Photoperiod influences reproduction in the prairie vole (Microtus ochrogaster)

Four experiments examined the role of photoperiod in the regulation of seasonal breeding in the prairie vole. Adult male voles maintained in short (8L:16D) as compared to long (16L:8D) photoperiods for 10 wk had reduced testicular and seminal vesicle weights, but fertility was not impaired. Male prairie voles reared from birth until 35 days of age in short as compared to long photoperiods also had reduced testicular and seminal vesicle weights, as well as diminished fertility. The incidence of pregnancy did not differ between long- and short-day female voles paired for 6 days with long- or short-day males (93%, 86%, 89%, and 88%, respectively). Photoperiod did not affect the incidence or the timing of postpartum pregnancies in long- or short-day females paired with long-day males through the birth of several litters. Adult male prairie voles retain only marginal sensitivity to short photoperiods, maturing males are highly responsive to short days, and adult females are insensitive to photoperiod. These data suggest that termination of the breeding season in the autumn may be due to the lack of fecund males in the population.     

Effects of methyl parathion (o,o-dimethyl o-4-nitrophenyl phosphorothioate) on rat sperm morphology and sperm count, but not fertility, are associated with decreased ascorbic acid level in the testis

Methyl parathion (MP; o,o-dimethyl o-4-nitrophenyl phosphorothioate) is an organophosphorous pesticide used world wide to spray agricultural crops. The present study was aimed to investigate the genotoxic and cytotoxic effects on male germ cells and their possible relation with testicular ascorbic acid levels. Adult male Wistar rats (n=5/group) received MP at 0, 0.5, or 1 mg/kg (experiments 1 and 2) for 12 days and 0, 0.75 or 1.5 mg/kg (experiment 3) for 25 days (i.p.) everyday at intervals of 24 h. The epididymal sperm count, sperm abnormalities and testicular ascorbic acid levels (by 2,4-dinitrophenyl hydrazine method) were estimated on days 130, 77 and 17 following the last exposure in experiments 1, 2, and 3, respectively. Virgin untreated female rats were mated with treated males from experiments 2 and 3 for a week effective from day 35 to 41 after the first treatment, and fertility indices were measured after the birth of pups. Sperm count was decreased in experiments 2 and 3 (P<0.01), and in all three experiments, the abnormal sperms increased (P<0.001). Concomitantly, the ascorbic acid levels decreased in the testis (P<0.05-0.001; one-way ANOVA and Bonferroni's post hoc test). The body weights of offspring of treated males did not show significant changes from those of the controls, although there were some decreases observed. MP reduced the lactation index in experiment 2 (P<0.001; Chi-square test). The number of pups/parent along with fertility indices showed some numerical decrease but without any statistical significance. The present findings suggest that MP is a weak genotoxic and cytotoxic agent in the rat exposed to human exposure dose-levels, and that these effects, except the fertility are well correlated with decreased ascorbic acid level in the testis. Furthermore, MP-induced changes in the germ cells do not have any significant effects on F1 generation.     

Effects of prenatal X-irradiation on postnatal testicular development and function in the Wistar rat: development/teratology/behavior/radiation

It is evident that significant permanent tissue hypoplasia can be produced following radiation exposure late in fetal development. Because two organs, brain and testes, are developmentally and functionally interrelated, it was of interest to determine whether fetal testicular hypoplasia was a primary or a secondary effect of fetal brain irradiation. Twenty-four pregnant Wistar strain rats were randomly assigned to one of four groups, and a laparotomy was performed on day 18 of gestation. The fetuses received sham irradiation, whole body irradiation, or only head/thorax or pelvic body irradiation at a dosage level of 1.5 Gy. Mothers were allowed to deliver and raise their offspring until postnatal day 30, when the offspring were weaned. At 60 days of age, 74 male offspring were allowed to mate with colony control females of similar age until successful insemination or until the males reached 90 days of age, when they were killed. Testes were weighed and processed for histologic examination. Direct radiation of testes, due to whole body or pelvic exposure, resulted in testicular growth retardation and significantly reduced spermatogenesis. Breeding activity of the males and the percent of positive inseminations were also slightly reduced. However, a significant percentage of male offspring receiving direct testicular radiation did produce offspring. Head/thorax-only irradiation did not adversely affect testicular growth or spermatogenesis. Therefore, the use of histologic analysis as the sole determinant of infertility may be misleading. This study indicates that testicular growth retardation and an increased infertility rate result from direct prenatal exposure of rat testes to X-radiation and are not necessarily mediated via X-irradiation effects on the central nervous system.     

Is embryonic mortality increased in normal female rats mated to subfertile males?

Normal female rats were mated to control males or males which were subjected to unilateral testicular heating (43 degrees C for 30 min), irradiation (500 R), efferent duct ligation, arterial ligation or castration; in all males, the contralateral ductus deferens was ligated. All treatments caused reduced fertility and eventually infertility, as judged by the percentage of females becoming pregnant; the infertility was temporary after heating and irradiation. During the periods of reduced fertility, the numbers of fetuses per pregnant female and the fetus/corpus luteum ratios were reduced. In subsequent experiments, after heating of the testis, there was not only failure of fertilization despite the presence of normal numbers of spermatozoa in the uterus, but also an increased rate of embryonic degeneration in normal females. These results provide evidence that the male, while still fertile, can affect the fecundity of the female and the rate of embryo mortality.     

Genetic variation in fertility of heat-stressed male mice

The damaging effects of heat stress on male fertility are evident in developing spermatozoa expressed in ejaculates 18-28 days post-stress in mice. Our objectives were to: (1) assess genetic variation in fertility of heat-stressed male mice and (2) determine response to selection for fertility after heat stress in male mice. Mature male mice were exposed to heat stress (35+/-1 degrees C; n=50) or control (21+/-1 degrees C; n=10) conditions for 24h (day 0) and then hemicastrated for tissue collection. Two periods of mating tests followed, period 1 (from days 3 to 11) when no reductions in fertility were anticipated, and period 2 (days 18-26) when variation in fertility was expected. Period 2 pregnant females were sacrificed in late gestation. Males were indexed by multiplying overall mean ovulation rate by pre-implantation survival and number of pregnant period 2 mates. The five highest and five lowest ranking males were identified as heat stress resistant and susceptible, respectively. Resistant males were 61.2units superior in the index, 57.5% greater in pregnancy rate, and 57.6 total fetuses greater than susceptible males. Progeny of resistant sires were superior to progeny of susceptible sires in estimated breeding value by 4.5units for the index, 4.1% for pregnancy rate, and 5.2 fetuses (P<0.0001). Heritability estimates for the index, pregnancy rate, and number of fetuses ranged from 0.09 to 0.13, suggesting male fertility following heat stress is heritable and responds to selection.     

Effects of extremely low frequency magnetic field on fertility of adult male and female rats.

To investigate the effects of an extremely low-frequency (ELF) magnetic field on their fertility, adult male and female Sprague-Dawley rats were exposed to a 50 Hz sinusoidal magnetic field of approximately 25 microT (rms) for 90 days before they were mated with unexposed counterparts. Exposure to a 50 Hz field reduced male rat fertility. The number of pregnant females was reduced when mated with exposed males, and the number of resorptions increased. The effects of magnetic field on male fertility were shown to be partly reversible, when the same exposed group of males were remated 45 and 90 days after being removed from the fields. Exposure of adult female rats to 50 Hz magnetic fields for 90 days before mating significantly reduced their fertility. The mean numbers of implantations and living fetuses per litter were statistically significantly decreased in the 50 Hz group. These results suggest that low frequency magnetic fields have some adverse effects on fertility of male and female rats.     

Evaluation of propylene oxide for mutagenic activity in 3 in vivo test systems

Propylene oxide (CAS No. 75-56-9) was tested for mutagenic activity following vapor exposure using 3 in vivo test systems. Rat dominant lethal and mouse sperm-head morphology assays were conducted using males exposed to propylene oxide at 300 ppm in a dynamic exposure chamber for 7 h per day on 5 consecutive days. A sex-linked recessive lethal test in Drosophila melanogaster employed a 24-h static exposure to propylene oxide at 645 ppm. Male mice were killed 1, 3, 5, 7, and 9 weeks post-exposure for evaluation of sperm-head morphology. Propylene oxide exposure did not result in an increase in abnormal forms. Male rats were mated with 2 virgin females per week for 6 weeks following exposure. A statistically significant increase in preimplantation losses and a statistically significant reduction in the number of living implants in the first post-exposure week did not appear to be treatment related. A highly significant increase in sex-linked recessive lethal mutations was observed in two germ cell stages (mature sperm and developing spermatocytes). These results warrant continued caution in potential human exposure to propylene oxide.     

Effects of expression of human or bovine growth hormone genes on sperm production and male reproductive performance in four lines of transgenic mice.

Reproductive performance was studied in transgenic males from lines expressing and transmitting four hybrid genes: mouse metallothionein-I/human growth hormone (GH) (MT/hGH), MT/hGH placental variant (MT/hGH.V), MT/bovine GH (MT/bGH) and phosphoenolpyruvate carboxykinase/bGH (PEPCK/bGH). Each male was exposed to three normal females for 1 week and to three different normal females for another week. Females were examined for vaginal plugs and necropsied on day 14 of pregnancy. Males were killed for analysis of organ weights, numbers of testicular spermatids, numbers of epididymal sperm and measurements of plasma glucose concentration. Fertility of MT/hGH and MT/hGH.V transgenic males was significantly lower than in normal males, primarily because most males failed to impregnate any females. In females that became pregnant, the numbers of corpora lutea, total fetuses and live fetuses did not differ from those in females mated to normal (nontransgenic) males. Fetal crown-rump length on day 14 of pregnancy did not differ between litters sired by normal or by transgenic males. Weights of testes and seminal vesicles were significantly greater in all four types of transgenic male, but daily sperm production per unit weight (g-1) of testis was not affected and epididymal sperm reserves were either normal or slightly higher than normal. Plasma glucose concentrations were significantly higher in PEPCK/bGH mice than in other mice. Average or individual reproductive performance of transgenic males from the various lines did not correlate with any of the parameters examined except for significantly heavier seminal vesicles in MT/hGH and MT/hGH.V males than in normal males; these transgenic males exhibited a high incidence of infertility. Since hGH and hGH.V, but not bGH, are lactogenic in rodents, it was concluded that chronic stimulation of GH and prolactin receptors by ectopically produced human GHs in transgenic mice compromises male fertility by an unknown mechanism. Reduced fertility of transgenic males with MT/hGH or MT/hGH.V hybrid genes is due to failure to inseminate or impregnate females rather than to reduced numbers of spermatozoa or gross changes in the male reproductive system.     

Time course of androgenic modulation of odor preferences and odor cues in male meadow voles, Microtus pennsylvanicus.

During the breeding season, male meadow voles prefer female over male odors and females prefer male over female odors. Testosterone control of males' odor preferences and production of odors attractive to females differ. A male meadow vole's preference for female versus male odor was still evident 1 week after castration, but not 1 week later. This preference was reinstated in testosterone-treated male voles 2 weeks after the onset of hormone replacement. The attractiveness of male odors to females did not disappear until 3 weeks after castration. The attractiveness of male odors was reinstated 1 week after castrated males were treated with testosterone. The time course for the androgenic modulation of production of odors attractive to females may facilitate breeding. For example, at the end of the breeding season males may emit an odor that is still attractive to females. Similarly, at the beginning of the breeding season males may emit an odor that is attractive to females.     

Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken

Transplantation of male germ cells into sterilized recipients has been widely used in mammals for conventional breeding and transgenesis purposes. This study presents a workable approach for germ cell transplantation between male chickens. Testicular cells from adult and prepubertal donors were dispersed and transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. In comparison to males transplanted with testicular cell preparations from adult donors, in which the first ejaculates with sperm were recovered about 5 wk after transfer, a substantial interval (about 10 wk) was necessary to obtain ejaculates after the transfer of testicular cells from prepubertal donors. However, in both cases, recipient males produced ejaculates capable of fertilizing ova and producing progeny expressing donor genes.     

Reproductive toxicity and pharmacokinetics of di-n-butyl phthalate (DBP) following dietary exposure of pregnant rats

Most rodent developmental toxicity studies of dibutylphthalate (DBP) have relied on bolus gavage dosing. This study characterized the developmental toxicity of dietary DBP. Pregnant CD rats were given nominal doses of 0, 100, or 500 mg DBP/kg/day in diet (actual intake 0, 112, and 582 mg/kg/day) from gestational day (GD) 12 through the morning of GD 19. Rats were killed 4 or 24 hr thereafter. DBP dietary exposure resulted in significant dose-dependent reductions in testicular mRNA concentration of scavenger receptor class B, member 1; steroidogenic acute regulatory protein; cytochrome P450, family 11, subfamily a, polypeptide 1; and cytochrome P450 family 17, subfamily a, polypeptide 1. These effects were most pronounced 4 hr after the end of exposure. Testicular testosterone was reduced 24 hr post-exposure in both DBP dose groups and 4 hr after termination of the 500-mg DBP/kg/day exposure. Maternal exposure to 500 mg DBP/kg/day induced a significant reduction in male offspring's anogenital distance indicating in utero disruption of androgen function. Leydig cell aggregates, increased cord diameters, and multinucleated gonocytes were present in DBP-treated rats. Monobutyl phthalate, the developmentally toxic metabolite of DBP, and its glucuronide conjugate were found in maternal and fetal plasma, amniotic fluid, and maternal urine. Our results, when compared to previously conducted gavage studies, indicate that approximately equal doses of oral DBP exposure of pregnant rats, from diet or gavage, result in similar responses in male offspring. Birth Defects Res (Part B), 86:345–354, 2009. © 2009 Wiley-Liss, Inc.     

Chronic effects of cadmium on kidney,liver, testis,and fertility of male rats

Male Wistar rats (n:20), at 5 wk of age, were given cadmium in drinking water (10 mg/L water) for 52 wk; 8 males and 20 female rats, as controls, were given tap water. At the end of 28 and 40 wk, some of the cadmium-treated males and control group male rats were sacrificed for the histopathological examination of testis, kidney, and liver. At the end of 56 wk, histopathological examinations were performed in the same way. Liver, kidney, and testis cadmium levels were also determined by atomic absorption spectrophotometry. All the cadmium-treated male rats showed pathological testicular alterations, and liver and kidney damage after chronic exposure. Cadmium levels were found to be highest in the kidney (1.009 +/- 0.034 microgram/g wet tissue in the infertile group). At the end of the 52-wk period, reproductive capacity of the cadmium-treated rats was investigated and was found to be lost in 39.89% of the animals.     

Termination of gonadal refractoriness in Turkish hamsters, Mesocricetus brandti

The Turkish hamster (Mesocricetus brandti) is a photoperiodic species. In this investigation, we characterized the photoperiodic requirements for termination of gonadal refractoriness, defined as the inability of the animal to respond to short-day treatment with gonadal regression. Paired testes weights were reduced to less than 20% of their original weight by 10 wk of 12L:12D treatment. This was followed by spontaneous testicular recrudescence (completed by Week 25 of 12L:12D treatment), the overt indication of refractoriness to short photoperiods. Next, the period of long-day exposure sufficient for termination of refractoriness was determined. Refractory males were exposed to 16L:8D for 5 to 20 wk. Ten weeks of 16L:8D treatment was enough for the animals to regain the sensitivity to a second challenge of 12L:12D treatment. Fifteen weeks of 20L:4D or 16L:8D terminated refractoriness in female Turkish hamsters; 20L:4D therefore was not interpreted as a short day by refractory hamsters. This was unexpected because in photosensitive animals this photoperiod acts like a short day, causing gonadal regression. These results suggest that Turkish hamsters are similar to Syrian hamsters in that both species require two or more months of long days in summer to recover sensitivity to the short days of the following fall.     

Tests of mutagenesis and reproduction in male rats exposed to 2,450-MHz (CW) microwaves

Tests of mutagenesis and reproduction were conducted in male rats which were irradiated by 2,450-MHz, continuous-wave (CW) microwaves, 4 hr/day from day 6 of gestation to 90 days of age at 5 mW/cm²; or 5 hr/day for five days beginning on the 90th day of age at 10 mW/cm²; or 4 hr/day, 5 days/ wk for four weeks, beginning on the 90th day of age. During selected weekly periods after treatment, the rats were bred to pairs of untreated, normal female rats that were examined in late pregnancy by means of the dominant lethal assay. The reproductive efficiency of these males, as reflected in their breeding, was also examined for changes relating to their microwave experience. No significant evidence of germ-cell mutagenesis was detected when data of microwave-exposed males were compared with those of sham-exposed males, even though there were significant increases in rectal and intra-testicular temperatures at a power density of 28 mW/cm². Temporary sterility, as indexed by fewer pregnancies, was seen at the highest power density.     

Relationship between longevity of spermatozoa after insemination and the percentage of normal embryos in brown marsupial mice (Antechinus stuartii)

Twenty-six female brown marsupial mice in a laboratory colony were mated at intervals ranging from 1 to 20 days between coitus and ovulation. The numbers of corpora lutea and normal embryos were counted. A multiple regression model examined the parabolic relationship between the proportion of normal embryos and the time from coitus to ovulation. The proportion of normal embryos increased until a mean of 9.5 days and decreased thereafter. This relationship was independent of the year of breeding and the number of corpora lutea. After survival of spermatozoa for up to 13 days in the female reproductive tract, the fertility levels of females was 88-92%. Low fertility levels after 13 days appeared to be due to a decrease in the number of spermatozoa. Reproductive tracts from 7 females killed after insemination and examined histologically showed many spermatozoa in the isthmus of the oviduct and the uterus at 5 days post coitum; spermatozoa confined to the isthmus between 6 and 13 days; and few spermatozoa in the isthmus at 14 days after copulation. A comparison between the fertility levels in the females which had been inseminated once and a further 17 females which had been inseminated 2 or 3 times suggested that spermatozoa from 2nd and 3rd inseminations can contribute spermatozoa for fertilization. In these females fertility levels did not decline with time after the first mating.     

Evaluation of bull fertility in dairy and beef cattle using cow field data

A successful outcome to a given service is a combination of both male and female fertility. Despite this, most national evaluations for fertility are generally confined to female fertility with evaluations for male fertility commonly undertaken by individual breeding organisations and generally not made public. The objective of this study was to define a pertinent male fertility trait for seasonal calving production systems, and to develop a multiple regression mixed model that may be used to evaluate male fertility at a national level. The data included in the study after editing consisted of 361,412 artificial inseminations from 206,683 cow-lactations (134,911 cows) in 2,843 commercial dairy and beef herds. Fixed effects associated with whether a successful pregnancy ensued (pregnant = 1) or not (pregnant = 0) from a given service were year by month of service, day of the week, days since calving, cow parity, level of calving difficulty experienced, whether or not the previous calving was associated with perinatal mortality, and age of the service bull at the date of insemination. Non-additive genetic effects such as heterosis and recombination loss as well as inbreeding level of the service bull, dam or mating were not associated with a successful pregnancy; there was no difference in pregnancy rate between fresh or frozen semen. Random effects included in the model were the additive genetic effect of the cow, as well as a within lactation and across lactation permanent environmental effect of the cow; pedigree group effects based on cow breed were also included via the relationship matrix. Temporal differences in the AI technician and service bull were also included as random effects. A difference in five percentage units in male fertility was evident between the average effects of different dairy and beef breeds. The correlation between raw pregnancy rates for bulls with more than 100 services (n = 431) and service bull solutions from the mixed model analysis was 0.66. The correlation between the raw pregnancy rates of 288 technicians with more than 100 services and their respective solutions from the mixed model was 0.35. These low to moderate correlations suggest considerable re-ranking among both service bulls and technicians and suggest possibly a benefit of using a statistical model to better estimate the performance of both service bulls and technicians.     

Suppression of testicular maturation and fertility following androgen administration to neonatal male rats

Administration of supraphysiologic doses of androgen to male rats within the first few neonatal days markedly suppresses subsequent testicular maturation; this effect diminishes as androgen is injected on succeeding postnatal days. Testosterone propionate (TP) administered neonatally at dosages up to 3.5 mg appreciably diminished postnatal testicular growth; postpubertal androgen secretion, as assessed by accessory sex organ weights and serum testosterone concentrations and as reflected by a castrationlike developmental pattern of the hepatic enzyme, histidase; spermatogenesis; and fertility. Beyond three mo of age testicular growth rates and androgen secretion--but not fertility--tended to be restored. These effects of neonatal androgen do not require aromatization to estrogen; indeed 5 alpha-dihydrotestosterone elicited more profound testicular suppression than TP, which was sustained until at least 100 days of age. Testes of neonatally androgenized rats were capable of responding to gonadotropins administered at three wk of age with increases in weight and androgen secretion. These findings suggest that a developmental event, suppressible by pharmacologic doses of androgen, occurs at a nontesticular site during the first few post partum days in the male rat; this event programs subsequent testicular maturation.     

Alpaca semen characteristics under free and directed mounts during a mating period

Most studies in alpaca reproductive biology have been focused on female physiology. Only recent research is being conducted in order to increase the knowledge on males. Semen characteristics during breeding periods will contribute to understanding the poor fertility rates in alpaca.

Ten adult male alpacas were distributed randomly into two groups and submitted alternatively to two regimens of semen collection of 12 days duration (day 1, initial day of semen collection). Semen samples were collected using an artificial vagina and a receptive, non-pregnant female. With regimen 1, males were maintained with females except for the days of sexual rest (6 and 7). Semen was collected on days 1, 5, 8 and 12. With regimen 2, males were exposed to females for daily semen collection only, before and after sexual rest. Mating duration, color and volume of ejaculates, spermatozoa concentration and morphology were evaluated.

No statistical differences for the variables were found between regimens that were used for semen collection. With respect to influence of day, however, the total numbers of spermatozoa ejaculated on days 1 and 5 of semen collection were statistically different (p < 0.05). Azoospermic samples increased on days 5 and 12 of semen collection. Partial recovery in spermatozoa concentration and number of spermatozoa ejaculated were observed after sexual rest. Although normal spermatozoa percentage was less on day 1 (p < 0.05) as compared with values found in the following ejaculates (days 5 and 12), the total number of normal spermatozoa was greater.

These results support the conclusion that when male alpaca have a daily ejaculation during five consecutive days, they might copulate without having enough spermatozoa for fertilization towards the end of the mating period.     

Sterilisation of the potato moth, Phthorimaea operculella (Lepidoptera: Gelechiidae) by fumigation with thiotepa

The chemosterilant thiotepa was used in fumigation chambers for sterilisation of male and female potato moths. Its effectiveness was registered by recording egg production, egg hatch, longevity of moths, and mating frequency. Also studied was the effect on the persistence of the chemical of temperature, exposure time and concentration. The tests showed that it was possible to sterilise fully male and female moths with thiotepa without adverse effects on mating and longevity. Generally, males were more susceptible to sterilisation than females. No definite relationship was found between dosage and egg production, longevity and mating frequency of moths. The compound did not lose its sterilising activity when used successively up to five times during the same day or once weekly for a total of 4 wk. The sterilising activity was related to temperature, exposure time, concentration and rates of airflow of the circulatory fumigation chambers. Some reduction of fertility occurred when F, adults paired with untreated moths.     

Leptin levels and body composition of mice selectively bred for high voluntary locomotor activity

Selective breeding produced four replicate lines of high-runner (HR) mice that run on wheels for approximately 2.7 times more revolutions per day than four unselected control lines. Previous studies found that HR mice of both sexes have lower body fat (isotope dilution at 15 wk of age) and that males (females not studied) have smaller retroperitoneal fat pads (17 wk). HR mice also exhibit elevated plasma corticosterone and insulin-stimulated glucose uptake by some hindlimb muscles but apparently do not differ in circulating insulin or glucose levels (males at 18 wk). Given their lower body fat and higher activity levels, we hypothesized that HR mice would have lower circulating leptin levels than controls. Female mice were given wheel access for 6 d at 7 wk of age, as part of the routine wheel testing for the selective breeding protocol, and then were killed after one additional week without wheels to reduce possible acute effects of activity on leptin. As hypothesized, serum leptin levels were significantly lower in HR mice. ANCOVA indicated that leptin was strongly positively correlated with both total body fat (measured by ether extraction) and body mass change from weaning, but HR mice still had significantly lower adjusted leptin levels (ANCOVA). Within HR lines but not within control lines, individual variation in leptin levels was negatively correlated with amount or speed of wheel running measured a week before being killed. Growth from weaning to euthanasia and body dry mass were lower in HR mice than in controls, but absolute dry masses of the ventricles, liver, gut, and uterus plus ovaries did not significantly differ, nor did percentage of the total dry mass as fat. HR mice offer a novel model for studying the causes and consequences of physiologically relevant variations in serum leptin.