Factors influencing tissue nitrate concentration in field-grown wild rocket (Diplotaxis tenuifolia) in southern England.

Wild rocket (Diplotaxis tenuifolia) is a leafy vegetable known for its high tissue nitrate concentration (TNC) which can exceed the limits set in the relevant European legislation designed to protect human health. The aim of this work was to understand the factors influencing TNC and to develop best practice guidelines to growers. Commercial crops of field-grown wild rocket were studied over two seasons. In 2010, ten separate crops were sampled representing a range of soil types and time periods during the summer. Two fields sampled using a 'W'- or 'X'-shaped sampling pattern demonstrated that 10 incremental samples bulked to make 1 kg of fresh material could be used to provide an adequate sample for determination of TNC in the wild rocket crop, as is the case for other leafy vegetables. Of eight commercial crops sampled in 2010 with an average nitrogen (N) fertiliser application of 104 kg N ha(-1), two exceeded the limit of 6000 mg NO3(-) kg(-1) set in the legislation. In 2011, six N response experiments were carried out, and only two sites showed a significant yield response to N fertiliser. The reason for the lack of response at the other sites was principally due to high levels of soil mineral N prior to drilling, meaning the crops' requirement for N was satisfied without additional fertiliser N. In the experimental situation at an N fertiliser application rate of 120 kg N ha(-1), 50% of crops would have exceeded the 6000 mg NO3(-) kg(-1) limit. In both seasons, low radiation levels in the 5 days prior to harvest were shown to increase TNC, although the relationship was also influenced by N supply. Strategies for optimising N nutrition of field-grown wild rocket are discussed.

Cloning and expression of porcine adiponectin, and its relationship to adiposity, lipogenesis and the acute phase response.

Adiponectin is an adipocyte-derived hormone that has been implicated recently in the regulation of inflammation in immunocytes, and in lipid metabolism and glucose homeostasis in liver, skeletal muscle and adipocytes. However, information in non-rodent models is limited. We have cloned and sequenced the porcine adiponectin open reading frame and evaluated the regulation of adiponectin in vivo following lipopolysaccharide (LPS) or E. coli administration. The porcine sequence shares approximately 88, 86, 85 and 83% homology with the dog, human, cow and mouse adiponectin respectively, and 79-83% similarity with dog, human, cow and mouse proteins at the amino acid level, based on the translated porcine sequence and GenBank submissions for the other species. Relative serum adiponectin concentrations were not altered in pigs infused with E. coli, and mRNA expression in adipose tissue was not responsive to LPS. However, analysis of serum from very lean vs a substantially fatter genotype of pig indicated that relative circulating adiponectin concentrations are higher (P<0.01) in the lean pigs than in the fatter genotype, and that the difference is established relatively early in the growth curve. Also, incubating pig adipocytes for 6 h with recombinant pig adiponectin resulted in an approximately 30% reduction (P<0.05) in lipogenesis compared with adipocytes under basal conditions and with those incubated in the presence of insulin. This is the first report in any species that adiponectin antagonizes the incorporation of glucose carbon into lipid in the adipocyte, and provides additional evidence that adiponectin acts as an autocrine regulatory factor to regulate energy metabolism.

Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: II. Effects on the hypothalamic-pituitary-adrenal axis of weaned pigs.

A study was conducted with 20 weaned barrows (14 d, 4.98 +/- 0.21 kg) to determine the effect of feeding spray-dried plasma (SDP) after weaning on the pig's stress response to a lipopolysaccharide (LPS) challenge. After weaning, pigs were fed a diet containing 0 or 7% SDP for 7 d. On d 6 after weaning, all pigs were nonsurgically fitted with a jugular catheter. On d 7 after weaning, the pigs were given i.p. injections of either saline or LPS (150 microg/kg BW) followed by serial blood collection every 15 min for a 3-h period. Following the 3-h blood collection, all pigs were killed and tissue was collected for mRNA analysis. Pig weight on d 7 after weaning was not affected by dietary treatment (P > 0.21). Pigs fed the diet with SDP had lower (P < 0.05) levels of hypothalamic corticotropin-releasing hormone (CRH) mRNA, pituitary gland CRH receptor mRNA, and adrenal gland adrenocorticotropin-releasing hormone (ACTH) receptor mRNA. Dietary treatment did not affect pituitary gland proopiomelanocortin (POMC) mRNA. No effect of LPS treatment was observed in any of the mRNA levels examined. For both serum ACTH and cortisol, there was a significant diet x LPS treatment interaction (P < 0.01) such that both the ACTH and cortisol responses to the LPS challenge were greater in the pigs fed the diet with SDP than in the pigs fed the diet without SDP. For pigs given the saline injection, diet did not affect basal serum cortisol concentration; however, basal serum ACTH concentration was lower in those pigs fed the diet with SDP (P < 0.0001). A diet x LPS treatment interaction (P < 0.024) was observed for adrenal gland mRNA expression for steroidogenic acute regulatory (StAR) protein such that the LPS-induced increase in StAR mRNA was greater in the pigs fed SDP than in pigs fed the diet without SDP. These results demonstrate that pigs fed a diet with SDP have an increased activation of the pituitary-adrenal axis following an LPS challenge compared to pigs fed a diet without SDP.

Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: I. Effects on the immune axis of weaned pigs.

A study was conducted with 20 weaned barrows (14 d, 4.98 +/- .21 kg) to determine the effect of spray-dried plasma (SDP) on the pig's immune response to a lipopolysaccharide (LPS) challenge. After weaning, pigs were fed a diet containing 0 or 7% SDP for 7 d. On d 6 postweaning, all pigs were fitted with a jugular catheter. On d 7 postweaning, the pigs were given an i.p. injection of either saline or LPS (150 microg/kg BW) followed by a 3-h blood collection every 15 min. Following blood collection, all pigs were killed and tissue was collected for mRNA analysis. Additionally, the small intestine was collected for measurement of villus height, crypt depth, and villus height:crypt depth ratio (VCR) at three sites (25, 50, and 75% of the total length). Feeding SDP resulted in reduced (P < 0.05) mRNA expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mRNA in the adrenal gland, spleen, hypothalamus, pituitary gland, and liver. Additionally, expression of IL-6 mRNA was reduced (P < 0.05) in the spleen and pituitary gland for pigs fed SDP. For pigs fed the diet with SDP, LPS administration did not affect (P > 0.10) cytokine mRNA expression, whereas LPS reduced expression of TNF-alpha mRNA in the spleen and IL-1beta mRNA in the adrenal gland, spleen, and thymus for pigs fed the diet without SDP. For pigs fed the diet with SDP, LPS caused serum TNF-alpha to increase 150-fold compared to a 60-fold increase for pigs fed the diet without SDP. Similarly, interferon-gamma (IFN-gamma) increased 110-fold for pigs fed the diet with SDP compared to a 16-fold increase for pigs fed the diet without SDP. For pigs fed the diet with SDP, LPS caused major villus atrophy, whereas for pigs fed the diet without SDP, LPS had no effect on intestinal morphology. These results demonstrate that the basal activation of the immune system appears to be less for pigs fed the diet with SDP compared to pigs fed the diet without SDP after weaning. Additionally, for pigs fed the diet with SDP, there appeared to be an overresponse of the immune system following LPS administration, which resulted in major damage to the mucosa of the gastrointestinal tract.

Impact of environmental temperature on response of neonatal pigs to an endotoxin challenge.

OBJECTIVE: To evaluate the effect of various environmental temperatures (ET) on the ability of neonatal pigs to cope with an endotoxin challenge. ANIMALS: 28 crossbred male pigs that were 24 hours old. PROCEDURE: At 24 hours of age, pigs were placed in environmentally controlled chambers maintained at 18 or 34 C (14 pigs/ET). Rectal temperatures (RT) were recorded at 15-minute intervals for 3 hours following an IP injection of 0.9% NaCl (7 control pigs/ET) or lipopolysaccharide (LPS; 150 microg/kg of body weight; 7 LPS-treated pigs/ET). Tissue specimens and blood samples were collected following the 3-hour challenge period. RESULTS: LPS-treated pigs exposed to 18 C had a period of hypothermia whereas RT for LPS-treated pigs at 34 C did not differ from control pigs. The LPS-treated pigs maintained at 18 C lost the most body weight during the 3-hour period and also had the greatest increase in serum cortisol concentration. Serum prolactin (PRL) concentration was decreased in pigs at 18 C, compared with pigs at 34 C. Challenge with LPS resulted in an increase in serum PRL concentration at 18 C but had no effect on serum PRL at 34 C. Challenge with LPS resulted in an increase in expression of tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 receptor mRNA in the hypothalamus. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to a cold ET can inhibit the ability of neonatal pigs to cope with an exogenous endotoxin challenge. When combined, cold stress and exposure to exogenous endotoxin induces a rapid and potentially dangerous loss of body temperature in neonatal pigs.

Effects of weaning on somatotrophic gene expression and circulating levels of insulin-like growth factor-1 (IGF-1) and IGF-2 in pigs.

The present study evaluated somatotrophic gene expression in liver, muscle and adipose tissue 4 d after weaning, a time point corresponding to greatly reduced serum concentrations of insulin-like growth factor (IGF)-1 and IGF-2 in pigs. Two-week-old barrows were either cross-fostered to a sow (SOW, n = 8) or weaned and fed a phase 1 diet containing either 0 or 7% spray-dried plasma (NP, n = 8 and SDP, n = 8; respectively). Piglets were allocated such that two size groups were equivalently represented in each experimental group (small, 3.5-4.3 kg and large, 4.6-5.7 kg). Animals were weighed daily and sacrificed 4 d after weaning for blood and tissue collection. Daily gains of the SOW piglets were significantly greater than those of the weaned pigs for the first 3 d of the experiment (P < 0.0001). Weight gains in the SOW and SDP pigs between d 3 and 4 were equivalently elevated relative to the NP pigs (P < 0.0001). Serum IGF-1 and IGF-2 concentrations were decreased in both NP and SDP compared to SOW (P < 0.0001). Serum IGF-2 levels were significantly lower in small piglets (P = 0.006). A Weaning Group X Size interaction was noted for liver IGF-2 mRNA (P < 0.03), reflecting a higher level of expression in large SOW piglets relative to small SOW piglets. Weaning did not affect IGF-1, IGF-2, or growth hormone (GH) receptor mRNA levels in liver, muscle, or fat (P > 0.05). Liver IGF-binding protein (IGFBP)-3 and acid-labile subunit (ALS) mRNA levels also were unaffected by weaning. Small pigs had lower levels of liver ALS (P = 0.0003), muscle IGF-2 (P = 0.02), and muscle GH receptor (P = 0.006) mRNAs. In contrast, adipose tissue IGF-1 and IGF-2 mRNA levels were greatest in the small piglets (P = 0.001 and 0.029, respectively).

Cloning of porcine prepro-orexin cDNA and effects of an intramuscular injection of synthetic porcine orexin-B on feed intake in young pigs.

Early growth is an important determinant of gain and efficiency in growing pigs. A major limiting factor of piglet growth is feed intake. Orexins, newly discovered neuropeptides, may be important regulators of appetite. The orexin gene, which encodes orexin-A and -B, was recently identified in rodents and man. The objectives of this study were to clone the cDNA for porcine orexin, utilize the cDNA sequence information to produce synthetic hormone, and evaluate the effect of orexin administration on feed intake in weanling pigs. Oligonucleotide primers were designed for reverse transcription polymerase chain reaction production of porcine orexin cDNA. The polymerase-chain-reaction products were cloned, sequenced, and found to be 88.5% homologous to the human orexin sequence. Predicted translation of porcine orexin cDNA revealed orexin-A and -B amino acid sequences that were 100% and 96% homologous to the known human peptides, respectively. Porcine orexin-B was synthesized according to the predicted sequence. Twenty-six cross-bred piglets were utilized in three replicates (n = 8-10/replicate). Piglets were weaned between 2-3 wk of age. One week after weaning, equal numbers of animals in each replicate received intramuscular (i.m.) injections of orexin-B (3 mg/kg body weight) or vehicle (sterile water). Feed intake was monitored from -24 to 24 h relative to injection (time 0). The orexin-injected pigs ingested an additional meal at 12 h when compared with the control animals (P = 0.02). Cumulative feed intake was increased by orexin-B administration from 12 to 24 h postinjection (P < or = 0.05). Total feed intake at 24 h was improved by 18% in orexin-treated pigs (P = 0.05). The ability to stimulate appetite during critical periods of early growth, particularly following weaning, could result in significant improvements in swine-production efficiency.

cDNA cloning and tissue-specific gene expression of ovine leptin, NPY-Y1 receptor, and NPY-Y2 receptor.

The physiological regulation of food intake is a critical factor in both the rate at which an animal grows and its reproductive activity. Recently, progress has been made in elucidating a complex system in which insulin, leptin, and neuropeptide Y function to monitor an animal's energy balance and regulate feed intake and fertility. RNA was extracted from ovine hypothalamic, anterior pituitary, pancreas, and adipose tissue. Using the reverse transcription-polymerase chain reaction. cDNAs were cloned and sequenced for leptin (350 base pairs [bp], GenBank accession number U62123 and 441 bp, GenBank accession number U84247), NPY-Y1 receptor (350 bp, GenBank accession no. U62122) and NPY-Y2 receptor (440 bp, GenBank accession no. U83458). Probes generated from these clones were used to detect mRNA expression within tissues thought to be involved in the coregulation of feed intake and reproduction. Leptin was found to be expressed in sheep adipose tissue. The ovine NPY-Y1 receptor mRNA was detected within the arcuate nucleus and paraventricular nucleus of the hypothalamus, the dentate gyrus of the hippocampus and in pancreatic, anterior pituitary, and adipose tissues. Expression of ovine NPY-Y2 receptor mRNA was detected in the hippocampus and within pancreatic tissue. These observations provide evidence of potential mechanisms that exist for mediating communication between peripheral and central tissues within the insulin-leptin-NPY pathway.

Effects of an intravenous injection of NPY on leptin and NPY-Y1 receptor mRNA expression in ovine adipose tissue.

Neuropeptide Y (NPY) is highly expressed in hypothalami of undernourished and genetically obese animals, and is a potent regulator of food intake and reproduction. Leptin, a protein expressed by adipocytes, has been reported to reduce hypothalamic NPY expression. We recently detected (by ribonuclease protection assay [RPA]) expression of the NPY receptor subtype Y1 (but not Y2) mRNA in adipose tissue. Based on these observations we hypothesized that NPY-Y1 receptors in adipose may represent a peripheral mechanism by which NPY can regulate leptin expression in a direct and rapid manner. To test this hypothesis, adipose samples were biopsied from the tailhead region of 48 +/- 3 kg wether lambs immediately before and 30 min after a single intravenous injection of 50 micrograms porcine NPY ("treated" animals, n = 5), or vehicle ("control" animals, n = 4). Injection of NPY resulted in an increase in expression (P = 0.013; as measured by RPA) of both leptin and NPY-Y1 mRNA. In treated animals, negative correlations were found between response in leptin expression and body weight (r = -0.82, P = 0.092), and between leptin response and initial leptin mRNA levels (r = -0.81, P = 0.097). These data provide evidence of a peripheral mechanism by which NPY may regulate adipocyte expression of both leptin and NPY-Y1 receptor mRNA.

Leptin receptor mRNA is expressed in ewe anterior pituitary and adipose tissues and is differentially expressed in hypothalamic regions of well-fed and feed-restricted ewes.

Infertility associated with suboptimal nutrition is a major concern among livestock producers. Recently, much effort has been put into understanding the role of the protein leptin in regulating feed intake and reproduction. Leptin, produced by adipocytes, has receptors in the hypothalamus, but more precise locations of leptin receptor-expressing cell bodies have not been reported in a livestock species. The leptin receptor transcript has several splice variants in the mouse and human, but only the "long-form" product (OBRL) is capable of signal transduction. A partial ovine long-form leptin receptor cDNA was cloned and used to evaluate OBRL mRNA expression within hypothalamic, anterior pituitary, and adipose tissues of ovariectomized adult ewes. Expression was detected in reverse transcription-polymerase chain reaction products of all tissues examined. OBRL mRNA was detected by in situ hybridization in the ventromedial and arcuate nuclei of the hypothalamus. In ewes that had been feed restricted for 3 wk before tissue collection, the expression of OBRL mRNA in these areas was greater (P < 0.05) than that found in well-fed ewes. These findings provide evidence that the full-length leptin receptor is expressed in hypothalamic, anterior pituitary, and adipose tissue (the latter proffering an autoregulatory mechanisms for leptin) and that within the hypothalamus, this receptor form is differentially expressed in well-fed vs. feed-restricted animals.

Sperm motion predicts fertility in male hamsters treated with alpha-chlorohydrin.

An understanding of the relationship between altered sperm motion and sperm function (fertility) is important when interpreting the biological significance of toxicant-induced changes in sperm velocity in rodent test species. Previous studies showed that a brief (4-day) exposure of male hamsters to the model chemical alpha-chlorohydrin (ACH) results in significant deficits in epididymal and uterine sperm velocity, which are associated with both a delay and a failure of fertilization in vivo. To characterize this effect in terms of fertility, similarly treated male hamsters were bred to untreated females and pups were counted the day before parturition. ACH treatment resulted in a dose-dependent decline in the percentage of sperm-positive females that were pregnant at the end of gestation (100, 78, 67, 22, and 0 where males were treated with 0, 33, 49, 66, and 83 mg ACH/kg/day, respectively). Cauda epididymal sperm from the same males were assayed for motion characteristics using computer-assisted sperm analysis (CASA), and for fertilizing ability in vitro. While the percentage of motile sperm was unaffected by ACH treatment, sperm velocity declined in a dose-dependent manner at all ACH treatment levels. Furthermore, the velocity of sperm from infertile males was shifted downward consistently across the entire velocity distribution. Since treated males tended to either be infertile (no pups) or have near normal litter size, the correlation between sperm velocity and litter size was nonlinear. Therefore, logistic regression models using velocity cut-off values were the most useful models for predicting fertility. These results support the contention that fertility relies on there being a sufficient number of sperm that exceed a velocity threshold. Sperm from treated males were also less likely to support in vitro fertilization (IVF), providing further evidence of impaired sperm function associated with acute exposure to ACH.

Method of euthanasia does not affect sperm motility in the laboratory rat.

To determine if anesthetic agents used in laboratory animal euthanasia affected sperm motion parameters, rats (n = 10 per group) were euthanized by one of 5 different methods: decapitation alone, or decapitation following either ether, halothane, or Nembutal anesthesia, or CO2 asphyxiation. Sperm were collected from the distal cauda epididymis, diluted, and videotaped for computer-aided sperm analysis (CASA; HTM-2030, Hamilton-Thorn Research, Beverly, MA). The percentage of motile sperm (MOT), their straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linear index (LINX), and linearity (LIN) were measured on > or = 200 motile sperm per sample. No significant differences in any of these 6 motion parameters were found among the treatment groups. Thus, none of these 5 methods of euthanasia affect sperm motion as assessed by CASA methods, making them equally suitable for use in reproductive toxicology studies.

Spermatotoxicity of dibromoacetic acid in rats after 14 daily exposures.

Halogenated acetic acids are major disinfection by-products of water chlorination and ozonation. Limited data in experimental animals indicate that repeated doses of dichloroacetic acid (DCA) or single doses of dibromoacetic acid (DBAA) cause testicular damage. In the present study, spermatotoxic effects were investigated in rats given oral doses of 0, 10, 30, 90, or 270 mg DBAA/kg/day for 14 days. In rats dosed with 270 mg/kg/day, there were marked effects on epididymal sperm motility and morphology including the flagellar fusion of 2 or more sperm. Testis weight, epididymis weight, and testicular sperm head counts were mildly reduced relative to control, whereas epididymal sperm counts were substantially decreased. Histologic changes in the testis included retention of Step 19 spermatids in Stages IX to XII, abnormal development of late spermatids, and the formation of atypical structures resembling residual bodies that were observed predominantly in Stages X to XIV and I of the cycle of the seminiferous epithelium. At the dosage of 90 mg/kg/day, effects on spermiation, spermatid development, epididymal sperm counts, sperm motility, and sperm morphology were less severe than at the higher dosage. Reduced caput sperm counts and mild effects on spermiation also occurred at 30 and 10 mg/kg/day. These studies indicate that subchronic exposure to DBAA has the potential to affect reproductive outcome in the rat. Compared to previous studies of DCA (12), DBAA, on a molar basis, appears to be a stronger testicular toxicant than the dichloro analogue.

Acute spermatogenic effects of bromoacetic acids.

Chlorine and bromine can react with natural organic substances in source waters to form haloacetic acids, major disinfection by-products of water chlorination. Several toxic effects including testicular damage have been attributed to the chloroacetic acids but little information is available on the bromine analogues. In this report we present the results of acute toxicity and acute spermatotoxicity studies of monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA). In adult male rats the acute oral toxicity of MBAA was 10-fold that of DBAA (LD50 177 vs 1737 mg/kg). No reproductive-related endpoints were affected in rats given a single dose of 100 mg MBAA/kg or 14 daily doses of 25 mg MBAA/kg/day. In rats dosed with DBAA, serum testosterone fell to 17% of control 2 days after a single dose of 1250 mg/kg but returned to control levels by Day 14. Marked effects on sperm motion were seen on post-treatment Days 14 and 28. Degenerative flagellar changes in cauda sperm were present on Day 14 while abnormal sperm head shapes and flagellar degeneration were observed in both caput and cauda sperm on Day 28. Histopathology indicated altered spermiation at all time-points as evidenced by retention of Step 19 spermatids beyond Stage VIII of the cycle of the seminiferous epithelium. Disorganization, distortion, and degeneration of late spermatids were also observed. On Day 14 structures resembling residual bodies were rarely seen in the testis but were numerous in the epididymis. Caput sperm counts were decreased on Day 2 and cauda sperm counts were decreased on Days 14 and 28.(ABSTRACT TRUNCATED AT 250 WORDS)