Successful cryopreservation of porcine embryos by vitrification.
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Animals↗
Biomedical subjects
P Holm
Publications and source records attributed to P Holm.
Significant reduction of the antiatherogenic effect of estrogen by long-term inhibition of nitric oxide synthesis in cholesterol-clamped rabbits.
The purpose of this study was to investigate whether endothelium-derived nitric oxide (NO) is involved in the plasma lipid-independent antiatherogenic effect of estrogen and levormeloxifene, a partial estrogen receptor agonist. 85 rabbits were ovariectomized and balloon-injured in the middle thoracic aorta. The rabbits were fed a cholesterol-enriched diet supplemented with 17beta-estradiol, levormeloxifene, or placebo, either alone, or together with 160 microg/ml NG-nitro- -arginine methyl ester (-NAME), an NO synthase inhibitor, in their drinking water for 12 wk. Plasma cholesterol was maintained at 25-30 mmol/liter by individualized cholesterol feeding. In the undamaged aorta, the extent of atherosclerosis in the estrogen group was only one-third that in the placebo group. Simultaneous administration of -NAME, however, significantly reduced the antiatherogenic effect of estrogen (P < 0.01). There was no significant difference between the placebo group given -NAME and the group treated with placebo alone. At the previously endothelium-denuded site, estrogen had no effect on atherosclerosis development, whereas -NAME combined with estrogen significantly increased atherogenesis (P < 0.05). The effects of levormeloxifene were almost similar to those of estrogen. Active vascular concentrations of -NAME were demonstrated in an additional study, in which maximal aortic/coronary endothelium-dependent relaxation was significantly inhibited in rabbits given -NAME. Thus, in this study a considerable part of the plasma lipid-independent antiatherogenic effect of estrogen was mediated through its effect on endothelial NO in cholesterol-fed rabbits. The results for levormeloxifene suggest a common mechanism of action for estrogen and partial estrogen receptor agonists on atherogenesis.
Acetylcholine↗
Post-thaw in vitro survival of vitrified cloned bovine embryos.
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Animals↗
The endothelin ETB receptor antagonist BQ-788 reduces the pulmonary vasodilator effect of endothelin-1 during acute hypoxia in pigs.
Pigs were subjected to acute, intermittent hypoxia (fraction of inhaled O2 0.1, n = 10). The increase in mean pulmonary artery pressure during hypoxia was not altered after i.v. administration of the selective endothelin ETB receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1- methoxycarbonyltryptophanyl-D-norleucine) (1 mg). However, the vasodilatory effect of endothelin-1 (25 ng/kg per min) infused into the pulmonary artery during hypoxia was attenuated by BQ-788. The present results suggest that the pulmonary vasodilator effect of exogenously administered endothelin-1 during acute hypoxia is mediated by endothelin ETB receptors in the pig. Furthermore, endothelin ETB receptor antagonism with BQ-788 does not influence the pulmonary vascular response to acute hypoxia.
Analysis of Variance↗
Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos.
The objective of this study was to compare the overall efficiency, measured by in vitro embryonic survival, and practical value of bovine in vitro embryo production, biopsy, vitrification, and direct transfer technology using 2 different manipulation methods for biopsy. Slaughterhouse-derived oocytes were matured in vitro, fertilized (Day 0) with frozen-thawed, Percoll-separated spermatozoa and cultured on a granulosa cell monolayer. In Experiment 1, one or two blastomeres were expelled from Day 4 embryos by mechanical force through a hole made by partial zona dissection. Using a darning needle hole system for individual culture of biopsied embryos from Day 4 to Day 7.5, the blastocyst per oocyte rate was 50%, and 76% of the blastocysts survived subsequent vitrification and direct in-straw rehydration. Attempts to increase the cell number of the biopsies by further in vitro culture were unsuccessful. In Experiment 2, Day 7 and Day 8 embryos were manually biopsied before or after vitrification. When biopsy was performed before vitrification, 98% of the embryos survived manipulation, and 86% of these re-expanded after vitrification and in-straw dilution. Biopsy after vitrification was less efficient, since only 69% of the embryos survived both processes. The cumulative efficiency of embryo production, Day 7.5 biopsy and vitrification--in-straw direct rehydration was lower (P < 0.001) than that of Day 4 biopsy and Day 7.5 vitrification (29 vs 38%, respectively). However, a Day 7.5 biopsy may have the more practical application since the size of the biopsy is larger and the process is not as time-consuming as the long-term individual culture of the biopsied embryos.
Journal Article↗
Preliminary data on a genome search in NIDDM siblings: the NIDDM1 locus on chromosome 2 is not linked to NIDDM in the Sardinian population. Study Group for the Genetics of Diabetes in Sardinia.
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Aged↗
Age-related changes in the peroxyl radical scavenging capacity of human plasma.
Aging and the diseases that typically follow with increasing age, notably atherosclerosis and cancer, are often proposed to be involved in increased oxidative stress. Animal studies, on the other hand, show no clear-cut pattern of age-related changes in enzymatic antioxidant defences. In this study we have demonstrated that total peroxyl radical scavenging antioxidant capacity (TRAP) in human plasma changes with age. We also found that among the antioxidants in human plasma there exists a major fraction of so far unidentified antioxidant(s). A chemiluminescent TRAP assay was used to determine the presence of peroxyl radical scavenging antioxidants in human plasma. The material consisted of 87 healthy volunteers, aged 20-96 years, who used no regular medication, vitamins, or trace elements. In females, total antioxidant capacity increased significantly during the life span. The increase in TRAP was mainly due to unidentified antioxidants. In males, TRAP increased until age 51-74, and then significantly decreased. The decrease observed among males was also due to the sharp decline in the concentration of unidentified antioxidants.
Adolescent↗
3-Morpholino-sydnonimine-induced suppression of human neutrophil degranulation is not mediated by cyclic GMP, nitric oxide or peroxynitrite: inhibition of the increase in intracellular free calcium concentration by N-morpholino-iminoacetonitrile, a metabolite of 3-morpholino-sydnonimine.
This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.
Acetonitriles↗
A partial estrogen receptor agonist with strong antiatherogenic properties without noticeable effect on reproductive tissue in cholesterol-fed female and male rabbits.
Estrogen replacement therapy retards the development of cardiovascular disease and osteoporosis in postmenopausal women. However, long-term unopposed use increases the risk of cancer in endometrium and possibly in breast. The racemic compound ormeloxifene, widely used in India as an antifertility agent, is a partial estrogen receptor agonist with antiosteoporotic properties. The present study was undertaken to investigate the effect of the L-enantiomer (levormeloxifene) and the d-enantiomer (d-ormeloxifene) on the development of atherosclerosis. In a short-term experiment (6 weeks), 4 x 10 ovariectomized female rabbits were fed a 0.25% cholesterol-enriched diet and the effect on plasma cholesterol levels was studied. In a long-term experiment (13 weeks), 4 x 15 ovariectomized female and 4 x 15 shamoperated male rabbits were maintained at a similar plasma cholesterol level of 25 mmol/L and the effect on undamaged and balloon-injured arterial wall was studied. In both experiments, the rabbits were treated with levormeloxifene, d-ormeloxifene, 17 beta-estradiol, or placebo, respectively. In the short-term experiment, levormeloxifene, in contrast to d-ormeloxifene, significantly reduced plasma cholesterol by 30% compared with the placebo group. In the long-term experiment, levormeloxifene, in contrast to d-ormeloxifene, significantly reduced atherosclerosis by 50% in the undamaged arterial wall of both female and male rabbits. Because these rabbits were cholesterol-clamped, the antiatherogenic effect was not mediated via plasma cholesterol lowering. Like estrogen, levormeloxifene did not inhibit atherosclerosis in the endothelium-denuded site of aorta. The antiatherogenic effects of levormeloxifene were thus similar to those of estrogen, but produced in the absence of any noticeable estrogenic effect on uterine or testicular tissue.
Animals↗
Dose-dependent suppression of transplant arteriosclerosis in aorta-allografted, cholesterol-clamped rabbits. Suppression not eliminated by the cholesterol-raising effect of cyclosporine.
Cyclosporine may suppress transplant arteriosclerosis; however, it also raises plasma cholesterol, which could promote the disease. Our aim was to test these hypotheses experimentally. In experiment 1 (n = 34), cholesterol was clamped at a human level of 5 to 7 mmol/L, and rabbits were given either saline or cyclosporine in a low, medium or high dose. In experiment 2 (n = 15), in which dietary cholesterol was fixed at 0.05 g.kg-1.d-1, and experiment 3 (n = 16), in which no dietary cholesterol was added to the chow, rabbits were given either medium-dose cyclosporine, saline, or vehicle. The duration of each experiment was 5 weeks. In experiment 1, cyclosporine attenuated the development of transplant arteriosclerosis dose dependently (trend test: P < .0001). Cyclosporine also suppressed, in a dose-dependent manner, the activation of the immune system (trend test: P < .05) and the presence of T lymphocytes (trend test: P < .0001) and macrophages in the intima (trend test: P < .01). Despite a higher plasma cholesterol level in cyclosporine-treated rabbits compared with saline-treated rabbits in both experiment 2 (4.9 versus 2.9 mmol/L) and experiment 3 (1.6 versus 0.8 mmol/L), transplant arteriosclerosis was significantly reduced by cyclosporine (Mann-Whitney U test; P < .05 and P < .05). These results suggest that cyclosporine suppresses experimental transplant arteriosclerosis dose dependently. Accordingly, in the assessment of the optimal cyclosporine dose to heart-transplanted patients, it should be taken into account that a dose reduction may promote transplant arteriosclerosis.
Animals↗
Antiatherogenic effect of estrogen abolished by balloon catheter injury in cholesterol-clamped rabbits.
The purpose of this study was to investigate the importance of an intact endothelial cell layer for the direct antiatherogenic effect of estrogen on the arterial wall. Thirty rabbits were bilaterally ovariectomized and subjected to mechanical injury of the endothelium by balloon catheterization of the upper thoracic aorta. Immediately after the operation, treatment was initiated with either 17 beta-estradiol or placebo given intramuscularly. All rabbits were clamped at a similar plasma cholesterol level from 1 week before the operation until the experiment was terminated 13 weeks later. In the undamaged aorta, ie, the aortic arch, the lower thoracic aorta, and the upper abdominal aorta, the estrogen-treated rabbits had one third (P = .06), one sixth (P = .002), and one seventh (P = .001), respectively, the accumulation of cholesterol of the placebo-treated rabbits. In the upper thoracic aorta that had been subjected to mechanical injury of the endothelium, however, aortic cholesterol accumulation was not significantly different between the two groups. Similar results were obtained by histological evaluation of the aortic tissues. Immunohistochemical staining with antibodies against macrophages, smooth muscle cells, and T lymphocytes revealed no significant differences in the intimal distribution of cells between estrogen- and placebo-treated rabbits, except for a higher number of T lymphocytes per unit intimal area of the undamaged aortic arch (P < .0005) in the estrogen-treated-rabbits than the placebo-treated rabbits. This is the first study to demonstrate that the antiatherogenic effect of estrogen is abolished by balloon catheter injury in cholesterol-clamped rabbits. These results may indicate that an intact endothelial cell layer is crucial for the direct antiatherogenic effect of estrogen on the arterial wall.
Animals↗
Survival and development of bovine blastocysts produced in vitro after assisted hatching, vitrification and in-straw direct rehydration.
The purpose of this study was to establish an efficient combination of assisted hatching and cryopreservation procedures for producing bovine embryos in vitro. A total of 1312 day 7 blastocysts were subjected randomly to 14 different combinations of three factors: osmotic stress, assisted hatching and vitrification. Re-expansion, initiation and completion of the hatching process, as well as attachment to the culture dish, were analysed by SAS Genmod procedure. Incubation with sucrose was found to decrease survival rates; among the assisted hatching procedures used, zona fenestration resulted in higher survival rates compared with partial zona dissection and controls; and vitrification decreased survival and further development. The combined effect of sucrose incubation and vitrification decreased further development markedly, as did partial zona dissection followed by vitrification. Partial zona dissection performed in medium containing sucrose severely lowered embryo survival. Zona fenestration without sucrose incubation followed by vitrification did not compromise further embryo development: 86%, 84% and 79% of the blastocysts initiated, completed hatching and attached to the bottom, respectively. These data were not different from the controls (80%, 76% and 63%, respectively; P > 0.05). Cell count analysis revealed a decrease in the total number of cells as a result of the assisted hatching and vitrification compared with controls (135 versus 202, respectively; P < 0.0001). Although embryo transfer results (36% pregnancy rate and 30% calving rate) require further improvement, this combination of methods may prove useful in the commercial production of bovine embryos in vitro.
Animals↗
Endothelin in the pulmonary circulation with special reference to hypoxic pulmonary vasoconstriction.
1. The experimental model using periods of ventilation with a gas mixture containing 10% oxygen in the anesthetized pig was found to induce HPV that was reproducible and remained stable for up to two hours. 2. Intrapulmonary infusion of ET-1 during normoxia resulted in a dose-dependent increase in the SVR with a concomitant decrease in CO and rise in PVR. Infusion of ET-3 and S6c evoked similar responses, but of a considerably smaller magnitude. The dose-dependent systemic vasoconstriction evoked by ET-1 infusion was reduced after administration of the combined ETA and ETB receptor antagonist bosentan as well as the selective ETA receptor blockers BMS-182874 and TBC-11251 indicating that this effect is primarily mediated by ETA receptors. ETA receptors are present in porcine pulmonary arteries, since BMS-182874 caused a rightward shift of the concentration-response curve to ET-1 in vitro. 3. Administration of selective ETA- or combined ETA and ETB antagonists but not of a selective ETB antagonist reduced the SVR in normoxic pigs, indicating that ET acting through ETA receptors contributes to systemic vascular tone in the pig. In addition, ETA selective and non-selective ETA and ETB antagonists produced a reduction of PVR, although this effect was less consistent than the influence on SVR. This indicates that ETA receptors may contribute to basal pulmonary vascular tone. The plasma levels of ET-1 increased following the non-selective ET receptor antagonist bosentan but were unaffected by selective ETA receptor antagonism. 4. Intrapulmonary infusion of ET-1 produced in low doses a pulmonary vasodilatation during HPV in the pig. This pulmonary vasodilatory effect was also evident when ET-3 or S6c was infused. The pulmonary vasodilatory effect of ET-1 infusion was abolished following administration of the selective ETB receptor antagonist BQ-788, indicating that the pulmonary vasodilatory effect of ET in HPV in the pig is mediated by ETB receptors. Higher doses of ET-1 infusion during HPV resulted in systemic and pulmonary vasoconstriction. 5. Both combined ETA and ETB blockade using bosentan and selective ETA receptor inhibition using BMS-182874 or TBC-11251 reduced the development of HPV in the pig. In addition, bolus injection of TBC-11251 reversed already established HPV. Selective ETB receptor antagonism had no effect on HPV. These findings suggest that ETA receptor activation contributes to HPV in the pig. 6. The concentration-dependent contraction evoked by ET-1 in human vessels in vitro (LAD, IMA, PA, SV) was reduced after incubation with BQ-123 and bosentan. Inhibition of NO- and prostaglandin-synthesis enhanced the contractions in the LAD and IMA, but not in the PA and SV. These findings are in concord with a predominance of ETA receptors in the investigated vessels. Nitric oxide and prostacyclin seem to be important determinants of the functional response to ET in human LAD and IMA, but of less importance in the PA and SV. 7. In the human vessels investigated, the tissue content of ET-1 was higher than that of ET-3 and resembled the distribution of the perivascular C-fiber peptide CGRP. Tissue content of ET-1 was considerably lower than that of sympathetically stored NPY. In human plasma, the arterial and venous concentration of ET-1 was higher in patients with chronic hypoxemia and borderline hypertension than in the venous samples drawn from healthy subjects. The arterial and venous levels of ET-1 did not differ significantly either at rest or during ET-1 infusion, indicating that the pulmonary circulation does not extract ET-1 in these patients. 8. The study in patients with borderline pulmonary hypertension and chronic hypoxaemia showed no pulmonary vasodilatory effects of intrapulmonary ET-1 infusion but rather systemic vasoconstriction and decreased CO. The AVO2 difference increased markedly during ET-1 infusion. 9. (ABSTRACT TRUNCATED)
Adult↗
Vitrification of porcine embryos using the Open Pulled Straw (OPS) method.
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Animals↗
Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration.
The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20-22 degrees C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4 degrees C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22 degrees C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zone dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS+albumin, TCM199 and TCM199+calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20-22 degrees C, the embryo survival rate decreased (PBS+albumin) or no embryo survived (TCM199+calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.
Animals↗
Isolated maize zygotes mimic in vivo embryonic development and express microinjected genes when cultured in vitro.
We established conditions for the regeneration of natural maize zygotes isolated from pollinated plants with the goal of investigating the molecular control of early embryogenesis in higher plants. Viable zygotes were excised from embryo sacs by minimal enzymatic digestion and microdissection. Viable zygotes transferred to coculture with androgenic microspores from barley developed into embryo-like structures in 61% of the cases. No development was observed when zygotes were cultured in the presence of maize anthers undergoing androgenetic embryogenesis. Zygote-derived embryo-like structures regenerated into fertile plants through secondary embryogenesis when transferred to solid medium. The first zygotic division was asymmetrical and bipolar structures similar to pretransitional embryos observed in planta were later produced as observed using light and electron microscopy. Conditions for efficient microinjection of DNA into zygotes were established. Calcofluor and PATAG staining of zygotes showed that cell wall regeneration occurred as early as 20 min after enzymatic isolation and that after 2 hr, each zygote was bordered with cell wall material. Through quantitative microphotometry, DNA synthesis during the first cell cycle of the zygote was shown to occur between isolation and 12 hr of culture. Microinjection of two types of reporter genes (GUS gene and anthocyanin regulatory genes) demonstrates transient expression in plant zygotes. On average, 3.5% of microinjected zygotes showed transgenic expression. Reporter gene expression was observed in zygotes at different time points of their first cell cycle.
Cell Cycle↗
Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos.
Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections. NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell stages, whereas in all late 8-cell stages, it was present. In NTE from nonactivated (MII phase) cytoplasts, there was a sharp decline in RNA synthesis at 1 hr and 3 hr after fusion and a total absence by 20 hr after fusion. In contrast, NTE from activated (S phase) cytoplasts exhibited continued high levels of RNA synthesis at 1 hr and moderate levels at 3 hr after fusion, although it had ceased by 20 hr after fusion. In all NTE (activated and nonactivated cytoplasts), there was no RNA synthesis seen at the 2-cell stage. However, at the 4-cell stage, weak RNA synthesis was seen in all NTE from activated cytoplasts, whereas none was observed in those from MII nonactivated cytoplasts. At the 8-cell stage, nearly all NTE from S-phase cytoplasts showed weak to moderate levels of RNA synthesis. We conclude that the nucleus reprogramming differs between NTE reconstructed from activated and nonactivated cytoplast with the former undergoing a slower cessation of RNA synthesis after fusion and earlier resumption of RNA synthesis, occurring as early as the 4-cell stage.
Animals↗
Overall efficiency of in vitro embryo production and vitrification in cattle.
In 5 replicates a total of 719 immature oocytes recovered from 94 slaughterhouse-derived bovine ovaries were matured and fertilized in vitro, then cultured for 7 to 9 d on a granulosa cell monolayer in TCM 199 supplemented with calf serum. Of 338 blastocysts (47% of oocytes cultured), 301 were vitrified in Hepes/bicarbonate buffered TCM-199 medium, 20% calf serum and dimethylsulfoxide and ethylene glycol as the cryoprotectants. After thawing in 1 M sucrose and subsequent culture in vitro, 237 (79%) of the blastocysts re-expanded and 177 (59%) hatched. Re-expansion and hatching rates differed between the blastocysts vitrified on Day 7 and Day 8 (84 and 69% vs 70 and 41%, respectively). We conclude that the applied methods are relatively simple and inexpensive to use, with an overall efficiency of the in vitro production/vitrification procedure being 1.9 hatched blastocyst/ovary. Therefore, this system seems suitable for large-scale production of cryopreserved bovine embryos for various purposes.
Journal Article↗