A field approach to observing changes in behavioural welfare indicators over 2 years in stabled horses.

In stabled horses, several behaviours are considered to be important indicators of the state of welfare at the individual level: stereotypies, aggressive behaviours towards humans and the "withdrawn", alert, sternal, lateral and observation postures. Until now, these behaviours have been extensively studied in relation to different horse management practices. However, little is known about their changes or consistency over time. This study aimed to investigate differences in the expression of these behaviours assessed on 44 stabled horses during an initial 3-month period and then again on the same individuals 2 years later. Out of the six behaviours studied, two showed significant differences between the 2 years with medium effect sizes: the levels of aggressiveness towards humans increased (Wilcoxon signed-rank test: V = 65, P = 0.005) and those of recumbent rest during the day decreased (V = 416, P < 0.001) over time. The results also suggested limited evidence of major changes over time in the expression of stereotypies, the "withdrawn", alert and observation postures. However, the principal component analyses carried out on all the behaviours showed that alert and observation postures may slightly decrease over time for some individuals. Regarding stereotypies and the "withdrawn" posture, the results mainly suggested a change at the individual level in the expression of these behaviours over time. This study provided new insights into the dynamic nature of several behaviours when the horses' living conditions are not fundamentally altered and raised hypotheses about the state of welfare of stabled individuals over a 2-year period.

Effect of luteinizing hormone overstimulation on equine follicle maturation.

There is evidence in several species that high circulating LH concentrations can interfere with normal follicle development and ovulation. In the mare, high LH levels after induction of luteolysis with PGF(2α) have been temporally associated with an increased incidence of anovulatory follicles. We hypothesized that a premature increase in LH levels during a follicular wave in mares would disrupt normal follicle maturation leading to ovulatory dysfunction. In experiment 1, all follicles >10 mm were ablated at midestrous cycle in pony mares followed by twice daily administration of equine LH (eLH; 1.6 µg/kg body weight) or saline (vehicle; N = 8 mares per group). When a dominant follicle reached >32 mm, an ovulatory dose of hCG was given. Treatment with eLH had no effects on ovulatory responses or progesterone levels during the posttreatment luteal phase. In experiment 2, after follicle ablation, mares were treated with eLH or vehicle (as above) or were given a single injection of PGF(2α) (N = 7 mares per group), followed by aspiration of a dominant follicle when it reached >32 mm. Administration of eLH induced an increase in circulating LH levels similar to that after PGF(2α) injection. Neither PGF(2α) nor eLH administration had significant effects on follicle growth or total number of follicles in the postablation wave. However, compared with mares treated with vehicle, the preovulatory follicle in the eLH and PGF(2α) groups had lower levels of androstenedione (P = 0.03) and higher levels of insulin-like growth factor I (P = 0.03). Further, levels of prostaglandin E2 in preovulatory follicles tended to be lower in the eLH and PGF(2α) groups (P = 0.06). In conclusion, exposure of developing follicles to high LH in mares did not have apparent effects on ovulation but it induced changes in follicular fluid factor levels which might reflect a disruption in follicle and/or oocyte maturation, indicating the need to further study the implications of using PGF(2α) for the control of fertility in farm animals.

Kisspeptins and the reproductive axis: potential applications to manage reproduction in farm animals.

Kisspeptins (Kp) are a family of neuropeptides produced mainly by two hypothalamic neuronal cell populations. They have recently emerged as a major regulator of the gonadotropin axis and their action is located upstream of the gonadotropin-releasing hormone (GnRH) cell population. In less than 10 yr a growing body of literature has demonstrated the involvement of these peptides in most, if not all, aspects of reproductive axis maturation and function. In contrast to these abundant basic research studies, few experiments have evaluated the potential application of Kp as tools to manipulate reproduction in domestic animals. In mammals, exogenous Kp administration potently stimulates gonadotropin secretion. This action is exerted mainly, if not exclusively, through the stimulation of GnRH release. Intravenous, intraperitoneal, or subcutaneous administration of Kp induced a robust and rapid increase in plasma gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]). However, this stimulatory effect is of short duration. Prolonged LH and FSH release over several hours can be achieved only when Kp are given as repeated multiple bolus or as an infusion. Kp administration was used in two experimental models, ewe and pony mare, with the aim of inducing well-timed and synchronized ovulations. During the breeding season, progesterone-synchronized ewes were given an intravenous infusion of Kp starting 30 h after the removal of progesterone implants. An LH surge was induced in all Kp-treated animals within 2 h of infusion onset. In contrast, in pony mares a constant infusion of Kp for 3 d in the the late follicular phase was unable to induce synchronized ovulation. Another set of studies showed that Kp could be used to activate reproductive function in acyclic animals. Pulsatile administration of Kp in prepubertal ewe lambs was shown to activate ovarian function, leading to enhanced ovarian steroidogenesis, stimulation of LH preovulatory surge, and ovulation. In anestrous ewes, an intravenous infusion of a low dose of Kp induced an immediate and sustained release of gonadotropins, followed a few hours later by an LH surge. This hormonal pattern mimicked hormonal changes normally observed during the estrous cycle follicular phase and was associated with a high percentage of ovulating animals (80%). In summary, exogenous administration of Kp appears to be a new tool to manipulate reproduction. However, optimal doses and periods of treatment should be defined for each species, and the development of powerful analogs or long-term release formulations is necessary before large-scale applications in domestic animals could be envisaged.

RF9 powerfully stimulates gonadotrophin secretion in the ewe: evidence for a seasonal threshold of sensitivity.

GPR147 and its endogenous ligands, RFRPs, are emerging as important actors in hypothalamic-pituitary axis control. The role of this system would be to inhibit gonadotrophin secretion. However, data on the subject are contradictory. The discovery of RF9 (adamantanecarbonyl-RF-2-NH(2)), a GPR147 antagonist, prompted us to use this new tool to further investigate this system in the ewe. Accordingly, we tested the effect of i.c.v. administration of RF9 on gonadotrophin secretion in the ewe during anoestrous and the breeding season. Intracerebroventricular injections of RF9 (from 50-450 nmol) caused a clear elevation in peripheral blood plasma luteinising hormone (LH) concentrations. The effect of RF9 on LH was more pronounced during the anoestrous season. Furthermore, peripheral administration of RF9 as a bolus (2.1, 6.2 and 12.4 µmol per ewe) or as a constant i.v. infusion (2.1, 6.2, 12.4 and 18.6 µmol/h per ewe) to anoestrous acyclic ewes induced a sustained increase in LH plasma concentrations. A pharmacokinetic study showed that RF9 (12.4 µmol bolus i.v.) has an effective half life of 5.5 h in the plasma. Conversely, RF9 is not detectable in the cerebrospinal fluid, suggesting that it does not cross the blood-brain barrier. The increase in LH plasma concentrations induced by RF9 was blocked by previous administration of 1.3 µmol per ewe of gondotrophin-releasing hormone (GnRH) antagonist Teverelix. This suggests that GnRH is involved in the stimulatory effect of RF9 on gonadotrophin secretion. Finally, no variation in LH plasma concentrations could be detected in ovariectomised ewes injected either i.c.v. or i.v. with RFRP3 (VPNLPQRF-NH(2)). The lack of effect of RFRP3 in our experimental setting suggests that the mechanisms involved in RF9 action are probably more complex than previously assumed. Our results indicate that delivery of RF9 in the ewe greatly increases gondadotrophin secretion in both the oestrus and anoestrus season, suggesting a potential new way of controlling reproduction in mammals.

Supplementation of equine early spring transitional follicles with luteinizing hormone stimulates follicle growth but does not restore steroidogenic activity.

This study was conducted to test the hypothesis that supplementation of growing follicles with LH during the early spring transitional period would promote the development of steroidogenically active, dominant follicles with the ability to respond to an ovulatory dose of hCG. Mares during early transition were randomly assigned to receive a subovulatory dose of equine LH (in the form of a purified equine pituitary fraction) or saline (transitional control; n = 7 mares per group) following ablation of all follicles >15 mm. Treatments were administered intravenously every 12 h from the day the largest follicle of the post-ablation wave reached 20 mm until a follicle reached >32 mm, when an ovulatory dose of hCG (3000 IU) was given. Saline-treated mares during June and July were used as ovulatory controls. In a preliminary study, injection of this pituitary fraction (eLH) to anestrus mares was followed by an increase in circulating levels of LH (P < 0.01) but not FSH (P > 0.6). Administration of eLH during early transition stimulated the growth of the dominant follicle (Group x Day, P < 0.00001), which attained diameters similar to the dominant follicle in ovulatory controls (P > 0.1). In contrast, eLH had no effect on the diameter of the largest subordinate follicle or the number of follicles >10 mm during treatment (P > 0.3). The numbers of mares that ovulated in response to hCG in transitional control, transitional eLH and ovulatory control groups (2 of 2, 3 of 5 and 7 of 7, respectively) were not significantly different (P > 0.1). However, after hCG-induced ovulation, all transitional mares returned to an anovulatory state. Circulating estradiol levels increased during the experimental period in ovulatory controls but not in transitional eLH or transitional control groups (Group x Day, P = 0.013). In addition, although progesterone levels increased after ovulation in transitional control and transitional eLH groups, levels in these two groups were lower than in the ovulatory control group after ovulation (Group, P = 0.045). In conclusion, although LH supplementation of early transitional waves beginning after the largest follicle reached 20 mm promoted growth of ovulatory-size follicles, these follicles were developmentally deficient as indicated by their reduced steroidogenic activity.

Insights into the mechanism by which kisspeptin stimulates a preovulatory LH surge and ovulation in seasonally acyclic ewes: potential role of estradiol.

We have previously demonstrated that a constant intravenous infusion of kisspeptin (Kp) for 48 h in anestrous ewes induces a preovulatory luteinizing hormone (LH) surge followed by ovulation in approximately 75% of animals. The mechanisms underlying this effect are unknown. In this study, we investigated whether Kp-induced preovulatory LH surges in anestrous ewes were the result of the general activation of the whole gonadotropic axis or of the direct activation of central GnRH neurons required for the GnRH/LH surge. In the first experiment, a constant iv infusion of ovine kisspeptin 10 (Kp; 15.2 nmol/h) was given to 11 seasonally acyclic ewes over 43 h. Blood samples were taken every 10 min for 15 h, starting 5h before the infusion, and then hourly until the end of the infusion. We found that the infusion of Kp induced a well-synchronized LH surge (around 22 h after the start of the Kp infusion) in 82% of the animals. In all ewes with an LH surge, there was an immediate but transient increase in the plasma concentrations of LH, follicle-stimulating hormone (FSH), and growth hormone (GH) at the start of the Kp infusion. Mean (+/- SEM) concentrations for the 5-h periods preceding and following the start of the Kp infusion were, respectively, 0.33 +/- 0.09 vs 2.83 +/- 0.49 ng/mL (P = 0.004) for LH, 0.43 +/- 0.05 vs 0.55 +/- 0.03 ng/mL (P = 0.015) for FSH, and 9.34 +/- 1.01 vs 11.51 +/- 0.92 ng/mL (P = 0.004) for GH. In the first experiment, surges of LH were observed only in ewes that also had a sustained rise in plasma concentrations of estradiol (E(2)) in response to Kp. Therefore, a second experiment was undertaken to determine the minimum duration of Kp infusion necessary to induce such a pronounced and prolonged increase in plasma E(2) concentration. Kisspeptin (15.2 nmol/h) was infused for 6, 12, or 24h in seasonally acyclic ewes (N = 8), and blood samples were collected hourly for 28 h (beginning 5h before the start of infusion), then every 2h for the following 22 h. Kisspeptin infused for 24h induced LH surges in 75% of animals, and this percentage decreased with the duration of the infusion (12h = 50%; 6h = 12.5%). The plasma concentration of E(2) was greater in ewes with an LH surge compared to those without LH surges; mean (+/- SEM) concentrations for the 5-h period following the Kp infusion were, respectively, 2.23 +/- 0.16 vs 1.27 +/- 0.13 pg/mL (P < 0.001). Collectively, our results strongly suggest that the systemic delivery of Kp induced LH surges by activating E(2)-positive feedback on gonadotropin secretion in acyclic ewes.

Kisspeptin immunoreactive neurons in the equine hypothalamus Interactions with GnRH neuronal system.

To determine if kisspeptin could be implicated in the control of reproduction in equine species, we studied the distribution of kisspeptin neurons and their anatomical interactions with GnRH neurons in the hypothalamus of pony mares. Brains were collected in three pony mares between 2 and 4h after ovulation. One major population of kisspeptin immunoreactive cell bodies was found in the arcuate nucleus (ARC), where they extended from the middle of the nucleus to the premammillary recess. Kisspeptin immunoreactive varicose fibers extended from the preoptic area to the mammillary nuclei, with important densities especially in the anterior periventricular area and the median eminence (ME). Rare close appositions of kisspeptin fibres on GnRH cell bodies were observed in the ARC. Close appositions between kisspeptin and GnRH fibres were also confirmed at a low incidence in the anterior basal periventricular area and at a high incidence in the ME. This work provides neuroanatomical bases for further investigations into the role of kisspeptin in equine reproduction.

Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes.

We determined whether kisspeptin could be used to manipulate the gonadotropin axis and ovulation in sheep. First, a series of experiments was performed to determine the gonadotropic responses to different modes and doses of kisspeptin administration during the anestrous season using estradiol-treated ovariectomized ewes. We found that: 1) injections (iv) of doses as low as 6 nmol human C-terminal Kiss1 decapeptide elevate plasma LH and FSH levels, 2) murine C-terminal Kiss1 decapeptide was equipotent to human C-terminal Kiss1 decapeptide in terms of the release of LH or FSH, and 3) constant iv infusion of kisspeptin induced a sustained release of LH and FSH over a number of hours. During the breeding season and in progesterone-synchronized cyclical ewes, constant iv infusion of murine C-terminal Kiss1 decapeptide-10 (0.48 mumol/h over 8 h) was administered 30 h after withdrawal of a progesterone priming period, and surge responses in LH occurred within 2 h. Thus, the treatment synchronized preovulatory LH surges, whereas the surges in vehicle-infused controls were later and more widely dispersed. During the anestrous season, we conducted experiments to determine whether kisspeptin treatment could cause ovulation. Infusion (iv) of 12.4 nmol/h kisspeptin for either 30 or 48 h caused ovulation in more than 80% of kisspeptin-treated animals, whereas less than 20% of control animals ovulated. Our results indicate that systemic delivery of kisspeptin provides new strategies for the manipulation of the gonadotropin secretion and can cause ovulation in noncyclical females.

Differential estradiol requirement for the induction of estrus behavior and the luteinizing hormone surge in two breeds of sheep.

For a better understanding of the mechanisms that lead to the preovulatory GnRH/LH surge and estrus behavior, the minimum estradiol (E) requirements (dose and duration) to induce each of these events were determined and compared between two breeds of ewes having either single (Ile de France) or multiple (Romanov) ovulations. The ewes were initially studied during a natural estrus cycle, and were then ovariectomized and run through successive artificial estrus cycles. For these artificial cycles the duration and amplitude of the follucular phase E increase were manipulated by E implants. Under all conditions, the onset of estrus behavior was similar in the two breeds, although its duration was longer in Romanov ewes. While a moderate E signal (6 cm for 12 h) induced an LH surge in 10/10 Ile de France ewes, a larger E signal (12 cm for 12 h) was minimally effective in Romanov ewes (4/10). Additional studies revealed that a small E signal (3 cm for 6 h) induced full estrus behavior in all Romanov ewes but was completely ineffective in Ile de France animals (0/10). Higher doses and mostly longer durations of the E signal (12 cm for 24 h) were required to induce a surge in all the Romanov ewes. These results demonstrate a clear difference in the E requirement for the induction of estrus behavior and the LH surge between breeds of ewes that have different ovulation rates. These data provide compelling evidence that, in one breed, the neuronal systems that regulate both events require different estrogen signals.

High strain rate testing of kidney stones.

Sections of struvite kidney stones were tested in compression at high strain rates ( approximately 3000s(-1)) using a Kolsky bar and at low strain rates ( < 0.001 s(-1)) using an Instron testing machine. The peak stress in both cases appeared to be similar. At high strain rates the values of flow stress measured were between 40 and 65 MPa and at low strain rates they were between 37 and 58 MPa. However, the morphology of the damage was dramatically different. Stones tested at low strain rates formed a small number of cracks but otherwise remained intact at the end of the test. In comparison, stones tested at high strain rates were reduced to a powder. Kidney stones are a two-phase material consisting of a crystalline ceramic phase and an organic binder. We speculate that in the high strain rate tests the large difference in the sound speed between the matrix and the crystalline grains leads to shear stresses that destroy the stone. These data indicate that shear stress induced by the internal structure may be a mechanism by which shock waves comminute kidney stones in lithotripsy.

Kinetic studies and production rate of equine (e) FSH in ovariectomized pony mares. Application to the determination of a dosage regimen for eFSH in a superovulation treatment.

The appropriate dosage regimen for equine FSH (eFSH) (dose, dosing interval) administration in a superovulation treatment in pony mares was determined by a kinetic approach using production rates and kinetic parameters of elimination of the hormone. Two dosage regimens were then tested in superovulation protocols. The eFSH production rates were determined by sampling four ovariectomized pony mares every 10 min for 8 h during the breeding season. Kinetic parameters were determined by administering four dose levels of a preparation of eFSH (4.4, 8.8, 17.6 and 35.2 micro g/kg) by the i.v. route to the same mares, in a randomized 4x4 Latin Square protocol. The overall mean plasma clearance was 0.256+/- 0.07 ml.kg(-1).min(-1), and was independent of the dose. The mean residence time ranged from 5.5 to 10.8 h and increased with the dose. The estimated FSH production rates were 8.6 to 15.3 micro g.kg(-1).day(-1) (i.e. 2.89 to 3.45 mg per day per mare). Two dosage regimens of eFSH were then tested in cyclic mares (ten treated mares in each trial): 3.45 mg per day (4.4 micro g/kg three times a day by the i.v. route), which corresponds to the maximal daily production rate of the native hormone in ovariectomized mares, and 1.72 mg per day (2.2 micro g/kg three times a day), which corresponds to half of that production rate. The dosage regimen of 2.2 micro g/kg three times a day gave satisfactory results in terms of efficacy (numbers of ovulations and embryos) with minimal unwanted effects (luteinized or anovulatory follicles).

Attempt to control the day of ovulation in cycling pony mares by associating a GnRH antagonist with hCG.

With the objective of controlling the day of ovulation, 40 mares were assigned to a control or three treated groups: A3d, A4d, and A5d. The treated groups received antarelix (Teverelix 0.01 mg/kg, i.v., twice a day) for 3, 4, or 5 days from the day the dominant follicle (F1) reached 28 mm (=D0), and one injection of hCG (1600 IU, i.v.) on D1, D2, or D3, respectively. Control mares received one injection of hCG when F1 reached 35 mm. Plasma LH, FSH, progesterone, and total estrogens were assayed. In the A3d, A4d, and A5d groups, 9 (90%), 6 (60%), and 5 (50%) out of 10 mares, respectively, ovulated on the expected day (i.e. between 24 and 48 h after hCG injection). In the control group, 7/10 (70%) presented the typical response to hCG. For 3 mares in both the A4d and A5d groups, the dominant follicle at the time the treatment was started did not ovulate and ovulation was postponed for between 11 and 15 days after the end of treatment. In the treated mares, the LH surge was abolished, and total estrogens were depressed during the preovulatory peak but the concentrations of FSH were not modified. Endocrine parameters were not altered in postponed cycles. Fertility did not differ in treated and control cycles. These results demonstrate that in mares: (1) ovulation can be programmed on a specific day of a 3-day period, with a success rate of 67%, by a treatment associating antarelix and one injection of hCG; (2) nevertheless in 20% of cases the dominant follicle regresses and does not ovulate; (3) for these mares ovulation is postponed by approximately 2 weeks; (4) terminal growth of the preovulatory follicle only requires low circulating concentrations of LH but atresia induced by a GnRH antagonist is significant when this treatment is administrated for more than 18 h.

Use of a GnRH antagonist, antarelix, associated or not with hCG, to control ovulation in cyclic pony mares.

The GnRH antagonist antarelix (Teverelix) was administered to mares (0.01 mg/kg, i.v., twice a day) during the periovulatory period. In Experiment 1, 20 mares were divided into a treated (A3d-) and a control (Control-) group. A3d- mares received antarelix for 3 days from the day when the dominant follicle (F1) reached 32 mm (D0). In Experiment 2, 10 mares were divided into a treated (A6d+) and a control (Control+) group. A6d+ mares received antarelix for 6 days from D0 and hCG was injected in all animals (1600 IU, i.v.) on D1. Pregnancies were determined 13 days after ovulation. In both experiments, antarelix interrupted or totally abolished the LH surge. In Experiment 1, 5/10 of the A3d- mares (with maximum LH concentrations of 11.6 ng/ml at the beginning of treatment) ovulated at the same time as the Control- mares; the other five mares (with LH concentrations under 5.4 ng/ml) ovulated 13.4+/-0.6 days later. In Experiment 2, all the A6d+ mares ovulated at the same time as the Control+ mares. In treated mares which ovulated during the treatment, progesterone concentrations and fertility did not differ from control mares. These results demonstrate that in mares: (1) a small elevation of endogenous LH can induce ovulation, (2) ovulation can be postponed approximately 13 days after a 3-day antarelix treatment if initiated just before the preovulatory LH surge, (3) ovulation can be induced by hCG on depressed levels of endogenous LH, (4) the inhibition of the post ovulatory LH surge has no effect either on the corpus luteum or on fertility.

A comparison of the effect of estrogen with or without progesterone on mood and physical symptoms in postmenopausal women.

Using a randomized, placebo-controlled design, this study assessed the effects of estrogen alone (ES) or in combination with cyclic progesterone (EP) on daily ratings of mood and physical symptoms before and after 6 months of daily hormone treatment. Fifty-four postmenopausal women were recruited from the community at large and specifically selected as being asymptomatic at the time of enrollment and without significant psychiatric history. Ratings were obtained every day for 30 days prior to treatment and again every day during the last 30 days of treatment. Results revealed that when compared with pretreatment levels, women randomized to EP (n = 16) exhibited statistically significant increases in daily depression, cramping, and breast tenderness and a marginally significant increase in daily anxiety. However, these increases were mild, not clinically significant, and did not interfere with normal functioning. Women randomized to ES (n = 20) showed no significant change in daily mood measures, although they did experience an increase in breast tenderness with estrogen. A significant placebo (n = 18) effect was observed, as there was a reliable reduction in daily irritability ratings (p < 0.05) with placebo. These findings suggest that for most postmenopausal women, the use of hormones will not be associated with clinically significant changes in mood or physical symptoms, which weighs favorably into the cost-benefit ratio for women considering hormone replacement therapy.

[Determination of reference temperatures for a cold test in digital plethysmography]. / Détermination des températures de référence pour un test au froid par pléthysmographie digitale.

BACKGROUND: The purpose of this study was to determine optimal warm and cold temperatures for performance of a cold test. METHODS: Thirty-six healthy volunteers (22 women and 14 men; mean age 40, 94 years, range: 20-58) were included in the study. Plethysmographic signal amplitudes were evaluated at different temperatures (47, 45, 43, 40, 35, 20, 15, 13, 11 and 9 degrees C) under standardized conditions, using digital strain-gauge plethysmographic (Perivein Janssen). Reproducibility was assessed by repetition in the same subject of six digital plethysmography signal measurements at 45 degrees C performed at different times and days. Clinical tolerance was estimated according to an analogue scale. RESULTS: Maximal warm amplitude was obtained at 45 degrees C and maximal vasoconstriction at 13 degrees C. The thermal test was well tolerated for the temperature range between 11 and 45 degrees C. Reproducibility of the measurements was satisfactory. CONCLUSIONS: 45 degrees C is the non-nociceptive reproducible vasoparalyzing reference temperature, allowing measurement of maximal digital circulatory capacities. 11 degrees C should be adopted as the optimal vasoconstriction temperature for performance of a cold test.

Shapes of benz[a]anthracenes: the crystal and molecular structure of 2-methylbenz[a]anthracene.

The crystal structure of 2-methylbenz[a]anthracene (2-MBA), the least carcinogenically active of the monomethylbenz[a]anthracenes, has been determined by application of direct methods to single-crystal X-ray diffractometric data and refined by least squares to R = 0.033 (Rw = 0.035). Deviations of the carbon atoms from the mean molecular plane are much smaller than in the rather more active 1-MBA; in 2-MBA, the benzo-ring A is inclined at about 2 degrees to each of the three rings in the anthracene moiety and even the methyl carbon atom is displaced by only 0.07 A from the ring-carbon atom plane of 2-MBA (and by 0.01 A from the ring-A plane). As in other MBA, the shortest C-C bond in this accurately determined structure is at the K-region (C(5)-C(6) = 1.330(3) A) but three other bonds are short; C(8)-C(9) = 1.347(4), C(10)-C(11) = 1.353(3) and the M-region bond C(3)-C(4) = 1.359(4) A (0.003 A longer if corrected for rigid-body librations). The 2-methyl group appears to take up two orientations with one trio of hydrogen positions more favored than the other.

Molecular structure of 5,10-dimethoxybenzo[j]fluoranthene.

The molecular and crystal structure of the synthetic 5,10-dimethoxy derivative of the carcinogen benzo[j]fluoranthene has been determined by direct methods from X-ray diffractometric data and refined to an R index 0.041 over 2788 independent reflections. The benzo ring is inclined at approximately 3 degrees to the almost planar fluoranthene moiety (carbon atoms have r.m.s. deviation of 0.02 A from the carbon-atom plane); the methoxy carbon atoms lie within 0.2 A of the molecular plane. Corresponding bond lengths (e.s.d. 0.004 A for carbon-oxygen and carbon-carbon) and angles lie within 3 sigma for the two independent molecules, with mean dimensions of methoxy groups: C-C = 1.422, C-O = 1.372 A, C-O-C = 117.4 degrees.

The molecular structure of 11-methylbenz[a]anthracene: an almost planar substituted benz[a]anthracene.

The molecular structure of the almost inactive 11-methylbenz[a]anthracene has been determined by single-crystal X-ray diffraction analysis and refined to a final discrepancy index R of 0.036 over 955 independent reflections. With the benzo-ring A inclined at only 1.8 degrees to the furthest anthracene ring D, the benz[a]anthracene nucleus is much more nearly planar than in other (and carcinogenically more active) benz[a]anthracenes with bay-methyl substituents. Several carbon atoms in rings D and A deviate by approximately 0.05 A from the mean plane through the ring-carbon atoms of the bay-ring B. The shortest carbon-carbon bonds are C5-C6 = 1.341(5) A (at the K-region), C3-C4 = 1.355(5) and C8-C9 = 1.360(5) A, while C10-C11 next to the substituent is also quite short at 1.367(5) A.

Field ion microscopy of biomolecules.

A new type of image, existing only at field strengths below the denaturation field strengths of molecules, has been discorvered. This type of image has structure, is not symmetrical and thus differs from previously reported low field strength images. The possibility that macromolecules adsorbed on tip surfaces produce such structured images has been exhaustively investigated. The result is that no observation has been found which disproves this hypothesis and many tests conducted in such attempts yielded correlations consistent with this hypothesis. It is therefore concluded that it is highly probable that biomolecules produce structured images and that it is highly improbable that these correlations represent a chance event. On the other hand, these correlations may be due to some cause unknown to the authors; but we consider this possibility to be unlikely. Some micrographs have been obtained which provide a reasonable basis for the hope that tertiary structure information may be defrived from low field strength imaging of partially embedded biomolecules or of biomolecules that are resistant to field denaturation during imaging. Information may presently be obtained from analysis of low field strength ion micrographs about the size and shape of some biomolecules.