Chromatin modifier developmental pluripotency associated factor 4 (DPPA4) is a candidate gene for alcohol-induced developmental disorders.

BACKGROUND: Prenatal alcohol exposure (PAE) affects embryonic development, causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neuronal disorders and birth defects. We hypothesize that early alcohol-induced epigenetic changes disrupt the accurate developmental programming of embryo and consequently cause the complex phenotype of developmental disorders. To explore the etiology of FASD, we collected unique biological samples of 80 severely alcohol-exposed and 100 control newborns at birth. METHODS: We performed genome-wide DNA methylation (DNAm) and gene expression analyses of placentas by using microarrays (EPIC, Illumina) and mRNA sequencing, respectively. To test the manifestation of observed PAE-associated DNAm changes in embryonic tissues as well as potential biomarkers for PAE, we examined if the changes can be detected also in white blood cells or buccal epithelial cells of the same newborns by EpiTYPER. To explore the early effects of alcohol on extraembryonic placental tissue, we selected 27 newborns whose mothers had consumed alcohol up to gestational week 7 at maximum to the separate analyses. Furthermore, to explore the effects of early alcohol exposure on embryonic cells, human embryonic stem cells (hESCs) as well as hESCs during differentiation into endodermal, mesodermal, and ectodermal cells were exposed to alcohol in vitro. RESULTS: DPPA4, FOXP2, and TACR3 with significantly decreased DNAm were discovered-particularly the regulatory region of DPPA4 in the early alcohol-exposed placentas. When hESCs were exposed to alcohol in vitro, significantly altered regulation of DPPA2, a closely linked heterodimer of DPPA4, was observed. While the regulatory region of DPPA4 was unmethylated in both control and alcohol-exposed hESCs, alcohol-induced decreased DNAm similar to placenta was seen in in vitro differentiated mesodermal and ectodermal cells. Furthermore, common genes with alcohol-associated DNAm changes in placenta and hESCs were linked exclusively to the neurodevelopmental pathways in the enrichment analysis, which emphasizes the value of placental tissue when analyzing the effects of prenatal environment on human development. CONCLUSIONS: Our study shows the effects of early alcohol exposure on human embryonic and extraembryonic cells, introduces candidate genes for alcohol-induced developmental disorders, and reveals potential biomarkers for prenatal alcohol exposure.

Gnawing bones as enrichment for farmed blue foxes ( Vulpes lagopus).

According to present acts and regulations, farmed foxes shall have a gnawing or other enrichment object in their cages. However, research on the welfare effects of gnawing objects has been scarce. We assessed physiology and health, that is weight development, urinary cortisol-creatinine ratio, serum cortisol level after adrenocorticotropic hormone administration, internal organ masses and incidence of gastric ulcerations as well as dental and overall oral health, in pair-housed juvenile blue foxes that were housed either with or without a possibility to interact with bones (cattle femur) during their growing season (July to December). The results show that the physiological effects of the possibility to interact with bones were either non-significant or suggested that competition for bones may jeopardize the welfare of subordinate individuals. However, the results clearly show that gnawing bones are beneficial for the dental health of farmed foxes.

A note on the reproductive success of primiparous blue fox vixens in social groups.

Our aim was to compare traditional breeding system, i.e. artificial insemination with singly-housing, to alternative breeding systems in farmed blue fox (Alopex lagopus or Vulpes lagopus) vixens. At the age of 7 weeks (i.e. at weaning), 48 randomly selected female blue fox cubs were divided into four experimental groups: (1) artificially inseminated singly-housed vixens in cages (AI-SC), (2) artificially inseminated pair-housed vixens in double-cages (AI-PC), (3) naturally bred pair-housed vixens with a male in triple-cages (NB-PC), and (4) naturally bred pair-housed vixens with a male in outdoor enclosures (NB-PE). The cubs were counted on days 1, 2, 3, 7, 14 and 49 postpartum and the reproductive performance per breeding (RPB) and per mated vixens (RPM) with its subcomponents were formed from these data. RPB was zero in both NB groups. In AI-PC and AI-SC, RPB was 1.3+/-2.5 and 4.1+/-4.7 cubs, respectively. RPB and the percentage of vixens that weaned cubs were lower in NB-PC and NB-PE than in AI-PC. In AI groups, both RPB and RPM at weaning tended to be better in singly-housed than in pair-housed blue fox vixens. No statistically significant differences were found between AI groups in the percentage of vixens without oestrus, barren vixens, vixens that lost all cubs or weaned at least one cub. Only in one pair (AI-PC) both vixens displayed communal breeding and weaned cubs. The present results show that vixens in traditional breeding system had better RPM than in any of the studied pair-housing systems. The current blue fox population has been effectively selected for cage-breeding and artificial reproduction, and therefore blue foxes generally reproduce well with artificial insemination after careful detection of oestrus in traditional breeding conditions.

Group housing in row cages: an alternative housing system for juvenile mink.

We studied a group housing system as an alternative to the traditional pair housing of juvenile mink. The focus was on both the welfare and production of mink. The pairs were housed in standard mink cages, whereas the groups were in row cage systems consisting of three standard mink cages connected to each other. The welfare of the mink was evaluated by behavioural observations (stereotypies and social contacts), evaluation of the incidence of scars assumed to be caused by biting, and adrenal function (serum cortisol level after adrenocorticotropic hormone (ACTH) administration and adrenal mass). Feed consumption, pelt length, quality and price were used for comparing the two housing systems from the economic point of view. Although the incidence of scars showed that there might have been more aggressive behaviour among the group-housed than among the pair-housed mink, this was not observed unambiguously in behavioural observations, and, at least, aggression did not cause mortality or serious injuries to the animals as has been observed in some earlier studies. In addition, the housing system did not affect pelt size, and, although the quality of the pelts was slightly lower in the group than in pair-housed mink, there was only a tendency for lower pelt prices. The lower pelt prices in the group-housed mink might even be partially compensated for by the group-housed mink eating 10% to 20% less in the late autumn, due to thermoregulatory benefits, than their pair-housed conspecifics. The results on the frequency of stereotypic behaviour (but not adrenal function) suggest that the group-housed animals were possibly less stressed than the pair-housed animals. Group housing of juvenile farmed mink in a row cage system cannot be recommended before the effects on welfare and production are clarified in further studies.

The effect of a combination of permanent breeding cage and low housing density on the reproductive success of farmed blue foxes.

Social factors are known to affect the reproduction of many canids both in the wild and in farms. For example, reproduction in farmed silver foxes is regulated by social stress; foxes seem to benefit from noncramped housing conditions and permanent breeding cages. However, no comparable studies have been carried out in farmed blue foxes. The aim of our experiment was to create an alternative, improved, economically viable and practical housing solution for blue foxes. Therefore, we compared reproductive performance of blue foxes in permanent breeding cages with low animal densities (L group, N=79) and traditional housing with its changing social environment with high animal density (H group, N=74). The reproductive data from the L and H groups were compared separately for primiparous and multiparous vixens because the reproductive performance in primiparous vixens was substantially lower (P<0.001) than in multiparous vixens. Altogether, 41 and 39% of the primiparous vixens in the H and L group whelped (P>0.05), but only 28 and 34%, respectively, weaned at least one cub (P>0.05), i.e., 72 and 66% of the primiparous vixens did not reproduce in the H and L group, respectively (P>0.05). The total reproductive performance, expressed as cubs at weaning per breeding female, was 1.7+/-3.5 for the H and 1.6+/-2.9 for the L group (P>0.05). In the primiparous vixens, the only statistically significant difference observed between the two housing systems was that the onset of oestrus occurred five days earlier in the H than in the L group (P<0.05). All multiparous vixens in the L group exhibited oestrus compared to 94% in the H group (P>0.05). Furthermore, there was a nonsignificant (ns) trend for fewer barren females (9% versus 17%), more successfully reproducing vixens (83% versus 74%) and a higher number of live-born cubs (10.9+/-4.7 versus 9.4+/-3.9) in the L than in H group in the multiparous vixens (for all P>0.05). This resulted in 1.7 and 1.4 cubs more per breeding and per mated vixen, respectively, at weaning in the L group (7.3+/-5.0) compared to the H group (5.6+/-4.2), but also this difference was nonsignificant. Although our present results lack statistical significance, they are promising enough to encourage field experiments with sufficiently large number of animals to prove or disprove these preliminary findings that lower housing density and permanent breeding cage, together or separately, may enhance reproduction particularly in multiparous blue fox vixens.

Determination of 17alpha-hydroxyprogesterone in serum by liquid chromatography-tandem mass spectrometry and immunoassay.

17Alpha-hydroxyprogesterone (17OHP) is the most important serum marker for congenital adrenal hyperplasia (CAH). 17OHP is usually measured by immunoassay but its detection by mass spectrometry (MS) is a potentially superior method. An LC-MS (liquid chromatography-mass spectrometry) method was developed which utilizes 0.5 ml serum spiked with 6-alpha-methylprednisolone (6-MP) or deuterated 17OHP (d8-IS) as the internal standard. The samples were extracted with ether/ethylacetate, and the extract was evaporated to dryness and analysed by LC-MS/MS operating in the positive mode after separation on a reversed-phase C18 column. The calibration curves for analysis of serum 17OHP exhibited consistent linearity and reproducibility in the range of 5-250 nmol/l. Interassay CVs were 8.5 and 9.2% at mean concentrations of 7.9 and 23 nmol/l, respectively. The detection limit was 1 nmol/l (signal-to-noise ratio=3). The mean recovery of 17OHP added to serum ranged from 76 to 89% and that of internal standards from 75 to 82%. The regression equation for the LC-MS/MS (x) and in-house radioimmunoassay (RIA) (y) methods was: y=0.87x+0.26 (r=0.97; n=100) and for a commercial RIA it was: y=1.32x+0.02 (r=0.97; n=26).

Evaluation of the DPC IMMULITE 2000 assay for total homocysteine in plasma.

The present report describes an evaluation in three laboratories of the completely automated total homocysteine immunoassay adapted to the IMMULITE 2000 from DPC. The precision depended on the control materials used, but with quality control materials and patient samples imprecisions were found to be in the range of 5.3 to 6.1% and 5.4 to 6.0%, respectively. Dilution experiments proved the assay to be linear and correlations with HPLC and GC-MS methods were close (r=0.98 and 0.97, respectively). In addition, the samples from the Nordic program for external quality assessment of methylmalonic acid and homocysteine for 2000 were assayed in the three laboratories. Imprecision evaluated from these samples was 5.3% and the recovery of the added L-homocystine was equivalent to the mean recovery of the 58 participants in the program. The precision is close to the quality goals. In conclusion, the method is an attractive alternative when coping with an increasing number of requests for the analysis of total homocysteine.

Trade-off between floor level and floor material in farmed silver foxes.

Farmed silver foxes (Vulpes vulpes) were allowed to balance their known preference for an elevated floor against their presumed preference for a sand floor. In Experiment 1, foxes had to choose between two identical cages, connected with an opening. One cage had a wire floor and the other had a sand floor, but the cages either were on the same (low or elevated) or on different levels (one cage 40 cm higher than the other). In Experiment 2, the cage pairs were connected with a 1.2 m long wire-mesh tunnel, one cage was always on a higher level (50 cm) than the other. In Experiment 1, foxes always preferred the sand floor during their active time. They also preferred the sand floor for resting, if it was on the same level as wire floor, but did not show any genuine preference if the floors were on different levels. In Experiment 2, foxes never preferred the lower floor. They preferred the elevated sand floor for activity and the elevated wire floor for lying. When two floors were identical they preferred the elevated one. Their rest consisted of 11-22 bouts, a major part of these being spent in the preferred cage. They also preferred a previous lying site to a new one, often exclusively and independently of floor material. In Experiment 1 foxes preferred the sand floor whereas in Experiment 2 they preferred the elevated floor indicating that the ability of a trade-off situation to rank resources depends on the method it is inflicted.

Lack of effect of alcohol on ethinylestradiol in premenopausal women.

An acute elevation in estradiol during alcohol intake has been reported in postmenopausal women on estrogen replacement therapy. The objective of the present study was to investigate the acute and long-term effect of alcohol on ethinylestradiol, the estrogen component found in most oral contraceptives. Nine healthy premenopausal women with regular use of an oral contraceptive containing 30 microg ethinylestradiol and 75 microg gestodene were challenged with alcohol (0.4 g/kg p.o., approximately 2-3 standard drinks) 2 h after intake of the oral contraceptive pill at menstrual cycle day 14. Blood samples were taken at 0, 2, 3, 4, 5, and 6 h from intake of alcohol. The challenge was repeated after a 7-day period of controlled alcohol intake (0.8 g/kg/day) at cycle day 21. The same experiments were carried out during placebo conditions. At day 21 an increase in the alcohol elimination rate was observed compared with day 14. No significant acute or long-term effect of alcohol on ethinylestradiol was found. The lack of an acute effect comparable to that reported for estradiol may be due to the protection of the ethinyl group at the 17-position of ethinylestradiol.

Validation of the feeding test as an index of fear in farmed blue (Alopex lagopus) and silver foxes (Vulpes vulpes).

The reliability and validity of the eating behaviour in the presence of man (Feeding test) as an index of fear were assessed in farmed blue (Alopex lagopus) and silver foxes (Vulpes vulpes). Repeatability of the Feeding test was good in both species. No further habituation occurred after the fourth successive test in either species. In addition, the behaviour of both species was independent of the person who performed the test. The normal feeding interval, i.e., 24 h, between feed deliveries, was long enough to provide reliable results. The presence of a cage mate did not influence the blue foxes' response in the Feeding test. A significant relationship between the results of the Feeding test and the Tit-bit test in both species and between the Feeding test and fearfulness score in silver foxes indicate that all these tests measure similar features, most probably foxes' fear of humans. Those silver foxes that did not eat in the Feeding test had higher base levels of cortisol than the animals that did eat, providing further support for the above conclusion. The present study demonstrates that the Feeding test is a reliable, i.e., repeatable and free of random errors, and fairly valid fear test for blue and silver foxes. The Feeding test seems likely to give good results in measuring fear in farmed blue and silver foxes, but further investigations will be needed to fully validate it, especially for blue foxes.

Estrogen-related acetaldehyde elevation in women during alcohol intoxication.

Alcohol is more often unpleasant and causes tissue damage more rapidly in women than men. The present study was designed to find out whether acetaldehyde, the primary metabolite of alcohol, could play a crucial role in these actions. Special emphasis was focused on the appropriate determination of blood acetaldehyde and hormonal factors. Occurrence of elevated blood acetaldehyde levels during alcohol oxidation was established in both normally cycling women and ones taking oral contraceptives, but not in men. An association between elevated acetaldehyde levels and high estrogen phases was observed in both groups of women. Estrogen-related acetaldehyde elevation is suggested to be the key factor explaining the gender differences of the adverse effects of alcohol.