Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct

The ovine oviduct was evaluated as a culture system for early bovine embryos. One- to two-cell embryos were collected from superovulated heifers killed 36 or 48 h after the onset of estrus, embedded in agar cylinders, and transferred to oviducts ligated at the uterotubal junction. After 5 d (6.5 to 7.0 d after donor estrus), embryos were recovered and evaluated for development to the late morula or blastocyst stage. In Experiment 1, 86 embryos were cultured in 10 ewes in which the onset of estrus was synchronized with that of the donors. Fifty-eight embryos (68%) were recovered; of these, 31 (53%) had continued normal development. In Experiment 2, development in ovariectomized versus intact cyclic ewes was compared. Recovery from ovariectomized ewes (26/39, 67%) did not differ from intact cyclic ewes (26/35, 74%) and the proportion developing normally also did not differ (ovariectomized: 7/26, 27%; intact cyclic: 11/26, 42%). In Experiment 3, embryo development was compared in anestrous versus ovariectomized ewes. Recovery rate (anestrous: 22/43, 51%; ovariectomized: 20/51, 39%) and the proportion developing normally (anestrous: 8/22, 37%; ovariectomized: 9/20, 45%) did not differ between treatments. Developmental competence of oviduct-cultured embryos was tested by transfer to 16 synchronous heifers, of which eight (50%) became pregnant; five delivered calves. Results indicate that the ovine oviduct provides an adequate site for the culture of early bovine embryos.     

Induction of Medicarpin Biosynthesis in Ladino Clover Callus by p-Chloromercuribenzoic Acid Is Reversed by Dithiothreitol

Treatment of Ladino clover (Trifolium repens L.) callus with 1 millimolar p-chloromercuribenzoic acid (PCMBA) stimulated the biosynthesis of the phytoalexin medicarpin. Increased medicarpin concentration was detected at 6 hours, and maximum level was achieved by 36 hours. Dithiothreitol (DTT), added to callus at 2, 6, 10, and 18 hours following addition of PCMBA, partially counteracted the effect of PCMBA in the cells. The greatest effect on reversal of medicarpin accumulation was found at the earlier times for DTT addition. Compared to a 24-hour PCMBA control, final medicarpin concentrations were 9 to 19%, 37 to 45%, and 92 to 105% of the control value when DTT was added at 0.5, 1 or 2 hours, 3 or 4 hours, and 6, 8, 10, or 12 hours, respectively, after PCMBA addition. The results indicate that stimulation of medicarpin biosynthesis is reversible, but once activated, the pathway is not shut down by removal of elicitor.     

Inseparability of X-Heterochromatic Functions Responsible for X:Y Pairing, Meiotic Drive, and Male Fertility in Drosophila melanogaster

Deficiencies encompassing part or all of the X heterochromatin of Drosophila melanogaster have been linked to three abnormalities in male meiosis and spermatogenesis: X-Y nondisjunction, skewed sperm recovery ratios favoring sperm with reduced chromatin content, and sterility in males carrying either a Y-autosome translocation or mal ⁺Y. In this study, 18 X heterochromatic deficiencies of varying sizes were tested in XY males for their spermatogenic phenotypes. All 18 proved to be either mutant for all three phenotypes or wild type for all three. Although variable among mutant deficiencies, expression levels of all three phenotypes were strongly correlated. Deficiencies that cause high levels of nondisjunction also cause severe recovery ratio distortion and are completely sterile in conjunction with mal⁺ Y. Low nondisjunction deficiencies cause comparable mild effects for the other phenotypes. The same deficiencies were also tested in males carrying a large heterochromatic free X duplication Dp(1; f)3. For all deficiencies which induce nondisjunction in XY males, the Y and free duplication pair regularly and the X fails to pair in XYDp males. Drive levels are constant across deficiencies in these males. Thus elimination of variability in the pairing phenotype also eliminates variability in sperm recovery ratios.     

Calcium bridges are not load-bearing cell-wall bonds in Avena coleoptiles

I examined the ability of frozen-thawed Avena sativa L. coleoptile sections under applied load to extend in response to the calcium chelators ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) and 2-[(20bis-[carboxymethyl] amino-5-methylphenoxy)methyl]-6-methoxy-8-bis [carboxymethyl]aminoquinoline (Quin II). Addition of 5 mM EGTA to weakly buffered (0.1 mM, pH 6.2) solutions of 2(N-morpholino) ethanesulfonic acid (Mes) initiated rapid extension and wall acidification. When the buffer strength was increased (e.g. from 20 to 100 mM Mes, pH 6.2) EGTA did not initiate extension nor did it cause wall acidification. At 5 mM Quin II failed to stimulate cell extension or wall acidification at all buffer molarities tested (0.1 to 100 mM Mes). Both chelators rapidly and effectively removed Ca²⁺ from Avena sections. These data indicate that Ca²⁺ chelation per se does not result in loosening of Avena cells walls. Rather, EGTA promotes wall extension indirectly via wall acidification.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - Quin II 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis(carboxymethyl)aminoquinoline - Mes 2(N-morpholino)ethanesulfonic acid     

Comparisons of eight commercial on-farm milk progesterone tests

To evaluate eight commercial on-farm milk progesterone kits, milk samples (50 ml each of foremilk and postmilk strippings) were collected during the estrous cycle from 10 cycling Holstein cows for 24 consecutive days. Relative concentrations of progesterone were classified as low or high by comparison with standard progesterone samples supplied with each kit. The concentration of progesterone in each milk sample was determined by radioimmunoassay (RIA). Accuracy of classification into low or high levels by commercial tests was determined by the percentage of similarity with RIA values using discriminant analysis. Accuracy of the eight tests ranged from 89.0 to 98.9% for low progesterone, 74.8 to 85.6% for high progesterone, and 80.3 to 87.3% for all samples (n = 238). The percentage of fat in milk or an interaction of the percentage of milkfat by day of estrous cycle influenced commercial test results for all tests except Accufirm and Calfcheck. Progesterone levels, estimated by the test-kits, were low from 1.5 +/- 0.5 to 2.8 +/- 0.9 days before estrus (X +/- SEM) and until 4.0 +/- 0.6 to 5.9 +/- 1.3 days after estrus. These data support the principle that a single low progesterone sample cannot be used to determine proper timing of insemination. All eight commercial kits can be used to determine accurately the relative concentrations of progesterone in milk samples.     

Synthesis and biological activity of an azido derivative of paclobutrazol, an inhibitor of gibberellin biosynthesis

A photolabile azido derivative of the kaurene oxidase inhibitor 1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl) pentan-3-ol (paclobutrazol) has been synthesized for use as a photoaffinity labeling agent. The compound was tested as an inhibitor of the oxidation of ent-kaurene catalyzed by cell-free preparations from endosperm of Cucurbita maxima. The I₅₀ of the azido derivative was 9.5 nanomolar, which compares well with that of paclobutrazol (6.3 nanomolar in our measurements). The azido compound bound to Cytochrome P-450 in microsomes from Cucurbita maxima, and induced a Type II spectral change, with an apparent binding constant of 0.24±0.04 micromolar.     

Changes in Abscisic Acid and Indoleacetic Acid before and after Anthesis Relative to Changes in Abscission Rates of Cotton Fruiting Forms

Cotton (Gossypium hirsutum L.) fruiting forms exhibit pronounced changes, with age, in their probability of abscission. Large floral buds rarely abscise, but after anthesis the young fruits (bolls) have a high probability of abscising. Abscission rate reaches a peak about 5 to 6 days after anthesis and then gradually decreases. An experiment was conducted to try to determine the reason for the rapid and pronounced increase in probability of abscission just after anthesis. Cotton was grown in the field and fruiting forms of various ages from 9 days before to 9 days after anthesis were all harvested the same day and subsequently analyzed for ABA and IAA. The concentration of ABA decreased slightly at anthesis and increased gradually thereafter. In contrast, the concentration of IAA was high before anthesis and then decreased at anthesis to about one-fifth the previous concentration. IAA remained low for at least 4 days after anthesis and then increased rapidly between 7 and 9 days after anthesis. The high concentration of IAA in floral buds before anthesis is probably a major factor in their resistance to abscission. Likewise, the low concentration of IAA at anthesis and for about 4 days thereafter may promote fruit abscission during the young boll stage.