstimulated prepubertal lambs (2/6; 33.3%, and 1/10; 10.0%, respectively), but was higher for fresh than frozen embryos from hormone-stimulated prepubertal ewes (7/16; 43.8%, and 2/14; 14.3%, respectively; P < 0.05). There were high rates of in vitro and in vivo development of oocytes from 3- to 5-week-old lambs, but in vitro development was lower than with oocytes from adult ewes. However, the speed of embryonic development in vitro and the in vivo development of fresh and frozen embryos were similar to those derived from adult and unstimulated prepubertal ewes. The present results were an improvement in the efficiency of producing embryos and offspring from hormone-stimulated 3- to 5-week-old lambs.     

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.     

Luteotrophic effect, growth and survival of whole versus half embryos and, their relationship with plasma progesterone concentrations of recipient dairy heifers

This prospective and randomised experiment was designed to compare the luteotrophic effect of whole versus half embryos and, to evaluate the relationship between the plasma progesterone (P4) profiles and the rates of early embryonic (from Days 7 to 25), late embryonic (Days 25-42) and foetal (Days 42-63) mortalities of whole and half embryo recipients. Within a single herd, 188 virgin, healthy, cyclic, reproductively sound, with adequate body condition score, Holstein dairy heifers were randomly allocated to receive one whole or one half embryo on Day 7 of the oestrous cycle (Day 0=estrus). In each embryo-transfer (ET) group, half of the recipients were treated with a CIDR (controlled internal drug releasing device) between Days 7 and 19. Pregnancy was evaluated by ultrasound on Days 25, 42 and 63 and plasma P4 profiles were obtained until Day 63 of pregnancy. CIDR-treated and untreated heifers had similar pregnancy rates on Days 25, 42 and 63 and, embryo size on Day 42 was also similar in treated and untreated recipients. Therefore, CIDR treatment failed to promote growth and survival of half and whole embryos. Half embryos presented a significantly higher rate of early and late embryonic mortality than whole embryos. In contrast, foetal mortality was similar in whole and half embryos and, this was coincidental to a similar embryo size on Day 42. Therefore, half embryos exhibited a compensatory growth until Day 42, irrespective of CIDR treatment, after which they presented a similar survival rate to that of whole embryos. Half embryo-derived pregnancies presented significantly lower plasma P4 concentrations on Day 25 than whole embryo-derived pregnancies, suggesting that this lower luteotrophic effect of half embryos could be related to their higher rate of late embryonic mortality. No significant relationship between the early luteal P4 concentrations and embryo survival was observed in whole and half embryo recipients. The first detectable luteotrophic effect of embryonic origin was observed on Day 14 and no detectable second luteotrophic effect was observed until Day 63 of pregnancy. Treatment with CIDR significantly increased plasma P4 concentrations during treatment but induced a significant decrease after removal of the device, suggesting that secretion of luteotropins was downregulated in the course of treatment.     

Cloned cattle fetuses with the same nuclear genetics are more variable than contemporary half-siblings resulting from artificial insemination and exhibit fetal and placental growth deregulation even in the first trimester

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome), and fetal overgrowth. Fetal and placental development was investigated at Day 50, during placentome formation; at Day 100, when placentation was completed; and at Day 150, when the hydrops syndrome frequently develops. The NT fetuses were compared with contemporary half-siblings generated from in vitro-produced embryos or by artificial insemination (AI). Fetal cotyledon formation and vascularization of the chorioallantoic membranes was initiated normally in NT conceptuses, but fewer cotyledons successfully formed placentomes. By Day 100, the mean number of placentomes was significantly lower in surviving NT fetuses. Only those with normal placentome numbers were represented in surviving NT pregnancies at Day 150. The mean total caruncle tissue weight of the placentomes was significantly higher in the surviving NT groups at Days 100 and 150, irrespective of the placentome numbers, indicating that increased NT placental weight was caused by excessive uterine tissue growth. By Day 100, NT fetuses exhibited growth deregulation, and those that survived to Day 150 were 17% heavier than contemporary AI controls. Placentome, liver, and kidney overgrowth accompanied the hydrops syndrome at Day 150. The NT fetal overgrowth was not a consequence of in vitro embryo culture and showed no correlation with placental overgrowth. However, in vitro culture and incomplete reprogramming of the donor genome are epigenetic effects that may override genetic traits and contribute to the greater variability in placental and fetal development in the NT group compared with AI half-siblings.