Effects of bovine serum albumin and estrous cow serum on development and ultrastructure of in vitro-produced porcine embryos

This study evaluated the effects of bovine serum albumin (BSA; 4 mg/ml) and estrous cow serum (ECS; 10%) in North Carolina State University (NCSU) 23 medium on the development of in vitro-matured and in vitro-fertilized porcine oocytes. Early cleavage rate was significantly (P < 0.05) higher in NCSU/ECS (71.3 +/- 14.7%) vs. NCSU/BSA (60.6 +/- 4.7%). Cleavage beyond the four-cell stage was not different between the two culture media (43.5 +/- 9.5% and 41.4 +/- 17.7%, respectively). The proportion of development to blastocysts was--with borderline significance (P = 0.05)--higher in NCSU/BSA (28.0 +/- 4.4%) than in NCSU/ECS (20.4 +/- 7.3%). Blastocysts produced in NCSU/BSA had significantly (P < 0.001) higher cell numbers than those cultured in NCSU/ECS (29.5 +/- 20.1 vs. 16.9 +/- 10.8). The ultrastructure of in vitro-produced blastocysts from both culture systems was compared vs. in vivo-derived blastocysts. The latter showed a clear differentiation between trophectoderm (TE) and inner cell mass (ICM) cells. The TE cells were anchored to other TE cells or ICM cells by long, well-developed junctional complexes. The apical membrane of trophoblast cells was covered with numerous microvilli. Mitochondria were abundant, round to elongated in shape, and showed clear transverse cristae. The ultrastructure of blastocysts cultured in NCSU/BSA mimicked that of in vivo-derived embryos closely. In contrast, blastocysts from the NCSU/ECS culture system displayed an irregular ultrastructure with reduced numbers of organelles and numerous cytoplasmic inclusions, such as lipid-yolk-vacuoles and vacuoles with lipid content. In some sections of these embryos, cellular debris was detected in cytoplasm. The shape of mitochondria was more ovoid and cristae were not visible. In summary, our results demonstrate a beneficial influence of ECS in the culture medium on initial cleavage of in vitro-produced porcine embryos. Clearly negative effects of ECS in the subsequent culture period are associated with marked ultrastructural changes of embryonic cells.     

Effect of pterostilbene on development,equatorial lipid accumulation and reactive oxygen species production of in vitro-produced bovine embryos

The pterostilbene (PT) molecule is a phytoalexin with a reducing effect on reactive oxygen species (ROS) and with a capacity to block lipogenesis. However, the potential reducing effects of PT on equatorial lipid accumulation and ROS have not yet been elucidated for in vitro-derived bovine embryos. The present study evaluated the effects of concentrations of 3, 1, 0.33, 0.11 μM PT, and a vehicle group on the percentage of cleaved embryos, embryos with more than 6 cells, percentage of blastocyst on Day 7 and 8, percentage of transferable embryos on Day 7, the cell count and relative concentration of lipids. In the second experiment, the effects of 0.33 μM PT and a vehicle group within two different O₂ environments (5% and 20%) were evaluated for ROS generation and the percentage of Day 8 blastocysts. In the first experiment, no significant differences were found between the treatments with PT and the vehicle group (p > .05) concerning the percentage of cleaved embryos and embryos with more than 6 cells. Lipid reduction was observed in the groups treated with PT versus the vehicle group (p < .05). The vehicle group showed a higher rate of blastocyst production on Days 7 and 8 (p < .05) and an increase in the percentage of transferable embryos on Day 7 compared to the PT treatment groups (p < .05). Cell counts were not significantly different between treatments with PT and the vehicle group (p > .05). In the second experiment, the O₂ concentration did not significantly affect ROS generation (p > .05); however, the groups treated with PT (0.33 μM) had a reduction in ROS (p < .05). The O₂ concentration also did not significantly affect the rate of blastocyst production on Day 8 (p = .7696). Future research should be conducted to ascertain whether the reduction of lipids could enhance the cryopreservation and post-thaw viability of PT-treated embryos.     

Effect of linoleic acid-albumin on post-thaw survival of in vitro-produced bovine embryos at the 16-cell stage

The effect of addition of linoleic acid-albumin (LAA) to culture medium before freezing on the survival rate of bovine 16-cell embryos after freezing-thawing was investigated. Embryos were incubated in CR1aa containing LAA (0.25 mg/ml) for 4 days after insemination. A conventional slow cooling method was used, in which embryos were cooled at a rate of 0.3 degrees C/min to -30 degrees C in medium supplemented with 1.5 M ethylene glycol and 0.2 M trehalose. The developmental rate to the blastocyst stage of thawed embryos that had been cultured with LAA-containing medium before freezing was higher than that of these cultured without LAA (P<0.05). However, with fresh, non-frozen, embryos that were incubated under the same culture conditions (with and without LAA), no such difference was found.     

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.     

Uterine tubal cells remain uninfected after culture with in vitro-produced embryos exposed to bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) has been isolated from washed and sonicated, in vitro-produced embryos, but the infectivity of BVDV associated with intact, developing, embryos has not been demonstrated. The objective of this study was to determine if a dose of BVDV infective for co-culture cells was associated with individual, developing embryos, following artificial exposure to the virus and washing. In 5 replicates, zona pellucida-intact, in vitro-produced embryos were assigned to a negative control embryo group, or were incubated in 10(5)-10(6) cell culture infective doses (50%, CCID50) per milliliter of a type I, noncytopathic (strain SD-1) BVDV for 2 h. Unexposed negative control embryos and exposed positive control embryos were washed, sonicated and assayed for BVDV using virus isolation with immunoperoxidase monolayer assay. Immediately or following cryopreservation, remaining virally-exposed, washed embryos were co-cultured individually with BVDV-negative cultures of bovine uterine tubal cells in a medium free of BVDV-neutralizing activity. After two days in culture, uterine tubal cells and embryos (including the zona pellucida) were separated and washed. The culture medium, uterine tubal cells and embryos were then assayed for BVDV. Bovine viral diarrhea virus was not isolated from any negative control embryo group, but was isolated from all positive control embryo groups. Although all uterine tubal cell populations were confirmed to be susceptible to BVDV, virus was never isolated from uterine tubal cells or embryos from post-exposure culture. In conclusion, although BVDV remains associated with washed in vitro-produced embryos, the virus associated with unsonicated embryos was not infective for uterine tubal cells in vitro.     

卵泡抑素对牛胚胎早期发育的影响

利用添加外源性卵泡抑素或抑制卵泡抑素基因表达的方法,以牛受精胚胎为研究对象,结果显示,胚胎阶段适当的添加卵泡抑素,可以加快胚胎发育,不但提高卵裂率,还可以在受精后7d提高囊胚发育率,这其中10μg/L的卵泡抑素为最佳添加量,达到35%的囊胚率。研究还发现,早期阶段沉默卵泡抑素基因,不会影响囊胚率,与处理组无显著差别。结果表明,可能在早期的胚胎发育的最初阶段,卵泡抑素并不是必须的,但如果外源性的添加适量的卵泡抑素可以在正常的基础上提高发育率。     

Effect of phosphonoformic acid in the development of bovine embryos in vitro

This research evaluated the ability of phosphonoformic acid to inhibit bovine herpesvirus 1 (BHV-1) in cumulus cells commonly used in co-culture with bovine in vitro-produced embryos. At 200 and 400 microg/ml, phosphonoformic acid inhibited 4 logs of BHV-1. Subsequently, phosphonoformic acid (200 and 400 microg/ml) added to both in vitro fertilization and culture medium resulted in a decrease in the proportion of developed blastocysts, and the number of cells per blastocyst was lower in the treated embryos. Therefore, while phosphonoformic acid can effectively inhibit replication of BHV-1 in co-culture cells, it also inhibits development of in vitro-produced bovine embryos.     

In vitro development of bovine embryos encapsulated in sodium alginate

The objective of this study was to evaluate the in vitro development of bovine embryos encapsulated in alginate. Day-4 embryos produced in vitro (n = 110) were encapsulated with 1.5% sodium alginate and co-cultured with oviduct cells. Unencapsulated embryos (n = 106) were used as controls. In vitro development rate to the blastocyst stage at Day 7 was similar between encapsulated, 42.7%, (47/110) and control. 34% (36/106). embryos. Although encapsulated embryos were able to hatch on Day 9, they did so in a lower proportion than controls (P < 0.05). In conclusion, alginate encapsulation of bovine embryos does not disturb the in vitro development up to the blastocyst stage but significantly reduces the hatching process.     

Blastocyst production from in vitro-produced day-2 bovine embryos classified by cleavage stage, and cytogenetical evaluation of the resultant day-8 blastocysts

The present study was conducted to determine the criteria for selecting good quality embryos on Day-2 post-insemination and at the blastocyst stage. Bovine oocytes were matured, fertilized and cultured in vitro. First, Day-2 embryos were classified based on the number of blastomeres into 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell stage embryos; chromosome samples were then prepared. In the second experiment, the Day-2 embryos classified according to the number of blastomeres were cultured separately for an additional 6 days (Day 8). The resultant Day-8 blastocysts from each group of Day-2 embryos were classified into the following 3 grades based on morphology and diameter: Grade A, hatched and hatching blastocysts; Grade B, expanded blastocysts; and Grade C, unexpanded blastocysts. Chromosome samples were then prepared. The 5- to 8-cell stage Day-2 embryos had the lowest incidence of chromosomal abnormalities (13.5%, P<0.05) and the highest development rate to blastocysts (59.2%, P<0.05). Furthermore, the blastocysts derived from the 5- to 8-cell stage embryos had the largest mean number of cells (102.8+/-42.4, P<0.05), largest number of metaphases per blastocyst (9.5+/-4.8, P<0.05) and lowest incidence of chromosomal abnormalities (24.6%, P<0.05). The Grade A blastocysts had the largest mean number of cells (136.6+/-33.4, P<0.05), a large number of metaphases per blastocyst (11.9+/-5.5, P<0.05) and a low incidence of severe chromosomal abnormalities (17.3%). The results showed that, at Day 2, the 5- to 8-cell stage embryos were of better quality since they had the lowest incidence of chromosomal abnormalities and the highest blastocyst rate and the resultant blastocysts had the largest number of cells and lowest incidence of chromosomal abnormalities. In particular, selection of Grade A blastocysts can improve the development rate to term.     

Production and development of calves from sexed-bisected bovine embryos

Sixty-five chromosomal preparations from bisected bovine embryos were examined and 43 embryos (66.2%) with metaphase plates were observed; however, only 25 embryos (38.5%) were sexed. Fifteen of the bisected embryos sexed by chromosomal analysis were transferred to each of the 15 recipients, and 5 recipients became pregnant. Female calves were born on December 28, 1985, August 2, 1986 and July 18, 1988. The first female calf born from a sexed-bisected embryo (December 28, 1985) was the first case in Japan. The gestation lengths and birth weights of these calves were 277, 278 and 274 days, and 42.0, 44.0 and 37.0 kg, respectively. These two calves grew within the range of the "Standard developmental growth curve of the Holstein heifer" of the Japanese Holstein Association. The milk yield from 2 sexed-bisected cows were recorded. In the first cow (Case 1), she produced 8,575 kg of milk, 5.0% fat and 9.4% solids-non-fat (SNF) per year. In the other cow (Case 2), her expected milk volume was 7,800 kg per year. Confirmation of parentage was done by blood typing, and certified by the Japanese Livestock Animal Improvement Association.     

Effect of co‐culture with intact embryos on development of bovine separated blastomeres

To improve embryo development in bovine separated blastomeres, we evaluated applicability of co‐culture with intact embryos. The morphological quality of blastocysts derived from separated blastomeres and rate of blastocyst formation were only slightly increased when the cells were co‐cultured with intact embryos, which did not provide significant differences when statistically analyzed. However, the cell count of inner cell mass (ICM), trophectoderm (TE) and total number of cells in Day 8 blastocysts were significantly higher when the cells were co‐cultured with the intact embryos than those with the cells cultured individually (P < 0.05). Transfer of four monozygotic pairs of blastocysts derived from the cells co‐cultured with intact embryos led to three pregnancies even when the blastomeres were produced by in vitro maturation and in vitro fertilization of oocytes collected by ovum pick‐up from elite cows. These results suggest that co‐culturing with intact embryos may enhance development of bovine separated blastomere.     

超数排卵对家兔早期胚胎体外发育的影响

为探讨超数排卵对家兔早期胚胎体外发育能力的影响,收集自然发情组和超数排卵处理组交配72 h后家兔胚胎,对经筛选合格的胚胎以小鼠成纤维细胞为饲养层进行体外培养,以自然发情交配的家兔胚胎为对照,观察培养96 h贴壁情况和内细胞团传代能力.超数排卵处理的家兔交配后经筛选所获得的可用胚胎数量增多;其96 h贴壁率和成功传代内细...     

Infection of early bovine embryos with bovine herpesvirus-1

Recently hatched bovine embryos were exposed in vitro to 1 of 4 strains of bovine herpesvirus-1 to determine whether the viruses would replicate in these embryos and, if so, what pathologic consequences would ensue. Exposure to each of the viruses resulted in embryonic infection and death, and replication of the agents was demonstrated by electron microscopy and titration of progeny virus. There were no dramatic differences between virus strains in pathogenicity or in the ultrastructural pathologic findings of infection.