Chilled Embryos Research Articles

Factors affecting the efficiency of equine embryo transfer (EET) in polo mares under subtropical conditions of Pakistan.

Equine embryo transfer (EET) is a prominent technology in the equine breeding industry, and its efficacy is affected by a number of factors. The current study aimed to determine the effects of the breed of donor/recipient mares, estrus/ovulation induction treatment, cooled transportation of embryos, and synchrony between donor and recipient mares on the efficiency of the EET under subtropical conditions of Pakistan. A total of eighty-four (n = 84) Polo-playing donor mares (Argentino-polo = 41 and Anglo-Arab = 43) and seventy (n = 70) recipient mares (light breed = 26 and heavy breed = 44) were used for EET. The donor mares exhibiting natural estrus (n = 28) were detected by teaser a stallion, and corpus luteum (CL) having mares (n = 56) were treated with prostaglandin (150 μg of Cloprostenol) for estrus induction. The mares' follicular growth was monitored through ultrasonography until the dominant follicle's size reached 35 mm or more with a moderate to obvious uterine edema score. Afterward, the mares were treated either with GnRH, i.e., 50 μg of Lecirelin acetate (n = 41) or Ovusyn, i.e., 1500 IU hCG (n = 43). Insemination with chilled semen was performed 24 hours later. The embryos were collected non-surgically, 7 or 8 days after ovulation, from the donor mares. The collected embryos were transferred into the well-synchronized recipient mares as fresh (n = 44) or chilled (n = 26) embryos. The pregnancy after ET was checked through ultrasonography. Statistical analysis revealed that the embryo recovery rate (ERR) remained significantly higher (P<0.05) for the Prostaglandin (PG) treated group of donors as compared to the natural heat group of donors. The breed of donor mares, type of ovulatory treatment given, and day of embryo collection did not significantly (P>0.05) affect the ERR. There was no significant effect of the type (fresh vs chilled), classification, and stage of development of embryo on pregnancy outcomes (P>0.05). ET pregnancy rate was significantly affected by the breed of recipient mares and ovulation synchrony between donor and recipient mares (P<0.05). In conclusion, under the subtropical conditions of Pakistan, PG-based estrus induction of donor mares, breed of recipient mares, and ovulation synchrony between the donor and recipient mares had a substantial effect on the efficiency of EET.

The application of different reproduction techniques for rare species waterlily tulip (Tulipa kaufmanniana Regel.) propagation under ex situ conditions

T. kaufmanniana is a threatened and endemic species of the Tien Shan Mountains with a complex of valuable ornamental features. However, the commercial usage of the plant is limited due to the restricted availability of the bulbs in nature, difficulties in overcoming seed dormancy and low efficiency of the species reproduction. Results of the investigation of the reproductive biology of this species in the culture conditions allowed us to characterize T. kaufmanniana as viable with successful seedage. In the work reported here the effect of low temperature on the proper development of embryos is addressed, and an attempt is made to clarify the effect of cytokinins (TDZ, BAP) and auxin (NAA) on the regeneration capacity of isolated T. kaufmanniana embryos for the adventitious shoots and bulblets formation. Direct shoot organogenesis was induced by the combined action of chilling and BAP or TDZ treatment. Among the variants tested, TDZ promoted the formation of adventitious bulbs in 48 % of the chilled embryos, compared to non-chilled embryos (0.0%) and embryos exposed to the same concentration of BAP (39%). The best culture condition for T. kaufmanniana embryos growth and bulblets formation consisted of chilling for 10 wk in the dark at 4 °C, 4 wk at 20 °C under 16 h light photoperiod and 10 wk at 4 °C in the dark. Sufficiently effective protocol for clonal micropropagation was developed for the first time for T. kaufmanniana by the use of immature zygotic embryos as primary explants.

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos.

BackgroundCryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked.MethodsIn the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested.ResultsOur results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality.ConclusionsBased on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development.

Use of methanol as cryoprotectant and its effect on sox genes and proteins in chilled zebrafish embryos

Methanol is a widely used cryoprotectant (CPA) in cryopreservation of fish embryos, however little is known about its effect at the molecular level. This study investigated the effect of methanol on sox gene and protein expression in zebrafish embryos (50% epiboly) when they were chilled for 3h and subsequently warmed and cultured to the hatching stages. Initial experiments were carried out to evaluate the chilling tolerance of 50% epiboly embryos which showed no significant differences in hatching rates for up to 6h chilling in methanol (0.2-, 0.5- and 1M). Subsequent experiments in embryos that had been chilled for 3h in 1M methanol and warmed and cultured up to the hatching stages found that sox2 and sox3 gene expression were increased significantly in hatched embryos that had been chilled compared to non-chilled controls. Sox19a gene expression also remained above control levels in the chilled embryos at all developmental stages tested. Whilst stable sox2 protein expression was observed between non-chilled controls and embryos chilled for 3h with or without MeOH, a surge in sox19a protein expression was observed in embryos chilled for 3h in the presence of 1M MeOH compared to non-chilled controls and then returned to control levels by the hatching stage. The protective effect of MeOH was increased with increasing concentrations. Effect of methanol at molecular level during chilling was reported here first time which could add new parameter in selection of cryoprotectant while designing cryopreservation protocol.

51 ARTIFICIAL DORMANCY OF BOVINE EMBRYOS FOR A MAXIMUM OF 7 DAYS USING A SIMPLE MEDIUM

The worldwide pregnancy rate using cryopreserved mammalian embryos has not improved over the past 2 decades, probably because the freeze-thawing processes cause significant damage. Therefore, it is now relevant to examine the feasibility of short-term non-freezing preservation, and whether this could be applied to embryos that have high vitality and are to be transferred into recipients within several days. We introduce here an artificial dormancy fluid that can extend the hypothermic storage period of bovine embryos for a maximum of 7 days. First, to examine the effect of different basal media and the optimal concentration of fetal bovine serum (FBS) for hypothermic preservation, bovine blastocysts produced in vitro were stored at 4°C in a plastic ministraw in 1 of the following 3 media: PBS, medium 199, or Leibovitz L15 with various amount of FBS (0, 5, 20, 50, or 100%) for 3 days. Second, to examine the effect of Good's buffers, bovine embryos produced in vivo (morula to blastocyst stages) were stored at 4°C in a plastic ministraw in medium 199 plus 50% FBS supplemented with various Good's buffers [HEPES, TES, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), MOPS, and 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS)] for 7 days. Following hypothermic preservation, the chilled embryos were squeezed out of the straw into PBS and washed 3 times in the same medium. Subsequently, the embryos were cultured in CR1aa medium supplemented with 5% FBS for 48 h at 38.5°C under 5% CO2 in air with high humidity. The viability rate of the embryos was assessed at the end of the culture period. Finally, to observe the pregnancy rate of chilled embryos, 32 embryos produced in vivo were stored at 4°C for 7 days in medium 199 plus 50% FBS supplemented with HEPES. Following hypothermic preservation, the chilled embryos were transferred into recipient heifers (1 embryo per recipient). Pregnancy was determined by real-time B-mode ultrasonography (Convex scanner HS-1500, Honda electronics Co. Ltd, Toyohashi, Japan) on Day 60 of gestation. Data were analysed using the chi-squared test. The viability rate of the embryos after hypothermic storage for 3 days was significantly increased for medium 199 plus 50% FBS [27/30 (90%)] compared with PBS [18/30 (60%)] or Leibovitz L15 [15/30 (50%)] plus 50% FBS (P &lt; 0.05). Chilled embryos stored for 7 days in medium 199 plus 50% FBS supplemented with HEPES had much higher survival than embryos stored in the same medium with other Good's buffers. The pregnancy rate of the chilled embryos stored for 7 days was extremely high [24/32 (75%)] and normal live calves were delivered at term. In conclusion, maintaining artificial dormancy of bovine embryos for 7 days using a simple medium appears to be feasible. This is the first documented success of storing chilled mammalian embryos in a viable state for 7 days. To be of practical value, bovine embryo preservation at hypothermic temperatures must be able to maintain viability for periods longer than 7 days. This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry.

46 HYPOTHERMIC STORAGE FOR 10 DAYS OF BOVINE EMBRYOS USING TYPE III ANTIFREEZE PROTEIN

Recently, we developed a medium that enabled bovine embryos to be held for up to 7 days at 4°C (Tsuchiya et al. 2014 IETS meeting). To be of practical value, mammalian embryo preservation at hypothermic temperatures must be able to maintain viability for periods longer than 7 days. Antifreeze proteins (AFP) were discovered in various organisms (such as fish, insects, plants, and bacteria) living in cold regions. They show a unique ability to protect cold-sensitive cells from hypothermic damage. Here, we found that a biomolecule known as type III AFP solubilised into an optimized solvent can keep alive bovine embryos for a maximum of 10 days at hypothermic temperatures. First, human hepatoma (HepG2) and rat insulinoma (RIN-5F) cells were stored at 4°C in Euro-Collins solution (Kobayashi Seiyaku, Osaka, Japan) supplemented with or without 10 mg mL–1 of type III AFP for 24 h. The viability rate of the cells was assessed by trypan blue (Dojindo, Kumamoto, Japan) dissolved in PBS. Second, high-quality blastocysts produced in vivo were stored at 4°C in a plastic ministraw in 25 mM HEPES medium 199 plus 20% fetal bovine serum (FBS) supplemented with or without 10 mg mL–1 of type III AFP for 10 days. Following hypothermic preservation, the chilled embryos were squeezed out of the straw into PBS and washed 3 times in the same medium. Subsequently, the embryos were cultured in CR1aa medium supplemented with 5% FBS for 48 h at 38.5°C under 5% CO2 in air with high humidity. The viability and hatching rate of the embryos were assessed at the end of the culture period. Finally, 4 embryos stored for 10 days with type III AFP were reloaded into plastic straws with the washing medium and transferred into recipient heifers (1 embryo per recipient). Pregnancy was determined by real-time B-mode ultrasonography (Convex scanner HS-1500, Honda Electronics Co. Ltd., Toyohashi, Japan) on Day 60 of gestation. Data were analysed using chi-squared and Student's t-tests. In the absence of type III AFP, the cell viability values of HepG2 and RIN-5F after hypothermic storage for 24 h were only 5 and 22%, respectively. However, in the presence of type III AFP, the cell viability values were dramatically increased (HepG2: 71%; RIN-5F: 59%) than those measured without type III AFP. To examine the effect of type III AFP for hypothermic preservation of bovine embryos, we used 80 high-quality embryos produced in vivo and assigned them randomly to 2 experimental groups. The viability and hatching rates of the chilled embryos stored with type III AFP for 10 days were significantly higher (58 and 30%, respectively) than those of without type III AFP (28 and 0%, respectively). The pregnancy rate of the chilled embryos stored with type III AFP was 50%: 2 pregnancies continue until now. In conclusion, prolongation of short-term preservation period with AFP-containing fluid will realise LN2-free storage of bovine embryos for a 10-day period. This work was supported by the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry.

Optimization Protocol for Storage of Goldfish (Carassius auratus) Embryos in Chilled State

A series of five experiments were conducted to explore suitable conditions for storing of goldfish embryos in a chilled state. The factors studied were embryo stage, storage temperature, physiological saline solutions and goldfish artificial coelomic fluid (GFACF) medium, antibiotics (penicillin and streptomycin), antioxidants (vitamin E, vitamin C), buffer (Hepes, Tris) and BSA (bovine serum albumin). First, goldfish embryos at eight developmental stages were incubated in aerated and dechlorinated tap water at 0 °C for 24 h. Result shows that early developmental stages were most sensitive to chilling. Heartbeat-stage goldfish embryos were chilled at 0, 4 or 8 °C for up to 72 h in water, and chilled storage was possible only for up to 18, 24 and 48 h at 0, 4 and 8 °C, respectively, without a decrease in viability. Chilling of goldfish embryos at 8 °C in GFACF medium and Dettlaff's solution instead of water and other physiological saline solutions prolonged their viability (p < 0.01). Nevertheless, viability of chilled embryos in GFACF medium was slightly, but non-significantly, higher than in Dettlaff's solution. Supplementation of the GFACF medium with antibiotics, Hepes or BSA increased the viability of chilled embryos, but the tested vitamin E analogue Trolox, vitamin C or Tris concentration had no effect on embryo viability. The outcome of this series of experiments shows that heartbeat-stage goldfish embryos could be chilled for 60 h in GFACF supplemented with 25 mm Hepes, 100 U/ml penicillin, 10 μg/l streptomycin and 1 g/l BSA in such a way that embryonic development does not proceed, and viability is not lost.

Factors affecting chilled storage of zebrafish ( Danio rerio) embryos

Chilled storage of zebrafish embryos was investigated at a temperature that arrests embryonic development as this technique might offer interesting practical applications. Five parameters played an important role for chilled storage: (a) storage temperature, (b) development stage of embryos, (c) storage solution (extender), (d) postchilling treatment, and (e) inhibition of growth of microorganisms by antibiotics. The optimal chilling temperature was 8 °C. Prim-5 stage (24 h postfertilization [hpf]) and prim-25 stage (36 hpf) embryos had similar high chilling resistance and could be chilled for 33 h without a loss in viability. Five-somite stage (12 hpf) embryos had a lower chilling resistance and could be chilled only for 14 h without a loss in viability. After longer incubation periods, the viability started to decrease. Under these conditions, chilling in physiologic saline solutions was superior to that in water. Fifty percent of the prim-5 stage and prim-25 stage embryos survived for 41 h at 8 °C in water but for 46 h in physiologic saline solution. A similar effect was observed for 5-somite stage embryos (50% survival rate in water, 28 h; 50% survival rate in physiologic saline solution, 35 h). When embryos were incubated in physiologic saline solution instead of water in the postchilling phase, the embryo viability was positively affected, too. Also, supplementation of the storage solution with antibiotics (penicillin and streptomycin) increased the viability of chilled embryos. In summary, the current study shows that chilled storage of zebrafish embryos is possible for sufficiently long periods to synchronize the development of embryos deriving from different spawning dates or to delay the development for experimental purposes. To prolong the storage periods, further development and standardization of the methodology is necessary.

Metabolic costs of heat solicitation calls in relation to thermal need in embryos of American white pelicans

Chilled embryos of pelicans,Pelecanus erythrorhynchos, begin to vocally solicit parental heat at the pipped-egg stage. Honest signalling models predict that if vocal heat solicitation is a true reflection of need, then solicitation should be costly and costs should increase with the embryo’s need for warmth. Using open-flow respirometry, we measured the metabolic costs associated with vocal heat solicitation by exposing embryos to either a decreasing or increasing series of body temperatures, ranging from 25 to 37.8°C. We measured baseline costs (stable temperature, embryo silent) and costs associated with cold-induced calling at each temperature. At natural incubation temperature (37.8°C), call rates and costs associated with calling were negligible, as was thermal need. Metabolic costs relative to baseline costs and costs per call increased with thermal need as body temperature declined. Absolute metabolic costs increased between 37.8 and 35°C, then remained stable down to 25°C. Call rates increased as embryos were chilled within the range of temperatures most frequently experienced in nature (35–37.8°C), then decreased significantly for all lower temperatures, probably owing to reduced overall metabolic rate at lower temperatures (25–37.8°C). The results generally support the honest signalling prediction that vocal heat solicitation is metabolically costly, and that costs increase with need.

Dormancy and germination of olive embryos as affected by temperature

Olive seeds do not germinate promptly when placed under favourable conditions, which is a problem in raising young plants for breeding or experimental purposes. In a series of experiments an investigation of the role of temperature in the germination of olive embryos was conducted. Naked, unchilled olive embryos (Olea europaea L. cv. Chalkidikis), cultured in vitro at 20°C, had a germination capacity of 73%, whereas that of embryos which had previously been chilled at 10°C for 2 or more weeks reached 96%. Intact seeds did not germinate at 20°C unless they had previously been subjected to 10°C for 3 or 4 weeks. Embryos chilled while in the intact seed and excised just before transfer to 20°C, reacted in a similar way to naked embryos, but reached their maximum germination capacity after 4 weeks at 10°C. Under constant temperature conditions the highest germination percentage of embryos was observed at 10 and 15°C and the highest germination rate at 15°C, while a moderate capacity and rate of germination occurred at 20°C, and a very low percentage and rate at 25 and 30°C. Prechilling at 10°C did not affect germination at 15°C, but improved the percentage and the rate of germination at 20, 25 and 30°C. The germination percentages of embryos chilled for 1 or 2 weeks at 10°C and then transferred to 25°C were lower than those of similarly chilled embryos transferred to 20°C. The chilling effect could not be reversed at 25°C when the embryos had been chilled for 3 or more weeks. The results show that olive seeds exhibit a state of dormancy that is caused by factors residing partly in the endosperm and partly within the embryo.

Measurement of endogenous ABA levels in chilled somatic embryos of carrot by immunoassay

Somatic embryos of carrot, Daucus carota L. 'Royal Chantenay', were chilled at 4°C for the last 3 days of development in order to harden torpedo stage embryos to increase embryo survival during desiccation. ABA levels in chilled and non-chilled embryos were measured using a polyclonal radioimmunoassay and a monoclonal enzyme-linked immunosorbent assay (ELISA). The monoclonal ELISA is the preferred technique due to superior sensitivity and specificity. ABA levels, measured by either technique, were similar in chilled and non-chilled embryos. The relative water content was lower in chilled embryos than in non-chilled embryos and chilling altered protein secretion of one cell line.

Effects of Chilling and ABA on [3H]Gibberellin A4 Metabolism in Somatic Embryos of Grape (Vitis vinifera L. × V. rupestris Scheele)

Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. x V. rupestris Scheele). We have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with [(3)H]GA(4) (of high specific activity, 4.81 x 10(10) becquerel per millimole) for 48 hours at 26 degrees C. Chilling had little effect on the total amount of free [(3)H]GA-like metabolites formed during incubation at 26 degrees C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble [(3)H] metabolites formed at 26 degrees C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogenous GA precursors (e.g., GA(12) aldehyde-, kaurene-, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with [(3)H]GA(4) treatment at 26 degrees C, reduced the uptake of [(3)H] GA(4) but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26 degrees C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water-soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs).

Endogenous Gibberellin-Like Substances in Somatic Embryos of Grape (Vitis vinifera × Vitis rupestris) in Relation to Embryogenesis and the Chilling Requirement for Subsequent Development of Mature Embryos

Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera x Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4 degrees C, and subsequently incubated at 26 degrees C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H(2)O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO(2) partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H(2)O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4 degrees C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H(2)O-soluble fraction declined throughout chilling. Activity in the GA-precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26 degrees C after chilling enhanced activity in the free GA and H(2)O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26 degrees C with (+/-) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H(2)O-soluble fractions.Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA(4) and GA(9) in ABA-treated, chilled embryos was also confirmed by GLC-MS.

Early Lesions in Chicken and Turkey Embryos Inoculated with Mycoplasma gallisepticum Strain S6 at Various Stages of Incubation

Much of the variability reported in earlier studies of the lesions induced in chick embryos by mycoplasma is probably referrable to the use of poorly characterized organisms. Another important discrepancy results from the use of dead and chilled embryos, which would also tend to cloud the histopathologic results. This study sought only to define the early lesions of Mycoplasma gallisepticum (Strain S6) infections in chicken and turkey embryos at various stages of incubation. The only previous embryo work with this strain of Mycoplasma is that of Casorso (1), who reported the failure of this organism to produce lesions in 7-day-old chicken embryos. No histopathologic studies seem to have been made of this infection in turkey embryos. The S6 strain of M. gallisepticum is a neurotropic strain isolated from a turkey brain which readily produces a severe cerebral arteritis when inoculated intravenously in poults aged 2 to 7 weeks (4,5,6). By this route it is much less pathogenic to chicks. Thomas et al. (6) found only a single case of very mild arteritis among 8 inoculated 3-day-old hatched chicks. However, they demonstrated that this organism produced a toxin that was probably responsible for the vascular changes. This strain, in addition to its neurotropic tendencies, will incite severe respiratory disease in turkeys and chickens, depending on the route of inoculation (9).

DIFFERENCES IN NITROGEN CONTENT IN EXTRACTS OF CHILLED AND NON-CHILLED TISSUES

In an attempt to elucidate the increase of growth promotion of tissue cultures by extracts obtained from tissues chilled before extraction, determinations of nitrogen content were made on (a) fresh, and chilled chick embryos, (b) extracts prepared from fresh and chilled chick embryos, (c) fresh and chilled human placenta, (d) extracts prepared from fresh and chilled human placenta.There was no difference in dry weight, nitrogen content, phosphorus content, and nitrogen: phosphorus ratio between chilled and fresh chick embryos and chilled and fresh human placenta. There was, however, a significant difference in the nitrogen content of the extracts prepared from fresh and from chilled tissues, It is suggested that chilling of tissues for 7 days at + 4 °C. prior to extraction may render the cells more easily destructible during extraction, and thus supply a better yield of nutritive substances.