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PubMed1998

Differentiating between parthenogenetic and "positive development" embryos in turkeys by molecular sexing.

In mated or inseminated turkeys, 5 to 15% of eggs set for incubation show only rudimentary development. Most of these embryos die during the first 24 to 48 h of incubation and contain only unorganized sheets of tissue. This abnormal development is termed "positive development" (PD). Turkey eggs also show incidence of parthenogenesis and the resulting progeny is believed to be always male. As both types of embryos are morphologically similar at the early stage of incubation, it has been speculated that PD embryos may in fact be parthenogens. By identifying the sex at the blastodermal stage with the help of DNA markers, we have differentiated between the PD embryos and parthenogens. Parthenogenetic embryos were obtained from eggs laid by uninseminated or virgin Beltsville Small White (BSW) hens, and the PD embryos were obtained from eggs of inseminated Nicholas and British United Turkeys of America (BUTA) hens. DNA was extracted from blastoderms of parthenogenetic and PD embryos. Turkey W-chromosome specific DNA probe and primers were used to detect females in all samples by Southern blot and polymerase chain reaction (PCR), respectively. No female was detected among the 35 parthenogens examined, whereas there were 3 females among the 11 PD embryos. The presence of both males and females among PD embryos suggests that they are products of fertilization, and that at least these 3 female embryos, if not all the 11 PD embryos, are not of parthenogenetic origin. It is concluded, therefore, that PD embryos result from errors in fertilization or from early embryonic mortality following successful fertilization, and that they are unlikely to be of parthenogenetic origin.

Animals↗

PubMedDate not supplied

Genetic control of survival of frozen mouse embryos.

Lines of mice selected for increased litter size (L+), increased body weight (W+), or randomly (K) were used to study genetic variation in embryo cryosurvival in response to standard cryopreservation protocols. A total of 60528-cell embryos from 400 females were used in two studies. In Study 1, embryos from L+, W+, and K were frozen by slow-cool and ultrarapid (direct-plunge) methods to evaluate effects of selection on cryosurvival and genotype X freezing method interaction. Post-thaw survival (PTS) was measured as percentage of recovered embryos developing in vitro to blastocyst per donor female. Nonfrozen control embryos developed similarly for each line. Within slow-cool freezing, lines differed (W+ greater than K, W+ = L+, L+ = K; p less than 0.05); no differences were observed within the ultrarapid freezing. However, line X method interaction effects on PTS were not significant. In Study 2, reciprocal crosses were made between L+ and K and between W+ and K. Hybrid and pure line embryos were frozen by slow-cooling. Control embryos developed similarly for all genotypes. Selection lines did not differ for overall PTS. However, hybrid embryos from L+ dams were superior to those from K dams (84 vs. 61%; p less than .001). No overall embryo heterosis was observed. Differences were not significant among embryo genotypes or treatments for cell number or in vivo survival. These results demonstrate significant correlated responses in embryo post-thaw cryosurvival due to selection, and implicate both maternal and embryonic genomes as controlling mouse embryo cryosurvival.

Animals↗

PubMed1999

Onset of nucleolar and extranucleolar transcription and expression of fibrillarin in macaque embryos developing in vitro.

Specific aims were to characterize the onset of nucleolar and extranucleolar transcription and expression of the nucleolar protein fibrillarin during preimplantation development in vitro in macaque embryos using autoradiographic and immunocytochemical techniques. Autoradiography was performed on whole embryos cultured with [3H]uridine for assessment of nucleolar (rRNA) and extranucleolar (mRNA) transcription. Expression of fibrillarin was immunocytochemically assessed in whole embryos using a primary antibody against fibrillarin and a fluorescein isothiocyanate-conjugated secondary antibody. Extranucleolar incorporation of [3H]uridine was first detected in 2-cell embryos cultured 6-10 h with [3H]uridine. Culture with alpha-amanitin prevented incorporation of label in 2-cell embryos, and treatment with ribonuclease reduced the signal to background levels, indicating that [3H]uridine was incorporated into mRNA and not rRNA or DNA. Nucleolar incorporation of [3H]uridine was not evident in pronucleate-stage or 2- to 5-cell embryos, but it was detected in one 6-cell embryo and in all 8-cell to blastocyst-stage embryos. Fibrillarin was first expressed in some 6- to 7-cell embryos, but it was consistently expressed in all 8-cell embryos. Fibrillarin was localized to the perimeter of the nucleolar precursor bodies, forming a ring that completely encapsulated these structures. Fibrillarin was not expressed in 8- to 16-cell embryos cultured with alpha-amanitin, indicating that it is transcribed, rather than recruited, at the 8-cell stage. In conclusion, in in vitro-fertilized macaque embryos developing in vitro, extranucleolar synthesis of mRNA is initiated at the 2-cell stage while the onset of nucleolar transcription occurs at the 6- to 8-cell stage, coincident with expression of fibrillarin.

Amanitins↗

PubMed2006

Biosecurity and the various types of embryos transferred.

The aim of the present paper was to review some features related to the risk analysis of three types of embryos to be transferred, namely the in vivo derived, the in vitro produced and the cloned ones. For in vivo-collected embryos, a considerable number of experiments and scientific investigations have been performed and hundreds of thousands of embryos are transferred annually with no contamination of associated diseases. Provided that the code of practice such as that published by the International Embryo Transfer Society is strictly followed by the embryo transfer practitioners, the statement made some 17 years ago saying that the in vivo-derived embryo transfer was the safest way of exchanging genes remains entirely true, thanks to the professionalism of the embryo transfer industry. For the in vitro-produced embryos, some particular rules have to be followed because of specific risks for some pathogens to strongly adhere to the zona pellucida of such embryos. There are some means to monitor and control those effects, and the transfer of in vitro-produced embryos can also be a very safe way to exchange genes around the world. The third type of embryos, the cloned ones, is a quite different category and the risk analysis to be soundly made still needs a lot of investigations so as to characterize the potential risks if there are, in terms not only of disease transmission but also in terms of public health, zoonotic risks as well as those related to quality and safety of food. The problem in this regard, is more directly addressed for offspring of clones than to the cloned embryos themselves. Published data on this issue are increasing in numbers so that progress in that area is expected in the few years to come.

Animals↗

PubMed2006

Blastocyst development of 4-cell mouse embryos after laser destruction of one blastomere with or without its microsurgical removal.

AIM: To study the rate of blastocyst formation in 4-cell mouse embryos after laser destruction of one blastomere, with or without microsurgical removal of the destroyed blastomere. METHODS: Mouse embryos were randomly allocated to two control and two experimented groups. Control embryos were either non-manipulated (117 embryos) or underwent laser ablation of zona only (114 embryos). Experimented embryos had laser destruction of zona and the adjacent blastomeres. Destroyed blastomeres were either left in situ (115 embryos) or were microsurgically removed (107 embryos). They were cultured in sequential media for 72 h and were assessed for cleavage/morula arrest and blastocyst formation rates. RESULTS: Embryos arrested at cleavage/morula stages were higher when destroyed blastomeres remained in situ (30.4%) than when they were immediately removed (15.0%, P < 0.05). Blastocysts in the group with immediate removal of the destroyed blastomeres (85%) were significantly higher than when destroyed blastomeres were left in situ (69.6%, P < 0.05). Blastocyst formation in the repaired embryos was significantly lower than the non-manipulated (91.5%) and the manipulated controls (94.8%, P < 0.05). Hatching blastocysts were highest in control embryos with zonal ablation (72.8%). Proportions of hatching/hatched blastocysts in embryos, with or without removal of destroyed blastomeres, were not significantly different (39.3% and 33.9%, respectively). The percentage of embryonic loss during an attempt at microsurgical repair was 6.1%. CONCLUSION: Microsurgical removal of destroyed blastomere was effective in restoring blastocyst development. It could reduce the rate of cleavage/morula arrest.

Animals↗

PubMed2004

Single or multiple embryo transfer following in vitro fertilisation for improved neonatal outcome: a systematic review of the literature.

BACKGROUND: The aim of the current review was to determine if single versus two or more embryos, or double versus three or more embryos, transferred to the woman of a subfertile couple at in vitro fertilisation (IVF) maximises the likelihood of pregnancy, while minimising the likelihood of multiple pregnancy and adverse sequelae. METHODS: Studies were identified that reported maternal, infant and cost outcomes following embryo transfer at IVF. RESULTS: Three randomised trials and 17 cohort studies were included. From two randomised trials, single embryo transfer was found to result in decreased incidence of clinical pregnancy, multiple pregnancy and low birthweight. In the cohort studies for single embryo transfer compared with transfer of two or more embryos the incidence of live birth and singleton pregnancies was unchanged, and the incidence of multiple pregnancies and low birthweight was reduced. For double embryo transfer compared with the transfer of three or more embryos, the incidence of clinical pregnancy, live birth, preterm birth and low birthweight babies was reduced. CONCLUSIONS: Information on neonatal and maternal outcomes following transfer of different numbers of embryos is limited. Transfer of one embryo does not alter the likelihood of a singleton pregnancy or birth when compared to transfer of two or more embryos. Transfer of one or two embryos decreases the risk of a multiple pregnancy, preterm birth and low birthweight. Further large, well-designed randomised trials are required to provide maternal and neonatal outcomes of relevance to a couple undergoing IVF.

Australia↗

PubMed1975

Incidence and origin of heteroploidy, especially haploidy, in chick embryos from intraline and interline matings.

Preparations for chromosomal analysis were made from 2107 chick embryos at 16 hours of incubation. The embryos resulted from intraline and reciprocal interline matings of two genetically different stocks (AG and D6). The two stocks had been previously characterized as producers of high (AG) and low (D6) frequencies of chromosomally aberrant embryos. The overall frequency of aberrant embryos was 4.0 plus or minus 0.42%. The types and frequencies of abnormalities were: haploidy and haploid-euploid mosaics, 57%; polyploidy and polyploid-diploid mosaics, 19%; aneuploidy, 17%; aneuploid-diploid mosaics, 5%; and structural aberrations, 2%. Although there were no significant differences among the four types of matings in the overall frequency of heteroploid embryos (P greater than 0.1), a significant difference (P smaller than 0.01) in the frequency of haploid and haploid-euploid mosaic embryos was found. The difference was entirely attributable to the line of dam; D6 dams had 1.2% and AG dams had 2.7% haploid and haploid-euploid mosaic embryos. The difference between lines of sires was not significant. In addition, there was evidence of nonrandom distribution, among dams of both lines, of haploid and haploid-euploid mosaic embryos. It was concluded that the genotype of dam is an important influencing factor in the production of haploid cell lines in embryos. The superfluous genome in triploid embryos is usually maternal in origin, resulting from retention of the second polar body in the ovum. Sex-chromosome aneuploidy in chick embryos apparently derives from nondisjunction at meiosis I of oogenesis. The sex proportion of 2023 chromosomally sexed, diploid embryos was 50.2 plus or minus 1.1% male. No significant heterogeneity was observed among the types of matings. Sex proportion was not significantly influenced by any of a number of nongenetic variables.

Aneuploidy↗

PubMed2006

Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions.

BACKGROUND: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined. METHODS: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6. RESULTS: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts. CONCLUSION: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

Animals↗

PubMed1989

Genetic differences in glucose phosphate isomerase activity among mouse embryos.

We have compared mouse embryos of three heterozygous, congenic genotypes (with high, medium and low levels of oocyte-coded glucose phosphate isomerase (GPI-1) activity respectively) to test whether 1) the survival time of oocyte-coded GPI-1 activity in the early embryo is affected by its activity level in the oocyte and 2) whether embryo-coded GPI-1 is detected earlier in embryos that inherit low levels of oocyte-coded GPI-1. The oocyte-coded GPI-1 was entirely GPI-1A allozyme in the high and medium groups but was the less stable GPI-1C allozyme in the low group. We determined total GPI-1 activity and the ratio of different GPI-1 allozymes in early embryos and calculated the activity of oocyte-coded and embryo-coded GPI-1. In all three groups, the oocyte-coded enzyme activity remained at a more or less constant level for the first 21 1/2 days. Some oocyte-coded GPI-1 remained in 4 1/2 day embryos from the high and medium groups but was gone by 5 1/2 days. Very little remained in 4 1/2 day embryos that inherited low levels of a less stable form of the enzyme (GPI-1C allozyme). Despite a 4- to 5-fold difference in initial oocyte-coded GPI-1 activity, no differences were seen among the three genotypically distinct groups of embryos in the time of activation of the embryonic Gpi-1s genes. The embryo-coded GPI-1 was first detectable in 3 1/2 day compacted morulae in all three groups. The level of oocyte-coded GPI-1, in the high group, when embryo-coded GPI-1 was first detected was higher than the level in the low group at any stage prior to detection of embryo-coded GPI-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles.

Early bovine embryos (1- to 8-cell stages) were recovered from superovulated heifers at slaughter on Days 2 or 3. Embryos were cultured for 3-4 days in Medium B2 supplemented with 15% (v/v) fetal calf serum in the absence (B2SS, 106 embryos) or presence of trophoblastic vesicles (B2SS + TV, 190 embryos). At the end of culture, there were more (P less than 0.001) morulae (greater than or equal to 16 cells) in B2SS X TV (46%) than in B2SS alone (18%) irrespective of the initial cell stage. More 8-cell embryos reached the 16-cell stage than did embryos with less than 8 cells (30% vs 15% in B2SS, P greater than 0.05; 70% vs 41% in B2SS + TV, P less than 0.005). After culture, 102 morulae were transferred non-surgically to temporary recipient heifers (84 embryos cultured in B2SS + TV and 18 in B2SS). After 2 or 3 days, 14 out of 58 embryos from the B2SS + TV group and 3 out of 10 embryos from the B2SS group were recovered as blastocysts. Most blastocysts were deep-frozen and stored for several weeks. After thawing, 10 apparently normal embryos from the B2SS + TV group were transferred non-surgically into 10 recipient heifers. Four pregnancies were induced, but only one embryo survived to term (birth of a normal male calf). It is concluded that trophoblastic vesicles release one or several unknown compound(s) normally present in vivo, promoting the cleavage of early bovine embryos.

Animals↗

PubMed1993

Quantitative analysis of morphological modifications of day 6.5 horse embryos after cryopreservation: differential effects on inner cell mass and trophoblast cells.

Sixteen embryos were recovered nonsurgically at day 6.5 after induced ovulation from Welsh pony mares and were evaluated for cellular changes that occur because of exposure to the cryoprotectant with or without the freeze and thaw process. Day 6.5 horse embryos were either (i) frozen and thawed using glycerol as cryoprotectant (n = 6), (ii) given only the glycerol treatment (n = 5), or (iii) washed in phosphate-buffered saline (PBS) the same number of times as in the glycerol treatment (n = 5). After treatments, embryos were incubated in Minimum Essential Medium (MEM), supplemented with BSA, glutamine, antibiotics and buffered with Hepes, for 1 h for one embryo per group and for 6 h for the others. After histological fixation, embryos were serially sectioned. On observation by light microscopy, the total numbers of interphasic, mitotic and pycnotic nuclei of each embryo were counted. Electron microscopy was used to evaluate the damage to the fine structure of intracellular organelles. The proportion of mitotic cells did not differ among groups (control: 2.3%; glycerol-treated: 1.8%; frozen-thawed: 1.3%). There were significant differences in the proportion of pycnotic cells both between control (12.8% +/- 5.6) and glycerol-treated embryos (39.4% +/- 15.9) (P < 0.05) and between control and frozen-thawed embryos (42.2% +/- 14.9) (P < 0.001), but no difference was found between treated embryos (glycerol-treated and frozen-thawed embryos). Degenerated cells were not localized in the same place in each embryo and no ultrastructural alteration was uniformly observed among every embryo of each group, but inner cell mass (ICM) cells were affected most by treatments (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

In vitro development of day 2 embryos obtained from young, fertile mares and aged, subfertile mares.

This study was designed to investigate the development of day 2 embryos obtained from young and aged mares, co-cultured with oviductal epithelial cells obtained from mares in each age group in a 2 x 2 crossover design. Young, fertile mares (n = 19; 2-7 years of age) and aged, subfertile, mares (n = 16; 17-24 years of age) were used as embryo and oviductal epithelial cell donors. Embryos (n = 37) were collected from the oviducts 2 days after ovulation and were paired (embryos obtained from young mares with embryos obtained from aged mares) so that eight pairs were co-cultured with young mare oviductal epithelial cells and eight pairs were co-cultured with aged mare oviductal epithelial cells. Five additional embryos obtained from young mares were co-cultured with oviductal epithelial cells from either young mares or aged mares but were not paired. Embryos were co-cultured for 7 days at 38.5 degrees C in 5% CO2 or until morphological degeneration was detected. The proportions of paired embryos that reached the blastocyst stage were similar for embryos obtained from young mares and embryos obtained from aged mares after co-culture with oviductal epithelial cells from young mares (6 of 8 versus 5 of 8) or from aged mares (6 of 8 versus 5 of 8), respectively. Although the overall rate of development of embryos to blastocyst from both young mares and aged mares was similar, blastocysts developing from embryos obtained from aged mares were inferior to blastocysts obtained from young mares in terms of number of cell nuclei, quality score, and diameter at day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed2006

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos.

This study indicated that prolonged exposure of donor cell nuclei to oocyte cytoplasm before activation results in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stage in vitro. However, after transfer of embryos to recipients, there was no difference in pregnancy rates throughout gestation. Chromatin morphology was evaluated for embryos held 2, 3, 4 and 5 h between fusion and activation. In embryos held 2 h, 15/17 (88.2%) embryos contained condensed chromosomes, while only 12/24 (50.0%) embryos held 3 h exhibited this characteristic. The proportion of embryos with elongated or fragmented chromosomes tended to increase with increased hold time. While 15/19 (78.9%) of embryos held 2 h developed a single pronucleus 6 h after activation, only 8/22 (36.4%) had one pronucleus after a 4-h hold. Embryos held 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h cleaved at rates of 207/281 (73.7%), 142/166 (85.5%), 655/912 (71.8%), 212/368 (57.6%), 406/667 (60.9%), 362/644 (56.2%) and 120/228 (52.6%) respectively. Further development to compacted morula/blastocyst stage occurred at rates of 78/281 (27.8%), 42/166 (25.3%), 264/912 (28.9%), 79/368 (21.5%), 99/667 (14.8%), 94/644 (14.6%) and 27/228 (11.8%) respectively. Embryos held less than 2.5 h between fusion and activation established pregnancies in 18/66 (27.3%) of recipients, while embryos held over 2.5 h established pregnancies at a rate of 17/57 (29.8%). This study indicates that holding bovine nuclear transfer embryos less than 2.5 h between fusion and activation results in improved nuclear morphology and increased development to compacted morula/blastocyst stage, and results in pregnancy rates equivalent to embryos held over 2.5 h.

Animals↗

PubMed1999

Influence of rat placental lactogen-I on the development of whole rat embryos in culture.

Rat placental lactogen-I (rPL-I), the first prolactin-like hormone expressed in the placenta during pregnancy in the rat, is known to influence maternal functions. In the present study, we have investigated the effects of rPL-I on the growth and development of cultured whole rat embryos. Rat embryos, with or without ectoplacental cone (EPC) attached, were explanted at day 9 of gestation. After 48 h of culture, the embryos, enclosed by the yolk sacs, were assessed by the presence of visible heart contractions ('heart beats'), crown-rump length (CRL) and yolk sac diameter (YSD). When intact embryos with EPC were cultured, the concentrations of rPL-I and rPL-II (products of EPC) in the medium were 850+/-841 and 92+/-181 ng/ml respectively (means+/-s.e.m.). In embryo cultures with the EPC removed, rPL-I levels decreased to</=10 ng/ml, and only 70% of the embryos were viable, with visible heart beats. In the viable embryos, both CRL and embryonic DNA synthesis were reduced compared with controls, and the addition of rPL-I (1 microg/ml) did not prevent this reduction. YSD and yolk sac DNA synthesis were also reduced compared with control embryos, and the addition of rPL-I significantly prevented this decrease by 45%. In embryos cultured without EPC in the presence of neutralizing rabbit anti-rat prolactin serum (anti-rPRL), embryonic and yolk sac DNA synthesis were reduced by 35% compared with embryos exposed to normal rabbit serum. Addition of rPL-I significantly increased (P<0.05) embryonic and yolk sac growth. Thus the effects of rPL-I on embryo growth could only be seen in the absence of prolactin. The addition of human prolactin in the presence of anti-rPRL also resulted in significant increases (P<0.05) in embryonic DNA synthesis and CRL. These results suggest that rPL-I may substitute for prolactin to influence the growth of the rat embryo.

Animals↗

PubMed1999

Risk of disease transmission by llama embryos.

An assessment was made of the risk of transmission of foot and mouth disease (FMD), vesicular stomatitis, bluetongue, tuberculosis and brucellosis by llama embryos. The study suggests that embryo transfer is a safe method for the international movement of llama embryos despite the special characteristics of these embryos, such as the absence of a zona pellucida, and despite the lack of data on pathogen-embryo interactions. For acute viral diseases such as FMD, vesicular stomatitis or bluetongue, embryo transfer reduces the risk of international embryo movement by a factor of 10(4). Therefore, if favourable epidemiological or ecological conditions exist in the region of origin of the embryos, the risk of contamination of a batch of llama embryos with the above agents is close to zero. The risk of contamination with Mycobacterium or Brucella depends on the incidence of these diseases, but under the most unfavourable prevalence levels, the risk does not exceed 10(-3.3), given that the results of diagnostic tests of the herd and of donor animals are negative before and after collection of the embryos. This study demonstrates that risk assessment can be a valuable tool to facilitate international movement of embryos, particularly for those species for which little or no data are available regarding embryo-pathogen interactions.

Animals↗

PubMed1991

Effect of serum-free co-culture and synchrony of recipients on development of cultured sheep embryos to fetuses.

The percentage of sheep embryos that continued to develop after collection and immediate transfer on d 2 after estrus was similar when phosphate-buffered saline with 10% fetal calf serum (PBSFCS, 45%), physiological saline (50%), or tissue culture medium 199 supplemented with 10% fetal calf serum (M199FCS, 47%) was used to flush embryos from oviducts. Co-culture of sheep embryos for 3 d with oviductal cells tended (P = .1) to reduce the percentage of embryos that developed to fetuses after transfer compared with those embryos transferred immediately. Tissue culture medium 199 supplemented with .3% BSA (M199BSA) was an adequate substitute for M199FCS for culture of sheep oviductal cells if tissue culture wells were pretreated with fibronectin. Estradiol in concentrations from 10 to 1,000 pg/ml and progesterone at concentrations of 1 or 10 ng/ml in M199BSA failed to stimulate embryo development during 3 d of co-culture beyond that seen in co-culture with M199FCS or M199BSA without added steroid. Transfer of sheep embyros co-cultured for 3 d in M199BSA or M199FCS to recipients synchronized with donors resulted in about 19% of the embryos developing to fetuses, whereas transfer to recipients that were in estrus 24 h after donors resulted in 33% of embryos developing to fetuses. The significant (P less than .05) improvement for delayed recipients may reflect the relatively lesser developmental rate of co-cultured embryos compared with that of embryos in vivo. Embryo development into fetuses was similar after co-culture in M199FCS or M199BSA co-cultures; therefore, serum is not required for the co-culture of sheep embryos.

Animals↗

PubMed1993

Transfer of split goat embryos without zonae pellucidae either fresh or after freezing.

Embryos in the morula to blastocyst stage were collected from superovulated goats either surgically or after slaughter. Embryos with good or fair morphology were bisected with a microblade without the aid of a holding pipette or other microinstruments. Of 103 morulae and 77 blastocysts that were split, 200 (97%) and 151 (98%) demi-embryos, respectively, with no major morphological aberrations were obtained. Zona-free demi-embryos derived from blastocysts were incubated in vitro for 2 h, and those derived from morulae for 24 h so that they would reach the blastocyst stage. Demi-embryos were transferred either fresh or after freezing and thawing. After 2 h of incubation, a significantly higher proportion of zona-free demi-embryos derived from blastocysts were of good quality than were zona-free demi- embryos derived from morulae (75 vs 45%). Seventy-six percent of zona-free demi-embryos derived from morulae developed to the blastocyst stage after 24 h in vitro. The quality of the embryos before splitting had a significant effect on the development of the demi-embryos in vitro. Transfer of 11 pairs of zona-free demi-embryos resulted in the birth of five twin and three singleton kids. Transfer of 11 pairs of demi-embryos frozen-thawed without zonae pellucidae resulted in the delivery of two singleton kids.

Animals↗

PubMed1987

Comparison of Ham's F10 with CO2 or Hepes buffer for storage of equine embryos at 5 C for 24 H.

Forty equine embryos collected 7 d post-ovulation were stored at 5 C for 24 h in one of two culture media (n = 20/group): 1) Ham's F10 + 10% heat-treated fetal calf serum (FCS) buffered by gassing with 5% CO2, 5% O2 and 90% N2 and 2) Ham's F10 + 10% FCS with Hepes buffer (25 mM). Embryos cultured in Ham's F10 + CO2 maintained a better quality score and had a larger average increase in diameter (+34.8 micron) than embryos stored in Hepes buffered Ham's F10 (-10.2 micron). Embryos were transferred surgically into recipient mares that ovulated -3 to +1 d in relation to the donor mare. Twenty embryos cultured in Dulbecco's phosphate buffered saline + 10% FCS and transferred less than 1 h after collection were used as controls. Pregnancy rates were higher (P less than .05) for embryos stored in Ham's F10 + CO2 (70%, 55%) than for embryos stored in Ham's F10 + Hepes (20%, 15%) at 14 and 35 d, respectively. At 14 d, pregnancy rates for control embryos (90%) were similar (P greater than .05) to pregnancy rates for embryos cultured in Ham's F10 + CO2 (70%); however, by 35 d, pregnancy rates were higher (P less than .05) for controls (80%) than for embryos stored in Ham's F10 + CO2 (55%). It was concluded that Ham's F10 + CO2 was superior to Ham's F10 + Hepes for short-term storage of equine embryos at 5 C, and that satisfactory pregnancy rates could be obtained from transfer of embryos stored in Ham's F10 + CO2 at 5 C for 24 h.

Animals↗