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Summary. Individual blastocysts from cows were cultured for 3 h under 5% CO2 in air, in 4 μl droplets of Ham's F-10 medium containing d-[5-3H]glucose, d-[1-14C]-glucose, d-[6-14C]glucose, [2-14C]pyruvate, or l-[U-14C]glutamine, and with or without 2,4-dinitrophenol (DNP) or phenazine ethosulphate (PES). The 14CO2 or 3H2O produced were collected by exchange with an outer bath of 400 μl 25 mm-NaHCO3. All combinations of substrate and treatment (control, DNP or PES) produced measurable quantities of labelled product except for d-[6-14C]glucose in the presence of PES. Untreated and DNP-treated embryos developed normally during a subsequent 48-h culture period in fresh medium, but PES-treated embryos degenerated. Pyruvate and glutamine metabolism both increased markedly in the presence of DNP, indicating that the Krebs' cycle is active, and that glutamine can be used as an energy substrate. Conversely, DNP has no significant effect on glucose metabolism, indicating that glycolysis is blocked in the bovine blastocyst due to a lack or inhibition of pyruvate kinase. The production of 14CO2 from d-[1-14C]glucose increased significantly in the presence of PES, indicating that the activity of the pentose shunt is less than maximal.
Keywords: embryo; cattle; energy metabolism; glutamine
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After maturation in vitro for 0, 6, 12, 18 or 24 h, the metabolism of radiolabelled glucose, glutamine, pyruvate and glycine by individual cattle oocytes was measured for 3 h. The metabolism of glucose through the Embden–Meyerhof (1.77–2.66 pmol per oocyte per 3 h) and pentose-phosphate (0.39–0.75 pmol per oocyte per 3 h) pathways was low and did not change over time. The oxidative metabolism of glucose carbon through the Krebs cycle was low throughout maturation, but increased significantly (P ≤ 0.05) at 6 h (0.41 pmol per oocyte per 3 h) and 18 h (0.69 pmol per oocyte per 3 h). Pyruvate, glutamine and glycine metabolism in the Krebs cycle increased during culture. Pyruvate metabolism increased significantly from 0 h (17.3 pmol per oocyte per 3 h) to 6 h (23.3 pmol per oocyte per 3 h) and reached a maximum at 12 h (30.8 pmol per oocyte per 3 h). Glutamine metabolism was unchanged from 0 to 12 h (0.89 pmol per oocyte per 3 h), and then increased significantly at 18 h (2.25 pmol per oocyte per 3 h). Glycine metabolism increased significantly from 6 h (0.21 pmol per oocyte per 3 h) to 12 h (0.46 pmol per oocyte per 3 h) and reached a maximum at 18 h (0.68 pmol per oocyte per 3 h). The results suggest that oxidative metabolism increases, and is the major site of cellular energy production, during maturation of the cattle oocyte in vitro.
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In a number of species, including rats and mice (Bartke, Steele, Musto & Caldwell, 1973), bulls (Katangole, Naftolin & Short, 1971), and rams (Purvis, Illius & Haynes, 1974), there is evidence of episodic fluctuation of circulating levels of testosterone. It has generally been concluded that testosterone is released from the testis in a pulsatile fashion, temporally related to the episodic release of LH (Katangole et al., 1971; Moor & YoungLai, 1975).
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Summary. Mature cyclic Holstein heifers were given a luteolytic dose of cloprostenol followed by two i.v. injections, 12 h apart, of various doses of [Ac-d-Nal1, d-p-Cl-Phe2, d-Trp3, d-Arg6, d-Ala10]-LHRH, beginning either at the time of first observation of behavioural oestrus, or 48 h after the cloprostenol injection. When treatment began at the first observation of oestrus, the time of ovulation, as determined by ultrasonic echography, was significantly delayed by total doses of 0·8 mg or more of the antagonist. When given at 48 and 60 h after cloprostenol injection, a total dose of 1·5 mg of the antagonist significantly delayed the growth of the ovulating follicle, the onset of oestrus, the preovulatory surges of oestradiol, LH and FSH, and ovulation. It is concluded that the LHRH antagonist can effectively suppress endogenous LH secretion and may therefore be useful in the study of follicular development, ovulation, and other events in the oestrous cycle of the cow.
Keywords: LHRH; antagonist; heifer; oestrus; ovulation
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Summary. The metabolism of radiolabelled glucose and glutamine was measured in individual cattle embryos produced by in vitro maturation and fertilization of oocytes, and culture with bovine oviductal epithelial cells. Metabolism of glucose through the pentose-phosphate pathway increased almost 15 times and the total metabolism of glucose 30 times, during development from the two-cell to the expanded blastocyst stage. The first marked increase in glucose metabolism did not occur until between the eight- and 16-cell stages, the time of activation of the embryonic genome. Conversely, the metabolism of glutamine was high in two- and four-cell embryos and then decreased to reach a minimum at the compacted morula to blastocyst stage, possibly because of degradation of maternally derived enzymes. Blastocyst expansion was accompanied by significant increases in the metabolism of glucose and glutamine, presumably reflecting the increased energy demands of Na+–K+ ATPase necessary for formation and maintenance of the blastocoel.
Keywords: cattle; embryo; energy metabolism; development; glutamine
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Summary. The ability of purified preparations of platelet-activating factor (PAF), from three different suppliers, to induce thrombocytopaenia in mice after splenectomy and to activate mouse platelets in vitro was examined. Although the PAF preparations were potent activators of horse and cow platelets in vitro, injections of up to 1 μg PAF failed to elicit thrombocytopaenic responses in either CD1 or Swiss Webster random-bred mice. However, when thrombin was injected into Swiss Webster mice, a dose-dependent decrease in the concentration of platelets was observed. Furthermore, isolated platelets from these strains and from 3 inbred lines (C3H/He, BALB/c, C57BL/6) of mice, were not aggregated by PAF in vitro but were sensitive to adenosine diphosphate and thrombin. No change in circulating platelet concentrations was observed over the initial 7 days of gestation in intact Swiss Webster and C57BL/6 or splenectomized C57BL/6 mice, suggesting either an absence of PAF production during early pregnancy in these strains or insensitivity of their platelets to PAF. These results suggest that many mouse strains are unsuitable for the bioassay of PAF.
Keywords: platelet-activating factor; thrombocytopaenia; aggregometry; bioassay; embryonic viability; mouse
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Summary. Individual Day-7 embryos (morulae to expanded blastocysts) were incubated with radiolabelled substrates and karyotyped to determine the sex. In Exp. 1, embryos were incubated for 3 h with d-[1-14C]glucose, as a measure of the activity of the pentose-phosphate pathway (PPP) and d-[5-3H]glucose, as a measure of total glucose metabolism. The labelled products 14CO2 and 3H2O were collected throughout the measurement period. Total glucose metabolism in male embryos was twice that in female embryos and increased between the morula and expanded-blastocyst stages. Relative to total glucose metabolism, PPP activity was four times greater in female than in male embryos. In Exp. 2, embryos were cultured with d-[1-14C]glucose, and l-[3,4-3H(N)]glutamine (a measure of Krebs cycle activity) in the presence of brilliant cresyl blue, a stimulator of the PPP. Glutamine metabolism increased from the morula to expanded-blastocyst stages. Relative to the metabolism of glutamine, the activity of the PPP was one-third greater in female than in male embryos.
Keywords: cattle; embryo; metabolism; development; sex
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The effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the maturation and subsequent development of cattle oocytes in vitro were evaluated in three experiments. Cumulus–oocyte complexes (COC) were collected from cattle ovaries and matured for 20–24 h in control medium or in medium containing 50 ng EGF ml−1, 100 ng IGF-I ml−1, EGF + IGF-I, or 10% (v/v) fetal calf serum plus 0.1 i.u. human menopausal gonadotrophin ml−1 (hMG). In Expt 1, treatment with EGF + IGF-I stimulated cumulus expansion, the metabolism of pyruvate and glutamine, and nuclear maturation. In Expt 2, only the metabolic measurements from oocytes that reached metaphase II were considered, and EGF + IGF-I stimulated pyruvate metabolism to the same extent as serum + hMG. In Expt 3, the oocytes were fertilized after maturation culture, and the resultant embryos cultured for up to 8 days. The cleavage was greater in the EGF and EGF + IGF-I groups than in the controls but less than in the serum + hMG group. Moreover, the number of blastocyst cells at 7 days after insemination and the proportion of cleaved embryos that developed to the blastocyst stage by day 8 was greater in the serum + hMG group than in the control group indicating that maturation treatment can affect early embryonic development. In conclusion, EGF + IGF-I can stimulate cumulus expansion, oxidative metabolism, nuclear maturation and cleavage after fertilization of bovine oocytes in vitro. The relative effects of the treatments on oocyte pyruvate metabolism in Expts 1 and 2 generally paralleled their effects on cleavage and subsequent development in Expt 3, suggesting that mitochondrial function is related to developmental potential. Further investigation is required to determine which component(s) of serum or gonadotrophin treatment is responsible for the effects on subsequent embryonic development.
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The objective of this study was to compare the development and metabolic activity of cattle embryos co-cultured with bovine oviductal cells or cultured in serum-free medium previously conditioned by bovine oviductal cells. Zygotes were produced by in vitro fertilization of oocytes from bovine ovaries obtained from an abattoir. Development to the four-cell stage occurred by 48 h after fertilization in both culture systems, but co-cultured embryos reached the 16-cell stage by 96 h, whereas those cultured in conditioned medium did not do so until 24 h later. Similarly, the morula and blastocyst stages were reached 24 h earlier in co-culture than in conditioned medium. There were significantly more cells in the blastocysts from co-culture (96.8 ± 6.1 versus 56.7 ± 3.3; P ≤ 0.0001). The metabolism of glutamine did not differ between embryos cultured in the two systems, but the metabolism of glucose was significantly greater in embryos cultured in conditioned medium. The first significant increase in glucose metabolism occurred between the four-cell and the 16-cell stages in embryos cultured in conditioned medium, but occurred between the 16-cell and morula stages in the co-cultured embryos, such that the glucose metabolism was significantly greater at the 16-cell stage in embryos cultured in conditioned medium compared with co-cultured embryos (6.5 ±1.0 versus 1.5 ± 0.4 pmol per embryo per 3 h, P ≤ 0.0001). The concentration of glucose was significantly less, and that of lactate significantly greater, in co-culture medium than in conditioned medium. The results suggest that the activity of enzymes involved in glucose transport or metabolism in the early cattle embryo can be affected by the prior culture conditions, and that a high rate of glucose metabolism may be unfavourable for development.
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In vitro produced bovine zygotes show substantial variation in the time required to complete the first cell cycle and in their in vitro development potential. A number of reports have highlighted the fact that the fastest developing embryos in vitro are most likely to be comparable with their in vivo counterparts. At 24 h after IVF, presumptive zygotes were cultured in droplets of synthetic oviduct fluid medium. Droplets were examined at regular intervals and all cleaved embryos at each time point were transferred into new droplets and cultured separately for the duration of the experiment. All uncleaved zygotes were returned to the incubator and re-examined at the successive time points until 48 h after insemination, at which time the remaining uncleaved oocytes were retained as a group. A representative number of day 7 blastocysts from zygotes that had cleaved by 30 or 36 h were transferred to synchronized recipients and pregnancy was diagnosed by ultrasonography at day 35. Glucose and glutamine metabolism was examined in zygotes and blastocysts and compared retrospectively with time of first cleavage. A representative number of blastocysts from each of the cleavage groups was sexed using PCR. Data were analysed by chi-squared and regression analysis. Development to the blastocyst stage decreased as the time from insemination to first cleavage increased (r = 0.97, P < 0.03). There was no difference in blastocyst hatching, number of blastocyst cells or pregnancy rate between the 30 and 36 h groups. The overall sex ratio was 62% males (n = 258, P < 0.0001) and was not different in the 30 and 36 h groups (61%, n = 155 versus 63%, n = 95, respectively). These results indicate that although time of first cleavage has a major influence on the probability of an embryo developing to the blastocyst stage, once that stage is attained, subsequent developmental characteristics are unrelated to the time of first cleavage.
