Luteolin protects against adipogenic and lipogenic potency induced by human relevant mixtures of persistent organic pollutants (POPs) in the 3T3-L1 model.

Human exposure to persistent organic pollutants (POPs) may contribute to obesogenic effects. We have previously shown that POP mixtures modelled on blood levels relevant to the Scandinavian population induces adipogenic effects in the mouse 3T3-L1 cell line. Luteolin is a flavone that has shown anti-lipogenic and anti-adipogenic effects on adipogenesis in in vitro models. In this study, luteolin has been applied to inhibit adipocyte formation and intracellular lipid content increase induced by a human relevant mixture of POPs. 3T3-L1 cells were exposed to a POP mixture consisting of 29 chemicals, including amongst others polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), perfluoroalkylated acids (PFAAs), and polybrominated diphenyl ethers (PBDEs). Rosiglitazone was applied as a positive lipogenic control. Luteolin was tested between 0.5 and 10 µM. High content analysis was used to assess changes in adipocyte formation and intracellular lipid content in the 3T3-L1 cell line. Luteolin significantly reduced POP-induced adipocyte formation at 2, 5 and 10 µM, and lipid accumulation at 10 µM. Interestingly, luteolin did not affect rosiglitazone induced adipo- and lipogenic effects, suggesting differences in mechanisms of action. In conclusion, this in vitro study shows that dietary polyphenols such as luteolin may protect against POP induced adipo- and lipogenic effects.

A mixture of persistent organic pollutants relevant for human exposure inhibits the transactivation activity of the aryl hydrocarbon receptor in vitro.

While humans are exposed to mixtures of persistent organic pollutants (POPs), their risk assessment is usually based on a chemical-by-chemical approach. To assess the health effects associated with mixed exposures, knowledge on mixture toxicity is required. Several POPs are potential ligands of the Aryl hydrocarbon receptor (AhR), which involves in xenobiotic metabolism and controls many biological pathways. This study assesses AhR agonistic and antagonistic activities of 29 POPs individually and in mixtures by using Chemical-Activated LUciferase gene eXpression bioassays with 3 transgenic cell lines (rat hepatoma DR-H4IIE, human hepatoma DR-Hep G2 and human mammary gland carcinoma DR-T47-D). Among the 29 POPs, which were selected based on their abundance in Scandinavian human blood, only 4 exerted AhR agonistic activities, while 16 were AhR antagonists in DR-H4IIE, 5 in DR-Hep G2 and 7 in DR-T47-D when tested individually. The total POP mixture revealed to be AhR antagonistic. It antagonized EC50 TCDD inducing AhR transactivation at a concentration of 125 and 250 and 500 fold blood levels in DR-H4IIE, DR-T47-D and DR-Hep G2, respectively, although each compound was present at these concentrations lower than their LOEC values. Such values could occur in real-life in food contamination incidents or in exposed populations. In DR-H4IIE, the antagonism of the total POP mixture was due to chlorinated compounds and, in particular, to PCB-118 and PCB-138 which caused 90% of the antagonistic activity in the POP mixture. The 16 active AhR antagonists acted additively. Their mixed effect was predicted successfully by concentration addition or generalized concentration addition models, rather than independent action, with only two-fold IC50 underestimation. We also attained good predictions for the full dose-response curve of the antagonistic activity of the total POP mixture.

Human blood-based exposure levels of persistent organic pollutant (POP) mixtures antagonise androgen receptor transactivation and translocation.

INTRODUCTION: Human exposure to persistent organic pollutants (POPs) has been linked to genitourinary health-related conditions such as decreased sperm quality, hypospadias, and prostate cancer (PCa). Conventional risk assessment of POPs focuses on individual compounds. However, in real life, individuals are exposed to many compounds simultaneously. This might lead to combinatorial effects whereby the global effect of the mixture is different from the effect of the single elements or subgroups. POP mixtures may act as endocrine disruptors via the androgen receptor (AR) and potentially contribute to PCa development. AIM: To determine the endocrine disrupting activity of a POP mixture and sub-mixtures based upon exposure levels detected in a human Scandinavian population, on AR transactivation and translocation in vitro. MATERIALS AND METHODS: The Total POP mixture combined 29 chemicals modelled on the exposure profile of a Scandinavian population and 6 sub-mixtures: brominated (Br), chlorinated (Cl), Cl + Br, perfluorinated (PFAA), PFAA + Br, PFAA + Cl, ranging from 1/10× to 500× relative to what is found in human blood. Transactivation was measured by reporter gene assay (RGA) and translocation activity was measured by high content analysis (HCA), each using stably transfected AR model cell lines. RESULTS: No agonist activity in terms of transactivation and translocation was detected for any POP mixtures. In the presence of testosterone the Cl + Br mixture at 100× and 500× blood level antagonised AR transactivation, whereas the PFAA mixture at blood level increased AR transactivation (P < 0.05). In the presence of testosterone the Cl and PFAA + Br mixtures at 1/10×, 1×, and 50× blood level antagonised AR translocation (P < 0.05). CONCLUSION: Taken together, some combinations of POP mixtures can interfere with AR translocation. However, in the transactivation assay, these combinations did not affect gene transactivation. Other POP combinations were identified here as modulators of AR-induced gene transactivation without affecting AR translocation. Thus, to fully evaluate the effect of environmental toxins on AR signalling, both types of assays need to be applied.

A mixture of Persistent Organic Pollutants (POPs) and Azoxymethane (AOM) show potential synergistic effects on intestinal tumorigenesis in the A/J Min/+ mouse model.

A multitude of cancer types, including breast, testicular, liver and colorectal cancer, have associations with exposure to Persistent Organic Pollutants (POPs). The present study aimed to investigate whether a mixture of POPs could affect intestinal tumorigenesis in the A/J Min/+ mouse, a model for human colorectal cancer (CRC). Pollutants were selected for their presence in Scandinavian food products and the mixture was designed based on defined human estimated daily intake levels. Mice were exposed through the diet, at control, low and high mixture concentrations, for 10 weeks. In a separate experiment, mice also received one subcutaneous injection of Azoxymethane (AOM) to explore whether this carcinogenic compound influenced the effect of the POPs. Intestinal tumorigenesis was examined by surface microscopy and histopathology. Moderate and dose-dependent increases in tumorigenesis were observed after dietary POP exposure. The AOM treatment alone stimulated the growth of colonic lesions, but did not increase the formation of new lesions. Combined AOM treatment and POP exposure demonstrated a synergistic effect on lesion formation in the colon, and to a lesser extent in the small intestine. This synergy was also evident by an increased number of malignant colonic tumors (carcinomas). In conclusion, the study shows that a mixture of POPs interacted synergistically with a known carcinogen (AOM), causing increased intestinal tumorigenesis in the A/J Min/+ mouse model.

Progressive retinal atrophy in Shetland sheepdog is associated with a mutation in the CNGA1 gene.

Progressive retinal atrophy (PRA) is the collective name of a class of hereditary retinal dystrophies in the dog and is often described as the equivalent of retinitis pigmentosa in humans. PRA is characterized by visual impairment due to degeneration of the photoreceptors in the retina, usually leading to blindness. PRA has been reported in dogs from more than 100 breeds and can be genetically heterogeneous both between and within breeds. The disease can be subdivided by age at onset and rate of progression. Using genome-wide association with 15 Shetland Sheepdog (Sheltie) cases and 14 controls, we identified a novel PRA locus on CFA13 (Praw  = 8.55 × 10(-7) , Pgenome  = 1.7 × 10(-4) ). CNGA1, which is known to be involved in human cases of retinitis pigmentosa, was located within the associated region and was considered a likely candidate gene. Sequencing of this gene identified a 4-bp deletion in exon 9 (c.1752_1755delAACT), leading to a frameshift and a premature stop codon. The study indicated genetic heterogeneity as the mutation was present in all PRA-affected individuals in one large family of Shelties, whereas some other cases in the studied Sheltie population were not associated with this CNGA1 mutation. To our knowledge, this is the first report of a mutation in CNGA1 causing PRA in dogs.

Gene expression during testis development in Duroc boars.

Androstenone is a steroid pheromone occurring in the pubertal Leydig cells. Breeding against androstenone can decrease pheromone odour in swine meat but appears to cause unwanted side effects such as delayed onset of puberty. To study causality, global gene expression in developing boar testes at 12, 16, 20 and 27 weeks was investigated using a porcine cDNA microarray. The morphological status and androgenic levels of the same individuals have been described in a previous publication. In the present paper, expression of genes and pathways has been analysed with reference to these findings. Nine clusters of genes with significant differential expression over time and 49 functional charts were found in the analysed testis samples. Prominent pathways in the prepubertal testis were associated with tissue renewal, cell respiration and increased endocytocis. E-cadherines may be associated with the onset of pubertal development. With elevated steroidogenesis (weeks 16 to 27), there was an increase in the expression of genes in the MAPK pathway, STAR and its analogue STARD6. A pubertal shift in genes coding for cellular cholesterol transport was observed. Increased expression of meiotic pathways coincided with the morphological onset of puberty. Puberty-related change in Ca(2+) pathway transcripts, neurosteroids, neuronal changes and signalling in redox pathways suggested a developmental-specific period of neuromorphogenesis. Several growth factors were found to increase differentially over time as the testis matured. There may be interactions between MAPK, STAR and growth factors during specific periods. In conclusion, pathways for neurogenesis, morphological pathways and several transcripts for growth factors, which have known modulating effects on steroidogenesis and gonadotropins in humans and rodents, act at specific ages and developmental stages in the boar testis. The age dependency and complexity shown for development-specific testis transcripts must be considered when selecting phenotypic parameters for genetic selection for low androstenone. The results of selection based on measurement of phenotypic maturation and androstenone (or other steroid) levels at one specific age may differ depending on the age used. More research is necessary to find the optimal phenotype to use in order to reduce the unwanted side effects.

Comparison of Holstein-Friesian and Norwegian Red dairy cattle for estrus length and estrous signs.

This study addressed the effect of breed on estrus length and estrous behavior by observing 20 Holstein-Friesian (HF) and 20 Norwegian Red (NRF) cows on an outdoor wood-chip pad through 1 estrous cycle (22d). Detailed behavioral data were collected by continuous (24 h) video monitoring of all cows. Accurate estimation of duration of estrous periods, behavioral signs (sum per period and counts per hour), and duration and number of sexually active groups were reported through all stages of mount estrus (prestand, standing estrus, and poststand). These dependent variables were analyzed with a basic statistical model that included fixed effects for breed and lactation group. Other independent variables (milk yield, body condition score, and number of cows in standing estrus) were added to the basic model one by one and included in an expanded model if they had an effect on the respective dependent variables. Estrus duration was considerably shorter in HF compared with NRF cows for all the major periods: mount estrus (11.2 ± 3.0 vs. 21.3 ± 2.7 h), standing estrus (7.1 ± 1.4 vs. 11.7 ± 1.3 h), mounting period (6.9 ± 2.7 vs. 18.2 ± 2.4 h), and mounted period (9.2 ± 2.8 vs. 17.5 ± 2.6 h). Additionally, the NRF cows spent more time in sexually active groups (36.1 ± 4.0 vs. 17.6 ± 4.8%) during standing estrus compared with HF cows. The NRF cows participated in a greater number of sexually active groups (9.6 ± 1.3 vs. 5.5 ± 1.3) with longer average duration (0.42 ± 0.04 vs. 0.20 ± 0.04 h) and continued to be more active in these groups through late stages of estrus (poststand) compared with the HF breed. Mounting activity differed between breeds as NRF mounted more times in total (46.3 ± 6.2 vs. 18.1 ± 6.3) and per hour (2.6 ± 0.4 vs. 1.5 ± 0.5) during mount estrus. In addition, NRF tended to express the primary estrous sign, standing when mounted, more often during standing estrus (32.4 ± 5.0 vs. 18.5 ± 5.2). The HF initiated more unsuccessful mounts (1.6 ± 0.3 vs. 0.6 ± 0.3) per hour than did NRF during mount estrus. A significant effect of milk yield was demonstrated only on this behavior. For other estrous signs, HF cows initiated chase-up (2.0 ± 0.5 vs. 0.5 ± 0.4) and anogenital sniff (3.7 ± 0.6 vs. 2.0 ± 0.5) more frequently (counts per hour), whereas NRF expressed more total head butt behavior (32.3 ± 4.7 vs. 14.2 ± 4.8) during mount estrus. Body condition score had a significant effect on receptive behavior. Measures of estrus duration, sexually active group activity, and behavior related to estrus should be subjected to larger studies for improved heat detection and possible implementation in breeding programs.

Sexually active groups in cattle-a novel estrus sign.

The current study presents a novel objective measure for characterizing sexually active groups (SAG 3-5) and relates this measure to other behaviors of lactating Holstein-Friesian cows. Cows in SAG 3-5 were required to participate in a minimum of 1 estrus behavior per 5min while staying within 3m (2 cow lengths) of its partner(s) for a minimum of 5min. Twenty Holstein-Friesian cows were video-monitored continuously through 1 complete estrous cycle (22d). Standing behavior, SAG 3-5, secondary estrus signs (SEC), and other social and agonistic behaviors were recorded continuously. The period of mounting estrus (MTE) was divided into the 3 parts: prestand, standing estrus (STE), and poststand. The mean durations of MTE, prestand, STE, and poststand period were 12.9±1.84, 4.0±1.93, 7.1±1.44, and 1.8±0.57h (n=13). The fractions of time spent in SAG 3-5 during MTE, prestand, STE, and poststand period were 13, 8, 19, and 1% (n=11). During MTE, cows participated, on average, in 5.8±1.24 SAG 3-5 and initiated 9.5±2.99 mounts, with mean durations of 0.25±0.03h and 4.00±0.36s, respectively. The novel measure SAG 3-5 was a sign of long duration not confined only to groups of STE cows. On one day when no cows were in estrus and during the periods 4 to 24h before and after MTE, no SAG 3-5 behaviors were observed. Luteal-phase cows participated in SAG 3-5 only when the partner was a single cow in estrus. The time spent in SAG 3-5 increased between 1 and 3h before MTE and the prestand period (3 vs. 8%) and reached a peak level during STE. From STE to poststand, time spent in SAG 3-5 decreased considerably (19 vs. 1%). The observed decrease in nonmutual agonistic behaviors 4 to 24h before MTE is suggested as an early sign of pre-estrus. Changes in SAG 3-5, agonistic behaviors, and SEC are suggested as indicators of the specific stages of MTE. Increased SEC initiated and SAG 3-5 were indicators of late pre-estrus and early estrus (prestand). Peak levels of SAG 3-5, SEC, and social agonistic behaviors were indicators of STE. A sudden decrease in behaviors, preceded by frequent interactions, was indicative of late estrus (poststand). On the basis of the findings reported here, we propose that SAG 3-5, as well as proceptive and receptive patterns of SEC and agonistic behaviors, be included in estrus detection protocols. Updated knowledge of these behavioral interactions may assist when determining the stage of estrus and the optimal time to breed dairy cows.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis.

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA. At the receptor level, OTA (within a concentration range of 0.25-2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect. At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1-1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117±14 ng/ml) over 3 times that of the solvent control (36±9 pg/ml). Western blotting confirmed an increase in aromatase protein. Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.

An in vitro investigation of endocrine disrupting effects of trichothecenes deoxynivalenol (DON), T-2 and HT-2 toxins.

Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins. H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1-1000ng/ml), T-2 toxin (0.0005-5ng/ml) or HT-2 toxin (0.005-50ng/ml) for 48h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes. Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P<0.05) by DON (100ng/ml), T-2 toxin (0.5ng/ml) and HT-2 toxin (5ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study. Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

In vitro and in vivo association of porcine hepatic cytochrome P450 3A and 2C activities with testicular steroids.

The aim of this study was to screen the inhibitory potential of several testicular steroids on cytochrome P450 3A (CYP3A) and 2C (CYP2C) activities in porcine liver microsomes. The microsomes used in this study were obtained from pubertal male pigs of two breeds, Landrace and Duroc. For the in vitro inhibition study, porcine microsomes were incubated in the presence of 17ß-estradiol, 17α-estradiol, androstenone, dehydroepiandrosterone and dihydrotestosterone. Both reversible and mechanism-based inhibitions were examined. 7-benzyloxyresorufin (BR) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were used as substrates for CYP3A, and diclofenac and tolbutamide (TB) as substrates for CYP2C. 7-benzyloxyresorufin O-dealkylase (BROD) activity was inhibited by all tested steroids in the microsomes from Landrace pigs via mechanism-based mode, but in the microsomes from Duroc pigs, BROD activities were inhibited only in the presence of 17ß-oestradiol. Mechanism-based inhibition of BFC metabolism by the tested steroids was observed in the microsomes from both breeds, but this inhibition was weak and did not exceed 20%. TB hydroxylase (TBOH) activity in the microsomes from Duroc pigs was inhibited by 17α-oestradiol through the mechanism-based mode of inhibition. None of the investigated steroids inhibited TBOH activity in Landrace pigs. For the in vivo study, male pigs were injected with a single dose of human chorionic gonadotropin (hCG) to stimulate testicular steroid production by the Leydig cells. In vivo stimulation with hGC did not alter BROD activity either in Landrace or in Duroc pigs. BFC metabolism was significantly induced by hCG stimulation in both breeds and TBOH activity only in Duroc pigs. Activity of diclofenac hydroxylase was not detected in either Landrace or Duroc pigs. Breed significantly affected BROD and TBOH activity with BROD being higher in Landrace and TBOH in Duroc pigs. This study improved our understanding of the role of testicular steroids in the regulation of porcine CYP450 activity.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL). α-ZOL exhibited the strongest estrogenic potency (EC(50) 0.022±0.001 nM), slightly less potent than 17-ß estradiol (EC(50) 0.015±0.002 nM). ZEN was ~70 times less potent than α-ZOL and twice as potent as ß-ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, α-ZOL or ß-ZOL. ZEN, α-ZOL or ß-ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 µM. At 100 µM, cell viability decreased and levels of hormones were significantly reduced except for progesterone. ß-ZOL increased estradiol concentrations more than α-ZOL or ZEN, with a maximum effect at 10 µM, with ß-ZOL (562±59 pg/ml)>α-ZOL (494±60 pg/ml)>ZEN (375±43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Steroidogenesis in primary cultures of neonatal porcine Leydig cells from Duroc and Norwegian Landrace breeds.

Breed differences in steroidogenic activity between primary Leydig cells derived from neonatal purebred Duroc and Norwegian Landrace boars were investigated in vitro. Concentrations of testosterone, estradiol, androstenone, cortisol and progesterone produced into the medium were determined. To explore underlying mechanisms the cellular expression of a suite of genes relevant in steroidogenesis was measured using reverse transcription and quantitative PCR (RT-qPCR). Basal steroid concentrations indicated a larger production capacity for steroids in unstimulated Duroc cells. Stimulation of the cells with LH increased steroid hormone secretion significantly in both breeds in a dose dependent manner. Testosterone and androstenone concentrations increased approximately 50- and 15-fold, respectively, whereas concentrations of estradiol, cortisol and progesterone increased to a lesser extent. At levels of maximal LH stimulation, absolute steroid concentrations were higher in Duroc. However, the relative increase in hormone concentrations was significantly lower in Duroc cells for estradiol, progesterone and cortisol when compared to basal levels. LH exposure was associated with a general up-regulation of mRNA levels for steroidogenic genes, stronger in Duroc than in Norwegian Landrace. This was in agreement with the higher absolute concentrations of steroid hormones measured in culture medium from the LH-stimulated Duroc Leydig cells, but did not concur with the fact that the relative increase in hormone production was lower in Duroc than in Norwegian Landrace Leydig cells for some hormones. It was concluded that breed differences in steroid hormone concentrations and gene expression between Norwegian Landrace and Duroc are complex and cannot be explained by a simple mechanism of action.

Effects of mixtures of persistent organic pollutants (POPs) derived from cod liver oil on H295R steroidogenesis.

Crude cod liver oil and liver oil supplements are consumed as a source of vitamin A, D and polyunsaturated fatty acids; during winter and early pregnancy. Crude cod liver oil however constitutes a considerable source of persistent organic pollutants (POPs). This paper aimed at characterizing and quantifying the influence of POP mixtures extracted from three different steps in the cod liver oil industrial process on hormone production and the expression of steroidogenesis-related genes in H295R cells. Exposure to extracts from crude cod liver oil and from its industrial waste increased progesterone (P4), cortisol (Cort), testosterone (T) and estradiol (E2) production; and among others, the expression of MC2R, CYP11B1 and HSD3B2 genes. Observed effects after exposure to pharmaceutical cod liver oil extract were considerably lower. The type of effects on gene expression and hormone production were similar to those induced by forskolin and PCBs, the latter being the major contaminants within the extracts. Additional research is required to further unveil the mechanisms behind the observed steroidogenic effects and to assess whether the potential risk might outweigh the potential benefits of crude and processed cod liver oil consumption.

Behavior of lactating Holstein-Friesian cows during spontaneous cycles of estrus.

The objectives of the present study were to describe, in detail, behavior associated with standing estrus (STE) in lactating dairy cows and behavioral changes during complete estrous cycles. Estrus signs were monitored by continuous video recording of 20 Holstein-Friesian (HF) cows housed on an outdoor wood-chip pad during 1 estrous cycle (22 d). Other social behavior was recorded during STE and, for comparison, during 1 selected day when none of the cows were in estrus. Standing stationary when mounted was defined as the primary estrus sign. Anogenital sniff, chin rest, attempt to mount, and mount were defined as secondary estrus signs. Ovarian cyclicity was confirmed by progesterone measurements. This study reports short mean duration of STE (7.1±1.44h) and estrus (mount period; 12.9±1.84h) of the 13 cows expressing these signs. All mounting activities involved at least one cow in, or within 4h of, STE. The most frequent sign during STE was anogenital sniff initiated, followed by chin rest received, chin rest initiated, chase up initiated, anogenital sniff received, mount initiated, head butt, mount received, attempt to mount initiated, push away received, play rub, attempt to mount received, follow initiated, threat received, flehmen, avoid, bellow, and social lick received. Standing and mounting activity in HF cows was inconsistent during estrus, indicating that other signs could be of greater use. The frequency of secondary estrus signs initiated and received increased gradually during the last 12h before STE, revealing significant differences between periods from 4 to 6 and 1 to 3h before STE. A considerable increase in receptive behavior (secondary estrus signs received) was identified between 1 to 3h prior to STE and STE. Both frequent initiated and received behaviors were associated with STE. A significant decrease in the frequency of secondary estrus signs initiated and received occurred 3h after STE. Cows in STE simultaneously predominantly chose the other standing cow as mate and expressed secondary estrus signs more frequently. Based on the results of this study, we suggest that chase up could be regarded as a reliable indicator of estrus and that the changes in proceptive (initiated) and receptive (received) behavior could be used as predictors of different stages in estrus. Knowledge of these behavioral signs may improve heat detection rates and the ability to predict the optimum breeding time for dairy cows.

Differences in testosterone, androstenone, and skatole levels in plasma and fat between pubertal purebred Duroc and Landrace boars in response to human chorionic gonadotrophin stimulation.

The concentrations of the boar taint compounds androstenone and skatole in plasma and fat, together with those of testosterone in plasma, were investigated in pubertal purebred Duroc and Landrace boars following stimulation with human chorionic gonadotrophin (hCG). Higher initial levels of androstenone and testosterone were found in Duroc than Landrace boars. Duroc boars, which were approximately ten days older than the Landrace boars, also showed a more advanced stage of spermatogenesis than Landrace boars. While Landrace boars had the highest skatole levels. Following stimulation with hCG the relative increases in testosterone, androstenone, and skatole concentrations were highest in Landrace boars. The level of androstenone in fat three days after hCG stimulation exceeded 1 microg/g fat in all stimulated boars. The decreases in plasma levels of androstenone and testosterone on Days 2 and 3 after hCG stimulation were more pronounced in Landrace than Duroc boars. However, unlike the plasma androstenone and testosterone levels, the plasma concentrations of skatole did not decrease on Days 2 and 3 following stimulation, but remained elevated on Day 3. These results indicate that the lower levels of testicular steroids in Landrace boars compared with Duroc boars was not due to a lower production capacity, but more likely to a faster disappearance of steroids in Landrace boars. In the present study, age, live weight, and testicular development did not significantly contribute to the variation in fat androstenone. The present data and previous reports on candidate genes related to androstenone biosynthesis and metabolism suggests that future selection against factors associated with boar taint remains a possible solution for the problem of boar taint in the swine industry.

Hepatic ethoxy-, methoxy- and pentoxyresorufin O-dealkylase activities in Landrace and Duroc pigs stimulated with HCG.

The effect of human chorionic gonadotropin (hCG) stimulation on the activities of ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD) and pentoxyresorufin O-depentylase (PROD) was studied in intact male pigs of purebred Landrace and Duroc breeds. Pigs were divided into four groups: two control groups of each breed, without hCG stimulation (n = 20 for each breed), and two experimental groups (n = 18 for each breed), with hCG stimulation (Pregnyl(®); N.V. Organon, Oss, The Netherlands, 30 IU/kg live weight). Pigs were slaughtered 3 days after hCG stimulation and enzyme activities were measured in hepatic microsomes using two approaches. First, only one substrate concentration was used for the analysis of each enzyme activity. We found that EROD activity was suppressed by hCG-stimulation in Landrace (p = 0.004), but not Duroc pigs (p > 0.05). Generally, EROD activity was higher in Duroc pigs compared with Landrace (p = 0.017). Methoxyresorufin O-demethylase and PROD activities did not differ between groups (p > 0.05). To further characterize EROD, MROD and PROD, enzyme kinetic studies were performed. V(max) values for EROD and MROD in both breeds were lower after hCG stimulation (p < 0.001 for Landrace and p < 0.05 for Duroc). Additionally, V(max) values for EROD significantly differed between Landrace and Duroc pigs being higher in Duroc pigs (p < 0.05). We concluded that both hCG stimulation and breed differences may be important in the regulation of EROD and MROD activities. This study provides the first data on the effect of hCG stimulation and thus high testicular steroids, on EROD, MROD and PROD activities. Further studies are needed to investigate individual CYP450 enzymes and their regulation in porcine tissues.

Comparison of accuracy of ultrasonography, progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer.

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n=195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n=33), December 2006 (n=92), or January 2007 (n=70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.

Differential effects of antiepileptic drugs on steroidogenesis in a human in vitro cell model.

OBJECTIVES: To better understand the interaction of antiepileptic drugs and production of sex hormones, possible effects of valproate (VPA), levetiracetam (LEV) and carbamazepine (CBZ) on steroidogenesis were investigated in the human adrenal carcinoma cell line H295R. MATERIALS AND METHODS: H295R cells were exposed to different concentrations of VPA, LEV or CBZ for 48 h. Sex hormone concentrations and mRNA expression levels were analyzed via radioimmunoassay and quantitative real time (RT)-PCR, respectively. RESULTS: In VPA-exposed cells estradiol levels decreased in a dose-dependent manner, while testosterone and progesterone levels were unaffected. Expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), steroidogenic acute regulatory protein (StAR), CYP11a, CYP17, CYP21, 3betaHSD2, 17betaHSD1 was downregulated and expression of CYP11beta2 was upregulated. No effect on sex hormone production was observed under influence of LEV or CBZ. Expression of StAR, CYP17, CYP19 and 3betaHSD2 was downregulated in LEV-exposed cells, and expression of HMGR, CYP11beta2 and CYP17 was downregulated in CBZ-exposed cells. CONCLUSIONS: VPA exposure resulted in a decrease in estradiol levels and a general downregulation of expression of genes encoding for enzymes early in steroidogenesis. No consistent changes were seen with LEV or CBZ exposure.

Commencement of luteal activity in three different selection lines for milk yield and fertility in Norwegian Red cows.

Relationships among commencement of luteal activity (C-LA), milk yield, and energy balance (EB) were investigated in 3 selection lines of Norwegian Red cows at the Norwegian University of Life Sciences from 1994 through 2001. The cows were selected for low genetic merit for milk yield (LMP), high genetic merit for milk yield (HMP), and a combination of high indices for milk yield and fertility (HI). Breeding values for fertility were based on 56-d nonreturn rate. The material included 268 lactations from 147 cows. Milk samples for progesterone analysis were drawn 3 times weekly from 1994 through 1998, and 2 times weekly from 1999 to 2001. Commencement of luteal activity was defined as the first 2 consecutive measurements of progesterone concentration >3 ng/mL not earlier than 10 d after calving. Selection line was significantly related to C-LA, so that the least squares mean days from calving to C-LA were 22.5, 30.4, and 27.2 d for LMP, HMP, and HI cows, respectively. The HMP cows produced more milk than the LMP cows. The average milk yield in the sixth week of lactation was 24.0, 27.1, and 25.3 kg for LMP, HMP, and HI cows, respectively. The interval to C-LA decreased for the HMP and HI cows after phenotypic adjustment for EB in the model. Least squares means for the interval to C-LA were 23.2, 29.7, and 25.6 d for the LMP, HMP, and HI cows, respectively, in a model that included parity, selection lines, and EB as covariates. Cumulated EB during the first 4 wk of lactation, which itself differed between selection lines, did not fully account for differences in interval to C-LA between selection lines. Thus, the results of the present investigation indicate that selection for milk yield negatively affects C-LA over and above the effects caused by concurrent changes in EB. The increase in days to C-LA caused by selection for high yields can be reduced if selection for milk yield is combined with fertility in the breeding program.