Perchlorate affects thyroid function in eastern mosquitofish (Gambusia holbrooki) at environmentally relevant concentrations

The purpose of this study was to determine the effects of perchlorate on thyroid function in mosquitofish. Adult mosquitofish were exposed to 0, 0.1, 1, 10, 100, and 1000 mg/L sodium perchlorate for 2, 10, and 30 d. Whole body thyroxin (T4) content and histological assessment of thyroid follicles (e.g., follicular epithelial height, hyperplasia, hypertrophy, and colloid depletion) were used to gauge alterations in thyroid function. Follicular epithelial cell height, hyperplasia, and hypertrophy increased with increasing perchlorate concentration, especially in fish exposed for 30 d, and these effects were statistically significantly different from control at concentrations as low as 0.1 mg/L (nominal concentration). The percent occurrence of follicles with depleted colloid decreased with increasing perchlorate concentration, which is contrary to what is expected with thyroid inhibition. There also was a decrease in whole body T4 concentration in fish exposed to perchlorate for 30 d, but clear dose-response relationships were less evident for whole body T4 than for histopathological endpoints. In conclusion, thyroid histopathology provides a sensitive biomarker for thyroid endocrine disruption at environmentally relevant concentrations of sodium perchlorate, and whole body T4 is a less sensitive indicator of perchlorate exposure than is histopathology.     

Surveillance of perchlorate level in wine, seafood, polished rice, milk, powdered milk and yogurt

Perchlorate, which may be naturally occurring or artificial in origin, inhibits iodide uptake into the thyroid gland and disturbs thyroid function. In order to investigate perchlorate contamination in foods in Japan, perchlorate levels in 28 wine samples, 20 seafood samples, 10 polished rice samples, 30 milk (include whole milk, composition modified milk, low fat milk, processed milk, milk drink) samples, 10 powdered milk samples and 10 yogurt samples were measured. Perchlorate was found in all wine, milk, powdered milk and yogurt samples tested. Perchlorate levels ranged from 0.2 ng/g to 103 ng/g in wine samples, from 2 ng/g to 11 ng/g in milk samples, from 3 ng/g to 35 ng/g in powdered milk samples, and from 2 ng/g to 11 ng/g in yogurt samples. Perchlorate levels in the seafood samples were under the LOQ (0.8 ng/g) in 8 samples and ranged from 0.8 ng/g to 72 ng/g in 12 samples. In all polished rice samples, perchlorate level was under the LOQ (1.0 ng/g).     

Perchlorate and iodide in dairy and breast milk

Perchlorate inhibits iodide uptake and may impair thyroid and neurodevelopment in infants. Recently, we unambiguously identified the presence of perchlorate in all seven brands of dairy milk randomly purchased from grocery stores in Lubbock, TX. How widespread is perchlorate in milk? Perchlorate in 47 dairy milk samples from 11 states and in 36 human milk samples from 18 states were measured. Iodide was also measured in a number of the samples. Perchlorate was detectable in 81 of 82 samples. The dairy and breast milk means were, respectively, 2.0 and 10.5 microg/L with the corresponding maximum values of 11 and 92 microg/L. Perchlorate is present in virtually all milk samples, the average concentration in breast milk is five times higher than in dairy milk. Although the number of available measurements are few at this point, for breast milk samples with a perchlorate content greater than 10 microg/L, the iodide content is linearly correlated with the inverse of the perchlorate concentration with a r2 of >0.9 (n = 6). The presence of perchlorate in the milk lowers the iodide content and may impair thyroid development in infants. On the basis of limited available data, iodide levels in breast milk may be significantly lower than it was two decades ago. Recommended iodine intake by pregnant and lactating women may need to be revised upward.     

Perchlorate, nitrate, and iodide intake through tap water

Perchlorate is ubiquitous in the environment, leading to human exposure and potential impact on thyroid function. Nitrate can also competitively inhibit iodide uptake at the sodium-iodide symporter and thus reduce thyroid hormone production. This study investigates the intake of perchlorate, nitrate, and iodide attributable to direct and indirect tap water consumption. The National Health and Nutrition Examination Survey collected tap water samples and consumption data from 3262 U.S. residents during the years 2005-2006. The median perchlorate, nitrate, and iodide levels measured in tap water were 1.16, 758, and 4.55 μg/L, respectively. Measured perchlorate levels were below the United States Environmental Protection Agency (U.S. EPA) drinking water equivalent level for perchlorate (24.5 μg/L). Significant correlations were found between iodide and nitrate levels (r = 0.17, p < 0.0001) and perchlorate and nitrate levels (r = 0.25, p < 0.0001). On the basis of 24 h recall, 47% of the study participants reported drinking tap water; 89% reported either direct or indirect consumption of tap water. For the adult population (age ≥ 20 yrs) the median tap water consumption rate was 11.6 mL/kg-day. Using individual tap water consumption data and body weight, we estimated the median perchlorate, nitrate, and iodide dose attributable to tap water as 9.11, 11300, and 43.3 ng/kg-day, respectively, for U.S. adults. This perchlorate exposure dose from tap water is relatively small compared to the total perchlorate exposure dose previously characterized for the U.S. adults (median 64 ng/kg-day) and the U.S. EPA reference dose (700 ng/kg-day).     

Uptake, elimination, and relative distribution of perchlorate in various tissues of channel catfish

This study was undertaken to determine the kinetics of uptake and elimination of perchlorate in channel catfish, Ictalurus punctatus. Perchlorate--an oxidizer used in solid fuel rockets, fireworks, and illuminating munitions--has been shown to effect thyroid function, causing hormone disruption and potential perturbations of metabolic activities. For the uptake study, catfish were exposed to 100 mg/L sodium perchlorate for 12 h to 5 d in the laboratory. Perchlorate in tissues was analyzed using ion chromatography. The highest perchlorate concentrations were found in the head and fillet, indicating that these tissues are the most important tissues to analyze when determining perchlorate uptake into large fish. To calculate uptake and elimination rate constants for fillet, gills, G-I tract, liver, and head, fish were exposed to 100 ppm sodium perchlorate for 5 days, and allowed to depurate in clean water for up to 20 days. The animals rapidly eliminated the perchlorate accumulated showing the highest elimination in fillet (Ke = 1.67 day(-1)) and lowest elimination in liver (Ke = 0.79 day(-1)).     

Perchlorate exposure and dose estimates in infants

Perchlorate is a naturally occurring inorganic anion used as a component of solid rocket fuel, explosives, and pyrotechnics. Sufficiently high perchlorate intakes can modify thyroid function by competitively inhibiting iodide uptake in adults; however, little is known about perchlorate exposure and health effects in infants. Food intake models predict that infants have higher perchlorate exposure doses than adults. For this reason, we measured perchlorate and related anions (nitrate, thiocyanate, and iodide) in 206 urine samples from 92 infants ages 1-377 days and calculated perchlorate intake dose for this sample of infants. The median estimated exposure dose for this sample of infants was 0.160 μg/kg/day. Of the 205 individual dose estimates, 9% exceeded the reference dose of 0.7 μg/kg/day; 6% of infants providing multiple samples had multiple perchlorate dose estimates above the reference dose. Estimated exposure dose differed by feeding method: breast-fed infants had a higher perchlorate exposure dose (geometric mean 0.220 μg/kg/day) than infants consuming cow milk-based formula (geometric mean 0.103 μg/kg/day, p < 0.0001) or soy-based formula (geometric mean 0.027 μg/kg/day, p < 0.0001), consistent with dose estimates based on dietary intake data. The ability of perchlorate to block adequate iodide uptake by the thyroid may have been reduced by the iodine-sufficient status of the infants studied (median urinary iodide 125 μg/L). Further research is needed to see whether these perchlorate intake doses lead to any health effects.     

Perchlorate: water and infant formulae contamination in France and risk assessment in infants

Perchlorate ions ClO₄^–, known to inhibit competitively the uptake of iodine by the thyroid, have been detected in drinking water in France as well as in infant formulae. A tolerable daily intake (TDI) has been established at 0.7 µg kg^–1 bw day^–1 based on the inhibition of iodine uptake. Due to this mechanism of action, the iodine status could strongly influence the biological effect of perchlorate. Perchlorate concentrations in water and infant formulae were measured and the exposure of children under 6 months of age calculated. It appeared that the TDI could be exceeded in some children. As the iodine status is not optimal within the entire French population, there appears to be a need to clarify the sources of perchlorate ultimately to decrease exposure.     

Perchlorate in dairy milk. Comparison of Japan versus the United States

Perchlorate has been considered a potential threat to human health, especially to developing infants and children due to its ability to inhibit iodide uptake by the sodium iodide symporter (NIS) of the thyroid. Although the U.S. has been the prime focus of perchlorate contamination, at least some of the similar sources of perchlorate exist across the world, and it has been detected in many types of foods and beverages worldwide. We present here perchlorate data from cow's milk samples from Japan (mean 9.4 +/-2.7 microg/L, n = 54), which are higher on average than those found in U.S. dairy milk samples reported by a 2004 Food and Drug Administration (FDA) study (5.9+/-1.8 microg/L, n= 104).     

Perchlorate in groundwater: a synoptic survey of "pristine" sites in the coterminous United States

Perchlorate is widely used as an oxidant in solid rocket propellants and energetic applications, and it has frequently been detected in groundwaters at concentrations relevant to human health. The possibility of naturally occurring perchlorate has only recently received significant attention. Relying primarily on domestic, agricultural, and recreational wells, we utilized a network of volunteers to help collect 326 groundwater samples from across the coterminous United States. Care was taken to avoid known, USEPA-documented sites of perchlorate use or release, as well as perchlorate contamination due to disinfection using hypochlorite. Using IC-ESI-MS and a Cl18O4- internal standard, we achieved a method detection limit (MDL) of 40 ng/L perchlorate and a minimum reporting level (MRL) of 120 ng/L. Of the 326 samples, 147 (45%) were below the MDL, while 42 (13%) were between the MDL and the MRL. Of the 137 samples that could be quantified, most (109) contained < 1000 ng/L perchlorate; the remaining 28 samples contained from 1000 to 10400 ng/L. Our results support the notion that perchlorate occurs naturally in many groundwaters, but the unusually high concentrations (> 10000 ng/L) previously reported for the west-central Texas area appear to be anomalous. Perchlorate concentrations were positively correlated with nitrate levels (P < 0.001) but not with chloride concentrations. Opportunities exist for follow-up studies of perchlorate's origins using isotope forensics and for further elucidation of the role of atmospheric processes in the formation or transport of perchlorate.     

Trace analysis of bromate, chlorate, iodate, and perchlorate in natural and bottled waters

A simple and rapid method has been developed to simultaneously measure sub-microg/L quantities of the oxyhalide anions bromate, chlorate, iodate, and perchlorate in water samples. Water samples (10 mL) are passed through barium and hydronium cartridges to remove sulfate and carbonate, respectively. The method utilizes the direct injection of 10 microL volumes of water samples into a liquid chromatography-tandem triple-quadrupole mass spectrometry (LC-MS/MS) system. Ionization is accomplished using electrospray ionization in negative mode. The method detection limits were 0.021 microg/L for perchlorate, 0.045 microg/L for bromate, 0.070 microg/L for iodate, and 0.045 microg/L for chlorate anions in water. The LC-MS/MS method described here was compared to established EPA methods 300.1 and 317.1 for bromate analysis and EPA method 314.0 for perchlorate analysis. Samples collected from sites with known contamination were split and sent to certified laboratories utilizing EPA methods for bromate and perchlorate analysis. At concentrations above the reporting limits for EPA methods, the method described here was always within 20% of the established methods, and generally within 10%. Twenty-one commercially available bottled waters were analyzed for oxyhalides. The majority of bottled waters contained detectable levels of oxyhalides, with perchlorate < or = 0.74 microg/L, bromate < or = 76 microg/L, iodate < or = 25 microg/ L, and chlorate < or = 5.8 microg/L. Perchlorate, iodate, and chlorate were detectable in nearly all natural waters tested, while bromate was only detected in treated waters. Perchlorate was found in several rivers and reservoirs where itwas not found previously using EPA 314.0 (reporting limit of 4 microg/L). This method was also applied to common detergents used for cleaning laboratory glassware and equipmentto evaluate the potential for sample contamination. Only chlorate appeared as a major oxyhalide in the detergents evaluated, with concentrations up to 517 microg/g. Drinking water treatment plants were also evaluated using this method. Significant formations of chlorate and bromate are demonstrated from hypochlorite generation and ozonation. From the limited data set provided here, it appears that perchlorate is a ubiquitous contaminant of natural waters at trace levels.     

Perchlorate in the United States. Analysis of relative source contributions to the food chain

Perchlorate has been considered by some a potential threat to human health, especially to developing infants and children because it may inhibit iodide uptake by the sodium iodide symporter (NIS) of the thyroid. In the United States, during the last several decades, environmental perchlorate has had three recognized sources stemming from (a) its use as an oxidizer (including in rocket propellants), (b) its presence in Chilean nitrate fertilizer (CNF), and (c) natural production. An analysis of the relative source strengths and how they may influence entry into the food chain has not been conducted. Averaged over the last --60 years, we estimate that the source strengths have been (a) 10.6, (b) 0.75, and (c) 0.13-0.64 Gg/y for the United States as a whole. Of this, while (b) and (c) represent actual dispersed amounts, the figure in (a) is the amount of perchlorate produced and only a fraction (f) of it has been dispersed and often in a more localized fashion. In addition, dispersal of (b) has taken place only over agricultural land. Considering that the total land area in the United States is 5.5 x the arable land area, in terms of incorporation into the food chain,the figure cited in (b) has a proportionately greater impact. Most estimates of fwill thus suggest that over the considered period, the contribution of CNF to incorporation of perchlorate in the food chain has likely been comparable to oxidizer perchlorate, with natural production being a lesser source. Fireworks presently constitute a potentially important source of increasing importance but a quantitative impact cannotyet be assessed.     

Effects of genotype and transpiration rate on the uptake and accumulation of perchlorate (ClO4-) in lettuce

Although evidence of perchlorate accumulation in plants exists, there is a scarcity of information concerning the key factors and mechanisms involved. To ascertain whether genotypic variation in perchlorate accumulation occurs within lettuce, hydroponic plant uptake experiments were conducted with five types of lettuce (Lactuca sativa L.), which were grown to market size atthree perchlorate (ClO4-) concentrations (1, 5, or 10 microg/L). Perchlorate accumulated in the leafy tissues to varying amounts, ranging from 4 to 192 microg/kg fresh weight (FW), and the ranking of perchlorate accumulation was crisphead > butter head > romaine > red leaf > green leaf. The effect of transpiration rate on perchlorate accumulation was further examined using crisphead, butter head, and green leaf lettuce. By growing lettuce in controlled-environment chambers with two climatic regimes, "cloudy, humid, cool" (80% RH, 18/15 degrees C, 250 micromol/m2s photosynthetic photon flux density (PPFD)) and "sunny, dry, warm" (approximately 50% RH, 28/18 degrees C, 500 micromol/m2s PPFD), up to 2.7-fold differences in transpiration rates were achieved. Across all three genotypes, the plants that transpired more water accumulated more perchlorate on a whole-head basis; however, the effect of transpiration rate on perchlorate accumulation was not as great as expected. Despite 2.0-2.7-fold differences in transpiration rate, there were only 1.2-2.0-fold differences in perchlorate accumulation. In addition to whole-head analysis, plants were sectioned into inner, middle, and outer leaves and processed separately. Overall, the ranking of perchlorate accumulation was outer leaves > middle leaves > inner leaves. Transpiration rate has a clear effect on perchlorate accumulation in lettuce, but other factors are influential and deserve exploration.     

Risk assessment of dietary exposure to perchlorate for the Austrian population

Perchlorate is frequently found as contaminant in a variety of food. Based on analytical data of perchlorate occurrence in food products from the Austrian market, this study calculated dietary perchlorate exposure of the Austrian population for the three age classes of adults, children and infants. Furthermore, a detailed risk assessment was conducted based on the tolerable daily intake (TDI) of 0.3 µg/kg body weight/day, established by the European Food Safety Authority in 2014. Calculations of a scenario of average food consumption did not indicate elevated health risks by dietary perchlorate uptake. Exposure estimates reached only 12%, 26% and 24% of the TDI for adults, children and infants, respectively. However, in a scenario of high consumption, the TDI was exceeded by all age classes with 132%, 161% and 156%. The major cause for this exceedance is the comparatively high perchlorate contamination of spinach, but also other leaf vegetables, legumes and pineapples, leading to elevated exposure of high consumers. Our calculations reveal that the current provisional intra-Union trade reference level for perchlorate in spinach of 0.2 mg/kg, advocated by the European Commission, is not sufficient to protect high consumers against possible health risks. In order to reduce health risks to a tolerable level for all consumers, lowering of the regulatory maximum perchlorate concentrations is indicated. Moreover, a generally diversified diet can also counteract excessive exposure to perchlorate as well as to other harmful food contaminants.     

Widespread occurrence of perchlorate in water,foodstuffs and human urine collected from Kuwait and its contribution to human exposure

Perchlorate is a thyroid hormone-disrupting compound and is reported to occur widely in the environment. Little is known on human exposure to perchlorate in Kuwait. In this study, 218 water samples, 618 commonly consumed foodstuffs and 532 urine samples collected from Kuwait were analysed to assess the exposure of the Kuwaiti population to perchlorate. For the estimation of daily intake of perchlorate, food consumption rates were obtained from the National Nutrition Survey in the State of Kuwait (NNSSK). The results showed that leafy vegetables accounted for a major share of perchlorate exposure among the Kuwaiti population at 0.062 µg kg^–1 bw day^–1 (36.2%), followed by fruits at 0.026 µg kg^–1 bw day^–1 (15.3%) and non-leafy vegetables at 0.017 µg kg^–1 bw day^–1 (10.1%). The urinary perchlorate geometric mean (GM) concentrations ranged from 8.51 to 17.1 µg l^–1 for the five age groups, which were higher than those reported in other countries. The estimated urinary perchlorate exposure for the Kuwaiti general population was 0.42 µg kg^–1 bw day^–1, which was higher than that reported for the United States. The dietary intake of perchlorate for the Kuwaiti population ranged from 0.14 to 0.67 µg kg^–1 bw day^–1 for the five age groups, with a mean total daily intake of 0.17 µg kg^–1 bw day^–1 for the general population. The highest estimated dietary mean daily intake of perchlorate (0.67 µg kg^–1 bw day^–1) was found for children at 3–5 years. The estimated dietary perchlorate exposure in Kuwait is higher than the recommended mean reference dose (RfD) but lower than that of provisional maximum tolerable daily intake (PMTDI) set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA).     

A steady-state biofilm model for simultaneous reduction of nitrate and perchlorate, part 2: parameter optimization and results and discussion

Part 1 of this work developed a steady-state, multispecies biofilm model for simultaneous reduction of nitrate and perchlorate in the H(2)-based membrane biofilm reactor (MBfR) and presented a novel method to solve it. In Part 2, the half-maximum-rate concentrations and inhibition coefficients of nitrate and perchlorate are optimized by fitting data from experiments with different combinations of influent nitrate and perchlorate concentrations. The model with optimized parameters is used to quantitatively and systematically explain how three important operating conditions (nitrate loading, perchlorate loading, and H(2) pressure) affect nitrate and perchlorate reduction and biomass distribution in these reducing biofilms. Perchlorate reduction and accumulation of perchlorate-reducing bacteria (PRB) in the biofilm are affected by four promotion or inhibition mechanisms: simultaneous use of nitrate and perchlorate by PRB and competition for H(2), the same resources in PRB, and space in a biofilm. For the hydrogen pressure evaluated experimentally, a low nitrate loading (<0.1 g N/m(2)-d) slightly promotes perchlorate removal, because of the beneficial effect from PRB using both acceptors. However, a nitrate loading of >0.6 g N/m(2)-d begins to inhibit perchlorate removal, as the competition effects become dominant.     

高氯酸盐对高脂膳食小鼠脂质代谢的影响

目的 探究高氯酸盐对高脂膳食小鼠脂质代谢的影响。方法 将C57BL/6J小鼠分为4组,分别为对照组(CG)、高氯酸盐低剂量组(LG,0.1 mg/kg bw/d)、中剂量组(MG,1.0 mg/kg bw/d)和高剂量组(HG,10.0 mg/kg bw/d),连续暴露12周,通过对肝脏油红O染色、血生化指标检测、脂代谢调控酶测定和脂肪酸组成及含量测定进行高氯酸盐对高脂膳食小鼠脂质代谢的评估。结果 高于1.0 mg/kg bw/d的暴露剂量可在小鼠肝脏内形成脂滴累积,总胆固醇、甘油三酯和高密度脂蛋白胆固醇含量均显著性升高(P<0.05),并且HG组脂质代谢关键调控酶乙酰辅酶A羧化酶(acetyl-CoA carboxylase,ACC)和脂肪酸合成酶(fatty acid synthase,FAS)含量显著性升高(P<0.05),表明高氯酸盐可能会促进脂肪的合成。随着高氯酸盐暴露浓度的增加,棕榈酸(C16:0)、珍珠酸(C17:0)、硬脂酸(C18:0)、花生四烯酸(C20:4)和二十二碳六烯酸(C22:6)体内水平显著下降,而棕榈亚酸(C16:1)、油酸(C18:1)和亚油酸(C18:2)体内水平显著上升,饱和脂肪酸显著下降,不饱和脂肪酸显著上升。结论 高氯酸盐可能会造成小鼠肝脏损伤和脂代谢紊乱,且随着暴露剂量的升高肝脏损伤越显著。本研究为高氯酸盐体内毒性损伤和健康效应的研究提供了理论基础。     

Gonadotrophin responsiveness, aromatase activity and insulin-like growth factor binding protein content of bovine ovarian follicles during the first follicular wave

The aim of this study was to examine the function of granulosa cells and hormone concentrations in follicular fluid in bovine ovarian follicles during selection of the first dominant follicle. Ovaries were obtained from beef heifers on days 1-5 after ovulation: follicles > 4 mm in diameter were dissected and follicular fluid and granulosa cells were collected from individual follicles. Oestradiol production by granulosa cells after culture with testosterone was used to determine aromatase activity and responsiveness to gonadotrophins was determined by cAMP production after culture with FSH or LH. Concentrations of oestradiol, progesterone and insulin-like growth factor binding proteins (IGFBPs)-4 and -5 were measured in follicular fluid. Follicles were classified as largest or smaller (days 1 and 2), or dominant or subordinate (days 3-5). Aromatase activity was greater in granulosa cells from the largest follicle than in granulosa cells from smaller follicles on days 1, 3, 4 and 5 (P < 0.05). Responsiveness to LH was not detected in granulosa cells on day 1, but from day 2 to day 5 cells from the largest follicle were significantly more responsive than cells from smaller follicles (P < 0.05). Responsiveness to FSH was detected in granulosa cells from all follicles from day 1 onwards and did not differ between cells from the largest follicle or smaller follicles on any day. Follicular fluid concentrations of oestradiol and the ratio of oestradiol:progesterone were greater and concentrations of IGFBP-4 and -5 were lower in the largest follicle than in smaller follicles from day 2 to day 5 (P < 0.05). In conclusion, selection of the dominant follicle is associated with increased granulosa cell aromatase activity followed by increased cAMP response to LH and follicular fluid oestradiol concentrations, and decreased follicular fluid concentrations of IGFBP-4 and -5 within 2 days after ovulation.     

Water, sodium chloride and acidified sodium chlorite effects on Escherichia coli O157:H7 and Staphylococcus aureus on beef briskets

Effectiveness of spray application of potable water wash (WW), 25% (w/v) sodium chloride (NaCl), and 0.1% (v/v) acidified sodium chlorite (ASC) was evaluated against Escherichia coli O157:H7 and Staphylococcus aureus inoculated onto beef briskets. The purpose was to identify antimicrobial treatments which may be applied to beef carcasses and more specifically in kosher meat facilities. Treatments were applied for 10-60 s at pressure of 419 kPa. Water wash, NaCl, and ASC significantly reduced E. coli O157:H7 as compared with the control, although, only ASC resulted in improved removal with increased exposure time. Water wash did not significantly reduce S. aureus counts throughout exposure and NaCl was only effective after 60 s of exposure, while ASC reduced counts throughout exposure. E. coli O157:H7 was twice as sensitive to WW and NaCl as S. aureus in terms of percent reduction in cell count.     

Effects of ovarian follicle ablation on FSH, oestradiol and inhibin A concentrations and growth of other follicles in sheep

The aim of this study was to examine the effect of removal of the largest follicle or all visible follicles during the first follicle wave on subsequent follicular growth, steroid, inhibin A and gonadotrophin secretion in sheep. On day 4.5 of a synchronized oestrous cycle, ewes (n = 18) were assigned to one of three groups which underwent either no treatment (control), ablation of the largest follicle (largest follicle aspirated and cauterized via laparotomy) or ablation of all follicles (all visible follicles ablated). Between day 0 and day 10 of the oestrous cycle, blood samples were collected every 8 h and ovaries were examined daily using transrectal ultrasonography. The lifespan of the second largest follicle (number of days > 3 mm in diameter) was longer (6.7 +/- 0.9 days; P < 0.05) and the maximum diameter tended to be greater (4.8 +/- 0.3 mm; P = 0.07) in ewes in which the largest follicle was ablated than in the control ewes (3.8 +/- 0.4 days; 4.2 +/- 0.3 mm). There was no difference in the day of emergence of the second follicular wave between groups (day 6.9 +/- 0.4). However, the peak of the transient increase in FSH concentrations after ablation was earlier (day 5.67 +/- 0.15; P < 0.05) in ewes in which all follicles were ablated than in control ewes (day 6.72 +/- 0.36); the timing in ewes that had only the largest follicle ablated was intermediate (day 6.11 +/- 0.28). Serum inhibin A concentrations were about three-fold lower (P < 0.05) in both follicle ablation groups than in the control group. The numbers of follicles 2-3 mm in diameter during the first 3 days of the second follicular wave were greater in 'ablated ewes' (both groups had 2.6 +/- 0.2 follicles day-1) than in control ewes (1.7 +/- 0.3 follicles day-1). It is concluded that: (i) transient increases in FSH concentrations precede the emergence of follicle waves; (ii) ablation of all follicles on day 4.5 after oestrus advanced the timing of the next peak in FSH concentrations and the numbers of small follicles associated with the development of the second follicular wave; and (iii) ablation of the largest follicle resulted in an increase in the lifespan of the second largest follicle, indicating a regulatory role of large dominant follicles over smaller subordinate follicles.     

Interactive effects of granulosa cell apoptosis, follicle size, cumulus-oocyte complex morphology, and cumulus expansion on the developmental competence of goat oocytes: a study using the well-in-drop culture system

Using a well-in-drop (WID) oocyte/embryo culture system that allows identification of follicular origin, we have investigated the effects of granulosa cells (GCs) apoptosis, follicle size, cumulus-oocyte complexes (COCs) morphology, and cumulus expansion on the developmental competence of goat oocytes matured and cultured individually following parthenogenetic activation. The WID system supported oocyte maturation and embryo development to a level similar to the conventional group system. The majority of goat oocytes acquired competence for development up to the 8-16 cell stage in follicles larger than 2 mm, but did not gain the ability to form morula/blastocyst (M/Bs) until follicles larger than 3 mm in diameter. The extent of atresia affected M/Bs formation. This effect varied according to the follicle size. Cumulus expansion increased with follicle size and decreased with increasing incidence of GCs apoptosis. Oocyte developmental potential was also correlated with cumulus expansion. Regardless of the degree of follicle atresia, 73-84% of the floating cells in the follicular fluid (FF) underwent apoptosis. Correlation between floating cell density in FF and oocyte developmental potency suggests the possibility to use the floating cell density as a simple and non-invasive marker for oocyte quality. It is concluded that the developmental potential of an oocyte is determined by multifactor interactions, and multiple factors must be considered together to accurately predict the quality of an oocyte.