Non-transferrin bound iron measurement is influenced by chelator concentration

Release of non-protein bound iron plays an important role in the toxicity inflicted by chemotherapy in cancer patients. Since large variations have been described for different methods measuring non-transferrin bound iron (NTBI), we aimed to obtain more accurate values. After binding to the chelator nitrilotriacetic acid disodium salt (NTA) and ultrafiltration, the NTBI can be measured spectrophotometrically by the addition of thioglycolic acid (TGA) and baptophenanthroline disulfonic acid (BPT). Results demonstrated that NTBI values increased with NTA concentration. In samples incubated with 80 mM NTA, >5-fold higher NTBI values were found compared to using 10 mM NTA. Optimal concentration of NTA was established by additions of iron to serum with known latent iron-binding capacity (LIBC). Iron addition curves showed that NTBI could be measured starting from the LIBC of the serum with optimal yield after incubation with 4 mM NTA in 5 mM Tris-HCl pH 6.5, with 3 mM TGA and 6.2 mM BPT for the colour reaction. The results showed excellent correlation with 195 samples measured also by HPLC. For the spectrophotometric method, significantly higher NTBI values were measured in patient samples with maximal iron saturation compared to patients with lower iron saturation.     

Iron loading induces cholesterol synthesis and sensitizes endothelial cells to TNFα-mediated apoptosis

In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non–transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2–mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.     

Pregnancy-associated changes in plasma concentration of the endocrine disruptor di(2-ethylhexyl) phthalate in a sheep model

The plasticizer di(2-ethylhexyl)phthalate (DEHP), used for producing polyvinyl chloride (PVC), acts as an endocrine disruptor with toxic effects on reproductive and developmental processes. Exposure to DEHP in humans is mainly by environment and food. Thus, our aim was to determine plasma levels in livestock animals using the ewe (Ovis aries) as a model. In a first trial, 150 samples from ewes of different ages (2 to 7 yr) and reproductive status (pregnant and nonpregnant) were analyzed by high-performance liquid chromatography (HPLC). DEHP was detected in 34.7% of the samples, with a mean level of 0.45 ± 0.01 μg/mL (range, 0.05 to 2.81 μg/mL). The percentage of nonpregnant animals with DEHP traces was higher in animals older than 4 yr (n = 66, 37.9%) than in younger animals (n = 69, 17.4%; P < 0.05), although the mean levels in ewes with residues were similar (0.16 ± 0.01 vs. 0.16 ± 0.02 μg/mL). All the pregnant ewes (n = 15) showed presence of DEHP, with higher plasma levels than that in nonpregnant females (1.42 ± 0.18 vs. 0.16 ± 0.01 μg/mL; P < 0.0001). For confirming the effect of pregnancy on mobilization of DEHP from body fat, 101 ewes of the same age were sampled in a second trial at a different farm. The percentage of animals with DEHP traces was higher in pregnant ewes (n = 32, 71.9%; P < 0.005) than in nonpregnant ewes (n = 37, 35.1%) or in ewes that recently gave birth (n = 32, 21.9%), although mean levels were similar (0.42 ± 0.02, 0.33 ± 0.02, and 0.34 ± 0.05 μg/mL, respectively). In conclusion, current results indicate a high incidence of ewes reared in the field showing accumulation of phthalates; percentage of animals with presence of DEHP increases with age, due to an extended period of exposure, but mainly during pregnancy, due to the mobilization of body reserves.     

Nontransferrin-bound Iron in Serum of Patients Receiving Bone Marrow Transplants

Nontransferrin-bound iron (NTBI) and other parameters of iron status were measured in 40 patients undergoing bone marrow transplantation (BMT) prior to conditioning therapy (between day −10 and −7), at the time of BMT (day 0), and 2 weeks later (day +14). Serum iron and transferrin saturation values were normal before conditioning therapy. At day 0 serum iron values were high and median transferrin saturation was 98% (changes in the values of both serum iron and transferrin saturation, p < .0001). Transferrin saturation values were still elevated 2 weeks posttransplant (day +14 vs. baseline values, p = .0001). Starting at low NTBI levels pretransplant (median 0.4 , range 0–4.2 , controls: ≤ 0.4 ), all patients revealed high levels on day 0 (median 4.0 , range 1.9–6.9 , p < .0001) and 2 weeks posttransplant (median 2.7 , range 0–6.2 , p < .0001). These observations indicate that the plasma iron pool in patients undergoing BMT increases to a level at which the normal ability to sequestrate iron becomes exhausted and considerable amounts of NTBI appear in serum. This “free” form of iron can mediate the production of reactive oxygen species and may cause organ toxicity in the early posttransplantation period. © 1997 Elsevier Science Inc.     

Nontransferrin-bound serum iron in thalassemia and sickle cell patients

Nontransferrin-bound iron (NTBI) was separated from transferrin bound iron (TBI) by DEAE-Sephadex-CDS filtration. TBI is eluted with Tris-NaCl buffer, NTBI that is retained on the column is eluted with citric acid. NTBI was identified in serum from thalassemia and sickle cell patients. Normal serum contained less than 6% NTBI as compared with 15-18% in patient's sera. NTBI levels were decreased significantly after 8 hr chelation with deferoxamine (DFO).     

Non-transferrin-bound iron in plasma following administration of oral iron drugs

Non-transferrin-bound iron (NTBI) was detected in serum samples from volunteers with normal iron stores or from patients with iron deficiency anaemia after oral application of pharmaceutical iron preparations. Following a 100 mg ferrous iron dosage, NTBI values up to 9 μM were found within the time period of 1–4 h after administration whereas transferrin saturation was clearly below 100%. Smaller iron dosages (10 and 30 mg) gave lower but still measurable NTBI values. The physiological relevance of this finding for patients under iron medication has to be elucidated.     

Diagnostic utility of HFE variants in Spanish patients: Association with HLA alleles and role in susceptibility to acute lymphoblastic leukemia

Two single nucleotide polymorphisms (SNPs) in the Human Hemochromatosis (HFE) gene, C282Y and H63D, are the major variants associated to altered iron status and it is well known that these mutations are in linkage disequilibrium with certain Human Leukocyte Antigen (HLA)-A alleles. In addition, the C282Y SNP has been previously suggested to confer susceptibility to acute lymphoblastic leukemia (ALL). We have aimed to assess the diagnosis utility of these polymorphisms in a population of Spanish subjects with suspicion of hereditary iron overload and to evaluate the effect of their associations with HLA-A alleles on the susceptibility to ALL. Both the 63DD [OR = 4.31 (1.7–11.2)] and 282YY (p for trend = 0.02) genotypes were more frequently found among subjects with suspicion of iron overload than among controls. 282YY carriers displayed significantly higher transferrin saturation index (TSI) values (p < 0.001) as well as serum iron (p = 0.01) and ferritin (p = 0.01) levels. In addition, transferrin levels were lower in these subjects (p = 0.01). Likewise, patients who were carriers of the compound heterozygous diplotype (282CY/63HD) showed significantly higher TSI and serum iron and ferritin concentrations. The H63D SNP did not significantly affect the analytical parameters measured. All 282YY carriers and 69.2% of compound heterozygotes showed an altered biochemical index. The frequencies of the HFE SNPs in ALL pediatric patients were lower than those found in controls, whereas the HLA-A*24 allele was significantly overrepresented in the patients group [OR = 3.76 (1.9–7.3)]. No HFE-HLA-A associations were found to modulate the ALL risk. These results suggest that it may be useful to test for both HFE H63D and C282Y polymorphisms in patients with iron overload, as opposed to just genotyping for the C282Y SNP, which is customary in some healthcare centers. These HFE variants and their associations with HLA-A alleles were not observed to be relevant for the susceptibility to ALL in our population.     

Aluminium and human breast diseases

The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 ± 28 μg/l) compared with control healthy subjects (mean 131 ± 10 μg/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 μg/l) compared with human serum (median 6 μg/l) or human milk (median 25 μg/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate.     

Quantification of non-transferrin-bound iron in the presence of unsaturated transferrin.

Non-transferrin-bound iron (NTBI) has been reported to be associated with several clinical states such as thalassemia, hemochromatosis, and in patients receiving chemotherapy. We have investigated a number of ligands as potential alternatives to nitrilotriacetic acid (NTA) to capture NTBI without chelating transferrin- or ferritin-bound iron in plasma. We have established, however, that NTA is the optimal ligand to chelate the different forms of NTBI present in sera and can be adopted for utilization in the NTBI assay. NTA (80 mM) removes all forms of NTBI, while only mobilizing a small fraction of the iron bound to both transferrin and ferritin. We have compared three different detection systems for the quantification of NTA-chelated NTBI: the established HPLC-based method, a simple colorimetric method, and a method based on inductive conductiometric plasma spectroscopy. The sensitivity and reproductibility of the colorimetric method were acceptable compared with the other two methods and would be more convenient as a routine laboratory screening assay for NTBI. However, the limitations of this method are such that it can only be utilized in situations where desferrioxamine is not used and when transferrin saturation levels are close to 100%. Only the HPLC-based method is applicable for patients receiving (desferrioxamine) chelation therapy. In some diseases such as hemochromatosis, transferrin may be incompletely saturated. In such cases, to avoid in vitro donation of iron onto the vacant sites of transferrin, sodium-tris-carbonatocobaltate(III) can be added to block the free iron binding sites on transferrin. If this step is not taken, there may be an underestimation of NTBI values.     

Effects of low versus physiologic plasma progesterone concentrations on ovarian follicular development and fertility in beef cattle

The objective of this study was to determine the effects of low versus physiologic plasma progesterone concentrations during the ovulatory wave on fertility in cattle. Suckled beef cows (Bos taurus; n = 129) and pubertal heifers (Bos taurus; n = 150) at random stages of the estrous cycle were given a luteolytic dose of prostaglandin F_2α (500 μg cloprostenol; PGF) twice, 11 d apart. Ten days after the second PGF treatment, cattle were given estradiol benzoate im (1.5 and 1.0 mg for cows and heifers, respectively) and a progesterone-releasing intravaginal device (Cue-Mate) with a single pod containing 0.78 g progesterone (Day 0). Cattle in the low-progesterone group (n = 148) received a luteolytic dose of PGF on Day 0, whereas those in the high-progesterone (i.e., physiologic plasma concentrations) group (n = 131) were allowed to retain their corpora lutea. On Day 8, the Cue-Mate was removed, and PGF was given to both groups. Fifty-four hours to 56 h later, cattle received 12.5 mg of porcine LH (pLH) im and were concurrently artificially inseminated. The dominant follicle in the low-progesterone group was larger (P < 0.001) than that in the high-progesterone group on the day of insemination (14.9 ± 0.3 mm vs. 12.7 ± 0.3 mm, mean ± SEM). At 7 d after ovulation, the low-progesterone group had a larger corpus luteum (24.5 ± 0.54 mm vs. 21.9 ± 0.64 mm, P < 0.01) and higher plasma progesterone concentration (4.0 ± 0.3 vs. 3.1 ± 0.2, P < 0.01) than that of the high-progesterone group. However, pregnancy rates did not differ (79 of 148, 53.4%, and 70 of 131, 53.4%) for low- and high-progesterone groups, respectively). In summary, low circulating progesterone concentrations during the growing phase of the ovulatory follicle resulted in a larger dominant follicle and a larger CL that produced more progesterone, with no significant effect on pregnancy rate.     

Interruption of the canine estrous cycle with a low and a high dose of the GnRH antagonist, acyline

To test the efficacy and clinical safety of a low and high dose of the GnRH antagonist, acyline, on estrous cycle interruption and anovulation in female dogs, 20 proestrous (<3 d) bitches were randomly assigned to one of the following pharmacological protocols (given sc): acyline 110 μg/kg (ACY-L; n = 6); acyline 330 μg/kg (ACY-H; n = 8); or placebo (PLACE, n = 6). The animals were monitored (clinical and vaginal cytology examinations) daily for 60 d. Blood samples for serum progesterone serum concentrations were collected 14 d after treatment to determine if ovulation had occurred. Appearance of side effects and days to the onset of the first spontaneous estrous cycle after treatment were also recorded. In both ACY groups, but not the PLACE group, estrous cycles were interrupted after treatment (P < 0.05). The interval from treatment to estrus interruption in ACY-L and ACY-H groups was 3.0 ± 0.6 and 3.2 ± 0.2 d, respectively (LSM ± SEM; P > 0.05). In the PLACE bitches, physical, behavioral and cytological proestrus slowly progressed to estrus and diestrus. Ovulation was absent in all ACY, but not in PLACE bitches (P < 0.05). None of the females manifested side effects related to the treatments (P > 0.05). Spontaneous return to a normal estrous cycle during the study period occurred in all ACY (ACY-L 19.5 ± 2.7 d vs ACY-H 24.8 ± 2.0 d; P > 0.05), but in none of the PLACE bitches (P < 0.05). In conclusion, acyline efficiently, safely and reversibly interrupted an early phase of the estrous cycle in bitches by preventing ovulation.     

Comparative effects of cationic triarylmethane, phenoxazine and phenothiazine dyes on horse serum butyrylcholinesterase

The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25 ^°C in 50 mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K_i values of 4.5 ± 0.50 μM, 0.41 ± 0.007 μM, 0.44 ± 0.086 μM and 0.050 ± 0.0074 μM. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K′ values (≡[I]²_0.5 extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20 ± 0.096 μM, 0.0018 ± 0.0015 μM, 0.92 ± 0.23 μM and 0.23 ± 0.08 μM. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features.     

Effect of feed restriction or feeding high-fibre diet during the rearing period on body composition, serum parameters and productive performance of rabbit does

This study evaluated the effect of different feeding regimes from 11 weeks of age to first parturition on feed intake, leptin, non-esterified fatty acids (NEFA) and total protein serum levels, as well as productive performance in young rabbit does. In addition, body composition was estimated by bioimpedance analysis. Thirty-six 11-week-old does were randomly distributed in three groups. The AL-C group was fed ad libitum a control diet containing 350 g neutral detergent fibre (aNDFom)/kg, 11.6 MJ digestible energy (DE)/kg and 173 g crude protein (CP)/kg, and the does were inseminated at 16 weeks of age. The R-C group was fed 150 g/d of the same diet until 16 weeks of age, one week before artificial insemination (AI) at 17 weeks of age, and then fed ad libitum. The AL-F group was fed a diet containing 475 g aNDFom/kg, 9.4 MJ DE/kg and 174 g CP/kg ad libitum, and was inseminated at 17 weeks of age. During rearing (11-16 weeks), does in the R-C group had the lowest DE (1.54 MJ/d; P<0.003) and digestible protein (DP, 17.9 g/d; P<0.001) intake, as well as the lowest protein (172 g/kg; P<0.05) and energy (5.9 MJ/kg) body contents, leptin concentration at 16 weeks of age (2.48 ng/ml; P<0.001) and fertility (P<0.02) at first AI. Daily feed intake during pregnancy and lactation, as well as prolificacy and litter weight, were similar among groups. The highest percentage of body fat was observed for all the does when were inseminated for the first time (135 g/kg; P<0.001), consistent with the highest leptin (4.48 ng/ml; P<0.001) and total protein serum levels (6.87 g/dl; P<0.001) at this time. Serum NEFA level around parturition was higher (P<0.05) in groups AL-C and R-C (1.11 and 0.85 mmol/l) than in group AL-F (0.71 mmol/l), suggesting a lower lipid mobilization that tended to improve fertility rate for AL-F does on day 11 post-parturition (P<0.09). In conclusion, feed restriction during the rearing period delays reproductive development in young rabbits. In nulliparous does, ad libitum feeding during rearing with a high-fibre diet allows a similar productive performance to that of feeding with a less fibrous diet. Nevertheless, the use of high fibre diets during rearing does not affect feed intake throughout the first pregnancy and lactation.     

Ovarian and endocrine responses in tropical sheep treated with reduced doses of cloprostenol

The present study aimed to assess the efficacy of reduced doses of cloprostenol for synchronizing estrus and ovulation in hair sheep. With the aim to evaluate the luteolytic activity of reduced cloprostenol doses, a first experiment was performed using a relatively large (group H: 126 μg; n = 8), medium (group M: 68.25 μg; n = 6) and small (group L: 38.5 μg; n = 6) cloprostenol dose. Luteolysis was assessed at Days 3 and 6 after injection (Day 0) by progesterone concentrations (P₄) and transrectal ultrasonography (US). In Experiment 2, sheep were randomly assigned to the same three doses to evaluate a protocol for estrous synchronization using two injections administered 9 days apart. A third trial was performed with ewes treated (9 days apart) with the large dose (H = 126 μg; n = 12) and with a small dose adjusted for facilitating volume management (LA = 43.75 μg; n = 12). Presence of estrous cycling was determined in all the ewes by US and P₄ assay, at Days −9, −6, −2, 0 (Day of second cloprostenol injection), 8 and 11. Bleeding and US were done every 4 h from 16 h of the beginning of the estrus during the third trial to assess the preovulatory LH surge and timing of ovulation. Additionally, blood samples were drawn at Days 0, 1, 2 and 3 to assess estradiol (Experiments 2 and 3) and P₄ (Experiment 2) concentrations during the ovarian follicular phase. In all experiments, percentage of animals showing luteolysis, preovulatory follicular dynamics and function and percentage of ewes showing behavioral estrus in response to treatment was similar among groups. Timing of estrus for group H was earlier than group L (28.6 ± 1.8 h compared with 37.1 ± 2.4 h; P < 0.05). In the third trial, the preovulatory LH peak was higher in the LA group than group H, in terms of maximum mean concentration during the surge (27.7 ± 1.8 ng/mL compared with 21.3 ± 2.2 ng/mL; P < 0.05) and area under the curve (AUC; 183.4 ± 12.7 ng/mL compared with 127.7 ± 10.9 ng/mL; P < 0.01). However, timing of ovulation was similar for H and LA groups. Thereafter, ovulation rate and luteal function at Day 11 were similar. Current results demonstrate that reduced doses of cloprostenol may be applied in a practical manner for reproductive management of sheep, with the additional advantage of reducing treatment costs.     

Photonic characteristics and ex vivo imaging of Escherichia coli-Xen14 within the bovine reproductive tract

The objectives of this study were to (1) characterize the photonic properties of Escherichia coli-Xen14 and (2) conduct photonic imaging of E. coli-Xen14 within bovine reproductive tract segments (RTS) ex vivo (Bos indicus). E. coli-Xen14 was grown for 24 h in Luria Bertani medium (LB), with or without kanamycin (KAN). Every 24 h, for an 8-d interval, inoculums were imaged and photonic emissions (PE) collected. Inoculums were subcultured and plated daily to determine the colony forming units (CFU) and ratio of photon emitters to nonemitters. In the second objective, abattoir-derived bovine reproductive tracts (n = 9) were separated into posterior and anterior vagina, cervix, uterine body, and uterine horns. Two concentrations (3.2 × 10⁸ and 3.2 × 10⁶ CFU/200 μL for relative [High] and [Low], respectively) of E. coli-Xen14 were placed in translucent tubes for detection of PE through RTS. The CFU did not differ (P = 0.31) over time with or without KAN presence; they remained stable with 99.93% and 99.98% photon emitters, respectively. However, PE were lower (P < 0.0001) in cultures containing KAN than in those containing no KAN (629.8 ± 117.7 vs. 3012.0 ± 423.5 relative lights units per second [RLU/sec], respectively). On average, the percentage of PE between RTS, for both concentrations, was higher (P < 0.05) in the uterine body. In summary, E. coli-Xen14 remained stable with respect to the proportions of photon emitters with or without KAN (used to selectively culture E. coli-Xen14). However, KAN presence suppressed photonic activity. The ability to detect PE through various segments of the reproductive tract demonstrated the feasibility of monitoring the presence of E. coli-Xen14 in the bovine reproductive tract ex vivo.     

Non-Transferrin-Bound Iron (NTBI) Uptake by T Lymphocytes: Evidence for the Selective Acquisition of Oligomeric Ferric Citrate Species

Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI). NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.     

Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows

Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TAI) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2 × 2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5 ± 2.3 d postpartum, received a 3 mg norgestomet ear implant sc, plus 3 mg norgestomet and 5 mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54 h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100 μg gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means ± SEM, 1.53 ± 0.1 vs. 0.48 ± 0.1 mm/d; P < 0.0001), its diameter on Day 11 (11.4 ± 0.6 vs. 9.3 ± 0.7 mm; P = 0.03), as well as ovulation rate (80.8% vs. 50.0%, P = 0.02), whereas GnRH improved the synchrony of ovulation (72.0 ± 1.1 vs. 71.1 ± 2.0 h). In Experiment 2 (n = 599 cows, 40 to 120 d postpartum), pregnancy rates differed (P = 0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control, GnRH, eCG, and eCG + GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 33.8%, P = 0.002; and 48.0% vs 37.6%, P = 0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol.     

Increased whole blood manganese concentrations observed in children with iron deficiency anaemia

A prospective observational study was carried out at Alder Hey Children's Hospital, Liverpool, England, UK on children aged 1–6 years attending the pathology department for routine blood tests (n = 225). Whole blood manganese concentrations were measured plus the following markers of iron status; haemoglobin, MCV, MCH, RBC count, ferritin, transferrin saturation and soluble transferrin receptors. Multiple regression analysis was performed, with blood manganese as the dependent variable and factors of iron status, age and gender as independent variables. A strong relationship between blood manganese and iron deficiency was demonstrated (adjusted R² = 34.3%, p < 0.001) and the primary contributing factors to this relationship were haematological indices and soluble transferrin receptors. Subjects were categorised according to iron status using serum ferritin, transferrin saturation and haemoglobin indices. Children with iron deficiency anaemia had higher median blood manganese concentrations (16.4 μg/L, range 11.7–42.4, n = 20) than children with iron sufficiency (11 μg/L, range 5.9–20.9, n = 59, p < 0.001). This suggests that children with iron deficiency anaemia may be at risk from manganese toxicity (whole blood manganese >20 μg/L), and that this may lead to neurological problems. Treatment of iron deficiency in children is important both to improve iron status and to reduce the risk of manganese toxicity.     

Endocrine effects of the GnRH antagonist, acyline, in domestic dogs

Gonadotrophin-releasing hormone (GnRH) antagonists may have a future role in the control of canine reproductive function. In this study, the effects of a single dose of the potent GnRH antagonist, acyline, on serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were evaluated in male dogs. Blood samples were drawn before (Day −1) and after (30, 60, and 90 min, 3, 6, 9, 12, and 24 h, and 3, 6, 9, 14, 22, and 29 d) treatment with acyline (330 μg/kg, sc); serum concentrations of FSH, LH, and T varied throughout the study period (P < 0.01, <0.05, and <0.01, respectively). Gonadotrophins decreased below pretreatment concentrations 60 min after injection, whereas T took 90 min to decrease below baseline (P > 0.05). Follicle-stimulating hormone, LH and T decreased until Day 9, when they reached their nadir at 2.0 ±1.1 ng/mL (P < 0.01), 1.2 ± 0.2 ng/mL (P > 0.05), and 0.5 ± 0.2 ng/mL (P < 0.05), respectively. Both gonadotrophins and T began increasing on Day 14 after treatment, although FSH and T serum concentrations still remained below baseline on that day (P > 0.05). Follicle-stimulating hormone and T rebounded above baseline on Day 29, whereas LH reached concentrations were similar to baseline at this time (P > 0.05). No local or systemic side effects were detected in any dog following acyline treatment. In conclusion, a single acyline treatment safely and reversibly decreased serum gonadotrophin and T concentrations in dogs for 9 d.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.