Development of bovine embryos on Vero/BRL cell monolayers (mixed co-culture)

The objective of this study was to test a new co-culture system of bovine embryos, which we call "mixed co-culture." This system consists of culturing embryos on cell monolayers composed of both Vero and BRL cells (Vero/BRL). Cumulus-oocyte complexes from ovaries of slaughtered cows were matured and fertilized in vitro. The presumptive zygotes were cultured with Vero/BRL (Group 1), BRL (Group 2) or Vero (Group 3) cell monolayers, in 40 microL drops of Menezo B2 medium supplemented with 10% FBS and antibiotics. The development of the presumptive zygotes was compared on Day 2 [48 h post insemination (pi)] and Day 7 (168 h pi). On Day 2, there was no difference between the groups. On Day 7, the highest percentage of compacted morulae/blastocysts was observed in mixed co-culture of Vero/BRL cells: 40% versus 36% on BRL versus 27% on Vero cell monolayers. The differences were statistically significant (P < or = 0.05). Among compacted morulae/blastocysts, blastocysts prevailed in mixed co-culture: 67% on Vero/BRL as compared with 55% on BRL and 27% on Vero cell monolayers. The differences were highly statistically significant (P < or = 0.01). The results suggest that Vero/BRL cells improve the development of bovine embryos.     

Viability of bovine blastocysts obtained after 7, 8 or 9 days of culture in vitro following vitrification and one-step rehydration

This study examined morphological appearance, viability and hatching rates in relation to the total cell number following vitrification of in vitro produced bovine blastocysts and expanded blastocysts. In Experiment 1, embryos obtained after 7, 8 or 9 d of culture were pooled and equilibrated in either 10% ethylene glycol (EG) or 10% EG plus 0.3M trehalose in Dulbecco's phosphate buffered saline (DPBS) supplemented with 10% calf serum and 0.6% BSA for 5 min each, at room temperature, and then vitrified together in precooled vitrification solutions consisting of 40% EG (Treatment 1), 40% EG plus 0.3M trehalose (Treatment 2), 40% EG plus 0.3M trehalose and 20% polyvinylpyrrolidone (PVP, Treatment 3) in DPBS. The embryo viability and hatching rates of Treatment 1 (19 and 3%) differed significantly (P < 0.05) from those of Treatment 2 (56 and 31%) and Treatment 3 (70 and 43%). There was a significant difference (P < 0.05) in embryo viability between Treatment 2 (31%) and Treatment 3 (43%). In Experiment 2, Day 7, 8 and 9 embryos were vitrified separately, with higher viability and hatching rates in Experiment 1 than in Experiment 2. The viabilities of Day 7 (87%), 8 (71%) and 9 (46%) embryos differed significantly (P < 0.05). Again, there were significant differences (P < 0.01) among the hatching rates of Day 7 (75%), 8 (38%) and 9 (9%) embryos. The total cell number of hatched blastocysts was then determined by differential fluorochrome staining. The total cell number of Day 7, 8 and 9 embryos differed significantly (P < 0.05).     

Cryopreservation of in vitro-derived bovine blastocysts microinjected with foreign DNA at the pronuclear stage

Days 6 and 7 bovine blastocysts derived from in vitro-fertilized and DNA-injected zygotes (day of IVF = Day 0) were cryopreserved either by conventional two-step freezing or by vitrification. Foreign DNA used for microinjection was the green fluorescent protein gene under the control of the immediate early promoter of human cytomegalovirus. All blastocysts were produced by an in vitro system and were harvested on Days 6 and 7. The proportion of DNA-injected zygotes developing into blastocysts on Days 6 and 7 (total 8%) was lower than that of nontreated zygotes (total 19%; P < 0.01). After cryopreservation in 1.5 M ethylene glycol, the survival rates of DNA-injected blastocysts assessed by re-expansion at 24 h of culture (Day 6: 59%, Day 7: 71%) were comparable with those of nontreated blastocysts (Day 6: 76%, Day 7: 71%). The post-thaw hatching rate within 72 h of culture of DNA-injected Day 7 blastocysts (38%) was not different from that of nontreated Day 7 blastocysts (40%), but the hatching rate of DNA-injected Day 6 blastocysts (23%) was lower than that of nontreated Day 6 blastocysts (47%; P < 0.05). After vitrification in 7.2 M ethylene glycol, 0.0026 M Ficoll-70 and 0.3 M sucrose, the survival and hatching rates of DNA-injected Day 7 blastocysts (61 and 28%, respectively) were similar to those of nontreated Day 6 (71 and 33%, respectively) and Day 7 (75 and 36%, respectively) blastocysts. However, the post-warming survival rate of DNA-injected Day 6 blastocysts was only 30%, and none of the blastocysts hatched (P < 0.01). The mean cell number of DNA-injected Day 6 blastocysts (100.3 +/- 36.4 cells) was lower than that of nontreated Day 6 blastocysts (130.5 +/- 37.1 cells; P < 0.01), while those of DNA-injected and nontreated Day 7 blastocysts were not different (111.2 +/- 42.8 and 119.6 +/- 31.4 cells, respectively). These results indicate that Day 7 IVMFC bovine blastocysts derived from DNA-injected zygotes can be successfully cryopreserved by conventional two-step freezing or vitrification.     

A high proportion of bovine blastocysts produced in vitro are mixoploid.

Fluorescence in situ hybridization with chromosome 6- and chromosome 7-specific probes was used to assess the extent of chromosome abnormalities in developing bovine blastocysts at 7-8 days after insemination in vivo or in vitro. Interphase nuclei (N = 10 946) were analyzed from 151 blastocysts produced in vitro and from 28 blastocysts recovered from superovulated animals. This revealed that 72% (109 of 151) of the in vitro-produced blastocysts were mixoploid, i.e., were a mixture of normal, diploid, and polyploid cells. However, only a small fraction of the total number of cells were chromosomally abnormal. Of the mixoploid blastocysts, 83% (91 of 109) contained less than 10% polyploid cells, 13% (14 of 109) contained 11-25% polyploid cells, and only 4% (4 of 109) of the blastocysts had more than 25% polyploid cells per blastocyst. In contrast, a significantly lower proportion (25%) of mixoploidy was found in 28 bovine blastocysts developed in vivo (p < 0.0001). All of the mixoploid blastocysts that had developed in vivo contained less than 10% polyploid cells. No entirely aneuploid blastocysts, i. e., blastocysts in which all cells had the same type of chromosome abnormality, were found in either of the groups. Taken together, the most common chromosome abnormalities observed were diploid-triploid mixoploidies and diploid-tetraploid mixoploidies. Thus, our results confirm earlier reports that morphologically normal bovine blastocysts developed in vivo are often mixoploids. We further show that in vitro-produced bovine blastocysts have a high rate of mixoploidy. Although the difference in mixoploidy rate detected in this study may not be general, it is an interesting phenomenon for further studies.     

A paucity of structural integrity in cloned porcine blastocysts produced in vitro

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P < 0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P < 0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P < 0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I: <20%; II: 20-40%; III: >40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.     

Numerical chromosome errors in day 7 somatic nuclear transfer bovine blastocysts

Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulation method: half-cytoplasts were made from zona-free oocytes by bisection, after which two half-oocytes and one granulosa cell (serum-starved primary culture) were fused together and activated. The NT embryos were cultured in modified synthetic oviductal fluid containing essential and nonessential amino acids, myoinositol, sodium citrate, and 5% cattle serum in microwells for 7 days, at which time nuclei from all blastocysts were extracted and chromosome aberrations were evaluated using dual-color fluorescent in situ hybridization with bovine chromosome 6- and 7-specific probes. Five embryo clone families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 +/- 4.5% and 73.7 +/- 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing or=5N chromosome complements, respectively) between two clone families were different (P < 0.01), as were blastocyst yields between other clone families (P < 0.01). Blastocyst yield was not correlated to % total ploidy error between clone families, but an inverse relationship (P < 0.01) between blastocyst total cell number and total % chromosome abnormality was observed within embryos. Categorization of the blastocysts into three quality grades (good, medium, and poor) and comparison of the distribution of ploidies when classified into 0%, 0.1-5.0%, 5.1-10.0%, 10.1-15.0%, and 15.1-100% errors within embryos indicated that medium- and poor-grade embryos were different (P < 0.05) from good-quality, in vitro-produced embryos. In a separate study, 11 different granulosa cell cultures (that did not correspond to those used for NT) were evaluated and found to possess only 0.23 +/- 0.12% ploidy errors. These results demonstrate that 1) the percentage of ploidy errors in bovine NT blastocysts is inversely related to total blastocyst cell number, 2) the mixoploid condition is representative of the majority of embryos, 3) 100% polyploid NT blastocysts can exist, and 4) the ploidy errors seem not to be derived from the donor cells.     

Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL

Programmed cell death (apoptosis) characteristically affects the single cells of blastocysts whereas necrosis affects cluster of cells in both the inner cell mass (ICM) and the trophectoderm (TE). This study uses the trophectodermrminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay as a way of evaluating the proportion of apoptotic cells and, thus, bovine blastocyst quality during in vitro culture at Days 6,7, and 8. Furthermore, parthenogenetic blastocysts were compared to in vitro fertilized blastocysts at Day 7. Confocal microscopy was used to generate three-dimensional reconstructions of the blastocysts. Apoptosis was observed in both early (Day 6) and late (Day 8) developing blastocysts. The dead cell index (DCI, total number of apoptotic nuclei/total number of nuclei) tend to increase as the in vitro culture time increases, and apoptosis is proportionately higher in the ICM than in the TE. The ratio of ICM to TE cells remains relatively constant even as the blastocysts cell number increases (Day 6 = 11.9 +/- 2.2, Day 7 = 11.2 +/- 0.5, Day 8 = 11.7 +/- 0.4). The overall cell number is significantly reduced in parthenogenetic blastocysts compared to Day 7 in vitro produced blastocysts (P = 0.037). The parthenogenetic blastocysts also show an increase of apoptosis over Day 7 controls. The decrease in cell number in the parthenogenetic blastocysts may be due to the increase of apoptotic nuclei observed. Based on these results we found the TUNEL assay to be a useful method for evaluating in vitro culture conditions of pre-implantation bovine embryos.     

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.     

Pentoxifylline improves in vitro fertilization and subsequent development of bovine oocytes

The purpose of this study was to examine whether pentoxifylline improves in vitro fertilization and developmental rates of bovine oocytes. In the first experiment, we examined the effects on the fertilization rate of various concentrations of pentoxifylline (0-7.5 mM) combined with heparin (10 IU/mL). In the second experiment, we examined fertilization cleavage and blastocyst rates after frozen-thawed spermatozoa, obtained from four different bulls, were incubated with heparin (10 IU/mL) with or without caffeine (5 mM) or pentoxifylline (5 mM). In the first experiment, a significantly higher fertilization rate was obtained in heparin containing 5 mM pentoxifylline compared to that in heparin alone or in heparin containing 7.5 mM pentoxifylline (86% vs 60% vs 64%, respectively). The percentage of monospermy in 5 mM pentoxifylline (81%) was significantly higher than in heparin alone (57%). In the second experiment, the interactions among Bulls A, B, C, and D; between treatments (pentoxifylline-with-heparin, caffeine-with-heparin and heparin alone), and between bulls and treatments were analyzed for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts. Among bulls, there was a significant difference in the number of oocytes penetrated (P < 0.01), monospermic oocytes (P < 0.05), cleaved oocytes (P < 0.001), and blastocysts (P < 0.001). Between treatments, there was a significant difference in the number of oocytes penetrated (P < 0.001), monospermic oocytes (P < 0.01) and cleaved oocytes (P < 0.001). Interaction between bulls and treatments was observed for the number of oocytes penetrated (P < 0.05). Individually, for Bulls A, C and D, the numbers of oocytes penetrated and monospermic oocytes in pentoxifylline-with-heparin were significantly higher than in heparin alone. For Bull D, significantly higher results were obtained for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts in pentoxifylline-with-heparin compared to caffeine-with-heparin and heparin alone (P < 0.05). These results suggest that treating sperm with 5 mM pentoxifylline in combination with heparin is effective for bovine in vitro fertilization and it that this treatment is effective even for bulls that produce low fertilization and blastocysts after sperm treatment with caffeine-with-heparin or heparin alone.     

In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells

To verify the importance of somatic cells upon in vitro embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 supplemented with estrous cow serum (10% v/v) and 0.25 mM sodium pyruvate (ECSTCM) under the following treatments: 1) ECSTCM alone; 2) together with bovine oviduct epithelial cells (BOEC); 3) with cumulus cells (CC); 4) in fresh BOEC conditioned ECSTCM; or 5) in frozen-thawed BOEC conditioned ECSTCM. Culturing zygotes encased in cumulus cells significantly reduced the cleavage rate (P<0.05). There was no difference between culture systems in the proportions of embryo development through the 8-cell stage (P=0.42) up to the morula/blastocyst stages (P=0.50) at Day 7 post insemination. However, co-culture with BOEC yielded the highest percentage (21.2% of zygotes; P<0.05) of quality Grade-1 and Grade-2 embryos with the number of blastomeres per embryo (114.4) comparable to that of 7-day-old in vivo-developed embryos of similar grades (102.5), and higher (P<0.05) than those of the other treatments. The ratio of blastocysts to total morulae/blastocysts obtained from frozen-thawed conditioned medium was lower (P<0.05) than that from ECSTCM or after co-culture with BOEC at Day 7 post insemination. On average, 7.5 to 17.5% of the zygotes developed to blastocyst, expanded blastocyst and hatched blastocyst stages by Day 10 post insemination, depending upon the culture system. The difference between treatments, however, was not significant (P=0.68). The results indicate that chronological development up to hatching of bovine IVM-IVF embryos is not favored by somatic cells; however, the presence of viable oviduct epithelial cells in culture significantly improves the quality of 7-day-old embryos.     

In vitro culture of bovine egg fertilized either in vivo or in vitro

Three-quarters of in vivo and one-third of in vitro fertilized bovine eggs reached blastocyst stage when cultured on tubal cell monolayers (TCM), but no hatching occurred in B2 medium supplemented with estrous cow serum. When after 3 days of culture on TCM, morulae were transferred on endometrial cell monolayers (UCM), the same proportion of blastocysts was obtained and one-third of them hatched. Histological studies of hatched blastocysts showed that the number of inner cells was significantly lower than in hatched blastocysts recovered in vivo 8-8.5 days after ovulation. Moreover, the number of pycnotic cells was higher than normal, although mitosis were present. On the contrary, there was no difference in either the number or the appearance of trophoblastic cells between blastocysts obtained in vitro and in vivo. The addition of transforming growth factor (TGF-beta) to either TCM or UCM co-cultures at the very beginning of blastocyst formation specifically stimulated growth of the inner cell mass (ICM). The number of cells at hatching was about double (120) and significantly higher than that found in 8-8.5-day blastocysts in vivo. Moreover, hatching percentages were similar to the controls, even when eggs were cultured for 8 days only on TCM. However the proportion of pycnotic cells remained higher than normal, although many mitotic cells were unevenly distributed in ICM) In vivo during hatching, there were always pycnotic cells in ICM, but their number was limited and approximately similar to the number of mitosis. The uterine factors which control both mitosis and pycnosis in ICM remain to be discovered.     

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age

The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P < 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5-13% vs. 2-4%; P < 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8.     

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.     

Reducing the amount of cytoplasm available for early embryonic development decreases the quality but not quantity of embryos produced by in vitro fertilization and nuclear transplantation

The effect of reducing the amount of cytoplasm available for early embryonic development was investigated in embryos produced by in vitro fertilization (IVF) and nuclear transplantation. In Experiment 1, approximately 1/2 or 1/20 of the cytoplasm was removed from bovine embryos at the pronuclear-stage of development. The percentage of embryos developing to the compact morula or blastocyst stage was significantly higher in non-manipulated controls (26%) than in embryos with 1/20 of the cytoplasm removed (16%), and those with 1/2 of the cytoplasm removed (10%; P < 0.05). There was also a significant difference in the average number of cells between blastocysts in which 1/20 of their cytoplasm was removed (67), those with 1/2 of their cytoplasm removed (55), and nonmanipulated controls (77; P < 0.05). In Experiment 2, nuclear transfer embryos were produced in which approximately 1/2 or 1/20 of the cytoplasm was removed during oocyte enucleation. The percentage of embryos developing to the blastocyst stage was 17% for both groups of nuclear transfer embryos compared to 44% for control embryos (P < 0.05). The mean number of cells in blastocysts produced by nuclear transfer in which 1/20 of the cytoplasm was removed during oocyte enucleation (61) was no different than that in control embryos (66), but significantly higher than the mean number of cells in blastocysts produced by nuclear transfer in which 1/2 of the cytoplasm was removed (42; P < 0.05). There was no indication that altering the amount of cytoplasm available for early embryonic development of IVF embryos affected the timing of differentiation events, including those of embryo compaction and blastocyst formation.     

Effect of human leukemia inhibitory factor on in vitro development of IVF-derived bovine morulae and blastocysts

This study was conducted to investigate whether human leukemia inhibitory factor (hLIF) improves the subsequent development of IVF-derived bovine morulae and blastocysts. To obtain IVF-derived bovine morulae, ova were matured and fertilized in vitro and cultured in 0.5 ml of synthetic oviduct fluid (SOF) medium supplemented with 10% human serum (HS) for 5 d at 39 degrees C under a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2). Morulae and early blastocysts at Day 5 of culture were cultured in 0.5 ml of SOF medium with or without 5000 U/ml recombinant hLIF for 2 or 3 d (2 groups). To investigate the effect of addition of hLIF on the subsequent development of morulae, SOF medium was supplemented with 8 mg/ml BSA instead of HS. To test whether hLIF affects the subsequent development of IVF-derived bovine blastocysts, only good blastocysts that developed from SOF medium with or without hLIF at Days 7 and 8 of culture were frozen by a conventinal slow freezing method and again cultured in SOF medium with or without the addition of hLIF for 3 d after thawing (4 groups). Survival of frozen-thawed bovine embryos was evaluated for re-expansion and hatching of blastocysts during 3 d of culture. There was no significant difference in the developmental rate of Day 5 embryos to blastocysts between those cultured with (47.8%) and without (47.6%) addition of hLIF. However, the addition of hLIF before freezing significantly increased the hatching rate of IVF-derived bovine morulae (P < 0.05), whereas addition of hLIF after thawing did not increase the subsequent development of blastocysts. These results suggest that hLIF added at the Day 5 morula stage may contribute to bovine embryonic development through the hatching process.     

Analysis of DNA fragmentation in mouse embryos exposed to an extremely low-frequency electromagnetic field

Effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on DNA damage in biological systems are still a matter of dispute. The aim of the present study was to investigate the possible effect of electromagnetic field exposure on DNA fragmentation in cells (blastomers) of mouse blastocysts. Eighty female NMRI mice were randomly divided into 2 groups of 40 animals each. The control group was left unexposed whereas the animals in the EMF-group were exposed to a 50-Hz EMF at 0.5?mT 4?h per day, 6 days a week for a duration of 2 weeks. After the 8(th) day of exposure, the female mice in both groups were superovulated (with injections of pregnant mare serum gonadotropin and human chorionic gonadotropin) and then mated overnight. At approximately 4 days after mating (102?h after the human chorionic gonadotropin treatment), blastocysts were obtained by flushing the uterus horns. The mean numbers of pregnant mice, blastocysts after flushing, blastomers within the blastocysts, and the DNA fragmentation index following staining in both groups were compared using statistical methods (SPSS, the Chi-square test, the Student's t-test and the Mann-Whitney U-test, P?相似文献   

Sucrose-induced shrinkage of in vitro produced bovine morulae: effect on viability, morphology and ease of evaluation

Sucrose (0.3 M) was used to cause artificial compaction of the embryonic cell mass of in vitro produced bovine embryos to facilitate morphological evaluation. Embryos were produced using routine in vitro maturation (IVM) and fertilization (IVF) techniques. The time necessary to induce shrinkage in 0.3 M sucrose to 75% of the original volume of Day 5 morulae was found to be less than l min, and 95% of the volume was regained in PBS after 2.5 min. No detrimental effect was observed after a 5- to 10-min sucrose treatment on subsequent blastocyst formation at Days 6 and 7 (P > 0.05). Furthermore, no significant differences were observed in the total number of cells, or in the mitotic and pycnotic cell index of blastocysts in different treatment groups. Agreement among 7 evaluators grading 40 Day 6 embryos was examined using the kappa coefficient of agreement (kappa). Overall agreement among evaluators for classification of quality grade was poor (48.2 %, kappa = 0.31) for embryos evaluated in PBS, but the rate improved when the same embryos were scored in sucrose (62.5 %, kappa = 0.49). Evaluating less compact in vitro produced bovine morulae in sucrose increases agreement among evaluators, since embryos in sucrose mimick the appearance of in vivo produced embryos. Thus, we conclude that scoring in vitro produced embryos in sucrose improves agreement among evaluators.     

Patterns of silver staining in cells of six-day blastocyst and kidney fibroblast of the domestic rabbit

The distribution pattern of silver-NORs was studied in cells of six-day blastocysts and kidney fibroblasts of the rabbit using the Ag-AS technique. At metaphase and interphase there was a binomial distribution of the number of stained sites in both populations but blastocysts had a greater percentage of cells with larger numbers of stained sites. Up to 7 of the 8 chromosomes known to bear NORs were stained in cells from blastocysts while a maximum of 6 were stained in fibroblasts. A significant difference was found between the mean numbers of chromosomal NORs per cell in metaphases from blastocysts and fibroblasts, where they were 4.2 and 3.3 respectively. Similarly, the mean number of NORs in interphase was significantly greater in cells from blastocysts. The distribution of staining on chromosome pair 13 was related to cell type. Significantly more cells in blastocysts than fibroblasts showed staining in this chromosome pair.     

Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.     

Utility of ultrasound stimulation for activation of pig oocytes matured in vitro

The present study was carried out to examine the development of pig oocytes after exposing to ultrasound under various conditions. When oocytes were exposed to ultrasound in the sorbitol medium, the blastocyst formation rate was significantly (P < 0.01) higher than that of oocytes exposed in HEPES-TLP-PVA. Optison, an echo-contrast microbubble, prevented the development into blastocysts of oocytes exposed to ultrasound in the sorbitol medium (P < 0.01). The mean number of cells in the blastocysts developed from oocytes exposed to ultrasound with 10% duty cycle was significantly (P < 0.05) higher than that obtained by using ultrasound with 50% duty cycle. The blastocyst formation rate of oocytes exposed to ultrasound for 30 sec was significantly (P < 0.05) higher than that exposed for 10 sec. There were no significant differences in the rates of oocytes developed to the blastocyst stage and the mean numbers of cells in the blastocysts among different intensities of ultrasound. The pronuclear formation and second polar body extrusion rates of oocytes exposed to ultrasound did not differ from eclectically activated oocytes. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from oocytes activated by exposing to ultrasound was significantly (P < 0.05) higher than that obtained by applying electric pulses. The results of the present study showed that ultrasound stimulation can induce the nuclear activation and parthenogenetic development of pig oocytes matured in vitro.