Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.     

Glycogen levels of rat ova during cleavage]

Cytophotometric measurements of PAS positive material in isolated rat eggs leads to a good evaluation of their glycogen content. Amounts of this polysaccharide in these eggs were found relatively constant during cleavage.     

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.     

Penetration of intact bovine ova with ram sperm in vitro

In culture, mature bovine ovarian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal-looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.     

Improved cryopreservation of in vitro-produced bovine embryos using a chemically defined freezing medium

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in bovine embryo cryopreservation solutions. During the experiment, fetal calf serum (FCS) and bovine serum albumin (BSA) were used as references. A combination of a thermodynamic approach using differential scanning calorimetry and a biological approach using in vitro-produced bovine embryo slow-freezing was used to characterize cryopreservation solutions containing CRYO3, FCS and BSA. The CRYO3 and fetal calf serum (FCS) slow-freezing solutions were made from Dulbecco's phosphate-buffered saline containing 1.5 m ethylene glycol, 0.1 m sucrose and 20% (v.v⁻¹) of CRYO3 or FCS. The bovine serum albumin (BSA) solution was made by adding 0.1 m sucrose to a commercial solution containing 1.5 m ethylene glycol and 4 g L⁻¹ BSA. These solutions were evaluated using three characteristics: the end of melting temperature, the enthalpy of crystallization (thermodynamic approach) and the embryo survival and hatching rates after in vitro culture (biological approach). The CRYO3 and FCS solutions had similar thermodynamic properties. In contrast, the thermodynamic characteristics of the BSA solution were different from those of the FCS and CRYO3 solutions. Nevertheless, the embryo survival and hatching rates obtained with the BSA and FCS solutions were not different. Similar biological properties can thus be obtained with slow freezing solutions that have different physical properties within a defined range. The embryo survival rate after 48 h of in vitro culture obtained with the CRYO3 solution (81.5%) was higher than that obtained with the BSA (42.2%, P = 0.000 12) and FCS solutions (58%, P = 0.016). Similarly, the embryo hatching rate after 72 h of in vitro culture was higher with the CRYO3 solution (61.1%) than with the BSA (31.1%, P = 0.0055) and FCS solutions (36%, P = 0.018). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in in vitro-produced bovine embryo cryopreservation solutions.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum

Bovine follicular oocytes were matured in TCM 199 supplemented with: (1) fetal calf serum (FCS, 20% v/v), Luteinizing Hormone (LH, 10 ug/ml), and Estradiol-17-beta (E(2), 1 ug/ml) in Experiment 1; (2) 20% cow serum recovered at standing estrus (Experiment 2); or (3) 20% FCS (Experiment 3). Maturation, fertilization, and initial cleavage development were evaluated at 16 and 48 h after in vitro insemination. The proportions of oocytes fertilized after maturation in the presence of added hormones (78.5%, Experiment 1) or estrous serum (71.3%, Experiment 2) were significantly higher (p < 0.01) than after use of FCS alone (39.3%, Experiment 3). Cleavage of zygotes within 48 h post-insemination differed significantly (p < 0.01) between maturation treatments, 27.3%, 75.5% and 6.6% for Experiments 1, 2, and 3, respectively. Results demonstrate a beneficial influence of estrous cow serum, characterized by an elevated concentration of LH, on bovine oocyte maturation in vitro.     

Effects of antisperm antibodies on early cleavage of fertilized ova.

Early cleavage of zygotes was abnormal after oocytes were fertilized with sperm cells from one of 64 infertile couples studied in our human in vitro fertilization and embryo development procedure. The abnormal cleavage, which did not affect fertilization, was not due to polyspermy or parthenogenetic activation of oocytes, but to antisperm antibodies present on the sperm cells. These antibodies did not react with zona-intact or zona-free human oocytes, although they reacted with the acrosomal (strongly) and tail (weakly) regions of human sperm. The antibodies recognized a double band comprising two glycoproteins of 14 +/- 3 and 18 +/- 3 kDa and a protein band of 22 +/- 3 kDa on the Western blot of lithium diiodosalicylate-solubilized human sperm extract. Antisera were raised in rabbits against the double band (14 +/- 3- and 18 +/- 3-kDa antigens) and the 22 +/- 3-kDa protein. Passive transfer of affinity-purified human or rabbit immunoglobulins directed against the double band (14 +/- 3- and 18 +/- 3-kDa antigens), but not those directed against the 22 +/- 3-kDa protein, caused a significant inhibition of early cleavage of oocytes without affecting pronuclear formation in mice. These results suggest that the sperm surface antigens of 14 +/- 3 and 18 +/- 3 kDa may provide an extranuclear signal to oocytes to divide in mice and humans. These antigens may also find clinical applications in the management of immunoinfertility and in the development of an antisperm contraceptive vaccine for humans.     

Ovum recovery in superovulated cows and cleavage rates in the fertilized ova

Four groups of 10 cows were given one, two, three, or four thousand i.u. of PMS on Day 16 of the estrous cycle. On Day 19, the cows were injected with 10–15 mg of estrogen. At the ensuing estrus, the cows were bred and given 2,000 i.u. of HCG. The cows were slaughtered for ovum recovery 3–7 days after breeding. On the average, the cows produced 10.3 ± 1.6 ovulations with a range from 0 to 41. Each 1,000 i.u. of PMS was associated with an increase of 5.83 ± 1.18 in number of ovulations. The variation among cows within treatment groups was not attributable to differences in age or body weight. On the average, 51.5% of the ovulated ova were recovered, with the recovery rate being higher (64.2%) for ova in the oviducts and lower (34.2%) for those in the uterus. Ovarian length at the time of slaughter was directly related to PMS dosage and a smaller proportion of the ova were recovered from animals having largest ovaries. Only 38.4% of 211 recovered ova were fertilized. It was estimated that cleavages occurred at intervals of 12.6 ± 23.6 hours.     

Fertilizability and developmental capacity of bovine oocytes cultured individually in a chemically defined maturation medium

This study investigated effects of adding hypotaurine (HT), beta-merocaptoethanol (beta-ME), or both into a chemically defined maturation medium (TCM-199 containing 0.1% polyvinyl alcohol: PVA) on maturation, fertilization and development of individually (single) cultured bovine oocytes. Mean GSH concentration in the oocytes cultured in the medium supplemented with either beta-ME (1.11 +/- 0.05 nM) or HT plus beta-ME (0.97 +/- 0.03 nM) was significantly (P < 0.05) higher than that in the medium containing PVA alone (0.75 +/- 0.03 nM). Adding beta-ME showed a significantly (P < 0.05) higher rate of the second metaphase stage (93.6 +/- 3.3%) than in the medium containing PVA alone (single-control) (65.2 +/- 7.9%). Adding both HT and beta-ME showed significantly (P < 0.05) higher rates (92.6 +/- 2.7%) of normal fertilization than did adding HT alone (63.5 +/- 4.6%). Also, adding both HT and beta-ME significantly (P < 0.05) lowered the polyspermy rate than did adding HT alone. Adding either beta-ME or both HT and beta-ME showed no significant difference in cleavage. Blastocyst development did not improve significantly adding either HT, beta-ME or both, although beta-ME alone or HT plus beta-ME tended to result in a higher rate of blastocysts (6.4 and 6.8%, respectively) than resulted without additives (1.6%). Our results show that adding beta-ME to a chemically defined maturation medium increased the intracellular GSH level of bovine oocytes cultured individually, and can improve the maturation rate leading to the blastocyst stage throughout in vitro production.     

Glucose requirement at different developmental stages of in vitro fertilized bovine embryos cultured in semi-defined medium

Three experiments were conducted in which 2-cell bovine embryos were prepared from oocytes, obtained from abattoir ovaries, by in-vitro maturation for 22 to 24 hours, followed by exposure to spermatozoa for 8 hours and culture for 40 hours within the cumulus. The cumulus cells were then removed, and the cleaved embryos were cultured for a further 120 hours or longer, in the presence or absence of glucose, pyruvate and lactate. Very few embryos developed in the complete absence of energy substrates. Lactate and pyruvate, alone or combined, supported development to the 8-cell stage, but pyruvate was required to support development to the morula stage (Experiment 1). When present throughout culture or when added at 48 or 96 hours postinsemination, 5.56 mM glucose was detrimental to development (Experiments 1 and 2). However, when added at 120 hours postinsemination, 5.56 mM glucose improved development to the blastocyst and expanded blastocyst stages, compared with no glucose or 11.12 mM glucose (Experiment 3).     

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.     

Serum-free medium conditions for steroidogenesis of bovine follicular thecal cells cultured on collagen gel matrix

Summary Thecal cells isolated from bovine ovarian follicles were cultured with a serum-free basal medium or a serum-free complete medium in the presence or absence of collagen gel matrix, and their cellular proliferation and steroidogenesis were compared with those of cells cultured with a serum-containing medium. The cells cultured with the serum-free basal medium produced larger amounts of progesterone, androstenedione, and estradiol than the cells cultured with the serum-containing medium, but no appreciable cell proliferation was observed in the serum-free medium. Response of thecal cells to 8 bromo-cAMP, a steroidogenic agent, varied according to the type of steroid production examined and the type of culture medium used. In a cultivation period of 4 d, progesterone production was stimulated about five-fold by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium without collagen matrix, whereas androstenedione production was stimulated about three- to fourfold in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium with or without collagen matrix. Estradiol production, however, was significantly suppressed by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and also in the serum-containing medium. Thus, among the conditions examined, the most suitable primary culture media for steroidogenesis of thecal cells were the serum-free media, especially serum-free complete medium on collagen gel matrix.     

Carotene cleavage activity in bovine ovarian follicles

Carotene cleavage activity has been demonstrated in bovine ovarian follicles by incubating cell-free homogenates from granulosa cells with 15, 15'-(14)C-beta-carotene. Enzyme activity was highly dependent on the quality of follicles studied. Highest cleavage activity was found in large, preovulatory follicles. The correlation between follicular fluid concentration of vitamin A and the conversion rate obtained with the corresponding enzyme preparations was significantly positive. No correlation, however, could be shown between cleavage activity and beta-carotene, alpha-tocopherol or cholesterol. The only form of vitamin A in follicular fluid of cattle was found to be retinol. The results support the hypothesis that a local conversion of beta-carotene into vitamin A in follicular structures is responsible for the increase of the intrafollicular concentration of vitamin A during follicular development.     

Improved embryoid induction and green shoot regeneration from wheat anthers cultured in medium with maltose

Summary Anthers from spring wheat (Triticum aestivum L.) genotypes, including six F₁ hybrids, were cultured in a modified liquid N6 medium containing either sucrose or maltose. In every case, use of maltose resulted in greater microspore callus induction and green shoot regeneration than culture in sucrose-containing medium. Induction in maltose medium also allowed green shoots to be recovered from crosses that showed only a poor response in other media and from two genotypes that did not respond to modified N6 medium with sucrose. Replacement of sucrose with maltose generally resulted in microspores having a more embryogenic mode of development in which distinct embryoids often formed. The most responsive genotype produced over 200 green shoots/100 anthers when cultured in medium with maltose.NRCC publication no. 31494     

Dynamics of morphofunctional changes in aging bovine ova during prolonged culture in vitro

In the absence of activating stimuli, aging processes are initiated in matured mammalian oocytes, which negatively affect the quality of ova and their capacity for further development. On the model of the prolonged culture of bovine oocytes, the dynamics of a number of morphofunctional changes associated with the postovulatory aging was investigated in the present work. In cumulus-enclosed oocytes, migration of the first polar body relative to metaphase II chromosomes started between 18 and 22 h of maturation. The angle of the body deviation from the metaphase plate rose as the culture time increased to 30 h. By 32 h of culture, a gain in the rate of ova with the abnormal chromosome morphology was observed that continued up to 56 h. Furthermore, after 56 h, signs of spontaneous parthenogenetic activation were revealed in 16% of matured oocytes. During the prolonged culture of oocytes deprived of cumulus cells after 20 h of maturation, an increase in the frequency of chromosomal abnormalities was found only by 44 h. At the same time, the cumulus elimination did not affect the maintenance of the meiosis II blockade in aging ova. Meanwhile, destructive chromosomal changes in oocytes were attended by a gradual rise in the level of apoptotic degeneration and reduction in the proliferative activity of surrounding cumulus cells. The results obtained point to the varying temporal dynamics of distinct morphofunctional changes and to the participation of cumulus cells in modulation of the speed of metaphase chromosome destructive modifications in aging bovine ova.     

Growth,antrum formation,and estradiol production of bovine preantral follicles cultured in a serum-free medium

The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17beta (E(2)) production in preantral follicles of bovine ovaries. Large preantral follicles (145-170 micro m in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.5 degrees C in 5% CO(2) in air. This culture environment allowed most preantral follicles to maintain a three-dimensional structure with the presence of a thecal layer and basement membrane surrounding the granulosa cells throughout the entire culture period. The effects of insulin, insulin-like growth factor (IGF)-I, IGF-II, FSH, and LH on preantral follicle growth were investigated in serum-free medium. Follicular diameters were significantly larger in the presence of insulin, IGF-I, IGF-II, or FSH after 13 days in culture. Oocyte diameters were also significantly larger in the presence of all hormones tested. The single addition of insulin, IGF-I, or FSH induced antrum formation between Days 7 and 13 of culture. Insulin progressively induced E(2) secretion by follicles after antrum formation, but IGF-I and FSH had no apparent effect. FSH and LH caused an increase in oocyte diameter in the presence of insulin. The addition of three hormones (insulin, FSH, and LH) initiated antrum formation and E(2) production earlier than insulin-containing medium alone. Furthermore, maximal E(2) secretion was maintained steadily between 7 and 13 days in this culture condition. From these results, we have demonstrated that insulin, FSH, and LH play substantial roles in the growth and development of bovine large preantral follicles in a serum-free medium.