Diffusion of fluoroquinolones into equine fetal fluids did not induce fetal lesions after enrofloxacin treatment in early gestation.

While recent work demonstrated that enrofloxacin and ciprofloxacin reach the fetoplacental unit without causing obvious lesions in the 9-month-old equine fetus or resulting foal, many practitioners still hesitate to prescribe a fluoroquinolone during pregnancy. Since early gestation is a critical time for fetal skeletal development, if fluoroquinolones are chondrotoxic to the fetus at any point during gestation, this period would be important. The aim of this study was to assess the effects of 2 weeks' exposure to enrofloxacin on the equine fetus between 46 and 60 days gestation. Twelve pregnancies from nine healthy mares were allocated into two groups: untreated (n=7), or treatment (7.5mg/kg enrofloxacin, PO×14days, n=6). Abortion was induced with prostaglandin 24h after the last enrofloxacin dose, or on the equivalent day of gestation for untreated mares. Four of nine mares were rebred for a second cycle and were assigned to the opposite treatment to serve as their own controls. Fetal fluids from treated mares were analysed for enrofloxacin and ciprofloxacin concentrations. Fetal organs (heart, lungs, spleen, kidney, and liver) and limbs were examined histopathologically. Enrofloxacin and ciprofloxacin diffused to the fetal fluids during early gestation and did not result in detectable abnormalities in the fetus after 14 days of treatment. While current research does not determine long-term foal outcomes, enrofloxacin may be useful for select bacterial infections in pregnant mares.

Dietary supplementation with L-arginine between days 14 and 25 of gestation enhances embryonic development and survival in gilts.

Embryonic loss is a major problem in mammals, but there are few effective ways to prevent it. Using a porcine model, we determined effects of dietary L-arginine supplementation between days 14 and 25 of gestation on embryonic growth and survival. Gilts were checked daily for estrus with boars in the morning and bred at onset of the second estrus and 12 h later (the time of breeding = day 0 of gestation). Between days 14 and 25 of gestation, 15 gilts/treatment were housed individually and fed twice daily 1 kg of a corn- and soybean meal-based diet supplemented with 0.0, 0.4, or 0.8 % L-arginine. All diets were made isonitrogenous by addition of L-alanine. On day 25 of gestation, gilts were hysterectomized to obtain conceptuses. Compared with controls, dietary supplementation with 0.4 or 0.8 % L-arginine increased (P ≤ 0.05) arginine concentrations in maternal plasma, total volume of amniotic fluid; total amounts of arginine in allantoic and amniotic fluids; total amounts of fructose and most amino acids in amniotic fluid; placental growth; and the number of viable fetuses per litter by 2. The numbers of total fetuses, fetal weight, corpora lutea, volume of allantoic fluid, maternal circulating levels of progesterone and estrogen, or total amounts of hormones in allantoic fluid did not differ among the three treatment groups. Reproductive performance of gilts did not differ between the 0.4 and 0.8 % L-arginine groups. Thus, dietary supplementation with 0.4 or 0.8 % L-arginine between days 14 and 25 of gestation enhances embryonic/fetal survival in swine.

The effect of dietary supplementation with linoleic acid to late gestation ewes on the fatty acid composition of maternal and fetal plasma and tissues and the synthetic capacity of the placenta for 2-series prostaglandins.

Linoleic acid (18:2n-6) is metabolised to arachidonic acid (20:4n-6), the precursor for 2-series prostaglandins (PGs). Increased consumption of 18:2n-6 during pregnancy may thus modify PG synthesis during labour. We have investigated whether increased 18:2n-6 composition during gestation altered the fatty acid consumption and PG synthesis of maternal and fetal tissues in the sheep. Ewes were fed a control diet or a diet providing 40% more 18:2n-6 from 96 days gestation. Half of each group received dexamethasone on day 136 to up-regulate the PG synthetic pathways promoting parturition. Maternal and fetal tissues were collected at 138 days. The 18:2n-6 diet significantly increased the 20:4n-6 content of maternal plasma, fetal plasma and allantochorion (51-81%) phosphatidylcholine, and fetal liver (40%) and maternal caruncular endometrium (57%) phosphatidylethanolamine. Increased 18:2n-6 intake increased production of PGF(2alpha) and PGE(2) in all placental tissues (maternal caruncular and intercaruncular endometrium and fetal allantochorion) by 23-98%, whereas dexamethasone increased it by 32-142%. This suggests that consumption of an 18:2n-6-enriched diet in late pregnancy enhanced placental PG production by increasing the supply of 20:4n-6. Variations in the extent to which the diet altered the polyunsaturated fatty acid (PUFA) content of the different tissues indicated complex interactions between nutrient availability and metabolic adaptation.

Angiogenesis and anti-angiogenesis activity of Chinese medicinal herbal extracts.

The aqueous extracts of 24 herbs traditionally used as curing ischemic heart disease in clinic in China were screened for their in vitro angiogenic activity, another twenty-four traditionally used as anti-tumor or anti-inflammatory remedies in China were screened for their in vitro anti-angiogenic activity. The activity of angiogenesis was determined by quantitation of vessels on chick embryo chorioallantoic membrane (CAM) model and cell proliferation of cultured bovine aortic endothelial cells (BAECs). Among the herbal extracts examined, the aqueous extracts of Epimedium sagittatum, Trichosanthes kirilowii and Dalbergia odorifera showed the strong angiogenetic activity both in CAM and BAECs models; and the aqueous extracts of Berberis paraspecta, Catharanthus roseus, Coptis chinensis, Taxus chinensis, Scutellaria baicalensis, Polygonum cuspidatum and Scrophularia ningpoensis elicited significant inhibition at a concentration of 1g dry herb /ml.

Effect of folic acid plus glycine supplement on uterine prostaglandin and endometrial granulocyte-macrophage colony-stimulating factor expression during early pregnancy in pigs.

The objective was to determine the effects of folic acid+glycine supplement on uterine metabolism of prostaglandin and mRNA expression of endometrial granulocyte-macrophage colony-stimulating factor (GM-CSF) in nulliparous (NYL) and multiparous Yorkshire-Landrace (YL) sows, and in multiparous Meishan-Landrace sows (ML). In each of these three groups, sows were randomly assigned to two treatments: 15 ppm folic acid+0.6% glycine or no supplement. The dietary supplement was given from the estrus before mating to slaughter on Day 25 of pregnancy. At slaughter, endometrial tissue was collected to determine endometrial expression levels of GM-CSF mRNA, cyclooxygenase-1 (COX1) and -2 (COX2) and to evaluate in vitro endometrial secretion of prostaglandin E2 (PGE2) secretion. Allantoic fluid samples were also collected to determine the concentration of PGE2, prostaglandin F2alpha (PGF2alpha), estradiol-17beta (E2), progesterone (P4), and transforming-growth factor-beta2 (TGF-beta2). The allantoic contents of PGF2alpha, E2 and P4, and endometrial in vitro secretion of PGE2 were not significantly influenced by the folic acid+glycine supplement. The folic acid+glycine supplement tended (P<0.07) to increase allantoic content of PGE2 and TGF-beta2 in all sows and increased (P<0.05) endometrial expression of COX2, especially in NYL sows. The endometrial expression of COX1 was decreased (P<0.05) by folic acid+glycine supplement, especially in multiparous YL sows. The allantoic contents of PGE2 and PGF2alpha were not significantly affected by sow type. However, NYL sows had higher (P<0.05) endometrial in vitro secretion of PGE2 and allantoic content of P4 than multiparous YL and ML sows. The allantoic content of E2 was also higher (P<0.05) in NYL sows than in multiparous ML sows only. The allantoic content of TGF-beta2 was lower (P<0.05) in multiparous ML than in multiparous YL only sows. Finally, in YL and NYL sows, folic acid+glycine supplement decreased (P<0.05) the endometrial expression of GM-CSF but not in ML sows. In summary, folic acid+glycine supplement altered endometrial expression of GM-CSF and uterine metabolism of prostaglandins during the post-attachment period of porcine embryos but some of these effects were manifest only in Meishan and nulliparous sows.

Effects of conjugated linoleic acid on prostaglandins produced by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion in late pregnant ewes.

The anticarcinogenic properties of conjugated linoleic acid (CLA) are, at least partially, attributed to its ability to interrupt the n-6 polyunsaturated fatty acid (PUFA) metabolic pathway for the biosynthesis of eicosanoids, including prostaglandins (PG). Both PGE(2) and PGF(2alpha) play key roles in parturition. In the present study, we compared the effects of CLA (a mixture of cis- and trans-9, 11- and -10, 12-octadecadienoic acid) and linoleic acid (LA) on PG production by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion from late pregnant ewes. The results demonstrated that supplementation of LA and CLA significantly affected both the proportions and the amounts of PGs produced by all three tissue types. The ability of the uterus and placenta to respond to oxytocin (OT, endometrium only) and lipopolysaccharide (LPS) was also affected. LA inhibited PGE(2) and PGF(2alpha) production in the absence or presence of either oxytocin or LPS. In endometrial cells with or without oxytocin or LPS, CLA dose-dependently suppressed PGF(2alpha) generation, whereas low doses of CLA (20 microM) increased PGE(2) generation. Supplementation with CLA therefore increased the PGE(2)/PGF(2alpha) ratio in the endometrial cells. These results suggest that dietary supplementation of LA or CLA may affect both the initiation and progression of parturition.

Effect of maternal glucocorticoid treatment on ovine fetal fluids at 0.6 gestation.

This study examined the effects of maternal dexamethasone treatment on the volume and composition of fetal fluids, and on placental morphology at 0.6 gestation (80-90 days). Nine pregnant ewes were infused with dexamethasone (D, 0.76 mg h-1 for 72 h) while an additional nine ewes received saline (S, 0.38 mL h-1 for 72 h). Allantoic fluid (ALF) volume was significantly greater (P < 0.01) in the D group (737 +/- 116 mL) than in the S group (190 +/- 55 mL), but there was no difference in amniotic fluid (AMF) volume. The urine flow rate was 11 times higher in three D fetuses. The 51Cr-EDTA infused into the bladders of four fetuses during the final 4-5 h of the 72 infusions was detected in both AMF and ALF. Dexamethasone treatment significantly altered the composition of the fetal fluids but had no affect on fetal body weight, organ weights and placental weight; however, there were fewer cotyledons under 5 g (P < 0.05). In the D group, 3% of cotyledons were of the 'bovine' type in morphology, whereas all cotyledons in the S group were of the 'ovine' type. These findings suggest that prolonged exposure to large doses of glucocorticoids during pregnancy would affect the volume and composition of the fetal fluids and placental morphology, with potentially detrimental effects on the fetus.

The role of vitamin D in chorioallantoic membrane calcium transport.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is essential for the transport of eggshell calcium to the embryo across the chorioallantoic membrane (CAM). CAM contains the vitamin D receptor that increases following 1,25-(OH)2D3 injection into embryos at day 10 of incubation. Further, a single injection of 100 ng of 1,25-(OH)2D3 into vitamin D-deficient quail eggs at day 10 of incubation resulted in a significant increase in both body and yolk calcium. This is accompanied by an increase in carbonic anhydrase from low levels in deficiency to normal levels. Acetazolamide (AZ), a specific carbonic anhydrase inhibitor injected into the quail embryos, caused hypocalcemia and hyperphosphatemia. This is similar to the hypocalcemia and hyperphosphatemia found in vitamin D-deficient embryos. These results suggest that one mechanism of action of vitamin D in the mobilization of eggshell calcium is the activation of carbonic anhydrase that acidifies the calcium carbonate shell.

A new member to the astacin family of metalloendopeptidases: a novel 1,25-dihydroxyvitamin D-3-stimulated mRNA from chorioallantoic membrane of quail.

1,25-Dihydroxyvitamin D-3 is essential for the utilization of eggshell calcium by avian embryo through the chorioallantoic membrane (CAM). A cDNA library was constructed from poly(A)+ RNA extracted from vitamin D-deficient CAMs given 1,25-dihydroxyvitamin D-3. Screening this library by differential hybridization yielded a full-length (approximately 1.8 kb) cDNA, whose corresponding mRNA is increased 3-fold 2.5 h after a single injection of 1,25-(OH)2D3. The complete nucleotide sequence for the full-length cDNA has been determined. An open-reading frame, corresponding to a 310 amino acid, 41 kDa protein was found. Searching protein sequence data bases revealed a strong similarity to the following proteases: astacin, a crayfish digestive protease, Oryzias latipes hatching enzyme constituent protease (Orz), Xenopus laevis developmentally regulated UVS.2 protein secreted by the hatching gland of embryos, the NH2-terminal domain of human bone morphogenetic protein (BMP-1) and Drosophila dorsal-ventral patterning tolloid. The cDNA has approximately 36% overall identity with astacin and BMP-1, and is more than 60% identical to either Orz or UVS.2. Moreover, multiple alignment analysis indicates that 37 residues, including 3 cysteine residues, are strictly conserved in the complete 200-amino acid astacin sequence. All 6 proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases. This motif is found within an extended sequence of HEXXHXXGFXHE that is unique to this subgroup of metalloendopeptidases. In addition, the 6 proteins have 50% identity (including the present cDNA) and 79% are conserved in 4 of these proteins in a 24-amino acid sequence that includes the putative active site. The level of mRNA for the new protein reaches a maximum at day 12 of embryonic life and declines thereafter. It is suggested that this clone corresponds to an mRNA encoding for a protease that may play a role in the degradation of eggshell matrix.

Regulation of extraembryonic calcium mobilization by the developing chick embryo.

During development, the chick embryo mobilizes the calcium it needs from two extraembryonic sources, first the yolk and then the eggshell. Since previous studies have strongly suggested that vitamins D and K may regulate chick embryonic calcium metabolism, we have examined here how these vitamins might be involved in regulating the calcium mobilization processes. We used as our experimental system chick embryos which were maintained in long-term in vitro culture in the absence of the eggshell. Our results showed that exogenous vitamin D3, in the form of the active 1,25-dihydroxylated metabolite, was hypercalcaemic in both control embryos and the calcium-deficient, shell-less embryos. Since the eggshell was absent in the latter, the vitamin D-induced hypercalcaemia must involve mobilization of calcium from the yolk and, or, the embryonic skeleton. The latter was unlikely since concomitant hyperphosphataemia was not observed. By radiolabelling the yolk with 45Ca2+ and subsequently monitoring its distribution, we showed that vitamin D3 stimulated yolk calcium mobilization. However, exogenous vitamin D3 did not appear to influence the calcium uptake activity of the chorioallantoic membrane (CAM), the tissue responsible for translocating eggshell calcium. On the other hand, when embryos were rendered vitamin K deficient by the administration of its antagonist, Warfarin, CAM calcium activity was significantly depressed, an effect which was remedied by vitamin K supplementation. We conclude that, during normal chick embryonic development, vitamin D is primarily involved in regulating yolk calcium mobilization whereas vitamin K is required for eggshell calcium translocation by the CAM.

Supplemented eggshell restores calcium transport in chorioallantoic membrane of cultured shell-less chick embryos.

It was previously reported (Tuan, 1980a) that the development-specific expression of calcium transport and related functions in the chick embryonic chorioallantoic membrane (CAM) requires the continuous presence of the eggshell, the calcium source of the embryo. To further understand the mechanism of action of the eggshell on the CAM functions, this study reports the effects of eggshell supplementation on chick embryos maintained in shell-less cultures. The cultured embryos were able to accumulate and utilize the exogenous shell calcium, applied directly onto the CAM, for skeletal formation. In the region of the CAM directly adhering to the added shell, calcium transport activity, calcium-binding protein (CaBP) activity, and vitamin K-dependent gamma-glutamyl carboxylase activity were significantly restored. These results strongly suggest that the proximity of shell calcium may regulate expression of calcium transport and related functions in the chick embryonic CAM.

Method for obtaining ovine uterine secretions from unilaterally pregnant ewes.

Development of the ovine conceptus was confined to the uterine horn ipsilateral to the corpus luteum (CL) by placing a ligature around that uterine horn at a point near the uterine body on day 5 of pregnancy. On day 140 of gestation, seven of 10 ewes were still pregnant and from 21 to 815 ml of uterine fluid (488 +/- 94 ml, X +/- SEM) were collected from the nongravid uterine horn. Total recoverable protein (X +/- SEM) was 13.4 +/- 3.4 grams. Polyacrylamide gel electrophoresis of the reduced proteins in presence of sodium dodecyl sulfate indicated that protein composition of uterine fluid was distinct from that of colostrum, serum, amniotic fluid, and allantoic fluid, and revealed the presence of two major polypeptides with molecular weights of about 57,000 and 58,500, respectively, plus numerous other minor components. Gel filtration on columns of Sephadex G-200 and Sepharose CL-6B suggested that these polypeptides formed a series of aggregates of high molecular weight when kept under nonreducing conditions. Glucose (.18 +/- .03 mg/ml), but not fructose, was present in uterine fluid. In addition, high levels of prostaglandin F (451.4 +/- 83.3 ng/ml) were present.

Variations in the distribution of calcium, magnesium and inorganic phosphorus within chronically catheterized sheep conceptuses during the last eight weeks of pregnancy.

The concentrations of calcium, magnesium and inorganic phosphorus were higher in foetal arterial plasma than in maternal jugular plasma in sheep examined between 90 and 145 days of gestation. During the same period the calcium and magnesium concentrations of foetal urine were usually less than amniotic fluid values which in turn were less than maternal plasma concentrations. In allantoic fluid, calcium concentrations were usually less and magnesium concentrations greater than maternal and foetal plasma values. A 2-5 fold increase in the calcium concentrations of allantoic fluid after superfical uterine surgery and in amniotic fluid from a group of foetuses that were exposed during operation, were considered to be artefacts of technique. Inorganic phosphorus concentrations in foetal urine, amniotic fluid and allantoic fluid were variable.