Production efficiency of Japanese black calves by transfer of bovine embryos produced in vitro

The objective of this study was to examine the production efficiency of Japanese Black beef calves after transfer of bovine embryos derived from an in vitro procedure. In vitro-produced (IVP) embryos were obtained from in vitro maturation and fertilization and in vitro development by co-culture with cumulus cells until 7 or 8 days after insemination. In vivo-developed (IVD) embryos from superovulated Japanese Black heifers and cows 7 days after artificial insemination were used as a control group. Bovine embryos were transferred nonsurgically to recipient cows on Day 7 +/- 1 of the estrous cycle. Pregnancy was diagnosed by palpation per rectum at Day 60 to 70 after estrus. Pregnancy, abortion, perinatal accident and birth rates were examined according to the origin of embryos (IVP or IVD), the number of transferred embryos (single or twin) and the storage status (fresh or frozen-thawed). In Experiment 1, production efficiency by twin transfer of fresh IVP embryos was examined. Higher pregnancy rates (52 1% vs 42 9%, P < 0.05) and birth rates (47.0% vs. 33.0%, P < 0.05) were obtained by twin transfer than by single transfer of fresh IVP embryos. Thus, the twin transfer of fresh IVP embryos was effective for production of calves, although the birth rates for single and twin transfers of fresh IVD embryos were still higher (55.5% and 76.1%, P < 0.05). But the abortion and perinatal accident rates for twin transfer of fresh IVP embryos were also significantly greater than those for single and twin transfer of fresh IVD embryos (P < 0.05). In Experiment 2, production efficiency by twin transfer of frozen-thawed IVP embryos was examined. Either single or twin transfer of frozen-thawed IVP embryos resulted in a similar pregnancy rate (41.3% vs. 46.7%, P > 0.05) and birth rate (34.1% vs. 41.1%, P>0.05). Thus, in combination with frozen-thawed IVP embryos, the twin transfer did not enhance production efficiency. In conclusion, Japanese Black beef calves could effectively produce calves by twin transfer to Holstein recipients when using fresh IVP embryos, and by single transfer when using frozen-thawed IVP embryos.     

Tubal transfer of bovine embryos: a simple endoscopic method reducing long-term exposure of in vitro produced embryos

Although numerous trials had shown the need to define a procedure to get free access to the bovine oviduct, there was no adequate report of a technique which was accepted for the routine transfer of early tubal-stage embryos. We have now report an endoscopically mediated transvaginal method for transferring embryos into the oviduct. The in vitro produced embryos were loaded into a curved glass capillary tube which was connected to a perfusor tube plus 1-mL syringe. The capillary tube was directly inserted via the infundibulum into the ampulla. After first having checked the ovaries for the presence of a corpus luteum the embryos were deposited under visual guidance in about 20 to 50 microL medium. Twenty-four Simmental and Brown Swiss heifers received 26 embryos and 9 animals became pregnant, of which 7 recipients delivered 8 live calves. With practice, the time used for endoscopic transfer was reduced to less than 10 min. The results demonstrate that the described technique is suitable for practical application. Especially for the early transfer of IVP-derived embryos this technique might be advantageous. In conclusion, this method is also of great potential interest for the recovery of tubal-stage embryos and for the in vivo culture of embryos followed by conventional flushing at Day 7.     

The developmental competence of bovine nuclear transfer embryos derived from cow versus heifer cytoplasts

Due to its economic importance, the production of cattle by nuclear transfer has been a primary research focus for many researchers during the past few years. While many groups have successfully produced cattle by nuclear transfer, and progress in this area continues, nuclear transfer remains a very inefficient technology. This study evaluates the effect of the oocyte source (cow and heifer) on the developmental competence of nuclear transfer embryos. In order for nuclear transfer to be successful, a differentiated donor cell must be reprogrammed and restored to a totipotent state. This reprogramming is probably accomplished by factors within the oocyte cytoplasm. This study indicates that oocytes derived from cows have a greater capacity to reprogram donor cell DNA following nuclear transfer as compared to heifer oocytes based on in vitro development to the 2-cell stage and to the compacted morula/blastocyst stages. Nuclear transfer embryos derived from cow oocytes resulted in significantly higher rates of pregnancy establishment than embryos derived from heifer oocytes and resulted in higher pregnancy retention at 90 and 180 days and a greater number of term deliveries. Following delivery more calves derived from cow oocytes tended to be healthy and normal than those derived from heifer oocytes. The differences in developmental efficiency between nuclear transfer embryos derived from cow and heifer cytoplasts demonstrate that subtle differences in oocyte biology can have significant effects on subsequent development of nuclear transfer embryos.     

The sex ratios of bovine embryos produced in vivo and in vitro

The male:female ratio of developing bovine embryos produced and allowed to develop in vitro and in vivo was determined retrospectively from the cytogenetic analysis of 804 embyos. The overall male:female ratio of the 307 (38%) embryos that could be sexed was 162 (52.8%):145 (47.2%) and did not differ (P>0.05) from the expected 1:1 ratio. Among premorula stage embryos produced in vivo (n = 66) and in vitro (n = 30), the ratios were 1.2:1 and 0.76:1, respectively. Among morulae and blastocysts produced in vivo (n = 74), produced and cultured in vitro (n = 106, and produced in vitro and cultured in vivo (n = 31), the ratios were 1.11:1, 1.3:1 and 0.94:1, respectively, none of which differed significantly from 1:1. There was no difference (P>0.05) in the number of cells or mitotic index between male and female morulae and blastocyst, respectively.     

Plasma concentrations of bovine pregnancy-associated glycoprotein (bPAG) do not differ during the first 119 days between ongoing pregnancies derived by transfer of in vivo and in vitro produced embryos

Calves derived from IVP embryos may suffer from the large offspring syndrome that has been related to effects of in vitro culture on the intrinsic quality of the embryo. Limited information is available on the role of the placenta in such cases. In this study, bovine pregnancy-associated glycoprotein (bPAG) was used as a marker to test whether placental function is influenced by the route of embryo production. Therefore, from day 7 until day 119 of ongoing gestations, resulting from transfer of MOET (n = 53), IVP-co-culture (n = 21) and IVP-SOF (n = 38) embryos, bPAG levels were compared in peripheral plasma of recipients. Plasma progesterone levels were compared as well. From day 25 of gestation onwards, bPAG could be detected in all recipients and the levels were significantly influenced by the day of gestation. Although IVP calves were significantly heavier than the in vivo produced calves, this difference was not reflected in the bPAG profiles of the embryo production groups. Yet, the mean bPAG level of the three last sampling moments (days 105-119) tended to be positively related to the birth weight of the calves, irrespective of the embryo production technique. Progesterone concentrations were not influenced by route of embryo production, but were significantly affected by parity of the recipient and day of gestation.     

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.     

Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization

The incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P < 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.     

Telomerase activity in early bovine embryos derived from parthenogenetic activation and nuclear transfer

This study examined the telomerase activity in preimplantation bovine embryos derived from either parthenogenetic activation or nuclear transfer. Telomeres are the DNA-protein structures located at the ends of eukaryotic chromosomes. Telomerase is the ribonuclear enzyme that helps to restore telomere length by synthesizing telomeric DNA repeat (5'-TTAGGG-3') from its own RNA template. Without telomerase activity, telomeres shorten with each cell division through conventional DNA replication. In most mammalian species, telomerase activity is present in germ cells but not in somatic cells. Previously, we reported the dynamics of telomerase activity in bovine in vitro fertilized (IVF) embryos. In the present study, we examined the telomerase activity in bovine embryos derived either from parthenogenetic activation or somatic cell nuclear transfer (i.e., cloning). Embryos from both sources were harvested at different stages, from zygote to blastocyst. Telomerase activity in embryos derived from parthenogenetic activation and nuclear transfer showed a dynamic profile similar to that of those derived from IVF. Telomerase activity was detected in embryos at all stages examined, with the highest level in the blastocyst stage, regardless of the method of embryo production.     

Pregnancy rates and births after unilateral or bilateral transfer of bovine embryos produced in vitro.

Late morulae and blastocysts produced in vitro were nonsurgically transferred to heifers by unilateral (n = 184) or bilateral (n = 94) transfer. Of the recipients, 58% had serum progesterone values greater than 1.4 ng ml-1 on day 21 and rectal palpation on day 35 showed that 50% (138 of 278) were pregnant. The embryonic mortality rate between days 21 and 35 was estimated to be about 14% and between days 36 and 90 to be about 12%. Of the animals, 8% aborted between days 91 and 250 of pregnancy. No difference was observed in pregnancy rates between unilateral transfer of one (47%) or two embryos (49%) and bilateral transfer (53%), or in the twinning rate between bilateral transfer (42%) and unilateral transfer of two embryos (33%). The pregnancy rate was 54% with embryos evaluated as morphologically excellent or good, 51% with fair embryos and 26% with poor ones. A higher pregnancy rate (60%) was obtained after embryo transfer when the synchrony between recipient and embryo was -1 day.     

牛体内,外受精胚胎玻璃化冷冻保存技术的研究初报

利用３种培养液即输卵管合成液（ＳＯＦ）、ＴＣＭ１９９和ＣＲｌａａ对牛体外受精后的卵母细胞进行培养,结果卵裂率分别达８５％、６７％和７２％,囊胚发育率分别为３７％、２１％和３０％。对所获得的囊胚利用ＥＦＳ玻璃化溶液进行冷冻保存。在１０％ＥＧ中预处理５ｍｉｎ后再移入ＥＦＳ４０平衡３０ｓ二步法冷冻保存的胚胎,其１解冻后继续发育率高达８６％,与对照组９１％相比无显性差异（Ｐ＞０．０５）。而ＥＦＳ３０二步     

Macromolecule absorption and cortisol secretion in newborn calves derived from in vitro produced embryos

Earlier reports indicate that calves derived from in vitro produced (IVP) embryos are more susceptible to neonatal disease than calves produced after artificial insemination (AI) or natural mating. The aims of the present study were to investigate whether calves born after IVP embryos show an altered macromolecule absorption (immunoglobulin G (IgG) and porcine serum albumin (PSA)) compared with AI calves and whether the macromolecule absorption could be related to the degree of acidosis or to the cortisol secretion around birth. Hence, IgG and PSA absorption in control AI calves (n=7) was compared with that in two groups of IVP calves (IVP-defined: SOFaa embryo culture with polyvinyl alcohol, n=6; IVP-serum: SOFaa embryo culture with serum and co-culture, n=8). The calves were fed colostrum (40ml/kg) at 2, 6 and 12h after birth. At 24h after birth, both AI and IVP calves had achieved a level of plasma IgG sufficient to provide passive immunization (>15mg/ml). When the values were adjusted for the varying colostral IgG contents and the degree of acidosis, the IVP-defined calves had significantly lower peak plasma IgG concentrations than the AI calves at 18-24h after birth (P<0.04). However, when the macromolecule marker (PSA), was fed to all calves at 2 and 12h after birth the resulting plasma PSA levels were significantly lower in the AI calves compared with the IVP calves during the whole observation period (P<0.0001). Calves with a moderate neonatal acidosis (mean pH<7.2 during the first 30min after birth) had reduced peak plasma IgG concentration at 18-24h after birth (P<0.02) compared to calves without acidosis. The basal and ACTH-stimulated cortisol levels were lower in the newborn IVP-defined calves than in the AI calves (P<0.05) and the IVP-serum calves (P<0.002). Cortisol levels shortly after birth correlated positively with birth weight (r=0.60, P<0.0001) and with gestation length (r=0.34, P<0.04). Since, the IVP calves absorbed sufficient amounts of IgG from colostrum to acquire sufficient passive immunity, we conclude that the lower viability described in IVP offspring probably is not caused by an impaired passive immunization. IVP-defined calves had significantly lower absorption efficiency of IgG compared with AI calves, whereas absorption of a non-Ig macromolecule (PSA) was higher for IVP than AI calves. This might indicate a more selective absorption in AI calves in favor of IgG. Acidosis around birth affected immunoglobulin absorption negatively. IVP-defined calves had significantly lower cortisol levels the first 3h after birth and during an ACTH-challenge and a lower IgG absorption efficiency, which might indicate a mild degree of organ dysmaturity in these calves.     

Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos.

Interferon-tau (IFNtau) is the pregnancy recognition signal of bovine embryos, inhibiting luteolysis. We studied trophoblastic growth and IFNtau secretion of embryos with different developmental potential, i.e., in vivo derived and in vitro produced embryos, cloned embryos and demi-embryos, to evaluate if the ability of secreting IFNtau might be responsible for differences in pregnancy rates after transfer of these categories of embryos to recipients. Day 8 embryos of excellent quality were individually placed in microdrops of buffalo rat liver cell-conditioned medium and maintained for up to 23 days. Embryos were observed on Days 11, 15, 19 and 23, the mean diameter (2r) of attached and spherical embryos was measured, and their trophoblastic area was calculated as r2pi or 4r2pi, respectively. Simultaneously, medium was changed and the IFNtau levels of conditioned media were determined using a bioassay of antiviral activity. Trophoblastic area was smaller (P < 0.05) in demi-embryos than in all other groups, which exhibited similar trophoblastic growth until Day 19. However, on Day 23 trophoblastic area of in vivo derived embryos was more than twice (P < 0.05) as large as those of in vitro produced and nuclear transfer (NT) embryos. IFNtau levels increased only slowly with time in culture of demi-embryos. By contrast, the level of IFNtau doubled from Day 11 to Day 15 in conditioned media from all other groups of embryos. The linear increase in IFNtau production of vivo and in vitro derived embryos continued until the end of the culture period, whereas conditioned media from NT embryos contained significantly (P < 0.05) less IFNtau activity on Days 19 and 23 than those of the former two groups. Our results demonstrate different capabilities of secreting IFNtau for in vivo derived and in vitro produced embryos vs. NT and demi-embryos, which may--at least part--be responsible for the differences in pregnancy rates after transfer to recipients.     

Few polyploid blastomeres in morphologically superior bovine embryos produced in vitro

Morphologically inferior bovine embryos developed in vivo have been shown by karyotyping to have a higher rate of chromosomally abnormal cells than morphologically normal embryos. The objective of this study was to re-examine this finding using interphase cytogenetics. A total of 155 IVP Day 8 bovine blastocysts were graded by their morphology (excellent, good, or poor) and timing of development (hatched, expanded, or non-expanded), and afterwards analysed for chromosome abnormalities by fluorescence in situ hybridization using differentially labelled probes for chromosomes 6 and 7. The overall frequency of diploid embryos was 7%, and did not differ according to grading. Although the frequency of mixoploidy was not correlated to the morphological grading, the blastocysts with excellent morphology displayed fewer polyploid nuclei in comparison to blastocysts with good (P=0.05) or poor morphology (P=0.01). There were however also prominent exceptions showing that a blastocyst with an excellent morphology can display a high degree of polyploidy. The results further demonstrate that the morphologically normal embryos contain a higher number of cells and develop more rapidly than the morphologically inferior embryos.     

Chronology of apoptosis in bovine embryos produced in vivo and in vitro

The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.