Evaluation of a bovine visual pregnancy test for the detection of pregnancy-associated glycoproteins in sheep.

As sheep produce similar pregnancy-associated glycoproteins (PAGs) to cattle, a commercially available bovine visual pregnancy test based on the detection of PAGs (visual-PAG-test) was evaluated in sheep. The test was performed with whole blood (WhB), plasma (P) and serum (S) of 163 pregnant and 153 non-pregnant ewes. Additionally, 11 pregnant ewes were tested weekly from day 14 to 49 of gestation and monthly from day 60 to day 149. Ten ewes were sampled weekly from the date of lambing until day 63 post-partum (p.p.). The sensitivity in mid-pregnancy (n = 163) was 98.16% (WhB), 99.39% (P) and 99.39% (S) compared to transabdominal ultrasonography as the gold standard while the specificity (n = 153) was 94.12% (WhB), 76.47% (P) and 93.46% (S), respectively. During early pregnancy, all 11 ewes were correctly identified as pregnant on day 42 (100%); however, the test sensitivity decreased to 54.6% (WhB) and 63.6% (S and P, respectively) at day 49. The ewes were again consistently identified as pregnant on day 63 (P) or on day 119 (S, WhB). The test was consistently negative from day 42 p.p. onwards in eight out of ten ewes. Two ewes remained consistently positive until the last sample on day 63 p.p. In conclusion, the test could be used to accurately select pregnant ewes at day 42 with a drop in sensitivity at day 49. The sensitivity of the visual-PAG-test was good in mid to late pregnancy, and early detection of pregnancy was possible in individual animals. In some ewes, the PAGs were however detectable for more than 63 days p.p.-the previous breeding history should therefore always be taken into account for correct interpretation of the test results.

Correlation of two radioimmunoassay systems for measuring plasma pregnancy-associated glycoproteins concentrations during early pregnancy and postpartum periods in water buffalo.

This is the first time that PAG determination using two different antisera raised against PAG molecules purified from both caprine (RIA-706) and bubaline placentas (RIA-860) is reported in water buffalo. Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) programme, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and 45. Pregnancy was confirmed by ultrasonography on days 28 and 45. The blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 postcalving. Differences in PAG concentrations were observed between pregnant and nonpregnant buffaloes starting from day 23 post AI using both RIA-706 and RIA-860 (p < 0.001). However, estimated mean concentrations of PAG measured by RIA-706 were higher than RIA-860 (p < 0.001) and Bland-Altman analysis showed biases ranged from 0.0 ng/ml at day 23 to 0.79 ng/ml at day 28 post AI. Moreover, RIA-706 showed greater sensitivity and accuracy both at 23 and 25 days of pregnancy. RIA-706 and RIA-860 decreased below 1 ng/ml from 40 and 30 days postpartum, respectively, suggesting that PAG are better recognized by the antisera raised against the caprine PAG in the postpartum period also. This is essential when using PAG as an appropriate marker of early pregnancy after postpartum for detecting new pregnancies. The results of this study show that the ability of RIA systems to recognize early PAG could be improved using antisera raised against PAG molecules isolated from caprine placenta.

REPRODUCTIVE HORMONE PATTERNS IN MALE AND FEMALE COWNOSE RAYS ( RHINOPTERA BONASUS) IN AN AQUARIUM SETTING AND CORRELATION TO ULTRASONOGRAPHIC STAGING.

Reproductive management of cownose rays ( Rhinoptera bonasus) under professional care plays an important role in conservation of the species, but hormone and ultrasonographic analyses of their 12-mo reproductive cycle have not been documented previously. Plasma reproductive hormone concentrations (17B-estradiol, progesterone, testosterone, and androstenedione) were measured monthly via radioimmunoassay for 1 yr in an aquarium-managed population of adult females ( n = 15) and males ( n = 5). Ultrasounds of the uterus were performed each month at the time of sample collection to identify gestation stage (0-5) based on a previously developed in-house staging system. Stages were correlated to hormone concentrations to track progression through pregnancy. Thirteen females were reproductively active, and each produced one pup in March-June, similar to timing for free-ranging populations. Female estradiol increased steadily throughout gestation from stages 0 to 5, while progesterone, testosterone, and androstenedione were increased only in early gestation (stages 1 and 2). Unlike month of year, gestation stage strongly predicted hormone concentration, but specific values to predict parturition date were not identified. Male testosterone and progesterone were higher in March-June (mating season) than July-January, while estradiol and androstenedione did not exhibit a seasonal pattern. Aquarium-managed cownose rays have similar reproductive patterns to what is reported in wild populations. Ultrasonographic monitoring with serial hormone analysis and accurate mating records will provide the most useful information for managing a reproductive population of cownose rays in an aquarium setting.

Diagnostic performance of the urinary canine calgranulins in dogs with lower urinary or urogenital tract carcinoma.

BACKGROUND: Onset of canine transitional cell carcinoma (TCC) and prostatic carcinoma (PCA) is usually insidious with dogs presenting at an advanced stage of the disease. A biomarker that can facilitate early detection of TCC/PCA and improve patient survival would be useful. S100A8/A9 (calgranulin A/B or calprotectin) and S100A12 (calgranulin C) are expressed by cells of the innate immune system and are associated with several inflammatory disorders. S100A8/A9 is also expressed by epithelial cells after malignant transformation and is involved in the regulation of cell proliferation and metastasis. S100A8/A9 is up-regulated in human PCA and TCC, whereas the results for S100A12 have been ambiguous. Also, the urine S100A8/A9-to-S100A12 ratio (uCalR) may have potential as a marker for canine TCC/PCA. Aim of the study was to evaluate the diagnostic accuracy of the urinary S100/calgranulins to detect TCC/PCA in dogs by using data and urine samples from 164 dogs with TCC/PCA, non-neoplastic urinary tract disease, other neoplasms, or urinary tract infections, and 75 healthy controls (nested case-control study). Urine S100A8/A9 and S100A12 (measured by species-specific radioimmunoassays and normalized against urine specific gravity [S100A8/A9USG; S100A12USG], urine creatinine concentration, and urine protein concentration and the uCalR were compared among the groups of dogs. RESULTS: S100A8/A9USG had the highest sensitivity (96%) and specificity (66%) to detect TCC/PCA, with specificity reaching 75% after excluding dogs with a urinary tract infection. The uCalR best distinguished dogs with TCC/PCA from dogs with a urinary tract infection (sensitivity: 91%, specificity: 60%). Using a S100A8/A9USG ≥ 109.9 to screen dogs ≥6 years of age for TCC/PCA yielded a negative predictive value of 100%. CONCLUSIONS: S100A8/A9USG and uCalR may have utility for diagnosing TCC/PCA in dogs, and S100A8/A9USG may be a good screening test for canine TCC/PCA.

Comparison of three different methods for the quantification of equine insulin.

BACKGROUND: Exact analysis of equine insulin in blood samples is the key element for assessing insulin resistance or insulin dysregulation in horses. However, previous studies indicated marked differences in insulin concentrations obtained from sample analyses with different immunoassays. Most assays used in veterinary medicine are originally designed for use in human diagnostics and are based on antibodies directed against human insulin, although amino acid sequences between equine and human insulin differ. Species-specific assays are being used more frequently and seem to provide advantages compared to human-specific assays. The aim of this study was to compare three immunoassays, one porcine-specific insulin enzyme-linked immunosorbent assay (ELISA), advertised to be specific for equine insulin, one porcine-specific insulin radioimmunoassay (RIA) and one human-specific insulin chemiluminescence immunoassay (CLIA), all three widely used in veterinary laboratories for the analysis of equine insulin. Furthermore, we tested their clinical applicability in assessing insulin resistance and dysregulation by analysis of basal blood and blood samples obtained during a dynamic diagnostic stimulation test (OGT) with elevated insulin concentrations. RESULTS: Insulin values obtained from the ELISA, RIA and CLIA, investigated for analyses of basal blood samples differed significantly between all three assays. Analyses of samples obtained during dynamic diagnostic stimulation testing with consecutively higher insulin concentrations revealed significantly (p < 0.001) lower insulin concentrations supplied by the CLIA compared to the ELISA. However, values measured by ELISA were intermediate and not different to those measured by RIA. Calculated recovery upon dilution, as a marker for assay accuracy in diluted samples, was 98 ± 4 % for ELISA, 160 ± 41 % for RIA and 101 ± 11 % for CLIA. CONCLUSIONS: Our results indicate that insulin concentrations of one sample measured by different methods vary greatly and should be interpreted carefully. Consideration of the immunoassay method and reliable assay-specific reference ranges are of particular importance especially in clinical cases where small changes in insulin levels can cause false classification in terms of insulin sensitivity of horses and ponies.

Effects of acute and chronic heat stress on plasma metabolites, hormones and oxidant status in restrictedly fed broiler breeders.

Heat tolerance can be improved by feed restriction in broiler chickens. It is unknown whether the same is true for broiler breeders, which are restrictedly fed. Therefore, the current study was conducted to study the effects of heat stress on plasma metabolites, hormones, and oxidative status of restricted fed broiler breeders with special emphases on the temperature and latency of heat exposure. In trial 1, 12 broiler breeders were kept either in a thermoneutral chamber (21°C, control, n = 6) or in a chamber with a step-wise increased environmental temperature from 21 to 33°C (21, 25, 29, 33°C, heat-stressed, n = 6). Changes in plasma total cholesterol, glucose, and triiodothyronine (T3) were closely related to the environmental temperature. When the temperature reached 29°C, plasma T3 (P < 0.05) was significantly decreased in acute heat-stressed birds, whereas plasma glucose (P < 0.001) and cholesterol (P = 0.002) increased only when the temperature reached 33°C. Plasma triglyceride (P = 0.026) and creatine kinase (CK, P = 0.018) were lower in heat-stressed birds than controls regardless of the temperatures applied. In Trial 2, 24 broiler breeders were divided into 2 groups and raised under 21°C and 32°C for 8 weeks, respectively. Total cholesterol was increased in chronic heat-stressed broiler breeders after 4 weeks. Plasma lactate dehydrogenase (LDH, P = 0.047) and glutamic-oxaloacetic transaminase (GOT, P = 0.036) was up-regulated after 6 weeks of thermal treatment, whereas plasma CK (P = 0.009) was increased at the end of thermal treatment. Plasma malonaldehyde, protein carbonyl content, activity of total superoxide dismutase (SOD), and corticosterone content were not altered after acute and prolonged heat challenges. Taken together, acute heat stress primarily resulted in disturbance of plasma metabolites, whereas chronic heat stress caused tissue damage reflected by increased plasma LDA, GOT, and CK. During acute heat stress, plasma metabolites were minimally disturbed in broiler breeders until the environmental temperature reached 33°C.

The onset of stress response in rainbow trout Oncorhynchus mykiss embryos subjected to density and handling.

The present study made an attempt to measure the cortisol content, as an indicator of stress response, in rainbow trout embryos which were exposed to different densities and handling stress (air exposure) during incubation. The three densities of experimental embryos at early development stages were considered as 2.55 embryos/cm(2) (low density), 5.10 embryos/cm(2) (normal density) and 7.65 embryos/cm(2) (high density). The cortisol content of eggs (5.09 ± 0.12 ng/g) decreased to 3.68 ± 0.14 ng/g in newly fertilized eggs. Resting level of cortisol dropped at three densities by day 18 of post fertilization. Then, cortisol increased at hatching stage to 1.16 ± 0.11, 1.20 ± 0.12 and 1.21 ± 0.14 ng/g at low, normal and high densities, respectively. There were no statistically significant differences between cortisol concentrations in three densities. The acute handling stress test (5-min out-of-water), conducted on embryos (48 h post fertilization, organogenesis and eyed stage) in three densities, revealed no differences in whole-body cortisol levels between stressed and unstressed experimental groups. At hatching stage in low-density group, level of cortisol increased but the difference with the pre-stress levels was not statistically significant. Furthermore, significant differences in cortisol levels of stressed and unstressed embryos were detected on hatching in normal and high density groups [1.20 ± 0.12 at time 0-1.49 ± 0.11 ng/g at 1 hps (hours post stress) and from 1.21 ± 0.14 at time 0 to 1.53 ± 0.10 ng/g at 3 hps, respectively]. The results showed no difference in profile of cortisol in different densities, but acute stress conducted on embryos, incubated in different densities, revealed differences in cortisol stress response at hatching between normal and high density, which lead to cortisol increase at hatching time. It indicates that the lag time in the cortisol response to stressors immediately after hatching does not occur when the siblings were stressed during the embryo stage. Results, finally, indicated that hypothalamus-pituitary-interrenal axis was active and responded to an acute stressor under normal and high density, but it is unresponsive to a stressor around hatching under low density.

Influence of breeding season on fecal glucocorticoid levels in captive Greater Rhea (Rhea americana).

Sex hormones and stress-related changes can be seasonally influenced. We investigate whether fecal glucocorticoid metabolite (FGM) levels can differ between male and female captive Greater Rheas during the breeding and non-breeding seasons. Over a 3-year-period, fresh fecal samples from 10 individuals (five of each sex) were collected during the breeding months (October, November, and December) and non-breeding months (April and June). A total of 960 samples were assayed using a commercial radioimmunoassay. Results showed that FGM levels (mean ± SE) were affected by the breeding season in a sex-dependent way. Male Greater Rheas showed significantly higher FGM levels in the breeding months than in the non-breeding months (13.44 ± 0.37 vs. 7.92 ± 0.1 ng/g feces, respectively). By contrast, females did not show FGM seasonal changes throughout the same sampling periods (7.55 ± 0.14 vs. 7.26 ± 0.73 ng/g feces). Moreover, during the breeding season months, males showed higher average FGM levels than females (13.44 ± 0.37 vs. 7.55 ± 0.14 ng/g feces, respectively), and no differences were found between sexes during the non-breeding season (7.92 ± 0.1 vs. 7.26 ± 0.73 ng/g feces, respectively). Our findings suggest that male Greater Rheas have a higher adrenocortical activity during the breeding season, which is probably indirectly related to the increased testosterone levels and agonist interactions that are also observed during that phase. Studies aimed to determine the appropriate sex ratio for captive rearing should be developed to minimize male agonist encounters and therefore improve welfare of the captive group.

A polymorphism in XKR4 is significantly associated with serum prolactin concentrations in beef cows grazing tall fescue.

Fescue toxicosis is a common syndrome of poor growth and reproductive performance of beef cattle grazing endophyte-infected tall fescue infected with Lolium arundinaceum Schreb. Together with decreased feed intake, decreased growth rates and tissue necrosis due to vasoconstriction, depressed circulating serum prolactin concentrations are typically observed in cattle afflicted with fescue toxicosis. Polymorphisms within the XK, Kell blood group complex subunit-related family, member 4 (XKR4) gene located on BTA14 have been previously reported to be associated with rump fat thickness, residual feed intake, average daily feed intake and average daily gain in cattle. Associations also have been reported between XKR4 genotype and effectiveness of the dopamine antagonist iloperidone as a treatment of schizophrenia in humans. Domperidone, a related dopamine antagonist, mediates effects of fescue toxicosis on livestock, including restoring depressed concentrations of prolactin. A mixed-breed population of 592 beef cattle grazing endophyte-infected tall fescue was used to examine the association between XKR4 genotype and circulating prolactin concentrations. The SNP rs42646708 was significantly (P = 0.0002) associated with serum prolactin concentrations and explained 2.45% of the phenotypic variation. Effect of genotype at the SNP was tested across five breeds, with significant associations within both Angus (P = 0.0275) and Simmental (P = 0.0224) breeds. These results suggest XKR4 may play a role in mediating the negative effects of fescue toxicosis, and polymorphisms within this gene may be useful markers for selection for genetic resistance to the debilitating effects of endophyte-infected tall fescue.

The effect of perch access during pullet rearing and egg laying on physiological measures of stress in White Leghorns at 71 weeks of age.

Egg laying strains of chickens have a strong motivation to perch. Providing caged chickens with perches allows them to perform their natural perching behavior and also improves their musculoskeletal health due to exercise. Little is known about the effect of perch access for hens on physiological measures of stress. Our hypothesis was that denying chickens access to perches would elicit a stress response. The objective of this study was to determine the effect of perch access during all or part of life cycle on physiological homeostasis in caged 71-wk-old White Leghorn hens. A total of 1,064 chicks were assigned randomly to cages with and without perches (n = 14 pullet cages/perch treatment) on day of hatch. As pullets aged, chicks were removed from cages to provide more space. At 17 wk of age, 324 chickens in total were assigned to laying cages consisting of 4 treatments with 9 replicates per treatment. Treatment 1 chickens never had access to perches during their life cycle. Treatment 2 chickens had access to perches only from 17 to 71 wk of age (laying phase). Treatment 3 chickens had access to perches only from hatch to 16.9 wk of age (pullet phase). Treatment 4 chickens always had access to perches during their life cycle. At 71 wk of age, chickens were sampled for measurement of plasma catecholamines (epinephrine, norepinephrine, and dopamine) and corticosterone; blood serotonin and Trp; fluctuating asymmetry of shank length and width; and adrenal weight. Only shank width differed among treatments. Chickens with previous exposure to perches during the pullet phase had wider shanks than chickens without access to perches (P = 0.006), suggesting that early perching promoted skeletal development. These results suggest that a stress response was not elicited in 71-wk-old White Leghorn hens that always had access to perches compared with hens that never had access to perches during all or part of their life cycle.

Stress mitigating and growth enhancing effect of dietary tryptophan in rohu (Labeo rohita, Hamilton, 1822) fingerlings.

An experiment was conducted to study the stress mitigation and growth enhancing role of dietary L-tryptophan (TRP) under thermal stress in rohu, Labeo rohita fingerlings for 45 days. Seven hundred and twenty fishes were distributed in three major groups that are ambient temperature (26 °C), 34 and 38 °C in triplicate following a complete randomized design. Acclimation of fishes to 34 and 38 °C over average ambient temperatures were carried out at 1 °C/day. Each group was fed with a diet supplemented with 0, 0.36, 0.72 or 1.42 % L-TRP. Results showed that blood glucose and serum cortisol level were found to be significantly higher (p < 0.05) in the higher temperature groups than the ambient temperature group. Similarly, aminotransferase, lactate dehydrogenase, malate dehydrogenase, CAT, superoxide dismutase activities were found to be significantly higher (p < 0.05) in the control groups (0 % L-TRP) and decreasing activities of these enzymes were observed with the increasing level of dietary L-TRP. In different temperature groups, L-TRP-supplemented groups were found to have higher (p < 0.05) growth, RGR and PER. The results obtained in the present study indicate that dietary L-TRP mitigates thermal stress and enhances growth. From the present study, we can conclude that dietary supplementation of L-TRP at the 0.72 % level in the diet is found to be optimum to reduce thermal stress even up to 38 °C in rohu, L. rohita. The baseline data obtained here could be useful for the farmers to formulate feeds to culture the fish in different agro-climatic zones.

Profiles of thyrotropin, thyroid hormones, follicular cells and type I deiodinase gene expression during ontogenetic development of tilapia larvae and juveniles.

The aims of the present study are to determine whether triiodothyronine (T3) and/or thyroxine (T4) in tilapia larvae is gifted through the mother, and to investigate the change profiles of thyrotropin (TSH), thyroid follicular cells and type I deiodinase (D1) gene expression following larval development. T3 and T4 contents were measured using radioimmunoassay, thyrotropin was observed using immunocytochemistry, and the D1 gene was cloned and measured using real-time PCR. Results indicated that the ß-TSH-immunoreactive cells (thyrotropin ICC) signals were detected at 9 dph (i.e., 9 days of post-hatching). Thyroid follicular cells were observed first at 3 dph, while the T3 contents of the whole body gradually decreased before 11 dph. T4 contents were detected until 13 dph, with higher secretion during 19-21 dph. In addition, the T3 synthesis was not inhibited by thiourea (TU) before 13 dph, but the TU response in the larvae appeared after 13 dph. Type I deiodinase (D1: GenBank accession number KC591724) was found to contain 2444 bases and encoded 248 amino acids. The D1 mRNA expression began to increase at 13 dph, with a higher expression during 15-19 dph. These results suggested that the T3 contents were maternally derived before 13 dph. Both thyroid hormonal changes and some parameters related to thyroid hormone synthesis in ontogenetic tilapia are discussed.

Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females.

BACKGROUND: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species. RESULTS: Amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n=33) and non-pregnant (n=26) water buffalo females. CONCLUSIONS: Our results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

Genotype and fetal size affect maternal-fetal amino acid status and fetal endocrinology in Large White × Landrace and Meishan pigs.

This study compared maternal plasma amino acid concentrations, placental protein secretion in vitro and fetal body composition and plasma amino acid and hormone concentrations in feto-placental units from the smallest and a normally-sized fetus carried by Large White × Landrace or Meishan gilts on Day 100 of pregnancy. Compared with Large White × Landrace, Meishan placental tissue secreted more protein and Meishan fetuses contained relatively more fat and protein, but less moisture. Fetal plasma concentrations of insulin, triiodothryonine, thyroxine and insulin-like growth factor (IGF)-II were higher in Meishan than Large White × Landrace fetuses. In both breeds, fetal cortisol concentrations were inversely related to fetal size, whereas concentrations of IGF-I were higher in average-sized fetuses. Concentrations of 10 amino acids were higher in Large White × Landrace than Meishan gilts, while glutamine concentrations were higher in Meishan gilts. Concentrations of alanine, aspartic acid, glutamic acid and threonine were higher in Meishan than Large White × Landrace fetuses. Average-sized fetuses had higher concentrations of asparagine, leucine, lysine, phenylalanine, threonine, tyrosine and valine than the smallest fetus. This study revealed novel genotype and fetal size differences in porcine maternal-fetal amino acid status and fetal hormone and metabolite concentrations.

Short communication: hair cortisol concentrations in Holstein-Friesian and crossbreed F1 heifers.

The aim of this study was to evaluate the hypothalamic-pituitary-adrenal (HPA) axis activity of Holstein-Friesian and crossbreed F1 heifers by analysis of the cortisol concentrations in hair samples. Cortisol, the primary hormone of the HPA axis, is the biological endpoint for the investigation of the HPA response. The study was conducted on 290 prepubertal heifers; 142 heifers were pure Holstein-Friesian and 148 were crossbreed F1 heifers obtained from the 3-way rotational system with Swedish Red and Montbéliarde breeds. Extraction was performed on the hair using methanol, and cortisol concentrations were determined by a radioimmunoassay method. Cortisol concentrations measured in regrown hair of crossbreed F1 heifers were significantly lower than those in hair of Holstein-Friesian heifers. This result helps us to better understand the differences in HPA activity and allostatic load between Holstein-Friesian and crossbreed F1 heifers and allows us to better assess the adaptability of these animals to the environment and the importance of crossbreed traits for profitability in dairy farming.

Plasma concentrations of pregnancy-associated glycoproteins measured using anti-bovine PAG-2 antibodies on day 120 of gestation predict abortion in dairy cows naturally infected with Neospora caninum.

The present study sought to determine: (i) the effects of Neospora caninum infection and twin pregnancy on plasma pregnancy-associated glycoprotein-2 (PAG-2) concentrations throughout pregnancy and (ii) whether plasma PAG-2 concentrations could predict abortion in N. caninum-infected cows. The study was performed on a commercial Holstein-Friesian dairy herd in northeastern Spain and the final data included those recorded in 53 non-aborting and 19 aborting animals. Blood samples were collected immediately before pregnancy diagnosis (on Days 40, 90, 120, 150, 180 and 210 post-insemination) in non-aborting cows or until the time of abortion detection in aborting cows. General lineal models (GLM) repeated measures anova revealed the different behaviour of PAG-1 and PAG-2, and significant effects of Neospora seropositivity, cool season and twin pregnancy on plasma PAG-2 concentrations throughout gestation (between-subject effects). In addition, based on the odds ratios, the likelihood of abortion increased in Neospora-seropositive cows (by a factor of 7.0) compared to seronegative animals and decreased in cows with a high plasma PAG-2 concentration (>4.5 ng/ml) on Day 120 of pregnancy (by a factor of 0.24), compared to the remaining cows. In conclusion, there is a relationship between plasma PAG-2 concentrations and the risk of abortion in Neospora-infected dairy cows. Thus, plasma PAG concentrations measured using anti-boPAG-2 antiserum on Day 120 of gestation could serve as an indicator of the abortion risk in N. caninum infected animals; values <4.5 ng/ml indicating a high risk of abortion in chronically infected animals.

Effects of perch access and age on physiological measures of stress in caged White Leghorn pullets.

The neuroendocrine system controls animals' adaptability to their environments by releasing psychotropic compounds such as catecholamines [epinephrine (EP), norepinephrine (NE), and dopamine (DA)], corticosterone (CORT), and serotonin (5-hydroxytryptamine or 5-HT). Changes of these neuroendocrine compounds have been used as biomarkers of animals' stress responses associated with their well-being. Assuming that pullets, like laying hens, are highly motivated to perch, we hypothesize that pullets with access to perches will experience less stress than pullets that never have access to perches. The objective of this study was to examine the effects of perch access and age on physiological measurements of stress in White Leghorn pullets housed in conventional cages. Hatchlings (n = 1,064) were randomly assigned to 28 cages. Two parallel metal round perches were installed in each of 14 cages assigned the perch treatment, whereas control cages were without perches. Two birds per cage were bled at wk 4, 6, and 12 wk of age. Plasma levels of CORT, DA, EP, and NE, blood concentrations of 5-HT and Trp, and heterophil to lymphocyte ratios were measured. Data were analyzed using a 2-way ANOVA. The perch treatment or its interaction with age did not affect any parameter measured in the study. The increase in the concentrations of circulating EP, NE, 5-HT (numerical increase at 4 wk), and Trp in 4- and 6-wk-old pullets compared with 12-wk-old pullets is unclear, but may have been due to acute handling stress at younger ages. In contrast, concentrations of DA were less at 4 wk compared with levels at 6 and 12 wk of age. Plasma CORT levels and the heterophil to lymphocyte ratio, indicators of long-term stress, were unaffected by age (P = 0.07 and 0.49, respectively). These results indicated that age, but not perch access, affects neuroendocrine homeostasis in White Leghorn pullets. Pullets that were never exposed to perches showed no evidence of eliciting a stress response.

Genetic variations alter production and behavioral responses following heat stress in 2 strains of laying hens.

Genetic differences alter the type and degree of hens' responses and their ability to adapt to a stressor. This study examined the effects of genotypic variations on the productivity and behavior of laying hens following heat stress (HS). Two strains of White Leghorn hens were used: DXL (Dekalb XL), a commercial strain individually selected for egg production and KGB (kind, gentle bird), a strain selected for high group productivity and survivability. Ninety hens (48 DXL and 42 KGB) at 28 wk of age were randomly assigned to either a hot (H: mean = 32.6°C) or control (C: mean = 24.3°C) treatment and housed in pairs by strain for 9 d. Egg production and quality, behavior, body and organ weights, and circulating hormone concentrations were measured. Heat-stressed hens had lower egg production [adjusted (adj) P < 0.001] than their respective controls. Among H-DXL hens, egg weight tended to be reduced at d 1 and was reduced at d 9 (adj P = 0.007), but was reduced only at d 9 among H-KGB hens (adj P = 0.007). Eggshell thickness was also reduced among H hens at d 9 (adj P = 0.007), especially among H-KGB hens (adj P = 0.01). Plasma triiodothyronine concentration was reduced among H-hens (adj P = 0.01), especially among H-DXL hens (adj P = 0.01). Neither temperature nor strain affected the plasma thyroxine and plasma and yolk corticosterone concentrations. Heat-stressed hens spent less time walking (adj P = 0.001) and more time drinking (adj P = 0.007) and resting (adj P = 0.001) than C-hens. The results indicate that although HS reduced production and caused behavioral changes among hens from both strains, the responses differed by genotype. The data provide evidence that genetic selection is a useful strategy for reducing HS response in laying hens. The results provide insights for conducting future studies to develop heat-resistant strains to improve hen well-being, especially under the current commercial conditions.

Release of avian neurotensin in response to intraluminal contents in the duodenum of chickens.

Peripheral and hepatic-portal plasma levels of neurotensin (NT) in fed and fasted chickens were determined using RIA. Portal levels of NT(1-13) (fed = 61.3 ± 3.9 fmol/mL; fasted = 44.5 ± 3.9 fmol/mL) were significantly higher than peripheral levels (fed = 8.2 ± 3.3 fmol/mL; fasted = 7.8 ± 3.0 fmol/mL) collected from the wing vein, indicating that some NT is metabolized in the liver. Portal plasma levels of NT collected from fed birds were also significantly higher than portal plasma levels of NT collected from fasted birds. Neurotensin, as identified by HPLC, exhibited a 2-fold increase in plasma extracts following perfusion of the proximal ileum with a 10-mg sample of oleic acid, as compared with control samples of plasma collected before oleic acid perfusion. In whole-animal studies, the injection of a micellar solution of oleic acid into isolated segments of the duodenum resulted in elevated plasma immunoreactive NT in blood collected from the pancreaticoduodenal vein. Injection of a 1,000 mOsm sodium chloride solution had a slightly lesser and delayed effect compared with oleic acid, but a greater effect than 0.1 N hydrochloric acid in isotonic saline solution. Injection of an amino acid solution (10% Travasol), 300 mOsm glucose solution, or pure corn oil had no effect. These results demonstrate that intraduodenal oleic acid is a potent stimulus for the release of NT from the duodenum into the hepatic-portal circulation of chickens.

Cerulenin upregulates heat shock protein-70 gene expression in chicken muscle.

Lines of evidence suggested that systems involved in the regulation of the stress responses and energy homeostasis are highly integrated. Because cerulenin, the natural antibiotic product of the fungus Cephalosporium ceruleans and a broad-spectrum fatty acid synthesis (FAS) inhibitor, has been shown to affect food intake and energy balance, and because the biomarker of stress Hsp-70 gene was found to interact directly with fatty acids, we hypothesized that cerulenin may regulate Hsp-70 gene expression. Therefore, the present study was undertaken to examine this issue. Cerulenin administration significantly (P < 0.05) decreased food intake and induced Hsp-70 mRNA levels in muscle, but not in liver or hypothalamus of 2-wk-old broiler chickens. These changes were accompanied by an unpregulation of muscle uncoupling protein and carnitine palmitoyltransferase 1 mRNA levels. This result indicated that the regulation of Hsp-70 gene expression in normal chickens, as estimated by oxidative stress indices [TBA reacting substances, ferric reducing/antioxidant power, and ceruloplasmin oxidase activity] levels, is tissue-specific. In attempt to discriminate between the effect of cerulenin and cerulenin-reduced food intake on Hsp-70 gene expression, we also evaluated the effect of food deprivation on the same cellular responses. Food deprivation for 16 h did not affect Hsp-70 gene expression in all tissues examined, indicating that the effect of cerulenin is independent of the inhibition of food intake. To ascertain whether the effect of cerulenin is direct or indirect, we carried out in vitro studies. Cerulenin treatment did not affect Hsp-70 gene expression in Leghorn male hepatoma and quail myoblast cell lines, suggesting that the observed effect in vivo may be mediated through the central nervous system.